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Tamoxifen inhibits proliferation and induces apoptosis in human hepatocellular carcinoma cell line HepG2 via down-regulation of survivin expression
Available online at
www.sciencedirect.com Biomedicine & Pharmacotherapy 63 (2009) 375e379
Original article
Tamoxifen inhibits proliferation and induces apoptosis in human hepatocellular carcinoma cell line HepG2 via down-regulation of survivin expression Renhua Guo a,*, Zuhu Huang b, Yongqian Shu a, Shidai Jin a, Hongmei Ge c a
Department of Oncology, First Affiliated Hospital of Nanjing Medical University, Guangzhou Road 300, Nanjing 210029, PR China b Department of Infectious Disease, First Affiliated Hospital of Nanjing Medical University, Nanjing, PR China c Laboratory of Molecular Biology, First Affiliated Hospital of Nanjing Medical University, Nanjing, PR China Received 20 August 2008; accepted 26 September 2008 Available online 23 October 2008
Abstract Tamoxifen has been used in patients with hepatocellular carcinoma (HCC). However, its inhibitory mechanism remains unknown. In current study, we evaluated the effect of tamoxifen on the estrogen receptor-a-negative HCC cell proliferation, apoptosis and expression of survivin which had been known to play an important role in promotion of cellular proliferation as well as inhibition of apoptosis in cancer cells. HepG2 cells were incubated with tamoxifen (0.1, 1, 10, or 20 mM) for up to 72 h. Cell proliferation was assessed, flow cytometric analysis was performed, and survivin expression was detected. Our results are showed as follows. Ten or 20 mM tamoxifen induced a reduction of cell proliferation. Basically reduction of proliferation was related to an increase in the fraction of G0/1-phase. When tamoxifen was administrated at higher concentration (20 mM), the increase of the relative apoptosis appeared with a delay, augmenting the effect of tamoxifen on cell proliferation. When apoptosis was induced, a significant depression of survivin expression preceded. In conclusion, the tamoxifen decreasing cell proliferation and induction of apoptosis of HepG2 cells depends on drug concentration, which is due to cytostatic and cytocide effects, the latter may be mediated by a down-regulation of survivin expression. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Estrogen receptor; Survivin; Apoptosis
1. Introduction Tamoxifen is a non-steroidal anti-estrogen which is commonly used in adjuvant treatment as well as in the firstline treatment of postmenopausal breast cancer. Furthermore, tamoxifen has been used in other malignant tumors, such as hepatocellular carcinoma (HCC) [1]. Tamoxifen treatment of advanced, inoperable HCC has been shown to prolong survival
Abbreviations: HCC, hepatocellular carcinoma; ER, estrogen receptor; PKC, protein kinase C; G0/G1, Gap0/Gap1; S, synthesis; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide. * Corresponding author. Tel.: þ86 25 83718836 6714; fax: þ86 25 83724440. E-mail address: [email protected] (R. Guo). 0753-3322/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2008.09.010
of some patients [2,3]. However, those estrogen receptor-alpha (ER-a)-negative [4], or variant ER-as in ER-a-positive [5] are insensitive to tamoxifen [6]. Therefore, the effects of tamoxifen in HCC are more likely ER-a-independent pathway than dependent pathway. The effects of tamoxifen in tumor cells by ER-a-independent pathways have been reported [7]. Anti-proliferation action of tamoxifen has been reported to be associated with an increase of Ca2þ influx [8], and an enhanced generation of reactive oxygen species [9], or an inhibition of protein kinase C activity [10]. But so far, the role of survivin in mediating tamoxifen effect in hepatoma cells remains unknown. Survivin, a member of inhibitor of apoptosis protein family, is expressed during the G2/M phase. It directly inhibits the terminal effector cell-death proteases caspase-3 and caspase-7
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[11]. It binds to the microtubules of the mitotic spindle, where it counteracts the induction of apoptosis [12]. Moreover, experimental data have shown survivin in promotion of cellular proliferation as well as inhibition of apoptosis in cancer cells [13]. Survivin overexpression has been identified as a negative prognostic factor in various cancer types [14e16] and is implicated in resistance to apoptosis induction by anticancer agents and ionizing radiation [17,18]. It has been demonstrated that down-regulation of survivin expression using conventional antisense or siRNA facilitated cancer cell apoptosis and sensitized cells to anti-cancer agents [19e25]. Studies have elucidated tamoxifen-induced growth inhibition and/or apoptosis in the ER-a-negative breast carcinoma cell line [26] as well as in the ER-a-negative human hepatoma cell line Hep3B [10] was related to the suppression of PKCa activity. This enzyme is involved in regulation of survivin expression [27,28]. Therefore, the inhibition of proliferation and induction of apoptosis of tamoxifen may, at least in part, be mediated by down-regulation of survivin expression. To investigate the ER-a-independent anti-proliferative effect of tamoxifen in HCC, we design cell culture studies using the human hepatoma cell line HepG2, which does not express ER-a [29] and determine the effects of tamoxifen on proliferation, cell cycle, apoptosis, and survivin expression in vitro. Meanwhile, the role of survivin in mediating the antiproliferative effect of tamoxifen in HepG2 cells is evaluated too. 2. Materials and methods 2.1. Cell cultures HepG2 cells (obtained from American Type Culture Collection) were cultured in RPMI-1640 media (Gibco/BRL, USA) containing 10% (w/v) fetal bovine serum, 100 ml/ml penicillin and 100 mg/l streptomycin at 37 C in a humidified atmosphere with 5% CO2 and 95% air. Subcultures were made by trypsinization and reseeded for experiments. Tamoxifen (Sigma, USA) (0.1, 1, 10, 20 mM) was administered and cell culture was continued for 24, 48 and 72 h, medium was exchanged daily. 2.2. Proliferation assay Effects on cellular proliferation were determined using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) staining method. Cells in the logarithmic growth phase were plated in a 96-well plate and incubated for 24 h in the fresh media. Then, they were treated with tamoxifen at various concentrations and times. At 4 h prior to the end of incubation, 20 ml MTT was added to each well, and 150 ml DMSO was added to stop the reaction. The optical density (OD) of each well was measured using a microculture plate reader with a test wavelength of 492 nm. The cell inhibition rate (IR) was calculated using the following equation: IR ¼ (1 OD value in the treated samples/OD value in the control samples) 100%.
2.3. Cell cycle phase analysis Cell cycle phase distribution was determined by flow cytometry. After treatment, cells were trypsinized, washed, and collected. And then, cells were fixed and stained with propidium iodide as described. Samples were analyzed by flow cytometry. At least 10,000 events were evaluated. Cell cycle phase distribution was determined using Modfit Software (Verity Software House, Topsham, ME) to analyze DNA content histograms. 2.4. Apoptosis assay After HepG2 cells had been exposed to tamoxifen for 24 or 48 and 72 h, cells were washed twice with ice-cold PBS and incubated with Annexin-VeFITC and propidium iodide according to the manufacturer’s protocol. After 15 min of incubation at room temperature in the dark, cells were resuspended in 1.0 ml incubation buffer and analyzed using a flow cytometer (CellQuest Software, Becton Dickinson). 2.5. Western blot analysis The harvested cells were washed twice in ice-cold PBS, lysed in lysis buffer at 0 C for 10 min, and centrifuged. The total protein concentration of the supernatants was determined by using micro bicinchoninic acid assay (Pierce, Rockford, IL). The lysates containing 60 mg proteins were boiled for 10 min with SDS sample buffer and then separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to cellulose nitrate membranes. Membranes were then blotted for 1 h at room temperature and then incubated with 1:1000 mouse anti-survivin antibody (Cell Signaling, USA) for overnight at 4 C. They were then probed with horseradish peroxidase-conjugated anti-mouse IgG for 1 h at room temperature. Immunoreactive bands were visualized by the enhanced chemiluminescence system (Santa Cruz, CA). GAPDH was used as an internal control. 2.6. Statistical assay Data were expressed as means standard deviation from three independent experiments. Comparisons among groups were performed with analysis of variance. A value of p < 0.05 was considered statistically significant. Statistical tests were performed using SAS 9.13 (SAS Institute, Cary, NC, USA). 3. Results 3.1. Effect on proliferation of HepG2 cells with tamoxifen Tamoxifen reduced proliferation of HepG2 cells in concentration- and time-dependent manner (Fig. 1). A 72-h time response curves are shown at various concentration of tamoxifen. Fig. 1 depicts a concentration-dependent decrease in proliferation in the presence of increasing concentrations of
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3.3. Effect on apoptosis in HepG2 cells with tamoxifen Fig. 2 shows the relative level of apoptosis in HepG2 cells treatmented with tamoxifen. An increase of apoptosis occurred in the treatment of 20 mM of tamoxifen in HepG2 cells. When HepG2 cells were incubated for 24 h, the relative level of apoptosis was still low, it was approximately seven-fold increase when the incubation time was prolonged to 72 h. 3.4. Effects of survivin expression with tamoxifen
Fig. 1. The effect of tamoxifen on proliferation of HepG2 cells. HepG2 cells were treated for 24e72 h with varying concentrations of tamoxifen. Proliferation assay was used to determine growth relative to control cells. Data are expressed as percentage of control. Values are means SD of three independent experiments in duplicate. *, Difference to control, p < 0.05. **, Significantly difference to control, p < 0.01. Similar results were obtained in at least two independent experiments.
tamoxifen. When tamoxifen treatment at a concentration of 1 mM, cell proliferation was slightly affected. However, at concentrations of 20 mM, tamoxifen induced a reduction of cell proliferation significantly. The decrease of cell proliferation started with a delay and was only transient when 10 mM tamoxifen was used, and the progression in cell proliferation inhibition was stopped, if tamoxifen was removed from the medium after 24 h treatment (data not shown). However, cell proliferation was completely exhausted during incubation in the presence of 20 mM tamoxifen. 3.2. Effect on cell cycle phase distribution in HepG2 cells with tamoxifen The cell cycle phase distribution was examined by flow cytometry after tamoxifen treatment and shown in Table 1. HCC cells were treated with tamoxifen for 48 h in different concentration, then were stained with propidium iodide and analyzed by flow cytometry. Tamoxifen treatment increased the percentage of cells in the Gap0/Gap1 (G0/G1) phase from 48.5% to 55%. Concomitant with this increase in the percentage of cells in the G0/G1 phase was a decrease in the percentage of cells in the synthesis (S) phase from 21.4% to 13.7%.
To assess the mechanism of cytostatic and cytotoxic action of tamoxifen in HepG2 cells, the effect of the drug on survivin expression was evaluated. There were no changes of survivin protein in 0.1, 1 and 10 mM, while 20 mM tamoxifen treatment resulted in reduction of survivin protein compared with that in controls (media) (Fig. 3). The expression of survivin protein was significantly reduced following treatment with tamoxifen at 20 mM for 24 h, a further reduction when the incubation was continued for 48 and 72 h. 4. Discussion This study examined the effects of tamoxifen on proliferation, cell cycle, apoptosis in human HCC in vitro, the results suggested tamoxifen to be more potent in HCC cells. Previous trials have been reviewed which evaluated the effect of tamoxifen on survival in advanced inoperable stages of HCC, the results were shown that in the case of no therapeutic response, the tamoxifen levels were not high enough to activate the ER-a-independent pathways [30]. Tamoxifen caused the significant proliferation inhibition of HepG2 cells during 72 h incubation in the presence of tamoxifen at 10 or 20 mM, but not at 0.1 and 1 mM. The findings are in accordance with proliferation studies on HepG2 cells, as reported previously [29]. As observed in other ER-anegative tumor cell lines [29], the anti-proliferative effect of tamoxifen on HepG2 cells occurs at micromolar concentrations. Whereas for ER-a-positive breast cancer cells, tamoxifen reduces cell growth in the nanomolar range [31]. The ER-a-independent effects on proliferation observed in the
Table 1 Cell cycle phase distribution of HepG2 cells after tamoxifen treatment for 24 h. Treatment
G0/G1 [%]
S [%]
G2/M [%]
Control 0.1 mM 1 mM 10 mM 20 mM
48.5 48.6 49.1 51.3 55
21.4 21.2 20.3 19.2 13.7
30.1 30.2 29.6 29.5 31.3
Values from a representative experiment are shown, and similar results were obtained in at least two independent experiments. G, gap; M, mitosis; and S, synthesis.
Fig. 2. Effect of tamoxifen on induction of apoptosis in HepG2 cells. Cells were treated with the different concentration of tamoxifen (0.1e20 mM) for 24e72 h. Bars represent means SD of three independent experiments in duplicate. *, Difference to control, p < 0.05. **, Significantly difference to control, p < 0.01. Similar results were obtained in at least two independent experiments.
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Fig. 3. Detection of survivin protein level from cells. HepG2 cells were treated with tamoxifen (0.1e20 mM), and cell lysates were prepared after 24, 48 and 72 h. Western blot analysis was then performed. Similar results were obtained in at least two independent Western blots.
hepatoblastoma cell lines may be clinically relevant, since the required concentrations (up to 20 mM) are achievable in the liver tissue of patients during steady-state tamoxifen treatment [32]. In various ER-a-negative cancers, tamoxifen has been shown to have therapeutic activity at doses approximately 4e 8-fold above those used for ER inhibition [1]. Our findings in hepatoma cell lines support the hypothesis that patients benefit from an enhancement of the tamoxifen dose in case of HCC. In current studies, we found that 10 mM of tamoxifen showed a decrease of cell proliferation with a delay and stopping during incubation for more than 48 h. The latter effect may display a recovery of growth rate in low drug potency. Recovery of growth rate may be due to the cells acquiring tamoxifen resistance, which has been shown previously for breast cancer cell lines [33]. We also found that 20 mM of tamoxifen induced a complete exhaustion of cell proliferation. The findings are in accordance with observations of Farinati [34]. The author concluded from clinical results that patients with HCC who enter the phase of cancer progression after an initial response to tamoxifen may respond again following an increase in dosage. FACS analysis indicated that the decrease of cell proliferation is basically related to an increase of cells in the G0/1-phase, suggesting an arrest of cells in this phase (Table 1). We suppose that reduction of proliferation may be attributed to this effect. Induction of apoptosis occurs in addition to cytostatic effects when the tamoxifen acts at 20 mM. The apoptosis increases with a delay (after 48 h incubation) and augments substantially the effect of tamoxifen on cell proliferation. Our results showed that tamoxifen reduced the expression of survivin protein in ER-independent pathway at 20 mM of drug potency. When the expression of survivin reduced, cell proliferation also reduced, while the relative apoptosis was still unchanged, suggesting cytostatic actions of tamoxifen. This observations show the survivin expression is not related to cytostatic effects of the drug, since at lower drug potency, similar cytostatic actions were observed without changes in survivin expression. Since both reduction of survivin expression and induction of apoptosis occurred exclusively when the drug potency was higher, the relationship between both effects was considered. We supposed that the tamoxifen-induced apoptosis is mediated by the down-regulation of survivin expression. We observed that down-regulation of survivin expression in HepG2 cells during tamoxifen treatment clearly preceded the occurrence of apoptosis. When a significant reduction of survivin expression was detectable (following 24 h treatment), the
relative apoptosis was still unchanged and the progression of cell proliferation reduction could still be stopped by withdrawal of the drug, suggesting that even an initiation of apoptotic pathways had not taken place this time. These results support the hypothesis that the suppression of survivin expression is a primary effect of tamoxifen at higher potency, resulting in apoptosis. Further experiment was progressed to examine the effects of siRNA of survivin on tamoxifeninduced apoptosis. In summary, our experimental results demonstrate that tamoxifen (10 mM) reduces proliferation in ER-a-negative HepG2 cells in concentration-dependent manner, the reduction of cell proliferation is only transitory and may be due to cytostatic effects, which are augmented by inducing apoptosis. A significant down-regulation of survivin expression precedes apoptosis, which suggested that the cytotoxic effects of tamoxifen are mediated by a suppression of survivin expression. Acknowledgment We thank Dr. P.C. Xun for excellent statistical assistance and generous help in the review of the manuscript. References [1] Gelmann E. Tamoxifen for the treatment of malignancies other than breast and endometrial carcinoma. Semin Oncol 1997;24:165e70. [2] Elba S, Giannuzzi V, Misciagna G, Manghisi OG. Randomized controlled trial of tamoxifen versus placebo in inoperable hepatocellular carcinoma. Ital J Gastroenterol 1994;26:66e8. [3] Martinez Cerezo FJ, Tomas A, Donoso L, Enriquez J, Guarner C, Balanzo J, et al. Controlled trial of tamoxifen in patients with advanced hepatocellular carcinoma. J Hepatol 1994;20:702e6. [4] Boix L, Bruix J, Castells A, Fuster J, Bru C, Visa J, et al. Sex hormone receptors in hepatocellular carcinoma. Is there a rationale for hormonal treatment? J Hepatol 1993;17:187e91. [5] Villa E, Camellini L, Dugani A, Zucchi F, Grottola A, Merighi A, et al. Variant estrogen receptor messenger RNA species detected in human primary hepatocellular carcinoma. Cancer Res 1995;55:498e500. [6] Villa E, Camellini L, Dugani A, Buttafoco P, Grottola A, Manenti F. Variant liver estrogen receptors and response to tamoxifen [letter]. Gastroenterology 1996;111:271e2. [7] Mandlekar S, Kong AN. Mechanisms of tamoxifen-induced apoptosis. Apoptosis 2001;6:469e77. [8] Kim JA, Young SK, Jung MW, Lee SH, Yong SL. Involvement of Ca2þ influx in the mechanism of tamoxifen-induced apoptosis in HepG2 human hepatoblastoma cells. Cancer Lett 1999;147:115e23.
R. Guo et al. / Biomedicine & Pharmacotherapy 63 (2009) 375e379 [9] Lee YS, Kang YS, Lee SH. Role of NADPH oxidase in tamoxifeninduced generation of reactive oxygen species and apoptosis in HepG2 human hepatoblastoma cells. Cell Death Differ 2000;7:925e32. [10] Cheng AL, Chuang SE, Fine RL, Yeh KH, Liao CM, Lay JD, et al. Inhibition of the membrane translocation and activation of protein kinase C, and potentiation of doxorubicin-induced apoptosis of hepatocellular carcinoma cells by tamoxifen. Biochem Pharmacol 1998;55:523e31. [11] Tamm I, Wang Y, Sausville E, Scudiero DA, Vigna N, Oltersdorf T, et al. IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas [CD95], Bax, caspases, and anticancer drugs. Cancer Res 1998;58:5315e20. [12] Li F, Ambrosini G, Chu EY, Plescia J, Tognin S, Marchisio PC, et al. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 1998;396:580e4. [13] Ambrosini G, Adida C, Sirugo G, Altieri DC. Induction of apoptosis and inhibition of cell proliferation by survivin gene targeting. J Biol Chem 1998;273:11177e82. [14] Monzo M, Rosell R, Felip E, Astudillo J, Sanchez JJ, Maestre J, et al. A novel anti-apoptosis gene: reexpression of survivin messenger RNA as a prognosis marker in non small cell lung cancers. J Clin Oncol 1999;17:2100e4. [15] Kawasaki H, Altieri DC, Lu CD, Toyoda M, Tenjo T, Tanigawa N. Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer. Cancer Res 1998;58:5071e4. [16] Nakagawara A. Molecular basis of spontaneous regression of neuroblastoma: role of neurotrophic signals and genetic abnormalities. Hum Cell 1998;11:115e24. [17] Zaffaroni N, Daidone MG. Survivin expression and resistance to anticancer treatments: perspectives for new therapeutic interventions. Drug Resist Updat 2002;5:65e72. [18] Asanuma K, Moriai R, Yajima T, Yagihashi A, Yamada M, Kobayashi D, et al. Survivin as a radioresistance factor in pancreatic cancer. Jpn J Cancer Res 2000;91:1204e9. [19] Olie RA, Simoes-Wust AP, Baumann B, Leech SH, Fabbro D, Stahel RA, et al. A novel antisense oligonucleotide targeting survivin expression induces apoptosis and sensitizes lung cancer cells to chemotherapy. Cancer Res 2000;60:2805e9. [20] Carvalho A, Carmena M, Sambade C, Earnshaw WC, Wheatley SP. Survivin is required for stable checkpoint activation in taxol-treated HeLa cells. J Cell Sci 2003;116:2987e98. [21] Daly C, Wong V, Burova E, Wei Y, Zabski S, Griffiths J, et al. Angiopoietin-1 modulates endothelial cell function and gene expression via the transcription factor FKHR [FOXO1]. Genes Dev 2004;18:1060e71.
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[22] Ling X, Bernacki RJ, Brattain MG, Li F. Induction of survivin expression by taxol (paclitaxel) is an early event, which is independent of taxolmediated G2/M arrest. J Biol Chem 2004;279:15196e203. [23] Song H, Xin XY, Xiao F, Wang DT, Yue QH, Han X. Survivin gene RNA interference inhibits proliferation, induces apoptosis, and enhances radiosensitivity in HeLa cells. Eur J Obstet Gynecol Reprod Biol 2008; 136:83e9. [24] Ma X, Wang S, Zhou J, Xing H, Xu G, Wang B, et al. Induction of apoptosis in human ovarian epithelial cancer cells by antisurvivin oligonucleotides. Oncol Rep 2005;14:275e9. [25] Cheng SQ, Wang WL, Yan W, Li QL, Wang L, Wang WY. Knockdown of survivin gene expression by RNAi induces apoptosis in human hepatocellular carcinoma cell line SMMC-7721. World J Gastroenterol 2005;11:756e9. [26] Gundimeda U, Chen ZH, Gopalakrishna R. Tamoxifen modulates protein kinase C via oxidative stress in estrogen receptor-negative breast cancer cells. J Biol Chem 1996;271:13504e14. [27] Xiao D, Wang K, Zhou J, Cao H, Deng Z, Hu Y, et al. Inhibition of fibroblast growth factor 2-induced apoptosis involves survivin expression, protein kinase C alpha activation and subcellular translocation of Smac in human small cell lung cancer cells. Acta Biochim Biophys Sin 2008;40:297e303. [28] Szla´vik V, Va´g J, Marko´ K, Demeter K, Madara´sz E, Ola´h I, et al. Matrigel-induced acinar differentiation is followed by apoptosis in HSG cells. J Cell Biochem 2008;103:284e95. [29] Jiang SY, Shyu RY, Yeh MY, Jordan VC. Tamoxifen inhibits hepatoma cell growth through an estrogen receptor independent mechanism. J Hepatol 1995;23:712e9. [30] Tan CK, Chow PK, Findlay M, Wong C, Machin D. Use of tamoxifen in hepatocellular carcinoma: a review and paradigm shift. J Gastroenterol Hepatol 2000;15:725e9. [31] Taylor CM, Blanchard B, Zava DT. Estrogen receptormediated and cytotoxic effects of the antiestrogens tamoxifen and 4-hydroxytamoxifen. Cancer Res 1984;44:1409e14. [32] Lien EA, Sohlheim E, Ueland PM. Distribution of tamoxifen and its metabolites in rat and human tissues during steady-state treatment. Cancer Res 1991;51:4837e44. [33] Johnston SR. Acquired tamoxifen resistance in human breast cancer e potential mechanisms and clinical implications. Anticancer Drugs 1997; 8:911e30. [34] Farinati F. Tamoxifen treatment in hepatocellular carcinoma. Gastroenterology 1996;111:272e4.
www.sciencedirect.com Biomedicine & Pharmacotherapy 63 (2009) 375e379
Original article
Tamoxifen inhibits proliferation and induces apoptosis in human hepatocellular carcinoma cell line HepG2 via down-regulation of survivin expression Renhua Guo a,*, Zuhu Huang b, Yongqian Shu a, Shidai Jin a, Hongmei Ge c a
Department of Oncology, First Affiliated Hospital of Nanjing Medical University, Guangzhou Road 300, Nanjing 210029, PR China b Department of Infectious Disease, First Affiliated Hospital of Nanjing Medical University, Nanjing, PR China c Laboratory of Molecular Biology, First Affiliated Hospital of Nanjing Medical University, Nanjing, PR China Received 20 August 2008; accepted 26 September 2008 Available online 23 October 2008
Abstract Tamoxifen has been used in patients with hepatocellular carcinoma (HCC). However, its inhibitory mechanism remains unknown. In current study, we evaluated the effect of tamoxifen on the estrogen receptor-a-negative HCC cell proliferation, apoptosis and expression of survivin which had been known to play an important role in promotion of cellular proliferation as well as inhibition of apoptosis in cancer cells. HepG2 cells were incubated with tamoxifen (0.1, 1, 10, or 20 mM) for up to 72 h. Cell proliferation was assessed, flow cytometric analysis was performed, and survivin expression was detected. Our results are showed as follows. Ten or 20 mM tamoxifen induced a reduction of cell proliferation. Basically reduction of proliferation was related to an increase in the fraction of G0/1-phase. When tamoxifen was administrated at higher concentration (20 mM), the increase of the relative apoptosis appeared with a delay, augmenting the effect of tamoxifen on cell proliferation. When apoptosis was induced, a significant depression of survivin expression preceded. In conclusion, the tamoxifen decreasing cell proliferation and induction of apoptosis of HepG2 cells depends on drug concentration, which is due to cytostatic and cytocide effects, the latter may be mediated by a down-regulation of survivin expression. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Estrogen receptor; Survivin; Apoptosis
1. Introduction Tamoxifen is a non-steroidal anti-estrogen which is commonly used in adjuvant treatment as well as in the firstline treatment of postmenopausal breast cancer. Furthermore, tamoxifen has been used in other malignant tumors, such as hepatocellular carcinoma (HCC) [1]. Tamoxifen treatment of advanced, inoperable HCC has been shown to prolong survival
Abbreviations: HCC, hepatocellular carcinoma; ER, estrogen receptor; PKC, protein kinase C; G0/G1, Gap0/Gap1; S, synthesis; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide. * Corresponding author. Tel.: þ86 25 83718836 6714; fax: þ86 25 83724440. E-mail address: [email protected] (R. Guo). 0753-3322/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2008.09.010
of some patients [2,3]. However, those estrogen receptor-alpha (ER-a)-negative [4], or variant ER-as in ER-a-positive [5] are insensitive to tamoxifen [6]. Therefore, the effects of tamoxifen in HCC are more likely ER-a-independent pathway than dependent pathway. The effects of tamoxifen in tumor cells by ER-a-independent pathways have been reported [7]. Anti-proliferation action of tamoxifen has been reported to be associated with an increase of Ca2þ influx [8], and an enhanced generation of reactive oxygen species [9], or an inhibition of protein kinase C activity [10]. But so far, the role of survivin in mediating tamoxifen effect in hepatoma cells remains unknown. Survivin, a member of inhibitor of apoptosis protein family, is expressed during the G2/M phase. It directly inhibits the terminal effector cell-death proteases caspase-3 and caspase-7
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[11]. It binds to the microtubules of the mitotic spindle, where it counteracts the induction of apoptosis [12]. Moreover, experimental data have shown survivin in promotion of cellular proliferation as well as inhibition of apoptosis in cancer cells [13]. Survivin overexpression has been identified as a negative prognostic factor in various cancer types [14e16] and is implicated in resistance to apoptosis induction by anticancer agents and ionizing radiation [17,18]. It has been demonstrated that down-regulation of survivin expression using conventional antisense or siRNA facilitated cancer cell apoptosis and sensitized cells to anti-cancer agents [19e25]. Studies have elucidated tamoxifen-induced growth inhibition and/or apoptosis in the ER-a-negative breast carcinoma cell line [26] as well as in the ER-a-negative human hepatoma cell line Hep3B [10] was related to the suppression of PKCa activity. This enzyme is involved in regulation of survivin expression [27,28]. Therefore, the inhibition of proliferation and induction of apoptosis of tamoxifen may, at least in part, be mediated by down-regulation of survivin expression. To investigate the ER-a-independent anti-proliferative effect of tamoxifen in HCC, we design cell culture studies using the human hepatoma cell line HepG2, which does not express ER-a [29] and determine the effects of tamoxifen on proliferation, cell cycle, apoptosis, and survivin expression in vitro. Meanwhile, the role of survivin in mediating the antiproliferative effect of tamoxifen in HepG2 cells is evaluated too. 2. Materials and methods 2.1. Cell cultures HepG2 cells (obtained from American Type Culture Collection) were cultured in RPMI-1640 media (Gibco/BRL, USA) containing 10% (w/v) fetal bovine serum, 100 ml/ml penicillin and 100 mg/l streptomycin at 37 C in a humidified atmosphere with 5% CO2 and 95% air. Subcultures were made by trypsinization and reseeded for experiments. Tamoxifen (Sigma, USA) (0.1, 1, 10, 20 mM) was administered and cell culture was continued for 24, 48 and 72 h, medium was exchanged daily. 2.2. Proliferation assay Effects on cellular proliferation were determined using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) staining method. Cells in the logarithmic growth phase were plated in a 96-well plate and incubated for 24 h in the fresh media. Then, they were treated with tamoxifen at various concentrations and times. At 4 h prior to the end of incubation, 20 ml MTT was added to each well, and 150 ml DMSO was added to stop the reaction. The optical density (OD) of each well was measured using a microculture plate reader with a test wavelength of 492 nm. The cell inhibition rate (IR) was calculated using the following equation: IR ¼ (1 OD value in the treated samples/OD value in the control samples) 100%.
2.3. Cell cycle phase analysis Cell cycle phase distribution was determined by flow cytometry. After treatment, cells were trypsinized, washed, and collected. And then, cells were fixed and stained with propidium iodide as described. Samples were analyzed by flow cytometry. At least 10,000 events were evaluated. Cell cycle phase distribution was determined using Modfit Software (Verity Software House, Topsham, ME) to analyze DNA content histograms. 2.4. Apoptosis assay After HepG2 cells had been exposed to tamoxifen for 24 or 48 and 72 h, cells were washed twice with ice-cold PBS and incubated with Annexin-VeFITC and propidium iodide according to the manufacturer’s protocol. After 15 min of incubation at room temperature in the dark, cells were resuspended in 1.0 ml incubation buffer and analyzed using a flow cytometer (CellQuest Software, Becton Dickinson). 2.5. Western blot analysis The harvested cells were washed twice in ice-cold PBS, lysed in lysis buffer at 0 C for 10 min, and centrifuged. The total protein concentration of the supernatants was determined by using micro bicinchoninic acid assay (Pierce, Rockford, IL). The lysates containing 60 mg proteins were boiled for 10 min with SDS sample buffer and then separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to cellulose nitrate membranes. Membranes were then blotted for 1 h at room temperature and then incubated with 1:1000 mouse anti-survivin antibody (Cell Signaling, USA) for overnight at 4 C. They were then probed with horseradish peroxidase-conjugated anti-mouse IgG for 1 h at room temperature. Immunoreactive bands were visualized by the enhanced chemiluminescence system (Santa Cruz, CA). GAPDH was used as an internal control. 2.6. Statistical assay Data were expressed as means standard deviation from three independent experiments. Comparisons among groups were performed with analysis of variance. A value of p < 0.05 was considered statistically significant. Statistical tests were performed using SAS 9.13 (SAS Institute, Cary, NC, USA). 3. Results 3.1. Effect on proliferation of HepG2 cells with tamoxifen Tamoxifen reduced proliferation of HepG2 cells in concentration- and time-dependent manner (Fig. 1). A 72-h time response curves are shown at various concentration of tamoxifen. Fig. 1 depicts a concentration-dependent decrease in proliferation in the presence of increasing concentrations of
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3.3. Effect on apoptosis in HepG2 cells with tamoxifen Fig. 2 shows the relative level of apoptosis in HepG2 cells treatmented with tamoxifen. An increase of apoptosis occurred in the treatment of 20 mM of tamoxifen in HepG2 cells. When HepG2 cells were incubated for 24 h, the relative level of apoptosis was still low, it was approximately seven-fold increase when the incubation time was prolonged to 72 h. 3.4. Effects of survivin expression with tamoxifen
Fig. 1. The effect of tamoxifen on proliferation of HepG2 cells. HepG2 cells were treated for 24e72 h with varying concentrations of tamoxifen. Proliferation assay was used to determine growth relative to control cells. Data are expressed as percentage of control. Values are means SD of three independent experiments in duplicate. *, Difference to control, p < 0.05. **, Significantly difference to control, p < 0.01. Similar results were obtained in at least two independent experiments.
tamoxifen. When tamoxifen treatment at a concentration of 1 mM, cell proliferation was slightly affected. However, at concentrations of 20 mM, tamoxifen induced a reduction of cell proliferation significantly. The decrease of cell proliferation started with a delay and was only transient when 10 mM tamoxifen was used, and the progression in cell proliferation inhibition was stopped, if tamoxifen was removed from the medium after 24 h treatment (data not shown). However, cell proliferation was completely exhausted during incubation in the presence of 20 mM tamoxifen. 3.2. Effect on cell cycle phase distribution in HepG2 cells with tamoxifen The cell cycle phase distribution was examined by flow cytometry after tamoxifen treatment and shown in Table 1. HCC cells were treated with tamoxifen for 48 h in different concentration, then were stained with propidium iodide and analyzed by flow cytometry. Tamoxifen treatment increased the percentage of cells in the Gap0/Gap1 (G0/G1) phase from 48.5% to 55%. Concomitant with this increase in the percentage of cells in the G0/G1 phase was a decrease in the percentage of cells in the synthesis (S) phase from 21.4% to 13.7%.
To assess the mechanism of cytostatic and cytotoxic action of tamoxifen in HepG2 cells, the effect of the drug on survivin expression was evaluated. There were no changes of survivin protein in 0.1, 1 and 10 mM, while 20 mM tamoxifen treatment resulted in reduction of survivin protein compared with that in controls (media) (Fig. 3). The expression of survivin protein was significantly reduced following treatment with tamoxifen at 20 mM for 24 h, a further reduction when the incubation was continued for 48 and 72 h. 4. Discussion This study examined the effects of tamoxifen on proliferation, cell cycle, apoptosis in human HCC in vitro, the results suggested tamoxifen to be more potent in HCC cells. Previous trials have been reviewed which evaluated the effect of tamoxifen on survival in advanced inoperable stages of HCC, the results were shown that in the case of no therapeutic response, the tamoxifen levels were not high enough to activate the ER-a-independent pathways [30]. Tamoxifen caused the significant proliferation inhibition of HepG2 cells during 72 h incubation in the presence of tamoxifen at 10 or 20 mM, but not at 0.1 and 1 mM. The findings are in accordance with proliferation studies on HepG2 cells, as reported previously [29]. As observed in other ER-anegative tumor cell lines [29], the anti-proliferative effect of tamoxifen on HepG2 cells occurs at micromolar concentrations. Whereas for ER-a-positive breast cancer cells, tamoxifen reduces cell growth in the nanomolar range [31]. The ER-a-independent effects on proliferation observed in the
Table 1 Cell cycle phase distribution of HepG2 cells after tamoxifen treatment for 24 h. Treatment
G0/G1 [%]
S [%]
G2/M [%]
Control 0.1 mM 1 mM 10 mM 20 mM
48.5 48.6 49.1 51.3 55
21.4 21.2 20.3 19.2 13.7
30.1 30.2 29.6 29.5 31.3
Values from a representative experiment are shown, and similar results were obtained in at least two independent experiments. G, gap; M, mitosis; and S, synthesis.
Fig. 2. Effect of tamoxifen on induction of apoptosis in HepG2 cells. Cells were treated with the different concentration of tamoxifen (0.1e20 mM) for 24e72 h. Bars represent means SD of three independent experiments in duplicate. *, Difference to control, p < 0.05. **, Significantly difference to control, p < 0.01. Similar results were obtained in at least two independent experiments.
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Fig. 3. Detection of survivin protein level from cells. HepG2 cells were treated with tamoxifen (0.1e20 mM), and cell lysates were prepared after 24, 48 and 72 h. Western blot analysis was then performed. Similar results were obtained in at least two independent Western blots.
hepatoblastoma cell lines may be clinically relevant, since the required concentrations (up to 20 mM) are achievable in the liver tissue of patients during steady-state tamoxifen treatment [32]. In various ER-a-negative cancers, tamoxifen has been shown to have therapeutic activity at doses approximately 4e 8-fold above those used for ER inhibition [1]. Our findings in hepatoma cell lines support the hypothesis that patients benefit from an enhancement of the tamoxifen dose in case of HCC. In current studies, we found that 10 mM of tamoxifen showed a decrease of cell proliferation with a delay and stopping during incubation for more than 48 h. The latter effect may display a recovery of growth rate in low drug potency. Recovery of growth rate may be due to the cells acquiring tamoxifen resistance, which has been shown previously for breast cancer cell lines [33]. We also found that 20 mM of tamoxifen induced a complete exhaustion of cell proliferation. The findings are in accordance with observations of Farinati [34]. The author concluded from clinical results that patients with HCC who enter the phase of cancer progression after an initial response to tamoxifen may respond again following an increase in dosage. FACS analysis indicated that the decrease of cell proliferation is basically related to an increase of cells in the G0/1-phase, suggesting an arrest of cells in this phase (Table 1). We suppose that reduction of proliferation may be attributed to this effect. Induction of apoptosis occurs in addition to cytostatic effects when the tamoxifen acts at 20 mM. The apoptosis increases with a delay (after 48 h incubation) and augments substantially the effect of tamoxifen on cell proliferation. Our results showed that tamoxifen reduced the expression of survivin protein in ER-independent pathway at 20 mM of drug potency. When the expression of survivin reduced, cell proliferation also reduced, while the relative apoptosis was still unchanged, suggesting cytostatic actions of tamoxifen. This observations show the survivin expression is not related to cytostatic effects of the drug, since at lower drug potency, similar cytostatic actions were observed without changes in survivin expression. Since both reduction of survivin expression and induction of apoptosis occurred exclusively when the drug potency was higher, the relationship between both effects was considered. We supposed that the tamoxifen-induced apoptosis is mediated by the down-regulation of survivin expression. We observed that down-regulation of survivin expression in HepG2 cells during tamoxifen treatment clearly preceded the occurrence of apoptosis. When a significant reduction of survivin expression was detectable (following 24 h treatment), the
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