Pancreatic cancer is one of the most lethal human cancers and a major unsolved health problem in the past century.1-3 Despite efforts in the past 50 years, conventional treatment approaches, such as surgery, radiation, chemotherapy, or combinations of these have had little effect to cure pancreatic cancer.1-4 Therefore, revealing the differences between normal and transformed pancreatic cells on molecular level became a promising direction to treat this disease.5-11 Compared with normal pancreatic cells, pancreatic cancer cells show significant differences in many signaling pathways including constitutive activation of kinases, cyclooxygenase, and inhibition of tumor suppressor proteins.12-14 Based on these alterations, serial strategies have been designed to treat pancreatic cancer and some of them show effective outcomes.15,16
Resveratrol was first isolated from the roots of white hellebore which is a natural existence plant antitoxin.17 Resveratrol attracted little interest until 1992, when it was postulated to explain some of the cardio-protective effects of red wine.18,19 Since then, dozens of reports have shown that resveratrol can enhance stress resistance and extend the lifespan of various organisms from yeast to vertebrates, as well as prevent or slow the progression of a wide variety of illnesses, including cancer, inflammation, cardiovascular disease and ischemic injuries.20-25 Furthermore, systemic administration of resveratrol shows slightly toxic effect.26 Resveratrol inhibits carcinogenesis at multiple stages through multiple molecular mechanisms.27 It is potent protein kinase inhibitor. Moreover, it can inhibit activity of cyclooxygenase 2.28-30
Considered the inhibitory effects of resveratrol to both kinases and cyclooxygenase, we evaluated the treatment effect of resveratrol on pancreatic cancer. Consistent with the results from gastric cancer and thyroid cancer cell lines, resveratrol induced apoptosis of capan-2 and colo357 pancreatic cancer cell lines, but not capan-1, miapaca-2 and bxpc-3. Resveratrol shows insignificant effect on normal pancreatic cells. To further confirm these results, we detected the activation of caspase-3 in capan-2 and colo357.
Cell culture and resveratrol treatment
The human pancreatic cancer cell lines (capan-1, capan-2, colo357, miapaca-2, bxpc-3) were cultured in Dulbecco's modified Eagle's medium (DMEM) or RPMI-1640 (Gibco BRL, China) with 10% inactivated fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco BRL, China). The human pancreatic duct epithelial cell lines (HPDE) was presented by Dr. Ming-Sound Tsao (University of Toronto, Canada) and cultured in Keratinocyte serum-free medium (SFM) + EGF+bovine pituitary extract (Invitrogen, China) supplemented with 1 × antibiotic-antimycotic (Gibco BRL, China). All of the cells were put in a 5% CO2 atmosphere at 37°C for generational culture. Human pancreatic cancer cell lines were provided by Helmut Friess except capan-1, which was obtained from the cell bank at Chinese Academy of Sciences. Resveratrol was obtained from Sigma and dissolved in dimethyl sulfoxide (DMSO) at concentration of 100 mmol/L as stock solution. Therefore, the final dilution of DMSO was more than 500 times which shows insignificant toxicity to cells. Stock solution was further diluted with medium when treating cells.
Western blotting analysis
The human pancreatic normal and cancer cell lines left untreated or treated were washed with cold phosphate buffer saline (PBS) and were lysed in Laemmli lysis buffer. Equal amounts of lysate were separated using 12% SDS-PAGE. Proteins were transferred to nitrocellulose membranes. The membranes were then blocked with PBS containing 5% low-fat milk and 0.05% Tween for 1 hour, then washed three times with PBS containing 0.05% Tween and incubated with specific primary antibodies for 2 hours at room temperature. After being washed with PBS containing 0.05% Tween, the membranes were incubated with peroxidase-conjugated secondary antibodies labeled horseradish peroxidase (HRP) and were developed on X-ray film. Mouse monocolonal anti-p21, anti-GAPDH and anti-p53 were purchased from BD Biosciences (USA) with 1:500 dilution. Rabbit polycolonal anti-phospho-ERK and anti-caspase-3 were from Cell Signaling Technology (USA) with 1:1000 dilution.
Cell viability was assessed using the MTT assay. Briefly, the human pancreatic cancer cells were cultured in 96-well plates at a density of 1×104 cells/ml and treated with various concentrations of resveratrol or equal volum DMSO for 24 hours. Each concentration was repeated for 6 equal wells. Twenty μl of MTT labeling reagent was added to each well containing cells in 150 μl of medium, and cells were incubated in a humidified incubator at 37°C to allow the MTT to be metabolized for 3 hours. The media was removed and cells were resuspended in formazan in 200 μl of DMSO. The absorbance of the samples was measured at a wavelength of 490 nm by Microplate Reader, and the viability values were expressed as percentages of control values.
Cell apoptosis measurement
For analysis of cell apoptosis, cells were seeded in 24-well plates at a density of 5×105 cells/ml with fresh complete culture medium and treated with 200 μmol/L resveratrol or not for 24 hours, after treated following protocol with and then transferred into tubes and stained with Propidium Iodide (PI) (Sigma). Cells were then used to detect apoptosis by flow cytometry.
Results are expressed as means ± standard deviation (SD). Statistical analyses were performed using SAS statistical software (SAS Institute Inc., USA). Student's t test followed by least significant difference t test (LSD-t) was used for group comparison. The P value <0.01 was regarded as statistically significant.
Sensitivity of human pancreatic normal and cancer cell lines to resveratrol
To evaluate the pro-apoptosis effect of resveratrol to human pancreatic cancer cell lines, we treated some pancreatic cancer cell lines with resveratrol on a moderate concentration of 200 μmol/L. After 24 hours, the apoptosis was detected. It shows that capan-1, miapaca-2 and bxpc-3 were insensitive to resveratrol treatment (Figure 1A, B and C); on the contrary, capan-2 and colo-357, especially latter, shows high sensitivity to resveratrol treatment (Figure 1 D and E). Resveratrol induced 4 times apoptotic capan-2 cells compared to control. Colo-357 shows more sensitivity to resveratrol, 10 times apoptotic cells were detected in colo-357 after resveratrol treatment compared with control. To further confirm the toxicity of resveratrol to normal pancreatic cells, we detected the apoptosis of normal pancreatic cells HPDE with the same treatment. The result shows that resveratrol has insignificant apoptotic effect on HPDE (Figure 1F). These data indicate that resveratrol as a promising molecule to treat human pancreatic cancer cells.
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Figure 1. Apoptosis analysis of human pancreatic normal and cancer induced by resveratrol. A: capan-1 was treated with 200 μmol/L resveratrol for 24 hours. B: Bxpc-3 was treated with 200 μmol/L resveratrol for 24 hours. C: Miapaca-2 was treated with 200 μmol/L resveratrol for 24 hours. D: capan-2 was treated with 200 μmol/L resveratrol for 24 hours. E: colo357 was treated with 200 μmol/L resveratrol for 24 hours. F: HPDE was treated with 200 μmol/L resveratrol for 24 hours.
Resveratrol significantly inhibits the viability of the capan-2 and colo357 cells in a concentration-dependent manner. The cell viabilities of capan-2 and colo357 in the 200 μmol/L resveratrol was significantly lower than in the control group (P <0.001) (Figure 2).
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Figure 2. Cell viability of human pancreatic cancer cell lines (assayed by MTT). A: Resveratrol significantly decreased cell viability in the capan-2 cells (P <0.01 or P <0.001 compared with control group). B: Resveratrol significantly decreased cell viability in the colo357 cells in a concentration-dependent manner (P <0.001 compared with control group).
Activation of caspases upon resveratrol treatment
As Figure 1 shown, the relationship between resveratrol and apoptosis in human pancreatic cancer cells has been established. To further explore the molecular mechanisms of resveratrol, we test the cleavage of caspase-3 upon resveratrol treatment. It shows a time-dependent manner of resveratrol to caspase-3 cleavage in capan-2 (Figure 3A). Consistent with the results from capan-2, colo-357 also exhibit higher cleavated caspase-3 upon resveratrol treatment (Figure 3B). These results suggest that resveratrol induce apoptosis of capan-2 and colo357 through activating caspase-3.
Resveratrol induce mitogen-activated protein kinase (MAPK) activation and p21 accumulation
It has been well established that resveratrol induced apoptosis in thyroid cancer cell via an MAPK dependent mechanism.31,32 Based on this conclusion, we tested the extracellular signal-regulated kinase (ERK) activation in capan-2 and colo-357 cells. Consistent with the results from thyroid cancer cell, resveratrol can also induce ERK activation in pancreatic cancer cells (Figure 4A and B). p21 was a well-known downstream effector of ERK and proved as an effective pro-apoptosis protein. We then detected the p21 alteration upon resveratrol treatment. Consistent with the results from pro-apoptosis effect and ERK activation, p21 was induced after resveratrol treatment in capan-2 and colo357 (Figure 4C and D).
Resveratrol induce p53 accumulation
As an oncogene suppressor protein, p53 was another important protein involved in resveratrol induced apoptosis in other cells.33-40 Thus we tested the protein level of p53 after resveratrol treatment. It shows resveratrol led to marked increase in capan-2 and colo357 (Figure 5A and B). The increased p53 phosphorylation on Ser6 was also detected (data not shown). Taken together, these data indicates that resveratrol promotes p53 accumulation in human pancreatic cancer cells.
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Figure 3. Caspase-3 activation in human pancreatic cancer cell lines upon resveratrol treatment. 3A: Resveratrol (200 μmol/L) significantly induced caspase-3 cleavage in a time-dependent manner in capan-2 cells. 3B: Resveratrol significantly induced caspase-3 cleavage in a time-dependent manner in colo357 cells.
Figure 4. MAPK activation and p21 accumulation in human pancreatic cancer cell lines upon resveratrol treatment. 4A: Resveratrol (200 μmol/L) significantly induced ERK phosphorylation in a dose-dependent manner in capan-2 cells. 4B: Resveratrol (200 μmol/L) significantly induced ERK phosphorylation in a dose-dependent manner in colo357 cells. 4C: Resveratrol (200 μmol/L) significantly induced p21 accumulation in a dose-dependent manner in capan-2 cells. 4D: Resveratrol (200 μmol/L) significantly induced p21 accumulation in a dose-dependent manner in colo357 cells.
Figure 5. p53 accumulation in human pancreatic cancer cell lines upon resveratrol treatment. 5A: Resveratrol (200 μmol/L) significantly induced p53 accumulation in a dose-dependent manner in capan-2 cells. 5B: Resveratrol (200 μmol/L) significantly induced p53 accumulation in a dose-dependent manner in colo357 cells. Loading control was shown in Figure 4.
Resveratrol has been proved effective in many tumors, such as mammary gland, skin, prostate, leukemia cancer and multiple myeloma.27 Moreover, its well known anti-tumor medicine prospects were also supported by its low toxicity.26 Resveratol targets many key intracellular proteins which are highly associated with transformation including receptor tyrosine kinase, protein kinases and cyclooxygenase.29 Interestingly, these proteins were reported crucial for development of human pancreatic cancer.3 Based on these reports, we explored the effect of resveratrol on human pancreatic cancer cells. Consistent with the effect on other type cancer cells, resveratrol also shows significant pro-apoptosis effect on human pancreatic cancer cell lines capan-2 and colo357 at a moderate concentration of 200 μmol/L. Further toxicity assay indicates that this concentration shows insignificant toxicity to normal pancreatic cell lines. These data indicate resveratrol as a safe and promising small molecule to treat pancreatic cancer.
As a multi-target phytoalexin, resveratrol activates several cellular signaling pathways. To explore the molecular mechanisms of resveratrol on pancreatic cancer cells, we determined the activation of apoptosis executer caspase-3 upon resveratrol treatment. Consistent with the result from apoptosis, caspase-3 was observed cleaveted in capan-2 and colo357 in a time-dependent manner upon resveratrol treatment. The relationship between resveratrol and apoptosis signaling has been well described in other cell lines.31,33 Some targets such as MAPK, CD95, p53, Bcl-2, Bax have been proposed to be potential targets to resveratrol.31,32,34,36 We established the regulatory role of resveratrol to caspase-3 lines here indicating a potent pro-apoptosis function of resveratrol in pancreatic cancer cells.
Based on the finding of apoptosis signaling activation in pancreatic cancer cells, we were interested in which signaling pathway was involved in the activation of caspase-3. Thus we further detected the accumulation of p53 and activated MAPK upon resveratrol treatment in pancreatic cancer cells. The results shows that resveratrol induce phosphorylation of ERK in time-dependent manner upon resveratrol treatment in capan-2 and colo357. Moreover, resveratrol treatment led to p53 accumulation in the two cell lines. These data indicates that resveratrol inducing apoptosis in pancreatic cancer cells may through activating these pathway. As a classical ERK downstream effector, p21 was thought critical to execute ERK-induced cell cycle arrest and apoptosis. We then tested p21 protein level upon resveratrol treatment in capan-2 and colo357. Consistent with the result from ERK activation, p21 was observed up-regulated upon resveratrol treatment. To further confirm these results, we performed the cell cycle analysis in the presence of resveratrol. More canpan-2 or colo357 in G1 phase was observed upon resveratrol treatment, suggesting that resveratrol can induce cell cycle arrest in pancreatic cancer cells. The data indicates p53 and MAPK were believed involved in resveratrol-induced apoptosis. The significance of these signaling pathways need to be further studied.
In this study, capan-2 and colo357 pancreatic cancer cells were sensitive to resveratrol-induced apoptosis, but other pancreatic cancer cells including miapaca-2, bxpc-3 and capan-1 were resistant to resveratrol. We suspected this discrepancy was induced by the p53 mutation status. It has been reported canpan-2 and colo357 contain wild-type p53, and mutant p53 was observed in miapaca-2, bxpc-3 and capan-1. Functional p53 pathway is critical for resistance of pancreatic cancer cells to anti-tumor agents. These results suggest resveratrol is effective to p53 wild type pancreatic cancer.
Taken together, we established a primary relationship between resveratrol and apoptosis in pancreatic cancer cells here. Furthermore, we explored the molecular mechanisms of resveratrol-induced apoptosis in pancreatic cancer cells. These data indicate resveratrol a potent medicine to treat human pancreatic cancer.
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