Mechanism Of Suppression On Proliferation Of Human Colon Carcinoma Cell By Eukaryotic Expression Vector Of FADD And 5-fluorouracil

Colon carcinoma is a common gastrointestinal cancer in China, and its incidence is raising in recent years. At present the treatment of colon carcinoma is comprehensive therapy including surgical excision and chemotherapy, however the conditions of unsensitive to chemotherapeutic drugs has been getting attention. 5-fluorouracil, as a cell DNA synthesis inhibitors, are most widely used in colon cancer chemotherapy. But there are many clinical reports of drug resistance of 5-fluorouracil. FADD(Fas-associated death domain protein), a pro-apoptotic protein, plays an important role in apoptosis pathway mediated by death receptor molecule and cytotoxicity by anti-cancer drug-induced. The reports of combined FADD and 5-fluorouracil to treat colon carcinoma are rare. In this study, eukaryotic expression vector pEGFP-Nl-FADD was firstly constructed using molecular cloning methods, then transferred to human colon cancer SW480 cells through gene transfection technology. So SW480/FADD cell which has high and stable expression of FADD gene would be obtained. Secondly,5-fluorouracil was used in SW480/FADD cells, SW480/neo cells and SW480 cells in vitro, changes and apoptosis of tumor cells was observed, its mechanism was preliminary explored. Finally, three kinds of colon carcinoma cells xenograft model in nude mice subcutaneously was established, tumor inhibition of FADD combined 5-fluorouracil was observed. Based on the above findings, a potential new way of clinical treatment of colon cancer was found.Methods1. Total RNA extracted from the human colon carcinoma cell SW480, and FADD cDNA was obtained by RT-PCR, and FADD cDNA was digested by the restriction endonuclease BamH I and Xho I before connection with pEGFP-N1. pEGFP-N1-FADD was to prove by digested with restriction endonuclease and analyse of DNA sequeneing. The combinant was transfected into SW480 cell by using LipofectamineTM2000. The stable transfected cell line was selected in medium containing antibiotic(G418) and was observed by inverted fluorescence microscope. The expression of FADD detected by RT-PCR and Western blotting. 2. SW480, SW480/neo and SW480/FADD cells were treated with 5-fluorouracil, survival and growth inhibition rate was measured by MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay; conditions of apoptosis were measured by Hoechst33258 and Annexin V-FITC/PI of Flow Cytomtry; activity of procaspase-8, procaspase-3, cleaved caspase-8 and cleaved caspase-3 were measured by Western blotting.3. SW480, SW480/neo and SW480/FADD cells,5×106, suspended in 100μL PBS, were subcutaneously inoculated into the lower right flank of the nude mice. When the tumors were 100-150 mm3 in size,5-fluorouracil (40 mg/kg) were administrated via intraperitoneal injection every 4 days. Tumor growth was monitored using calipers every 4 days. The nude mice were killed at 44 days. Tumor volume (V) was calculated by using the formula:tumor volume V (mm3)=π/6×length (mm)×width (mm)2. Status of nude mice and inhibitory effect of tumor growth was observed, the tumor volume was calculated, and growth curve was plotted. Representative histological sections of TUNEL(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)were taken from mice bearing transplantation tumor were detected.Results1. FADD cDNA fragment(about 651bp size) was obtained by PCR from human colon cancer SW480 cells. The combinant, pEGFP-N1-FADD, was confirmed correctly by digested with restriction endonuclease and analyse of DNA sequeneing. At the 14 days, the resistanted monoclonal cells of SW480/FADD and SW480/neo were found by inverted fluorescence microscope. Expression of FADD in the stable transfected cell line, SW480/FADD, were higher than untransfective cell line, SW480 and transfected with blank plasmid, SW480/neo(P<0.05).2. After 5-fluorouracil worked on SW480/FADD, SW480/neo and SW480 cells, the result of MTT test showed cell growth rate of was increased in a dose-dependent and the number of cell survival was reduced in a time-dependent. S W480/FADD cells showed less cell viability than SW480 and SW480/neo cells(P<0.05). There was no significant difference between SW480/neo and SW480 cells. The results of Hoechst33258 and Annexin V-FITC/PI of Flow Cytomtry showed more apoptosis cells of SW480/FADD cells than SW480 and SW480/neo cells(P<0.05). The results of Western blotting showed activity of cleaved caspase-8 and cleaved caspase-3 of SW480/FADD cells were higher than SW480 and SW480/neo cells(P<0.05), and activity of procaspase-8 and procaspase-3 of SW480/FADD cells were less than SW480 and SW480/neo cells(P<0.05).3. In nude mice, the tumor growth rates of SW480/FADD cells were significantly suppressed than SW480 and SW480/neo cells(P<0.05). Representative histological sections of the tumor of SW480/FADD showed more cells of apoptosis. Representative TUNEL sections of the tumor of SW480/FADD cells showed more brown nucleus of cells than SW480 and SW480/neo cells(P<0.05).ConclusionsThe construction of the eukaryotic expression vector for FADD was successful, and the stable transfective cell line for overexpression FADD was obtained by G418 selections. Simultaneously, FADD expressed in SW480/FADD cells were higher than those SW480 and SW480/neo cell(P<0.05). This work may pave the way for further study of the functions of FADD. And the result of study showsed FADD gene promotes the therapy of 5-fluorouracil for colon carcinoma cells in vitro and in vivo. Based on the above findings, a potential new way of clinical treatment of colon cancer was found.