Study On The Expression And Function Of C-FLIP(L) In The TRAIL Pathway Of Breast Cancer

Objective1.To study the expression of c-FLIP(L) and TRAIL-DR4/DR5 in human breastcancer tissue and cell lines and analysis the correlation between c-FLIP(L) expressionand clinicopathologic parameters.2.To observe the association of c-FLIP(L) expression and apoptotic sensitivity inbreast cancer tissue and cell lines and primarily confirm the exact regulation ofc-FLIP(L) in TRAIL pathway in breast cancer.3.To study the co-operation between c-FLIP(L) and caspase-8 and make sure themechanism of c-FLIP(L) in TRAIL pathway in breast cancer primarily.Methods1.Use the technique of IHC to detect the expression of c-FLIP(L) and DR4/DR5 inbreast cancer tissue and its counterparts (150 cases each).Analysis the association ofthese factors and the correlation between c-FLIP(L) and clinicopathologic parameters.2.Detect c-FLIP(L) in mRNA and protein level by RT-PCR and Western blot andevaluate the difference of DR4/DR5 by indirect immunofluorescence FCM in breastcell lines.3.Calculate the apoptosis index and proliferate index by TUNEL and Ki-67immunohistochemistry and evaluate their correlation.4.Establish c-FLIP(L) high-expressed MDA-MB-231 breast cancer cell line bytransfecting c-FLIP(L) plasmids.Under TRAIL inducing,map out dose-dependentand time-dependent curve and select the perfect condition by MTT analysis.Detectapoptosis by Annexin V-FITC,DNA Ladder and phase contrast microscope inc-FLIP(L) high-expressed MDA-MB-231 cells.5.Use caspases assay plates,Western blot and Co-immunoprecipitation (Co-IP) toanalysis the co-operation between c-FLIP(L) and caspase-8.Results1.c-FLIP(L) is low or moderate in breast invasive ductal cancer tissue (36.36%),while it is high-expressed in normal counterparts (75%) (P＜0.01).The positive rate ofexpression level of+++ is 14.29% in malignant and 57.58% in normal breast tissue (P＜0.01).c-FLIP(L) expression is positively associated with age,menopausal status,distant metastasis and pathologic stage (P＜0.05).Still c-FLIP(L) is positive in the ERand/or PR positive cases and it has no correlation with Her-2 expression.2.c-FLIP(L) and DR4/DR5 are present in four breast cancer cell lines.Although thereare different levels of these factors,DR4 and DR5 are consistent in single cell line.3.DR4 is high-expressed in the cases which has high level of c-FLIP(L) and thecorrelation of them is 0.452 (P=0.035).As to DR5 and c-FLIP(L),the association hasno statistic significance (r_s=0.419,P=0.066).There is positive correlation betweenc-FLIP(L) expression and apoptotic index (r_s=0.554,P=0.005).When c-FLIP(L) ishigh-expressed,proliferate index changed into low or moderate and they are notstatistic correlated.4.The mRNA and protein level of c-FLIP(L) are increased after transfecting plasmids24h,and the mRNA level is still after 48h and 72h,while protein level is decreasedslightly.The growth inhibition rate begins to change after TRAIL induction 4h and ishigh after 24h.Under each time point,the rate increases obviously when TRAIL is10ng/ml compared with other doses.5.After TRAIL inducing,apoptotic rate increases to 51.56% in c-FLIP(L)high-expressed MDA-MB-231 cells.DNA Ladder can be detected and apoptoticmorphologic changes can be observed by phase contrast microscope.6.Under TRAIL inducing,the caspase-8 activity increases to 5-folds and caspase-3to 3-folds in c-FLIP(L) high-expressed MDA-MB-231 cell line,while the activitiesofcaspase-2 and caspase-9 do not change obviously.7.Compared with cells transfected with pEGFP-N1,there are some changes in thecells with c-FLIP(L) plasmids:the p55 segment of caspase-8 decreases and the p43,p 18,and p 10 increase;the p55 of c-FLIP(L) also decreases and p43 and p 12 increase.c-FLIP(L) can co-operate with caspase-8 by Co-IP analysis,and the p43 segment ofthese two factors can be detected in their complex.Conclusion1.We confirm that c-FLIP(L) protein can promote TRAIL pathway in breast cancerby ditecting the expression of c-FLIP(L) in cancer and its counterparts and observingthe exact regulation of c-FLIP(L) in apoptosis.We also report that the low-expressed c-FLIP(L) indicats good prognosis in breast cancer.2.c-FLIP(L) may have different expression level and apoptotic modulation underdifferent conditions,which needs further research to confirm.3.We should consider the different of c-FLIP(L) in diverse cancer types and distinctindividuals before use it as a significative target for prognosis and therapy.