Abstract
Background: Survivin is a negative regulator of apoptosis. We evaluated the efficacy of YM155, a selective suppressant of survivin, in combination with gemcitabine in the pancreatic cancer cell line MiaPaCa-2. Materials and Methods: Expression of survivin was demonstrated by immunoblotting. Cell cycle progression was determined by flow cytometric analysis. Cell viability was assayed using the trypan blue exclusion assay. Results: Gemcitabine up-regulated survivin expression, whereas treatment with YM155 suppressed the expression of survivin. Concomitant treatment with YM155 enhanced chemosensitivity to gemcitabine, which was accompanied by a decrease in the expression of survivin. Knockdown of endogenous survivin via RNA interference also enhanced the sensitivity to gemcitabine. In addition, YM155 potentiated the antitumor effect of gemcitabine in xenograft tumors of MiaPaCa-2. Conclusion: YM155 potentiates chemosensitivity to gemcitabine in pancreatic cancer cells by suppressing the induction of survivin. Combination treatment with gemcitabine and YM155 may be a potential therapeutic strategy for the treatment of pancreatic cancer that warrants further clinical investigation.
Pancreatic cancer has an exceptionally high mortality rate, making it one of the most common causes of cancer mortality in developed countries (1). Although chemotherapy, typically with the nucleoside analog gemcitabine, is the standard of care in patients with advanced or recurrent pancreatic cancer, the median overall survival in patients with advanced pancreatic cancer is less than one year, with response rates of around 20% to 40% (2, 3). There is, therefore, an unmet need for more efficacious and less toxic treatment options for patients with advanced pancreatic cancer.
Targeted therapy has changed the paradigm in the treatment of advanced cancer. Imatinib, a small-molecule tyrosine kinase inhibitor that inhibits BCR-ABL, c-KIT, and platelet-derived growth factor receptor alpha, has revolutionized the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors. In addition, the recent development of molecularly targeted agents has added to the armamentarium of systemic therapeutic agents used to treat breast, colon, lung, and gastric cancers.
Angiogenesis and the epidermal growth factor receptor (EGFR) pathway are the major molecular targets that have been actively investigated in the treatment of advanced pancreatic cancer. Despite numerous trials with diverse targeted agents, erlotinib is the only agent that, in combination with gemcitabine, has shown statistically significant improvements in survival. However, even this therapy disappointingly provides just 10 days of overall survival gain (4).
Defective apoptosis (programmed cell death) is a major causative factor in the development and progression of cancer (5). Survivin, a member of the inhibitor of apoptosis (IAP) family, functions as inhibit-or of caspase activation, thereby leading to negative regulation of apoptosis or programmed cell death, and also plays a central role in cell division (6, 7). Although it is expressed at high levels during fetal development, survivin is rarely expressed in normal healthy adult tissues. It is, however, up-regulated in the majority of cancer types making it a potential target for anticancer therapy (6). Survivin is also involved in the development of human pancreatic duct cell tumors (8) and is expressed in the majority of pancreatic carcinomas where its expression correlates with both cellular proliferation and apoptosis (9).
YM155 is a small-molecule suppressant of survivin that was developed by cell-based high-throughput screening and lead optimization (10). YM155 selectively suppresses survivin expression, resulting in activation of caspases and induction of apoptosis. Continuous infusion of YM155 has also been found to induce intratumoral survivin suppression and tumor regression in established human hormone refractory prostate cancer (HRPC) and non-Hodgkin lymphoma (NHL) tumor xenografts (10, 11). In clinical settings, YM155 was shown to be tolerable in phase I studies with advanced cancer patients and showed antitumor activity in those with NHL and HRPC (12, 13). Multicenter phase II trials demonstrated the safety and tolerability of YM155 in patients with unresectable melanoma (14) and advanced refractory non-small cell lung cancer (15). Given that survivin is intimately involved in cell cycle and apoptosis, and YM155 showed efficacy against human cancers in both preclinical and clinical settings, we postulated that a combination of YM155 and gemcitabine might show promising efficacy against pancreatic cancer.
Here, we evaluated the therapeutic potential of YM155 alone and in combination with gemcitabine in in vitro and in vivo preclinical pancreatic cancer models.
Materials and Methods
Cell culture, plasmids, reagents, and transfection. Human pancreatic cancer cell line MiaPaCa2, was purchased from the American Type Culture Collection. The cells were maintained at 37°C in an atmosphere with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Grand Island, NY, USA) with 10% heat-inactivated fetal bovine serum. YM155 monobromide was purchased from SelleckChem (Houston, TX, USA). For in vitro experiments, gemcitabine was purchased from Sigma-Aldrich (St. Louis, MO, USA) and was dissolved and diluted in saline immediately prior to use. For in vitro and in vivo experiments, the dose levels of YM155 are expressed as the cationic moiety of the drug.
In vitro cell proliferation and cytotoxicity assay. Briefly, after drug treatment for 48 h, the cell count was determined using a sulforhodamine B (SRB) colorimetric assay (16). Cell viability was determined by trypan blue exclusion, by counting at least 300 cells of each culture. The number of trypan blue-stained cells was recorded after incubation with the drug for 72 h.
Cell cycle analysis. For each analysis of DNA content, 1×106 treated cells were harvested by trypsinization and were fixed by rapid submersion in 1 ml cold 70% ethanol. After fixation at −20°C for at least 1 h, cells were pelleted, resuspended in 1 ml staining solution (50 μg/ml propidium iodide, 50 μg/ml RNase, 0.1% Triton X-100 in citrate buffer, pH 7.8), and were then washed with phosphate-buffered saline. Stained cells were transferred to polystyrene tubes with cell-strainer caps (Falcon), sorted using a fluorescence-activated cell sorter (BD FACSCalibur), and analyzed with the Cell Quest 3.2 software (Becton Dickinson, NJ, USA) (17).
Immunoblot analysis of treated cells. Cell lysates were prepared with radioimmune precipitation assay (RIPA) lysis buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 μM EGTA, 1% Triton X-100) containing a protease inhibitor cocktail (Sigma-Aldrich, St.Louis, MO, USA). Protein concentrations in extracts were determined using the Bradford assay, and 30 μg of total cell protein/sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to PolyScreen membranes (PerkinElmer Life Sciences, Waltham, MA, USA). Membranes were blocked with 5% nonfat dry milk in TBST buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) and were probed with one of the following antibodies: anti-survivin (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-cleaved poly ADP ribose polymerase (PARP), anti-phospho-H2AX (p-H2AX), and anti-phospho-CHK1 (p-Chk1) (Cell Signaling Technology, Danvers, MA, USA). Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies were used in conjunction with enhanced chemiluminescence detection (Amersham Biosciences, Piscataway, NJ, USA) to develop the western blots.
Reverse transcription-polymerase chain reaction (RT-PCR) analysis. For conventional RT-PCR, total RNA was extracted from MiaPaCa-2 cells after treatment with YM155 using TRIzol® (Invitrogen, Carlsbad, CA, USA) and cDNA was synthesized using avian myeloblastosis virus reverse transcriptase with oligo (dT15) as a primer. Targets were amplified from cDNA using the following primers: survivin: sense: 5’-CAG ATT TGA ATC GCG GGA CCC-3’, and antisense: 5’-CCA AGT CTG GCT CGT TCT CAG-3’; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sense: 5’-AGA AGG CTG GGG CTC ATT TG-3’, and antisense: 5’-AGG GGC CAT CCA CAG TCT TC-3’. The PCR conditions were 35 cycles of 30 s at 94°C, 30 s at 60°C, and 30 s at 72°C for survivin and 30 cycles of 30 s at 94°C, 30 s at 53°C and 30 s at 72°C for GAPDH. PCR products were analyzed by standard electrophoresis in agarose gels.
RNA interference. MiaPaCa-2 cells were transiently transfected with scrambled small interfering RNA (siRNA) (Genolution Pharmaceuticals, Inc., Korea) or survivin siRNA (5’-AAG GAC CAC CGC AUC UCU ACA-3’) using Lipofectamine™ RNAiMAX (Invitrogen) according to the manufacturer's instructions.
Tumor xenograft model. Five-week-old female BALB/c-nu/nu mice were obtained from Japan SLC Inc. and maintained for one week in our animal facility before tumor inoculation. Animals were housed on a 12-h light/dark cycle with food and water provided ad libitum in a barrier facility that is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Suspensions of MiaPaCa-2 tumor cells (1×107 viable cells/mouse) were inoculated subcutaneously into the bilateral flank region of the BALB/c-nu/nu mice. Analyses of these human tumor xenografts were initiated when the tumor volumes reached 50 mm3. Four mice were randomly allocated to each of the four treatment different groups: (i) vehicles: sterile normal saline administered by intraperitoneal (i.p.). injection twice weekly for 3 weeks (on days 1, 4, 8, 11, 15, and 18); (ii) 40 mg/kg or 120 mg/kg gemcitabine dissolved in saline administered by i.p. injection on the same schedule as i.p. saline; (iii) 10 mg/kg YM155 administered as a 3-day continuous infusion per week for 2 weeks using an implanted micro-osmotic pump (Alzet model 1003D; Durect, Cupertino, CA, USA); (iv) 10 mg/kg YM155 and/or 40 mg/kg or 120 mg/kg gemcitabine administered by infusion as described above. The tumor volumes were estimated by V=ab2/2, where a and b are tumor length and width, respectively. Antitumor effects of gemcitabine and YM155 were determined by measurement of tumor size and body weight three times per week. Carcass body weight was calculated by subtracting the tumor weight, estimated from the tumor volume, from the body weight.
Statistical analysis. The Student t-test was used for in vivo studies. p-Value less than 0.05 was considered significant.
Results
Sensitivity of the human pancreatic cancer cell line MiaPaCa-2 to gemcitabine, and/or YM155 alone. Firstly, we examined the effects of gemcitabine on cell viability in the pancreatic cancer cell line MiaPaCa-2. Following treatment with up to 40 nM gemcitabine, the proportion of dead cells was less than 10% (Figure 1A), whereas the proportion of cells in the S-phase dramatically increased from approximately 10% to 55% (Figure 1B), indicating that these concentrations of gemcitabine induced cell cycle arrest rather than cell death. This cell cycle arrest paralleled increased expression of survivin protein and increased levels of p-CHK1 and p-H2AX following treatment with gemcitabine (Figure 1C). Thus, induction of cell cycle arrest by gemcitabine in pancreatic cancer cells seemed to correlate with increased survivin expression.
Next, we investigated whether suppression of survivin expression allows cells to bypass the cell cycle arrest induced by gemcitabine using YM155, a selective survivin suppressant. We initially assayed the cytotoxic effect of YM155 alone on the pancreatic cancer cell line MiaPaCa-2. YM155 induced cell death in a dose-dependent manner (Figure 2A); however, marked cell cycle arrest was not noted, merely an increased proportion of cells in the subG1 fraction and a decrease in the G1 fraction (Figure 2B). YM155 treatment successfully suppressed the mRNA and the protein expression of survivin, as confirmed by RT-PCR and western blot analysis (Figure 2C and D). In addition, the cleavage of PARP increased in accordance with the increase in the proportion of dead cells induced by YM155 (Figure 2D).
Suppression of survivin by YM155 confers sensitivity to gemcitabine. We next investigated whether suppression of survivin by YM155 can bypass the cell cycle arrest induced by gemcitabine and sensitize cancer cells to gemcitabine treatment. For this purpose, we evaluated chemosensitivity and cell cycle distribution at doses of YM155 that induce cell death accompanied by suppression of survivin and at doses of gemcitabine that inhibit cell growth without induction of cell death. Co-treatment with YM155 noticeably decreased the proportion of cancer cells in S-phase arrest following gemcitabine treatment (Figure 3A). Moreover, YM155 sensitized the cancer cells to gemcitabine (Figure 3B). At the same time, we also examined the effects of the two drugs on survivin expression in MiaPaCa-2 cells and showed that the induction of survivin by gemcitabine was markedly inhibited by YM155 (Figure 3C), indicating that YM155 sensitizes cells to gemcitabine by suppressing the induction of survivin.
To validate the role of survivin in resistance to gemcitabine, we silenced survivin expression in MiaPaCa-2 cells using siRNA. Transfection with survivin siRNA successfully knocked-down the expression of survivin in MiaPaCa-2 cells (Figure 4A). In addition, cleavage of PARP, a substrate of caspase-3, and levels of p-H2AX clearly increased in cells transfected with survivin siRNA (Figure 4A). The proportion of cells in S-phase following gemcitabine treatment also decreased in survivin siRNA-transfected cells (Figure 4B). Furthermore, cell death increased in cells that were co-treated with gemcitabine and survivin siRNA compared with cells treated with either gemcitabine or survivin siRNA alone (Figure 4C), suggesting that YM155 sensitizes pancreatic cancer cells to gemcitabine through down-regulation of survivin and by bypassing the cell cycle arrest induced by gemcitabine.
Co-treatment with YM155 and gemcitabine dramatically reduces tumor growth. Based on the above in vitro results, we examined the effect of YM155 with and without gemcitabine on the growth of MiaPaCa-2 tumors in nude mice. Tumors grown in nude mice were treated with 10 mg/kg YM155 and/or 40 mg/kg or 120 mg/kg gemcitabine as described in the Materials and Methods. The growth rates of the xenograft MiaPaCa-2 tumors were significantly lower in mice that were co-treated with 10 mg/kg YM155 and 120 mg/kg gemcitabine than in those treated either 10 mg/kg YM155 or 120 mg/kg gemcitabine alone (p<0.05, Figure 5). In addition, there was no increase in body weight loss in the combination treatment arm compared with both gemcitabine and YM155 single agent treatment groups. Thus, the xenograft experiment showed that parenteral co-treatment with YM155 and gemcitabine significantly inhibited tumor growth.
Discussion
In this study we examined the validity of targeting survivin and the therapeutic potential of the survivin suppressor YM155, alone and in combination with gemcitabine, using a preclinical pancreatic cancer model. We found that YM155 exhibited antiproliferative activity and induced spontaneous apoptosis in the pancreatic cancer cell line MiaPaCa2 at nanomolar concentrations and induced tumor regression in an established xenograft model. In an in vitro study of combination treatment using a pancreatic cancer cell line, YM155 relieved gemcitabine-induced cell cycle arrest at the S-phase and the accompanying accumulation of survivin, and synergistically enhanced the cytotoxic activity of gemcitabine. Furthermore, in an established xenograft model, concomitant treatment with YM155 and gemcitabine resulted in greater tumor reduction than each single treatment.
Our present findings suggest that pancreatic cancer cells may acquire a cytoprotective phenotype involving cell cycle arrest through overexpression of survivin, thus mitigating the induction of apoptosis. Similar observations were reported in melanoma cells treated with docetaxel (18). In addition, pancreatic cancer cells are known to become sensitive to apoptosis induction when survivin is down-regulated by either siRNA or YM155 (19).
Pancreatic cancer is notorious for its intrinsic resistance to chemotherapy and targeted therapy and is known to be associated with many somatic mutations (1). Activating mutations of the KRAS2 oncogene are the most common genetic abnormality in pancreatic cancer, present in virtually all cases (2). In addition, the tumor suppressor genes CDKN2A (also known as p16 or INK4A), p53, and SMAD4 are also commonly inactivated in pancreatic cancer (3, 5, 20). These mutations are presumed to be the cause of resistance to systemic treatment and poor prognosis in patients with advanced pancreatic cancer.
Here, we showed that gemcitabine induced cell-cycle arrest at the S-phase and elevated the expression of survivin, which has not been previously reported. When YM155 was combined with gemcitabine, the accumulation of survivin and arrest of cells at the S-phase decreased and more intense apoptosis was observed than with each agent separately. In addition, YM155 potentiated the antitumor effect of gemcitabine without an increase in body weight loss in the xenograft model.
Although our observations were made for a single pancreatic cancer cell line, MiaPaCa2, they are consistent with previous reports suggesting survivin as a potential therapeutic target in pancreatic cancer. Vitamin E succinate and emodin were also shown to potentiate the effect of gemcitabine through inhibition of survivin (21, 22). Furthermore, siRNA directed against survivin enhanced the chemosensitivity of pancreatic cancer cells Panc-1 and BxPC3 to gemcitabine (19, 23), and short hairpin (sh) RNAs specific to survivin also had gene silencing effects and inhibited the proliferation of Patu8988 pancreatic cancer cells (24).
Taken together, these findings identify survivin as a potential therapeutic target in the treatment of pancreatic cancer. Through its ability to suppress survivin and induce apotosis, YM155 is a novel candidate for pancreatic cancer therapy. We have shown that YM155, both alone and in combination with gemcitabine, is effective in an in vitro and in vivo pancreatic cancer model. Further clinical investigation of YM155 in combination with gemcitabine for the treatment of advanced pancreatic cancer is warranted.
Acknowledgements
This study was supported by grants of the Korean Health Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A102059 and A100330).
Footnotes
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↵* These Authors contributed equally to this work.
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Conflict of Interest
There is no conflict of interest.
- Received February 21, 2012.
- Revision received March 20, 2012.
- Accepted March 21, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved