The Biological Effects Of Phorbol Esters Combined With Gemcitabine On Pancreatic Cancer Cells

Background and ObjectivesPancreatic cancer is the fourth leading cause of cancer-related mortality in the USA, and seventh in China. Despite some progress in treatment strategies recently, less than 5% of patients with pancreatic cancer are expected to survive for 5 years after diagnosis. Currently, the front-line treatment for pancreatic cancer is the pyrimidine antimetabolite gemcitabine and it has shown promise as a chemotherapeutic drug. However, the majority of patients with cancer of the pancreas show a poor response to chemotherapy. Accordingly, there is a need to develop better treatment regimens for pancreatic cancer.In studies with cultured cells from pancreas cancer, phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) was shown to inhibit the growth with low concentrations and was not involved in protein kinase C. Some reports showed that TPA could enhance the effects of gemcitabine (GEM) on ovarian cancer cells and protein kinase C was regulated during the treatment. The exact mechanism is not clear. Therefore the effects and mechanism of the combination of TPA and GEM on pancreatic cancer cells is very interesting. We also tested another phorbol ester, 12-O-acetylphorbol-13-decanoate (APD) which could not stimulate protein kinase C. (?) the present report, we provide data on the inhibitory effect of TPA or APD in combination with GEM on the growth, apoptosis, and invasion of Panc-1 human pancreatic cancer cells.Methods1 The effects of phorbol esters combined with GEM on Panc-1 cells1.1 Cell culture Panc-1 cells lines were cultured in PRMI-1640 medium supplemented with 10% bovine calf serum. Cells were grown at 37℃in humidified 5% CO2 atmosphere and were routinely subcultured every 3 days.1.2 Assessment of cell growth and morphology The cells were exposed to GEM, TPA or APD alone, combinations of GEM with TPA or APD for 96 hours. Cell growth effects were measured by MTT method. The morphological changes of Panc-1 cells were observed under inverted microscope at 24,48,72,96 hours.1.3 Flow cytometry Cell cycle distribution, apoptosis, and the express of surface antigen CXCR4 after treating by GEM, TPA or APD alone, combinations of GEM with TPA or APD on Panc-1 cells were evaluated by flow cytometry. Apoptosis evaluated by flow cytometry with Annexin V-FITC and PI double staining method.1.4 Invasion ability The invasion potency were studied in vitro by transwell matrigel invasion assay.2 Statistical AnalysisThe analyses were performed by SPSS 10.0 statistical software. All date were expressed by mean±tandard deviation (x±S). The differences of the data between the multiple groups were analyzed with one-way ANOVA. A value of P<0.05 was considered to be statistically significant.Results1 The effects of phorbol esters combined with GEM on human pancreatic cancer Panc-1 cells. 1.1 Morphological changesAfter 96 hours treatment of TPA, cell aggregation, refractive index reduced. The cell size became larger and pseudopods extended around. Some cells were round and cell fragments appeared. In higher concentrations, there were many small intracellular vacuoles observed with high-power microscope. The cell morphological changes of APD were similar to TPA.Treated with GEM, the cells showed a larger spread, lower refractive index, pseudopods extending which were out of line. There were more cell fragments. These effects were dose-dependent and time-dependent of GEM. The morphological changes in present of GEM combination with TPA or APD were significantly larger than those alone.1.2 The dosage effects of TPA, APD or GEMTPA, APD and GEM inhibited the growth of Panc-1 cells significantly with IC50 of 1.43nmol·L-1,22.91 nmol·L-1,42.40n mol·L-1 for 96 hours respectively.1.3 The effects of inhibition proliferation by GEM with TPA or APDTPA(0.32nmol·L-1,0.48nmol·L-1) with GEM inhibited the growth of Panc-1 cells with IC50 of 17.38nmol·L-1, 11.OOnmol·L-1. APD (3.56nmol·L-1, 8.90nmol·L-1) with GEM IC50 were 20.60nmol·L-1, 12.11,nmol·L-1. TPA (0.32nmol·L-1,0.48nmol·L-1) or APD (3.56nmol·L-1,8.90nmol·L-1) were able to synergistically inhibit proliferation within a certain concentration GEM (6.68nmol·L-1～66.80nmol·L-1) on Panc-1 cells.1.4 The effects of cell cycle distribution on Panc-1 cellsTPA, APD and GEM reduced cell numbers in G0/G1 phase. TPA or APD increase cell numbers in G2/M phase, and GEM increase in the proportion of cells in S phase. TPA (0.32nmol·L-1) or APD (3.56nmol·L-1) in combination with GEM (16.70nmol·L-1, 33.40nmol·L-1) resulted in significantly decrease the proportion of cells in Go/G phase and increase in S, G2/M phase.1.5 Apoptosis by GEM with TPA or APD on Panc-1 cellsAnnexin V-FITC/PI double staining analysis showed that both drug combinations or alone could induce apoptosis on Panc-1 cells in a dose-denpendent manner (compared with the control group, P<0.01). Compared with the control group, TPA 8.00nmol·L-1, APD 35.60nmol·L-1 and GEM 213.76nmol·L-1 induce apoptosis about 39.05%,37.09%,58.08%, respectively. GEM (16.70nmol?L-1,33.40nmol?L-1) combination with TPA (0.32nmol?L-1) or APD (3.56nmol?L-1) induce apoptosis about 55.32%,65.01%or 48.42%,64.43%, respectively.1.6 The expression of CXCR4 induced by TPA or APD combined GEMTPA, APD reduced the expression of CXCR4 in a dose-dependence manner (P<0.01). Compared with the control group, TPA, APD decreased the expression of CXCR4 about 62.93%,45.85% at 8.00nmol-L-1,35.60nmol?L-1 respectively. GEM, GEM combined with TPA or APD did not have any effect on the expression.1.7 The changes of invasion ability after treated by GEM with TPA or APD on Panc-1 cells.Compared with the control group, TPA, APD decreased the penetrated cell about 47.62%,42.41% at 8.00nmol?L-1,35.60nmol?L-1 respectively. There were no significant differences between GEM alone or with TPA or APD compared with the control group.Conclusion1 TPA, APD, GEM inhibite proliferation, induce apoptosis and reduce cell numbers of G0/G1 phase on Panc-1 cell. TPA, APD combined with gemcitabine could enhance these effects synergisticly.2 TPA, APD could reduce the expression of CXCR4 and the invasion capacity of Panc-1 cells.3 TPA, APD inhibit proliferation of Panc-1 cell could be partially eliminated by PKC inhibitor GF 109203X, other non-PKC phorbol ester recepter may involved.