Journal of Women’s Medicine Vol. 3 No. 3 September 2010
Original Articles
Isoliquiritigenin inhibited cell proliferation and triggered apoptosis in human endometrial cancer cell line Yoon Geon Kim, OMD1, Sabarish Ramachandran, PhD2, Dong-chul Kim, OMD1, Young Bin Hong, PhD3, Eun Ha Kim, OMD1, Sang-Hoon Kwon, MD2, So-Jin Shin, MD2, Soon-Do Cha, MD2, Insoo Bae, PhD3, 2,4
and Chi-Heum Cho, MD 1
Department of Oriental Medicine, Daegu Haany University Graduate School; 2Department of Obstetrics and Gynecology, Keimyung University School of Medicine, Daegu, Korea; 3Department of Oncology, Lombardi Cancer Center, Georgetown University, 4 Washington, DC, USA; Institute for Medical Genetics, Keimyung University School of Medicine, Daegu, Korea
Objective: To determine the anti-tumor effect of isoliquiritigenin (ISL) on endometrial cancer cell and to evaluate its effect on apoptosis in Hec1A endometrial cancer cell lines. Methods: Human endometrial cancer cell lines (Hec1A) and Ishikawa, and normal endometrial cell line (T-HESCS) were cultured in vitro. The viabilities of three cell lines on ISL were measured. Cell cycle distribution and induction of apoptosis were measured in Hec1A cells after ISL treatment. Results: ISL significantly reduced cell viabilities of endometrial cancer cell lines but not normal cell line in a dose-dependent manner. Cell cycle analysis indicated that ISL treatment increased the proportion of cells in the sub-G0/G1 phase. DNA fragmentation and fluorometric TUNEL assays also revealed apoptotic cell death after ISL incubation. ISL treatment markedly up-regulated the expression of cyclin-dependent kinase inhibitor, p21Cip1/Waf1 in a p53 independent manner and down regulated the expressions of cyclins and CDKs, with concomitant increase in FAS and cleavage of caspase 7, caspase 8, and caspase 9. In addition, elevation of caspase 3 activity also observed in a dose and time dependent manner. Conclusion: ISL inhibited cell proliferation and triggered apoptosis in human endometrial cancer cell line Hec1A. Hence, ISL can be used as a potentially potent clinical chemotherapeutic agent for treating endometrial cancer. Key words: Endometrial cancer cell line; Hec1A; Isoliquiritigenin; Apoptosis
Introduction Endometrial carcinomas are divided into two pathogenetic 1 groups based on their molecular and clinical characteristics. Type I, endometrial cancer, represents the majority of cases
∙ Received: 2010. 4. 20. ∙ Accepted: 2010. 6. 24. ∙ Corresponding author: Chi-Heum Cho, MD Department of Obstetrics and Gynecology, Keimyung University School of Medicine, 194 Dongsan-dong, Choong-gu, Daegu 700-712, Korea Tel: +82-53-250-7518 Fax: +82-53-250-7599 E-mail:
[email protected] *Kim YG and Ramachandran S contributed equally to this work.
and occurs in most pre- and peri-menopausal women exposed 2 to excess levels of estrogen. Type II, endometrial cancers, arises from atrophic endometrium in older women, and is not 2 associated estrogen exposure. In recent years endometrial cancer has become the most common gynecologic ma3 lignancy in many developed countries. Although the primary treatment in endometrial cancer is surgical dissection, adjuvant chemotherapy has also been accepted as an effective therapeutic strategy in high risk or advanced endo4 metrial cancer. In recent years, herbal therapies are commonly used in Western countries, but little is known about their effectiveness, mode of action and side effects. A lot of reports have shown the anti-carcinogenic property of plant polyphe-
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nols. Flavonoids, a plant pigment, are a group of naturally occurring polyphenolic substances found in fruits and vege7 tables and reported to have chemotherapeutic property. Over 4,000 flavonoids have been discovered and categorized, such as flavon, flavonol, flavanone, flavanol, isoflavone, chalcone, anthocyanin, and catechin according to its structure. Isoliquiritigenin (ISL) is a chalcone flavonoid, richly present in licorice (a legume) and shallot (a lil8 iaceae). Our previous study demonstrated that Spatholobus subrectus Dunn possess inhibitory property in human uterine leiomyoma cells (unpublished data). ISL is one of the active components in Spatholobus subrectus Dunn and involved in biochemical pathways such as antioxidative and superoxide scavenging, antiplatelet aggregation, and anti-estro9-11 genic activity. ISL is also reported to have anti-carcinogenic potencies such as anti-angiogenic and apoptosis in8,12-15 ducing activity. In the present study, we examined the anti-tumor effect of ISL on an endometrial cancer cell lines using cell cycle distribution and apoptosis analysis. This may offers a new promising therapeutic approach in overcoming endometrial cancer.
Materials and Methods 1. Cell culture Human endometrial cancer cell line (Hec1A) and normal endometrial cell line (T-HESCS) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Ishikawa, human endometrial cancer cell line, was purchased from Sigma (St. Louis, MO, USA). Cells were cultured in Dulbecco's Modified Eagle Medium, DMEM/F-12, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin antibiotic mixture and grown to confluence. All the cell culture media and supplements were obtained from GibcoBRL (Grand Island, NY, USA). 2. Cell viability assay To measure viability of ISL treated Hec1A cell, Tritiated Thymidine Incorporation (MTS) colorimetric assay (Promega, Madison, WI, USA) was performed. Hec1A cells seed3 ed in a 96 well plate (2×10 cells/well) were treated with ISL (Sigma) which dissolved in dimethyl sulfoxide (DMSO). For control treatment (0 µM ISL) in overall experiment, 0.1% DMSO was used. After 48 hours incubation, MTS reagent was added and incubated for 4 hours at 37℃. Optical
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density was measured at 495 nm. Cell viability values were calculated relative to the control cells (100%) and expressed as means±standard error of three independent experiments. 3. Determination of cell cycle distribution To determine cell distribution of the ISL treated Hec1A cells, FACS analysis was performed. After 48 hours of 50 µM ISL treatment, cells were harvested, washed with cold phospate buffered solution (PBS), and were fixed in ice-cold 70% ethanol at 4℃ for 24 hours. Then cells were treated with 0.1% RNaseA for 30 min at 37℃ and were stained with propidium iodide and incubated for 30 min at room temperature. DNA fluorescence was measured by flow cytometer (FACSCalibur↗, Becton Dickinson, Franklin Lakes, NJ, USA). The percentage of cells in each cell cycle phase was determined using the ModFit LT↗ software (Becton-Dickinson) based on the DNA histogram. 4. DNA fragmentation assay Apoptotic cell death by ISL was determined using the Cellular DNA fragmentation ELISA kit (Roche Diagnostics, Penzberg, Germany), which determines the amount of cyto3 plasmic histone-associated DNA fragments. Cells (2×10 / well) seeded on a 96 well plate were incubated with ISL for 48 hours then lyzed. The total lysates containing BrdU-labeled DNA fragments were transferred to anti-DNA antibody-coated microplates. Immobilized BrdU-labeled DNA fragments were measured at 370 nm after binding with anti-BrdU antibodies. Values were expressed as the mean of triplicate measurements. 5. Fluorometric TUNEL assay DNA strand breaks due to apoptosis were also determined by DeadEnd Fluorometric TUNEL System assay kit (Promega). Hec1A cells were treated with 50 µM ISL for 48 hours, then fixed with formaldehyde and washed with PBS. Terminal Deoxynucleotidyl transferase was added to cells to incorporate fluorescein-12-dUTP at 3’-OH into DNA ends. Fluorescence-positive cells were analyzed by flow cytometer (FACSCalibur↗). 6. Western blot analysis Cell extracts were prepared in lysis buffer (10 mM Tris [pH 7.4], 5 mM EDTA, 130 mM NaCl, 1% Triton X-100, serine protease inhibitor phenylmethylsulphonyl fluoride [10 µg/mL], leupeptin [10 µg/mL], aprotinin [10 µg/mL], 5
Yoon Geon Kim, et al. Isoliquiritigenin induces apoptosis in human endometrial cancer cell line
mM phenanthroline, and 28 mM benzamidine-HCl). Protein concentrations were measured using Bio-Rad Protein Assay Reagent (Bio-Rad, Richmond, CA, USA). Proteins were separated by 8~15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidine difluoride membrane (Millipore, Bedford, MA, USA). Then membrane was incubated with primary antibodies of p53, p21, p27, BAX, Bcl2, Bid, pro-caspase 3, caspase 7, 8, and 9, cdk2, cdk4, and β-actin (mouse monoclonal antibody; Cell Signaling Technology Inc., Beverly, MA, USA), cyclin A, cyclin E, cyclin D, cyclin B1, E2F, FAS (rabbit polyclonal antibody; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After following incubation with horseradish peroxidase conjugated secondary antibodies (Amersham Lifescience, Buckinghamshire, England), bands on the membranes were visualized by an enhanced chemiluminescence system (Amersham Lifescience).
experiment. Statistical significance was determined by using the Student’s t test. A P-value of 0.05 or less was considered to be of statistical significance.
Results 1. ISL affects cell viability of endometrial cancer cells but not normal cell The cytotoxicity of ISL on the endometrial cell lines was investigated using MTS analysis. ISL reduced cell viability of Hec1A and Ishikawa in a dose dependent manner (Fig. 1A, 1B). Cell viability of Hec1A and Ishikawa incubated with 20 µM of ISL significantly reduced to 89% and 73%, respectively. Incubation with 50 µM of ISL greatly reduced cell viability of Hec1A and Ishikawa to 37% and 25%, respectively (Fig. 1A, 1B). However, ISL did not show effective cytotoxicity to normal endometrial cell line, T-HESCS, at 20 µM. Moreover, 76% of T-HESCS cells remained viable after incubation with 50 µM ISL (Fig. 1C). Thus, ISL selectively affects endometrial cancer cell line compared with normal cell line in cell viability.
7. Determination of caspase 3 activity Caspase 3 activity assay was performed using the Caspase-3 Colorimetric Assay kit (R&D Systems, Minneapolis, MN, USA), which detects caspase 3 activity based on the cleavage of a chromogenic peptide substrate (DEVDpNA). Hec1A cells incubated with ISL for 48 hours were harvested and lyzed. Caspase-3 colorimetric substrate (DEVD-pNA) was added to cell lysates. After 2 hours incubation, releasesed chromophore pNA by caspase 3 was spectrophotometrically quantitated at a wavelength of 405 nm.
2. ISL augments sub-G0/G1 phase of cell cycle To determine whether reduced cell viability in Hec1A was due to perturbation of a cell cycle, we analyzed cell cycle distribution using flow cytometry. DNA histograms showed that ISL changed cell cycle distribution at high concentration (Fig. 2). Incubation with 50 µM ISL increased the ratio of sub-G0/G1 fraction from 12% (control) to 54% (50 µM ISL). There was no significant change in S and G2/M fraction, while the ratio of G1 fraction decreased from 55% (control) to 13 % (50 µM ISL). After incubation with low dose of ISL (10 and 20 µM), there was no significant alter-
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8. Statistical analysis All measurements were made on triplicate samples, and the results expressed as the mean±SD of a representative
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Fig. 1. Ctyotoxicity of isoliqiritigenin on human endometrial cancer cell lines. Viability of human endometrial cancer cell lines (Hec1A) (A) and Ishikawa (B) cells, and human normal endometrial cell line, (T-HESCS [C]) treated with an indicated dose for 48 hr. Cell viability was measured by Tritiated Thymidine Incorporation (MTS) assay and expressed as % cell survival against control * ** cells. Values represent means±standard error of experiments. P
