Effects Of Regulation Of Smac-IAPs Apoptosis Pathway On Chemotherpaeitic Sensitivity Of Gastric Cancer Cells

Part One Expression and correlation of XIAP,Smac and caspase-3 in gastric cancerObjective To study the expression of XIAP,Smac and caspase-3 in human gastric cancer tissues and its correlation with clinical and pathological characteristics.Methods Immunohistochemical staining was applied to detect the expression of XIAP,Smac and caspase-3 in 80 cases of gastric cancer and 40 cases of adjacent normal gastric mucosa.Results In 80 cases of gastric cancer,the expression rates of XIAP,Smac and caspase-3 were 90%(72/80),67.5%(54/80),and 62.5%(50/80),respectively.The XIAP expression was negatively correlated with tissue differentiation(P＜0.05),while expression of Smac and caspase-3 was positively correlated with tissue differentiation(P＜0.05).The expression of XIAP,Smac and caspase-3 was significantly different between intestinal-type and diffuse-type gastric cancer(P＜0.05),and correlated with lymph node metastasis (P＜0.05),but not with location,volume and invasion.The expression of XIAP and Smac in gastric cancer was negatively and positively correlated with caspase-3,respectively.In 40 cases of normal gastric mucosa,6 cases(15%) presented weak expression of XIAP,and the expression rates of Smac and caspase-3 were 92.5%(37/40) and 90%(36/40),respectively.Conclusions XIAP expression increased along with the malignant degree of gastric cancer,while expression of Smac and caspase-3 decreased,which may participate in the development and progression of gastric cancer.These proteins may serve as potential biomarkers for evaluating the prognosis of gastric cancer. Part Two Effects of transient transfection of Smac on chemotherapeutic sensitivities of gastric cancer cell lineObjective Abnormal apoptosis is one of key factors for the drug resistance of neoplasms.Smac is a novel gene involved in the regulation of apoptosis,which plays an important role in the inducing apoptosis effects of chemotherapeutic drugs on tumor cells. This study was designed to explore the effects of over-expression of Smac gene on chemotherapeutic sensitivities of gastric cancer cell line,in order to establish a basis for further improving chemotherapy of gastric cancer.Methods Under induction of liposome GeneSHUTTLE-40,Smac gene was transfected into gastric cancer cell line MKN-45.Cellular Smac gene expression was determined by reverse transcription-polymerase chain reaction(RT-PCR) and western blot analysis.Cisplatin and MMC were administrated to un-transfeted and transfected MKN-45 cells.Cellular proliferation activities were assayed by tetrazolium bromide(MTT) colorimetry.The morphological changes of cancer cells were observed under an inversion microscope.Cellular apoptosis was determined by Annexin V-FITC and propidium iodide staining flow cytometry.Results Compared with un-transfected control group,Smac mRNA and protein levels in transfected MKN-45 cells were significantly improved(P＜0.01).The growth inhibition rates of various concentrations of cisplatin and MMC on MKN-45 cells were improved by 10.10%-23.80%(P＜0.01) and 10.01%-15.86%(P＜0.01),respectively,with the cells presenting roundness,obvious refraction,and cellular fragment.Cellular apoptosis rates were improved by 6.7%-20.2%(P＜0.01) and 5.4%-13.2%(P＜0.01),respectively.Conclusions Transfection of extrinsic Smac gene resulted in its over-expression in gastric cancer cells,which could improve the chemotherapeutic sensitivities of MKN-45 cells.This is a potential strategy for ameliorating chemotherapeutic effects of gastric cancer. Part Three Establishment of gastric cancer cell line stably overexpressing Smac gene and its chemotherapeutic sensitivitiesObjective To explore the effects of stable over-expression of Smac gene on chemotherapeutic sensitivities of gastric cancer cell line.Methods Under the induction of liposome,the eukaryotic expression vector pcDNA3.1-Smac for Smac gene and its control vector pcDNA3.1 were transfected into gastric cancer cell line MKN-45.The subclone cell lines were obtained by persistent G₄₁₈ selection.Smac gene expression of cancer cells was detected by RT-PCR and western blot methods.The growth inhibition effects of MMC on cancer cells were also observed by MTT colorimetry and clone formation assay.Results The subclone gastric cancer cell lines,stably expressing Smac and neo gene respectively,were successfully selected,and named as MKN-45/Smac and MKN-45/neo. RT-PCR and western blot results demonstrated that Smac mRNA and protein levels of MKN-45/Smac cells were significantly higher than those of MKN-45 and MKN-45/neo (P＜0.01).After incubation with 10μg/ml MMC for 24 h,the growth inhibition rates of MKN-45 and MKN-45/neo were 27.85%,28.12%respectively,while that of MKN-45/Smac cells was 43.71%(P＜0.01).When compared with MKN-45 and MKN-45/neo cells,the clone formation abilities of MKN-45/Smac were reduced by 14.07%(P＜0.01) and 15.13%(P＜0.01),respectively.Conclusions Stable transfection of Smac gene and its over-expression in gastric cancer cell line could significantly improve their chemotherapeutic sensitivities to MMC, which established an experimental basis for ameliorating chemotherapy of gastric cancer.Part Four Regulatory effects of over-expression of Smac gene on apoptosis activities of gastric cancer cell lineObjective To explore the effects of over-expression of Smac gene on apoptosis activities of gastric cancer cell line. Methods The gastric cancer cell lines MKN-45,MKN-45/neo and MKN-45/Smac were cultured in vitro.After teatment with MMC as an apoptosis inducer,cellular growth activities were investigated by trypan blue staining method.Apoptosis of cancer cells was detected by electronic microscopy,AO-EB fluorescent staining and TUNEL methods. Cellular protein levels and its activities of caspase-3 were assayed by western blot and colorimetry.Results After incubation with 10μg/ml MMC for 24 h,growth activities of MKN-45/Smac were reduced by 10.0%～30.8%(P＜0.01),when compared with those of MKN-45. Partial MKN-45/Smac cells presented characteristic morphological changes of apoptosis under the electric and fluorescence microscopes,with apoptosis rates being 36.4%,which was significantly higher than that of MKN-45(15.2%,P＜0.01).After treatment with MMC, caspase-3 expression levels in MKN-45/Smac cells were significantly improved than those of MKN-45(P＜0.01),with caspase-3 activities enhanced by 3.25 times(P＜0.01).Conclusions Transfection of extrinsic Smac gene and its over-expression in gastric cancer cell line could significantly improve expression and activity levels of caspase-3 induced by MMC,resulting in obvious apoptosis-inducing effects,which established a novel strategy for regulating apoptosis activities of gastric cancer.Part Five The effects of downregulating XIAP expression on the apoptosis of gastric cancer cells induced by chemotherapeutic drugsObjective To observe the effects of downregulating XIAP expression on the chemotherapeutic sensitivities of gastric cancer cells.Methods The antisense eukaryotic vector for XIAP was constructed and stably transferred into gastric cancer cell line MKN-45.RT-PCR and western blot were applied to detect the XIAP gene expression.Cisplatin and MMC were administrated to untransfected and transfected gastric cancer cells.MTT colorimetry and clone formation assay were performed to measure the in vitro cell viability.Apoptosis and its rates were detected by electronic microscopy,AO-EB fluorescent staining and TUNEL.Cellular caspase-3 protein expression and its activities were assayed by western blot and colorimetry.Results RT-PCR and western blot indicated that the mRNA and protein levels of XIAP within gastric cancer MKN-45 cells stably transfected with antisense XIAP vector were significantly decreased by 84.75%(P＜0.01) and 89.75%(P＜0.01),respectively.After treatment with various concentrations of cisplatin and MMC for 24 hours,the cell growth inhibition of MKN-45 cells stably transfected with antisense XIAP was enhanced by 7.3%～25.3%(P＜0.01) and 12.3%～16.3%(P＜0.01),respectively.Partial cancer cells presented characteristic changes of apoptosis under an electronic microscope,while the apoptosis rates were 34.1%and 32.5%,respectively,which were significantly higher than that of untransfected control MKN-45 cells(14.2%,P＜0.05).Compared with MKN-45 cells,the caspase-3 expression within cells stably transfected with antisense XIAP was significantly increased by 2.45 times(P＜0.01),while the activity of caspase-3 was enhanced by 3.68 times(P＜0.01).Conclusions Downregulation of XIAP expression via antisense RNA could increase the expression and activity of caspase-3,enhance the apoptosis of cancer cells induced by chemotherapeutic drugs.Part Six Effects of curcumin on IAPs and Smac gene expression of gastric cancer cell lineObjective To explore the effects of curcumin on gene expression of IAPs and Smac in gastric cancer cell line,in order to explore the inducing apoptosis mechanisms of curcumin.Methods After being treated with 10～40μmol/L curcumin for 6～24 h,growth activities of MKN-45 cells were detected by MTT colorimetry.Cellular apoptosis was assayed by TUNEL and DNA Ladder methods.IAPs(Survivin,XIAP) and Smac gene expression was detected by RT-PCR and western blot.Cellular caspase-3 activities were detected by colorimetry method. expression was detected by RT-PCR and western blot.Cellular caspase-3 activities were detected by colorimetry method.Results When compared with untreated control group,cellular growth of antisense XIAP-transfected cells was significantly inhibited by various concentrations of curcumin, with inhibitory ratios being 12.18%～68.15%(P＜0.01).Partial MKN-45 cells presented characteristic morphological changes of apoptosis,with trapezia bands on gel electrophoresis.The apoptosis rates were 9.24%～28.12%(P＜0.01).Cellular mRNA and protein levels of Survivin and XIAP were significantly down-regulated(P＜0.01),with those of Smac being up-regulated(P＜0.01).Cellular caspase-3 activities were improved by 2.27～6.67 times(P＜0.01).Conclusions Curcumin could significantly induce apoptosis of gastric cancer cell line MKN-45,one of its mechanisms was through up-regulating Smac and down-regulating Survivin and XIAP expression,leading to activation of caspase-3.