The Effects Of Smad-4 Gene Targeted Therapy On Proliferation And Apoptosis Of Human Pancreatic Cells

Background:Human pancreatic cancer belongs to gastrointestinal cancers,with which the early diagnosis and clinical treatment remain difficult all over the world.Clinically,the single treatment of surgery has limited value in the treatment of pancreatic cancer.On the other side,chemotherapeutic options are also not very effective mainly due to drug resistance.In order to improve the survival rate of patients with pancreatic cancer,alternatively or supplemental treatments,such as interventional therapies or gene therapies are under investigation.A better understanding of etiopathogenesis and molecular/genetic biology of pancreatic cancers will help to identify novel molecular-targeted agents and potential therapeutic approaches for this disease.Smad-4/DPC-4 is a specific tumor suppressor gene of pancreatic cancer.It has been identified that:(1)Homozygous deletion or mutation of Smad-4/DPC-4 gene may lead to defective TGF-β/ Smad-4 signal transduction,(2)Tumor cells carry Smad-4 mutants might resist to chemotherapy-induced apoptosis,(3)The expression of Smad-4/DPC-4 can be induced by p21 wafl,and result in the inhibition of tumor cell growth by inhibiting cell cycle progression.In this study,the effects of Smad-4 gene intervention on biological behaviors including cell proliferation and apoptosis of pancreatic tumor were evaluated both at the cellular level and tumor implantation nude mice level.The results revealed that the cells stably expressed Smad-4/DPC-4 were more sensitive to the chemotherapy of gemcitabine and went through apoptosis. The expression of Smad-4/DPC-4 was related to less cell migration.The nude mice implanted Smad-4/DPC-4 pancreatic tumor cells had better outcome upon chemotherapy.The results indicate that the Smad-4/DPC-4 targeted gene therapy might be considered as a supplemental treatment of chemotherapy and explored to the patients with pancreatic cancer.Objectives:Smad-4 gene is a specific tumor suppressor gene of pancreatic cancer,belonging to transforming growth factor-βsuperfamily.Its deletion,mutation can lead to excessive cell proliferation,and it plays an important role in the growth,development and transfomation process of pancreatic cancer.In this study,the effects of Smad-4 gene intervention on biological behaviors including cell proliferation and apoptosis of pancreatic tumor were evaluated by using Smad-4 gene deficient cell lines and tumor implantation nude mice model.Methods:The pancreatic cancer cells CFPAC-1(wild-type),CFPAC-1(stable expression Smad-4)[DPC-4],CFPAC-1(blank expression vector)[TJM]were treated with gemcitabine at various concentrations(0～30μM)in this study.The MTT assay was used to assess cytotoxic activity induced by different concentrations of gemcitabine. Flow cytometry was adopted to measure the cell apoptosis after gemcitabine treatment.The migrations of tumor cells were observed in scratch experiments;the pancreatic cancer cells were implanted into nude mice by skin inoculation;and the effects of chemotherapy on those animals were observed by measuring the tumor size. All the experiments included in this study were repeated at least 3 times.SPSS software was applied for statistical analyses.The data was expressed as mean±SD and the statistical significance of the differences between control and parthenolide-treated cells was determined by a two-tailed Student's t test.The Spearman correlation test was used to analyze the correlation between reagents' concentrations and inhibition rates.P-value＜0.05 was considered as significant.Results:MTT assays revealed that the proliferation of DPC-4 cells was inhibited by gemcitabine in a dose-dependent manner.The EC50 was 14.5μM.Flow cytometry analysis showed that gemcitabine could significantly promote the apoptosis of DPC-4 cells.Scratch experiments suggested that the expression of Smad-4 could significantly affect the ability of cell migration.Animal experiments indicated that the chemotherapy by gemcitabine had better effects on Smad-4/DPC cell implanted nude mice.Conclusions:The results indicate that upon the treatment of chemotherapeutic drugs,the Smad-4 targeting gene intervention can inhibit the proliferation of pancreatic cancer cells and promote tumor cell apoptosis.The Smad-4/DPC-4 targeted gene therapy might be considered as a supplemental treatment of chemotherapy and explored to the patients with pancreatic cancer.