Phytochemicals in Cancer Prevention and Therapy

BioMed Research International

Phytochemicals in Cancer Prevention and Therapy Guest Editors: Poyil Pratheeshkumar, Young-Ok Son, Preethi Korangath, Kanjoormana Aryan Manu, and Kodappully Sivaraman Siveen

Phytochemicals in Cancer Prevention and Therapy


Phytochemicals in Cancer Prevention and Therapy Guest Editors: Poyil Pratheeshkumar, Young-Ok Son, Preethi Korangath, Kanjoormana Aryan Manu, and Kodappully Sivaraman Siveen

Copyright © 2015 Hindawi Publishing Corporation. All rights reserved. This is a special issue published in “BioMed Research International.” All articles are open access articles distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Contents Phytochemicals in Cancer Prevention and Therapy, Poyil Pratheeshkumar, Young-Ok Son, Preethi Korangath, Kanjoormana Aryan Manu, and Kodappully Sivaraman Siveen Volume 2015, Article ID 324021, 2 pages Black Rice Anthocyanins Suppress Metastasis of Breast Cancer Cells by Targeting RAS/RAF/MAPK Pathway, Xiang-Yan Chen, Jie Zhou, Li-Ping Luo, Bin Han, Fei Li, Jing-Yao Chen, Yan-Feng Zhu, Wei Chen, and Xiao-Ping Yu Volume 2015, Article ID 414250, 11 pages Coevolution between Cancer Activities and Food Structure of Human Being from Southwest China, Yawen Zeng, Juan Du, Xiaoying Pu, Jiazhen Yang, Tao Yang, Shuming Yang, and Xiaomeng Yang Volume 2015, Article ID 497934, 10 pages Systematic Review of the Use of Phytochemicals for Management of Pain in Cancer Therapy, Andrew M. Harrison, Fabrice Heritier, Bennett G. Childs, J. Michael Bostwick, and Mikhail A. Dziadzko Volume 2015, Article ID 506327, 8 pages Antiproliferative and Antiestrogenic Activities of Bonediol an Alkyl Catechol from Bonellia macrocarpa, Rosa Moo-Puc, Edgar Caamal-Fuentes, Sergio R. Peraza-Sánchez, Anna Slusarz, Glenn Jackson, Sara K. Drenkhahn, and Dennis B. Lubahn Volume 2015, Article ID 847457, 6 pages Curcumin Enhanced Busulfan-Induced Apoptosis through Downregulating the Expression of Survivin in Leukemia Stem-Like KG1a Cells, Guangyang Weng, Yingjian Zeng, Jingya Huang, Jiaxin Fan, and Kunyuan Guo Volume 2015, Article ID 630397, 16 pages PLK-1 Targeted Inhibitors and Their Potential against Tumorigenesis, Shiv Kumar and Jaebong Kim Volume 2015, Article ID 705745, 21 pages Garcinia dulcis Fruit Extract Induced Cytotoxicity and Apoptosis in HepG2 Liver Cancer Cell Line, Mohd Fadzelly Abu Bakar, Nor Ezani Ahmad, Monica Suleiman, Asmah Rahmat, and Azizul Isha Volume 2015, Article ID 916902, 10 pages Effect and Mechanism of Total Flavonoids Extracted from Cotinus coggygria against Glioblastoma Cancer In Vitro and In Vivo, Gang Wang, JunJie Wang, Li Du, and Fei Li Volume 2015, Article ID 856349, 9 pages Antiproliferative Activity of T. welwitschii Extract on Jurkat T Cells In Vitro, Batanai Moyo and Stanley Mukanganyama Volume 2015, Article ID 817624, 10 pages HPTLC Analysis of Bioactivity Guided Anticancer Enriched Fraction of Hydroalcoholic Extract of Picrorhiza kurroa, Md. Nasar Mallick, Mhaveer Singh, Rabea Parveen, Washim Khan, Sayeed Ahmad, Mohammad Zeeshan Najm, and Syed Akhtar Husain Volume 2015, Article ID 513875, 18 pages

The Ethanolic Extract of Taiwanofungus camphoratus (Antrodia camphorata) Induces Cell Cycle Arrest and Enhances Cytotoxicity of Cisplatin and Doxorubicin on Human Hepatocellular Carcinoma Cells, Liang-Tzung Lin, Chen-Jei Tai, Ching-Hua Su, Fang-Mo Chang, Chen-Yen Choong, Chien-Kai Wang, and Cheng-Jeng Tai Volume 2015, Article ID 415269, 10 pages Anti-Inflammatory and Anticancer Activities of Taiwanese Purple-Fleshed Sweet Potatoes (Ipomoea batatas L. Lam) Extracts, Marcelia Sugata, Chien-Yih Lin, and Yang-Chia Shih Volume 2015, Article ID 768093, 10 pages

Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 324021, 2 pages http://dx.doi.org/10.1155/2015/324021

Editorial Phytochemicals in Cancer Prevention and Therapy Poyil Pratheeshkumar,1 Young-Ok Son,2 Preethi Korangath,3 Kanjoormana Aryan Manu,4 and Kodappully Sivaraman Siveen5 1

Department of Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, KY 40536, USA Cell Dynamics Research Center and School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju 61005, Republic of Korea 3 Breast Cancer Program, School of Medicine, Johns Hopkins University, Baltimore, MD 21231, USA 4 Division of Cancer and Stem Cell Biology, DUKE-NUS Graduate Medical School, Singapore 169857 5 Translational Research Institute, Academic Health System, Hamad Medical Corporation, P.O. Box 3050, Doha, Qatar 2

Correspondence should be addressed to Poyil Pratheeshkumar; [email protected] Received 25 October 2015; Accepted 25 October 2015 Copyright © 2015 Poyil Pratheeshkumar et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Despite advances in modern medicine, cancer is still the major cause of mortality in both developing and developed countries. Search for safer and more effective chemoprevention and treatment strategy is a need for the improvement of patient care in the field. Prevention may be more effective and less costly because cancer is largely a preventable disease which could be attributed to a greater extent to lifestyle. Dietary phytochemicals have been used for the treatment of cancer throughout history due to their safety, low toxicity, and general availability. Population based studies suggest that a reduced risk of cancer is associated with high consumption of vegetables and fruits. Promising phytochemicals not only disrupt aberrant signaling pathways leading to cancer but also synergize with chemotherapy and radiotherapy. Thus, the cancer chemoprevention and therapeutic potential of naturally occurring phytochemicals are of great interest. In this special issue we have collected many interesting original research articles and reviews that provide solid evidence to support the application of phytochemicals or dietary agents in prevention and treatment of cancer. This special issue contains 3 review articles and 9 original peer-reviewed papers. A. M. Harrison et al. performed a systematic review of the biomedical literature for the use of phytochemicals for management of cancer therapy pain in human subjects; X.-Y. Chen et al. explored the molecular mechanisms underlying the antimetastatic activity of black

rice anthocyanins and identified its molecular targets in HER2+ breast cancer cells; the study by Y. Zeng et al. reports that Southwest China (especially Yunnan and Tibet) is the center of lowest mortality of cancers in China based on coevolution between human’s anticancer activities and functional foods from crop origin center; M. Sugata et al. studied the anti-inflammatory and anticancer activities of Taiwanese purple-fleshed sweet potatoes (Ipomoea batatas L. Lam.) extracts; R. Moo-Puc et al. investigated the antiproliferative activity of bonediol, an alkyl catechol isolated from the Mayan medicinal plant Bonellia macrocarpa against human prostate tumor cells; B. Moyo and S. Mukanganyama demonstrate the antiproliferative potential of T. welwitschii extract on Jurkat T cells; C.-J. Tai et al. report the potential of ethanolic extract of Taiwanofungus camphoratus (Antrodia camphorata) to enhance the cytotoxicity of cisplatin and doxorubicin on human hepatocellular carcinoma cells; G. Wang et al. explored the molecular mechanism of total flavonoids extracted from Cotinus coggygria against glioblastoma cancer in vitro and in vivo; M. N. Mallick et al. studied the anticancer activity of hydroalcoholic extract of Picrorhiza kurroa and its fractions; M. F. Abu Bakar et al. demonstrate that the Garcinia dulcis fruit extract induced cytotoxicity and apoptosis in HepG2 liver cancer cells; G. Weng et al. reported the curcumin enhanced busulfan-induced apoptosis in leukemia stem-like KG1a cells via downregulating the expression of

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survivin; S. Kumar and J. Kim in their review discuss potency and selectivity of PLK-1-targeted inhibitors and their molecular interactions with PLK-1 domains. In conclusion, this special issue discussed the potential anticancer phytochemicals and dietary agents, their molecular targets, and their mechanisms of actions. The understanding of molecular mechanism of a specific plant derived compound against a particular type of cancer will lead to the invention of novel drug and drug targets for therapeutic intervention.

Acknowledgments The guest editorial team would like to thank all external reviewers for their expert assistance and all authors who submitted their work to the issue. Poyil Pratheeshkumar Young-Ok Son Preethi Korangath Kanjoormana Aryan Manu Kodappully Sivaraman Siveen


Research Article Black Rice Anthocyanins Suppress Metastasis of Breast Cancer Cells by Targeting RAS/RAF/MAPK Pathway Xiang-Yan Chen, Jie Zhou, Li-Ping Luo, Bin Han, Fei Li, Jing-Yao Chen, Yan-Feng Zhu, Wei Chen, and Xiao-Ping Yu Department of Public Health, Chengdu Medical College, Chengdu 610500, China Correspondence should be addressed to Xiao-Ping Yu; [email protected] Received 29 July 2015; Revised 13 October 2015; Accepted 18 October 2015 Academic Editor: Pratheeshkumar Poyil Copyright © 2015 Xiang-Yan Chen et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Overexpression of human epidermal growth factor receptor 2 (HER2) drives the biology of 30% of breast cancer cases. As a transducer of HER2 signaling, RAS/RAF/MAPK pathway plays a pivotal role in the development of breast cancer. In this study, we examined the molecular mechanisms underlying the chemopreventive effects of black rice anthocyanins (BRACs) extract and identified their molecular targets in HER2+ breast cancer cells. Treatment of MDA-MB-453 cells (HER2+ ) with BRACs inhibited cell migration and invasion, suppressed the activation of mitogen-activated protein kinase kinase kinase (RAF), mitogen-activated protein kinase kinase (MEK), and c-Jun N-terminal kinase (JNK), and downregulated the secretion of matrix metalloproteinase 2 (MMP2) and MMP9. BRACs also weakened the interactions of HER2 with RAF, MEK, and JNK proteins, respectively, and decreased the mRNA expression of raf, mek, and jnk. Further, we found combined treatment with BRACs and RAF, MEK, or JNK inhibitors could enhance the antimetastatic activity, compared with that of each treatment. Transient transfection with small interfering RNAs (siRNAs) specific for raf, mek, and jnk inhibited their mRNA expression in MDA-MB-453 cells. Moreover, cotreatment with BRACs and siRNA induces a more remarkable inhibitory effect than that by either substance alone. In summary, our study suggested that BRACs suppress metastasis in breast cancer cells by targeting the RAS/RAF/MAPK pathway.

1. Introduction Breast cancer has the highest incidence rate of cancers among females in China [1]. Previous studies have shown that the human epidermal growth factor receptor 2 (HER2) was amplified or overexpressed in about 20–30% of breast cancers [2]. Furthermore, an epidemiological study found that HER2-overexpressing breast cancer is associated with a particularly aggressive form of the disease and poor prognosis [3]. Progress in this field in recent years has uncovered a plethora of mechanisms leading to the downstream signaling pathways of the HER2/neu receptor, including the phosphatidylinositol 3-kinase (PI-3K)/Akt, mitogenactivated protein kinase (MAPK), and the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathways [4]. Simultaneous, expression and activation of the RAS/RAF/MAPK pathway (Mitogen activated protein kinase pathway) play an important role in the development and progression of breast cancer [5].

Anthocyanins are natural phytochemicals, which are abundantly found in black rice and are bioactive dietary agents. They have received considerable attention owing to their numerous potential health benefits including interference with several processes involved in cancer development and progression [6]. In addition, our previous studies have revealed the antiangiogenic effects of black rice anthocyanins (BRACs) extract using in vitro and in vivo model systems [7]. We recently showed that BRACs suppressed HER2+ breast cancer lung metastasis in a mouse model, and similar antimetastasis effects were seen in HER2+ breast cancer MDA-MB-453 cells treated with 200 𝜇g/mL BRACs [8]. However, the molecular mechanisms underlying the antimetastatic effects of BRACs have not been explored. Therefore, the goal of the present study was to determine the effects of BRACs on cell migration and invasion in MDA-MB453 cells and evaluate the molecular mechanisms and possible involvement of RAS/RAF/MAPK in underlying the effects of the extract.

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2. Materials and Methods 2.1. Chemicals and Reagents. BRA-90 anthocyanins (BRACs) extract was purchased from New Star (Jilin, China). Antibodies against total HER2, total K-RAS, total extracellular signal-regulated kinase 1/2 (ERK1/2), total RAF1, and phosphorylated (p)-RAF1 (Ser259) were obtained from Abcam (Cambridge, UK). Antibodies against total mitogenactivated protein kinase 1 (MEK1), p-MEK1/2 (Ser218/222), p-ERK (Thr202/Tyr204), total c-Jun N-terminal kinase 1/2 (JNK1/2), p-JNK1/2 (Thr183/Tyr185), matrix metalloproteinase (MMP2), and MMP9 were obtained from Millipore (Billerica, MA, USA). Polyethylene terephthalate membrane (8 𝜇m pore size) Millicell hanging cell culture inserts were purchased from Millipore. BD Matrigel Basement Membrane Matrix was obtained from Becton and Dickinson (Franklin Lakes, NJ, USA). The RAF inhibitor (ZM336372) was purchased from Santa Cruz Biotech (Dallas, TX, USA), MEK inhibitor (U0126) was purchased from Cell Signaling Technology (Danvers, MA, USA), and JNK inhibitor (SP600125) was purchased from Tocris (Bristol, UK). The SYBR Select Master Mix (category number 4472908), raf, mek, jnk, and glyceraldehyde 3-phosphate dehydrogenase (gapdh) universal primers, Trizol Reagent, and Lipofectamine 3000 were purchased from Invitrogen (Carlsbad, CA, USA), and the cDNA Synthesis Kit was purchased from TaKaRa Bio (Otsu, Japan). 2.2. Cell Culture. The human breast cancer cell lines MCF10A and MCF-7 (HER2− ) were kindly provided by Dr. Man-Tian Mi (Third Military Medical University of China, Chong Qing, China). MDA-MB-453 (estrogen receptor [ER]− , HER2/neu+ ) cells were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). MCF-7 and MDA-MB-453 cells were separately cultured in Dulbecco’s modified Eagle’s medium (DMEM)/high glucose or L-15 medium containing 10% (v/v) fetal bovine serum (FBS). MCF-10A cells were cultured in DMEM/F12 medium containing 5% (v/v) horse serum in the presence of 10 𝜇g/mL insulin, 20 ng/mL epidermal growth factor (ECF), 100 ng/mL cholera toxin, and 0.5 𝜇g/mL hydrocortisone. All cells were incubated at 37∘ C in a humidified atmosphere with 5% carbon dioxide (CO2 ). 2.3. Wound Healing (Scratch) Assay. MDA-MB-453 and MCF-10A cells were grown on 35 mm dishes to 100% confluence and then scratched using sterile pipette tips to form a wound. The cells were then treated with BRACs (0 or 200 𝜇g/mL) in the presence or absence of the RAF/MAPK inhibitors (ZM336237, U0126, and SP125600) and siRNA (raf -siRNA, mek-siRNA, or jnk-siRNA) for 24 h. The images were recorded using a photomicroscope (Olympus, Tokyo, Japan). 2.4. Cell Invasion Assay. The cell invasion assay was performed using a polyethylene terephthalate membrane (8 𝜇m pore size) with Millicell hanging cell culture inserts and BD Matrigel. BRACs (200 𝜇g/mL) and RAF/MAPK pathway inhibitors were used alone or combined with treat the cells (MDA-MB-453 and MCF-10A cells loaded into the chamber).

BioMed Research International Conditioned medium containing 10% (v/v) FBS was added to each of the wells, and the cells were allowed to invade by penetrating through the membrane during a 24 h incubation at 37∘ C. Then, the invading cells at the bottom of the membrane insert were detached, and the invasive potential of the cells was evaluated microscopically by counting the number of invading cells. 2.5. Western Blot and Immunoprecipitation. For the western blotting analysis, 100 𝜇g of protein was resolved on a 10% (v/v) trisglycine polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF). The membrane was then blocked in blocking buffer containing 5% (w/v) nonfat dry milk and 1% (v/v) Tween 20 (T) in 20 mM trisbuffered saline (TBS, pH 7.6) for 1 h at 37∘ C, followed by incubation with the appropriate monoclonal or polyclonal primary antibody in blocking buffer for 1 h to overnight at 4∘ C. This was followed by 1 h of incubation with anti-mouse or anti-rabbit secondary antibodies conjugated to horseradish peroxidase (HRP, BioRad, Hercules, CA, USA), several washes, and detection using chemiluminescence (enhanced chemiluminescence, ECL Kit, Millipore), and then autoradiography using ChemiDoc XRS with the Quantity One software. For the immunoprecipitation, IgG beads (Millipore) were incubated with antibodies against HER2, MMP2, or MMP9 for 10 min at room temperature (20∼25∘ C), followed by treatment with equal amounts of protein (about 1000 𝜇g) for 24 h. Then, the samples were washed thrice with TBS-T buffer consisting of 187.5 mM Tris-HCl (pH 6.8), 6% (w/v) sodium dodecyl sulfate (SDS), 30% (v/v) glycerol, 150 mM dithiothreitol (DTT), 0.03% (w/v) bromophenol blue, and 0.1% (v/v) Tween 20 and then boiled in loading buffer at 80∘ C for 10 min. Proteins were finally resolved using western blotting as described above. 2.6. siRNA-Mediated Silencing of raf, mek, and jnk Genes. Three small interfering RNAs (siRNAs) designed to knock down the expression of the murine raf, mek, and jnk genes and a control siRNA with a scrambled sequence that did not specifically degrade any known cellular mRNA were purchased from Life Technologies (Carlsbad, CA, USA). MDA-MB-453 cells were transfected with the siRNAs using Lipofectamine 3000 (Life Technologies). The final siRNA concentration used for the transfection was 20 nM. 2.7. Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Gene expression was evaluated by using quantitative real-time reverse transcriptionpolymerase chain reaction (qRT-PCR) analysis. Total RNA (2 𝜇g) was reverse transcribed to single-stranded cDNA using a cDNA Synthesis Kit (TaKaRa Bio, Otsu, Japan) according to the manufacturer’s instructions. The qRT-PCR was performed with 20 ng of retrotranscribed RNA, 1 × SYBR Select Master Mix (Invitrogen), and the predesigned primers mix in a final volume of 10 𝜇L. Amplification reactions were carried out using a CFX Connect real-time PCR system with the following thermal profile settings: an initial step of 2 min at 95∘ C to activate the FastStart Taq DNA polymerase and then 40 cycles at 95∘ C for 15 s and 55∘ C for

BioMed Research International 15 s. Three independent PCR amplification experiments were performed for each transcript. The fluorescence intensities were converted into threshold cycles (Ct) using the Bio-Rad CFX Manager software; the baseline and threshold values were set automatically. Relative quantification of target gene expression was performed by 2−ΔΔCt method with the average Ct values of basal samples as the calibrator for each gene. 2.8. Statistical Analysis. Data were expressed as mean ± standard error of the mean (SEM), either Student’s 𝑡-test or one-way analysis of variance (ANOVA) was used for statistical analysis. All analyses were performed using the statistical package for the social sciences (SPSS) 13.0, and 𝑃 < 0.05 was considered to be statistically significant.

3. Results 3.1. BRACs Suppressed Migration and Invasion of MDA-MB453 HER2+ Breast Cancer Cells. To evaluate the potential antimetastatic effects of BRACs, we analyzed the ability to inhibit the migration and invasion of the MDA-MB-453 cell. BRACs inhibited migration and invasion of MDA-MB-453 cells while their effect against MCF-10A cells was much less potent (Figure 1). 3.2. BRACs Inhibited the Migration and Invasion of MDAMB-453 through RAF/MAPK Pathway. The addition of the RAF/MAPK inhibitors to the cell migration and invasion assay cultures reduced the migration and invasion of the MDA-MB-453 cells (Figures 2(a) and 2(b)). Furthermore, the RAF, MEK, and JNK inhibitors increased the antimetastatic effect of BRACs. In addition, transfection of MDA-MB-453 cells with raf -, mek-, and jnk-siRNA was performed to block the RAF/MEK/ERK pathway; we found that this decreased the invasion of MDA-MB-453 cells. Treatment with BRACs further increased the inhibitory effect of the siRNA-mediated RAF/MEK/ERK pathway blockade on the invasion of MDAMB-453 cells (Figure 3). These results indicated that RAF, MEK, and JNK are important molecular targets of BRACs in the inhibition of cell metastasis. 3.3. BRACs Decreased mRNA Expression of raf, mek, and jnk in HER2+ Breast Cancer Cells. To confirm the molecular mechanisms underlying the antimetastatic effects of BRACs, we investigated whether treatment with BRACs inhibited the mRNA expression of raf1, mek, and jnk in HER2+ breast cancer cells. As shown in Figure 4, we observed significant inhibition of raf1, mek, and jnk expression in siRNA-treated MDA-MB-453 cells. More interestingly, the expression of these genes was further reduced significantly when BRACs was applied in combination with the siRNAs (Figure 5). 3.4. BRACs Treatment Inhibited Protein Expression of K-RAS and Phosphorylation of RAF and MAPKs in HER2+ Breast Cancer Cells. We investigated the effects of BRACs on the kinase activity of K-RAS, an upstream kinase of RAF1. The western blot assay revealed that BRACs strongly suppressed K-RAS activity in MDA-MB-453 cells (Figure 6). BRACs also

3 decreased the phosphorylation of MEK1/2, ERK1/2, JNK, and RAF1, which was affected most. These data indicate that BRACs inhibited the phosphorylation of upstream kinases more strongly than that of downstream kinases. To determine whether the inhibitory effect of BRACs on RAF/MAPK signaling is due to the direct physical interaction of HER2 and RAF/MAPK proteins, we performed an in vitro immunoprecipitation (IP) assay. The results indicated that BRACs inhibited the interactions between HER2 and RAF1, MEK, and JNK (Figure 7). These results suggested that BRACs might bind to HER2 as well as RAF1, MEK, or JNK or all the three at allosteric sites. 3.5. Cotreatment with Inhibitors or siRNA and BRACs Suppressed Metastasis in HER2+ Breast Cancer Cells. As shown in Figure 8, treatment with inhibitors attenuated the phosphorylation of RAF1, MEK1/2, ERK1/2, and JNK. Furthermore, RAF/MAPK pathway inhibitors suppressed the expression of the various proteins in the RAF/MAPK pathway. We subsequently evaluated the effects of BRACs and RAF/MAPK pathway inhibitors on the activation of the same proteins in MDA-MB-453 cells. Strikingly, cotreatment with BRACs and MAPK inhibitors downregulated the phosphorylation of RAF/MAPK pathway proteins with a significantly greater potency than that shown by BRACs alone in MDA-MB-453 cells. In addition, we detected phosphorylation of RAF1, MEK, and JNK1/2 kinase in cells, while treatment with their respective siRNAs downregulated p-RAF, p-MEK, and pJNK protein expression in cell lysates. Interestingly, the cotreatment decreased the phosphorylation of RAF, MEK, and JNK more significantly than using either substance alone (Figure 9). 3.6. BRACs Treatment Inhibited the Protein Expression of MMP2 and MMP9 in HER2+ Breast Cancer Cells. To confirm that BRACs inhibit metastatic signaling, we examined their potential inhibition of MMP2 and MMP9. Treatment of MDA-MB-453 cells with BRACs completely blocked the expression of MMP2 and MMP9 proteins (Figure 10). Next, to examine the mechanism by which BRACs inhibited the interactions of MMP2 and MMP9 with HER2, ERK, and JNK, we pretreated MDA-MB-453 cells with or without BRACs for 24 h and then performed an immunoprecipitated assay with MMP2 and MMP9 antibodies followed by analysis using immunoblotting. We observed that BRACs inhibited the interaction of MMP2 and MMP9 with HER2, ERK, and JNK (Figures 10(b)–10(d)). In particular, BRACs markedly inhibited the interaction between MMP9 and JNK (Figure 10(d)).

4. Discussion Anthocyanins are a class of flavonoids, which include reddish natural pigments that are extensively distributed in fruits and flowers. The BRACs contain anthocyanins such as cyanins, peonidins, cyanidin-3-glucose, and peonidin-3-glucose. Epidemiological studies have positively correlated the dietary consumption of anthocyanins with reduced cardiovascular

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Figure 1: Black rice anthocyanins (BRACs) extract inhibits migration and invasion of human epidermal growth factor receptor 2 (HER2+ ) breast cancer MDA-MB-453 cell line. MCF-10A and MDA-MB-453 cells were exposed to BRACs (0 or 200 𝜇g/mL) for 24 h. (a) Cell migration was determined using wound healing migration assay (×20). (b) MCF-10A and MDA-MB-453 cells were plated in upper compartments of Matrigel invasion chambers and exposed to BRACs (0 or 200 𝜇g/mL). Then, invasive potential of treated cells was evaluated microscopically. (c) Migrating cell numbers were counted using a microscope. ∗∗ 𝑃 = 0.005 < 0.01. (d) Number of invading cells was determined by counting using a microscope. ∗∗ 𝑃 = 0.004 < 0.01. Data are mean ± SEM of three independent experiments.


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Figure 2: Black rice anthocyanins (BRACs) extract and rapidly RAF/MEK/JNK inhibitors decrease migration and invasion of MDA-MB-453 cells. (a) MDA-MB-453 cells were exposed to BRACs (0 or 200 𝜇g/mL) with or without RAF/MAPK inhibitors ZM33672 (70 nM), U0126 (10 𝜇M), or SP600125 (50 nM) for 24 h. Cell migration was determined using wound healing migration assay and numbers of migratory cells was determined by counting using a microscope; ∗ 𝑃 < 0.05 versus control and # 𝑃 < 0.05 versus BRACs groups. (b) MDA-MB-453 cells were plated in upper compartments of Matrigel invasion chambers and exposed to BRACs (0 or 200 𝜇g/mL) with or without RAF/MAPK inhibitors ZM33672 (70 nM), U0126 (10 𝜇M), or SP600125 (50 nM). ∗ 𝑃 < 0.05 versus control; # 𝑃 < 0.05 versusBRACs groups. Data are mean ± SEM of three independent experiments.

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Figure 3: Black rice anthocyanins (BRACs) extract and small interfering RNAs (siRNA) blocked migration and invasion of MDA-MB-453 human epidermal growth receptor 2 (HER2+ ) breast cancer cells. (a) MDA-MB-453 cells were exposed to BRACs (0 or 200 𝜇g/mL) with or without transfection with raf -, mek-, or jnk-siRNAs for 24 h. Cell migration was determined using wound healing migration assay and number of migratory cell was counted using a microscope. ∗ 𝑃 < 0.05 versus control and # 𝑃 < 0.05 versus BRACs groups. (b) MDA-MB453 cells were plated in the upper compartments of Matrigel invasion chambers and exposed to BRACs (0 or 200 𝜇g/mL) with or without transfection with raf -, mek-, or jnk-siRNAs. ∗ 𝑃 < 0.05 versus control and # 𝑃 < 0.05 versus BRACs groups. Data are mean ± SEM of three independent experiments.

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Figure 4: Black rice anthocyanins (BRACs) extract inhibits the mRNA expression of raf, mek, and jnk in MDA-MB-453 cells. MDA-MB-453 cells were treated with BRACs (0 or 200 𝜇g/mL) for 24 h. The mRNA expression of (a) raf, (b) mek, and (c) jnk measured using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). ∗ 𝑃 < 0.05; data are mean ± SEM of three independent experiments.

disease-associated mortality [9] and certain types of cancer [6, 7, 10]. Previous findings have suggested the potential of anthocyanins or anthocyanin-derived pigments for use in the chemotherapy of breast cancer [6–10]. Metastatic dissemination is an extremely complex and highly organized and organ-specific process that involves numerous reciprocal interactions between cancer cells and the normal cells of the host. Breast cancer progression is dependent on the capacity of cancer cells to metastasize to distant organs; dysregulation of gene expression associated with the RAS/RAF/MAPK pathway plays a key role in this process [11]. Importantly, HER2/Neu overexpression is

closely linked to the dysregulation of this pathway. In this study, we determined whether BRACs inhibited breast cancer cell invasion by suppressing RAS/RAF/MAPK signaling, and the possible involvement of MMP2 and MMP9, which are regulated by JNK, was involved in the antimetastatic activity of BRACs. Previous reports have suggested that K-RAS kinase plays an important role in regulating HER2+ breast cancer cell metastasis [12]. We previously found that BRACs downregulated the expression of K-RAS in MDA-MB-453 cells. Furthermore, RAF kinases play a central role in the RAS/RAF/MEK/JNK signaling pathway, and RAF is activated


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Figure 5: Black rice anthocyanins (BRACs) extract combined with raf -, mek-, or jnk-small interfering RNAs (siRNA) decreased mRNA expression of respective genes in MDA-MB-453 cells. MDA-MB-453 cells were treated with BRACs (0 or 200 𝜇g/mL) with or without raf -, mek-, or jnk-siRNAs for 24 h. mRNA expression of (a) raf, (b) mek, (c) jnk was measured using quantitative real-time reverse transcriptionpolymerase chain reaction (qRT-PCR). ∗ 𝑃 < 0.05; data are mean ± SEM of three independent experiments.

by guanosine triphosphate- (GTP-) bound RAS [13]. Following activation, RAF phosphorylates MEK, which on activation subsequently phosphorylates and activates MAPK, which has multiple targets and leads to changes in gene transcription, resisting apoptosis, and enhancing metastasis [14]. In our study, we showed that oncogenic signaling by RAF1 is linked to the activation of the MEK/JNK pathway in metastatic breast cancer cells. In these experiments, BRACs were shown to decrease invasiveness, phosphorylation of RAF1, and raf1 mRNA expression in MDA-MB-453 cells. Furthermore, cotreatment with BRACs and an RAF inhibitor or raf -siRNA induced a stronger inhibitory effect than that obtained with any of these substances alone. In agreement with our results, Jong-Eun Kim has also implicated the RAF1 pathway in the MAPK signaling in skin cancer cells and found that cyanidin suppresses ultraviolet B-induced skin cancer by targeting RAF1 [15]. Our findings are also consistent with, but not limited to, this result. New discoveries from our previous research showed that BRACs act synergistically with

the RAF inhibitor and siRNA to further inhibit invasiveness and potentiate the antitumorigenic activity against HER2overexpressing breast cancer cells. MEK1 is a tyrosine (Y-) and S/T-dual specificity protein kinase [16], and its activity is positively regulated by RAF phosphorylation on serine residues in the catalytic domain. Our data also showed that BRACs inhibited the phosphorylation of MEK1 and mek1 mRNA expression. Furthermore, cotreatment with BRACs and inhibitors or siRNAs exhibited stronger antimetastatic effects in MDAMB-453 cells than any treatment alone did. These findings are correlated with a reduced pulmonary metastatic burden. Moreover, the predominant downstream target of MEK1 is ERK, and BRACs treatment decreased the phosphorylation of ERK1/2. In individual tumor types, JNK may not play a role in tumor development or may contribute (positively or negatively) to tumor pathology. Previous studies have established that JNK signaling is required for normal mammary gland

8


K-RAS Anti-RAF1 p-RAF1 Anti-MEK1 p-MEK1/2 p-ERK1/2 Anti-JNK p-JNK 𝛽-actin

BRACs 0 200 0 200 0 200 (𝜇g/mL) MCF-10A MCF-7 MDA-MB-453

Figure 6: Effects of black rice anthocyanins (BRACs) extracts on phosphorylation of RAS, RAF, MEK, ERK, and JNK. MCF-10A, MCF-7, and MDA-MB-453 cells were treated with BRACs (0 or 200 𝜇g/mL) for 24 h. Phosphorylation of RAF, MEK, ERK, and JNK was analyzed by immunoblotting. Expression of 𝛽-actin served as an internal control. IP: HER2 IB: anti-HER-2 IB: anti-RAF1 IB: p-RAF1 IB: anti-MEK1 IB: p-MEK1/2 IB: p-ERK1/2 IB: anti-JNK IB: p-JNK BRACs (𝜇g/mL)

0 200 0 200 0 200 MCF-10A MCF-7 MDA-MB-453

Figure 7: Effects of BRACs on the interactions of HER2 with RAF, MEK, ERK, and JNK. MCF-10A, MCF-7, and MDA-MB-453 cells were treated with BRACs (0 or 200 𝜇g/mL) for 24 h. Cell lysates were collected and immunoprecipitated with anti-HER2 antibody and then immunoblotted with antibodies against HER2, RAF/phosphorylated (p)-RAF, MEK/p-MEK/p-ERK, and JNK/p-JNK.

development and that it has a suppressive role in mammary tumorigenesis [17]. In contrast, we discovered that JNK promoted the invasion of breast cancer cells. Our findings are also consistent with recent discoveries of Cellurale et al. [18], which demonstrated that JNK plays a key role in K-RAS-induced tumorigenesis. These results may explain the association between K-RAS mutations and HER2+ breast cancer. We reported that BRACs suppressed the phosphorylation of JNK and jnk mRNA expression. Similarly, SP600125 (JNK inhibitor) or jnk-siRNA downregulated JNK phosphorylation and jnk mRNA expression in both the presence and the absence of BRACs.

HER2 is a well-known upstream kinase of RAF/MAPK, and, therefore, we examined whether BRACs affected the interaction of HER2 with RAF1, MEK1, ERK1/2, and JNK. We found that BRACs inhibited the binding between each of these kinases with HER2. Therefore, we concluded that the BRACs-mediated inhibition of interactions between HER2 and RAF, MEK, ERK, and JNK subsequently inhibited the metastasis of MDA-MB-453 cells. MMP2 and MMP9 play vital roles in tumor metastasis and invasion via the degradation of various proteins of the extracellular matrix and destruction of histological barriers [19]. Our study investigated whether HER2+ breast cancer cell metastasis was affected by the RAF/MAPK signaling


9

Anti-MEK p-MEK1/2 Anti-RAF p-RAF 𝛽-actin

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(b)

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Figure 8: Effects of black rice anthocyanins (BRACs) extract and RAF/MAPK inhibitors on phosphorylation of RAF, MEK, ERK, and JNK. MDA-MB-453 cells were treated with BRACs (0 or 200 𝜇g/mL) with or without inhibitors ZM33672 (70 nM), U0126 (10 𝜇M), and SP600125 (50 nM) for 24 h. Phosphorylation of (a) RAF, (b) MEK1/2, ERK1/2, and (c) JNK was determined using immunoblotting. Expression of 𝛽-actin served as an internal control.

Anti-RAF

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Figure 9: Effects of black rice anthocyanins (BRACs) extract and small interfering RNA (siRNA) on the activation of RAF/MAPK in MDA-MB-453 cells. MDA-MB-453 cells were treated with BRACs (0 or 200 𝜇g/mL) with or without raf -, mek-, or jnk-siRNAs for 24 h. Phosphorylation of (a) RAF, (b) MEK1, and (c) JNK was determined using immunoblotting (IB). Expression of 𝛽-actin served as an internal control.

pathway targeted by BRACs treatment. We found that treatment with BRACs decreased the expression of MMP2 and MMP9. Furthermore, the immunoprecipitation assay revealed interactions between each of the HER2, ERK, and JNK proteins and MMP9, which is in agreement with the results of Cho et al. [20]. This finding suggests that HER2 can regulates the expression of MMP9 via the RAF/MAPK signaling pathway. Moreover, BRACs treatment inhibited the HER2/MAPK/MMP9 signaling pathway, leading to the suppression of metastasis in HER2+ breast cancer cells. The ERK signaling pathway is known to upregulate the expression of MMPs [21]. However, we found that JNK showed the highest level of binding to MMP2 and MMP9 among the proteins we investigated. Collectively, these results demonstrate the potential antimetastatic effect of BRACs treatment mediated via RAS/RAF/MAPK signaling in HER2+ breast cancer cells. In addition, BRACs treatment inhibited the activation and mRNA expression of key components of the RAF/MAPK

signaling pathway. Furthermore, it decreased the interactions of HER2 with downstream signaling components as well as those of MMP2 and MMP9 with their upstream regulators.

Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper.

Authors’ Contribution Xiang-Yan Chen and Jie Zhou contributed equally to this work.

Acknowledgments This work was supported by National Natural Science Foundation of China (81273074, 81573154), Innovation Research

10

BioMed Research International IP: HER2 MMP2

IB: MMP2

MMP9

IB: MMP9

𝛽-actin

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IB: MMP9

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0 200 MDA-MB-453 (c)

BRACs (𝜇g/mL)

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Figure 10: Effects of black rice anthocyanins (BRACs) extract on MMP2 and MMP9. (a) MDA-MB-453 cells were treated with BRACs (0 or 200 𝜇g/mL) for 24 h. Cells were harvested for analysis of MMP2 and MMP9 expression using immunoblotting. Expression of 𝛽-actin served as a loading control. (b) MDA-MB-453 cells were treated with BRACs (0 or 200 𝜇g/mL) for 24 h. Cell lysates were collected and immunoprecipitated with anti-HER2 antibody and then immunoblotted with antibodies against MMP2, MMP9, and HER2. (c) MDA-MB453 cells were treated with BRACs (0 or 200 𝜇g/mL) for 24 h. Cell lysates were collected and immunoprecipitated with an anti-ERK antibody and then immunoblotted with antibodies against MMP2, MMP9, and ERK. (d) MDA-MB-453 cells were treated with BRACs (0 or 200 𝜇g/mL) for 24 h. Cell lysates were collected and immunoprecipitated with an anti-JNK antibody and then immunoblotted with antibodies against MMP2, MMP9, and JNK.

Team Project of Sichuan Province Youth Science & Technology Foundation (2014TD0021), and Scientific Research Innovation Team Project of Sichuan Provincial University Foundation (14TD00234).

[5] K. S. Saini, S. Loi, E. De Azambuja et al., “Targeting the PI3K/AKT/mTOR and Raf/MEK/ERK pathways in the treatment of breast cancer,” Cancer Treatment Reviews, vol. 39, no. 8, pp. 935–946, 2013.

References

[6] L.-S. Wang and G. D. Stoner, “Anthocyanins and their role in cancer prevention,” Cancer Letters, vol. 269, no. 2, pp. 281–290, 2008.

[1] Z.-Z. Huang, W.-Q. Chen, C.-X. Wu et al., “Incidence and mortality of female breast cancer in China—a report from 32 Chinese cancer registries, 2003–2007,” Tumor, vol. 32, no. 6, pp. 435–439, 2012. [2] D. J. Slamon, G. M. Clark, S. G. Wong, W. J. Levin, A. Ullrich, and W. L. McGuire, “Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene,” Science, vol. 235, no. 4785, pp. 182–191, 1987. [3] M. Brown, A. Tsodikov, K. R. Bauer, C. A. Parise, and V. Caggiano, “The role of human epidermal growth factor receptor 2 in the survival of women with estrogen and progesterone receptor-negative, invasive breast cancer: the California Cancer Registry, 1999–2004,” Cancer, vol. 112, no. 4, pp. 737–747, 2008. [4] B. P. Zhou and M.-C. Hung, “Dysregulation of cellular signaling by HER2/neu in breast cancer,” Seminars in Oncology, vol. 30, no. 5, pp. 38–48, 2003.

[7] C. Hui, Y. Bin, Y. Xiaoping et al., “Anticancer activities of an anthocyanin-rich extract from black rice against breast cancer cells in vitro and in vivo,” Nutrition and Cancer, vol. 62, no. 8, pp. 1128–1136, 2010. [8] L.-P. Luo, B. Han, X.-P. Yu et al., “Anti-metastasis activity of black rice anthocyanins against breast cancer: analyses using an ErbB2 positive breast cancer cell line and tumoral xenograft model,” Asian Pacific Journal of Cancer Prevention, vol. 15, no. 15, pp. 6219–6225, 2014. [9] T. C. Wallace, “Anthocyanins in cardiovascular disease,” Advances in Nutrition, vol. 2, no. 1, pp. 1–7, 2011. [10] M. H. Sehitoglu, A. A. Farooqi, M. Z. Qureshi, G. Butt, and A. Aras, “Anthocyanins: targeting of signaling networks in cancer cells,” Asian Pacific Journal of Cancer Prevention, vol. 15, no. 5, pp. 2379–2381, 2014.

BioMed Research International [11] W. Kolch, “Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions,” Biochemical Journal, vol. 351, no. 2, pp. 289–305, 2000. [12] S. Vranic, Z. Gatalica, and Z.-Y. Wang, “Update on the molecular profile of the MDA-MB-453 cell line as a model for apocrine breast carcinoma studies,” Oncology Letters, vol. 2, no. 6, pp. 1131–1137, 2011. [13] K. Terai and M. Matsuda, “Ras binding opens c-Raf to expose the docking site for mitogen-activated protein kinase kinase,” The EMBO Reports, vol. 6, no. 3, pp. 251–255, 2005. [14] B. Cseh, E. Doma, and M. Baccarini, “‘RAF’ neighborhood: protein-protein interaction in the Raf/Mek/Erk pathway,” FEBS Letters, vol. 588, no. 15, pp. 2398–2406, 2014. [15] J.-E. Kim, J. Y. Kwon, S. K. Seo et al., “Cyanidin suppresses ultraviolet B-induced COX-2 expression in epidermal cells by targeting MKK4, MEK1, and Raf-1,” Biochemical Pharmacology, vol. 79, no. 10, pp. 1473–1482, 2010. [16] D. R. Alessi, Y. Saito, D. G. Campbell et al., “Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1,” The EMBO Journal, vol. 13, no. 7, pp. 1610–1619, 1994. [17] C. Cellurale, N. Girnius, F. Jiang et al., “Role of JNK in mammary gland development and breast cancer,” Cancer Research, vol. 72, no. 2, pp. 472–481, 2012. [18] C. Cellurale, G. Sabio, N. J. Kennedy et al., “Requirement of cJun NH2 -terminal kinase for Ras-initiated tumor formation,” Molecular and Cellular Biology, vol. 31, no. 7, pp. 1565–1576, 2011. [19] A. John and G. Tuszynski, “The role of matrix metalloproteinases in tumor angiogenesis and tumor metastasis,” Pathology and Oncology Research, vol. 7, no. 1, pp. 14–23, 2001. [20] H.-J. Cho, Y.-J. Jeong, K.-K. Park et al., “Bee venom suppresses PMA-mediated MMP-9 gene activation via JNK/p38 and NF𝜅B-dependent mechanisms,” Journal of Ethnopharmacology, vol. 127, no. 3, pp. 662–668, 2010. [21] S. Chakraborti, M. Mandal, S. Das, A. Mandal, and T. Chakraborti, “Regulation of matrix metalloproteinases: an overview,” Molecular and Cellular Biochemistry, vol. 253, no. 1-2, pp. 269–285, 2003.

11


Review Article Coevolution between Cancer Activities and Food Structure of Human Being from Southwest China Yawen Zeng,1 Juan Du,1 Xiaoying Pu,1 Jiazhen Yang,2 Tao Yang,1 Shuming Yang,1 and Xiaomeng Yang1 1

Biotechnology and Genetic Resources Institute, Yunnan Academy of Agricultural Sciences, Kunming 650205, China Kunming Tiankang Science & Technology Limited Company, Agricultural Biotechnology Key Laboratory of Yunnan Province, Kunming 650223, China

2

Correspondence should be addressed to Yawen Zeng; [email protected] Received 9 June 2015; Accepted 26 July 2015 Academic Editor: Pratheeshkumar Poyil Copyright © 2015 Yawen Zeng et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Yunnan and Tibet are the lowest cancer mortality and the largest producer for anticancer crops (brown rice, barley, buckwheat, tea, walnut, mushrooms, and so forth). Shanghai and Jiangsu province in China have the highest mortality of cancers, which are associated with the sharp decline of barley.

1. Introduction Natural products are very popular to combat various physiological threats [1]. Vegetables, fruits, spices, herbs, and beverages have opened up new avenue for the role of phytochemicals in the prevention of human chronic diseases like cancer [2]. Functional foods not only are natural bioactive products with food value and promising cancer prevention and therapy [3], but also prevent diseases, suppress aging, enhance biodefense, bioregulation, and so forth [4]. The biopolymers of edible mushrooms make them very good candidates for formulation of novel functional foods for anticancer and so forth [5]. Greater consumption of fruits and vegetables, as well as whole grain products, appears to lower the risk of multimorbidity [6]. Cancer is becoming the most important public health burden in the world. Its incidence is varying among geographical regions, for example, esophageal cancer high in China, lung cancer in USA, and gallbladder cancer in Chile [7]. Each year 11,844 to 121,442 additional cases of lung cancer, 9,129 to 119,176 cases of bladder cancer, and 10,729 to 110,015 cases of skin cancer worldwide are attributable to inorganic arsenic in food [8]. A diet with higher total diversity may reduce the risk of bladder cancer [9]. The dietary factors

are the primary cause of nasopharyngeal cancer [10]. The phytochemicals for anticancer drug design from the green husk of Juglans regia L. have gained attention worldwide [11]. 𝛽-glucans from the cell walls of barley, oat, mushrooms, yeast, seaweeds, algae, and bacteria are essential for new therapeutic strategies against cancer [12]. Southwest China is the only geographical area where functional crop production has significant anticancer effects on humans [13]. Yunnan has not only the largest biodiversity center (higher plants >18,000 species; over 500 cultivated plants and 650 species of wild crops), but also the largest reserves of Al, Pb, Zn, Ti, Sn, Cu, and Ni in China [14] and the lowest incidence and mortality of cancers in China. Modern humans originated from Africa between ∼100 and 200 Ka, Asia as early as ∼130 Ka, and Northern Eurasia by ∼50 Ka [15]. Hunger due to food shortage with climate change is the cause of early humans’ evolution from Africa to Asia and later into Eurasia. Hunger was the cause of migration for early humans’ evolution; however, disease prevention of early human migrations was associated with food structure from centers of crop origin, but coevolution between anticancer activities and food structure of human based on crop origin center from Southwest China is unclear.

2


2. Southwest Is the Lowest Mortality of Cancers in China

3. The Lowest Cancer Mortality Associated with Anticancer Crops in Yunnan

The age-standardized incidence rate for cancers in China during 1998∼2007 was 1.8586‰, 2.0205‰ in men and 1.5915‰ in women in urban areas, but in rural areas it was 2.4434‰ and 1.3790‰, respectively [16]. Cancer mortality in China during 2004-2005 was 1.3587‰; cancer mortalities of seven regions of China are stated in Table 1: East China (1.6688‰) > Northeast China (1.3921‰) > North China (1.3126‰) > Central China (1.2426‰) > South China (1.2281‰) > Northwest China (1.1433‰) > Southwest China (0.9471‰) [17] (see Table 1). There are significant differences among East China and five other regions (I, IV, V, VI, or VII) for mortality of major cancer; the age-standardized rates of liver cancer in China and world mortalities were 0.184‰ and 0.180‰, respectively [18]. There are significant differences among South China and six other regions (I, II, III, IV, VI, or VII) for mortality of nasopharyngeal cancer. Nasopharyngeal carcinoma incidence and mortality were obviously higher in South China than in other regions and lowest in North China, and its crude incidence and mortality were 0.0316‰ and 0.0153‰, but world age-standardized incidence and mortality were 0.0244‰ and 0.0118‰, respectively [19]. There are significant differences among Northwest China and six other regions (I, II, III, IV, V, or VI) for mortality of cervical cancer and gastric cancer. Dietary habit was an important factor contributing to gastric cancer, especially regular consumption of fried, grilled, high-salt, high-fat, spicy food and drinking boiled brick tea [20]; Wuwei, a city in Northwest China, has high incidence of gastric cancer, which is because of the carcinogens due to lack of Vc, infection of Hp, atrophic gastritis, and heritage [21]. There are significant differences among Northwest China and three other regions (II, V, or VI) for esophageal cancer (𝑝 < 0.05); intake of dietary fiber is associated with a reduced risk of esophageal cancer for adults [22], whereas the risk is increased with red meat [23]. Millet exhibits multiple biological activities, including anticancer, antioxidant, immunomodulatory, antifungal, and antihyperglycemia effects [24]; the major genetic split for broomcorn millet divided the accessions into an eastern and a western grouping in Northwestern China [25]. There are significant differences among Southwest China and four other regions (I, II, III, or V) for mortality of breast cancer. Mortality rates increased from Southwest to Northeast and from West to East in China from 1991 to 2011 [26]. There are significant differences among Southwest China and three other regions (I, III, or VII) for mortality of leukemia. There is a significant inverse association between high tea consumption and leukemia risk [27]. The patterns of somatic mutation suggest relevant connections between the functional categories of genes driving acute myeloid leukemia [28]. Xuanwei and Fuyuan of Southwest China have strikingly high incidence of lung cancer, due to food with higher Cd and Ti contents [29]. Southwest China is not only the region with lowest incidence and mortality of cancers in China [17] (see Table 1), but also a geographical area richest in anticancer crop.

Yunnan province in China spans approximately 394,000 Km2 . It borders Vietnam, Laos, and Myanmar. Kunming is the provincial capital of Yunnan, and its elevation is 1894 m. Yunnan is not only the cradle of human childhood, a transitional region among East Asia continent, South Asian subcontinent, and Indo-China Peninsula, but also a core integration area of Chinese culture, Indian culture, and Mid-south Peninsula culture which all merge with the local culture [30]. Yunnan province is renowned for three kingdoms of plants, animal, and nonferrous metals, parallel evolution of crop adaptation to nonferrous metals, and anticancer foods for human being are mostly cultivated in this region. Yunnan is not only the region with lowest incidence and mortality of cancers (0.541‰) in China, especially esophageal cancer, gastric cancer, liver cancer, leukemia, female breast cancer, and cervical cancer [17] (see Table 1), but also the largest center of origin and diversity of cultivated crops [13]. The foods/plant extracts of turmeric and Chinese goldthread are more likely to be beneficial against cancer [31]. The cysteine-conjugated metabolites of shogaols are novel dietary colon cancer preventive agents [32]. Consumption of herbal tea is associated with reduced risk of colon cancer, but iced coffee increases rectal cancer risk [33]. Yunnan is not only a core integration area of Vavilov’s three centers of crop origin, including Chinese Center, Indian Center, and Central Asiatic Center, but also the largest center of origin and diversity of anticancer crop. Major anticancer food structures for crop are as follows. 3.1. Association of Brown Rice with Anticancer Activities. Rice originates from a single domestication 8.2–13.5 Kya in the Southwest China [34]. In 2013, global rice yield was 700.7 million tons, but Chinese rice yield was 203.3 million tons. Yunnan is a region not only presenting great genetic diversity, but also the center of genetic differentiation of indica and japonica subspecies of Asian cultivated rice; however, pigmented rice with similar wild rice with 2384 accessions accounts for 45.1% of rice landrace in Yunnan [35], but white rice for present day human consumption accounts for 95% rice cultivars in China. MGN-3 from rice bran may represent a novel adjuvant for the treatment of metastatic breast cancer [36]. The momilactone B in rice bran caused G1 cell cycle arrest and apoptosis in U937 cells, which may be related to anticancer activity [37]. Atractylenolide I might contribute to the anticancer effect of germinated brown rice [38]. The purple rice extract could be developed for functional foods for colon cancer prevention [39]. Human HepG2 cell apoptosis induced by Methanolic-Payao-Purple rice extracts and vinblastine was mediated through a mitochondrial pathway [40]. 𝛾-Oryzanol, proanthocyanidin, and 𝛾-tocotrienol in red rice extract may have a potential to serve as food-derived chemotherapeutic agents for cancer patients [41]. Thai purple rice cooked under sterilization could be a potential source of protocatechuic acid exerting high antiproliferative activity [42]. Treatments with peonidin-3-glucoside and cyaniding3-glucoside from black rice extracts significantly reduced

Beijing Tianjin Hebei Shanxi Inner Mongolia I = North China Liaoning Jilin Heilongjiang II = Northeast China Shanghai Jiangsu Zhejiang Anhui Fujian Jiangxi Shandong III = East China Henan Hubei Hunan IV = Central China Guangdong Guangxi Hainan V = South China Chongqing Sichuan Guizhou Yunnan Tibet VI = Southwest China Shanxi Gansu Qinghai Ningxia Xinjiang VII = Northwest China China

Province/municipality/region

Major cancer ‰ 1.3132 1.2443 1.3880 1.4831 1.1343 1.3126 1.4498 1.1325 1.5939 1.3921 2.1707 1.9363 1.7273 1.5738 1.4119 1.1986 1.6633 1.6688 1.4440 1.2895 0.9943 1.2426 1.2788 1.1717 1.2339 1.2281 1.4632 1.3322 0.7557 0.5410 0.6433 0.9471 1.1652 1.2771 1.2303 1.1674 0.8764 1.1433 1.3587

Esophageal cancer (‰) 0.0876 0.0670 0.2962 0.2439 0.1438 0.1677 0.0914 0.0271 0.0600 0.0595 0.1432 0.2834 0.1122 0.2388 0.2736 0.0505 0.1062 0.1726 0.3132 0.0893 0.0437 0.1487 0.1441 0.0411 0.0299 0.0717 0.3004 0.1682 0.0195 0.0139 0.0386 0.1081 0.1339 0.1572 0.1156 0.1008 0.1941 0.1403 0.1521

Gastric cancer (‰) 0.0944 0.1141 0.2827 0.4242 0.1955 0.2222 0.2065 0.1891 0.1724 0.1893 0.3008 0.4452 0.3377 0.4133 0.1824 0.1726 0.3478 0.3143 0.3031 0.2188 0.0977 0.2065 0.1419 0.1536 0.2492 0.1816 0.2148 0.2659 0.1130 0.0829 0.2830 0.1919 0.2836 0.4420 0.3232 0.2233 0.1950 0.2934 0.2471

Liver cancer (‰) 0.1699 0.1699 0.2099 0.1606 0.1795 0.1780 0.2237 0.3172 0.3512 0.2974 0.2465 0.3524 0.3506 0.2688 0.3190 0.2523 0.3177 0.3010 0.2759 0.3299 0.1871 0.2643 0.3175 0.3377 0.2811 0.3121 0.3189 0.2890 0.1415 0.1023 0.2070 0.2117 0.2038 0.1946 0.1982 0.2268 0.1041 0.1855 0.2626

Colon Lung cancer cancer (‰) (‰) 0.1173 0.4009 0.0782 0.4437 0.0516 0.2854 0.0672 0.3141 0.0473 0.2810 0.0723 0.3450 0.0941 0.4614 0.0478 0.4382 0.0940 0.5101 0.0786 0.4699 0.2328 0.5151 0.0828 0.3695 0.1121 0.4184 0.0739 0.2776 0.0781 0.2627 0.0945 0.2963 0.0824 0.4565 0.1081 0.3709 0.0572 0.2551 0.0786 0.2798 0.0765 0.2514 0.0708 0.2621 0.0965 0.2748 0.0581 0.2607 0.0857 0.2113 0.0801 0.2489 0.0714 0.3244 0.0908 0.2709 0.0599 0.1693 0.0487 0.0996 0.0077 0.0309 0.0557 0.1790 0.0383 0.1875 0.0524 0.1568 0.0354 0.1994 0.0679 0.2345 0.0201 0.1005 0.0428 0.1757 0.0752 0.3083

Nasopharyngeal cancer (‰) 0.0092 0.0079 0.0028 0.0028 0.0051 0.0056 0.0046 0.0037 0.0075 0.0053 0.0251 0.0119 0.0165 0.0136 0.0293 0.0301 0.0088 0.0193 0.0044 0.0171 0.0228 0.0148 0.0529 0.0492 0.0718 0.0580 0.0080 0.0143 0.0134 0.0077 0.0026 0.0092 0.0059 0.0030 0.0000 0.0028 0.0036 0.0031 0.0146

Table 1: Geographical distribution of cancer mortality in China [17]. Leukemia (‰) 0.0442 0.0602 0.0337 0.0470 0.0360 0.0442 0.0374 0.0303 0.0420 0.0366 0.0483 0.0485 0.0382 0.0355 0.0411 0.0409 0.0479 0.0429 0.0347 0.0312 0.0393 0.0351 0.0359 0.0347 0.0279 0.0328 0.0322 0.0357 0.0310 0.0320 0.0026 0.0267 0.0363 0.0444 0.0507 0.0462 0.0379 0.0431 0.0385

Female breast cancer (‰) 0.0956 0.0851 0.0547 0.0416 0.0703 0.0695 0.0775 0.0762 0.0784 0.0774 0.1218 0.0574 0.0728 0.0440 0.0428 0.0579 0.0795 0.0680 0.0556 0.0637 0.0617 0.0603 0.0507 0.0633 0.0760 0.0633 0.0460 0.0445 0.0320 0.0288 0.0299 0.0362 0.0542 0.0463 0.0412 0.0509 0.0326 0.0450 0.0590

Cervical cancer (‰) 0.0199 0.0309 0.0264 0.0422 0.0172 0.0273 0.0162 0.0231 0.0210 0.0201 0.0177 0.0159 0.0303 0.0327 0.0191 0.0479 0.0155 0.0269 0.0273 0.0275 0.0539 0.0362 0.0205 0.0282 0.0296 0.0261 0.0117 0.0174 0.0379 0.0167 0.0349 0.0237 0.0468 0.0756 0.0291 0.0382 0.0889 0.0557 0.0280

BioMed Research International 3

4 the tumor size and volume in vivo [43]. China and India are the world’s largest producers of rice, which account for 26% and 20% of all world rice production, respectively. Therefore, Southwest China and North India have the lowest incidence and mortality of cancers associated with origin center of rice, especially pigmented rice. 3.2. Association of Barley with Anticancer Activities. Barley is the most important crop among functional foods [13]. Yunnan is the center of second origin for two naked barleys and the largest diversity center in China [14], as well as the largest Chinese producer. All 𝛽-glucans differ by their length and branching structures, which are considered biological response modifiers with health beneficial effects including anticancer activities [44]. The genotypes of vitamin E 31.5 𝜇g/g dry weight while being of ascorbic acid equivalent antioxidant capacity 158.1 mg AEAC/100 g fresh weight are potential candidates for breeding of barley cultivars with high vitamin E content or antioxidant capacity at harvest, even after storage [45]. The content (mg/kg) of tocotrienols for anticancer activities in barley is higher; that is, barley (910) > rice bran (465) > oat (210) > maize (200) > wheat germ oil (189) > rye (92) [46]. Green barley extract induced preferential antiproliferative and proapoptotic signals within B-lineage leukemia/lymphoma cells [47]. The bioactive compounds in germinated barley and other cereals may reduce the risk of diabetic agents and colon cancer [48]. Protocatechualdehyde present in barley suppressed cell growth and induced apoptosis, which may be a result of deacetylase 2-mediated cyclin D1 suppression [49]. Lunasin present in barley has been observed to prevent skin cancer, which could play an important role in the prevention of cancer in humans [50]. Remarkably high reduction of tumorigenesis and induction of apoptosis in the liver section were achieved in the mouse models with barley-Shochu distillation remnants [51]. Germinated barley foodstuff significantly increased the production of a tumor suppressor gene, which showed promising antineoplastic effects [52]. 3.3. Association of Buckwheat with Anticancer Activities. Yunnan is not only the center of origins and evolution for buckwheat [13, 14], but also the largest Chinese producer of Tartary buckwheat, which accounts for 60.1% in China. Rutin (2215.5 mg/100 g at 7 days) and quercitrin (2301.0 mg/100 g at 8 days) contents after sowing of buckwheat sprouts were approximately 35 and 65 times higher than those of buckwheat seeds [53]. Yunnan golden buckwheat has unique anticancer effects, and its product, “Weimaining” capsules, is the national second-class anticancer drug [54]. TBWSP31 from Tartary buckwheat water-soluble extracts is a novel antitumor protein and apoptosis inducer [55], and also quercetin from its seeds and bran exhibited the strongest cytotoxic effects against the human hepatoma cell line [56]. Tatariside G may be an effective candidate for chemotherapy against cervical cancer [57]. 3.4. Association of Tea with Anticancer Activities. After water, tea is the most widely consumed beverage [58]; tea is

BioMed Research International cultivated in Asia which is producing more than 91% of the world. Green tea and quercetin enhanced the therapeutic effect of docetaxel in castration-resistant prostate cancer cells [59]. Green tea polyphenols have strong antioxidants and the inhibition of 16 cancers [13], such as (−)-epigallocatechin-3O-gallate, induces apoptosis in acute myeloid leukemia cells [60]. The articles on the association of tea with cancer are 3214 according to PubMed literature database. Yunnan province in China is center of origin for Camellia sinensis, which has 35 species and 3 varieties, accounting for 76.6% of Camellia sinensis in the world [13, 14, 61]. There are more than 500 compounds identified. Tea in Yunnan has 15 accessions for 35.0%–46.8% polyphenols and 14 accessions for 5% caffeine [61]. Epigallocatechin-gallate in green tea has been shown to have antiproliferative activity in colon cancer cells [62]. Catechins of green tea are flavanols, which have many health related characteristics; they especially lower the cytotoxicity and cost of anticancer treatment, inhibit proliferation of breast cancer cells, and block carcinogenesis [1, 63]. Humans would be able to achieve consistent cancer prevention effects provided there is timely intervention of green tea catechins at appropriate high-dose levels [58]. Green tea and coffee consumption has protective effects on esophageal cancer [64]. Therefore, green tea is the most economic and effective method for anticancer treatment. 3.5. Association of Walnut with Anticancer Activities. Yunnan is not only the center of origins and evolution for fruits, which has 66 families, 134 genus, and 499 species [65], but also the Chinese largest producer of walnut (2679,000 ha), which accounts for 50.2% of China in 2013. Walnuts are rich in 𝜔-3 fatty acids, tocopherols, 𝛽-sitosterol, and pedunculagin, which slow down the growth of prostate, colon breast, and renal cancers [66]. The tumor size in mice having walnut in their diet was one-fourth than that of the control diet [67]. The 𝛼-linolenic acid and 𝛽-sitosterol from walnut oil decreased proliferation of MCF-7 cells [68]. Changes in the miRNA expression profiles likely affect target gene transcripts involved in pathways of anti-inflammation, antivascularization, antiproliferation, and apoptosis [69]. Walnuts decrease the risk of these chronic diseases (cancer, type 2 diabetes, cardiovascular disease, and visceral obesity via inflammation) [70]. Dietary walnut can reduce cancer growth and development by its anticancer mechanism of suppressing the activation of NF𝜅B [71]. The dihydroxy3,4󸀠 -dimethoxyflavone and regiolone from walnut leaves can induce apoptosis in human breast adenocarcinoma cells [72]. The bioactive compounds in walnut green husks are capable of killing prostate carcinoma cells by inducing apoptosis [11]. 3.6. Association of Mushrooms with Anticancer Activities. Yunnan is richest in having species of wild mushrooms in China, which accounts for 90.2% in China, and 44.1% in the world. It has 670 species of edible mushrooms, which accounts for 72.4% in China, including boletes (224 species) and edible boletes (144 species) accounting for 57.4% and 72.4% in China, respectively [73]. The export of boletes and Tricholoma matsutake, having anticancer activities, from

BioMed Research International Yunnan province is up to 91,780,000 and 57,380,000 USD in 2011 [13]. Medicinal mushrooms have been used to treat cancer, fungal infections, hypertension, diabetes, inflammation, and renal disorders [74]. The most potent extract identified from Ganoderma lucidum inhibited the growth of a gastric cancer cell line by interfering with cellular autophagy and cell cycle [75]. Intake of mushrooms seems to be inversely associated with the epithelial ovarian cancer [76]. Polysaccharides from mushrooms have been widely used in far Asia as antitumor, immunostimulating, antimicrobial, hypocholesterolemic, hypoglycemic, and health-promoting agents [5]. The water extract of Umbilicaria esculenta has a great potential to be developed into an anticancer agent that targets telomerase [77]. 3.7. Association of Panax notoginseng with Anticancer Activities. Yunnan is the Chinese largest producer of Panax notoginseng, which accounts for 97% of the production of China, and more than 400 products were made from it by 1302 companies in China. P. notoginseng is a promising candidate in preventing and treating inflammation-associated colon carcinogenesis [78]. Macroporous resin from the leaves of P. notoginseng is enriched with low polarity PPD group saponins of 85% ethanol fraction, which is a new alternative source of anticancer saponins [79]. A new protopanaxadioltype ginsenoside, 6-O-𝛽-d-glucopyranosyl-20-O-𝛽-d-glucopyranosyl-20(S)-protopanaxadiol-3-one (1), was isolated from the roots of P. notoginseng, which exhibited cytotoxic activity against five human cancer cell lines [80]. An arabinogalactan RN1 from flowers of Panax notoginseng had an antiangiogenic effect via BMP2 signaling and could be a potential novel inhibitor of angiogenesis [81]. The major saponins in P. notoginseng saponin extract were ginsenosides Rg1 (31.1%) and Rb1 (34.4%), which may provide significant natural defense against human colon cancer [82]. In addition, Yunnan is the richest in species of Amorphophalms knojac (66.7%) and its largest producer in China. The konjac glucomannans are associated with a range of health applications which include decreasing the risk of gut cancer and colon carcinogenesis through reduced toxicity of faecal water and precancerous risk factors of human colon cancer [83].

4. Lower Cancer Mortality Associated with Anticancerous Food in Tibet Tibet in China spans approximately over 1,200,000 Km2 . It borders Myanmar, Bhutan, and Nepal. Tibetans have been adapted to an altitude exceeding 3,500 m in the early Upper Paleolithic [84]. Tibet is one of the regions with lowest cancer incidence and mortality of cancers (0.643‰) in China, especially colon cancer, lung cancer, nasopharyngeal cancer, and leukemia [17] (see Table 1). Major anticancer food structures are as follows. 4.1. Association of Naked Barley with Anticancer Activities. Tibet and its vicinity are not only the centers of domestication of cultivated barley [85, 86], but also the world’s largest producer of naked barley for major food. Barley and its products

5 are good sources of antioxidants [87], especially anticancer. The most common anthocyanin in the purple barley is cyanidin 3-glucoside, whereas delphinidin 3-glucoside is the most abundant anthocyanin in the blue and black groups [87]. Himalaya 292 for barley mutant has high contents of 𝛽glucan (9.7%) and protein (16.4%), as well as amylose starch (81.6%) [88]. The intrinsic differences of 𝛽-glucans in barley and other cereals will elicit variable immune and anticancer responses. The molecular mechanisms of 𝛽-glucan-induced signaling in immune cells are essential for the design of new therapeutic strategies against cancer [12, 89]. 𝛽-D-glucan from barley regulates breast cancer-relevant gene expression and may be useful for inhibiting endocrine-resistant breast cancer cell proliferation [90]. Whole hulless barley is a functional food that can reduce the prevalence of metabolic syndrome [91]. 4.2. Association of Milk with Anticancer Activities. Milk consumption is prevalent in daily diets, and its lactase persistence is likely to have an independent origin in Tibetans [92]. The milk protein 𝛼-casein would provide a more natural and nontoxic approach to the development of novel anticancer therapies [93]. In addition, Guizhou is not only one of the regions of lower cancer incidence and mortality of cancers (0.756‰) in China, but also one of the cradles of human childhood.

5. The Highest Cancer Mortality Associated with Food in Some Province of China Human chronic disease outbreak owes its origin to consumption of brown rice and barley, which were the staple diet of the ancient people, whereas white rice and wheat white flour are now consumed as staple foods of modern people [13]. Yunnan Province is the lowest mortality of cancers (0.541‰) in China [17] or all over the world [94], which associated with the largest production base for barley and tea as well as walnut, and so forth in China. East China includes Shanghai, Jiangsu, Zhejiang, Shandong, Anhui, Fujian, and Jiangxi 7 provinces/city, and it is the highest mortality of cancers (1.6688‰) and the most cancer villages (76) in China, which mainly covers some lower reaches of rivers including the Yellow River, Huaihe River, and Yangtze River and also appears near the Dongting Lake and Poyang Lake; the number of cancer villages in China is in turn East China (76) > Central China (28) > North China (24) = South China (24) > Southwest China (12) > Northwest China (3) = Northeast China (3) [95]. The highest cancer mortality associated with barley dropped in some provinces of China, especially Shanghai, Jiangsu, and Zhejiang. Nanjing (107.3 mg/kg) and Shanghai (95.59 mg/kg) as well as Hanzhou (75.7 mg/kg) cities are the highest soil Pb2+ concentrations in 35 cities for China [96]. The lipidtransfer protein from barley grain has an affinity to bind Co2+ and Pb2+ but has no affinity towards Cd2+ , Cu2+ , Zn2+ , and Cr3+ [97]. Barley 𝛽-glucan is a radioprotective agent, and it can enhance radioprotection in the human hepatoma cell line HepG2 [98]. Barley with polyphenols possesses many

6 other anticarcinogenic activities, and high epicatechin may be related to a reduced risk of breast cancer [99] and colon cancer. Consumption of lunasin from barley could play an important role in cancer prevention [100], but the barley cultivated areas in China in 1935 (6,380,000 ha) were 5.1 times than that in 2012. Shanghai city in 1986 was 6.0 times barley cultivated more than in 2012; Jiangsu province in 1957 (1,401,400 ha) was 7.5 times than in 2012; however Zhejiang province in 1935 (283800 ha) was 9.5 times cultivated than in 2012. Shanghai city has not only the highest cancer mortality (2.171‰) in China, especially colon cancer, lung cancer, and female breast cancer [17] (see Table 1) but also has the lowest diversity of cultivated crops, and its elevation is 4.0 m. Colorectal cancer increases by 4.2% annually in Shanghai, which is faster than the average increasing rate of the world [101]. Lunasin from soybean cotyledon and barley, a peptide with 43 amino acid residues, demonstrated chemopreventive and anticancer properties against colon and breast cancers [100, 102]. The consumption of barley rice has certain prevention and adjunctive dietary therapy functions for diabetes mellitus, cardiovascular disease, and cancers [103]. Breast cancer is the most common cancer among women in urban China, such as Shanghai, and so forth; however, soy food consumption is significantly associated with decreased risk of breast cancer and lung cancer [104], but cultivated area of soybean in Shanghai is the lowest in China. Fruit intake is inversely associated with the risk of colorectal cancer [105], but cultivated area of fruits in Shanghai is the lowest in China. The high intake of fruits, vegetables, milk, and eggs may reduce the risk of breast cancer, whereas high animal food intake may increase the risk [106]. Age seems to contribute to increased morbidity and mortality of colorectal carcinoma in Yangpu district of Shanghai, but the mortality of colorectal carcinoma appeared higher than the incidence [107]. Jiangsu province has not only the highest cancer mortality (1.936‰) in China, especially gastric cancer and liver cancer [17] (see Table 1), but also a province of lowest altitude ( Shangdong (16) > Henan (15) > Jiangsu (14) >Hebei (12) [95]. There are significant correlations between topsoil Pb concentration and gastric cancer, as well as grain Hg concentration with liver cancer in humans [108]. Natural lycopene shows a potential anticancer activity and reduces gastric cancer incidence [109]. The tricin from young green barley leaves on melanin production in B16 melanoma cells inhibits melanin biosynthesis with higher efficacy than arbutin, and it could be used as a whitening agent [110]. The annual average crude incidence and age-standardized incidence by world population were 2.52‰ and 1.79‰, respectively, but Jiangsu being an area with relatively low risk of female breast cancer presented cancer registry areas from 2006 to 2010 as 0.703‰ and 0.481‰, respectively [111].

BioMed Research International Zhejiang province has very high cancer mortality (1.727‰) in China, especially for liver cancer [17] (see Table 1). The crude incidence of cancer registered in Zhejiang province in 2009 was 3.202‰; however, age-standardized incidence by Chinese and world standard population was separately 1.6199‰ and 2.0792‰; meanwhile, the crude mortality rate was 1.7697‰ and the age-standardized mortality by Chinese and world standard population were 0.7917‰ and 1.0702‰, respectively [112]. The highest soil concentrations in Zhejiang province were 70.36 mg/kg for Pb, 47.49 mg/kg for Cr, 13.51 mg/kg for As, 0.73 mg/kg for Cd, and 0.67 mg/kg for Hg, while Cd caused the greatest cancer risk [113]. The bioaccumulation of heavy metals in food tubers carries a considerable risk for human cancers [114]. The inhibition of cancer cell viability and apoptosis by protocatechualdehyde in barley leaves may be result of activating transcription factor 3 expression through ERK1/2 and p38-mediated transcriptional activation [115].

6. Conclusion and Future Prospects Chronic disease prevention of early human migrations was associated with food structures from crop origin centers, especially from Asia with four centers of crop origin, which account for 58% in the world population. The early modern human of Southwest China was related to many ancestors of Asians. Southwest China, richest in anticancer crop, not only is the most important evolution base of Asian and anticancer crops, but also has the lowest mortality and incidence of cancers in China. Yunnan, richest in anticancer crops, is the cradle of human childhood and the lowest cancer incidence as well as mortality of cancers (0.541‰) in China, especially esophageal cancer, gastric cancer, liver cancer, leukemia, female breast cancer, and cervical cancer, and also the largest center of origin and diversity of functional crops with anticancer activities (brown rice, barley, buckwheat, tea, walnut, mushrooms, Panax notoginseng, Knojac, etc.). Tibet is not only one of the regions of lowest incidence and mortality of cancers (0.643‰) in China, especially colon cancer, lung cancer, nasopharyngeal cancer, and leukemia, but also the largest center of origin and diversity of naked barley for major functional foods. Shanghai and Jiangsu, in China, have the highest mortality of cancers (1.936∼2.171‰), which are associated with barley cultivated areas dropped about 6.5 times and 7.5 times, respectively. These results further support that Southwest China (especially Yunnan and Tibet) is the center of lowest mortality of cancers (0.643‰) in China based on coevolution between human’s anticancer activities and functional foods from crop origin center.

Abbreviations ALA: EPIYA: HepG2: Hp: Ka: Km2 : Kya:

Alpha-linolenic acid Glu-Pro-Ile-Tyr-Ala Liver hepatocellular cells Haptoglobin 1000 years Square kilometres Thousand years ago

BioMed Research International MCF-7 cells: Human breast adenocarcinoma cell line MGN-3: Arabinoxylan rice bran NF𝜅B: Nuclear factor kappa-light-chain-enhancer of activated B cells SNPs: Single nucleotide polymorphisms TBWSP31: Tartary Buckwheat Protein Fraction USA: United States of America USD: United States Dollar Vc: Vitamin C.

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Conflict of Interests

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The authors declare that they have no conflict of interests whatsoever to declare.

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Acknowledgments This research was supported by the National Natural Science Foundation of China (no. 31260326), China Agriculture Research System (CARS-05), the Science and Technology to Benefit the People (2014RA060), and the Exploit of Emphases New Production (2014BD001) as well as the Yunnan Introduction and Foster Talent Program (2012HB050, 2011CI059) from Yunnan Provincial Scientific and Technology Department. Thanks are due to the professional editing team at Eureka Science for language improvement.

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Review Article Systematic Review of the Use of Phytochemicals for Management of Pain in Cancer Therapy Andrew M. Harrison,1 Fabrice Heritier,2 Bennett G. Childs,3 J. Michael Bostwick,4 and Mikhail A. Dziadzko5 1

Medical Scientist Training Program, Mayo Clinic, Rochester, MN 55905, USA Department of Anesthesiology, CH du Forez, 42600 Montbrison, France 3 Mayo Graduate School, Mayo Clinic, Rochester, MN 55905, USA 4 Department of Psychiatry & Psychology, Mayo Clinic, Rochester, MN 55905, USA 5 Department of Anesthesiology, Mayo Clinic, Rochester, MN 55905, USA 2

Correspondence should be addressed to Mikhail A. Dziadzko; [email protected] Received 10 August 2015; Accepted 1 October 2015 Academic Editor: Sung-Hoon Kim Copyright © 2015 Andrew M. Harrison et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Pain in cancer therapy is a common condition and there is a need for new options in therapeutic management. While phytochemicals have been proposed as one pain management solution, knowledge of their utility is limited. The objective of this study was to perform a systematic review of the biomedical literature for the use of phytochemicals for management of cancer therapy pain in human subjects. Of an initial database search of 1,603 abstracts, 32 full-text articles were eligible for further assessment. Only 7 of these articles met all inclusion criteria for this systematic review. The average relative risk of phytochemical versus control was 1.03 [95% CI 0.59 to 2.06]. In other words (although not statistically significant), patients treated with phytochemicals were slightly more likely than patients treated with control to obtain successful management of pain in cancer therapy. We identified a lack of quality research literature on this subject and thus were unable to demonstrate a clear therapeutic benefit for either general or specific use of phytochemicals in the management of cancer pain. This lack of data is especially apparent for psychotropic phytochemicals, such as the Cannabis plant (marijuana). Additional implications of our findings are also explored.

1. Introduction Pooled prevalence of pain in cancer is greater than 50% [1]. This data is based on a systematic review of 52 research articles (out of a possible 4,737 articles) that spans 40 years of literature and includes patients after curative treatment, during cancer therapy, characterized as advanced/metastatic/ terminal disease, and/or at all disease stages. One reason for the low article inclusion rate in this systematic review (1.1%) is the complex, multifactorial nature of cancer pain, which includes different mechanisms and targets [2]. In the context of this multifactorial nature and poor definition of cancer pain, stringent inclusion criteria were another reason for this low article inclusion rate. Although phytochemical therapy has historically been used as a treatment for cancer, treatment of cancer pain in general is challenging [3].

The use of phytochemical therapy for the treatment of cancer pain is further confounded by historical folklore and phytochemical isolates of poorly defined chemical composition. Specific plants and the phytochemicals from these plants have been investigated for their anti-inflammatory properties [4]. One example is the dried fruits of flowering shrub Carissa carandas. In this case, a specific plant containing potentially numerous compounds active against pain, as opposed to a specific phytochemical, was investigated. Numerous plants have also been used in traditional South African medicine for the treatment of pain [5]. However, rigorous scientific investigation into any specific phytochemicals these plants may contain for the specific treatment of pain is lacking. These examples are in contrast with examples of progress in chemotherapeutics.

2 The need for new chemotherapeutic agents with increased efficacy and decreased toxicity has led to the development of novel biologicals, such as antibody therapy, for the treatment of cancer [6]. This movement has resulted in some success, such as rituximab for the treatment of B cell-mediated lymphomas and leukemias. An extract of Toxicodendron vernicifluum (also known as Rhus verniciflua Stokes and the Chinese lacquer tree) has been shown to induce growth inhibition and apoptosis of hepatic tumor cells in cell culture [7]. Likewise, ursolic acid—found in the waxy peels of fruits, as well as some herbs and spices—has been demonstrated to induce apoptosis of melanoma cells in cell culture [8]. Thus, evidence exists for the use of phytochemicals as novel chemotherapeutic agents. The molecular signaling pathways and other mechanisms explaining these observations are slowly being elucidated [9, 10]. For example, a variety of natural inhibitors of the STAT3 signaling pathway, which results in the induction of apoptosis in both hematological and solid tumor cells, have been identified. Examples include betulinic acid, butein, caffeic acid, and capsaicin. However, the need for more agents with this level of success for the management of chemotherapeutic pain has resulted in other avenues of investigation [11], including phytochemicals and other natural products [12]. The rationale for this investigation includes epidemiological data from dietary intakes and in vitro experimentation. While investigation into the use of phytochemicals for cancer chemotherapy is limited, exploration of phytochemicals for management of pain in cancer therapy is even more lacking. Specific extracts of the plant Swertia corymbosa have been isolated, identified, and shown to have dosedependent therapeutic effects in mouse and rat models for the management of convulsions, sedation, and anxiety [13]. Numerous traditional herbs and phytochemicals have also already been investigated at the level of in vivo experiments and some clinical trials for neurodegenerative diseases, such as Alzheimer’s disease [14]. In the context of these largely preclinical results, our objective was to perform a systematic review of the biomedical literature for the specific use of phytochemicals for the management of pain in cancer therapy in human subjects.

2. Methods 2.1. Background Definitions. One popular definition of phytochemicals (of the Kingdom Plantae) is “bioactive nonnutrient plant compounds in fruits, vegetables, grains, and other plant foods that have been linked to reducing the risk of major chronic diseases” [15]. For the purpose of this systematic review, as described in the search strategy below and the Discussion, fungochemicals (of the Kingdom Fungi) were also included. For the definition of management of pain in cancer therapy, both the direct and indirect antinociceptive effects of pain associated with antineoplastic treatment (oral mucositis, burns, neuropathy, enteritis, and proctitis)—as well as coanalgesic effects—were considered. The search strategy employed in this systematic review allowed for the inclusion of both whole plant products and specific plant extracts [16].

BioMed Research International 2.2. Data Sources and Searches. The internationally accepted Preferred Reporting Items for Systematic Reviews and MetaAnalyses (PRISMA) standard was used for this systematic review [17, 18]. A comprehensive search of 4 databases was conducted: PubMed (the National Library of Medicine), Ovid/MEDLINE (Wolters Kluwer), Scopus (Elsevier), and Web of Science (Thomson Reuters). English abstracts were searched from each database’s inception through July 01, 2015. The search strategy was designed and conducted with a controlled vocabulary supplemented with keywords to search for studies of phytochemicals (“phytotherap*”, “phytochemical*”, “plant”, and “plants”), fungochemicals (mushr*), pain (“pain” and “nociception”), and cancer (“cancer” and “neoplas*”). Case reports, case series, case studies, controlled trials, and comparative studies were included in this search strategy. Meta-analysis, reviews, commentaries, and letters were excluded. All abstracts were screened by 1 reviewer (Mikhail A. Dziadzko) and potentially relevant articles in human subjects were identified for full-text review by 2 reviewers (Mikhail A. Dziadzko and Bennett G. Childs). 2.3. Study Selection. A study was eligible for inclusion if it examined the use of any phytochemical for the management of pain in cancer therapy in human subjects. Only interventional (nonobservational) studies with controls were included. Phytochemical derivatives were excluded, as were abstracts and articles not available in English. 2.4. Data Extraction and Quality Assessment. The primary outcome of this systematic review was response to phytochemicals in the management of pain in cancer therapy. The Cochrane Collaboration tool for assessing risk of bias was utilized to rank the quality of these papers [19]. Briefly, this tool includes scoring for (1) sequence generation, (2) allocation concealment, (3) blinding of participants and personnel, (4) blinding of outcome assessors, (5) incomplete outcome data, (6) selective outcome reporting, and (7) other sources of bias. The review of all full-text articles for inclusion based on this Cochrane Collaboration tool was performed by 2 reviewers (Mikhail A. Dziadzko and Fabrice Heritier). 2.5. Data Synthesis and Analysis. Data abstraction was coordinated and performed using the online systematic review software Covidence (Alfred Health, Monash University, Melbourne, Australia) [20]. For each study, relative risk was calculated by extracting the number of patients with dichotomous (binary) pain outcomes and comparing between the control and exposure groups. Statistical analysis, as well as forest plot and stacked bar chart generation, was performed using JMP (SAS, Cary, North Carolina). All confidence intervals (CIs) are reported at the 95% level. Final full-text article review was performed by 3 reviewers (Mikhail A. Dziadzko, Fabrice Heritier, and Bennett G. Childs).

3. Results 3.1. General Characteristics of Included Studies. A total of 1,603 abstracts were identified through initial database search


3

Full-text articles assessed for eligibility (N = 32)

Full-text articles included (N = 7)

Other sources of bias

Selective outcome reporting

Abstracts excluded (N = 70)

Incomplete outcome data

Abstracts screened (N = 102)

Blinding of outcome assessors

Records excluded (N = 985)

Blinding of participants and personnel

Records screened against title (N = 1087)

Allocation concealment

Records after duplicates removed (N = 516)

100 90 80 70 60 50 40 30 20 10 0

Sequence generation

(%)

Records identified through database searching (N = 1603)

High Unclear Low

Figure 2: Stacked bar chart representation of results of the Cochrane Collaboration tool for assessing risk of bias across all studies. Full-text articles excluded (N = 25) 3—Outcome (pain) not reported 4—Not controlled study 3—Not phytochemical 8—Wrong setting/study design 6—Wrong language 1—Not article

Figure 1: PRISMA flow chart.

(Figure 1). After removal of duplicate records (𝑁 = 516) and record screening against title (𝑁 = 985), a total of 102 abstracts remained for screening. Based on screening, 70 of these abstracts were excluded, leaving 32 full-text articles eligible for further assessment. A total of 25 of these fulltext articles were excluded for wrong study design/setting (such as review or commentary) (𝑁 = 8), not being in the English language (𝑁 = 6), study not controlled (𝑁 = 4), no pain outcome reported (𝑁 = 3), not being a phytochemical (𝑁 = 3), or not being a research article (𝑁 = 1), leaving 7 full-text articles included in this systematic review. 3.2. Applied Methodology and Quality Assessment. The Cochrane Collaboration tool for assessing risk of bias was utilized to rank the quality of these 7 full-text articles to review author judgment (Table 1). For 6 of these 7 articles [21– 26], at least 3 of the 7 Cochrane Collaboration tool scoring categories were ranked as “Low” risk of bias. In only 1 of these 7 articles were more than 2 of these scoring categories ranked as “High” risk of bias [27]. For all 7 of these full-text articles, at least 1 of these scoring categories was ranked as “Unclear” risk of bias. For better graphical representation across all

studies, these results are also represented as a stacked bar chart (Figure 2). 3.3. Response to Phytochemicals for Management of Pain in Cancer Therapy. This systematic review synthesizes data for a different phytochemical in each of the 7 full-text articles examined (total 𝑁 = 827) (Table 2). Briefly, 6 of the 7 studies used a placebo as the control. Study duration ranged from immediate effect to 12 months. Delivery methods included oral, ointment, oral solution, and subcutaneous injection. The only fungochemical examined was in the study by Costa Fortes and colleagues [24]. None of these research studies were performed in the United States and none of these 6 phytochemicals or 1 fungochemical is known to have a psychotropic effect. The 1 study of SAMITAL by Pawar and colleagues [27] was excluded from relative risk analysis due to low 𝑁 and methodological uncertainty, as this resulted in an inability to calculate an accurate relative risk (Table 3). The average relative risk of phytochemical compared to control for the included studies (total 𝑁 = 800) was 1.03 [95% CI 0.59 to 2.06]. In other words (although not statistically significant), this relative risk indicates patients treated with phytochemicals were slightly more likely than patients treated with control to obtain successful management of pain in cancer therapy. To graphically assess response to phytochemicals in the management of pain in cancer therapy, a forest plot of relative risk for these 6 studies was generated (Figure 3).

4. Discussion In this systematic review of the use of phytochemicals for management of pain in cancer therapy, we identified a lack

4

BioMed Research International Table 1: The Cochrane Collaboration tool for assessing risk of bias; review of author judgment of risk of bias for each item.

Bao et al., 2010 [21] Belcaro et al., 2014 [22] Brooker et al., 2006 [23] Costa Fortes et al., 2010 [24] Pawar et al., 2013 [27] Pommier et al., 2004 [25] Tröger et al., 2014 [26]

Sequence generation

Allocation concealment

Blinding of participants and personnel

Blinding of outcome assessors

Incomplete outcome data

Selective outcome reporting

Other sources of bias

Low

Low

High

High

Low

Low

Unclear

Unclear

Unclear

Low

Low

Low

Unclear

High

Unclear

Low

Low

Low

Low

Low

Low

Unclear

Unclear

Low

High

Low

Low

Low

Unclear

High

Unclear

High

High

High

High

Low

Low

Unclear

High

Low

Low

Unclear

Low

Low

High

Unclear

Unclear

Low

Unclear

Table 2: Overview of the 7 full-text articles and associated phytochemicals included in this systematic review. Study

𝑁

Study design

Study duration Immediate intervention

Bao et al. [21] 124

RCT, open label

Belcaro et al. [22]

80

RCT, open label

60 days

Brooker et al. [23]

66

RCT, blinded

12 months

Costa Fortes et al. [24]

56

RCT, blinded

6 months

Pawar et al. [27]

27

RCT, blinded

50 days

Pommier et al. [25]

254

RCT, open label

6 weeks

Tröger et al. [26]

220

RCT, open label

12 months

Total

827

Phytochemical Xiaozheng Zhitong Paste Meriva (lecithin delivery system of curcumin) IH636 grape seed proanthocyanidin extract Agaricus silvaticus fungus extract

Delivery Ointment Oral

Oral

Patients Multiple cancers with metastases Chemo/radiotherapy postsurgical and multiple cancers Pain after high-dose radiotherapy for early breast cancer

Postsurgical patients with colorectal cancer and pain Pain from oral SAMITAL mucositis in patients (three botanical Oral solution treated for neck/head extracts) cancer Pain after Calendula Ointment radiotherapy for (plant) breast carcinoma S/C Mistletoe Pancreatic cancer injections

of quality research literature on this subject (𝑁 = 7). While we were able to demonstrate a slight therapeutic benefit use of phytochemicals in the management of cancer pain, this benefit did not achieve statistical significance, which is a function of both the quality and marginal number of the studies that were acceptable for inclusion in a systematic review. The average relative risk of phytochemical compared to control for the included studies (total 𝑁 = 800) was 1.03 [95% CI 0.59 to 2.06]. None of these research studies

Oral

Country China Italy

Major study design bias Control is not a placebo Heterogeneous cancer study population

UK

None

Brazil

Integrity of double-blinding unclear

India

Control versus exposure group inequity

France

Integrity of blinding questionable

Serbia

Not blinded

were performed in the United States and none of these 6 phytochemicals or 1 fungochemical is known to have a psychotropic effect. Over 1,500 research articles were identified potentially examining the use of phytochemicals for management of pain in cancer therapy in initial database screening in this systematic review. However, only 32 research studies reached the level of full-text article assessment for eligibility. Ultimately, only 7 of these studies met final inclusion criteria.


5

Table 3: Relative risk results of the systematic review. Note: Pawar and colleagues (the SAMITAL study) were excluded due to low 𝑁 and methodological uncertainty. Study Bao et al. [21] Belcaro et al. [22] Brooker et al. [23] Costa Fortes et al. [24] Pawar et al. [27] Pommier et al. [25] Tröger et al. [26] Average

𝑁 Lower CI Upper CI Relative risk 124 0.54 3.29 1.34 80 0.75 5.33 2.00 66 0.80 1.13 0.95 56 0.25 0.81 0.45 (27) N/A N/A N/A 254 0.49 0.89 0.66 220 0.70 0.90 0.79 800 0.59 2.06 1.03

Forest plot 01 Bao 02 Belcaro 03 Brooker 04 Costa Fortes 05 Pommier 06 Troger 07 average 0

1

2 3 Relative risk

4

5

6

Figure 3: Forrest plot results of the systematic review. Note: Pawar and colleagues (the SAMITAL study) were excluded due to low 𝑁 and methodological uncertainty.

For example, although small (𝑁 = 27) [27], the study by Pawar and colleagues of SAMITAL, an oral solution of three botanical extracts (Vaccinium myrtillus, Macleaya cordata, and Echinacea angustifolia) for the relief of oral mucositis induced by chemotherapy and/or radiotherapy in oncological patients [28], has an elegant randomized, placebo-controlled, single-blind Phase II study design. However, due to low 𝑁 and methodological uncertainty, this study could not be included in the relative risk analysis. Broadly, these results made formal efficacy score analysis for clinical practice recommendations with a standardized scoring system—such as the United States Preventive Services Task Force (USPSTF) grade (A, B, C, D, and I) and level of certainty (high, moderate, and low) or modified American Heart Association (AHA) class (I, IIa, IIb, and III) and level of evidence (A, B-R, B-NR, C, and E)—impossible [29, 30]. The lack of inclusion of any research study for any phytochemical from plants with potentially beneficial psychotropic effects, such as Cannabis sativa or Cannabis indica (marijuana), also raises

concerns regarding the quality and comprehensiveness of current phytochemical literature and research related to the management of pain in cancer therapy. In the case of the Cannabis plant, which contains the psychotropic chemical tetrahydrocannabinol (THC) and the weakly psychotropic chemical cannabinol (CBD), the potential benefits of this plant and these chemicals in management of pain, including for cancer therapy, have already been extensively explored [31]. One isomer of THC, dronabinol (trade name Marinol), has been approved by the United States (US) Food and Drug Administration (FDA) since 1985. Although beyond the immediate scope of this paper, a synthetic version of THC, nabilone (trade name Cesamet), has also been approved since 1985. In the US, another THCrich Cannabis “extract,” nabiximols (trade name Sativex), and pure CBD isolate (trade name Epidiolex) are currently under review by the FDA for approval in the US [32]. For example, Sativex has been in Phase III trials since 2006. However, in the case of the Cannabis plant, its Schedule I drug status in the US (declared to have dangerous addictive potential and no redeeming medical value) since 1970 has made it difficult to study. Currently, much of the social and political controversy that surround the use of the Cannabis plant for pain is being fought out between the federal government and individual states, many of which have legalized the drug for medical use, recreational enjoyment, or both. For reference, several research articles on medical marijuana reach the level of the 32 full-text articles assessed for eligibility in the systematic review but were eventually excluded for one or more of the reasons described in the Results [33–36]. In the case of marijuana, some of the molecular signaling pathways and other mechanisms explaining the potential therapeutic benefit of marijuana in pain management in general have been partially elucidated through in vitro, in vivo, and human studies [37]. However, the lack of rigor of many of the research publications regarding the clinical efficacy of marijuana for the management of pain in cancer therapy is the result of social and political controversy surrounding its illicit drug status in the US and many other countries around the world [38]. In the case of the US state of Minnesota (where the main campus of Mayo Clinic is located), the production and distribution of medical marijuana were recently legalized at the state level which is currently in production in the form of oral whole plant extracts (containing THC, CBD, other cannabinoids, and other potentially psychotropic chemicals from the Cannabis plant) by at least one of the two stateapproved manufacturers [39]. For reference, of the 1,500+ research abstracts examined in this systemic review, no other psychotropic phytochemical reached the level of more than 1 abstract and none were included for full-text review. Whether any of the phytochemicals examined in this systematic review will ultimately prove valuable for the management of pain in cancer therapy remains to be determined [40]. However, the additional sociological and political barriers to scientific investigation must be considered in the case of psychotropic phytochemicals, such as marijuana. It is important to consider the potential application of phytochemicals from a holistic approach to medicine, as

6 opposed to a disease-centric model for the treatment of medical illness. For example, the molecular signaling pathways and other mechanisms regulating the natural aging process, as well as chronic illnesses of aging, have been partially elucidated through an understanding of cellular senescence [41]. However, small molecules able to directly target senescent cells have yet to be identified. Beyond concerns regarding the quality and comprehensiveness of the current phytochemical literature and research regarding the management of pain in cancer therapy, as well as the specific case of psychotropic phytochemicals, there is also a need to consider the potential use of nonphytochemical fungochemicals in the management of pain in cancer therapy [42]. For example, the chaga mushroom (Inonotus obliquus) has longstanding historical value as a nonpsychotropic medicinal mushroom and is currently the subject of active research studies for its potential antioxidant, immuno-stimulating, anti-inflammatory, antinociceptive/pain, and anticancer properties [43–46]. Along these lines, the mechanisms of the 7 phytochemicals examined in this systematic review are thought to be primarily nociceptive, but the effect of these phytochemicals on the perception of pain is poorly understood. Limitations. There are several limitations to this study. (1) As “filtered information” at the top of the evidence-based medicine pyramid, the conclusions of all systematic reviews are subject to the biases and confounders of the results of the research studies on which these conclusions are based [47]. (2) The word “phytochemical” or any of its permutations is used infrequently in the case of the Cannabis plant (marijuana). Thus, it is likely that this systematic review failed to identify relevant studies for consideration due to this controlled vocabulary [48]. (3) The lack of quality research studies regarding the specific use of phytochemicals for management of pain in cancer therapy limits the statistical power and conclusions that can be ascertained from any systemic review of this subject. For all of these reasons, more high-quality human research studies of the phytochemicals explored in this systematic review, as well as phytochemicals in general, are needed to determine the value of these individual phytochemicals and/or plant extracts in the management of pain in cancer therapy.

5. Conclusion A lack of quality research literature on the subject of phytochemicals for management of pain in cancer therapy is identified in this systematic review. It is not currently possible to demonstrate a clear therapeutic benefit for either general or specific use of phytochemicals in the management of cancer pain. This lack of data is apparent for the psychotropic phytochemical-containing Cannabis plant (marijuana) but may only be a representative example of this problem due to the social and political controversy that surround this plant. There is also a need to consider the potential use of phytochemicals and nonphytochemical fungochemicals for applications ranging from holistic medicine to the natural aging process.


Disclaimer The contents of this paper are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.


Acknowledgments The authors gratefully acknowledge the Medical Scientist Training Program and Mayo Graduate School at Mayo Clinic. The authors also thank Mayo Clinic’s Center for Clinical and Translational Science, CTSA Grant no. UL1 TR000135 from the National Center for Advancing Translational Science (NCATS).

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Research Article Antiproliferative and Antiestrogenic Activities of Bonediol an Alkyl Catechol from Bonellia macrocarpa Rosa Moo-Puc,1 Edgar Caamal-Fuentes,2 Sergio R. Peraza-Sánchez,2 Anna Slusarz,3 Glenn Jackson,4 Sara K. Drenkhahn,5 and Dennis B. Lubahn3 1

Unidad de Investigación Médica Yucatán, Unidad Médica de Alta Especialidad, Centro Médico Ignacio Garc´ıa Téllez, Instituto Mexicano del Seguro Social (IMSS), Calle 41 No. 439, Colonia Industrial, 97150 Mérida, YUC, Mexico 2 Unidad de Biotecnolog´ıa, Centro de Investigación Cient´ıfica de Yucatán (CICY), Calle 43 No. 130, Colonia Chuburná de Hidalgo, 97200 Mérida, YUC, Mexico 3 Department of Biochemistry, University of Missouri, 117 Schweitzer Hall, Columbia, MO 65211, USA 4 Nebraska College of Technical Agriculture Veterinary Technology Program, 404 East 7th Street, Curtis, NE 69025, USA 5 Lindenwood University Belleville, 2600 W. Main, Belleville, IL 62226, USA Correspondence should be addressed to Rosa Moo-Puc; [email protected] and Dennis B. Lubahn; [email protected] Received 10 July 2015; Revised 24 September 2015; Accepted 30 September 2015 Academic Editor: Kanjoormana A. Manu Copyright © 2015 Rosa Moo-Puc et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The purpose of this study was to investigate antiproliferative activity of bonediol, an alkyl catechol isolated from the Mayan medicinal plant Bonellia macrocarpa. Bonediol was assessed for growth inhibition of androgen-sensitive (LNCaP), androgeninsensitive (PC-3), and metastatic androgen-insensitive (PC-3M) human prostate tumor cells; toxicity on normal cell line (HEK 293) was also evaluated. Hedgehog pathway was evaluated and competitive 3H-estradiol ligand binding assay was performed. Additionally, antioxidant activity on Nrf2-ARE pathway was evaluated. Bonediol induced a growth inhibition on prostate cancer cell lines (IC50 from 8.5 to 20.6 𝜇M). Interestingly, bonediol binds to both estrogen receptors (ER𝛼 (2.5 𝜇M) and ER𝛽 (2.1 𝜇M)) and displaces the native ligand E2 (17𝛽-estradiol). No significant activity was found in the Hedgehog pathway. Additionally, activity of bonediol on Nrf2-ARE pathway suggested that bonediol could induce oxidative stress and activation of detoxification enzymes at 1 𝜇M (3.8-fold). We propose that the compound bonediol may serve as a potential chemopreventive treatment with therapeutic potential against prostate cancer.

1. Introduction Prostate cancer is the second most frequently diagnosed cancer and the sixth leading cause of cancer death in males. Approximately, one man in five will be diagnosed with prostate cancer during his lifetime, and 1 man in 33 will die of this disease [1]. Treatment for this disease may include radiation therapy and androgen suppression; surgery and/or chemotherapy are often used. However, adverse effects have been described, decreasing the quality of life of patients [2]. Furthermore, in 15% of the patients, the cancer recurs within a few years as an advanced “hormone-refractory” and often metastatic disease. For these patients, there are few treatment options available [3], and the 5-year survival rate decreases to 28% [4].

Chemotherapy is a good option for the treatment of hormone-independent and hormone-dependent prostate cancer; however, few therapies in clinical phase of development are available [5]. In addition, some cancers have proven to be resistant to chemotherapy drugs [6]. Therefore, identification of new drugs for prostate cancer treatment has significant clinical implications. Currently one of the signaling pathways that has been of great interest because of its importance in the development and progression of prostate cancer is the sonic Hedgehog (Shh) signaling pathway. The Shh pathway involves the autocleavage of full length Shh into an active 20 kD N-terminal fragment (ShhN), which binds to its 12-pass transmembrane receptor, Patched (Ptc1), reversing (relieving) its inhibitory effect on Smoothened (Smo). In prostate

2 cancer, Shh pathway can produce malignant transformation of primitive prostate epithelial progenitor cells; this may be initiated by trapping of a normal stem cell in a Shh-dependent state of continuous renewal, which promotes tumor growth [7–10]. The estrogen and Hedgehog signaling pathways are crucial for physiological proliferation, differentiation, and development of the mammary and prostate glands [11, 12]. It has also been found that activation of both Shh and ER𝛼 can lead to the growth of cancerous tumors (insert references here that show Shh and ERa in breast and prostate cancer). Moreover studies suggest that ER𝛼 regulates the Shh pathway and promotes cancer development [13–17]. Recently a study using in vitro and in vivo models suggested estrogen, mediated through ER𝛼 and ER𝛽, could induce carcinogenesis and various types of toxicity in a normal prostate [18]. Investigations searching for new compounds that can inhibit the Hedgehog pathway and regulate the ERs to treat or prevent prostate cancer could have significant implications [19, 20]. A direct relationship between an increase in reactive oxygen species (ROS) and the induction of the Shh pathway has also been documented. This induction promotes the expression of the antiapoptotic gene Bcl-2 and inhibits the expression of the proapoptotic gene Bax [21]. In addition, Paschos et al. [22] suggest that androgens and estrogen play an important role in the generation of reactive oxygen species leading to the progression of prostate cancer and that the antioxidant activity of certain small molecules may prevent the progression of prostate cancer. NF-E2 Related Factor 2 (Nfr2) is an important transcription factor responsible for stimulating the transcription of genes in response to oxidative or electrophilic stress [23]. This process involves the binding of Nrf2 with Maf protein in the nucleus to form a heterodimer, subsequently interacting with antioxidant responsive element (ARE) to activate gene transcription [24]. The Nfr2-ARE pathway induces transcription of antioxidant proteins and phase II detoxifying enzymes, which are important for protection of cells against ROS damage [23]. Thus this pathway may serve as a marker of oxidative stress damage. The three ERs family members, ER𝛼, ER𝛽, and ER𝛾, play a novel functional role in the inhibitor of Nrf2 transcriptional activity. It is also the modulation of ER𝛼 and ER𝛽 that may be useful as a therapeutic target in cancer chemoprevention studies or for the development of selective estrogen receptor modulators with a lower risk of causing cancer [25–28]. In our continuous effort to search for novel anticancer agents from Mayan medicinal plants of the Yucatan peninsula, we recently isolated a novel compound from the medicinal plant Bonellia macrocarpa (Cavanilles) St˚ahl and Källersjö. This new alkyl catechol, called bonediol, has been demonstrated to have interesting antiproliferative activities in vitro on cancer cell lines [29]. Accordingly, this study evaluated the antiproliferative properties of bonediol in various lines of prostate cancer and also explored its effect on the Shh signaling pathway, interactions with the Nrf2 antioxidant response element, and potential binding to estrogen receptors (ER𝛼 and ER𝛽).


2. Material and Methods 2.1. Isolation of Bonediol. Root bark of B. macrocarpa was collected from Telchac Puerto, Yucatan (Mexico). The plant material was identified and authenticated by taxonomists from the Department of Natural Resources of the Scientific Research Center of Yucatan (CICY). Specimens under the voucher number P. Simá 2979 were deposited at CICY’s U Najil Tikin Xiw herbarium. The obtaining and characterization of the compound were performed as previously described [29]. The pure compound was dissolved in DMSO and stored at −20∘ C. 2.2. Cell Culture. Cell lines of human prostate cancer adenocarcinoma (PC-3), a metastatic variant of PC-3 (PC-3M), hormone sensitive human prostate carcinoma (LNCaP), and one normal human cell line (HEK-293) were obtained from the American Type Culture Collection (ATCC). Shh Light II (JHU-68) and COS-1 cells lines were used to evaluate Shh and Nrf2-ARE pathways, respectively. The cells line PC3 was propagated in F-12K medium (Gibco) and LNCaP in RPMI-1640 medium (Gibco). COS-1, HEK-293, and PC3M cells lines were propagated in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco). Shh Light II cells were maintained in DMEM containing 4 mmol/L of L-glutamine adjusted with 1.5 g/L sodium bicarbonate and 4.5 g/L glucose, supplemented with 0.4 mg/mL G-418 and 0.15 mg/mL zeocin (Invitrogen). All cell lines were cultured in sterile Costar T75 flasks containing fetal bovine serum (10% v/v), 100 U/mL penicillin G, and 100 mg/mL streptomycin at 37∘ C under a humid atmosphere containing 5% CO2 . 2.3. Antiproliferative Activity. Cells were cultured in 96-well plates at a concentration of 5 × 104 cells per well; after being cultured for 24 h at 37∘ C in an atmosphere of 5% CO2 (95% humidity) cells were incubated with appropriate dilutions of the test compound for 48 h. The growth inhibition of the cell lines was evaluated by the sulforhodamine B method [30]. Results are expressed as the concentration of agent that reduces cell growth by 50% (IC50 ). Docetaxel was used as a positive control. All determinations were performed in triplicate. In addition, the degree of toxicity to normal cells was evaluated, by determining the selectivity index (SI) [31]. 2.4. Assay of Inhibition from Hedgehog Pathway. Gli activity in the Shh Light II cell line was assayed after 48 h of treatment with bonediol compound in phenol red-free DMEM supplemented with 0.5% charcoal-stripped serum using the Dual Luciferase Reporter Assay System (Promega). Each experiment was performed at least thrice in duplicate. Mouse recombinant Shh was obtained from R&D Systems. Shh was dissolved in PBS with 0.1% bovine serum albumin. In each experiment, the controls and all treatments contained all vehicles used. All treatments were conducted in phenol red-free medium with charcoal-stripped serum [32]. Each experiment was performed at least thrice in duplicate.


2.6. Plasmid. The vectors containing Gal4-luciferase, the 4x mouse GST Ya subunit ARE (4 copies of the 41 bp GST Ya element) reporter, hemagglutinin (HA) tagged Nrf2, and the Nrf2 transactivation domain-Gal4 DNA binding domain fusion vector have been described previously [25, 28, 33]. phRG-TK control renilla luciferase vector was obtained from Promega. 2.7. Regulation of Antioxidant Response Element (Nrf2-ARE) Assay. The transcriptional activity of NF-E2 Related Factor 2 (Nrf2) on antioxidant response element (ARE) was monitored as previously described [25]. Briefly, COS-1 cells were seeded in 24-well plates in phenol red-free medium with 10% dextran-coated charcoal-stripped fetal bovine serum, for transient transfection with 150 ng 4x mouse GST Ya subunit ARE firefly luciferase reporter or Gal4-luciferase reporter, 10 ngphRGTK control renilla luciferase vector, and different expression vectors using plus and Lipofectamine reagents (Invitrogen, Carlsbad, CA). Constant transfected DNA amount was compensated by empty vector— pcDNA3.1(+)zeo (Invitrogen, Carlsbad, CA). After 12–16 h, transfected cells were then treated with bonediol or vehicle. After 24 h of incubation, cells were rinsed with PBS twice and lysed to measure the luciferase level using the Dual Luciferase assay kit (Promega, Madison, WI). Data were normalized to the cotransfected phRG-TK control renilla luciferase activity. All experiments were performed at least three times with duplicate samples per experiment. 2.8. Statistical Analysis. Graph Pad Prism 4 (Graph Pad Software, La Jolla, CA) was used to calculate 𝑃 values of 𝑃 < 0.05 which were considered significant in all cases. The IC50 were calculated using doses-response nonlinear fit curve. One-way analysis of variance (ANOVA) was used to assess significant differences among treated groups followed by Dunnett’s test.

3. Results In order to explore the possible antiproliferative effect of the compound bonediol, a SRB assay was performed to determine whether this molecule was able to inhibit the growth of prostate cancer cells. A typical dose-response behavior was observed in all cell lines tested, with IC50 in the various cell lines tested ranging from 8.5 to 20.6 𝜇M (Figure 1). Table 1 shows the median concentration that inhibited cell growth (IC50 ) and the selectivity index of bonediol towards all cell

Table 1: Antiproliferative activity IC50 (𝜇M) and selective index of bonediol from B. macrocarpa. Compound Bonediol Docetaxel

Hek-293 33.2 1.10

Cell lines IC50 𝜇M (SI) LNCaP PC-3 18.1 (1.8) 15.5 (2.1) 0.23 (4.78) 0.20 (5.50)

PC3-M 13.3 (2.5) 0.08 (13.75)

100 Cell growth inhibition (% control)

2.5. Competitive Binding Assay. Proteins were synthesized using the TNT Coupled Reticulocyte Lysate System from Promega. In vitro transcription/translation products were treated individually with various concentrations of [3H]17-𝛽-E2 in the absence or presence of various doses of unlabeled competitors or unlabeled 17-𝛽-E2 overnight at 4∘ C in order to achieve equilibrium binding. Bound and free ligand were separated by dextran-coated charcoal. Relative binding affinity (RBA) was determined by dividing the IC50 of the unlabeled 17-𝛽-E2 by the IC50 of the unlabeled competitor.

3

80 60 40 20 0

0.45

0.70 0.95 1.20 1.45 1.70 Log concentration bonediol (𝜇M)

Hek 293 IC50 33.2 𝜇M LNCaP IC50 18.1 𝜇M

1.95

2.20

PC-3 IC50 15.5 𝜇M PC3-M IC50 13.3 𝜇M

Figure 1: Bonediol inhibits cell growth on HEK-293, LNCaP, PC-3, and PC-3M cells. Various concentrations of bonediol were used for 48 h and the effects were examined using SRB colorimetric assay. Each experiment was performed at least thrice in duplicate. IC50 values represent the concentration of the compound at which halfmaximal inhibition we observed.

lines. Bonediol inhibited the growth of PC-3, LNCaP, and metastatic PC-3M cell lines with selectivity compared with Hek-293. Shh Light II cell, an NIH 3T3 cell line stably transfected with Gli1-dependent firefly luciferase and constitutive renilla luciferase reporters, was used to explore the ability of bonediol to inhibit Shh pathway activation. When we tested the model with various concentrations of bonediol (0.1, 0.5, 1, and 5 𝜇M) no significant inhibition was observed compared with the control cyclopamine (data not shown). We next analyzed the potential of bonediol to bind to estrogen receptors (ER𝛼 and ER𝛽) (Figure 2). Bonediol binds to both receptors in a dose-dependent manner; 2.5 𝜇M and 2.1 𝜇M displaced 50% of estradiol binding on ER𝛼 and ER𝛽, respectively. As mentioned above, the activation of the Nrf2 signaling pathway governs the expression of ARE-driven genes. This pathway has been associated with induction of oxidative stress and in this study was used as a possible marker of oxidative damage from bonediol to the cells. Bonediol induces activation of Nrf2-ARE in transfected COS-1 cells at 1 𝜇M (3.8-fold) and 5 𝜇M (2.8-fold) (Figure 3). The results show that bonediol activates Nrf2-ARE signaling possibly through induction of oxidative stress.

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BioMed Research International ER𝛼

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Bonediol (2504 nM) E2 (1.53 nM) (a)

(b)

Figure 2: Dose-response curves of 17𝛽-estradiol and bonediol in the radio ligand receptor binding assay using [3H]17b-estradiol and human estrogen receptor expressed in TNT Coupled Reticulocyte Lysate System: (a) ER𝛼 and (b) ER𝛽.

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Figure 3: Bonediol activate NRF2-ARE. Relative Nrf2-ARE activation from three assays is normalized with the control group. Each symbol is the mean ± SD. ∗∗ 𝑃 < 0.01 versus control.

4. Discussion This study evaluated for the first time the activity of bonediol on binding to the estrogen receptors, inhibition of the Shh pathway, and activation of Nrf2-ARE signaling. Previous work demonstrated the potential antiproliferative effect of bonediol [29] and that this compound has no significant cytotoxic activity [34]. Furthermore, bonediol does not induce apoptosis at low doses [34]. We explored here the antiproliferative potential of bonediol against three prostate cancer lines (hormone sensitive (LNCaP), hormone insensitive (PC-3), and high metastatic hormone insensitive PC3M) and the possible pharmacologic mechanisms. Bonediol has an antiproliferative effect in the three prostate cancer cell lines, indicating that this compound could have various pharmacologic mechanisms, both hormone-dependent and

hormone-independent. Prostate cancer has been reported to have an altered Shh signaling as a pathway of importance in advanced growth and this pathway has recently been shown of interest in the search for new compounds that can inhibit cancer [32, 35]. However, bonediol did not demonstrate an inhibition of this signaling pathway to a concentration of 10 𝜇M (data not shown). No further evaluations were performed at doses, since we observed damage to the cells without concomitant inhibition of Shh signaling. Interestingly, bonediol displaced 50% of estradiol on ER𝛼 and ER𝛽 at concentrations of 2.1 and 2.5 𝜇M, respectively. No studies on the estrogenic potential of alkyl catechols have been reported previously, but there are reports of related synthetic alkyl phenols compounds that have antiestrogenic effect [36]. Furthermore, some studies indicate that the main pharmacophore for recognition by the estrogen receptor is the presence of at least one phenolic alcohol and hydrophobic long chain [37, 38]. We do not know precisely how bonediol is binding to the ERs, but the presence of the two phenolic alcohols and long chain hydrophobic may be involved. The binding of the compound bonediol to the ERs does not indicate whether bonediol could be acting as an antagonist or as an agonist. Further studies are required to observe the way in which this compound could regulate either receptor. It is known that preferential ER𝛽 activation has an antiproliferative effect in breast and prostate cells and is viewed as a protective balance against ER𝛼 activation, which is associated with proliferation [39–41]. Bonediol may be regulating both ER𝛼 and ER𝛽, resulting in an antiproliferative effect on prostate cancer. Moreover, Nelles et al. [19] remarked the importance of development of new selective ER modulators with therapeutic potential. Finally, we found that bonediol activates Nrf2-ARE signaling at a concentration of 1 𝜇M (3.8-fold induction), which is indicative of oxidative stress and may be a mechanism of damage to the cell lines tested. However, another explanation

BioMed Research International for the activation of this pathway could be that some chemical compounds with antioxidant properties have the ability to be redox active and activate the Nrf2-ARE pathway [42]. In this context, some related alkyl phenols have shown antioxidant and prooxidant activity [43, 44]. Antioxidants from plants have been studied for the prevention of several cancer types, including prostate cancer, and it is currently believed that small doses of these compounds could have a beneficial effect by inducing the activation of antioxidant proteins and detoxifying enzymes, which would act against carcinogenic insults [23]. At a concentration of 5 𝜇M bonediol had a slightly lower induction (2.8-fold), compared with the inductive effect at 1 𝜇M (3.8-fold). This effect could be due to the compound exhibiting toxic effects in cell lines at the higher concentration. In the course of our research we found that the compound bonediol is able to bind ER𝛼 and ER𝛽. This is the first report of the potential estrogenic activity of these compounds (alkyl catecohols), isolated from plants. Additionally, bonediol induces activation of Nrf2-ARE, possibly functioning as an antioxidant and generating oxidative stress. Future studies aimed at elucidating how this compound binds to the estrogen receptors and the exact mechanism by which bonediol activates Nrf2-ARE will be beneficial to development of a potential new prostate cancer therapeutic.

5. Conclusion In summary, we found that bonediol binds to both ER𝛼 and ER𝛽 in the low micromolar range, has potential estrogenic activity, and can induce Nrf2 signaling. Furthermore, we propose that the compound bonediol may serve as a potential chemopreventive treatment with therapeutic potential against prostate cancer.


Acknowledgments This project was made possible by Grant no. P50AT006273 from the National Center for Complementary & Alternative Medicine (NCCAM), the Office of Dietary Supplements (ODS), and the National Cancer Institute (NCI) and additionally a Grant no. CB 2010-01-156755 from CONACYT Mexico. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NCCAM, ODS, NCI, or the National Institutes of Health. Rosa E. Moo-Puc received a postdoctoral Fellowship from CONACYT by the Project 2008/78749.

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Research Article Curcumin Enhanced Busulfan-Induced Apoptosis through Downregulating the Expression of Survivin in Leukemia Stem-Like KG1a Cells Guangyang Weng,1 Yingjian Zeng,2 Jingya Huang,3 Jiaxin Fan,2 and Kunyuan Guo1 1

Department of Hematology, Zhujiang Hospital, Southern Medical University, Guangzhou 510000, China Department of Hematology, Jiangmen Wuyi Traditional Chinese Medicine Hospital, Jiangmen 529000, China 3 Department of hemodialysis, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen 518000, China 2

Correspondence should be addressed to Kunyuan Guo; [email protected] Received 28 February 2015; Accepted 24 April 2015 Academic Editor: Pratheeshkumar Poyil Copyright © 2015 Guangyang Weng et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Leukemia relapse and nonrecurrence mortality (NRM) due to leukemia stem cells (LSCs) represent major problems following hematopoietic stem cell transplantation (HSCT). To eliminate LSCs, the sensitivity of LSCs to chemotherapeutic agents used in conditioning regimens should be enhanced. Curcumin (CUR) has received considerable attention as a result of its anticancer activity in leukemia and solid tumors. In this study, we investigated the cytotoxic effects and underlying mechanisms in leukemia stem-like KG1a cells exposed to busulfan (BUS) and CUR, either alone or in combination. KG1a cells exhibiting BUS-resistance demonstrated by MTT and annexin V/propidium iodide (PI) assays, compared with HL-60 cells. CUR induced cell growth inhibition and apoptosis in KG1a cells. Apoptosis of KG1a cells was significantly enhanced by treatment with CUR+BUS, compared with either agent alone. CUR synergistically enhanced the cytotoxic effect of BUS. Seven apoptosis-related proteins were modulated in CURand CUR+BUS-treated cells analyzed by proteins array analysis. Importantly, the antiapoptosis protein survivin was significantly downregulated, especially in combination group. Suppression of survivin with specific inhibitor YM155 significantly increased the susceptibility of KG1a cells to BUS. These results demonstrated that CUR could increase the sensitivity of leukemia stem-like KG1a cells to BUS by downregulating the expression of survivin.

1. Introduction Hematopoietic stem cell transplantation (HSCT) is currently one of the most effective methods of curing hematopoietic malignances [1–3]. In 1977, Thomas reported long-term survival in 13 patients with leukemia who underwent HSCT [4]. However, leukemic patients who received allo-HSCT are still susceptible to relapse and to nonrecurrence mortality (NRM) associated with the toxicity of the chemotherapeutic agents used for conditioning [5, 6], such as busulfan (BUS), cytoxan, and etoposide. Leukemia stem cells (LSCs) are considered to be responsible for leukemia relapse and drug resistance [7, 8]. Complete elimination of LSCs and reduced doses of chemotherapeutic agents are thus essential strategies for improving the prognosis in these patients [9]. Lapidot et al. demonstrated that acute myeloid LSCs possessed the cell

phenotype of CD34+ CD38− [10]. Notably, KG1a cells with a similar phenotype have demonstrated self-renewal potential and chemotherapy and immunotherapy resistance [11, 12]. KG1a cells are thus considered as leukemia stem-like cells and provide an ideal cells model for studying LSCs. The alkylating agent BUS is commonly applied in different conditioning regimens for HSCT, to eliminate the underlying leukemia cells and exert an immunosuppressive effect. However, BUS is associated with severe toxicities, including liver, lung, and skin toxicities, hemorrhagic cystitis, diarrhea, and mucositis [13, 14]. The ability of BUS to inhibit or effectively kill LSCs also remains unclear, leaving the potential for leukemia relapse after HSCT. Curcumin (CUR) is a polyphenol derived from the rhizomes of turmeric, which has received considerable attention as a result of its chemopreventive, chemotherapeutic, and

2 chemosensitizing activities in leukemia and various solid tumors, via targeting multiple signaling pathways [15–19]. CUR thus represents a potential sensitizing agent when combined with chemotherapeutic drugs for treating LSCs. In this study, we explored the cytotoxic efficiencies and molecular mechanisms of CUR and BUS alone and in combination in KG1a cells.

2. Materials and Methods 2.1. Reagents. Reagents include RPMI-1640 (Hyclone, SH30809.01B), fetal bovine serum (Hyclone, SH30084.03), penicillin and streptomycin (PAA, P11-010), CUR (Sigma, 458-37-7), DMSO (Amresco, 67-68-5), BUS (Sigma, 5598-1), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Seebio, 298-93-1), hydroxypropyl methylcellulose (Amresco, 9004-65-3), anti-CD34-PE/CD38-FITC (BD Biosciences, USA), FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, USA), CycleTEST Plus DNA Kit (BD Biosciences, USA), anti-PARP (BD, USA, 1 : 500), anti-caspase-3 (CST, USA, 1 : 5000), anti-survivin (BD, USA, 1 : 5000), ym155 (SELLECK, 781661-94-7), Human Apoptosis Antibody Array Kit (RayBio, USA), electrophoresis apparatus trophoresis (Tanon EPS200), and LI-COR Odyssey Scanner (USA). 2.2. Cell Lines and Culture. Human acute myeloid leukemia KG1a cells and human acute promyelocyte leukemia HL-60 cells were cultured in RPMI-1640 with 10% inactivated fetal bovine serum, penicillin, and streptomycin at 37∘ C under 5% CO2 , which were kindly presented by Miaorong She (Department of Hematology, Guangdong General Hospital, Guangzhou, China). 2.3. Cell Viability Assay. Cells viability was estimated by MTT assay. KG1a and HL-60 cells in logarithmic phase at 5 × 105 cells/mL were incubated in 96-well plates in the presence or absence of the indicated test samples in a final volume of 0.2 mL for 24 h or 48 h at 37∘ C under 5% CO2 . 20 𝜇L MTT solution (5 mg/mL in phosphate-buffered saline (PBS)) was then added to each well and incubated for 4 h at 37∘ C, followed by the addition of 200 𝜇L DMSO. Finally the plates were shaken and examined at 490 nm using a microplate reader (MK3, Shanghai). Each assay was performed in triplicate. Cells viability was calculated as follows: survival ratio (%) = (OD value of experimental samples/OD value of control samples) × 100%.

BioMed Research International 1640 medium supplemented with 0.9% methylcellulose and 20% fetal bovine serum (FBS) in a final volume of 1 mL at 37∘ C under 5% CO2 . Colonies (>50 cells) were counted and photographs were taken under a light microscopy after 14 days. All the samples were analyzed in triplicate. 2.6. Measurements of Apoptosis. The apoptotic rates of KG1a and HL-60 cells were determined by annexin V binding assays, according the manufacturer’s instructions. Briefly, approximately 1.0 × 106 cells in 6-well plates were treated with various concentrations of the indicated test samples at 37∘ C under 5% CO2 for 48 h. The cells were then harvested to analyze apoptosis. Cells were washed twice with cold PBS and then resuspended in 1x Binding Buffer at a concentration of 1 × 106 cells/mL and 100 𝜇L of the solution (1 × 105 cells) was transferred to a 5 mL culture tube and then 5 𝜇L of FITC annexin V and 5 𝜇L PI were added and the cells were gently vortexed, followed by incubation for 15 min at room temperature (25∘ C) in the dark. Finally, 400 𝜇L of 1x Binding Buffer was added to each tube and the cells were then analyzed by flow cytometry. 2.7. Cell Cycle Analysis. Approximately 1.0 × 106 cells in 6well plates were treated with various concentrations of the indicated test samples at 37∘ C under 5% CO2 for 48 h. Cell cycle analysis was performed by flow cytometry using the CycleTEST Plus DNA Kit (BD Biosciences), according to manufacture’s instructions. 2.8. Western Blot Analysis. Total cellular proteins were isolated with lysis buffer (RIPA). Equal amounts of protein were subjected to 10% or 15% polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skim milk, the membranes were incubated with primary antibodies (anti-PARP, anti-caspase-3, and anti-survivin) over night at 4∘ C and then incubated with horseradish peroxidase-conjugated antimouse secondary antibody at room temperature for 1-2 h. The protein bands were imaged using a chemiluminescence reagent (CTB, USA) and densities value of the bands was analyzed using Image J software, with glyceraldehyde 3phosphate dehydrogenase (GAPDH; HC301; 1 : 5000) as the internal reference.

2.4. Flow Cytometry Analysis for Immunophenotyping. Single-cell suspensions of 1.0 × 106 of KG1a and HL-60 cells were washed in PBS containing 2% fetal calf serum (FCS). The cells were resuspended in PBS and incubated for 30 min at 4∘ C with antibodies to surface antigens CD34 and CD38. Mouse IgG isotype was used as a control. The cells were then analyzed by flow cytometry.

2.9. Analysis of Apoptosis-Related Proteins by RayBio Arrays. The expression of 43 apoptosis-related proteins was analyzed using a Human Apoptosis Antibody Array Kit (RayBio, USA). Briefly, according to instructions, each of the capture antibodies was printed on the membranes, followed by addition of the treated or untreated cell lysate. After extensive washing, the membranes were incubated with a cocktail of biotin-conjugated anti-apoptotic protein antibodies. After incubation with the infrared fluorescent agent-streptavidin, the fluorescence signals were visualized using a LI-COR Odyssey Scanner.

2.5. Methylcellulose Colony Formation Test. Approximately 500 treated or untreated cells per well were cultured in RPMI

2.10. Statistical Analysis. The data ware represented as the mean ± standard deviation (SD) and analyzed using SPSS


3 120

KG1a

104

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40 80 160 320 640 BUS (𝜇M)

KG1a IC50 = 22523.1 𝜇M HL-60 IC50 = 354.5 𝜇M

(a)

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106

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10 10 93.6 102 86.8 102 93.2 2.6 2.7 90.4 3.2 3.8 1.4 1.4 1.4 10 10 10 101.4 102.1 103 104 105 106 107.2 102.1 103 104 105 106 107.2 102.1 103 104 105 106 107.2 102.1 103 104 105 106 107.2 104 104 104 104 0.7 17.4 0.6 0.6 11.2 20.5 1.1 1.2

∗∗

20

∗∗

15 10 5 0 Control

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KG1a HL-60 (c)

Figure 1: CD34+ CD38− KG1a cells were insensitive to BUS. (a) KG1a cells were stained with FITC-conjugated CD38 antibody and PEconjugated CD34 antibody and subjected to flow cytometry to analyze the purity of the CD34+ CD38− cells population. (b, c) KG1a cells were exposed to different concentrations of BUS for 24 or 48 h (c). MTT assay was performed (b) and apoptosis (c) was detected by annexin V/PI assay. Cells in the lower right quadrant represent early apoptosis and cells in the upper right quadrant represent late apoptosis. The graph displays the means ± SD of three independent experiments. ∗∗ 𝑃 < 0.01, ∗∗∗ 𝑃 < 0.001 (compared with untreated KG1a cells).

4


100 80

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102 67.6 11.4 101.4 2.1 3 4 5 10 10 10 10 106 107.2 Annexin V-FITC

Figure 2: Continued.

32


5 30

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10 5 0 Control

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CUR (𝜇M) (d)

Figure 2: CUR suppressed cell growth, induced S phase arrest, and induced cell apoptosis in KG1a cells. (a) KG1a cells were treated with different concentrations of CUR for 24 or 48 h. MTT assays were performed. (b) KG1a cells were treated with different concentrations of CUR for 48 h and analyzed for DNA content by flow cytometry. (c) KG1a cells were treated with CUR and inoculated in methylcellulose for 14 days and then observed under a right microscope (magnification ×40). The graph displays means ± SD of three independent experiments. ∗ ∗∗ ∗∗∗ 𝑃 < 0.05, 𝑃 < 0.01, and 𝑃 < 0.001 (compared with control). (d) KG1a cells were treated with different concentrations of CUR for 48 h and analyzed by flow cytometry. The graph displays means ± SD of three independent experiments. ∗ 𝑃 < 0.05, ∗∗ 𝑃 < 0.01 (compared with control).

13.0 and Graphpad Prism 5 software. Means of different groups were compared using one-way ANOVA followed by Bonferroni multiple comparison to evaluate the differences between two groups under multiple conditions. If the date failed the normality test, the Kruskal-Wallis one-way ANOVA on ranks was used for data that failed the normality test. A value of 𝑃 < 0.05 was considered statistically significant. Compusyn software was used to evaluate the synergistic effects of drug combinations. The combination index (CI) was generated by Compusyn software, where CI < 1, CI = 1, and CI > 1 indicated synergism, additive effect, and antagonism, respectively.

3. Results 3.1. CD34+ CD38− KG1a Cells Were Insensitive to BUS. The percentages of CD34+ CD38− cells were 92.3% in KG1a cells, but no CD34+ CD38− cells were detected among the HL-60 cells (Figure 1(a)). KG1a and HL-60 cell lines were treated with various concentrations of BUS for 48 h followed by cell viability and apoptosis analyses. BUS suppressed proliferation and induced apoptosis in more mature HL-60 cells, but not in KG1a cells (Figures 1(b) and 1(c)). The IC50 values for BUS were 22523.1 𝜇M in KG1a cells and 354.5 𝜇M in HL-60 cells, respectively. The apoptotic rate was significantly higher in HL-60 cells, compared with KG1a cells. These results indicated that leukemia stem-like KG1a cells were insensitive to BUS and exhibited drug resistance. 3.2. CUR Inhibited Cell Growth and Induced Cell Apoptosis in KG1a Cells. KG1a cells were treated with various concentrations of CUR (0–32 𝜇M) for 24 and 48 h and the cytotoxic effects were detected by MTT assay. CUR exhibited dose- and time-dependent cytotoxic effects in KG1a cells (Figure 2(a)). The IC50 values at 24 and 48 h were 51.3 𝜇M and

18.4 𝜇M, respectively. The antiproliferation effect of CUR in KG1a cells was confirmed further by colony formation assays. CUR suppressed colony formations in a dose-dependent manner (Figure 2(c)). To determine if CUR-induced growth inhibition was related to the cell cycle arrest, KG1a cells were exposed to CUR for 48 h followed by detection by flow cytometry. CUR induced S phase arrest in KG1a cells (Figure 2(b)). Treatment with 32 𝜇M CUR significantly increased the percentage of cells in S phase from 24.14% to 40.08%. We investigated the effect of CUR for 48 h on early and late apoptosis in KG1a cells by annexin V analysis. CUR induced apoptosis in a dose-dependent manner in KG1a cells (Figure 2(d)). These results demonstrated that CUR could inhibit cell growth and induce apoptosis in KG1a cells. 3.3. CUR Increased BUS-Induced Apoptosis by Downregulating Procaspase-3 followed by PARP Degradation in KG1a Cells. We determined if CUR could increase BUS-induced apoptosis in KG1a cells by examining proapoptotic effects of CUR and BUS alone and in combination (CUR + BUS) using annexin V/PI. Apoptosis was significantly increased in CUR + BUS group, compared with CUR- or BUS-alone groups (Figure 3(a)). For instance, apoptotic rates in cells treated with 16 𝜇M CUR, 80 𝜇M BUS, and the combination groups were 15.6 ± 1.5%, 5.7 ± 0.7%, and 28.3 ± 0.8%, respectively. Western blot analysis also demonstrated that the markers of apoptosis procaspase-3 cleaved PARP were significantly regulated in combination groups (Figure 3(b)). These results indicated that CUR significantly enhanced BUS-induced apoptosis. 3.4. CUR Synergistically Enhanced the Cytotoxic Effect of BUS in KG1a Cells. We investigated the ability of CUR to enhance the cytotoxic effect of BUS by treating KG1a cells with combinations of the two drugs at different doses but

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1.7

106

1.7

PI

105 104 103 102 101.4 10

107.1

106

4

5

107.2

6

10 10 10 Control Annexin V-FITC 107.1

4.8

1.7

106

1.3

107.1

2.5

106

105

105

4

4

104

PI

103

103 102 75.1 101.4

102 101.4

18.4

102.1 103 104 105 106 107.2 16

102.1 103 104 105 106 107.2 32

CUR (𝜇M) Annexin V-FITC

CUR (𝜇M) Annexin V-FITC

107.1

PI

104 103

104

106

10

43.1

46.5

102.1 103 104 105 106 107.2 32 + 80 CUR + BUS (𝜇M)

Annexin V-FITC

Annexin V-FITC

80 ∗∗ ∗∗∗

∗∗∗ ∗∗∗

∗∗ ∗∗∗

40

∗ ∗∗

20

160

80

0 CUR (𝜇M) BUS (𝜇M)

8.7

104 103

10 101.4

60

1.0

105

4

2

102 56.9 30.6 101.4 7.2 2.1 3 4 5 10 10 10 10 106 107.2 10 16 + 160 CUR + BUS (𝜇M)

Annexin V-FITC

6.3

102.1 103 104 105 106 107.2 160

107.1

9.2

103

22.7

102.1 103 104 105 106 16 + 80 CUR + BUS (𝜇M)

86.2

BUS (𝜇M) Annexin V-FITC

105

105

32

69.7

1.2

106

103

Apoptosis (%)

102 101.4

107.1

32 + 80

105

106

5.6

BUS (𝜇M) Annexin V-FITC

10.7

1.8

102 101.4

3.6

102.1 103 104 105 106 107.2 80

PI

5.9

16

106

1.7

Control

107.1

92.7

PI

8.8

1.9

103

16 + 160

83.3

16 + 80

102 101.4

10

32 + 160

103

10

PI

105

4

10

PI

10

3

105 PI

PI

106

107.1

6.7

1.2

3.0

93.7 2.1

CUR + BUS (𝜇M) (a)


102 101.4

34.3

56.2

102.1 103 104 105 106 107.2 32 + 160 CUR + BUS (𝜇M)

Annexin V-FITC


7 CUR (𝜇M) 16

Control

CUR + BUS (𝜇M)

BUS (𝜇M)

32

80

160

16 + 80

16 + 160

32 + 80

32 + 160

Cleaved PARP

Caspase-3 GAPDH Caspase-3

4

CUR + BUS (𝜇M)

∗ ∗∗

32 + 160

32 + 80

CUR (𝜇M) BUS (𝜇M)

16 + 80

Control

32 + 160

32 + 80

16 + 160

16 + 80

160

0

80

0.0 32

1

16

0.2

CUR (𝜇M) BUS (𝜇M)

∗∗ ∗∗

∗

2

160

0.4

∗

3

80

0.6

32

Relative density

0.8

Control

Relative density

1.0

5

16 + 160

∗ ∗

16

1.2

Cleaved PARP

∗∗ ∗

CUR + BUS (𝜇M) (b)

Figure 3: CUR increased BUS-induced apoptosis by downregulating procaspase-3 followed by PARP degradation in KG1a cells. (a, b) KG1a cells were treated with different concentrations of CUR or BUS alone or CUR + BUS for 48 h and analyzed by flow cytometry (a) and western blot (b). The graphs represent means ± SD of three independent experiments. ∗ 𝑃 < 0.05, ∗∗ 𝑃 < 0.01, and ∗∗∗ 𝑃 < 0.001.

in a constant ratio (CUR to BUS: 8 𝜇M to 80 𝜇M, 16 𝜇M to 160 𝜇M, and 32 𝜇M to 320 𝜇M, resp.) for 48 h. Synergistic effects were estimated using Compusyn software. Cotreatment with all doses exhibited synergistic effects in KG1a cells (Figures 4(a) and 4(b)). For example, 16 𝜇M CUR plus 80 𝜇M BUS resulted in a proliferation inhibition of 60.20% (Figure 4(c)), compared with CUR (44.40%) and BUS alone (4.53%), indicating a synergistic effect (CI = 0.733), in accord with the result of apoptosis assays. Cotreatment with 16 𝜇M CUR and 80 𝜇M BUS for 48 h also induced S and G2/M phase arrest in KG1a cells (Figure 4(d)), which may represent one of the mechanisms responsible for the synergism. 3.5. Effects of BUS and CUR on Protein Expression in KG1a Cells. We investigated the molecular mechanisms responsible for CUR-induced apoptosis and enhanced BUS-induced apoptosis in KG1a cells treated with 16 𝜇M CUR, 80 𝜇M BUS, and their combination by detecting expression levels of 43 apoptosis-related proteins using RayBio human apoptosis arrays. The threshold values of fold-change were usually set at ≤0.667 or ≥1.5. Three proteins (Bcl-2-associated death promoter (BAD), caspase-3, and HTRA) were upregulated and four proteins (Bcl-2, cellular inhibitor of apoptosis2 (cIAP-2), survivin, and X-linked inhibitor of apoptosis (XIAP)) were downregulated in CUR group and combination group (Table 1; Figure 5(a)). Survivin was significantly more

downregulated in the combination group compared with the CUR group. This result was further confirmed by western blot analysis (Figure 5(b)). Survivin is known to be an important antiapoptosis protein that participates in the modulation of apoptosis by various signal pathways. We therefore considered that survivin was a likely key factor in CUR-induced apoptosis and BUS sensitivity in KG1a cells. 3.6. Suppression of Survivin with YM155 Could Induce Apoptosis and Increase the Susceptibility to BUS in KG1a Cells. We clarified the role of CUR-induced survivin downregulation in sensitization of KG1a cells to BUS by suppressing survivin expression using the specific inhibitor YM155. The proapoptotic effect and sensitivity to BUS were evaluated by flow cytometry. The cytotoxic activity of YM155 in KG1a cells was detected by MTT assays. YM155 exhibited time- and dose-dependent growth-inhibitory effects in KG1a cells (Figure 6(a)). The IC50 values of 24 and 48 h were 8.86 ng/mL and 2.43 ng/mL, respectively. The YM155 IC50 of 2.43 ng/mL was used in subsequent experiments. KG1a cells were exposed to 2.43 ng/mL YM155 and 80 𝜇M BUS alone or in combination for 48 h and early and late apoptotic rates were then examined. YM155-induced apoptosis (14.90%) (Figure 6(b)) was similar to CUR-induced apoptosis in KG1a cells (15.50%, 16 𝜇M, Figure 3(a)). Suppression of survivin

8


Median-effect plot

Log(Fa ) 2

Combination index plot

CI

Log(D) CI = 0.733

A

B

3

CI = 0.579

0.5

0

−2

1

CUR + BUS

CUR + BUS

CUR BUS

Fa (a)

Inhibition rate (MTT, %)

100 ∗

80

∗

(b)

∗ ∗∗∗

∗∗

60 ∗∗

40 20

32 + 160

BUS (𝜇M)

16 + 80

160

8 + 40

CUR (𝜇M)

80

40

32

16

8

Control

0

CUR + BUS (𝜇M) Control

400

G0/G1

CUR = 16 𝜇M

300

300 200

(c)

S

G2/M

100

Cell number

Cell number

400

200 100

0

0 500 k 1 M 1.5 M 2 M DNA content BUS = 80 𝜇M

500 k 1 M 1.5 M 2 M DNA content CUR + BUS (16 𝜇M + 80 𝜇M) 300

Cell number

300 Cell number

0

−3

C

CI = 0.722

200 100 0

200

100

0 500 k 1 M 1.5 M 2 M DNA content


100 k 400 k 700 k 1 M DNA content

1.3 M


9 CUR + BUS (16 𝜇M + 80 𝜇M) BUS = 80 𝜇M CUR = 16 𝜇M Control 0

20

G2/M S

40 60 Ratio (%)

80

100

G0/G1 (d)

Figure 4: CUR synergistically enhanced the cytotoxic effect of BUS in KG1a cells. KG1a cells were exposed to CUR + BUS at different doses but in a constant ratio (CUR to BUS: 8 𝜇M to 80 𝜇M, 16 𝜇M to 160 𝜇M, and 32 𝜇M to 320 𝜇M, resp.) for 48 h examined by MTT assay. (a, b) CI-effect plots and median-effect plots were generated using Compusyn software. The points A, B, and C represent CI values for the three combination groups, respectively. (c) The graph displays means ± SD of three independent experiments. ∗ 𝑃 < 0.05, ∗∗ 𝑃 < 0.01, and ∗∗∗ 𝑃 < 0.001. (d) KG1a cells were treated with CUR or BUS alone or CUR + BUS for 48 h and analyzed with flow cytometry. The percentages of cells in S and G2/M phases were significantly higher in CUR + BUS group compared with the CUR- or BUS-alone group.

by YM155 increased the susceptibility to BUS, with a BUSinduced apoptotic rate of 40.36%, compared with 8.67% for BUS alone. These results revealed that suppression of survivin could contribute to CUR-induced apoptosis and the synergistic effect of CUR and BUS in KG1a cells.

4. Discussion LSCs were a rare population of cells in patients with leukemia. They possess characteristics of self-renewal, chemotherapy resistance, and immune resistance [20–22]. LSCs were thus commonly regarded as the origin of leukemia relapse and refractory [12, 23]. LSCs have been reported to demonstrate a CD34+ CD38− phenotype [10, 12, 24, 25], reflected by the acute immature myeloid leukemia cells KG1a cell line, which expresses high level of CD34 and lacks CD38. We also provided the first demonstration that leukemia stemlike KG1 cells were insensitive to BUS according to MTT assays and annexin V/PI assays, compared with the more mature acute promyelocyte leukemia HL-60 cells. KG1a cells have previously been shown to be resistant to the common chemotherapeutic agent daunorubicin [12]. CD34+ CD38− KG1a cells maybe thus provide an ideal model of LSCs, in accord with previous studies [12, 26]. CUR and its analogs have been showed to suppress the growth of various leukemia cells, including U937 cells [27, 28], K562 chronic myeloid leukemia cells [27], and HL-60 acute promyelocyte leukemia cells [29, 30], but its effects on LSCs have not been determined. CUR inhibited proliferation and induced S phase arrest and apoptosis in leukemia stemlike KG1a cells. CUR was previously shown to target cancer cells or cancer stem cells by several mechanisms, including autophagy, G2/M phase arrest, and apoptosis in hepatoma cells (HepG2, SMMC-7721, and BEL-7402) [31], reducing

the expression of stem cell markers (DCLK1/Lgr5/CD44) in colon cancer stem-like HCT-116 [32], and reducing microtentacles and preventing reattachment in breast cancer stemlike cells [33]. CUR has thus demonstrated indeed extensive anticancer effects in various tumors and has been shown to modulate numerous targets including the activation of transcription factors (NF-kB, STAT3, and AP-1), receptors (CXCR-4, HER-2, and IL-8), kinases (EGFR, ERK, and JAK), cytokines (TNF, IL), and others (cyclin-D1/E,XIAP-1)[15, 34]. Unfortunately, the mechanism of S phase arrest induced by CUR was not explored further in depth in this study. In a word, CUR exhibited an inhibitory effect on leukemia stemlike KG1a cells, which was particularly worthy of attention. Insensitivity of LSCs to conditioning chemotherapeutic drugs such as BUS is a major reason for leukemia relapse after HSCT. In this study, KG1a cells displayed resistance to BUS, indicated by a lack of apoptosis induction. We there explored the effects of the combination of CUR and BUS on apoptosis in KG1a cells. Encouragingly, CUR markedly enhanced BUS-induced apoptosis, as confirmed by annexin V/PI and western blot analysis. Similarly, the combination of various concentrations of CUR and BUS produced a synergistic antiproliferation effect in KG1a cells. Accumulating evidence suggests that CUR potentiates the effect, including enhancing the antiapoptotic effects of chemotherapeutic drugs such as 5-fluorouracil, bortezomib, FOLFOX, and paclitaxel in vitro or in vivo [35–39]. The results of the current study suggested that CUR has the potential to be a powerful chemosensitizing agent in various cancer cells, including cancer stem cells (CSCs). Notably, Yu et al. demonstrated that CUR either alone or together with FOLFOX could efficiently eliminate FOLFOX-resistant colon cancer stem cells [36]. However, the effects of the combination of CUR with BUS on cancer stem cells, especially LSCs, have not been reported. BUS is

10


Survivin

Survivin

CUR

Control

Survivin

Survivin

CUR + BUS

BUS (a) Control

CUR

BUS

CUR + BUS

Survivin

GAPDH

(b)

Figure 5: Expression of antiapoptosis protein survivin in KG1a cells. (a, b) KG1a cells were treated with CUR (16 𝜇M), BUS (80 𝜇M), or CUR + BUS for 48 h tested by protein arrays kit (a) as described in “methods.” The intensities of green fluorescence spots represent survivin expression. Survivin expression was significantly decreased in CUR and CUR + BUS groups, compared with controls, the same as the results analyzed by western blot analysis.

well-known conditioning agent for HSCT, and its ability to eliminate LSCs is vital for the successful cure of leukemia in patients undergoing this treatment. Gerber et al. pointed out that minimal residual disease detected during complete remission was enriched for CD34+ CD38− ALDHint leukemia cells, which were highly correlated with subsequent clinical relapse [25]. Combined treatment with CUR may allow a reduction in the clinical dose of BUS for HSCT, with the potential for reducing NRM. Nakane et al. showed that reduced-intensity conditioning by BUS was associated with lower NRM in patients undergoing unrelated bone marrow transplantation [40]. The results of the current study showed a significant reduction in the percentage of cells in G0/G1 phase in the combination group (Figure 4(d)), suggesting that cells in G0/G1 phase were more sensitive to this drug combination. Interestingly, cancer stem cells (including LSCs) tend to remain in quiescent phase and possess drug resistance [41–44]. The discovery that CUR could sensitize leukemia

stem-like KG1a cells to BUS suggested that further studies are warranted, especially with a view to elucidating the mechanism responsible for this effect. The results of apoptosis arrays showed that seven apoptosis-related proteins were significantly modulated in KG1a cells treated with CUR and CUR + BUS (Figure 5(a); Table 1). A mechanistic diagram was thus presented in Figure 7. Activated caspase-3 is the common effector caspase of the intrinsic and extrinsic pathways of apoptosis and is thus a marker of apoptosis [45]. Activated caspase-9 is an upstream protein effector that may stimulate caspase-3 [45]. XIAP inhibits caspases, including caspase-3 and caspase-9, by direct physical interactions [46]. Interestingly, we found that XIAP expression in KG1a cells was downregulated by CUR and especially by CUR + BUS. cIAP-2, another member of inhibitor of apoptosis (IAP) family, was downregulated in the same two groups. cIAP-2 could bind caspase-3 and mark it for proteasomal degradation rather than inhibit it


11

Table 1: Expression of apoptosis-related proteins in various treated groups. Name BAD BAX Bcl-2 Bcl-w BID BIM Caspase-3 Caspase-8 CD40 CD40L cIAP-2 CytoC DR6 Fas FasL HSP27 HSP60 HSP70 HTRA IGF-I IGF-II IGFBP-1 IGFBP-2 IGFBP-3 IGFBP-4 IGFBP-5 IGFBP-6 IGF-1sR Livin p21 p27 p53 SMAC Survivin sTNF-R1 sTNF-R2 TNF-alpha TNF-beta TRAILR-1 TRAILR-2 TRAILR-3 TRAILR-4 XIAP

Control

CUR

BUS

CUR + BUS

9754.372 11665.057 5201.059 3934.754 1241.608 8791.171 5414.356 6636.880 7229.618 16087.024 1971.306 8302.835 3370.080 25854.867 7155.526 2395.653 24408.943 6055.367 10052.830 1772.604 7991.872 3060.239 3750.645 5321.179 2012.843 11366.442 2354.116 5755.629 7478.838 17207.390 8486.943 9829.587 9838.568 76507.100 3284.761 3504.793 2641.505 6946.720 3835.964 7488.942 4649.857 4613.933 5465.996

11939.256 11534.453 4032.165 3354.182 1159.421 7763.556 9883.863 7681.166 5707.767 12956.135 1308.320 6758.989 2669.250 22694.082 6020.454 2109.392 20936.090 7532.267 20991.713 1407.585 8066.316 2144.135 2977.966 3985.511 1573.358 9202.906 1950.567 4286.285 6829.468 15850.718 7790.358 9303.164 10157.840 31134.629 2747.670 2428.035 1771.889 4871.952 3031.569 6315.272 3717.494 3900.142 2412.352

8854.383 11072.484 4793.070 3464.131 1473.127 8225.960 4966.096 7505.017 6661.514 15884.779 1917.709 8156.269 3008.736 24157.601 7420.907 2279.382 30040.496 6146.040 12382.184 1857.630 10003.085 2680.707 3901.504 5547.657 1736.271 10929.497 2340.662 5791.576 7818.628 18463.352 8890.430 11354.853 11915.987 81877.505 3781.346 3118.079 2802.065 6715.585 4114.182 7505.017 4614.036 4694.541 6570.195

21865.952 10875.680 2454.999 3789.188 1702.301 8295.705 12514.137 7338.462 5874.868 14392.403 702.301 7832.756 2958.532 23693.574 7047.913 2807.833 23881.647 6971.961 26027.247 1717.974 9495.273 2416.014 3569.769 5313.061 1873.496 10428.404 2413.603 5056.269 7778.504 17993.517 8879.213 11263.882 12549.047 11691.497 3136.960 2815.066 2499.200 6430.648 4102.643 7691.701 4605.376 4706.646 1011.597

CUR/control (fold-change) 1.224 0.989 0.775 0.852 0.934 0.883 1.825 1.157 0.789 0.805 0.664 0.814 0.792 0.878 0.841 0.881 0.858 1.244 2.088 0.794 1.009 0.701 0.794 0.749 0.782 0.810 0.829 0.745 0.913 0.921 0.918 0.946 1.032 0.407 0.836 0.693 0.671 0.701 0.790 0.843 0.799 0.845 0.441

BUS/control (fold-change) 0.908 0.949 0.922 0.880 1.186 0.936 0.917 1.131 0.921 0.987 0.973 0.982 0.893 0.934 1.037 0.951 1.231 1.015 1.232 1.048 1.252 0.876 1.040 1.043 0.863 0.962 0.994 1.006 1.045 1.073 1.048 1.155 1.211 1.070 1.151 0.890 1.061 0.967 1.073 1.002 0.992 1.017 1.202

CUR + BUS/control (fold-change) 2.242 0.932 0.472 0.963 1.371 0.944 2.311 1.106 0.813 0.895 0.356 0.943 0.878 0.916 0.985 1.172 0.978 1.151 2.589 0.969 1.188 0.789 0.952 0.998 0.931 0.917 1.025 0.878 1.040 1.046 1.046 1.146 1.275 0.153 0.955 0.803 0.946 0.926 1.070 1.027 0.990 1.020 0.185

KG1a cells were treated with CUR (16 𝜇M), BUS (80 𝜇M) alone, or CUR + BUS for 48 h tested by protein arrays kit. The data represent fluorescence intensities of 43 apoptosis-related proteins. The bold bands indicate proteins that were modulated by CUR or CUR + BUS. The threshold values of fold-change were usually set at ≤0.667 or ≥1.5.

12


Survival ratio (%)

100 80

# ∗∗

60

# ∗∗∗

40

# ∗∗∗

20 0

0.1 1 10 YM155 (ng/mL)

0

100

24 h IC50 = 8.86 ng/mL 48 h IC50 = 2.43 ng/mL (a) 1.34

10

1.23

10.24

2.92

102

102

50

101

40

100 100

104 10

93.74 10

1

3.69 2

10 Control

10

3

10

100 100

4

104

5.39

1.45

3

10 PI

102 101 100 100

82.18 1

4.66 2

3

10 10 10 YM155 (2.43 ng/mL)

2.98

104

8.39

Apoptosis (%)

103

101

PI

4

103 PI

PI

10

4

∗∗ ∗∗∗

30 ∗∗

20 10

3

0

Control

102

YM155

BUS

YM155 + BUS

101 3.28

89.89 1

2

10 10 10 BUS (80 𝜇M)

3

Annexin V-FITC

10

4

100 100

56.66 1

31.97 2

10 10 103 YM155 + BUS

104

Annexin V-FITC (b)

Figure 6: Suppression of survivin with YM155 could induce apoptosis and increase the sensitivity to BUS in KG1a cells. (a) KG1a cells were treated with different concentrations of YM155 for 24 and 48 h and examined by MTT assay. ∗∗ 𝑃 < 0.01 and ∗∗∗ 𝑃 < 0.001 (compared with control) and # 𝑃 < 0.05 (compared with 48 h group). (b) KG1a cells exposed to YM155 (2.43 ng/mL) and BUS (80 𝜇M) alone or CUR + BUS were analyzed by flow cytometry. The graph displays means ± SD of three independent experiments. ∗∗ 𝑃 < 0.01, ∗∗∗ 𝑃 < 0.001.

by physical interaction [47]. These results suggest that the downregulation of XIAP and cIAP-2 was closely related to the CUR-induced enhancement of apoptosis in KG1a cells. Notably, we provide the first evidence to demonstrate that CUR alone, and especially in combination with BUS, increased the expression of proapoptotic serine protease HTRA-2 in leukemia cells, particularly in leukemia stem-like cells (Table 1). HTRA2 plays a pivotal role in the induction of apoptosis in the response to various stressors, mediating interactions with a variety of inhibiter of IAPs, such as

XIAP and cIAP-1/2, through their BIR domains [48–50]. The neutralization of IAPs causes the activation of caspases 3/7/9 and thus contributes to the induction of apoptosis [48, 49]. Hence, the increase in HTRA-2 observed in the current study may thus be an important mechanism in the downregulation of XIAP and cIAP-2, finally, leading to apoptosis induction and enhancement of apoptosis in CUR + BUS-treated KG1a cells. This study also demonstrated that survivin expression was downregulated by CUR and CUR + BUS (Figures


13

CUR, CUR + BUS

Cell membrane

Survivin

BAD Cytochrome C Apaf-1

SMAC

Caspase-9 Apaf-1 Caspase-9 Cytochrome C

Bcl-2 XIAP Survivin

HTRA-2

Active caspase-9

HTRA-2

Survivin Caspase 3/6/7 XIAP

SMAC

Active caspase 3/6/7

cIAP-2

Apoptosis

Direct stimulatory modification by CUR or CUR + BUS Direct inhibitory modification by CUR or CUR + BUS Direct stimulatory modification Direct inhibitory modification

Figure 7: Mechanisms of CUR-induced apoptosis and enhanced sensitivity to BUS in KG1a cells, indicating the potential role of survivin.

5(a) and 5(b); Table 1). Survivin is an important IAP that tends to be overexpressed in cancer cells [51], including cancer stem cells [52, 53], which exerts antiapoptotic effects via various mechanisms. For example, survivin inhibits caspase-dependent apoptosis through cooperation with XIAP, inhibits the SMAC-XIAP complex, and interferes with caspase-3/caspase-9 [51] (Figure 7). Our results showed that KG1a cells overexpressed survivin protein (Figure 5(a)), in accord with the characteristics of leukemia stem-like cells. CUR alone and especially CUR + BUS decreased survivin expression in KG1a cells (consistent with the results of apoptosis showed in Figures 3(a) and 3(b)). Growing

evidence has demonstrated that downregulating or inhibiting survivin could induce apoptosis and eradicate cancer stem cells or LSCs [52, 54–56]. This suggests that CUR may induce apoptosis and enhance BUS-induced apoptosis by downregulating the expression of survivin in KG1a cells. This was confirmed by treating KG1a cells with survivin inhibitor YM155 alone or in combination with BUS. YM155 induced apoptosis and enhanced BUS-induced apoptosis in KG1a cells, in a similar manner to CUR (Figure 6(b)). Survivin appears to act as a key protein in the mechanisms whereby CUR sensitizes KG1a cells to BUS. BAD and Bcl-2 proteins were also shown to be modulated by CUR and CUR + BUS,

14 and further studies are warranted to explore their roles in the CUR-induced effects in KG1a cells. In summary, this study demonstrated underlying new mechanisms whereby CUR may overcome BUS insensitivity by downregulating survivin in leukemia stem-like KG1a cells. CUR, alone or in combination with BUS, could be a potential anti-LSCs agent for preventing leukemia relapse and reducing the NRM after HSCT. BUS is currently still widely used in the pretreatment of HSCT, but it shows significant side effects and carcinogenicity in patients undergoing HSCT, resulting in danger of being replaced by other conditioning regimens. CUR may solve these issues by combining BUS in the conditioning regimen.

Conflict of Interests


[7]

[8]

[9]

[10]

[11]

The authors have no competing interests to declare.

Authors’ Contribution Guangyang Weng and Yingjian Zeng contributed equally to this work.

[12]

[13]

Acknowledgments The authors would like to thank Yanjie HE (Laboratory of Hematology, Zhujiang Hospital) for assistance with experimental techniques, Pingfang Xia for revising the paper, and Miaorong She for KG1a cells. This work was supported by the National Natural Science Foundation of China (Grant no. 30973454).

[14]

[15]

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Review Article PLK-1 Targeted Inhibitors and Their Potential against Tumorigenesis Shiv Kumar and Jaebong Kim Department of Biochemistry, Institute of Cell Differentiation and Aging, College of Medicine, Hallym University, Chuncheon, Gangwon-do 200-702, Republic of Korea Correspondence should be addressed to Jaebong Kim; [email protected] Received 27 February 2015; Revised 8 May 2015; Accepted 14 May 2015 Academic Editor: Kanjoormana A. Manu Copyright © 2015 S. Kumar and J. Kim. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mitotic kinases are the key components of the cell cycle machinery and play vital roles in cell cycle progression. PLK-1 (Pololike kinase-1) is a crucial mitotic protein kinase that plays an essential role in both the onset of G2/M transition and cytokinesis. The overexpression of PLK-1 is strongly correlated with a wide spectrum of human cancers and poor prognosis. The (si)RNAmediated depletion of PLK-1 arrests tumor growth and triggers apoptosis in cancer cells without affecting normal cells. Therefore, PLK-1 has been selected as an attractive anticancer therapeutic drug target. Some small molecules have been discovered to target the catalytic and noncatalytic domains of PLK-1. These domains regulate the catalytic activation and subcellular localization of PLK-1. However, while PLK-1 inhibitors block tumor growth, they have been shown to cause severe adverse complications, such as toxicity, neutropenia, and bone marrow suppression during clinical trials, due to a lack of selectivity and specificity within the human kinome. To minimize these toxicities, inhibitors should be tested against all protein kinases in vivo and in vitro to enhance selectivity and specificity against targets. Here, we discuss the potency and selectivity of PLK-1-targeted inhibitors and their molecular interactions with PLK-1 domains.

1. Introduction Polo-like kinase-1 (PLK-1), a serine/threonine (Ser/Thr) protein kinase, is highly conserved from yeast to humans and is reported to play a role in the mitotic events of the fruit fly. Five PLK family members are known in humans: PLK-1, -2, -3, -4, and -5 [1, 2]. All members contain an N-terminal ATPbinding catalytic domain known as the kinase domain, and two C-terminal noncatalytic domains known as the Polo-box domains (PBDs) as shown in Figure 1 [3]. In contrast, PLK-4 contains only one C-terminal PBD domain, whereas PLK-5 has no N-terminal catalytic domain. In addition, PLK-5 is a distantly related member of the PLK family and exhibits different functions as well as a different tissue distribution. PLK5 plays a role in neurobiology and DNA damage response [4, 5]. In mitotic phase, Aurora-A-Borealis phosphorylates the T210-loop to activate PLK-1. Thus, PLK-1 interacts with PBD-bound substrate and progresses the cell cycle. PBD also plays a pivotal role in the subcellular localization and substrate interaction of PLK-1. PLK-1 is the most characterized member of PLK family because of its strong association with

many regulatory events progressing during mitosis, such as G2/M transition, spindle assembly maturation, chromosome segregation, and mitotic exit [6, 7]. Therefore, PLK-1 is one of the key players in mitosis, ensuring the proper regulation of G2/M onset; nevertheless, the deregulation of PLK-1 leads to multiple defects in metaphase, termed mitotic defects, and favors the promotion of aberrant cell survival. These defects lead to aneuploidy and genomic instability within the cells and cause tumorigenesis (aneuploidy, a hallmark of cancer) [8]. Furthermore, the overexpression of PLK-1 is strongly associated with many types of human cancers because upregulated PLK-1 causes the inactivation and/or degradation of tumor suppressor gene p53 in a G2-and S-phase-expressed1 (GTSE1) and Topo-1 binding protein- (TOPORS-) dependent manner, respectively [9, 10]. Moreover, in the absence of p53, the ATR-ATM checkpoint machinery fails to recognize DNA damage, causing cells to enter mitotic phase with a high load of genomic defects. In addition, the overexpression of PLK-1 inactivates CDK-1 in a CDC25C-dependent manner and triggers tumorigenesis [11]. Many studies have documented that PLK-1 is considered a mitotic proto-oncogene [12]. A wide

2

BioMed Research International Kinase domain

Polo-box domain PB1

221 305

53 K82 E131 D194 ATP binding sites

345

T210 T-loop/ activation loop

411

PB2

489 W413

511

592

603aa

H538 K540

Figure 1: PLK-1 polypeptide sequence of human. PLK-1 gene encodes a polypeptide sequences with 603 amino acids. PLK-1 consists of two types of domains (1) the conserved ser/thr N-terminal kinase domain (53-305aa). There are three ATP-binding cassettes in kinase domain: Lys82, Glu131, and Asp194, responsible for ATP-binding and T-loop (Thr210). (2) Two C-terminal polo-box domain (411-592aa), three key residues at PBD: Trp414, His538, and Lys540 are responsible for phosphopeptide binding.

range of human cancers have been screened to validate PLK1 inhibition during tumor growth. Thus, the inhibition of PLK-1, negatively affects cancer cell proliferation and reduces tumor growth [11, 13–17]. Furthermore, many studies have proven that overexpression of PLK-1 is not only related to tumorigenesis but also highlighted in the poor prognosis of cancer [18, 19]. Additionally, numerous studies have been published examining the inhibitory potential of PLK-1 as an antitumor drug target by using different approaches, including antisense oligonucleotides, small interfering (si) RNA and small molecules targeting the catalytic and/or PB domains in-vivo and in-vitro [20–22]. These studies suggested that PLK-1 is a promising, validated, attractive therapeutic drug target. These insights have initiated the design of various types of small molecules to downregulate and/or inhibit the overexpression of PLK-1 and regress tumor growth (Figure 4). PLK-1-targeted inhibitors are categorized into various classes on the basis of many properties such as the source of origin, biochemical properties, targeted domains, and interaction properties [23–29]. These inhibitors target PLK-1 as the Achilles heel of tumors [30, 31]. Although, most of the inhibitors show potent therapeutic capability in treating cancer, they also have a high risk of toxicity, owing to weak or no target selectivity against targeted domains because of the high similarity in the ATP-binding pocket and conformation. Therefore, PLK-1-targeted inhibitors are a point of emphasis for understanding the mechanism of action/s and interaction specificity with targeted residue/s. This knowledge will help reduce the toxicity level and increase the selectivity and specificity of the inhibitors to develop a safer, higher potency, and more selective drug-like molecules. Bioinformatics approaches have become an essential part of drug discovery to validate the efficacy and binding specificity of small molecules and to understand the mechanism of action against targets. These in silico tools help to improve the therapeutic value of the inhibitors and reduce their toxicity level to provide better therapeutic agents. In this study, we will discuss the potential, selectivity, and specificity profiling of PLK-1 targeted inhibitors against binding sites in an attempt to provide more selective and potent antitumor therapeutic agents.

Classification (a) Depend upon the Source (i) Natural source: Aristolactam AIIIa, Scytonemin, Wortmannin, (ii) Synthetic source: ON01910.Na, BI 2536, BI 6727, GSK461364A, HMN-176, SBE13, ZK-thiazolidinone (TAL), Compound 36, Compound 15, Compound 38, NMS-P937, LFM- A13, RO3280, TAK-960, (iii) Peptides: MQSpTPL or MAGPMQSpTPLNGAKK, LLCSpTPNG, PLHSpT, (iv) RNAi: TKM-080301. (b) Binding Site Based Classification (1) Inhibitor Interaction with kinase domain: (i) Non ATP-competitive type: ON01910.Na, Cyclapolin 1, (ii) ATP-competitive type: BI 2536, BI 6727, GSK461364A, HMN-176, SBE13, ZK-thiazolidinone (TAL), Scytoemin, Wortmannin, Compound 36, Compound 15, Compound 38, NMS-P937, LFM-A13, RO3280, TAK-960, (iii) RNAi: TKM-080301, (iv) Others: DAP81, PHA680626. (2) Inhibitor Interaction with PBD domain: (i) Natural or semisynthetic: Poloxin, Poloxipan, Purpurogallin, Aristolochia AIIIa, (ii) Peptide: MQSpTPL or MAGPMQSpTPLNGAKK, LLCSpTPNG, PLHSpT.

2. Targeting against the PLK-1 Kinase Domain The protein kinases catalyze the transfer of the 𝛾-phosphate group of ATP to substrates containing Ser/Thr/Tyr amino acid residues. Many studies have documented that the kinase


3

PLK-1 inhibitors in different phases of development

Inhibitors

Screening

Preclinical

References

1st

2nd

BI 2536

[11, 16, 31, 64]

BI 6727

[34, 65, 84]

ON 019190.Na

[11, 15, 66]

HMN-176

[19, 21, 22]

TKM-080301

[69]

NMS-P937

[20]

DAP-81

[43]

Cyclapolin1

[7]

TAL

[44]

SBE13

[45]

COM-36

[27, 46]

LFM-A13

[48]

Scytonemin

[11]

Figure 2: PLK-1 inhibitors are ongoing in different phase of clinical trials.

domain is one of the most promising drug targets [27]. During the last decade, PLK-1 catalytic domain targeted inhibitors have been identified from the screening of natural and synthetic compound libraries. These inhibitors interfere with the catalytic activity of PLK-1 and diminish its expression. Many of the inhibitors are ongoing in different phases of clinical trials, shown in Figure 2, and show promising therapeutic value, but also exhibit poor selectivity and specificity to PLK-1. The catalytic cleft for a large group of protein kinases is not only highly conserved among kinases but also similar in sequence and conformation. Subsequently, inhibitor development after lead optimization with better selectivity against the kinase domain remains a significant challenge [27]. Structure-guided drug design is an improved strategy to design more selective and potent drug like molecules. Various groups of researchers have determined the crystal structure of the kinase domain. The first kinase domain was crystallized by using ankyrin repeat proteins (DARPins) to address the active confirmation [32]. Meanwhile, another study addressed the binding mechanism of wortmannin by using the cocrystal structure of zebrafish PLK-1 [33]. Furthermore, the crystal structure of the T-loop mutant T210V interacted with PLK-1 inhibitors, termed nonhydrolyzable ATP analog adenylyl imidodiphosphate (AMPPNP) and PHA˚ resolutions, respectively [34]. Later, 680626, at 2.4 and 2.1 A

a structure-activity relationship study of BI 2536 indicated that a methoxy group is the main entity determining the specificity and selectivity of PLK-1 and non-PLK-1 kinases through interaction with Leu 132, which is present in a small pocket of the hinge region of PLK-1 [35]. In addition, NMSP937, a potent ATP-mimetic inhibitor, inhibits the methylated crystals of a PLK-1aa36-345 construct [34, 35]. The kinase domain targeted inhibitor is described in Table 1. 2.1. Scytonemin. Initially, scytonemin was identified as a PLK-1 inhibitor and was isolated from many strains of Cyanobacteria, Calothrix sp., and Lyngbya aestuarii [1, 2]. The structure of scytonemin was determined in 1993 [3]. PLK1 activates the M-phase inducer phosphatase 3 (CDC25C) by phosphorylation and leads to the CDC25C-dependent inhibition of WEE/MYT1 to initiate mitosis during the G2/M transition [4–6]. Scytonemin inhibits PLK-1 with an IC50 of 2 ± 0.1 𝜇M in a concentration-dependent manner in vitro and disrupts the activation of CDC25C [7, 8]. Another study has reported that scytonemin failed to inhibit PLK-1 up to IC50 of 3-4 𝜇M [12]. It was later found to be a nonselective inhibitor of PLK-1 due to the inhibition of other mitotic factors, including myelin transcription factor (MYT1), CDK1, checkpoint kinase-1 (Chk-1), and protein kinase C (PKC), with a similar half-inhibitory concentration [7]. Scytonemin is currently undergoing preclinical trials.

4


P GTSE1

PLK-1

p53

TOPORS

P p53 Ubiquitination U U

Inactivation

U

U

26s proteasome

Small peptides of p53

Figure 3: Model of regulation the Topo-1 binding protein- (TOPORS-) mediated degradation of p53 by PLK-1 in the response of long time cell cycle arrest. PLK-1-mediated phosphorylation of TOPORS at Ser-718, leads the ubiquitination of p53 to proteasomal degradation. PLK-1 mediated phosphorylation of GTSE1 inactivates the tumor suppressor gene p53. Consequently, PLK-1-mediated inactivation and/or degradation of p53 causes the tumorigenesis.

UV radiation

3

en equ h fr e g i 1, h amag d LK↓ P DNA of

cy

−p5 ↑P 3 LK of D -1, hig h NA dam freque nc y age

DNA fragmentation and apoptosis Cell cycle arrest and repair

Adaptation Check point failure due to lack of p53

+p5

+p53 ↓ PLK-1, low frequency of DNA damage

In DNA damage response

Mitotic entry with genomic defects PLK-1 overexpression No p53 Inhibition of WEE1/MYT1 Phosphorylated CDC25C Dephosphorylation of CDK-1 Phosphorylation of cyclinB Tumorigenesis

Figure 4: Role of PLK-1 in DNA damage based induction of tumorigenesis. In DNA damage response, overexpression of PLK1 degrades and/or inactivates the p53 in TOPORS and GTSE1 dependent manner (Figure 3). Consequently, cell enters in mitotic phase with high load of genomic defects. G2/M transition, PLK-1 dephosphorylates CDK-1 by activated CDC25C and also inhibits the CDK-1 activator WEE1/MYT1 to onset the mitotic entry with genomic defects and cause the tumorigenesis.

O

HN

O

HN

N

N

N

N

N

NH

S

Na

O

O

ON01910.Na

H

NH

O

H3 C O O

H3 C

O

O

N

N

H

N

N

H

CH3

O

O

H

H

O

CH3

N O NS NH2 F F O F H3 C

H3 C

N

H

N

NH

N

GSK 461364A

H N

BI 6727

H

O

BI 2536

H

O

N

Compounds

Undefined by Onconova Therapeutics

Thiophene benzimidazole developed by Glaxo SmithKline

A dihydropteridinone developed by Boehringer Ingelheim

A dihydropteridinone developed by Boehringer Ingelheim

Chemical class

Rigosertib sodium’Novanex, UNII-406FL5G00V, Estybon

UNII-8QO27TK6Q4, GSK-461364, 2-Thiophenecarboxamide, 5-(6-((4-methyl-1piperazinyl)methyl)-1Hbenzimidazol-1-yl)-3-((1R)-1-(2(trifluoromethyl)phenyl)ethoxy)-

Volasertib (USAN), UNII-6EM57086EA

UNII-4LJG22T9C6, BI-2536

Synonyms

9-10 nM,

2 nM

0.87 nM

0.83 nM

IC50 values for PLK-1a Selectivity

Selectivity index 𝑋 = b/a, 𝑌 = c/a, 𝑍 = d/a Interacting residues

A non-ATPcompetitive Plk1inhibitor; Affects microtubule dynamics

Also inhibits PDGFR, ABL, FLT1, CDK-2, PLK-2, Src, and Fyn. Efficacious both as a single agent and in combination with cytotoxic drugs in xenograft models

Has 400-fold greater potency for PLK1 than ATP-competitive for PLK2 and PLK3, Inhibitor, EC50 < 50 nM in > 83% of the 120 cancer cell lines tested

ND

𝑋 and 𝑌 = 400-fold 𝑋 = ND

ND

Glu140 (Homology Model)

(i) No inhibitory activity against a wide panel of Cys133 (hPlk1 Kinase more than 50 protein 𝑋 = 5.7-fold domain 13–345, ATP-competitive kinases 𝑌 = 64.36-fold T210V) inhibitor (ii) PLK2 IC50 = 5 nM 𝑍-ND PDB ID: 3fc2 (iii) PLK3 IC50 = 56 nM (iv) EC50 = 11–37 nM

Cys133, Leu132, (i) Exhibited 1,000-fold Leu59, Arg136, Arg57, selectivity against a wide Glu140, Cys67, Lys82, panel of tyrosine and 𝑋 = 4.21-fold Ala80, Leu130, Gly60, ATP-competitive serine/threonine 𝑌 = 10.84-fold Phe183, Asp194, inhibitor kinases 𝑍 = ND Val114 PDB ID: 2rku (ii) PLK2b IC50 = 3.5 nM (hPlk1 KD 13–345, (iii) PLK3c IC50 = 9.0 nM T210V) (iv) EC50 = 2–25 nM

Mechanism of action

Table 1: PLK-1 kinase domain-targeted inhibitors.

[11, 14, 15, 67]

[24–27, 66]

[34, 65]

[11, 16, 31, 64]

References


F

F

O

O

O

N

O

NH2

H

O S NH N O

N

NH H

F F NH

F

N N H2 N OH

O

S

O

ZK-Thiazolidinone

N

N

N

N

NMS-P937

H3 C O

NH2

N

O

NH O

HN

DAP-81

H

HN

HN

N

NH

O

Cyclapolin 1

F

N

O

N

S

HMN-176

H

O

O

Compounds

Synonyms

Developed by Bayer Schering Pharma AG, Berlin, Germany

Pyrazolequinazoline by Nerviano Medical Science

Diaminopyrimidine Derivative by R0ckefeller University, New York

Benzthiazole-3-oxide derivative developed by Cyclacel Ltd., Cambridge, UK

TAL

UNII-67RM91WDHQ NMS-1286937

Dialkylphthalate-810, DAP-810

Calthor, Citosarin, Cyclapen, Noblicil, Orfilina, Ultracillin, Cyclapen-W, Vastcillin,Vipicil,Wypicil Ciclacillinum, Cyc-800

Stilbazole compound by (E)-4-((2-N-(4Nippon Shinyaku Methoxybenzenesulfonyl)amino) Co. Ltd. stilbazole)1-oxide

Chemical class

19 ± 12 nM

20 nM

0.9 nM

20 nM

118 nM


Destabilized kinetochore microtubules. Dose-dependent Predicted to target reduction of CDC25C the nucleotide phosphorylation in cells pocket and recapitulation of key aspects of the loss-of-function phenotype for PLK1 More than 100 cell lines and 200 protein kinases have been tested Shows prolonged M ATP-competitive phase and induce apoptosis inhibitor Active in Xenograft tumor model IC50 < 100 nm on solid tumor Induced arrest in prometaphase-like arrest and finally cytokinesis ATP-competitive failure and Inhibitor multinucleation IC50 = 0.2–1.3 𝜇M on human and mouse tumor cell lines

Noncompetitive with respect to ATP

Inhibits PLK1; other family members were not determined Inhibits C-terminal Src kinase; IC50 ∼100 𝜇M Cell cycle may also be affected in G1/S

Shows potent antitumor activity in gastric, breast, and lung human tumor xenografts and so forth. Better activity compared ATP-competitive to known drugs such as cisplatin, doxorubicin, inhibitor vincristine, and tegafur-uracil Inhibits the expression of NF-Y and induces the cell cycle arrest

Mechanism of action

Table 1: Continued.

ND

ND

ND

ND

ND

Selectivity index 𝑋 = b/a, 𝑌 = c/a, 𝑍 = d/a

[43]

[42]

[19, 21, 22]

References

ND

[44]

Giu131, Cyc133, Lys82, Asp194, Cys67, [20, 23, 29] Phe183, Arg57, Leu132-Cys133Arg134

ND

ND

ND

Interacting residues

6 BioMed Research International

N

C

O

O

O

O H

O

O

[+X]

OH

N Cl

O

CH3 N CH3 HO CH3O N N+ NH N N NH

RO3280

F F

O

H O

H

O

CH3

S

N

N NH

O

N

NH

O

NH

O

Wortmannin

O

H

O

H

Scytonemin

HO

N

−

O

NH

N+

LFM-A13

H3 C

OH

SBE13

O

O H3 C

CH3

PHA-680626

HN

Compounds

NA

NA

NA

Synonyms

Pyrimidodiazepines derivatives

Steroidal furanoids, originally isolated from Penicillium wortmannii

NA

BRN 0067676, NSC 627609, SL-2052, UNII-XVA4O219QW, Wartmannin

Subunit derived from (3,3󸀠 -Bis((4tryptophan and hydroxyphenyl)methylene)-(1,1󸀠 Phenylpropanoid bicyclopent(b)indole)isolated from many 2,2󸀠 (3H,3󸀠 H)-dione) strains of cyanobacteria.

𝛼-cyano-𝛽-hydroxy-𝛽methylN-(2,5-dibromophenyl)propenamide

Vanillin derivative

Pyrrolo-pyrazole derivative

Chemical class

0.09 nM

24 nM

2.0 ± 1 𝜇M

Plx1 32.5 𝜇M using GST-CDC25 as a substrate

EC50 = 12–39 𝜇M

0.53 nM

IC50 values for PLK-1a

Shows 1000-fold selectivity within the PLK family

PLK-2 (IC50 = 0.07 𝜇M) PLK-3 (IC50 = 1.61 𝜇M) Weaker inhibition was detected on few kinases

Selectivity

ND

ND

ND

𝑋 or 𝑌 or 𝑍 = 1000-fold

𝑋 = 132-fold 𝑌 = more than 3000-fold


500- greater 318 wild type and binding affinity mutants protein kinases with PLK-1 ATP-competitive tested compared to Inhibitor More than 85% protein tested penal of kinases inhibits at 1 mM protein kinases

Also inhibits the other member of PLK family ATP-competitive and interacts with Inhibitor similar binding affinity Inhibits the PI3K

PLK-3 IC50 = 61 𝜇M Also inhibits human BTK with an IC50 of 17.2 ± 0.81 𝜇M The activity is 3–15 fold greater against a panel of protein kinases Also inhibits the transcriptional factor ATP-competitive MYT1 CDK-1, Chk-1, Inhibitor and PKC Does not directly inhibit PLK-1 up to 3-4 𝜇M

ND

ND

Mechanism of action

Table 1: Continued.

ND

Lys68 Cys119 PDB ID: 3d5x (zebrafish Plk1 kinase domain, 1–312 wild type and 13–312 T196D)

ND

ND

Arg93, Asp194, Cys133, Phe195, Phe183 (By homology model)

Glu131, Cys133, Lys82, His105 PDB ID: 2owb (hPlk1 KD, T210V)


[49]

[13, 17, 18]

[7, 12]

[47, 48, 68]

[45]

[15]

References


H

NH

O

O

O N

N N

H

O

O

Lipid nanoparticle based formulation developed by Tekmira Phaarmaceuticals Corp.

Derivative of 2-aminopyrazolopyridines

2-Aminoisoxazopyridine

NA

NA

NA

NA

TAK 960,

[4-[(9-cyclopentyl-7,7difluoro-5-methyl-6oxo-6,7,8,9 tetrahydro-5H pyrimido[4,5b][1,4]diazepin-2yl)amino]-2-fluoro-5methoxy-N-(1methylpiperidin-4-yl) benzamide]

Imidazopyridine derivative

Synonyms

Chemical class

ND

0.042 𝜇M

0.051 𝜇M

9.8 nM

0.8 nM


𝑋 = 2-fold 𝑌 = 18-fold 𝑍 = ND

𝑋 = 21-fold 𝑌 = 62-fold 𝑍 = ND


Silencing PLK-1 mRNA

Showed antiproliferative and gene silencing activity against human cancer cell lines Antitumor activity against human cancer xenografts

HCT116 colorectal ATP-competitive cancer cell lines showed G2/M arrest and Inhibitor induced apoptosis

ND

𝑋 and 𝑌 = 50-fold 𝑍 = ND

Treated cells showed monopolar phenotype 𝑋 = 3.37-fold ATP-competitive and mitotic arrest in 𝑌 = 27-fold Inhibitor colorectal carcinoma cell 𝑍 = ND lines

No inhibitory activity against 212 protein ATP-competitive kinases at 1 𝜇M. Inhibitor Tolerated toxicity observed against WBC

No inhibitory activity against 282 protein kinases ATP-competitive Anti-tumor activity Inhibitor against TP53, KRAS, MDR mutated cell lines Monopolar spindle and G2/M phase arrest

Mechanism of action

PLK-1 mRNA silencing

Phe169, Cys53, Cys119, Lys68 (hPlk1Lys82), Asp180 (hPlk1Asp 194), PDB ID: 3dbc (zPlk1KD, T196D)

ND

Cyc133, Lys82, Asp194

ND


[69]

[52]

[51]

[27, 46]

[53]

References

a = PLK-1, b = PLK-2, c = PLK-3, d = PLK-4, ND = not determined, PLK = polo-like kinase, IC50 = half-maximal inhibitory concentration, EC50 = effector concentration for half-maximum response, BTK = Bruton’s tyrosine kinase, PBD = Polo box domain, NIMA-interacting 1, Plx1 = Xenopus homologue of PLK-1, MYT1 = myelin transcription factor 1, PDGFR = platelets derived growth factor receptor, ABL = Abelson murine leukemia viral oncogene homolog 1, FLT1 = vascular endothelial growth factor receptor 1, CDK-1/2 = cyclin-dependent kinase-1/2, PKC = protein kinase C, PI3K = phosphoinositide-3-kinase, KRAS = Kirsten rat sarcoma viral oncogene homolog, TP53 = tumor suppressor p53, NF-Y = nuclear transcription factor Y subunit alpha, CDC25C = M-phase inducer phosphatase 3, Chk-1 = checkpoint kinase-1, and NA = Not available.

Cl

NH2

TKM-080301 RNAi

Compound 38

R

NH

H

Compound 15

Cl

H N NH

N

S

H2 N

Compound 36

H3 C

F

H3 C F

N

F NH N N N N F OH F H O

O

TAK-960

N

H N

Compounds

Table 1: Continued.

8 BioMed Research International

BioMed Research International 2.2. ON01910.Na. ON018910.Na is a hydrophilic benzyl styryl sulphone analog that was first reported as a PLK-1 inhibitor that functions in an ATP-independent manner. ON01910.Na treated cells exhibited inhibition of the PI3K/AKT pathways and downregulation of cyclin D1 with activation of the apoptosis-related genes NOXA and BIM. Furthermore, another study demonstrated that ON01910.Na treated cells showed inhibition of phosphorylation of CDC25C, a downstream protein of cyclin B, and activated the caspase pathway by the downregulation of Bid, Bcl-xl and Mcl-1 in B-cell lymphoma [36, 37]. A wide range of cancer cell lines have been screened to evaluate the anticancer potential and selectivity of ON01910.Na. ON01910.Na-treated cells displayed effects on microtubule dynamics and caused mitotic defects such as multipolar spindles and centrosome abnormalities. As a result, ON01910.Na treated cells exhibited the mitotic arrest and spindle abnormalities and caused the apoptosis [16]. Moreover, ON01910.Na also inhibited drug-resistant cell lines with an IC50 ranging from 50 to 250 nM in-vivo and invitro [14]. However, an additional study demonstrated that ON01910.Na did not inhibit PLK-1 enzymatic activity up to a concentration of 30 𝜇M in an in vitro kinase assay and was unable to show RNAi-induced cellular phenotypic resemblance [15]. Furthermore, the clinical development phase I studies of ON01910.Na have been successfully completed with some common adverse effects, including fatigue, pain, nausea, vomiting, and abdominal pain [14]. Moreover, an additional phase I study has been completed against advanced cancers or B-cell chronic lymphocytic leukemia with mild toxicities, including abdominal, skeletal, and tumor pain, nausea, and fatigue with mild hematological toxicities. Furthermore, the phase I study recommended a 3120 mg dose for the phase II studies, and the pharmacokinetic study showed that the half-life of distribution and elimination is 1 hour and 27 hrs, respectively [38]. ON01910.Na is now undergoing phase III clinical trials for myelodysplastic syndromes (MDS) correlated with misexpression of cyclin D1 [37]. 2.3. Wortmannin. Wortmannin is a steroid metabolite of the fungi Penicillium funiculosum and Penicillium wortmannin and was initially characterized as a phosphatidylinositol-3kinase (PI3K) inhibitor. However, a later study observed that wortmannin also inhibited PLK-1 with an IC50 of 24 nM [17]. According to the crystal structure analysis, wortmannin interacts with the Lys68 residue of the PLK-1 kinase domain [13]. Furthermore, PLK-1 is also inhibited by wortmannin in a time-dependent manner without inhibiting the remaining PLKs family members. However, wortmannin also interacts with PLK-1/2/3 with a similar binding affinity (1.5–3.0 𝜇M) [18]. 2.4. HMN-176. HMN-176 is an active metabolite of the synthetic antitumor oral prodrug, HMN-214, and was developed by the Japan-based company Nippon Shinyaku Co. Ltd. HMN-176 did not show direct catalytic inhibition of PLK-1. A wide range of human cancers have been screened to validate the potential of HMN-176 as an anticancer chemotherapeutic agent with an IC50 value of 118 nM in vitro [19]. HMN-176

9 interferes with the subcellular localization of PLK-1 in centrosome and cytoskeleton structure. Furthermore, HMN-176 also downregulates (multidrug resistant) MDR1 by inhibiting the transcription factor Nuclear Transcription Factor Y subunit alpha (NF-Y) and inducing mitotic cell cycle arrest [21, 22]. HMN-176 showed potent activity against various human tumor xenografts (Table 3). Furthermore, a phase I pharmacokinetic study of HMN-176 demonstrated that the maximum tolerated dose (MTD) of HMN-176 (8 mg/m2/day) is well tolerated, with some modest adverse effects, including myalgia/bone pain syndrome, hyperglycemia, neutropenic sepsis, and neuropathy. The adverse effects depend on the oral dose schedule and the degree of treatment [21]. 2.5. NMS-P937 1H-Pyrazolo(4,3-H)quinazoline-3-carboxamide,4,5-dihydro-1-(2-hydroxyethyl)-8-((5-(4-methyl-1-piperazinyl)-2(trifluoromethoxy)phenyl)amino). NMS-P937 is a derivative of pyrazolo quinazoline that was developed by Nerviano Medical Science, Milano, Italy. NMS-P937 is an oral available, selective ATP-competitive inhibitor [20]. NMS-P937 showed high selectivity among a panel of more than 250 protein kinases and no cross-reactivity with other PLK-1 family members. More than 100 cell lines related to hematological and solid cancer have been treated to evaluate the potential of NMS-P937, which has an inhibitory concentration less than 0.02 𝜇M. Another study has documented that NMS-P937 is well-tolerated with good potency and efficacy in preclinical xenograft tumor models. Moreover, the 2󸀠 -trifluoro-methoxy moiety of NMS-P937 determined the molecular selectivity for PLK-1 and the rest of the kinases, and the 2󸀠 -trifluoro-methoxy moiety fits in the ATP-binding pocketsformed by Arg57 and the hinge residues Leu132-Cys133-Arg134 [23]. NMS-P937 has been validated as an anticancer agent against different preclinical rodent, and nonrodent models of acute myelogenous leukemia (AML), exhibiting inhibition of PLK-1-mediated phosphorylation of TCTP at Ser46 and triggering the apoptosis induction. NMS-P937 also showed good oral bio-availability upon the combination with the white blood cell cancer drug Cytarabine and prolonged survival [29]. Furthermore, a phase I dose-escalation against advanced or metastatic solid tumors study has been completed successfully (https://clinicaltrials.gov/ct2/show/record/NCT01014429). 2.6. GSK461364A. GSK461364A is a selective thiophene amide derivative PLK-1 inhibitor that was designed by Glaxo Smith Kline [26]. It is an ATP-competitive inhibitor. GSK461364A has been screened against more than 120 cancer cell lines to validate its chemotherapeutic potency and selectivity. It showed at least 400-fold greater inhibitory potential for PLK-1 over other family members, including PLK-2/3, with an IC50 value of 50 nM. Moreover, GSK461364A was also screened against a panel of 260 protein kinases and exhibited an IC50 value of 1), or synergistic (