Critical role for c-FLIP(L) on Fas resistance in colon carcinoma cell line HT-29.
The cellular Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein-long form (c-FLIP(L)) is a key regulator of Fas signaling, although owing a dominant-negative homologue of caspase-8, the role of c-FLIP(L) remains controversial. In the present study, two pairs of small interfering RNA (siRNA) directed against c-FLIP(L) were used to assess the effect of c-FLIP(L) on Fas-mediated apoptosis of colon carcinoma in vitro. HT-29 cell line was selected for overexpression of c-FLIP(L) and Fas with RT-PCR and flow cytometry analyses. After electroporation, the mRNA level of c-FLIP(L) was significantly decreased (control siRNA versus c-FLIP(L) siRNA, 77.97+/-5.61% versus 26.22+/-3.79%) and the maximum interfering efficiency was around 66.49% using semi-quantitative RT-PCR analysis. Knockdown of c-FLIP(L) with the specific siRNA sensitized colon carcinoma cells to Fas-mediated apoptosis (control siRNA versus c-FLIP(L) siRNA, 5.68+/-2.11% versus 29.50+/-2.27%) using DNA content analysis and Annexin V-FITC analysis. In conclusion, our study indicated that c-FLIP(L) might be a suppressor of Fas-mediated apoptosis in Fas antigen expressing colon carcinoma and therefore a potential target for novel anticancer therapies.
Zang
Fenglin
F
Key Laboratory of Cancer Prevention and Therapy, Tianjin Cancer Institute and Hospital, Tianjin Medical University, Tianjin 300060, PR China.
Sun
Baocun
B
Zhao
Xiulan
X
Niu
Ruifang
R
Zhang
Shiwu
S
Yu
Man
M
Wei
Xiyin
X
Zhang
Lin
L
D017209:Apoptosis; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000235:genetics / Q000378:metabolism*; D003110:Colonic Neoplasms / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D018274:Electroporation; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D005838:Genotype; D019073:HT29 Cells; D006801:Humans; D010641:Phenotype; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors; D019014:fas Receptor / Q000378:metabolism*
D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; C491901:FAS protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D019014:fas Receptor