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Inhibition of mTOR enhances chemosensitivity in hepatocellular carcinoma
Cancer Letters 273 (2009) 201–209
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Inhibition of mTOR enhances chemosensitivity in hepatocellular carcinoma q Ka Ho Tam a, Zhen Fan Yang a, Chi Keung Lau a, Chi Tat Lam a, Roberta W.C. Pang b, Ronnie T.P. Poon a,* a b
Center for Cancer Research, Department of Surgery, The University of Hong Kong, Queen Mary Hospital, 102 Pokfulam Road, Pokfulam, Hong Kong, China Center for Cancer Research, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, 102 Pokfulam Road, Pokfulam, Hong Kong, China
a r t i c l e
i n f o
Article history: Received 25 March 2008 Received in revised form 25 March 2008 Accepted 4 August 2008
Keywords: mTOR p53 RAD001 HCC Chemosensitivity Chemoresistance
a b s t r a c t The present study investigated the effect of mammalian target of rapamycin (mTOR) inhibition on HCC cells in vitro and in vivo, either alone or in combination with cytotoxic agents. In vitro, HCC cell lines were exposed to RAD001, an mTOR inhibitor, either alone or in combination with cisplatin. Alone, RAD001 suppressed cell proliferation in all cell lines tested, but did not induce apoptosis. RAD001 in combination with cisplatin induced a significant increase in the number of apoptotic cells, downregulated the expression of pro-survival molecules, Bcl-2, survivin and cyclinD1, and increased the cleavage of PARP, compared to RAD001 or cisplatin alone. Transfection of p53 into the Hep3B cell line increased the sensitivity of tumor cells to cisplatin. The suppression of HCC tumor growth in vivo was enhanced by RAD001 combined with cisplatin, accompanied by a significant increase in the number of apoptotic cells in tumor tissues. This study demonstrates that inhibition of mTOR suppresses tumor growth and sensitizes tumor cells to chemocytotoxic agents. Ó 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Hepatocellular carcinoma (HCC) is the third most common cause of cancer death with a 5-year survival rate of 7% [1]. Hepatic resection and liver transplantation are the two mainstays of curative treatment for HCC, but can only be q Financial support: The study was supported by the Seed Funding Program for Basic Research 2006 and Sun Chieh Yeh Research Foundation for Hepatobiliary and Pancreatic Surgery of the University of Hong Kong. * Corresponding author. Tel.: +852 2855 3641; fax: +852 2817 5475. E-mail address: [email protected] (R.T.P. Poon). Abbreviations: HCC, hepatocellular carcinoma; TACE, transarterial chemoembolization; S6K1, ribosomal p70S6 kinase; 4E-BP1, eukaryotic initiation factor eIF4E binding protein; P-, phosphorylated-; wt, wildtype; DMEM, Dulbecco’s modified Eagle medium; FBS, fetal bovine serum; CO2, carbon dioxide; DMSO, dimethyl sulfoxide; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate buffered saline; RIPA, radioimmunoprecipitation assay; i.p., intra-peritoneal; H&E, hematoxylin and eosin; TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling; ANOVA, analysis of variance between groups; SD, standard deviation.
0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.08.018
applied to the early stage of HCC [2,3]. The majority of patients with HCC are diagnosed at a late stage when curative treatment options are not applicable. Currently, there is no proven effective systemic chemotherapy for HCC due to resistance of tumor cells to cytotoxic drugs [4,5]. The efficacy is limited for local chemotherapy, using agents such as cisplatin by means of transarterial chemoembolization (TACE), due to the poor response rate of tumor cells to treatment and tumor re-growth after treatment [6,7]. These reasons exemplify the need to fully understand the mechanism underlying chemoresistance in HCC to allow the design of more effective therapeutic strategies. Signaling pathways regulating cell growth and survival, such as those involving p53 and PTEN/PI3K/AKT, are often dysregulated in HCC [8–10]. Molecules targeting these abnormalities may suppress tumor progression and restore chemosensitivity of HCC cells. The mammalian target of rapamycin, mTOR, is a potential target for anti-cancer therapy as it is a key molecule in the PTEN/PI3K/AKT/mTOR signaling pathway and plays a critical role in controlling
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cell proliferation and survival. Cap-dependent translation is regulated by mTOR through ribosomal p70S6 kinase (S6K1) and the eukaryotic initiation factor eIF4E binding protein (4E-BP1), respectively. This control has important implications in tumorigenesis, cell survival and apoptosis. Abnormal activation of the PTEN/PI3K/AKT/mTOR signaling pathway frequently occurs in human cancers, including HCC [11]. A recent study showed that P-mTOR was expressed in 41% of HCC tumor tissue but not in adjacent cirrhotic liver tissue [12], suggesting that mTOR might be a specific therapeutic target for HCC. Several mTOR inhibitors, including rapamycin/sirolimus (Wyeth) and derivatives, such as RAD001/everolimus (Novartis), CCI-779/temsirolimus (Wyeth) and AP23573 (Ariad), are being developed as anti-cancer agents against various types of malignancies. Therapeutic approaches focused on mTOR inhibition alone can retard tumor growth, but cannot eradicate the tumor. This is because mTOR inhibitors are cytostatic not cytotoxic and arrest cancer cells in G1 phase of the cell cycle. Based on the role of mTOR in chemoresistance, the combination of an mTOR inhibitor and a cytotoxic agent may achieve an ideal therapeutic outcome by combining cancer cell cycle arrest and death. Inhibition of mTOR can enhance the efficacy of a broad range of chemocytotoxic agents, including cisplatin, doxorubicin, paclitaxel, carboplatin, dexamethasone, mitoxantrone and docetaxel, in various types of human cancers [13–16]. The mechanism underlying mTOR mediated enhancement of chemosensitivity remains unclear. It is postulated that chemosensitization depends on the activity of Akt, PTEN and p53, and loss of PTEN and aberrant Akt activity correlates with the upregulation of mTOR activity, thus rendering tumor cells sensitive to mTOR inhibitors [13,17,18]. The presence of functional p53 is essential for the sensitization of tumor cells to cisplatin by mTOR inhibitors [14]. A large proportion of HCC tumors present with a p53 mutation or deletion. Furthermore, the effect of mTOR inhibition on the chemosensitivity of HCC remains unknown. For these reasons, the present study was designed to investigate the anti-tumor effect of RAD001, an mTOR inhibitor, either alone or in combination with cisplatin, a conventional chemocytotoxic agent commonly used for the treatment of HCC, with a focus on p53-related signaling pathways. 2. Materials and methods 2.1. Cell lines, reagents and culture conditions Human hepatocellular carcinoma cell lines, HepG2 (wt p53), PLC (mutant p53) and Hep3B (p53 null), were purchased from the American Type Culture Collection (ATCC, Manassas, VA). A Hep3B cell line with wild-type p53 stable transfection was kindly provided by Prof. B.S. Lai, Chinese University of Hong Kong [19]. All cell lines were cultured in DMEM medium with 10% FBS and 1% penicillin and streptomycin (Life Technologies, Carlsbad, CA), and incubated at 37 °C in a humidified atmosphere with 5% CO2 in air. RAD001 (everolimus) was kindly provided by Novartis Institutes for Biomedical Research, Basel, Switzerland. A 10 mM RAD001 stock solution for cell culture was dis-
solved in DMSO (Sigma–Aldrich, St. Louis, MO), stored at 20 °C and diluted in fresh culture medium immediately before use. For in vivo studies, RAD001 and placebo microemulsion stocks were stored at 20 °C and diluted with 5% glucose immediately before use. Cisplatin was obtained from Amersham and Upjohn, Buckinghamshire, United Kingdom. 2.2. Determination of cell viability by 3,[4,5-dimethylthiazol2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay HCC cells were seeded onto a 96-well plate at a density of 1 104 cells per well. After 24 h, cells were exposed to different concentrations of RAD001, with or without cisplatin, in growth medium. After incubation for indicated time points, MTT was added to each well and incubated for another 4 h. The reaction was stopped with 0.01 N hydrochloric acid in 10% SDS solution and the absorbance was measured at 540/650 nm using a Vmax kinetic microplate reader (Bio-Rad Laboratories, Hercules, CA). Each sample was assayed in triplicate, and three independent experiments were performed for each group. 2.3. Cell cycle and cytofluorometric apoptosis analysis For cell cycle analysis, treated cells were harvested and fixed in absolute ethanol at 4 °C for 12 h. Cells were washed twice with PBS then stained with propidium iodide at 37 °C for 30 min. Cell cycle analysis was performed on a FACSCalibur (Becton Dickinson immunocytometry Systems, San Jose, CA). Cytofluorometric apoptosis analysis was performed by harvesting cells, labeling with the Annexin V-FITC antibody (BD Biosciences PharMingen, San Diego, CA) at room temperature for 15 min, and then analyzing apoptosis on a FACSCalibur. 2.4. Western blot analysis Total protein was extracted from treated cells using radioimmunoprecipitation assay (RIPA) buffer. Protein levels of P-S6 (Ser235/236), P-p70S6K (Thr389), P-4E-BP1 (Ser65), Bcl-2, PARP, P-p53 (Ser15), cyclin D1, p53, p21, survivin and actin were detected using standard Western blot analysis on 8–15% SDS–PAGE. Rabbit anti-human PS6 [Ser235/236], P-p70S6K [Thr389], P-4E-BP1 [Ser65], Bcl-2 and PARP polyclonal antibodies, mouse anti-human P-p53 [Ser15], p21 and cyclin D1 monoclonal antibodies were from Cell Signaling Technology (Beverly, MA). Mouse anti-human p53 monoclonal antibody was from Dako Corp. (Carpenteria, CA). Rabbit anti-human survivin polyclonal antibody was from Oncogene Research Products (Calbiochem, San Diego, CA). Mouse anti-human actin monoclonal antibody was from Sigma. 2.5. HCC xenograft in nude mice Four-week old male athymic nude mice were purchased from the Laboratory Animal Unit of the University of Hong Kong and housed in microisolator cages under positive air pressure maintained at a constant temperature (22 °C) and humidity, in a 12 h light/dark cycle. Sterilized rodent food
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Optical density (540nm)
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Treatment time (hours) Fig. 1. RAD001 inhibits proliferation of human hepatocellular carcinoma (HCC) cell lines. (A) Cells were treated with indicated concentrations of RAD001. At different time points after exposure, cell proliferation was quantified by the MTT assay. Data represents means ± standard deviation of three independent experiments (**P < 0.01, compared with control by One-way ANOVA). (B) PLC cells were treated with either DMSO (0.1%) or 10 nM RAD001 for 48 h, harvested, permeabilized with ethanol and labeled with propidium iodide. Cell cycle fractions were measured by flow cytometric analysis. (C) RAD001 inhibited activation of mTOR targets. Cells were treated with 10 nM RAD001 for 24 h. The expression of P-S6 (Ser235/236), P-p70S6K (Thr389), P-4E-BP1 (Ser65) and b-actin was assessed by Western blot analysis.
and water were available ad libitum. All animal experiments were approved by the Committee on the Use of Live Animals in Teaching and Research, the University of Hong Kong. For generation of HCC xenografts, 3 106 Hep3B cells were injected subcutaneously into the nude mice using a 23-gauge needle. Mice were then placed randomly into four groups (n = 7 in each group). Treatment was initiated when tumor nodules reached 100–200 mm3 and persisted for 4 weeks. Experimental groups included: (1) vehicle alone, (2) cisplatin alone (3 mg/kg per week, i.p.), (3) RAD001 alone (5 mg/kg/day, orally), (4) a combination of cisplatin and RAD001. Tumor size was monitored continuously using three-dimensional caliper measurements and tumor volume was calculated using the following formula [20].
Volume ¼ Length Width Depth 3:14=6
mination of histology by H&E staining and detection of apoptotic cells by terminal deoxynucleotidyl transferase– mediated dUTP nick end labeling (TUNEL, Roche, Basel, Switzerland). The apoptotic index was determined by counting the total number of positive nuclei in 20 randomly selected fields at 200 magnification. 2.7. Statistical analysis Statistical analyses were performed using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA). Comparisons of optical density values, percentage of viable cells and number of apoptotic cells between different groups were performed using the two-tailed Student’s t test or ANOVA. P < 0.05 was considered statistically significant. 3. Results
2.6. Histological studies 3.1. Inhibition of HCC cell proliferation and mTOR activity by RAD001
Mice were sacrificed when treatment was completed. Tumor tissue was fixed in 10% buffered formalin, embedded in paraffin and cut into sections 5 lm thick for deter-
To examine the effects of RAD001 on HCC cell proliferation, three HCC cell lines (HepG2, PLC and Hep3B) were treated with 0.01–10 nM RAD001 for 72 h. Significant dose-dependent inhibition of cell proliferation was
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observed in all three cell lines with 1–10 nM RAD001. RAD001-induced inhibition of cell proliferation was more pronounced in PLC cells than in HepG2 or Hep3B cells. Cell proliferation was inhibited only in PLC cells at 0.1 nM RAD001. Cell proliferation was inhibited by 60% in PLC cells and by 40% and 20% in HepG2 and Hep3B cells, respectively, after 72 h exposure to 10 nM RAD001 (Fig. 1A). Cell apoptosis assessed by flow cytometry or cellular morphology was not induced in any of the three HCC cell lines by RAD001. RAD001 was shown to inhibit cell cycle progression due to an increase in the proportion of cells in G0-G1 phase and decreases in the proportion of cells in S phase (Fig. 1B). Western blot analysis of HCC cells exposed to vehicle (DMSO) or 10 nM RAD001 for 24 h was used to investigate the molecular mechanisms induced by RAD001 (Fig. 1C). RAD001 induced a decrease in the phosphorylation of three downstream molecules of mTOR, including p70S6K (Thr389), ribosomal S6 (Ser235/236) and 4E-BP1 (Ser65). This result confirms that mTOR activity was inhibited by RAD001. The inhibitory effect of RAD001 on the phosphorylation of p70S6K (Thr389) was most prominent in PLC cells (Fig. 1C).
platin administration induced the proteolytic cleavage of PARP, and when cisplatin was combined with RAD001, a further increase in the expression of cleaved PARP was detected in all the three cell lines (Fig. 2C). Exposure to cisplatin inhibited the phosphorylation of p70S6K (Thr389) and 4E-BP1 (Ser65) in all the three HCC cell lines. This inhibitory effect was enhanced when cisplatin was combined with RAD001 in HepG2 cells. Exposure to cisplatin upregulated the expression of P-p53 (Ser15) in HepG2 and PLC cells, and the expression of p53 (Ser15) was further augmented in PLC cells when RAD001 was combined with cisplatin (Fig. 2D). To further analyze the molecular mechanism of RAD001-mediated chemosensitization, Western blot analysis was used to evaluate the expression of pro-survival molecules. Exposure to cisplatin alone inhibited the expression of pro-survival molecules, Bcl-2, cyclin D1 and survivin, in all the three HCC cell lines. Cisplatin alone decreased the expression of Bcl-2. Synergistic inhibition of expression of the above molecules was detected when RAD001 was combined with cisplatin (Fig. 2E).
3.2. Enhancement of cisplatin-induced cell apoptosis by RAD001
3.3. Enhancement of RAD001-mediated chemosensitization of Hep3B cells by p53
The synergistic effect of RAD001 and cisplatin on cell viability and apoptosis was examined in HepG2, PLC and Hep3B cell lines. Cell proliferation was inhibited in HepG2 cell line after exposure to cisplatin alone for 48 h. Cell proliferation was further inhibited when 10 nM RAD001 was combined with different doses of cisplatin, particularly in HepG2 and PLC cell lines (Fig. 2A). Cytofluorometric apoptosis analysis showed no alteration in the number of apoptotic cells in all the three cell lines exposed to RAD001 alone. By comparison, a significant increase in the number of apoptotic cells was detected in all the three cell lines when RAD001 was combined with cisplatin (Fig. 2B). RAD001 treatment did not induce proteolytic cleavage of PARP (86 kDa), a marker for cell apoptosis. This result is consistent with our finding using the cytofluorometric apoptosis assay that RAD001 does not induce cell apoptosis. On the contrary, cis-
To further characterize the role of p53 in RAD001-mediated chemosensitization, wild-type p53 was transfected into Hep3B cells, which have a deleted form of p53. Hep3B cells (Hep3B-p53) and Hep3B-p53 stable transfectants (Hep3B-p53+) were exposed to 10 nM RAD001 in the presence or absence of cisplatin. Cytofluorometric apoptosis analysis showed a significant increase in the number of apoptotic cells in Hep3B-p53+ cells compared to Hep3B-p53 cells when RAD001 was combined with cisplatin. The expression of p53 (Ser15) is induced by cisplatin in Hep3Bp53+ cells, consistent with the results for HepG2 cells ( Fig. 3A and B). The expression of p21 was downregulated by RAD001 in Hep3B-p53+ cells. However, there was no significant difference in the effect of RAD001 on the expression of Bcl-2, survivin and cyclin D1 between Hep3B-p53+ and Hep3B-p53 cells.
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K.H. Tam et al. / Cancer Letters 273 (2009) 201–209
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Fig. 2. RAD001 sensitized HCC tumor cells with different p53 status, HepG2 (wt p53), PLC (mutant p53) and Hep3B (p53 null), to cisplatin cytotoxicity. (A) and (B). Cells were treated with either DMSO control or 10 nM RAD001 in combination with cisplatin for 48 h. Cell viability and apoptosis were measured by the MTT assay and cytofluorometric apoptosis analysis, respectively. Data represented means ± standard deviation of three independent experiments (*P < 0.05, compared with control by two-tailed t tests and One-way ANOVA). (C), (D) and (E). Cells were treated with either DMSO control or 10 nM RAD001 in combination with 5 lg/ml cisplatin for 24 h. The expression level of PARP, P-p70S6K (Thr389), P-4E-BP1 (Ser65), P-p53 (Ser15), p53, cyclin D1, survivin, Bcl-2 and b-actin was assessed by Western blot analysis.
3.4. Synergistic anti-tumor activity by RAD001 and cisplatin The Hep3B xenograft model in nude mice was employed to investigate the anti-tumor effect of RAD001 and cisplatin in vivo. Treatment of mice with cisplatin or RAD001 alone suppressed tumor growth (Fig. 4A). When RAD001 was combined with cisplatin, tumor growth was inhibited synergistically. Tumor regression was observed in two out of seven animals administered both RAD001 and cisplatin. The TUNEL assay was performed to assess apoptotic cells in tumor tissue after different treatment regimens to determine whether the anti-tumor effect of RAD001 and cisplatin in vivo was mediated by the induction of cell apoptosis. RAD001 alone did not induce cell apoptosis in tumor tis-
sue, whereas cisplatin alone induced an increase in the number of apoptotic cells (24.7 ± 8.9 versus 12.4 ± 8.5 in non-treatment control). A further augmentation of cell apoptosis was observed when mice were treated with RAD001 together with cisplatin (44.9 ± 10.9 versus 16.45 ± 10.2 for RAD001 alone, and 24.7 ± 8.9 for cisplatin alone) (Fig. 4B).
4. Discussion The present study demonstrates that RAD001 inhibits HCC tumor growth in vivo, and enhances the sensitivity of HCC tumor cells in vitro and in vivo to cisplatin-induced
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Fig. 2 (continued)
cytotoxicity. Although the effect of RAD001 is independent of the p53 pathway, modification of p53 expression further enhances cisplatin-induced cytotoxicity in a p53 transfection background. Our study shows that RAD001 alone suppresses tumor cell proliferation in three HCC cell lines, HepG2, Hep3B and PLC, with different p53 characteristics. RAD001 inhibits tumor cell proliferation and arrests cells in G0/G1 phase in a dose-dependent manner. Surprisingly, although RAD001 alone did not induce apoptosis in HCC cells, RAD001 enhances cisplatin-mediated cytotoxicity in HCC cells both in vitro and in vivo. RAD001 combined with cisplatin induces a significant increase in apoptotic cell number compared to cisplatin alone. This was confirmed by the synergistic induction of PARP cleavage, and downregulation of pro-survival molecules, cyclin D1, survivin and Bcl-2. This synergistic effect was observed irrespective of
p53 status, as combined treatment had a similar effect on cell death and pro-apoptotic and anti-apoptotic pathways in all the three HCC cell lines, despite differing p53 characteristics. The above findings suggest that RAD001 enhances cisplatin-induced cytotoxicity in a p53 independent manner. In cell lines with wild-type or mutant forms of p53 (HepG2 and PLC), an upregulation of P-p53 was detected when RAD001 was combined with cisplatin, suggesting that RAD001 can also enhance cisplatin-mediated cytotoxicity through p53-dependent pathways. This effect was confirmed when wild-type p53 was transfected into Hep3B cells harboring a deletion mutant form of p53, before the cells were treated with RAD001 and cisplatin. RAD001 and cisplatin together induced a higher level of apoptosis in Hep3B-p53+ cells compared with Hep3B-p53 cells, indicating that the presence of p53 can also enhance RAD001-mediated chemosensitization. Expression of p21
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Fig. 3. Transfection of wild-type p53 enhanced RAD001-induced chemosensitization in Hep3B cells. (A). Hep3B cells were transfected with either vector control (Hep3B-p53) or wt p53 (Hep3B-p53+), before treated with either DMSO control or 10 nM RAD001 in combination with cisplatin for 48 h. Cell apoptosis was measured by cytofluorometric apoptosis analysis. Data represented means ± standard deviation of three independent experiments (*P < 0.05; **P < 0.01, compared with Hep3B-p53 cells by Two-way ANOVA). (B). The expression of P-S6 (Ser235/236), Pp53(Ser15), p53, p21, cyclin D1, survivin, Bcl-2 and b-actin was assessed by Western blot analysis.
was also detected in Hep3B-P53+ cells but not in Hep3Bp53 cells and RAD001 inhibited p21 expression in Hep3B-P53+ cells. p21 is one of the mechanisms to induce cell cycle arrest in DNA damaged cells, and this mechanism is important to allow subsequent DNA repair to protect cells from apoptosis. It has been reported that p21 modulated the nucleotide excision repair process to facilitate the repair after DNA damage [21]. Our data, as well as those by Beuvink and colleagues [14], have shown that RAD001 suppressed the expression of p21 in cells express-
Fig. 4. In vivo effect of RAD001 combined with cisplatin treatment in HCC xenografts. (A). Mice bearing Hep3B xenografts were treated with vehicle alone, RAD001 alone (5 mg/kg/day for 4 weeks), cisplatin alone (3 mg/kg per week for 4 weeks), or a combination of the two drugs. Mice were sacrificed after 4 weeks of treatment. Tumor growth was monitored twice a week. (B). TUNEL staining of tumor sections was performed after treatment. The apoptotic index was determined by counting the total number of positive nuclei in 20 randomly selected fields at 200 magnification. Data represented means ± standard deviation (**P < 0.01, compared with control group using One-way ANOVA).
ing wild-type p53. Combing with the report that antisense p21 sensitizes tumor cells to cisplatin-induced apoptosis [22], it is reasonable to suggest that p21 might participate in the enhanced effect of p53 on RAD001-mediated chemosensitization. Our data has shown that RAD001 enhanced the sensitivity of HCC cells to cisplatin in both p53-dependent and -independent manner. On the other hand, it seems that the effects of p53 on RAD001-mediated chemosensitization are tumor type specific [14,23], as various factors may also affect the function of mTOR, such as Akt, PTEN and S6K1. A recent study has shown that introduction of lesions on apoptosis into lymphomas reversed the effect of rapamycin mediated chemosensitization [24]. The response of mTOR inhibition on pro-survival molecules may depend on cellular context. Interestingly, our data has shown that the effect of RAD001 on the expression of Bcl-2, survivin and cyclin D1 was p53 independent. Downregulation of these pro-survival molecules could lower the
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apoptotic threshold and therefore sensitize HCC cells to cisplatin-induced apoptosis. RAD001 also enhanced the effects of cisplatin in vivo in a HCC xenograft model induced by Hep3B (p53) cells. By continuously monitoring the size of tumor nodules in nude mice, retardation of tumor growth was observed when animals were treated with cisplatin alone, RAD001 alone, or a combination of RAD001 and cisplatin. A significant observation was tumor regression in some animals administered the combination of RAD001 and cisplatin. Histological studies supported the finding that the combination of RAD001 and cisplatin induced more tumor cells to undergo apoptosis, compared with either RAD001 or cisplatin alone. This result suggests that the potent synergistic effect of RAD001 and cisplatin in vivo is also mediated by the induction of cell death. It was not possible to examine the effect of RAD001 on Hep3B-p53+ induced xenografts, due to the extremely slow rate of tumor growth when Hep3B cells were transfected with the tumor suppressor gene p53. The present study demonstrates that mTOR inhibition by RAD001 enhances cisplatin-mediated cytotoxicity in HCC through downregulation of pro-survival pathways, leading to a shift from anti-apoptotic pathways to proapoptotic signaling cascades in tumor cells. This is the first study to show that RAD001 enhances chemosensitivity of HCC to cisplatin through both p53-dependent and -independent pathways. This is an important finding for future treatment strategies for HCC since a large proportion of patients with HCC present with a mutant or deleted form of p53 in the HCC tumors. A therapeutic strategy combining cytotoxic agents with RAD001 is potentially effective for the treatment of HCC. After promising phase I studies, the therapeutic efficacy of various rapamycin derivatives, in monotherapy or in combination with interferon, is currently being assessed in several phase II/III studies with some showing long-lasting objective tumor responses [25–27]. Our data support a clinical trial of combining RAD001 with chemotherapy in patients with HCC. In conclusion, this study demonstrates that mTOR inhibition by RAD001 suppresses HCC tumor growth and sensitizes tumor cells to cisplatin-induced cytotoxicity. The RAD001-induced enhancement of cisplatin-induced cytotoxicity occurs via downregulation of pro-survival and pro-proliferation molecules. The involvement of both p53-dependent and -independent pathways in this process suggests that a strategy combining inhibition of mTOR with cytotoxic agents is a promising therapeutic approach for the treatment of HCC. Conflicts of interest statement The authors declare that they have no conflicts of interest to be disclosed regarding to this work.
Acknowledgements The Hep3B-p53+ cell line was kindly provided by Professor Paul BS Lai, Department of Surgery, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China. We also thank Prof. She-
ung Tat Fan, Department of Surgery, The University of Hong Kong, Hong Kong SAR, China, for his support and discussion in this project. References [1] F.X. Bosch, J. Ribes, M. Diaz, R. Cleries, Primary liver cancer: worldwide incidence and trends, Gastroenterology 127 (2004) S5– S16. [2] M. Schwartz, Liver transplantation for hepatocellular carcinoma, Gastroenterology 127 (2004) S268–S276. [3] R.T. Poon, S.T. Fan, C.M. Lo, I.O. Ng, C.L. Liu, C.M. Lam, J. Wong, Improving survival results after resection of hepatocellular carcinoma: a prospective study of 377 patients over 10 years, Ann. Surg. 234 (2001) 63–70. [4] J.M. Llovet, M. Sala, L. Castells, et al, Randomized controlled trial of interferon treatment for advanced hepatocellular carcinoma, Hepatology 31 (2000) 54–58. [5] J.C. Barbare, O. Bouche, F. Bonnetain, et al, Randomized controlled trial of tamoxifen in advanced hepatocellular carcinoma, J. Clin. Oncol. 23 (2005) 4338–4346. [6] C.M. Lo, H. Ngan, W.K. Tso, et al, Randomized controlled trial of transarterial lipiodol chemoembolization for unresectable hepatocellular carcinoma, Hepatology 35 (2002) 1164–1171. [7] Z.F. Yang, R.T. Poon, J. To, D.W. Ho, S.T. Fan, The potential role of hypoxia inducible factor 1a in tumor progression after hypoxia and chemotherapy in hepatocellular carcinoma, Cancer Res. 64 (2004) 5496–5503. [8] D.F. Calvisi, S. Ladu, A. Gorden, et al, Ubiquitous activation of Ras and Jak/Stat pathways in human HCC, Gastroenterology 130 (2006) 1117–1128. [9] K. Nakanishi, M. Sakamoto, S. Yamasaki, S. Todo, S. Hirohashi, Akt phosphorylation is a risk factor for early disease recurrence and poor prognosis in hepatocellular carcinoma, Cancer 103 (2005) 307–312. [10] M. Olivier, R. Eeles, M. Hollstein, M.A. Khan, C.C. Harris, P. Hainaut, The IARC TP53 database: new online mutation analysis and recommendations to users, Human Mutat. 19 (2002) 607–614. [11] S. Faivre, G. Kroemer, E. Raymond, Current development of mTOR inhibitors as anticancer agents, Nat. Rev. Drug Discov. 5 (2006) 671– 688. [12] W. Sieghart, T. Fuereder, K. Schmid, et al, Mammalian target of rapamycin pathway activity in hepatocellular carcinomas of patients undergoing liver transplantation, Transplantation 83 (2007) 425– 432. [13] H. Yan, P. Frost, Y. Shi, B. Hoang, S. Sharma, M. Fisher, J. Gera, A. Lichtenstein, Mechanism by which mammalian target of rapamycin inhibitors sensitize multiple myeloma cells to dexamethasoneinduced apoptosis, Cancer Res. 66 (2006) 2305–2313. [14] I. Beuvink, A. Boulay, S. Fumagalli, et al, The mTOR inhibitor RAD001 sensitizes tumor cells to DNA-damaged induced apoptosis through inhibition of p21 translation, Cell 120 (2005) 747–759. [15] W.H. Mondesire, W. Jian, H. Zhang, J. Ensor, M.C. Hung, G.B. Mills, F. Meric-Bernstam, Targeting mammalian target of rapamycin synergistically enhances chemotherapy-induced cytotoxicity in breast cancer cells, Clin. Cancer Res. 10 (2004) 7031–7042. [16] L. Wu, D.C. Birle, I.F. Tannock, Effects of the mammalian target of rapamycin inhibitor CCI-779 used alone or with chemotherapy on human prostate cancer cells and xenografts, Cancer Res. 65 (2005) 2825–2831. [17] H.G. Wendel, E. De Stanchina, J.S. Fridman, et al, Survival signaling by Akt and eIF4E in oncogenesis and cancer therapy, Nature 428 (2004) 332–337. [18] V. Grunwald, L. DeGraffenried, D. Russel, W.E. Friedrichs, R.B. Ray, M. Hidalgo, Inhibitors of mTOR reverse doxorubicin resistance conferred by PTEN status in prostate cancer cells, Cancer Res. 62 (2002) 6141–6145. [19] T.Y. Chi, G. Chen, L.K. Ho, P. Lai, Establishment of a doxycyclineregulated cell line with inducible, doubly-stable expression of the wild-type p53 gene from p53-deleted hepatocellular carcinoma cells, Cancer Cell Int. 5 (2005) 27. [20] R.L. Aft, J.S. Lewis, F. Zhang, J. Kim, M.J. Welch, Enhancing targeted radiotherapy by copper(II)diacetyl-bis(N4-methylthiosemicarbazone) using 2-deoxy-D-glucose, Cancer Res. 63 (2003) 5496–5504. [21] M.S. Sheikh, Y.Q. Chen, M.L. Smith, A.J. Fornace Jr., Role of p21Waf1/ Cip1/Sdi1 in cell death and DNA repair as studied using a tetracycline-inducible system in p53-deficient cells, Oncogene (1997) 1875–1882.
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Contents lists available at ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Inhibition of mTOR enhances chemosensitivity in hepatocellular carcinoma q Ka Ho Tam a, Zhen Fan Yang a, Chi Keung Lau a, Chi Tat Lam a, Roberta W.C. Pang b, Ronnie T.P. Poon a,* a b
Center for Cancer Research, Department of Surgery, The University of Hong Kong, Queen Mary Hospital, 102 Pokfulam Road, Pokfulam, Hong Kong, China Center for Cancer Research, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, 102 Pokfulam Road, Pokfulam, Hong Kong, China
a r t i c l e
i n f o
Article history: Received 25 March 2008 Received in revised form 25 March 2008 Accepted 4 August 2008
Keywords: mTOR p53 RAD001 HCC Chemosensitivity Chemoresistance
a b s t r a c t The present study investigated the effect of mammalian target of rapamycin (mTOR) inhibition on HCC cells in vitro and in vivo, either alone or in combination with cytotoxic agents. In vitro, HCC cell lines were exposed to RAD001, an mTOR inhibitor, either alone or in combination with cisplatin. Alone, RAD001 suppressed cell proliferation in all cell lines tested, but did not induce apoptosis. RAD001 in combination with cisplatin induced a significant increase in the number of apoptotic cells, downregulated the expression of pro-survival molecules, Bcl-2, survivin and cyclinD1, and increased the cleavage of PARP, compared to RAD001 or cisplatin alone. Transfection of p53 into the Hep3B cell line increased the sensitivity of tumor cells to cisplatin. The suppression of HCC tumor growth in vivo was enhanced by RAD001 combined with cisplatin, accompanied by a significant increase in the number of apoptotic cells in tumor tissues. This study demonstrates that inhibition of mTOR suppresses tumor growth and sensitizes tumor cells to chemocytotoxic agents. Ó 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Hepatocellular carcinoma (HCC) is the third most common cause of cancer death with a 5-year survival rate of 7% [1]. Hepatic resection and liver transplantation are the two mainstays of curative treatment for HCC, but can only be q Financial support: The study was supported by the Seed Funding Program for Basic Research 2006 and Sun Chieh Yeh Research Foundation for Hepatobiliary and Pancreatic Surgery of the University of Hong Kong. * Corresponding author. Tel.: +852 2855 3641; fax: +852 2817 5475. E-mail address: [email protected] (R.T.P. Poon). Abbreviations: HCC, hepatocellular carcinoma; TACE, transarterial chemoembolization; S6K1, ribosomal p70S6 kinase; 4E-BP1, eukaryotic initiation factor eIF4E binding protein; P-, phosphorylated-; wt, wildtype; DMEM, Dulbecco’s modified Eagle medium; FBS, fetal bovine serum; CO2, carbon dioxide; DMSO, dimethyl sulfoxide; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate buffered saline; RIPA, radioimmunoprecipitation assay; i.p., intra-peritoneal; H&E, hematoxylin and eosin; TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling; ANOVA, analysis of variance between groups; SD, standard deviation.
0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.08.018
applied to the early stage of HCC [2,3]. The majority of patients with HCC are diagnosed at a late stage when curative treatment options are not applicable. Currently, there is no proven effective systemic chemotherapy for HCC due to resistance of tumor cells to cytotoxic drugs [4,5]. The efficacy is limited for local chemotherapy, using agents such as cisplatin by means of transarterial chemoembolization (TACE), due to the poor response rate of tumor cells to treatment and tumor re-growth after treatment [6,7]. These reasons exemplify the need to fully understand the mechanism underlying chemoresistance in HCC to allow the design of more effective therapeutic strategies. Signaling pathways regulating cell growth and survival, such as those involving p53 and PTEN/PI3K/AKT, are often dysregulated in HCC [8–10]. Molecules targeting these abnormalities may suppress tumor progression and restore chemosensitivity of HCC cells. The mammalian target of rapamycin, mTOR, is a potential target for anti-cancer therapy as it is a key molecule in the PTEN/PI3K/AKT/mTOR signaling pathway and plays a critical role in controlling
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cell proliferation and survival. Cap-dependent translation is regulated by mTOR through ribosomal p70S6 kinase (S6K1) and the eukaryotic initiation factor eIF4E binding protein (4E-BP1), respectively. This control has important implications in tumorigenesis, cell survival and apoptosis. Abnormal activation of the PTEN/PI3K/AKT/mTOR signaling pathway frequently occurs in human cancers, including HCC [11]. A recent study showed that P-mTOR was expressed in 41% of HCC tumor tissue but not in adjacent cirrhotic liver tissue [12], suggesting that mTOR might be a specific therapeutic target for HCC. Several mTOR inhibitors, including rapamycin/sirolimus (Wyeth) and derivatives, such as RAD001/everolimus (Novartis), CCI-779/temsirolimus (Wyeth) and AP23573 (Ariad), are being developed as anti-cancer agents against various types of malignancies. Therapeutic approaches focused on mTOR inhibition alone can retard tumor growth, but cannot eradicate the tumor. This is because mTOR inhibitors are cytostatic not cytotoxic and arrest cancer cells in G1 phase of the cell cycle. Based on the role of mTOR in chemoresistance, the combination of an mTOR inhibitor and a cytotoxic agent may achieve an ideal therapeutic outcome by combining cancer cell cycle arrest and death. Inhibition of mTOR can enhance the efficacy of a broad range of chemocytotoxic agents, including cisplatin, doxorubicin, paclitaxel, carboplatin, dexamethasone, mitoxantrone and docetaxel, in various types of human cancers [13–16]. The mechanism underlying mTOR mediated enhancement of chemosensitivity remains unclear. It is postulated that chemosensitization depends on the activity of Akt, PTEN and p53, and loss of PTEN and aberrant Akt activity correlates with the upregulation of mTOR activity, thus rendering tumor cells sensitive to mTOR inhibitors [13,17,18]. The presence of functional p53 is essential for the sensitization of tumor cells to cisplatin by mTOR inhibitors [14]. A large proportion of HCC tumors present with a p53 mutation or deletion. Furthermore, the effect of mTOR inhibition on the chemosensitivity of HCC remains unknown. For these reasons, the present study was designed to investigate the anti-tumor effect of RAD001, an mTOR inhibitor, either alone or in combination with cisplatin, a conventional chemocytotoxic agent commonly used for the treatment of HCC, with a focus on p53-related signaling pathways. 2. Materials and methods 2.1. Cell lines, reagents and culture conditions Human hepatocellular carcinoma cell lines, HepG2 (wt p53), PLC (mutant p53) and Hep3B (p53 null), were purchased from the American Type Culture Collection (ATCC, Manassas, VA). A Hep3B cell line with wild-type p53 stable transfection was kindly provided by Prof. B.S. Lai, Chinese University of Hong Kong [19]. All cell lines were cultured in DMEM medium with 10% FBS and 1% penicillin and streptomycin (Life Technologies, Carlsbad, CA), and incubated at 37 °C in a humidified atmosphere with 5% CO2 in air. RAD001 (everolimus) was kindly provided by Novartis Institutes for Biomedical Research, Basel, Switzerland. A 10 mM RAD001 stock solution for cell culture was dis-
solved in DMSO (Sigma–Aldrich, St. Louis, MO), stored at 20 °C and diluted in fresh culture medium immediately before use. For in vivo studies, RAD001 and placebo microemulsion stocks were stored at 20 °C and diluted with 5% glucose immediately before use. Cisplatin was obtained from Amersham and Upjohn, Buckinghamshire, United Kingdom. 2.2. Determination of cell viability by 3,[4,5-dimethylthiazol2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay HCC cells were seeded onto a 96-well plate at a density of 1 104 cells per well. After 24 h, cells were exposed to different concentrations of RAD001, with or without cisplatin, in growth medium. After incubation for indicated time points, MTT was added to each well and incubated for another 4 h. The reaction was stopped with 0.01 N hydrochloric acid in 10% SDS solution and the absorbance was measured at 540/650 nm using a Vmax kinetic microplate reader (Bio-Rad Laboratories, Hercules, CA). Each sample was assayed in triplicate, and three independent experiments were performed for each group. 2.3. Cell cycle and cytofluorometric apoptosis analysis For cell cycle analysis, treated cells were harvested and fixed in absolute ethanol at 4 °C for 12 h. Cells were washed twice with PBS then stained with propidium iodide at 37 °C for 30 min. Cell cycle analysis was performed on a FACSCalibur (Becton Dickinson immunocytometry Systems, San Jose, CA). Cytofluorometric apoptosis analysis was performed by harvesting cells, labeling with the Annexin V-FITC antibody (BD Biosciences PharMingen, San Diego, CA) at room temperature for 15 min, and then analyzing apoptosis on a FACSCalibur. 2.4. Western blot analysis Total protein was extracted from treated cells using radioimmunoprecipitation assay (RIPA) buffer. Protein levels of P-S6 (Ser235/236), P-p70S6K (Thr389), P-4E-BP1 (Ser65), Bcl-2, PARP, P-p53 (Ser15), cyclin D1, p53, p21, survivin and actin were detected using standard Western blot analysis on 8–15% SDS–PAGE. Rabbit anti-human PS6 [Ser235/236], P-p70S6K [Thr389], P-4E-BP1 [Ser65], Bcl-2 and PARP polyclonal antibodies, mouse anti-human P-p53 [Ser15], p21 and cyclin D1 monoclonal antibodies were from Cell Signaling Technology (Beverly, MA). Mouse anti-human p53 monoclonal antibody was from Dako Corp. (Carpenteria, CA). Rabbit anti-human survivin polyclonal antibody was from Oncogene Research Products (Calbiochem, San Diego, CA). Mouse anti-human actin monoclonal antibody was from Sigma. 2.5. HCC xenograft in nude mice Four-week old male athymic nude mice were purchased from the Laboratory Animal Unit of the University of Hong Kong and housed in microisolator cages under positive air pressure maintained at a constant temperature (22 °C) and humidity, in a 12 h light/dark cycle. Sterilized rodent food
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Treatment time (hours) Fig. 1. RAD001 inhibits proliferation of human hepatocellular carcinoma (HCC) cell lines. (A) Cells were treated with indicated concentrations of RAD001. At different time points after exposure, cell proliferation was quantified by the MTT assay. Data represents means ± standard deviation of three independent experiments (**P < 0.01, compared with control by One-way ANOVA). (B) PLC cells were treated with either DMSO (0.1%) or 10 nM RAD001 for 48 h, harvested, permeabilized with ethanol and labeled with propidium iodide. Cell cycle fractions were measured by flow cytometric analysis. (C) RAD001 inhibited activation of mTOR targets. Cells were treated with 10 nM RAD001 for 24 h. The expression of P-S6 (Ser235/236), P-p70S6K (Thr389), P-4E-BP1 (Ser65) and b-actin was assessed by Western blot analysis.
and water were available ad libitum. All animal experiments were approved by the Committee on the Use of Live Animals in Teaching and Research, the University of Hong Kong. For generation of HCC xenografts, 3 106 Hep3B cells were injected subcutaneously into the nude mice using a 23-gauge needle. Mice were then placed randomly into four groups (n = 7 in each group). Treatment was initiated when tumor nodules reached 100–200 mm3 and persisted for 4 weeks. Experimental groups included: (1) vehicle alone, (2) cisplatin alone (3 mg/kg per week, i.p.), (3) RAD001 alone (5 mg/kg/day, orally), (4) a combination of cisplatin and RAD001. Tumor size was monitored continuously using three-dimensional caliper measurements and tumor volume was calculated using the following formula [20].
Volume ¼ Length Width Depth 3:14=6
mination of histology by H&E staining and detection of apoptotic cells by terminal deoxynucleotidyl transferase– mediated dUTP nick end labeling (TUNEL, Roche, Basel, Switzerland). The apoptotic index was determined by counting the total number of positive nuclei in 20 randomly selected fields at 200 magnification. 2.7. Statistical analysis Statistical analyses were performed using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA). Comparisons of optical density values, percentage of viable cells and number of apoptotic cells between different groups were performed using the two-tailed Student’s t test or ANOVA. P < 0.05 was considered statistically significant. 3. Results
2.6. Histological studies 3.1. Inhibition of HCC cell proliferation and mTOR activity by RAD001
Mice were sacrificed when treatment was completed. Tumor tissue was fixed in 10% buffered formalin, embedded in paraffin and cut into sections 5 lm thick for deter-
To examine the effects of RAD001 on HCC cell proliferation, three HCC cell lines (HepG2, PLC and Hep3B) were treated with 0.01–10 nM RAD001 for 72 h. Significant dose-dependent inhibition of cell proliferation was
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Actin Fig. 1 (continued)
observed in all three cell lines with 1–10 nM RAD001. RAD001-induced inhibition of cell proliferation was more pronounced in PLC cells than in HepG2 or Hep3B cells. Cell proliferation was inhibited only in PLC cells at 0.1 nM RAD001. Cell proliferation was inhibited by 60% in PLC cells and by 40% and 20% in HepG2 and Hep3B cells, respectively, after 72 h exposure to 10 nM RAD001 (Fig. 1A). Cell apoptosis assessed by flow cytometry or cellular morphology was not induced in any of the three HCC cell lines by RAD001. RAD001 was shown to inhibit cell cycle progression due to an increase in the proportion of cells in G0-G1 phase and decreases in the proportion of cells in S phase (Fig. 1B). Western blot analysis of HCC cells exposed to vehicle (DMSO) or 10 nM RAD001 for 24 h was used to investigate the molecular mechanisms induced by RAD001 (Fig. 1C). RAD001 induced a decrease in the phosphorylation of three downstream molecules of mTOR, including p70S6K (Thr389), ribosomal S6 (Ser235/236) and 4E-BP1 (Ser65). This result confirms that mTOR activity was inhibited by RAD001. The inhibitory effect of RAD001 on the phosphorylation of p70S6K (Thr389) was most prominent in PLC cells (Fig. 1C).
platin administration induced the proteolytic cleavage of PARP, and when cisplatin was combined with RAD001, a further increase in the expression of cleaved PARP was detected in all the three cell lines (Fig. 2C). Exposure to cisplatin inhibited the phosphorylation of p70S6K (Thr389) and 4E-BP1 (Ser65) in all the three HCC cell lines. This inhibitory effect was enhanced when cisplatin was combined with RAD001 in HepG2 cells. Exposure to cisplatin upregulated the expression of P-p53 (Ser15) in HepG2 and PLC cells, and the expression of p53 (Ser15) was further augmented in PLC cells when RAD001 was combined with cisplatin (Fig. 2D). To further analyze the molecular mechanism of RAD001-mediated chemosensitization, Western blot analysis was used to evaluate the expression of pro-survival molecules. Exposure to cisplatin alone inhibited the expression of pro-survival molecules, Bcl-2, cyclin D1 and survivin, in all the three HCC cell lines. Cisplatin alone decreased the expression of Bcl-2. Synergistic inhibition of expression of the above molecules was detected when RAD001 was combined with cisplatin (Fig. 2E).
3.2. Enhancement of cisplatin-induced cell apoptosis by RAD001
3.3. Enhancement of RAD001-mediated chemosensitization of Hep3B cells by p53
The synergistic effect of RAD001 and cisplatin on cell viability and apoptosis was examined in HepG2, PLC and Hep3B cell lines. Cell proliferation was inhibited in HepG2 cell line after exposure to cisplatin alone for 48 h. Cell proliferation was further inhibited when 10 nM RAD001 was combined with different doses of cisplatin, particularly in HepG2 and PLC cell lines (Fig. 2A). Cytofluorometric apoptosis analysis showed no alteration in the number of apoptotic cells in all the three cell lines exposed to RAD001 alone. By comparison, a significant increase in the number of apoptotic cells was detected in all the three cell lines when RAD001 was combined with cisplatin (Fig. 2B). RAD001 treatment did not induce proteolytic cleavage of PARP (86 kDa), a marker for cell apoptosis. This result is consistent with our finding using the cytofluorometric apoptosis assay that RAD001 does not induce cell apoptosis. On the contrary, cis-
To further characterize the role of p53 in RAD001-mediated chemosensitization, wild-type p53 was transfected into Hep3B cells, which have a deleted form of p53. Hep3B cells (Hep3B-p53) and Hep3B-p53 stable transfectants (Hep3B-p53+) were exposed to 10 nM RAD001 in the presence or absence of cisplatin. Cytofluorometric apoptosis analysis showed a significant increase in the number of apoptotic cells in Hep3B-p53+ cells compared to Hep3B-p53 cells when RAD001 was combined with cisplatin. The expression of p53 (Ser15) is induced by cisplatin in Hep3Bp53+ cells, consistent with the results for HepG2 cells ( Fig. 3A and B). The expression of p21 was downregulated by RAD001 in Hep3B-p53+ cells. However, there was no significant difference in the effect of RAD001 on the expression of Bcl-2, survivin and cyclin D1 between Hep3B-p53+ and Hep3B-p53 cells.
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Fig. 2. RAD001 sensitized HCC tumor cells with different p53 status, HepG2 (wt p53), PLC (mutant p53) and Hep3B (p53 null), to cisplatin cytotoxicity. (A) and (B). Cells were treated with either DMSO control or 10 nM RAD001 in combination with cisplatin for 48 h. Cell viability and apoptosis were measured by the MTT assay and cytofluorometric apoptosis analysis, respectively. Data represented means ± standard deviation of three independent experiments (*P < 0.05, compared with control by two-tailed t tests and One-way ANOVA). (C), (D) and (E). Cells were treated with either DMSO control or 10 nM RAD001 in combination with 5 lg/ml cisplatin for 24 h. The expression level of PARP, P-p70S6K (Thr389), P-4E-BP1 (Ser65), P-p53 (Ser15), p53, cyclin D1, survivin, Bcl-2 and b-actin was assessed by Western blot analysis.
3.4. Synergistic anti-tumor activity by RAD001 and cisplatin The Hep3B xenograft model in nude mice was employed to investigate the anti-tumor effect of RAD001 and cisplatin in vivo. Treatment of mice with cisplatin or RAD001 alone suppressed tumor growth (Fig. 4A). When RAD001 was combined with cisplatin, tumor growth was inhibited synergistically. Tumor regression was observed in two out of seven animals administered both RAD001 and cisplatin. The TUNEL assay was performed to assess apoptotic cells in tumor tissue after different treatment regimens to determine whether the anti-tumor effect of RAD001 and cisplatin in vivo was mediated by the induction of cell apoptosis. RAD001 alone did not induce cell apoptosis in tumor tis-
sue, whereas cisplatin alone induced an increase in the number of apoptotic cells (24.7 ± 8.9 versus 12.4 ± 8.5 in non-treatment control). A further augmentation of cell apoptosis was observed when mice were treated with RAD001 together with cisplatin (44.9 ± 10.9 versus 16.45 ± 10.2 for RAD001 alone, and 24.7 ± 8.9 for cisplatin alone) (Fig. 4B).
4. Discussion The present study demonstrates that RAD001 inhibits HCC tumor growth in vivo, and enhances the sensitivity of HCC tumor cells in vitro and in vivo to cisplatin-induced
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Fig. 2 (continued)
cytotoxicity. Although the effect of RAD001 is independent of the p53 pathway, modification of p53 expression further enhances cisplatin-induced cytotoxicity in a p53 transfection background. Our study shows that RAD001 alone suppresses tumor cell proliferation in three HCC cell lines, HepG2, Hep3B and PLC, with different p53 characteristics. RAD001 inhibits tumor cell proliferation and arrests cells in G0/G1 phase in a dose-dependent manner. Surprisingly, although RAD001 alone did not induce apoptosis in HCC cells, RAD001 enhances cisplatin-mediated cytotoxicity in HCC cells both in vitro and in vivo. RAD001 combined with cisplatin induces a significant increase in apoptotic cell number compared to cisplatin alone. This was confirmed by the synergistic induction of PARP cleavage, and downregulation of pro-survival molecules, cyclin D1, survivin and Bcl-2. This synergistic effect was observed irrespective of
p53 status, as combined treatment had a similar effect on cell death and pro-apoptotic and anti-apoptotic pathways in all the three HCC cell lines, despite differing p53 characteristics. The above findings suggest that RAD001 enhances cisplatin-induced cytotoxicity in a p53 independent manner. In cell lines with wild-type or mutant forms of p53 (HepG2 and PLC), an upregulation of P-p53 was detected when RAD001 was combined with cisplatin, suggesting that RAD001 can also enhance cisplatin-mediated cytotoxicity through p53-dependent pathways. This effect was confirmed when wild-type p53 was transfected into Hep3B cells harboring a deletion mutant form of p53, before the cells were treated with RAD001 and cisplatin. RAD001 and cisplatin together induced a higher level of apoptosis in Hep3B-p53+ cells compared with Hep3B-p53 cells, indicating that the presence of p53 can also enhance RAD001-mediated chemosensitization. Expression of p21
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Fig. 3. Transfection of wild-type p53 enhanced RAD001-induced chemosensitization in Hep3B cells. (A). Hep3B cells were transfected with either vector control (Hep3B-p53) or wt p53 (Hep3B-p53+), before treated with either DMSO control or 10 nM RAD001 in combination with cisplatin for 48 h. Cell apoptosis was measured by cytofluorometric apoptosis analysis. Data represented means ± standard deviation of three independent experiments (*P < 0.05; **P < 0.01, compared with Hep3B-p53 cells by Two-way ANOVA). (B). The expression of P-S6 (Ser235/236), Pp53(Ser15), p53, p21, cyclin D1, survivin, Bcl-2 and b-actin was assessed by Western blot analysis.
was also detected in Hep3B-P53+ cells but not in Hep3Bp53 cells and RAD001 inhibited p21 expression in Hep3B-P53+ cells. p21 is one of the mechanisms to induce cell cycle arrest in DNA damaged cells, and this mechanism is important to allow subsequent DNA repair to protect cells from apoptosis. It has been reported that p21 modulated the nucleotide excision repair process to facilitate the repair after DNA damage [21]. Our data, as well as those by Beuvink and colleagues [14], have shown that RAD001 suppressed the expression of p21 in cells express-
Fig. 4. In vivo effect of RAD001 combined with cisplatin treatment in HCC xenografts. (A). Mice bearing Hep3B xenografts were treated with vehicle alone, RAD001 alone (5 mg/kg/day for 4 weeks), cisplatin alone (3 mg/kg per week for 4 weeks), or a combination of the two drugs. Mice were sacrificed after 4 weeks of treatment. Tumor growth was monitored twice a week. (B). TUNEL staining of tumor sections was performed after treatment. The apoptotic index was determined by counting the total number of positive nuclei in 20 randomly selected fields at 200 magnification. Data represented means ± standard deviation (**P < 0.01, compared with control group using One-way ANOVA).
ing wild-type p53. Combing with the report that antisense p21 sensitizes tumor cells to cisplatin-induced apoptosis [22], it is reasonable to suggest that p21 might participate in the enhanced effect of p53 on RAD001-mediated chemosensitization. Our data has shown that RAD001 enhanced the sensitivity of HCC cells to cisplatin in both p53-dependent and -independent manner. On the other hand, it seems that the effects of p53 on RAD001-mediated chemosensitization are tumor type specific [14,23], as various factors may also affect the function of mTOR, such as Akt, PTEN and S6K1. A recent study has shown that introduction of lesions on apoptosis into lymphomas reversed the effect of rapamycin mediated chemosensitization [24]. The response of mTOR inhibition on pro-survival molecules may depend on cellular context. Interestingly, our data has shown that the effect of RAD001 on the expression of Bcl-2, survivin and cyclin D1 was p53 independent. Downregulation of these pro-survival molecules could lower the
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apoptotic threshold and therefore sensitize HCC cells to cisplatin-induced apoptosis. RAD001 also enhanced the effects of cisplatin in vivo in a HCC xenograft model induced by Hep3B (p53) cells. By continuously monitoring the size of tumor nodules in nude mice, retardation of tumor growth was observed when animals were treated with cisplatin alone, RAD001 alone, or a combination of RAD001 and cisplatin. A significant observation was tumor regression in some animals administered the combination of RAD001 and cisplatin. Histological studies supported the finding that the combination of RAD001 and cisplatin induced more tumor cells to undergo apoptosis, compared with either RAD001 or cisplatin alone. This result suggests that the potent synergistic effect of RAD001 and cisplatin in vivo is also mediated by the induction of cell death. It was not possible to examine the effect of RAD001 on Hep3B-p53+ induced xenografts, due to the extremely slow rate of tumor growth when Hep3B cells were transfected with the tumor suppressor gene p53. The present study demonstrates that mTOR inhibition by RAD001 enhances cisplatin-mediated cytotoxicity in HCC through downregulation of pro-survival pathways, leading to a shift from anti-apoptotic pathways to proapoptotic signaling cascades in tumor cells. This is the first study to show that RAD001 enhances chemosensitivity of HCC to cisplatin through both p53-dependent and -independent pathways. This is an important finding for future treatment strategies for HCC since a large proportion of patients with HCC present with a mutant or deleted form of p53 in the HCC tumors. A therapeutic strategy combining cytotoxic agents with RAD001 is potentially effective for the treatment of HCC. After promising phase I studies, the therapeutic efficacy of various rapamycin derivatives, in monotherapy or in combination with interferon, is currently being assessed in several phase II/III studies with some showing long-lasting objective tumor responses [25–27]. Our data support a clinical trial of combining RAD001 with chemotherapy in patients with HCC. In conclusion, this study demonstrates that mTOR inhibition by RAD001 suppresses HCC tumor growth and sensitizes tumor cells to cisplatin-induced cytotoxicity. The RAD001-induced enhancement of cisplatin-induced cytotoxicity occurs via downregulation of pro-survival and pro-proliferation molecules. The involvement of both p53-dependent and -independent pathways in this process suggests that a strategy combining inhibition of mTOR with cytotoxic agents is a promising therapeutic approach for the treatment of HCC. Conflicts of interest statement The authors declare that they have no conflicts of interest to be disclosed regarding to this work.
Acknowledgements The Hep3B-p53+ cell line was kindly provided by Professor Paul BS Lai, Department of Surgery, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China. We also thank Prof. She-
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