Fas-associated death domain protein enhance inhibitory effect of 5-fluorouracil on growth of human colorectal carcinoma cells
YIN An-ning, JIANG Ying-an, ZHANG Xian-feng, LUO He-sheng
Published 2009-12-15
Cite as Chin J Dig, 2009,29(12): 829-833. DOI: 10.3760/cma.j.issn.0254-1432.2009.12.011
Abstract
Objective To examine the sensitivity of human colorectal carcinoma cells to 5-fluorouracil treatment by stable transfection of extrinsic Fas-associated death domain protein(FADD) gene,both in vitro and in vivo,so as to investigate the feasibility of combination therapy of FADD gene and 5-fluorouracil in human colorectal carcinoma. Methods ①RT-PCR and Western blotting were used to detect the expressions of both mRNA and protein of FADD gene in SW480/FADD (stably transfected with FADD),SW480/neo and SW480 cells.②After treatment with 5-fluorouracil as an apoptotic inducer,in vitro cell growth activities were investigated by MTT assay.Cell apoptosis and its rates were determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)assay and flow cytometry of annexin V-FITC/PI staining.The expressions of caspase-8 and caspase-3 were examined by Western blotting. ③To examine the inhibitory effect of FADD gene combined with 5-fluorouracil, tumor xenograft model was prepared for in vivo study.Results ① Compared with SW480 and SW480/neo cells, FADD mRNA and protein levels of SW480/FADD cells were higher (P<0.05). ② Inhibitory rate of SW480/FADD cells was remarkably higher than SW480 and SW480/neo cells (P<0.05 ). ③ Forty-eight hours after treatment with 5-fluorouracil (10 mg/L), the apoptotic rate of SW480/FADD cells was (33.3 ± 4.5)%, which was higher than SW480 [(13. 9 ± 3. 2)%3 and SW480/neo [(14. 1 ± 3. 4)%], with significant difference (P< 0.05). ④ Forty-eight hours after treatment with 5-fluorouracil (10 mg/L),procaspase-8 and procaspase-3 expressions of SW480 and SW480/neo cells were higher than SW480/FADD cells, whereas their cleaved caspase-8 and cleaved caspase-3 expressions were lower than SW480/FADD cells (P<0. 05).⑤ In in vivo study, SW480/FADD cells increased the efficacy of fluorouracil-induced inhibition of tumor growth in nude mice. Conclusions Stable overexpression of FADD increases sensitivity of the cells to 5-fluorouracil and combination of FADD with 5-fluorouracil will he a promising alternative in colorectal cancer treatment.
Key words:
Colonic neoplasms; Fas-associated death domain protein; 5-Fluorouracil
Contributor Information
YIN An-ning
Department of Gastroenterology,Renmin Hospital of Wuhan University,Wuhan 430060,China
JIANG Ying-an
Department of Gastroenterology,Renmin Hospital of Wuhan University,Wuhan 430060,China
ZHANG Xian-feng
Department of Gastroenterology,Renmin Hospital of Wuhan University,Wuhan 430060,China
LUO He-sheng
Department of Gastroenterology,Renmin Hospital of Wuhan University,Wuhan 430060,China