| Pancreatic cancer is one of the most malignant tumor.At the time of diagnosis, only less than 10%of pancreatic cancer can be resected.Its 5-year survival rate is the lowest one among all of the cancers.Because of its high degree of malignancy,rapid progress,late to be diagnosised,short-duration,early metastasis and poor prognosis, pancreatic cancer is known as "King of Cancer".According to the clinical guidelines of NCCN and ESMO,gemcitabine is the first-line chemotherapy drug for the treatment of metastatic pancreatic cancer.However,the effect of gemcitabine is quite limited.The median survival time was only 5.65 months and the one year survival rate was only 16%～19%after the treatment of gemcitabine.To improve the gemcitabine effect for the advanced pancreatic cancer,many drugs have been tried to be combined with it,such as 5-fluorouracil,cisplatin,capecitabine,docetaxel and so on.Even though the effect of gemcitabine combined regiments improved the efficiency,the survival benefit has not been found in the clinical studies.The chemotherapy for pancreatic cancer has reached a plateau.In recent years,molecular target therapy is a research hotspot in the treatment of tumor because of its high specificity and less adverse effect.Epidermal growth factor receptor(EGFR),a member of the ErbB family,can promote tumor cell proliferation,inhibit its apoptosis, improve its ability of movement and thereby increase tumor invasion and distant metastasis.Previous studies have shown that the EGFR is over-expressed in the pancreatic cancer cells.Erlotinib is one of the tyrosine kinase inhibitors of EGFR(EGFR-TKIs).A Phaseâ…¢clinical trial showed that the combined use of erlotinib with gemcitabine prolonged median survival time(6.24 months vs 5.91 months,P=0.034) and 1-year survival rates(23%vs 17%,P=0.023) in the patients with advanced pancreatic cancer.Based on that study,the FDA of United States has approved erlotinib for the first-line treatment of advanced pancreatic cancer in November 2005.Although the use of Erlotinib combined with Gemcitabine has already extended the survival period,the improvement of the median survival time and the one-year survival rate is limited.Previous studies showed that sequential administration of EGFR-TKIs and chemotherapy is superior to simultaneous drug administration in non small cell lung cancer.However,how to improve the effect of Erlotinib combined with gemcitabine on pancreatic cancer remained to be explored. So,the first aim of this study was to investigate the sequence-dependent effect of gemcitabine and erlotinib on BXPC-3 and PANC-1 pancreatic cancer cells and probed its possible cellular mechanism.Pemetrexed is an antifolate aiming at several targets.Previous basic research showed that it could inhibit the proliferation of PANC-1 pancreatic cancer cells and exert an additive effect when pemetrexed was combined with gemcitabine.Phaseâ…¡clinical studies showed that pemetrexed improved a prominent effect for the advanced pancreatic cancer with little side effects.Because there is a timing effect when erlotinib is combined with pemetrexed for the treatment of non-small cell lung cancer and there is still lack of any reports about the effect of combined regiment of pemetrexed and erlotinib on pancreatic carcinoma,the second purpose of this study was to investigate the sequence-dependent effects of gemcitabine,pemetrexed and erlotinib on BXPC-3,PANC-1 and their cellular mechanism.We hoped that this basic study could provide evidence for the better clinical treatment of advanced pancreatic cancer.Partâ… EGFR mRNA and its protein in BXPC-3 and PANC-1 cellsMTT assay was used to measure the cell number.The expression of EGFR mRNA mad protein was examined by RT-PCR and western blotting respectively.Data are presented as mean±S.E.M,and was analyzed by SPSS13.0.Experiments between two groups were analyzed by t test.The following was the results of partâ… :1.The two cells grew logarithmicly in the first 2-7 days after inoculation and this stage was most suitable for cell growth experiments.2.There were EGFRmRNA and protein expression in both of the cells.3.The expression of EGFRmRNA(t=9.571,P=0.001)and its protein(t= 10.935,P=0.000) in BXPC-3 cells was higher than that in the PANC-1 cells.Partâ…¡The sequence-dependent effect of the combination of egemcitabine,pemetrexed and erlotinib on the proliferation of human pancreatic cancer cells,BXPC-3 and PANC-1MTT assay was used to mesure the tumor cell number.Data are presented as mean±S.E.M.Experiments between two groups were analyzed by t test.Multiple groups were analyzed by one-way ANOVA followed by LSD multiple comparison test.The results were shown as follows:1.Gemcitabine(3×10-10～3×10-2mol/L) significantly inhibited the cell proliferation of BXPC-3 and PANC-1 cells in a dose-dependent(F=86.765,P= 0.000;F=85.260,P=0.000)and time-dependent manner(F=135.385,P=0.000;F =134.132,P=0.000).Pemetrexed(1.7×10-10～1.7×10-2mol/L)significantly inhibited the cell proliferation of BXPC-3 and PANC-1 cells in a dose-dependent(F= 70.120,P=0.000;F=81.573,P=0.000)and time-dependent manner(F=70.025,P =0.000;F=56.772,P=0.000).Erlotinib(10-6-10-4mol/L)significantly inhibited the cell proliferation of BXPC-3 and PANC- 1 cells in a dose-dependent(F=22.180,P =0.000;F=15.517,P=0.000)and time-dependent manner(F=44.354,P= 0.000;F=7.813,P=0.000).The inhibitive effects of gemcitabine(3×10-9～3×10-6mol/L)(t=2.821,P=0.018;t=2.146,P=0.048;t=2.274,P=0.046;t= 2.278,P=0.046),pemetrexed(1.7×10-4～1.7×10-2mol/L)(t=3.120,P=0.011;t= 2.677,P=0.038;t=2.440,P=0.035)and erlotinib(10-6～10-4mol/L)(t=3.390,P= 0.008;t=3.896,P=0.005;t=5.777,P=0.000) were stronger for the BXPC-3 cells with high expression of EGFRmRNA and protein than that of PANC-1 cells with low expression of EGFRmRNA and protein.The IC50 values of gemcitabine on BXPC-3 and PANC-1 were 31.57±1.79μmol/L and 59.17±1.78μmol/L respectively.The IC50 values of pemetrexed on BXPC-3 and PANC-1 were 39.86±1.68μmol/L and 83.76±1.75μmol/L respectively.The IC50 values of erlotinib on BXPC-3 and PANC-1 could not be achieved at the 10-6～10-4mol/L concentration range.2.The effects of different combination of gemcitabine,pemetrexed and erlotinib were sequence-dependent.The inhibitory effect on the two cells was enhanced when they were used simultaneously(P=0.034;P=0.049)or erlotinib was added before gemcitabine(P=0.001;P=0.025);The inhibitory effect was enhanced when erlotinib was administrated in combination with pemetrexed simultaneously(P= 0.016;P=0.049 ) or at 24h intervals(P=0.000;P=0.000).24h sequential administration of pemetrexed following erlotinib exerted more obvious effect on the cell proliferation than that of simultaneous administaation(P=0.038;P=0.029); The inhibitory effect was enhanced when gemcitabine was administrated in combination with pemetrexed simultaneously(P=0.049;P=0.044)or at 24h intervals(P=0.000;P=0,000).24h sequential administration of gemcitabine following pemetrexed exerted more obvious effect on the cell proliferation than that of simultaneous administration(P=0.038;P=0.022).Partâ…¢The effect of gemcitabine,pemetrexed and erlotinib on the cell cycles of BXPC-3Cell cycles were detected by flow cytometry.Data are presented as mean±S.E.M. and analyzed by SPSS13.0.Multiple groups were analyzed by one-way ANOVA followed by LSD multiple comparison test.The results were shown as follows:1.Compared with control group,gemcitabine and erlotinib monotherapy, gemcitabine and erlotinib administrated simultaneously and 24 hours sequentially would block the BXPC-3 cells at G1 phase(P=0.002);Erlotinib and pemetrexed administrated simultaneously(P=0.046) or pemetrexed following erlotinib at 24 hours interval(P=0.004) would block the BXPC-3 cells at G1 phase.Pemetrexed following erlotinib at 24 hours interval would block the BXPC-3 cell at S phase(P= 0.026);Pemetrexed monotherapy(P=0.025) and gemcitabine following pemetrexed at 24 hours interval(P=0.001)would block the BXPC-3 cell at S phase.Pemetrexed following gemcitabine at 24 hours interval(P=0.003) or simultaneously(P=0.048) would block the BXPC-3 cell at G1 phase.Conclusion:There were EGFRmRNA and protein expression in both BXPC-3 and PANC-1 cells.The expression of EGFRmRNA and protein in BXPC-3 cells is higher than that in PANC-1 cells.Gemcitabine,pemetrexed and erlotinib inhibited the cell proliferation of BXPC-3 and PANC-1 cells in a dose-dependent and time-dependent manner.The effect of combination of gemcitabine,pemetrexed and erlotinib was sequence-dependent.The inhibitory effect on cell proliferation was enhanced when gemcitabine and erlotinib were used simultaneously or erlotinib was added 24h before gemcitabine.The inhibitory effect was enhanced when gemcitabine or erlotinib was administrated in combination with pemetrexed simultaneously or 24h intervals.24h sequential administration of gemcitabine or erlotinib following pemetrexed exerted more obvious effect on the cell proliferation than simultaneously or 24h sequential administration of pemetrexed following gemcitabine or erlotinib.The enhanced inhibitory effect of gemcitabine or erlotinib following pemetrexed may related with the effect on cell cycle.The additive effects of other groups might not be induced by their influence on the cell cycle.
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