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Involvement of mTOR and survivin inhibition in tamoxifen-induced apoptosis in human hepatoblastoma cell line HepG2
Biomedicine & Pharmacotherapy 64 (2010) 249–253
Original article
Involvement of mTOR and survivin inhibition in tamoxifen-induced apoptosis in human hepatoblastoma cell line HepG2 Renhua Guo a, Tongshan Wang a, Hua Shen a, Hong-mei Ge b, Jing Sun a, Zu-hu Huang c,1,*, Yong-qian Shu a,1,* a b c
Department of oncology, First Affiliated Hospital of Nanjing Medical University, Nanjing Guangzhou road 300, Nanjing 210029, China Laboratory of molecular biology, First Affiliated Hospital of Nanjing Medical University, Nanjing Guangzhou road 300, Nanjing 210029, China Department of Infectious disease, First Affiliated Hospital of Nanjing Medical University, Nanjing Guangzhou road 300, Nanjing 210029, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 May 2009 Accepted 7 June 2009 Available online 22 October 2009
Patients with advanced hepatocellular carcinoma (HCC) have shown to benefit from tamoxifen treatment. The mechanisms of tamoxifen effects in HCC, however, are not yet clearly understood. The PI3K/Akt/mTOR signal pathway is involved in cell proliferation, tumorigenesis, and apoptosis. Overexpression of survivin has played an important role in leading to antiapoptosis. The current study investigated changes in mTOR pathway and survivin expression in hepatocarcinoma cell line HepG2 treated with tamoxifen. We detected apoptosis of hepatocarcinoma cells by flow cytometry assay. Survivin transcription level and p70S6k was demonstrated by PCR, dual-luciferase reporter assay and western blot analysis respectively. Our results are showed as follows: tamoxifen leads to apoptosis of the cells, and reduction in survivin expression, as well as a dramatic reduction in the activated form of p70S6 kinase. Twenty micromoles tamoxifen treatment signifcantly depresses transcription of survivin mRNA. Treating HepG2 cells with rapamycin, a specific mTOR inhibitor, significantly reduce survivin protein level but did not affect survivin transcription, which indicated that tamoxifen and rapamycin were synergistic in regards to down-regulation of survivin expression in hepatocellular carcinoma cells. Our results suggest that tamoxifen down-regulate survivin expression in HepG2 cells is mediated by transcriptional and posttranscriptional level via PI3K/Akt/mTOR pathway to induce apoptosis. ß 2009 Elsevier Masson SAS. All rights reserved.
Keywords: Tamoxifen HepG2 cells Apoptosis Survivin mTOR
1. Introduction The liver is a hormone-sensitive organ, several lines of evidence suggest that sex hormones and their receptors may play a role in liver carcinogenesis [1]. Tamoxifen is a non-steroidal anti-estrogen that has been used in the treatment of breast cancer for >25 years [2] and lately as a long-term chemopreventive agent for breast cancer in healthy women at high risk of developing breast cancer [2,3]. Furthermore, tamoxifen has been used in other malignant tumors, such as hepatocellular carcinoma [4] and shown to prolong survival of some patients in advanced, inoperable HCC [5,6]. Cell culture experiments on the human hepatoblastoma cell lines Hep3B and HepG2 have shown that tamoxifen at concentra-
Abbreviations: HCC, hepatocellular carcinoma; ER, estrogen receptor; PI3K, phosphatidylinositol-3-kinase; mTOR, mammalian target of rapamycin. * Corresponding author. Tel.: +8625 83718836 6714; fax: +8625 83724440. E-mail addresses: [email protected], [email protected] (Z.-h. Huang), [email protected] (Y.-q. Shu). 1 These two authors contribute equally to this article. 0753-3322/$ – see front matter ß 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2009.06.007
tions in the nanomolar range induces growth inhibition and/or apoptotic cell death via an ER-a-independent mechanism [7,8]. This is in line with the low ER-a-expression in most HCC tissues. The apoptosis effects of tamoxifen on HepG2 cells are at least in part mediated by a down-regulation of survivin expression [9]. The PI3K/Akt pathway has been implicated in tumorigenesis [10]. PI3K activates a number of signaling molecules, among which the Akt/mTOR pathway is of particular interest because of its role in inhibiting apoptosis and promoting cell proliferation [11]. mTOR is a 289-kDa serine/threonine kinase that is a downstream target of Akt [12]. It has been shown to regulate mitogen stimulated protein synthesis and cell cycle progression [13–15]. Cell culture studies have demonstrated that one of the mechanisms by which mTOR controls protein synthesis is through phosphorylating downstream substrates, including p70s6 kinase (p70S6K1) and eukaryotic initiation factor (eIF) 4E binding protein 1 (4E-BP1) [16–18]. Therefore, by inhibiting mTOR, rapamycin causes a decrease in phosphorylation of these effectors, and a decrease in protein synthesis, effectively blocking the progrowth, pro-proliferative, and prosurvival actions of mTOR [19].
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Survivin, a member of the inhibitor of apoptosis proteins (IAP) family [20,21] is not present in most normal adult differentiated tissues. Its mRNA and the protein are present in large amounts in fetal tissue and most cancers [22,23]. Survivin protein suppresses apoptosis and stimulates cell division [24]. Our data have shown PI3K/Akt pathway regulating the expression of survivin in HCC cells [25]. Since inhibiting mTOR activity leads to cancer cell death while over-expression of survivin is associated with anti-apoptosis, it is possible that mTOR may directly or indirectly regulate the expression of survivin. In the current study, we investigate the pathway of the tamoxifen-induced down-regulation of survivin in HepG2 cells, which does not express ER-a [7]. We show here that in HepG2 cells tamoxifen could down-regulate both mTOR and survivin expression, and inhibiting mTOR leads to further downregulation of survivin expression at the post-transcriptional levels. 2. Materials and methods 2.1. Cell cultures HepG2 cell was obtained from American Type Culture Collection and maintained in RPMI-1640 media (Gibco/BRL, USA) supplemented with 10% fetal bovine serum, 100 ul/ml penicillin and 100 mg/l streptomycin at 37 8C in a humidified atmosphere containing 5% CO2. Subcultures were made by trypsinization and reseeded for experiments. 2.2. Cell growth assay HepG2 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 for 24 h. 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 of each well was measured using a microculture plate reader with a test wavelength of 492 nm. Each assay was performed three times and the average results were calculated. 2.3. Apoptosis assay HepG2 cells were washed twice with ice-cold PBS and incubated with Annexin V-FITC 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. RT-PCR for survivin Expression of survivin mRNA was analyzed by RT-PCR amplification. Total RNA was isolated using Trizol1 (Invitrogen) according to the manufacturer’s protocol. One microgram of total RNA was reverse transcribed at 37 8C for 45 min in the presence of random hexamer and Moloney murine leukemia virus reverse transcriptase (Gibco-BRL). Analysis of the expression of the survivin was performed by RT-PCR amplification. The primers used for the survivin reactions were 50 -GGCTCTTTCTCTGTCCAGTT30 (forward) and 50 -ACCACCGCATCTCTACATTC-30 (reverse). 2.6. Western blot analysis The harvested cells were washed twice in ice-cold PBS, lysed in lysis buffer at 0 8C for 10 min, and centrifuged. The total protein concentration of the supernatants was determined by using microbicinchoninic acid assay (Pierce, Rockford, IL). The lysates containing 60 ug 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 antibody (Cell Signaling, USA) for over night at 4 8C. 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. Antibodies specific for survivin and p70s6k Thr 389 were used. 2.7. Cell proliferative assay HepG2 cells (1 104) were plated in triplicate onto 6-well culture plates. The following day, the medium was changed. Cell culture was carried out in the presence of 20 mM tamoxifen (Sigma) or rapamycin (10 nM 1 h prior to 20 mM tamoxifen) tamoxifen for 48 h, respectively. All drugs were added to the medium after solution in 100% ethanol. Corresponding amounts of ethanol were also added as a media control for each experiment. 2.8. Statistical analysis Data were expressed as mean standard deviation from three independent experiments. Differences in luciferase activity between tamoxifen treatment and control group were analyzed by Student’s t-test. A value of p < 0.05 was considered statistically significant.
2.4. Plasmid and transient transfections 3. Results pLuc plasmid DNA containing 158 bp survivin core promoter gene and firefly luciferase was a gift from Dr. Jian Lu. We used dualluciferase reporter system, where the Renilla luciferase vector provides an internal control that serves as the baseline response. Transient transfections of HepG2 cells were performed in 24-wells plates using Lipofectamin 2000 from Invitrogen according to the manufacturer instructions. HepG2 cells were cultured in 24-well plates until they reached 85–90% confluence. One microliter of Lipofectamin 2000 reagent and 0.4 mg pLuc plasmid DNA were used to transfect each well of cells in the absence of serum. After 4– 6 h, the medium were replaced with 10% FBS RPMI-1640. Approximately 24 h after the beginning of the transfection, the cells were exposed to tamoxifen and, or rapamycin. The cells luciferase activity was then analyzed by dual-luciferase reporter assay system.
3.1. Tamoxifen inhibits growth and induces apoptosis of HepG2 cells Tamoxifen reduced proliferation of HepG2 cells in concentration-dependent manner (Fig. 1A). Fig. 1 depicts a concentrationdependent decrease in proliferation in the presence of increasing concentrations of tamoxifen. When at concentrations of 20 mM, the inhibition on cell viability was 50%. Ten micromoles or 20 mM tamoxifen induced a reduction of cell viability. Basically reduction of viability was related to an increase in the fraction of G0/1-phase (data not shown). When tamoxifen was present at higher concentration (20 mM), an additional increase of the rate of apoptotic cells occurred with a delay, augmenting the effect of tamoxifen on cell viability substantially (Fig. 1B).
R. Guo et al. / Biomedicine & Pharmacotherapy 64 (2010) 249–253
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Fig. 3. Tamoxifen inhibits mTOR activity. HepG2 cells were treated with or without tamoxifen (20 mM) for 48 h. Expression of P-p70S6K protein was detected by western blot. Cell lysates were immunoblotted with P-p70S6K (Thr389) antibody.
Fig. 1. Inhibition of human hepatocarcinoma cells growth by tamoxifen is due to increased apoptosis. A. HepG2 cells were treated with different concentrations of tamoxifen for 48 h, and cell viability was measured by MTT assay. B. Cell apoptosis was assayed by flow cytometry.
3.2. Tamoxifen down-regulates of survivin expression We determined the expression level of survivin mRNA and protein following incubation of the cells with tamoxifen at 20 mM for 48 h using RT-PCR and western blot. Survivin mRNA (Fig. 2A) and protein (Fig. 2B) levels were substantially reduced by tamoxifen treatment. To confirm that down-regulation of survivin expression by tamoxifen treatment occurs primarily at the transcriptional level, a plasmid carrying a firefly luciferase reporter gene driven by the 158 bp survivin core promoter was transfected into HepG2 cells and cells were cultured with or without tamoxifen treatment. The survivin promoter activity was drastically reduced by tamoxifen treatment, indicating that in tamoxifen-induced apoptosis of HCC cells, survivin expression was down-regulated because of reduced survivin expression at the transcription level. 3.3. Tamoxifen inhibits mTOR activity HepG2 cells were treated with or without tamoxifen at the concentration of 20 mM for 48 h, and then cell lysates were
prepared, and western blot were performed with an antibody specific for the phosphotylated form of p70s6 kinase at Thr389, the position known to bephosphotylated by mTOR. The amount of Thr389 phosphotylated p70s6 kinase in the tamoxifen-exposed cells was much less than that in the untreated cells (Fig. 3), indicating that tamoxifen down-regulated mTOR activity. HepG2 cells were cultured in the presence of rapamycin (10 nM 1 h prior to tamoxifen) or tamoxifen (20 mM) or both for 48 h. As shown in Fig. 4, rapamycin alone reduced cell growth rate by 20%, tamoxifen alone reduced cell growth rate by 45% and the two drugs together reduced cell growth rate by nearly 65%, suggesting synergistic or additive effect of tamoxifen and rapamycin in inhibiting growth of HCC cells in vitro. 3.4. Rapamycin reduces survivin protein expression in synergy with tamoxifen The effect of rapamycin on survivin mRNA transcription and protein expression was examined. Treatment of HepG2 cells with either rapamycin or tamoxifen alone resulted in significant reduction of survivin protein expression compared with that of the untreated cells, and cotreatment resulted in a more augmenting reduction in the survivin protein level than either single drug treatment (Fig. 5B). HepG2 cells transiently transfected with the reporter plasmid were treated with rapamycin (10 nM) or tamoxifen (20 mM) or both agents (10 nM rapamycin 1 h prior to tamoxifen) for 48 h. Tamoxifen nearly depresses the activity of the survivin promoter (Fig. 5C) or mRNA expression (Fig. 5A). On the other hand, rapamycin treatment had no effect at all on survivin promoter activity and survivin mRNA level, indicating that rapamycin probably reduced survivin protein expression at the posttranscriptional level. 4. Discussion Tamoxifen treatment has been shown to benefit patients with HCC. Since most HCCs do not express the ER-a, the role of ER-a-
Fig. 2. Tamoxifen down-regulates survivin expression. A. Survivin mRNA assay. HepG2 cells were harvested after incubation with 20 mM of tamoxifen for 48 h. The survivin mRNA was measured by RT-PCR. B. Survivin protein determined with western blot.
Fig. 4. Rapamycin and tamoxifen synergistically inhibit proliferation of HepG2 cells. HepG2 cells were cultured in the presence of rapamycin (10 nM), or tamoxifen (20 mM), or both drugs (10 nM rapamycin 1 h prior to tamoxifen) for 48 h. The results are shown as the mean S.D. of triplicate samples and are representative of three independent experiments. Statistical significance shown as * and ** was determined by t test, with p < 0.05 defined as significant.
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Fig. 5. Rapamycin reduced survivin protein expression but did not reduce survivin transcription. A. Survivin mRNA expression determined with RT-PCR. B. The effect of rapamycin on survivin protein expression was detected by western blot. HepG2 cells were treated with rapamycin, or tamoxifen or both for 48 h, cell lysates were immunoblotted with survivin antibody. C. The effect of rapamycin and tamoxifen on survivin promoter activity. HepG2 cells were transient transfected with pLuc plasmid DNA, which contains the luciferase reporter driven by the core survivin promoter, whole cell extract was then prepared from the cells and luciferase activity was measured by dual-luciferase reporter assay system (lands are as labeled on the figure). The mean S.D. of three experiments is shown. Statistical significance shown as * and ** was determined by t test, with p < 0.05 defined as significant.
independent antiproliferative tamoxifen action in HCC has to be evaluated. For this purpose, the human hepatoblastoma cell line HepG2 provides an excellent in vitro system. In HepG2 cells, ER-a is not detectable [7] or expressed at low levels which are insufficient for ligands to affect transactivation of ER-regulated genes [26]. On the other hand, HepG2 cells have been shown to retain many of the different characteristics of normal hepatocytes, although the cell line performs uraegenesis, glucuronidation, sulfation, and oxidative metabolism at least by a factor 10 lower than primary rat hepatocytes, whereas the rate of albumin synthesis is higher [27]. We demonstrated previously that tamoxifen at 20 mM reduces viability and induces apoptosis in HepG2 cells. The effects of the drug may be related to an increase of Ca2+ influx [28] or an enhanced generation of reactive oxygen species [29]. Further, we suggested that the observed antiproliferative effects of tamoxifen were, at least in part, due to a reduction of survivin which was detected simultaneously. Survivin is expressed during the G2/M phase. It directly inhibits the terminal effector cell-death proteases caspase-3 and caspase-7 [30]. It binds to the microtubles of the mitotic spindle, where it counteracts the induction of apoptosis [31]. Moreover, experimental data have shown survivin in promotion of cellular proliferation as well as inhibition of apoptosis in cancer cells [32]. Survivin overexpression has been identified as a negative
prognostic factor in various cancer types [33–35] and is implicated in resistance to apoptosis induction by anti-cancer agents and ionizing radiation [36,37]. 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 [38–44]. In the present study, we showed that tamoxifen treatment of HepG2 cells induces apoptosis and down-regulation of survivin mRNA and survivin protein. Increasing evidences implicate mTOR as a central player in cell proliferation, migration, and survival [45–47]. The mTOR protein is involved in the regulation of cyclins D1/A, cyclin-dependent kinases, cyclin-dependent kinase inhibitors (p21Cip1 and p27Kip1), retinoblastoma protein, RNA polymerase I/II/III-transcription and translation [48–51]. Suppression of these mTORmediated survival signals provides the opportunity to reactivate default apoptotic pathways in cancer cells and allow them to proceed on the path of death [52]. mTOR inhibitor rapamycin has been proposed as a potential therapeutic for cancer treatment and for restenosis prevention [53,54]. Our present study attempts to link these two important factors of human tumorigenesis in HCC, which is one of the leading causes of cancer and cancer-related death in China. We have demonstrated that in tamoxifen induced apoptosis of HCC cells HepG2 in a dose-dependent fashion, and tamoxifen reduced survivin expression of these cells primarily through reduction in survivin transcription. The tamoxifen treatment also reduced mTOR activity. Inhibition of mTOR activity with its specific inhibitor rapamycin resulted in decrease of HepG2 cells viability and downregulation of survivin protein expression, although rapamycin did not affect survivin transcription. The two effects, reduction of cell viability and down-regulation of survivin protein, caused by tamoxifen and rapamycin were synergistic or additive and that this would be a potential therapeutic combination for hepatocellular cancer. Our findings indicated that mTOR signal molecule might be involved in the regulation of survivin expression at posttranscriptional level. PI3K pathway has been reported as an important intracellular mediator frequently activated in cancer cells. PI3K activates a number of signaling molecules, such as Akt. Recent studies show mTOR is an important substrate of Akt. The down-regulation of mTOR activity may be through the inhibition of PI3K/Akt signal pathway. Thus, tamoxifen induce cell apoptosis by down-regulating survivin through two distinct mechanisms: (i) direct effect at the transcriptional level; (ii) indirectly by down-regulating mTOR, which leads to reduced survivin protein level. 5. Conclusion In summary, tamoxifen down-regulated survivin at both transcriptional level and PI3K/Akt/mTOR pathway-dependent post-transcriptional level to promote hepatocellular carcinoma cell apoptosis. Thus, we have identified survivin as a functionally important downstream element in the classical mTOR pathway of ER independent tamoxifen action in vitro. Acknowledgments We thanks Dr. PC. Xun for excellent statistical assistance and generous help in the review of the manuscript. We thank Dr. Jian Lu for providing us plasmid. This work was supported by the Natural science foundation of Jiangsu Province and high college of Jiangsu Province, China (BK2009443, 08KJB310025).
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Original article
Involvement of mTOR and survivin inhibition in tamoxifen-induced apoptosis in human hepatoblastoma cell line HepG2 Renhua Guo a, Tongshan Wang a, Hua Shen a, Hong-mei Ge b, Jing Sun a, Zu-hu Huang c,1,*, Yong-qian Shu a,1,* a b c
Department of oncology, First Affiliated Hospital of Nanjing Medical University, Nanjing Guangzhou road 300, Nanjing 210029, China Laboratory of molecular biology, First Affiliated Hospital of Nanjing Medical University, Nanjing Guangzhou road 300, Nanjing 210029, China Department of Infectious disease, First Affiliated Hospital of Nanjing Medical University, Nanjing Guangzhou road 300, Nanjing 210029, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 May 2009 Accepted 7 June 2009 Available online 22 October 2009
Patients with advanced hepatocellular carcinoma (HCC) have shown to benefit from tamoxifen treatment. The mechanisms of tamoxifen effects in HCC, however, are not yet clearly understood. The PI3K/Akt/mTOR signal pathway is involved in cell proliferation, tumorigenesis, and apoptosis. Overexpression of survivin has played an important role in leading to antiapoptosis. The current study investigated changes in mTOR pathway and survivin expression in hepatocarcinoma cell line HepG2 treated with tamoxifen. We detected apoptosis of hepatocarcinoma cells by flow cytometry assay. Survivin transcription level and p70S6k was demonstrated by PCR, dual-luciferase reporter assay and western blot analysis respectively. Our results are showed as follows: tamoxifen leads to apoptosis of the cells, and reduction in survivin expression, as well as a dramatic reduction in the activated form of p70S6 kinase. Twenty micromoles tamoxifen treatment signifcantly depresses transcription of survivin mRNA. Treating HepG2 cells with rapamycin, a specific mTOR inhibitor, significantly reduce survivin protein level but did not affect survivin transcription, which indicated that tamoxifen and rapamycin were synergistic in regards to down-regulation of survivin expression in hepatocellular carcinoma cells. Our results suggest that tamoxifen down-regulate survivin expression in HepG2 cells is mediated by transcriptional and posttranscriptional level via PI3K/Akt/mTOR pathway to induce apoptosis. ß 2009 Elsevier Masson SAS. All rights reserved.
Keywords: Tamoxifen HepG2 cells Apoptosis Survivin mTOR
1. Introduction The liver is a hormone-sensitive organ, several lines of evidence suggest that sex hormones and their receptors may play a role in liver carcinogenesis [1]. Tamoxifen is a non-steroidal anti-estrogen that has been used in the treatment of breast cancer for >25 years [2] and lately as a long-term chemopreventive agent for breast cancer in healthy women at high risk of developing breast cancer [2,3]. Furthermore, tamoxifen has been used in other malignant tumors, such as hepatocellular carcinoma [4] and shown to prolong survival of some patients in advanced, inoperable HCC [5,6]. Cell culture experiments on the human hepatoblastoma cell lines Hep3B and HepG2 have shown that tamoxifen at concentra-
Abbreviations: HCC, hepatocellular carcinoma; ER, estrogen receptor; PI3K, phosphatidylinositol-3-kinase; mTOR, mammalian target of rapamycin. * Corresponding author. Tel.: +8625 83718836 6714; fax: +8625 83724440. E-mail addresses: [email protected], [email protected] (Z.-h. Huang), [email protected] (Y.-q. Shu). 1 These two authors contribute equally to this article. 0753-3322/$ – see front matter ß 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2009.06.007
tions in the nanomolar range induces growth inhibition and/or apoptotic cell death via an ER-a-independent mechanism [7,8]. This is in line with the low ER-a-expression in most HCC tissues. The apoptosis effects of tamoxifen on HepG2 cells are at least in part mediated by a down-regulation of survivin expression [9]. The PI3K/Akt pathway has been implicated in tumorigenesis [10]. PI3K activates a number of signaling molecules, among which the Akt/mTOR pathway is of particular interest because of its role in inhibiting apoptosis and promoting cell proliferation [11]. mTOR is a 289-kDa serine/threonine kinase that is a downstream target of Akt [12]. It has been shown to regulate mitogen stimulated protein synthesis and cell cycle progression [13–15]. Cell culture studies have demonstrated that one of the mechanisms by which mTOR controls protein synthesis is through phosphorylating downstream substrates, including p70s6 kinase (p70S6K1) and eukaryotic initiation factor (eIF) 4E binding protein 1 (4E-BP1) [16–18]. Therefore, by inhibiting mTOR, rapamycin causes a decrease in phosphorylation of these effectors, and a decrease in protein synthesis, effectively blocking the progrowth, pro-proliferative, and prosurvival actions of mTOR [19].
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Survivin, a member of the inhibitor of apoptosis proteins (IAP) family [20,21] is not present in most normal adult differentiated tissues. Its mRNA and the protein are present in large amounts in fetal tissue and most cancers [22,23]. Survivin protein suppresses apoptosis and stimulates cell division [24]. Our data have shown PI3K/Akt pathway regulating the expression of survivin in HCC cells [25]. Since inhibiting mTOR activity leads to cancer cell death while over-expression of survivin is associated with anti-apoptosis, it is possible that mTOR may directly or indirectly regulate the expression of survivin. In the current study, we investigate the pathway of the tamoxifen-induced down-regulation of survivin in HepG2 cells, which does not express ER-a [7]. We show here that in HepG2 cells tamoxifen could down-regulate both mTOR and survivin expression, and inhibiting mTOR leads to further downregulation of survivin expression at the post-transcriptional levels. 2. Materials and methods 2.1. Cell cultures HepG2 cell was obtained from American Type Culture Collection and maintained in RPMI-1640 media (Gibco/BRL, USA) supplemented with 10% fetal bovine serum, 100 ul/ml penicillin and 100 mg/l streptomycin at 37 8C in a humidified atmosphere containing 5% CO2. Subcultures were made by trypsinization and reseeded for experiments. 2.2. Cell growth assay HepG2 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 for 24 h. 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 of each well was measured using a microculture plate reader with a test wavelength of 492 nm. Each assay was performed three times and the average results were calculated. 2.3. Apoptosis assay HepG2 cells were washed twice with ice-cold PBS and incubated with Annexin V-FITC 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. RT-PCR for survivin Expression of survivin mRNA was analyzed by RT-PCR amplification. Total RNA was isolated using Trizol1 (Invitrogen) according to the manufacturer’s protocol. One microgram of total RNA was reverse transcribed at 37 8C for 45 min in the presence of random hexamer and Moloney murine leukemia virus reverse transcriptase (Gibco-BRL). Analysis of the expression of the survivin was performed by RT-PCR amplification. The primers used for the survivin reactions were 50 -GGCTCTTTCTCTGTCCAGTT30 (forward) and 50 -ACCACCGCATCTCTACATTC-30 (reverse). 2.6. Western blot analysis The harvested cells were washed twice in ice-cold PBS, lysed in lysis buffer at 0 8C for 10 min, and centrifuged. The total protein concentration of the supernatants was determined by using microbicinchoninic acid assay (Pierce, Rockford, IL). The lysates containing 60 ug 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 antibody (Cell Signaling, USA) for over night at 4 8C. 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. Antibodies specific for survivin and p70s6k Thr 389 were used. 2.7. Cell proliferative assay HepG2 cells (1 104) were plated in triplicate onto 6-well culture plates. The following day, the medium was changed. Cell culture was carried out in the presence of 20 mM tamoxifen (Sigma) or rapamycin (10 nM 1 h prior to 20 mM tamoxifen) tamoxifen for 48 h, respectively. All drugs were added to the medium after solution in 100% ethanol. Corresponding amounts of ethanol were also added as a media control for each experiment. 2.8. Statistical analysis Data were expressed as mean standard deviation from three independent experiments. Differences in luciferase activity between tamoxifen treatment and control group were analyzed by Student’s t-test. A value of p < 0.05 was considered statistically significant.
2.4. Plasmid and transient transfections 3. Results pLuc plasmid DNA containing 158 bp survivin core promoter gene and firefly luciferase was a gift from Dr. Jian Lu. We used dualluciferase reporter system, where the Renilla luciferase vector provides an internal control that serves as the baseline response. Transient transfections of HepG2 cells were performed in 24-wells plates using Lipofectamin 2000 from Invitrogen according to the manufacturer instructions. HepG2 cells were cultured in 24-well plates until they reached 85–90% confluence. One microliter of Lipofectamin 2000 reagent and 0.4 mg pLuc plasmid DNA were used to transfect each well of cells in the absence of serum. After 4– 6 h, the medium were replaced with 10% FBS RPMI-1640. Approximately 24 h after the beginning of the transfection, the cells were exposed to tamoxifen and, or rapamycin. The cells luciferase activity was then analyzed by dual-luciferase reporter assay system.
3.1. Tamoxifen inhibits growth and induces apoptosis of HepG2 cells Tamoxifen reduced proliferation of HepG2 cells in concentration-dependent manner (Fig. 1A). Fig. 1 depicts a concentrationdependent decrease in proliferation in the presence of increasing concentrations of tamoxifen. When at concentrations of 20 mM, the inhibition on cell viability was 50%. Ten micromoles or 20 mM tamoxifen induced a reduction of cell viability. Basically reduction of viability was related to an increase in the fraction of G0/1-phase (data not shown). When tamoxifen was present at higher concentration (20 mM), an additional increase of the rate of apoptotic cells occurred with a delay, augmenting the effect of tamoxifen on cell viability substantially (Fig. 1B).
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Fig. 3. Tamoxifen inhibits mTOR activity. HepG2 cells were treated with or without tamoxifen (20 mM) for 48 h. Expression of P-p70S6K protein was detected by western blot. Cell lysates were immunoblotted with P-p70S6K (Thr389) antibody.
Fig. 1. Inhibition of human hepatocarcinoma cells growth by tamoxifen is due to increased apoptosis. A. HepG2 cells were treated with different concentrations of tamoxifen for 48 h, and cell viability was measured by MTT assay. B. Cell apoptosis was assayed by flow cytometry.
3.2. Tamoxifen down-regulates of survivin expression We determined the expression level of survivin mRNA and protein following incubation of the cells with tamoxifen at 20 mM for 48 h using RT-PCR and western blot. Survivin mRNA (Fig. 2A) and protein (Fig. 2B) levels were substantially reduced by tamoxifen treatment. To confirm that down-regulation of survivin expression by tamoxifen treatment occurs primarily at the transcriptional level, a plasmid carrying a firefly luciferase reporter gene driven by the 158 bp survivin core promoter was transfected into HepG2 cells and cells were cultured with or without tamoxifen treatment. The survivin promoter activity was drastically reduced by tamoxifen treatment, indicating that in tamoxifen-induced apoptosis of HCC cells, survivin expression was down-regulated because of reduced survivin expression at the transcription level. 3.3. Tamoxifen inhibits mTOR activity HepG2 cells were treated with or without tamoxifen at the concentration of 20 mM for 48 h, and then cell lysates were
prepared, and western blot were performed with an antibody specific for the phosphotylated form of p70s6 kinase at Thr389, the position known to bephosphotylated by mTOR. The amount of Thr389 phosphotylated p70s6 kinase in the tamoxifen-exposed cells was much less than that in the untreated cells (Fig. 3), indicating that tamoxifen down-regulated mTOR activity. HepG2 cells were cultured in the presence of rapamycin (10 nM 1 h prior to tamoxifen) or tamoxifen (20 mM) or both for 48 h. As shown in Fig. 4, rapamycin alone reduced cell growth rate by 20%, tamoxifen alone reduced cell growth rate by 45% and the two drugs together reduced cell growth rate by nearly 65%, suggesting synergistic or additive effect of tamoxifen and rapamycin in inhibiting growth of HCC cells in vitro. 3.4. Rapamycin reduces survivin protein expression in synergy with tamoxifen The effect of rapamycin on survivin mRNA transcription and protein expression was examined. Treatment of HepG2 cells with either rapamycin or tamoxifen alone resulted in significant reduction of survivin protein expression compared with that of the untreated cells, and cotreatment resulted in a more augmenting reduction in the survivin protein level than either single drug treatment (Fig. 5B). HepG2 cells transiently transfected with the reporter plasmid were treated with rapamycin (10 nM) or tamoxifen (20 mM) or both agents (10 nM rapamycin 1 h prior to tamoxifen) for 48 h. Tamoxifen nearly depresses the activity of the survivin promoter (Fig. 5C) or mRNA expression (Fig. 5A). On the other hand, rapamycin treatment had no effect at all on survivin promoter activity and survivin mRNA level, indicating that rapamycin probably reduced survivin protein expression at the posttranscriptional level. 4. Discussion Tamoxifen treatment has been shown to benefit patients with HCC. Since most HCCs do not express the ER-a, the role of ER-a-
Fig. 2. Tamoxifen down-regulates survivin expression. A. Survivin mRNA assay. HepG2 cells were harvested after incubation with 20 mM of tamoxifen for 48 h. The survivin mRNA was measured by RT-PCR. B. Survivin protein determined with western blot.
Fig. 4. Rapamycin and tamoxifen synergistically inhibit proliferation of HepG2 cells. HepG2 cells were cultured in the presence of rapamycin (10 nM), or tamoxifen (20 mM), or both drugs (10 nM rapamycin 1 h prior to tamoxifen) for 48 h. The results are shown as the mean S.D. of triplicate samples and are representative of three independent experiments. Statistical significance shown as * and ** was determined by t test, with p < 0.05 defined as significant.
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Fig. 5. Rapamycin reduced survivin protein expression but did not reduce survivin transcription. A. Survivin mRNA expression determined with RT-PCR. B. The effect of rapamycin on survivin protein expression was detected by western blot. HepG2 cells were treated with rapamycin, or tamoxifen or both for 48 h, cell lysates were immunoblotted with survivin antibody. C. The effect of rapamycin and tamoxifen on survivin promoter activity. HepG2 cells were transient transfected with pLuc plasmid DNA, which contains the luciferase reporter driven by the core survivin promoter, whole cell extract was then prepared from the cells and luciferase activity was measured by dual-luciferase reporter assay system (lands are as labeled on the figure). The mean S.D. of three experiments is shown. Statistical significance shown as * and ** was determined by t test, with p < 0.05 defined as significant.
independent antiproliferative tamoxifen action in HCC has to be evaluated. For this purpose, the human hepatoblastoma cell line HepG2 provides an excellent in vitro system. In HepG2 cells, ER-a is not detectable [7] or expressed at low levels which are insufficient for ligands to affect transactivation of ER-regulated genes [26]. On the other hand, HepG2 cells have been shown to retain many of the different characteristics of normal hepatocytes, although the cell line performs uraegenesis, glucuronidation, sulfation, and oxidative metabolism at least by a factor 10 lower than primary rat hepatocytes, whereas the rate of albumin synthesis is higher [27]. We demonstrated previously that tamoxifen at 20 mM reduces viability and induces apoptosis in HepG2 cells. The effects of the drug may be related to an increase of Ca2+ influx [28] or an enhanced generation of reactive oxygen species [29]. Further, we suggested that the observed antiproliferative effects of tamoxifen were, at least in part, due to a reduction of survivin which was detected simultaneously. Survivin is expressed during the G2/M phase. It directly inhibits the terminal effector cell-death proteases caspase-3 and caspase-7 [30]. It binds to the microtubles of the mitotic spindle, where it counteracts the induction of apoptosis [31]. Moreover, experimental data have shown survivin in promotion of cellular proliferation as well as inhibition of apoptosis in cancer cells [32]. Survivin overexpression has been identified as a negative
prognostic factor in various cancer types [33–35] and is implicated in resistance to apoptosis induction by anti-cancer agents and ionizing radiation [36,37]. 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 [38–44]. In the present study, we showed that tamoxifen treatment of HepG2 cells induces apoptosis and down-regulation of survivin mRNA and survivin protein. Increasing evidences implicate mTOR as a central player in cell proliferation, migration, and survival [45–47]. The mTOR protein is involved in the regulation of cyclins D1/A, cyclin-dependent kinases, cyclin-dependent kinase inhibitors (p21Cip1 and p27Kip1), retinoblastoma protein, RNA polymerase I/II/III-transcription and translation [48–51]. Suppression of these mTORmediated survival signals provides the opportunity to reactivate default apoptotic pathways in cancer cells and allow them to proceed on the path of death [52]. mTOR inhibitor rapamycin has been proposed as a potential therapeutic for cancer treatment and for restenosis prevention [53,54]. Our present study attempts to link these two important factors of human tumorigenesis in HCC, which is one of the leading causes of cancer and cancer-related death in China. We have demonstrated that in tamoxifen induced apoptosis of HCC cells HepG2 in a dose-dependent fashion, and tamoxifen reduced survivin expression of these cells primarily through reduction in survivin transcription. The tamoxifen treatment also reduced mTOR activity. Inhibition of mTOR activity with its specific inhibitor rapamycin resulted in decrease of HepG2 cells viability and downregulation of survivin protein expression, although rapamycin did not affect survivin transcription. The two effects, reduction of cell viability and down-regulation of survivin protein, caused by tamoxifen and rapamycin were synergistic or additive and that this would be a potential therapeutic combination for hepatocellular cancer. Our findings indicated that mTOR signal molecule might be involved in the regulation of survivin expression at posttranscriptional level. PI3K pathway has been reported as an important intracellular mediator frequently activated in cancer cells. PI3K activates a number of signaling molecules, such as Akt. Recent studies show mTOR is an important substrate of Akt. The down-regulation of mTOR activity may be through the inhibition of PI3K/Akt signal pathway. Thus, tamoxifen induce cell apoptosis by down-regulating survivin through two distinct mechanisms: (i) direct effect at the transcriptional level; (ii) indirectly by down-regulating mTOR, which leads to reduced survivin protein level. 5. Conclusion In summary, tamoxifen down-regulated survivin at both transcriptional level and PI3K/Akt/mTOR pathway-dependent post-transcriptional level to promote hepatocellular carcinoma cell apoptosis. Thus, we have identified survivin as a functionally important downstream element in the classical mTOR pathway of ER independent tamoxifen action in vitro. Acknowledgments We thanks Dr. PC. Xun for excellent statistical assistance and generous help in the review of the manuscript. We thank Dr. Jian Lu for providing us plasmid. This work was supported by the Natural science foundation of Jiangsu Province and high college of Jiangsu Province, China (BK2009443, 08KJB310025).
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