Linkage of Curcumin-Induced Cell Cycle Arrest and Apoptosis by ...

[Cell Cycle 6:23, 2953-2961, 1 December 2007]; ©2007 Landes Bioscience

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Linkage of Curcumin-Induced Cell Cycle Arrest and Apoptosis by Cyclin-Dependent Kinase Inhibitor p21/WAF1/CIP1 Rakesh K. Srivastava* Qinghe Chen Imtiaz Siddiqui Krishna Sarva Sharmila Shankar*

Abstract

Key words

Introduction

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curcumin, cell cycle, cyclin, cyclin-dependent kinase inhibitor, prostate cancer

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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/4951

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Original manuscript submitted: 07/27/07 Revised manuscript submitted: 08/23/07 Manuscript accepted:08/30/07

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*Correspondence to: Sharmila Shankar and Rakesh K. Srivastava; Department of Biochemistry; The University of Texas Health Science Center at Tyler; 11937 US Highway 271; Tyler, Texas 75708-3154; Tel.: 903.877.7559; Fax: 903.877.5320. Email: Sharmila.shankar@uthct.edu/ rakesh.srivastava@uthct.edu

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Department of Biochemistry; University of Texas Health Science Center at Tyler; Tyler, Texas USA

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We have recently shown that curcumin induces apoptosis in prostate cancer cells through Bax translocation to mitochondria and caspase activation, and enhances the therapeutic potential of TRAIL. However, the molecular mechanisms by which it causes growth arrest are not well understood. We studied the molecular mechanism of curcumin-induced cell cycle arrest in prostate cancer androgen-sensitive LNCaP and androgen-insensitive PC-3 cells. Treatment of both cell lines with curcumin resulted in cell cycle arrest at G1/S phase and that this cell cycle arrest is followed by the induction of apoptosis. Curcumin induced the expression of cyclin-dependent kinase (CDK) inhibitors p16/INK4a, p21/WAF1/CIP1 and p27/KIP1, and inhibited the expression of cyclin E and cyclin D1, and hyperphosphorylation of retinoblastoma (Rb) protein. Lactacystin, an inhibitor of 26 proteasome, blocks curcumin-induced down-regulation of cyclin D1 and cyclin E proteins, suggesting their regulation at level of posttranslation. The suppression of cyclin D1 and cyclin E by curcumin may inhibit CDK-mediated phosphorylation of pRb protein. The inhibition of p21/WAF1/CIP1 by siRNA blocks curcumin-induced apoptosis, thus establishing a link between cell cycle and apoptosis. These effects of curcumin result in the proliferation arrest and disruption of cell cycle control leading to apoptosis. Our study suggests that curcumin can be developed as a chemopreventive agent for human prostate cancer.

Acknowledgements

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The project was funded by the Department of Defense (W81XWH-04-1-0821 to RS) and the National Institutes of Health (CA12585701 to SS). We thank all the lab members for critically reading the manuscript.

Inappropriate and/or accelerated rates of cell proliferation are hallmark of cancer. The molecular regulatory network of the cell cycle and apoptosis are tightly intertwined.1,2 The known molecular regulatory networks of the cell cycle and apoptosis are quite complex and can overlap. During malignant transformation, a number of genetic and epigenetic alterations occurs as a result of increasing genomic instability caused by defects in checkpoint controls.1,2 These alterations allow cancer cells to acquire the capabilities to become self‑sufficient in mitogenic signals, deregulate the control of cell cycle, escape from apoptosis, and obtain unlimited replication potential. The transition from one cell cycle phase to another occurs in an orderly fashion and is regulated by different cellular proteins. Key regulatory proteins are the cyclin‑dependent kinases (CDK), a family of serine/threonine protein kinases that are activated at specific points of the cell cycle. Until now, nine CDK have been identified and, of these, five are active during the cell cycle, i.e., during G1 (CDK4, CDK6 and CDK2), S (CDK2), G2 and M (CDK1). When activated, CDK induce downstream processes by phosphorylating selected proteins.3,4 CDK protein levels remain stable during the cell cycle, in contrast to their activating proteins, the cyclins. Cyclin protein levels rise and fall during the cell cycle and in this way they periodically activate CDK.5 Different cyclins are required at different phases of the cell cycle. The three D type cyclins (cyclin D1, cyclin D2, cyclin D3) bind to CDK4 and CDK6, and CDK‑cyclin D complexes are essential for entry in G1.6 Unlike the other cyclins, cyclin D is not expressed periodically, but is synthesized as long as growth factor stimulation persists.7 Another G1 cyclin is cyclin E which associates with CDK2 to regulate progression from G1 into S phase.8 Cyclin A binds with CDK2 and this complex is required during S phase.9,10 In late G2 and early M, cyclin A complexes with CDK1 to promote entry into M. Mitosis is further regulated by cyclin B in complex with CDK1.11,12 Cyclins A and B contain a destruction box and cyclins D and E contain a PEST sequence [segment rich in proline (P), glutamic acid (E), serine (S) and threonine

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required for cell cycle progression and S phase entry. It has been shown that CDK4‑cyclin D1, but not CDK2‑cyclin E, specifically phosphorylated Ser780 in pRB, which can not bind to E2F‑1.36 Prostate cancer is characterized by an initial period during which the tumor growth is androgen dependent and localized disease responds to a variety of therapies (radical prostatectomy, radiotherapy and brachytherapy). Advanced prostate cancer initially responds to hormonal therapy, but the response usually lasts only about 18 months in the metastatic setting. After the failure of first‑line hormone therapy, standard treatment options are limited and survival is between 6 and 12 months for patients with androgen‑independent hormone‑refractory prostate cancer.37 Currently available chemotherapies do not confer a significant survival benefit in these cases. As a result there is a demand for new therapy that more specifically target the cellular events involved in the development of malignancy and in normal host processes required for tumor progression. Chemopreventive agent curcumin [1,7‑bis(4‑hydroxymethoxyphenyl)‑1,6‑hepatadiene‑ 3,5‑dione; diferulolylmethane] is such compound which has been shown to induce apoptosis in both androgen‑sensitive and ‑insensitive prostate cancer. It acts as antitumor, anti‑inflammatory, antiangiogenic and antioxidant, and thus offers numerous medical benefits against cancer.38‑44 Although curcumin has been shown to cause growth arrest and induce apoptosis in cancer cells, its molecular mechanisms are not well understood. The purpose of our studies was to investigate the molecular mechanisms by which curcumin induced growth arrest in prostate cancer cells.

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(T) residues]; these are protein sequences required for efficient ubiquitin‑mediated cyclin proteolysis at the end of a cell cycle phase.13 In addition to cyclin binding, CDK activity is also regulated by phosphorylation on conserved threonine and tyrosine residues. Full activation of CDK1 requires phosphorylation of threonine 161 (threonine 172 in CDK4 and threonine 160 in CDK2), brought about by the CDK7‑cyclin H complex, also called CAK. These phosphorylations induce conformational changes and enhance the binding of cyclins.14,15 The Wee1 and Myt1 kinases phosphorylate CDK1 at tyrosine‑15 and/or threonine‑14, thereby inactivating the kinase. Dephosphorylation at these sites by the enzyme Cdc25 is necessary for activation of CDK1 and further progression through the cell cycle.16 Alterations of CDK molecules in cancer have been reported, although with low frequency. CDK4 overexpression, that occurs as a result of amplification, has been identified in cell lines, melanoma, sarcoma and glioma.17 CDK1 and CDK2 have been reported to be overexpressed in a subset of colon adenomas, a greater overexpresion was seen in focal carcinomas in adenomatous tissue.18,19 CDK activity can be counteracted by cell cycle inhibitory proteins, called CDK inhibitors (CKI) which bind to CDK alone or to the CDK‑cyclin complex and regulate CDK activity. Two distinct families of CDK inhibitors have been discovered, the INK4 family and CIP/KIP family.20 The INK4 family includes p15 (INK4b), p16 (INK4a), p18 (INK4c), p19 (INK4d), which specifically inactivate G1 CDK (CDK4 and CDK6). These CKI form stable complexes with the CDK enzyme before cyclin binding, preventing association with cyclin D.21 The second family of inhibitors, the CIP/KIP family, includes p21 (WAF1/CIP1), p27 (KIP1), p57 (KIP2). These inhibitors inactivate CDK‑cyclin complexes.22,23 They inhibit the G1 CDK‑cyclin complexes, and to a lesser extent, CDK1‑cyclin B complexes.24 CKI are regulated both by internal and external signals: the expression of p21/WAF1/CIP1 is under transcriptional control of the p53 tumour suppressor gene.25 p27/KIP1 binds to CDK2 and cyclin E complexes to prevent cell cycle progression from G1 to S phase.22,23,26 Cell cycle deregulation associated with cancer occurs through mutation of proteins important at different levels of the cell cycle. In cancer, mutations have been observed in genes encoding CDK, cyclins, CDK‑activating enzymes, CKI, CDK substrates, and checkpoint proteins.27,28 Mutations in CDK4 and CDK6 genes resulting in loss of CKI binding have also been identified.29 The retinoblastoma tumor suppressor protein (pRB) is a negative regulator of cell proliferation.30 The antiproliferative activity of pRB is mediated by its ability to inhibit the transcription of genes that are required for cell cycle progression. This transcriptional regulatory function of pRB is achieved through several distinct mechanisms, which are best illustrated by its interaction with the E2F family and the inhibition of E2F‑regulated gene expression. The binding of E2F to pRB requires the large pocket of pRB (amino acids 379‑870). The ability of pRB to inhibit cellular proliferation is counterbalanced by the action of CDKs.31,32 pRB is phosphorylated in a cell cycle‑dependent manner by CDKs. In quiescent and early G1 cells, pRB exists in a predominantly unphosphorylated state. As cells progress toward S phase, pRB becomes phosphorylated. The initial phosphorylation of pRB is most likely catalyzed by CDK4‑cyclin D or CDK6‑cyclin D complexes. Subsequently, CDK2‑cyclin E and CDK2‑cyclin A phosphorylate pRB.33,34 pRB is rapidly dephosphorylated during mitosis.35 Inactivation of pRB by phosphorylation leads to the dissociation and activation of E2F, allowing the expression of many genes

Materials and Methods

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Reagents. Antibodies against p16/INK4a, p21/WAF1/CIP1, p27/KIP1, cyclin D1, cyclin E, retinoblastoma (phospho and total), CDK4, CDK6 and b‑actin were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Penicillin, streptomycin, RPMI‑1640 medium, and fetal bovine serum (FBS) were obtained from Invitrogen Corporation (Carlsbad, CA). Tris‑HCl, glycine, sodium chloride, sodium dodecyl sulfate (SDS), lactacystin, 4',6‑diamidino‑2‑ phenylindole (DAPI) and bovine serum albumin (BSA) were obtained from Sigma‑Aldrich (St. Louis, MO). Annexin‑V‑FITC / propidium iodide (PI) kit was purchased from BD Biosciences (San Jose, CA). Enhanced chemiluminescence (ECL) Western blot detection reagents were from Amersham Life Sciences Inc. (Arlington Heights, IL). Curcumin, with purity ≥ 95% was purchased from LKT Laboratories, Inc. (St. Paul, MN). Cell culture. Androgen‑sensitive LNCaP and androgen‑insensitive PC‑3 cell lines from human prostate cancer were obtained from the American Type Culture Collection (Manassas, VA). Cultures were maintained in RPMI 1640 supplemented with 10% heat‑inactivated fetal bovine serum (FBS) and 1% antibiotic‑antimycotic (Invitrogen) at 37˚C in a humidified atmosphere of 95% air and 5% CO2. Cells were counted and plated at the same initial density for treatments with curcumin. Measurement of apoptosis. Changes in the phospholipid bilayers of cell membranes are also observed early in the apoptosis process. The phosphotidylserine (PS) component of the phospholipid bilayers are externalized and can be detected by fluorescence labeling. Annexin V is a member of the annexin family of calcium‑dependent phospholipid‑binding proteins. Annexin V has a high affinity for PS‑containing phospholipid bilayers. Staining with FITC‑conjugated

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annexin V and propidium iodide (PI) can identify subpopulations of cells with membrane changes and the associated loss of membrane integrity. Cells were washed twice with cold PBS and resuspended in buffer at a concentration of 106 per ml. Cells were mixed with 10 ml of fluoresceine isothiocyanate (FITC)‑conjugated annexin V reagent and 10 ml of 3 mM propidium iodide (PI). After a 15 min incubation at room temperature in the dark and further washings, samples were analysed by flow cytometry. Flow cytometry was performed with a FACScan analyzer (Becton Dickinson) with 15 mW argon ion laser (488 nm) and Cell Quest software. Annexin V staining was detected in the FL1 (green) channel, whereas PI staining was monitored in the FL2 (red) channel: appropriate quadrants were set and the percentage of cells negative for both stains (viable cells), positive for annexin V (apoptotic cells) and positive for PI (dead cells) were acquired. Morphological analysis with DAPI. Cells were plated in 24‑well plates and treated with various doses of curcumin. After incubation, cells were resuspended in ice‑cold PBS and fixed with ice‑cold 20% paraformaldehyde containing 6% BSA for 30 min at room temperature, and washed with ice‑cold PBS. Thereafter, DAPI (1 mg/ml) solution 1:1000 dilution was added, and cells were further incubated for 20 min at room temperature. Finally, cells were washed thrice with PBS, mounted and analyzed under a fluorescence Olympus microscope (Olympus America Inc., Melville, NY). Pictures were captured using a Photometrics Coolsnap CF color camera (Olympus) and SPOT software (Diagnostic Instruments Inc., Sterling Heights, MI). Penetration of DAPI into the nucleus was evident, as it was possible to distinguish between the nuclei of healthy and apoptotic cells in view of the chromatin structure and condensation. Cell cycle analysis. Cell cycle distribution and ploidy status of cells after treatment with curcumin were determined by flow cytometry DNA analysis. At the end of treatments, cells were detached from the plates by the addition of 0.25% trypsin, washed in PBS, fixed in 70% ethanol at 4˚C and treated with 10 mg/ml RNAse for 30 min at 37˚C. The DNA content was evaluated in a FACScan flow cytometer (Becton‑Dickson, NJ, USA) after staining cells with propidium iodide buffer (0.1 mM EDTA, 0.1% Triton X‑100, 50 mg/ml propidium iodide, PBS pH 7.4) for 15 min in the dark at room temperature. For cell cycle analysis, only single cells were considered. A pass filter of 585 nm was used to collect PI fluorescence, acquiring 10,000 events for each sample. Transient transfection. Cells were plated in 60‑mm dishes in RPMI 1640 containing 10% FBS and 1% penicillin‑streptomycin mixture at a density of 1 X 106 cells/dish. The next day transfection mixtures were prepared. Cells were transfected with expression constructs encoding p21/WAF1/CIP1 siRNA plasmids or scrambled control plasmids (Psilencer) in the presence of an expression vector pCMV‑LacZ (Invitrogen life technologies) expressing b‑galactosidase. For each transfection, 2 mg of DNA was diluted into 50 ml of medium without serum. After the addition of 3 ml of LipofectAMINE (Invitrogen life technologies) into 50 ml Opti‑MEM medium, the transfection mixture was incubated for 10 min at room temperature. Cells were washed with serum‑free medium, the transfection mixture was added, and cultures were incubated for 24 h in the incubator. The next day, culture medium was replaced with fresh RPMI 1640 containing 10% FBS and 1% penicillin‑streptomycin mixture and curcumin was added for desired times. At the end of incubation, cells were washed with ice‑cold PBS and stained with DAPI to measure apoptosis.

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Figure 1. Effects of curcumin on cell cycle distribution of prostate cancer cells. PC‑3 cells were treated with or without curcumin (20 mM) for 24 h. After harvesting, cells were stained with propidium iodide and cell cycle distribution was analyzed by flow cytometry.

Western blot analysis. At the end of treatments with curcumin, cells were washed with cold PBS and lysed in ice‑cold lysis buffer (50 mM Tris‑HCl pH 7.5, 2 mM EDTA, 2 mM EGTA, 10 mM b‑glycerophosphate, 150 mM NaCl, 0.5% NP‑40, 1 mM phenyl‑methyl sulfonyl fluoride, 1 mM NaF, 1 mM DTT, 1% b‑mercaptoethanol and 4 mg/ml complete protease inhibitor cocktail (EMD Biosciences). Cell lysates were centrifuged at 15,000 g for 15 min at 4˚C and protein concentration was determined in the supernatants using the Coomassie Plus protein assay reagent (Pierce, Rockford, IL) using bovine serum albumin as standard. About 40 mg of crude proteins were mixed with SDS sample buffer, denatured and electrophoresed in 12% SDS‑PAGE gels. After electrophoresis, gels were transferred

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Figure 2. Effects of curcumin on apoptosis in prostate cancer cells. A) PC‑3 cells were treated with or without curcumin (5, 10, 20 mM) for 48 h. After harvesting, cells were stained with DAPI, and apoptosis was measured by fluorescence microscopy. Data represent mean ± SD. * = significantly different from respective control, p < 0.05. (B) PC‑3 cells were treated with or without curcumin (20 mM) for 0, 12, 24 and 48 h. After harvesting, cells were stained with DAPI, and apoptosis was measured by fluorescence microscopy. Data represent mean ± SD. *significantly different from respective control, p < 0.05. (C) LNCaP cells were treated with or without curcumin (5, 10, 20 mM) for 48 h. After harvesting, cells were stained with annexin‑V‑FITC/ PI, and apoptosis was measured by flow cytometry.

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to nitrocellulose membranes by electroblotting and blocked for two hours at room temperature in TBST (50 mM Tris‑HCl pH 7.5, 150 mM NaCl, 0.2% Tween‑20) containing 5% nonfat milk. Blots were sequentially incubated with the primary and secondary antibodies, washed in TBS‑T. Membranes were developed by enhanced chemiluminescence (ECL‑Plus, Amersham Pharmacia Biotech, Piscataway, NJ) and exposed to Kodak Biomax Light films for 1‑10 min. In order to detect a second protein, some blots were stripped by incubation with 100 mM Tris‑HCl, pH 7.4, 100 mM b‑mercaptoethanol, and 2% SDS at 60˚C for 30 min. Statistical analysis. The mean and standard deviation (SD) were calculated for each experimental group. Differences between groups were analyzed by one or two way ANOVA using PRISM statistical analysis software (GrafPad Software, Inc., San Diego, CA). Significant differences among groups were calculated at p < 0.05.

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Results

Curcumin induces cell cycle arrest at G1/S phase. In order to examine the molecular mechanisms of cell cycle arrest by curcumin, we first determined the effects of curcumin on various phases of cell cycle. Androgen‑insensitive PC‑3 cells were treated with curcumin for 24 h and cell cycle analysis was performed by flow cytometry (Fig. 1). In control PC‑3 cells, the distribution of cells in G1, S and G2 phases of cell cycle were 54.9%, 36.58%, and 8.52%, respectively. Treatment of PC‑3 cells with curcumin resulted in increased number 2956

of cells in G1 phase (54.9% to 64.99%) with concomitant reduction of cell numbers in S (36.58% to 32.84%) and G2 (8.52% to 2.17%) phases. Similarly, curcumin induced growth arrest at G1/S phase of cell cycle in androgen‑dependent LNCaP cells (data not shown). Curcumin induces apoptosis in prostate cancer cells. In order to establish a link between cell cycle and apoptosis, we next measured apoptosis by two complementary approaches i.e., annexin‑V‑FITC/ propidium iodide, and DAPI staining. In PC‑3 cells, curcumin induced apoptosis in a dose‑ and time‑dependent manner (Fig. 2A and B). Similarly, treatment of LNCaP cells with curcumin resulted in apoptosis in a dose‑dependent manner at 48 h (Fig. 2C). Curcumin had no significant effects on apoptosis in LNCaP cells up to 24 h (data not shown). It is interesting to note that the growth arrest was observed in both PC‑3 and LNCaP cells at 24 h of curcumin treatment, whereas a slight induction of apoptosis was noticed in these cell lines at 24 h. Based on the time course studies, it appears that curcumin‑induced growth arrest occurs prior to induction of apoptosis. Effects of curcumin on the expression of cell cycle regulatory proteins in androgen‑sensitive and ‑insensitive prostate cancer cells. In order to investigate the role of cyclin/CDK complexes in the antiproliferative activity of curcumin, the expression of G1/S regulators cyclin D1, cyclin E, pRb, CDK4, and CDK6 was analyzed (Fig. 3). Since inhibitory mechanism of curcumin on cell proliferation might also affect the expression of negative regulators of the cell cycle, we evaluated protein expression of CDK inhibitors p21/WAF1/CIP1 and

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Figure 3. Effects of curcumin on cell cycle regulatory proteins in prostate cancer cells. (A) PC‑3 cells were treated with curcumin (0, 10 or 20 mM) for 12, 24 and 48 h. Cells were harvested and the expressions of p16/INK4a, p21/WAF1/CIP1, p27/ KIP1, cyclin D1, cyclin E, pRB, RB, CDK4 and CDK6 were measured by Western blot analysis. b‑actin antibody was used as a loading control. (B) LNCaP cells were treated with curcumin (0, 10 or 20 mM) for 12, 24 and 48 h. Cells were harvested and the expressions of p16/INK4a, p21/ WAF1/CIP1, p27/KIP1, cyclin D1, cyclin E, pRB, RB, CDK4 and CDK6 were measured by Western blot analysis. b‑actin antibody was used as a loading control.

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observed only at 48 h of 10 mM or 20 mM curcumin treatment. Curcumin inhibited the expression of G1 phase proteins cyclin D1 and cyclin E in LNCaP cells. Treatment of LNCaP cells with curcumin resulted in an inhibition of hyper‑phosphorylation of pRb, and an induction of hypo‑phosphorylation of pRb. The expression of total Rb did not change in these cells upon curcumin treatment. Unlike PC‑3 cells, curcumin had no significant effect on CDK4 and CDK6 expression in LNCaP cells. Lactacystin blocks curcumin-nduced downregulation of cyclin D1 and cyclin E proteins. We next examined the mechanisms by which curcumin inhibited the expression of cyclin D1 and cyclin E in prostate cancer cells (Fig. 4). The inhibition of these cyclins by curcumin could be either by enhancing their degradation or inhibiting synthesis. Here, we explored the possibility of posttranslation modification i.e., degradation. It has been shown that cyclin D1 and cyclin E undergo ubiquitin‑dependent proteasomal degradation,45‑47 and lactacystin inhibits the 26 S proteasome.48 Prostate cancer PC‑3 cells were pretreated with lactacystin for 2 h followed by treatment with curcumin for 4 h. Curcumin induced degradation of cyclin D1 (Fig. 4A) and cyclin E in PC‑3 cells (Fig. 4B). Lactacystin prevented curcumin‑induced degradation of cyclin D1 and cyclin E. Similarly, lactacystin blocked curcumin‑induced degradation of cyclin D1 and cyclin E in LNCaP cells (data not shown). Curcumin‑induced apoptosis is blocked by CDK inhibitor p21/WAF1/CIP1 siRNA. We have recently shown that curcumin induces apoptosis by engaging mitochondrial pathway.49,50 Since curcumin induced cell cycle arrest and apoptosis, and CDK inhibitors p21/WAF1/CIP1 plays a major role at G1 stage of cell cycle,51 we sought to examine whether cell cycle is linked to apoptosis. Overexpression of p21/WAF1/CIP1 has been shown to induce apoptosis in cancer cells. PC‑3 and LNCaP cells were transfected with either p21/WAF1/CIP1 siRNA expression plasmids or scrambled control siRNA expression plasmids and treated with curcumin for 48 h (Fig. 5). Curcumin induced apoptosis in PC‑3/control siRNA and LNCaP/control siRNA cells. By comparison, curcumin had no effect on apoptosis in PC‑3/p21 siRNA and LNCaP/p21 siRNA cells. These data suggest that inhibition of growth arrest by CDK inhibitor p21/WAF1/CIP1 can block apoptosis induction by curcumin, and cell cycle arrest precedes apoptosis.

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Figure 4. Lactacystin blocks curcumin‑induced down‑regulation of cyclin D1 and cyclin E protein expression. PC‑3 cells were pretreated with 10 mM of lactacystin for 2 h and then exposed to 30 mM of curcumin for 3 h. Thereafter, whole cell extracts were prepared, subjected to SDS‑PAGE analysis, and immunoblotted with antibody against cyclin D1 (A), and cyclin E (B). b‑actin antibody was used as a loading control.

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p27/KIP1. The p21/WAF1/CIP1 and p27/KIP1 are inhibitors of cyclin/ CDK complexes mainly involved in G1/S transition. Curcumin induced a significant increase in protein expression of p21/WAF1/CIP1 and p27/KIP1 in androgen‑insensitive PC‑3 cells (Fig. 3A). We also noticed an induction of p16/INK4a with both doses of curcumin in PC‑3 cells at 48 h. Curcumin also inhibited hyper‑phosphorylation of pRb and induced hypo‑phosphorylation of pRb which reached a maximum at 12 h with 20 mM dose, whereas total protein expression of Rb did not change. Furthermore, curcumin inhibited the expression of cyclin D1, cyclin E, CDK4 and CDK6 in PC‑3 cells. We next examined the effects of curcumin on cell cycle regulatory proteins in androgen‑sensitive LNCaP cells (Fig. 3B). Curcumin induced a significant increase in protein expression of p21/WAF1/CIP1 and p27/KIP1 starting at 12 h and remained high at 24 h and 48 h. Unlike p21/WAF1/CIP1 and p27/KIP1, the induction of p16 was www.landesbioscience.com

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Figure 5. Inhibition of p21/WAF1/CIP1 by siRNA blocks curcumin‑induced apoptosis. (A) PC‑3 cells were transfected with either scrambled control siRNA plasmids (pSilencer) or p21/WAF1/CIP1 siRNA plasmids, in the presence of control plasmid (pCMV‑LacZ) encoding the b‑galactosidase (b‑Gal) enzyme. There was no difference in transfection efficiency among groups. After 24 h of incubation, the culture medium was replaced with fresh medium containing FBS and cells were treated with various doses of curcumin (0–30 mM) for 48 h. Cells were stained with DAPI and visualized under a fluorescence microscope. Data represent mean ± SD. *significantly different from respective control, p < 0.05. (B) LNCaP cells were transfected with either scrambled control siRNA plasmids (pSilencer) or p21/WAF1/CIP1 siRNA plasmids, in the presence of control plasmid (pCMV‑LacZ) encoding the b‑galactosidase (b‑Gal) enzyme. The transfection efficiency was about 80%. However, there was no difference in transfection efficiency among groups. After 24 h of incubation, the culture medium was replaced with fresh medium containing FBS and cells were treated with various doses of curcumin (0–30 mM) for 48 h. Cells were stained with DAPI and visualized under a fluorescence microscope. Data represent mean ± SD. *significantly different from respective control, p < 0.05.

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increase in cell number at G0/G1 phase with the corresponding decrease in the other phases in both prostate cancer cell lines. The G1/S phase arrest by curcumin was associated with the induction of CDK inhibitors p16/INK4a, p21/WAF1/CIP1 and p27/KIP1, and inhibition of hyper‑phosphorylated state of pRb protein in vitro. The ability of curcumin to induce CDK inhibitors p21/WAF1/CIP1 and p27/KIP1 was also confirmed in our xenograft experiment.52 Most importantly, we have demonstrated a link between cell cycle and apoptosis as CDK inhibitor p21/WAF1/CIP1 blocked curcumin‑ induced apoptosis. The p21/WAF1/CIP1 is a transcription target of p53 after DNA damage.25 Overexpression of p21/WAF1/CIP1 results in G1, G2 or S phase arrest.55,56 Conversely, p21‑deficient cells fail to undergo cell cycle arrest in response to p53 activation.57 Furthermore, p21/WAF1/CIP1 and p53 are essential to sustain the G2 checkpoint after DNA damage.58 The regulation of p21/WAF1/CIP1 is complex, which includes trascriptional regulation, epigenetic silencing, mRNA stability, and ubiquitin‑dependent and ‑independent deregulation of the protein.59 In the present study, inhibition of p21/WAF1/CIP1 by siRNA caused apoptosis in androgen‑sensitive and ‑insensitive prostate cancer cells, suggesting its role in linking curcumin‑induced cell cycle arrest and apoptosis. The p27/KIP1 binds to CDK2 and cyclin E complexes to prevent cell cycle progression from G1 to S phase. p27/KIP1 also acts as a tumor suppressor and its expression is often disrupted in human cancers. Studies in mice have shown that loss of p27/KIP1 increases tumor incidence and tumor growth rate in either specific genetic backgrounds, or when mice are challenged with carcinogens.60,61 Decreased p27/KIP1 levels have been correlated with tumor aggressiveness and poor patient survival.62‑67 Although p27/KIP1 is characterized as a tumor suppressor, inactivating point mutations with loss of heterozygosity are rarely observed in human cancer. The abundance of p27/KIP1 protein is largely controlled through a variety of post‑transcriptional regulatory mechanisms,68‑71 among which are sequestration by cyclin D/CDK4 complexes, accelerated protein destruction and cytoplasmic retention.23 In certain types of cancers, such as colorectal cancer, high expression levels of Skp2 and Cks1, specific p27/KIP1 ubiquitin ligase subunits, were strongly associated with low p27/KIP1 expression and aggressive tumor behavior.72 p27/KIP1 protein level changes during cell cycle progression, accumulating when cells progress through G1 and sharply decreasing just before cells enter S phase.73 Additionally, p27/KIP1 protein levels rise when cells exit cell cycle to G0, and decreases when cells enter the cell cycle again.73 These alterations in p27/KIP1 levels are mainly caused by regulation at the protein degradation level.68‑71 However, several studies have indicated that p27/KIP1 can also be regulated at the level of translation.74‑77 Similarly, induction of p19(INK4d) expression contributed to cell cycle arrest by vitamin D(3) and retinoids.78 Cyclins are tightly regulated in different stages of cell cycle. In support of our data, it has been demonstrated that curcumin induces the degradation of cyclin E expression through ubiquitin‑dependent pathway.54 Moreover, deregulated expression of cyclin E was found to be correlated with chromosome instability,79 malignant trasformation.80 tumor progression81 and patient survival.82 Cyclin E expression increases with increasing stage and grade of the cancers including breast, head and neck, prostate, colon, and lung cancer, and acute lymphoblastic and acute myeloid leukaemias,81‑87 suggesting its potential use as a prognostic marker. The downregulation of

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Discussion

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Curcumin, a well‑known chemopreventive agent, has been shown to suppress the proliferation of a wide variety of tumor cells through a mechanism that is not fully understood. In the present study, curcumin induced growth arrest at G1/S phase of the cell cycle, which was followed by apoptosis. Curcumin induced the expression of CDK inhibitors p16/INK4a, p21/WAF1/CIP1 and p27/KIP1 in both androgen‑sensitive LNCaP and androgen‑insensitive PC‑3 cells. The growth arrest at G1/S phase by curcumin was correlated with inhibition of cyclin D1 and cyclin E protein expression. Curcumin inhibited hyper‑phosphorylation of pRB and enhanced hypo‑phosphorylation of pRb in both PC‑3 and LNCaP cell lines. Similarly, we showed that curcumin induced the expression of p16/INK4a, p21/WAF1/CIP1 and p27/KIP1, and inhibited the expression of cyclin D1 in LNCaP xenografts implanted in nude mice.52 Curcumin also inhibited LNCaP tumor growth, metastasis and angiogenesis in vivo,52 suggesting its clinical utility for anticancer therapy and/or prevention. Proliferation arrest is one of the main features of curcumin in cancer cells. Using different prostate cancer cell lines, it has been suggested that curcumin induces disruption of the G1/S transition of cell cycle.53,54 In the present study, curcumin caused a growth arrest at G1/S phase in both androgen‑sensitive LNCaP and androgen‑insensitive PC‑3 cells. Curcumin induced a marked 2958

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be considered for cancer therapy or prevention. Furthermore, CDK inhibitors p21/WAF1/CIP1 plays a critical role in linking cell cycle arrest and apoptosis. References

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1. Hahn WC, Weinberg RA. Modelling the molecular circuitry of cancer. Nat Rev Cancer 2002; 2:331‑41. 2. Nowak MA, Komarova NL, Sengupta A, Jallepalli PV, Shih IM, Vogelstein B, Lengauer C. The role of chromosomal instability in tumor initiation. Proc Natl Acad Sci USA 2002; 99:16226‑31. 3. Morgan DO. Principles of CDK regulation. Nature 1995; 374:131‑4. 4. Pines J. Cyclins and cyclin‑dependent kinases: A biochemical view. Biochem J 1995; 308(Pt 3):697‑711. 5. Evans T, Rosenthal ET, Youngblom J, Distel D, Hunt T. Cyclin: A protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division. Cell 1983; 33:389‑96. 6. Sherr CJ. G1 phase progression: Cycling on cue. Cell 1994; 79:551‑5. 7. Assoian RK. Control of the G1 phase cyclin‑dependent kinases by mitogenic growth factors and the extracellular matrix. Cytokine Growth Factor Rev 1997; 8:165‑70. 8. Ohtsubo M, Theodoras AM, Schumacher J, Roberts JM, Pagano M. Human cyclin E, a nuclear protein essential for the G1‑to‑S phase transition. Mol Cell Biol 1995; 15:2612‑24. 9. Girard F, Strausfeld U, Fernandez A, Lamb NJ. Cyclin A is required for the onset of DNA replication in mammalian fibroblasts. Cell 1991; 67:1169‑79. 10. Walker DH, Maller JL. Role for cyclin A in the dependence of mitosis on completion of DNA replication. Nature 1991; 354:314‑7. 11. King RW, Jackson PK, Kirschner MW. Mitosis in transition. Cell 1994; 79:563‑71. 12. Arellano M, Moreno S. Regulation of CDK/cyclin complexes during the cell cycle. Int J Biochem Cell Biol 1997; 29:559‑73. 13. Glotzer M, Murray AW, Kirschner MW. Cyclin is degraded by the ubiquitin pathway. Nature 1991; 349:132‑8. 14. Jeffrey PD, Russo AA, Polyak K, Gibbs E, Hurwitz J, Massague J, Pavletich NP. Mechanism of CDK activation revealed by the structure of a cyclinA‑CDK2 complex. Nature 1995; 376:313‑20. 15. Paulovich AG, Hartwell LH. A checkpoint regulates the rate of progression through S phase in S. cerevisiae in response to DNA damage. Cell 1995; 82:841‑7. 16. Lew DJ, Kornbluth S. Regulatory roles of cyclin dependent kinase phosphorylation in cell cycle control. Curr Opin Cell Biol 1996; 8:795‑804. 17. Wolfel T, Hauer M, Schneider J, Serrano M, Wolfel C, Klehmann‑Hieb E, De Plaen E, Hankeln T, Meyer zum Buschenfelde KH, Beach D. A p16INK4a‑insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science 1995; 269:1281‑4. 18. Yamamoto H, Monden T, Miyoshi H, Izawa H, Ikeda K, Tsujie M, Ohnishi T, Sekimoto M, Tomita N, Monden M. Cdk2/cdc2 expression in colon carcinogenesis and effects of cdk2/cdc2 inhibitor in colon cancer cells. Int J Oncol 1998; 13:233‑9. 19. Kim JH, Kang MJ, Park CU, Kwak HJ, Hwang Y, Koh GY. Amplified CDK2 and cdc2 activities in primary colorectal carcinoma. Cancer 1999; 85:546‑53. 20. Sherr CJ, Roberts JM. Inhibitors of mammalian G1 cyclin‑dependent kinases. Genes Dev 1995; 9:1149‑63. 21. Carnero A, Hannon GJ. The INK4 family of CDK inhibitors. Curr Top Microbiol Immunol 1998; 227:43‑55. 22. Harper JW, Elledge SJ, Keyomarsi K, Dynlacht B, Tsai LH, Zhang P, Dobrowolski S, Bai C, Connell‑Crowley L, Swindell E, et al. Inhibition of cyclin‑dependent kinases by p21. Mol Biol Cell 1995; 6:387‑400. 23. Koff A. How to decrease p27Kip1 levels during tumor development. Cancer Cell 2006; 9:75‑6. 24. Hengst L, Reed SI. Inhibitors of the Cip/Kip family. Curr Top Microbiol Immunol 1998; 227:25‑41. 25. el‑Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell 1993; 75:817‑25. 26. Lees E. Cyclin dependent kinase regulation. Curr Opin Cell Biol 1995; 7:773‑80. 27. Sherr CJ. Cancer cell cycles. Science 1996; 274:1672‑7. 28. McDonald IIIrd ER, El‑Deiry WS. Cell cycle control as a basis for cancer drug development (Review). Int J Oncol 2000; 16:871‑86. 29. Easton J, Wei T, Lahti JM, Kidd VJ. Disruption of the cyclin D/cyclin‑dependent kinase/ INK4/retinoblastoma protein regulatory pathway in human neuroblastoma. Cancer Res 1998; 58:2624‑32. 30. Classon M, Harlow E. The retinoblastoma tumour suppressor in development and cancer. Nat Rev Cancer 2002; 2:910‑7. 31. Taya Y. RB kinases and RB‑binding proteins: New points of view. Trends Biochem Sci 1997; 22:14‑7. 32. Sherr CJ, Roberts JM. CDK inhibitors: Positive and negative regulators of G1‑phase progression. Genes Dev 1999; 13:1501‑12. 33. Ortega S, Malumbres M, Barbacid M. Cyclin D‑dependent kinases, INK4 inhibitors and cancer. Biochim Biophys Acta 2002; 1602:73‑87. 34. Sherr CJ, McCormick F. The RB and p53 pathways in cancer. Cancer Cell 2002; 2:103‑12.

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cyclin E by curcumin correlates with the decrease in the proliferation of human prostate cancer cells. Our data showed that the suppression of cyclin E expression was not cell type dependent as downregulation occurred in androgen‑sensitive LNCaP and ‑insensitive PC‑3 prostate cancer cells. Curcumin‑induced downregulation of cyclin E was reversed by proteasome inhibitor lactacystin, an inhibitor of 26S proteasome, suggesting the role of ubiquitin‑dependent proteasomal pathway. Cyclin D acts as a growth sensor and provides a link between mitogenic stimuli and the cell cycle. Cyclin D1 binds to CDK4 and CDK6 in early G1. Aberrant cyclin D1 expression has been reported in many human cancers. Cyclin D1 gene amplification occurs in breast, esophageal, bladder, lung and squamous cell carcinomas,88 and parathyroid adenomas.89 Cyclin D2 and cyclin D3 have also been reported to be overexpressed in some tumours.90‑92 The suppression of cyclin D1 by curcumin led to inhibition of CDK4‑mediated phosphorylation of Rb protein. The present study has demonstrated that curcumin‑induced downregulation of cyclin D1 was inhibited by lactacystin, suggesting that curcumin represses cyclin D1 expression by promoting proteolysis. Similarly, recent studies have demonstrated that curcumin can regulate cyclin D1 expression through transcriptional and posttranslational modifications,40,93 and this may contribute to the antiproliferative effects of curcumin against various cell types. Overall, these data suggest that downregulation of cyclins by genetic or pharmacological means may be useful for cancer therapy and prevention. The transition of the G1 to the S phase of the cell cycle marks an irreversible commitment to DNA synthesis and proliferation and is strictly regulated by positive and negative growth‑regulatory signals. The G1‑S transition is controlled by the Rb‑E2F pathway, which links growth‑regulatory pathways to a transcription program required for DNA synthesis, cell cycle progression and cell division.94‑96 This transcription program is activated by the E2F transcription factors and repressed by E2F‑Rb complexes.97 E2F overexpression or Rb inactivation is sufficient to induce S phase entry, whereas Rb overexpression can arrest cycling cells in G1, suggesting that the Rb‑E2F pathway is central to the control of the G1‑S transition.94,95 Mitogenic signal causes sequential activation of the CDK‑cyclin complexes CDK4/6‑cyclin D and CDK2‑cyclin E, which hyper‑phosphorylate pRb and thereby cause the release of active E2F.94,95 pRB‑deficient cells are hypersensitive to DNA damage‑induced apoptosis.98,99 On the other hand, E2F‑1 has a role distinct from other E2Fs in the regulation of apoptosis.100 Loss of E2F‑1 reduces tumorigenesis and extends the lifespan of Rb1+/‑ mice.101 E2F‑1 has been found to induce the expression of many apoptotic genes102 and thus mediate the response of chemopreventive agents. Overall, our results provide the molecular mechanisms through which curcumin contributes to the antiproliferative and antitumor activities. The down‑regulation of cyclin E, cyclin D1, and hyper‑phosphorylation of pRb, and up‑regulation of CDK inhibitors p16/INK4a, p21/WAF1/CIP1 and p27/KIP1 may contribute to the antiproliferative effects of curcumin against prostate cancer. These events may be responsible for growth arrest at G1/S phase of cell cycle followed by apoptosis in cancer cells. The strength of our studies is also confirmed by a recent report where curcumin chemosensitizes and radiosensitizes the effects by down‑regulating the MDM2 oncogene through the PI3K/mTOR/ETS2 pathway.103 Thus, targeting of cyclins and/or CDKs by pharmacological or genetic means may www.landesbioscience.com

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