The Expression Of C-FLIP(L) In Breast Cancer Cell Lines And Its Effect Of Sensitivity Of TRAIL-induced Apoptosis

Objective:Cellular Fas-associated death domain-like interleukin-1β-converting enzyme-like inhibitory protein(c-FLIP),which contain death effect domains (DED),block TRAIL mediated apoptosis.the long form of c-FLIP protein overexpressed in a variety of cancers, c-FLIP(L) and caspase-8coregulate apoptosis and involve carcinogenesis and development. In our study we detect expression of c-FLIP(L) and caspase-8of breast cancer cell lines, then used RNAi technology to knockdown the expression of c-FLIP(L). We investgate the effects of c-FLIP(L) down-regulation on TRAIL-induced apoptosis of breast cancer and its possible mechanism.Method:1. Real time PCR and Western blot were used to examine the basic expression level of c-FLIP(L) and caspase-8in a variety of breast cancer cell lines.2. We used RNA interference technology to knockdown the expression of c-FLIP(L) of SK-BR-3and MDA-MB-231cells and the expression of c-FLIP(L) was detected by Western blot.3. After down-regulating the expression of c-FLIP(L), under TRAIL inducing, cell viability was examined by WST-1analysis, then protract dose-dependent curve.4. Flow cytometry analysis was used to examine sensitivitys of apoptosis of SK-BR-3and MDA-MB-231,compare the difference of the control group and the transfection group.5. After TRAIL treating,western blot was used to detect the expression of subunit of caspase-8.Result:1. Compared with normal mammary epithelium cell HBL100,mRNA and protein of c-FLIP(L) and caspase-8varied in different breast cancer cell lines. We select c-FLIP(L) higher expression cell lines which could mediate TRAIL signaling, SK-BR-3and MDA-MB-231,to do next experiments.2. Transfect SK-BR-3and MDA-MB-231cells with c-FLIP(L) siRNA, lower expression of c-FLIP(L) were detected by western blot.3. After knockdown the expression of c-FLIP(L) and TRAIL inducing.compared with control, Under TRAIL500ng/ml and1000ng/ml, SK-BR-3and MDA-MB-231cell viability decreased significantly. The growth inhibition rate begin to change in dose-dependent style.4. After down-regulating c-FLIP(L) and TRAIL inducing for24h,the percentage of apoptotic of SK-BR-3was up to57.91%, MDA-MB-231increased to32.97%, the percentage of apoptosis increased significantly,sensitivity of apoptosis increased.5. Compared with control, after TRAIL treating, subunit of caspase-8,including p43and p18was found.It indicate that after c-FLIP(L) down-regulating,caspase-8was activated.Conelusion:1. mRNA and protein of expression level of cFLIP(L) and caspase-8varied in different breast cancer cell lines,It indicate the expression of c-FLIP(L) may associate with difference of TRAIL-induced apoptosis.2. Using WST-1and flow cytometry analysis, after transfecting c-FLIP(L) siRNA,compared with control, cell viability decreased,the percentage of apoptosis increased significantly. It suggest sensitivity of TRAIL-induced apoptosis increased after c-FLIP(L) downexpression.We speculate c-FLIP(L) inhibit TRAIL-mediated apoptosis signaling in c-FLIP(L) highly-expressed breast cancer cell lines.3. Compared with control,p43and p18subunit of caspase-8was detected by Western blot, active caspase-8subunit was found, knockdown the expression of c-FLIP(L),sensitivity of TRAIL increased, its possible mechanism may be cleavage and activation of caspase-8.