Induction of Growth Inhibition and Apoptosis in ...

Induction of Growth Inhibition and Apoptosis in Human Uterine Leiomyoma Cells by Isoliquiritigenin Dong-chul Kim, OMD, Sabarish Ramachandran, MS, Seung-hee Baek, OMD, Sang-Hoon Kwon, MD, Kun-Young Kwon, MD, Soon-Do Cha, MD, Insoo Bae, PhD, and Chi-Heum Cho, MD

Isoliquiritigenin(ISL), a calchone flavonoid, has cancer-preventing properties and is often used in Chinese medicine. In the present study, the authors use ISL to determine its effect on cell proliferation and cell cycle progression in primary cultured human uterine leiomyoma cells. Cell viability and cell proliferation assays were conducted. Flow cytometry, annexin V apoptosis assay, and DNA fragmentation assay were performed to determine the effect of ISL on cell cycle and apoptosis. The expression of cell cycle regulatory–related proteins was evaluated by Western blot. The cell viability and proliferation of uterine leiomyoma cells were significantly reduced by ISL treatment in a dose-dependent manner. Flow cytometry results showed that ISL induced subG1 and G2/M arrest. DNA fragmentation assay and annexin V apoptosis assays revealed apoptosis induction. ISL-induced growth inhibition in uterine leiomyoma cells was associated with increased p21Cip1/ Waf1 expression in a p53dependent manner. Activation of caspase-3 and downregulation of Bcl-2, cdk 2/4, and E2F, with a concomitant increase in dephosphorylation of Rb and poly–ADP-ribose polymerase cleavage, were observed. This study demonstrates that ISL inhibits cell proliferation by initiating apoptosis in human uterine leiomyoma cells coupled with increased cell cycle arrest. These results indicate that ISL could prove to be a promising chemopreventive and therapeutic agent against human uterine leiomyoma. KEY

WORDS:

Isoliquiritigenin, human uterine leiomyoma, apoptosis.

U

terine leiomyoma, also known as myomas or fibroids, is the most common benign tumor in the human female pelvis and the leading indicator of hysterectomies.1 Common symptoms associated with these tumors are pain, discomfort, menstrual disturbances, and infertility.2 The cause of uterine leiomyomas is unknown, but the growth of tumors is thought to be modulated by From the Department of Oriental Medicine, Graduate School, Daegu Haany University, Kyungbuk, Korea (D-cK, S-hB); the Department of Obstetrics and Gynecology (SR, S-HK, S-DC, C-HC) and the Department of Pathology (K-YK), School of Medicine, Keimyung University, Daegu, Korea; and the Department of Oncology, Lombardi Cancer Center, Georgetown University, Washington, DC (IB). D-cK and SR contributed equally to this work. Address correspondence to: Chi-Heum Cho, MD, Department of Obstetrics and Gynecology, School of Medicine, Keimyung University, 194 Dongsan-Dong, Choong-Ku, Daegu, Korea, 700-712. E-mail: [email protected] Acknowledgment:This work was supported by Korea Research Foundation Grant (KRF-2004-002-E00092).

Reproductive Sciences Vol. 15 No. 6 July 2008 552-558 DOI. 10.1177/1933719107312681 © 2008 by the Society for Gynecologic Investigation

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the ovarian hormones estrogen and progesterone. This hormone-dependent growth is demonstrated by the fact that most of these tumors are diagnosed during reproductive periods, change in size during pregnancy, or regress after the onset of menopause, which coincides with dramatic changes in the hormonal milieu.2 In recent years, herbal therapies have been commonly used in Western countries, but little is known about their effectiveness, mode of action, or side effects. The literature cites that certain plants possess anticarcinogenic properties. Flavonoids, plant pigments, are a group of naturally occurring polyphenolic substances found in fruits and vegetables and are reported to have chemotherapeutic properties. More than 4000 flavonoids have been discovered and categorized according to their structures, such as flavon, flavonol, flavanone, flavanol, isoflavone, chalcone, anthocyanin, and catechin. Isoliquiritigenin (ISL) is a chalcone flavonoid richly present in licorice (a legume) and shallot (a liliaceae). Our previous study demonstrated that Spatholobus subrectus Dunn possesses an inhibitory property in human uterine leiomyoma cells (unpublished

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Isoliquiritigenin’s Effects on Human Uterine Leiomyoma Cells

data). ISL is one of the active components of S subrectus Dunn3 and is involved in biochemical pathways such as antioxidative and superoxide scavenging activities,4 an antiplatelet aggregation effect,5 an inhibitory effect on aldose reductase activity,6 and antiestrogenic properties.7 There are also reports on the anticarcinogenic effects of ISL,8,9 mediated via its antiangiogenic10 and apoptosisinducing nature.11,12 In the present study, we examine the growth-inhibitory effect of ISL on human uterine leiomyoma cells. We also investigate its influence on cell cycle progression and cell cycle–associated gene expression.

MATERIALS AND METHODS Materials and Solutions

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Patients ranged in age from 35 to 44 years, with a mean age of 38.5 years, and had received no hormonal therapy for at least 6 months before surgery. Four samples were collected from the proliferative phase of the menstrual cycle, and 4 samples were obtained from the secretory phase of the menstrual cycle. Fresh uterine leiomyoma tissue samples were minced and digested by incubation in Hanks solution for 4 hours at 37°C, containing HEPES 0.0065 g/mL, collagenase 0.0015 g/mL, and DNase 0.0002 g/mL with periodic agitation. A portion of each tissue was stored at –70°C for isolation of mRNA and proteins. The dispersed uterine leiomyoma and myometrial cells were plated in D-MEM/F12 and grown to confluence.

Cell Viability Assay

Dulbecco’s Modified Eagle Medium, F12 nutrients mixture, and other supplements for cell culture were obtained from GibcoBRL (Grand Island, NY). ISL (Sigma Chemical Co, St Louis, MO) was dissolved in dimethyl sulfoxide (DMSO) as a stock solution (100 mM) and stored at –20°C. For all experiments, ISL was used in a dose-dependent manner, in which the final concentrations of the compound were 10 μM, 20 μM, and 50 μM. Control cultures received the carrier solvent (0.1% DMSO).

Establishment of Uterine Leiomyoma Cultures Uterine leiomyoma and their adjacent normal myometrium tissues for control were obtained from hysterectomies that were conducted on benign diseases at Keimyug University Dongsan Medical Center, Daegu, Korea. Informed written consent was obtained from patients, and sample collection and handling were performed based on the approval and guidance of the Ethical Committee of Keimyung University. Uterine leiomyoma tissues were obtained from central to peripheral parts of the biggest fibroid from women undergoing elective hysterectomy for uterine leiomyoma.The central part refers to the volume limited to 5 mm from the center of the tumor, and the peripheral part refers to the outer 5 mm under the capsule. The diagnosis of uterine leiomyoma was confirmed by histopathological analysis. All intramural leiomyomas were included in this study and calcified; hydrophic degenerative leiomyomas were excluded.

To measure the viability of ISL-treated uterine leiomyoma cells, MTS colorimetric assays were performed.The intensity of the purple color formed by this assay is proportional to the number of viable cells. Uterine leiomyoma cells were seeded in a 96-well plate (1 × 105 cells/well). Twenty-four hours postseeding, cells were treated with ISL at various concentrations for 48 hours, and then MTS reagent was added and incubated for an additional 4 hours at 37°C.The optical density (OD) was measured at 495 nM. Cell survival was calculated by subtracting the background OD of media alone and then normalized by dividing the OD of test wells by the OD of the control (untreated) wells.

Cell Proliferation Assay The ability of ISL to inhibit DNA synthesis was determined by estimating the amount of BrdU incorporation into DNA by a colorimetric immunoassay, using a cell proliferation enzyme-linked immunosorbent assay (ELISA), BrdU-colorimetric kit (Roche Diagnostics GmbH, Penzberg, Germany). Uterine leiomyoma cells were cultured in 48-well plates (1 × 104 cells/well). Twenty-four hours postseeding, cells were treated with ISL at various concentrations, and the assay was carried out according to the manufacturer’s instructions. The developed assay color was measured at 490 nm.The color intensity and the absorbance values directly correlate with the amount of BrdU incorporated into the DNA. The results are expressed as the percentage inhibition of


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BrdU incorporation by ISL over the control. Data were calculated as the percentage inhibition using the formula Inhibition (%) = (100 – (ODt/ODs) × 100,

where ODt and ODs are the optical density of the test substance and solvent control, respectively.

Apoptosis Assay Apoptosis inducted by ISL treatment on uterine leiomyoma cells was detected by performing the apoptosis assay, a flow cytometry–based experiment using Vybrant Apoptosis Assay kit (Molecular Probes, Eugene, OR), which contains 3 apoptosis-based staining dyes, allophycocyanin annexin V, C12-resazurin, and SYTOX green. Uterine leiomyoma cells were treated with ISL, and the assay was carried out following the manufacturer’s instructions and analyzed by flow cytometry.

Fluorescence-Activated Cell Sorting Analysis To determine the cell distribution, uterine leiomyoma cells were treated with various doses of ISL or DMSO alone (control).After 48 hours, cells were harvested, washed with phosphate-buffered saline (PBS), and fixed in cold 70% ethanol. Fixed cells were suspended in 0.1% RNase A and propidium iodide (50 μg/mL in PBS) to determine cell cycle dynamics. DNA fluorescence was measured by flow cytometer (FACS Calibur; Becton Dickinson, Franklin Lakes, NJ).The percentage of cells in each cell cycle phase was determined using ModFit LT software (Becton Dickinson) based on the DNA histogram.

DNA Fragmentation Assay Induction of apoptosis by ISL was determined using the cellular DNA fragmentation ELISA kit (Roche Diagnostics GmbH).The kit determines the amount of formation of cytoplasmic histone-associated DNA fragments (mononucleosomes and oligonucleosomes) due to apoptosis by using a photometric enzyme-linked immunoassay procedure. The assay was performed following the manufacturer’s instructions; the amount of cells used for the assay was 1 × 105 cells/well of a 96-well plate, and the developed assay color was measured at 370 nm.The results are expressed as the percentage detection of nucleosomes

Kim et al

in the cytoplasmic fractions of uterine leiomyoma cells treated with varied concentrations of ISL.

Western Blot Analysis ISL-treated cell extracts were prepared in lysis buffer (10 mM Tris [pH 7.4], 5 mM EDTA, 130 mM NaCl, 1% Triton X-100, PMSF [10 μg/mL], leupeptin [10 μg/mL], aprotinin [10 μg/mL], 5 mM phenanthroline, and 28 mM benzamidine-HCl). Protein concentrations were measured using Bio-Rad Protein Assay Reagent (Bio-Rad, Richmond, CA) following the manufacturer’s suggested procedure. Aliquots of protein were separated by 8% to 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidine difluoride membrane (Millipore, Bedford, MA).The membrane was blocked with Tris-buffered saline with 5% skim milk and 0.2% Tween 20, reacted with primary antibodies, and washed. The following antibodies were used: anti-Rb (Santa Cruz Biotechnology, Santa Cruz, CA), antip21Cip1/Waf1 (Santa Cruz Biotechnology), anti-p27Kip1 (Santa Cruz Biotechnology), anti-p53 (Calbiochem, San Diego, CA), anti–cyclin E (Santa Cruz Biotechnology), and anticdk2 (Santa Cruz Biotechnology) antibodies. β-Tubulin (Santa Cruz Biotechnology) was measured as the internal control. After reaction with horseradish peroxidase– conjugated secondary antibodies (Amersham Lifescience, Buckinghamshire, UK), immune complexes were visualized by using an enhanced chemiluminescence system (Amersham Lifescience) following the manufacturer’s suggested procedure.

Statistical Evaluation Data are expressed as means ± SEM for all experiments. A Student t test was used to assess the statistical significance between means. P < .05 was considered significant.

RESULTS Growth Inhibition of Human Uterine Leiomyoma Cells by ISL This study investigated the effects of ISL on the growth of uterine leiomyoma cells.As shown in Figure 1, in contrast with the control, cells treated with ISL showed decreased cell growth in a dose-dependent manner. Treatment with 50 μM ISL blocked .05; Figure 5). It is intriguing that no significant DNA fragmentation was seen, even though data from other experiments were indicative of apoptosis. It is possible that whole

We analyzed the change of expression of cyclin-dependent kinase (CDK) inhibitors after the indicated dose of ISL treatment in uterine leiomyoma cells by Western blot analysis, and ISL increased the p21Cip1/Waf1 protein level in a dosedependent manner (Figure 6A, B). In contrast, the protein levels of p53 were not changed at 10- and 20-μM concentrations of the ISL used. Increasing levels of the p21Cip1/Waf1 protein correlated well with a decline in cdk2 and cyclin B1 levels, a decrease in pro–caspase-3, poly–ADP-ribose polymerase (PARP) cleavage, enhanced dephosphorylation of Rb, and decline in E2f level. Furthermore, ISL induced an increased expression of FAS ligand. Taken together, these results demonstrate that caspase-3 is involved in mediating the induction of apoptosis by ISL and may take place in part through the induction of p21Cip1/Waf1 and FAS ligand.

DISCUSSION Uterine leiomyoma (fibroids or myomas) is an extremely common problem in women. Indeed, fibroids are known to be the leading cause for hysterectomy in the world. Despite the frequency of these tumors in women and the morbidity that they cause, leiomyomas remain a true frontier for gynecologic investigation.


Isoliquiritigenin’s Effects on Human Uterine Leiomyoma Cells

Figure 6. Effect of isoliqiritigenin (ISL) on the expression of cell cycle–related regulatory proteins. (A) Protein analysis was performed by Western blotting using antibodies against p21Cip1/Waf1, p27Kip1, Rb, Bcl2, cyclin B1, cdk2, cdk4, E2f1, pro–caspase-3, poly–ADP-ribose polymerase (PARP), p53, and FAS ligand. Human uterine leiomyoma cells were treated for the indicated dose of ISL, and total protein was isolated for Western blotting; β-tubulin was used as an internal control. (B) Protein expressions were quantified, normalized to internal control, and expressed as fold change in comparison to control untreated leiomyoma cells.Values are the means (±SEM) of 3 experiments with triplicate determinations. *P < .05; **P < .01.

Although the initiating factors that lead to development of leiomyomas are not known, there is a great deal of evidence showing that the ovarian steroids, estrogen and progesterone, are important factors for tumor growth.13-16 Biochemical and molecular studies have shown that leiomyomas have significantly increased levels of both estrogen and progesterone receptors when compared with normal myometrium.17,18 Studies have shown that estradiol stimulates the proliferation of uterine smooth muscle cells (SMCs).19,20 The SMCs of leiomyomas show both a higher mitotic activity than corresponding myometrium and decreased apoptosis.20 Associations between diet and uterine leiomyomata, although not consistently observed, raise the possibility that both dietary and exercise habits may also be related to the development of these tumors. The risk of uterine leiomyomata appeared to be reduced by approximately 50% among women who reported a high intake of green vegetables.21 These associations were independent of one another and other characteristics of the women. Notably, the risk of uterine leiomyomata was not related to an increase in body mass index in this population.Thus, the observed dietary associations may reflect unmeasured dietary lifestyle characteristics that are related to uterine leiomyoma development.

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Because of increased public interest in alternative medicine and disease prevention, use of herbal preparations containing high doses of flavonoids for health maintenance has become very popular, raising the potential for interactions with conventional drug therapies.22 ISL is a flavonoid belonging to the chalcone family and has several biochemical activities similar to other flavonoids. Recently, studies on the biological effects of ISL have expanded, especially concerning antitumor activities. ISL is found to promote antitumor activity on 2-stage mouse skin carcinogenesis8 and oral administration of ISL has an inhibitory effect on azoxymethane-induced murine colonic tumorigenesis.9 Other investigators have also reported on its anitiangiogenic activity10 and induction of apoptosis.11,12 In the present study, ISL showed the potential to inhibit cell proliferation of human uterine leiomyoma cells, with a specific alteration in the cell cycle. Many flavonoids are known to perturb the cell cycle progression that is a fundamental activity in the process of cell proliferation. The present study demonstrates that ISL restrained cell cycle progression at the subG1 and G2/M phase and inducted apoptosis. Therefore, growth inhibition by ISL may be, at least in part, related to cell cycle arrest at the G1 phase. It is known that p21Cip1/Waf1 inhibits the activities of cyclin-CDK complexes that regulate cell cycle progression and that overexpression of p21Cip1/Waf1 inhibits proliferation of mammalian cells.23-25 In addition, several reports have indicated p53-independent regulation of the p21Cip1/Waf1 gene.26-28 In the present study, ISL induced increased expression of p21Cip1/Waf1 without increasing the p53 protein level in leiomyoma cells. These results suggest that p21Cip1/Waf1 is involved, at least in part, with leiomyoma growth inhibition. This reflects that ISL-induced growth inhibition and apoptosis in uterine leiomyoma cells through the induction of p21Cip1/Waf1 may not be mediated by p53. Apoptosis has been well established to play a critical role in carcinogenesis and cancer progression. In this study, we determined the direct effect of ISL on cell proliferation of uterine leiomyoma cells; it suppressed cell growth in a concentration-dependent manner in uterine leiomyoma cells and evidently induced apoptosis. Our data showed that ISL possesses the capability to prohibit cell proliferation by inducting apoptosis. Caspase-3, one of the effector caspases, was activated, thereby inducing PARP cleavage, Rb dephosphorylation, and DNA fragmentation, the classical markers in the apoptosis cascade. Moreover, increased FAS ligand expression levels indicate the possibility that induction of apoptosis by ISL is mediated by the death receptor.These results suggest that ISL suppresses cell proliferation through an apoptosis-dependent mechanism.


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CONCLUSIONS We have demonstrated that ISL inhibited cell proliferation by initiating apoptosis in human uterine leiomyoma cells coupled with increased cell cycle arrest at the subG1 and G2/M phase and that this induction is associated with enhanced p21Cip1/Waf1 and FAS ligand expression. These data suggest that ISL may be a promising agent in human uterine leiomyoma treatment; p21Cip1/Waf1 and FAS ligand may be important factors in ISL-induced growth inhibition and death.

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