Resistance To Fas-mediated Apoptosis In Malignant Tumors Is Rescued By KN-93 And Cisplatin Via Down-regulation Of C-FLIP Expression And Phosphorylation

Apoptosis , programmed cell death, is a prosses died initiatively and programmly on the condition that control of gene physiology or pathology.It is known apoptosis has two methods one of which is death receptors path.The occurence of the malignant tumor is a Long-term, Multi-stage, multi-genes change accumulate process,in which the change of relating gene take part.Why malignant tumor accurs is the lose activity of apoptosis gene and the excessive expresstion restricted .Apoptosis pathway can be triggered by external signals through death receptors. The well known death receptor, Fas (CD95 or APO-1), belongs to TNFR (tumour necrosis factor receptor) family and triggers apoptosis upon binding of its cognate ligands (FasL, CD95L or APO-1L) or specific agonistic antibody (CH-11). Fas L or CH-11 can bind and activate their receptors by inducing receptor trimerization. Through their intracellular death domain, activated receptors recruit adaptor molecules such as Fas-associating protein with death domain (FADD) . FADD has a carboxyl-terminal DD and an amino-terminal death effecter domain (DED). Through its DED, FADD recruits the DED-containing apoptosis-initiating proteases caspase-8 and caspase-10 to the Fas receptor to assemble a DISC . In the DISC, caspase-8 undergo autocatalytic cleavage to release active subunits into cytoplasm . Activated caspase-8 can activate caspase 3 . c-FLIP, an antiapoptotic protein, was reported recently to be recruited to the DISC, interrupting DED-DED interaction between FADD and caspase-8 and inhibiting Fas-mediated apoptosis.Many tumour cells express resistence to apotosis still.In early observation,c-FLIP of melanoma tumours are expressed excessively that indicates c-FLIP of these cells inhibt apoptosis.The research recently in the organism had already revealed the excessive expession of c-FLIP in tumour cells transfection ,having promoted the increase of tumour,and the fail of immunity,which goes further to strengthen on c-FLIP regulation exists in tumour pathology. To investigate the molecular mechanisms in c-FLIP-mediated regulation of Fas signalling, we examined Fas-mediated DISC and its modulation in glioma and melanoma cells and showed that the expression and phosphorylation of c-FLIP could regulate Fas signalling through its recruitment to the Fas-mediated DISC. Furthermore, we tested the effects of CaMK II inhibitor KN-93 and a chemotherapeutical drug cisplatin on c-FLIP expression and phosphorylation in glioma and melanoma cells.Latest discovery the research shows indicates it brings new hope for biological treatment of malignant tumour though resisting the expression of copoptosis protein and its function.To investigate the molecular mechanisms that control tumour cell resistance and search for molecules that could overcome the FasL or CH11-resistance in certain tumours including glioma and melanoma. Tumour cell lines were examined for their sensitivity to CH11-induced apoptosis, and some of sensitive and some of resistant tumour cell lines were analyzed for Fas-mediated death-inducing signalling complex (DISC). CaMK II inhibitor KN-93 and chemotherapeutical drug cisplatin were used to treat resistant cells and their effects on CH-11-resistant tumour cells were tested. In sensitive tumour cells, apoptosis-initiating caspase-8 and caspase-10 were recruited to the DISC where they became activated through autocatalytical cleavage, leading to apoptosis through cleavage of downstream substrates such as caspase-3 and DFF45. In CH11-resistant cells, c-FLIP proteins were recruited to the DISC, resulting in the inhibition of caspase-8 and caspase-10 cleavage. c-FLIP protein expression and phosphorylation, calcium/calmodulin dependent protein kinase II (CaMK II) protein and enzymatic activity were up-regulated in resistant cells. Treatment of resistant cells with KN-93 and cisplatin down-regulated c-FLIP expression, inhibited c-FLIP phosphorylation, and rescued CH-11 sensitivity. This experiment is done carefully,the material used in the experiment are normally,methods used are as follows : Human Glioma and Melanoma Cell Lines, DISC Analysis by Immunoprecipitation, Western Blot and Two-dimensional PAGE Immunoblot, CaMK II Activity Assay.The results observed are as follows: Differential susceptibility of malignant tumour cells to Fas-mediated cell death, caspase-8 and caspse-10 are recruited to the Fas-mediated-DISC to initiate apoptosis, Differential kinetics and expression of c-FLIP in CH-11-sensitive and–resistant tumour cell lines,CaMK II inhibitor KN-93 can resume receptor apoptosis,Chemotherapeutic drug Cisplatin can rescue Fas-mediated cell death.The thoughts coming from the experiments are we should and have to find a way to prevent,control,cure malignant tumor.It has a broad area to develop.We can come to a conclusion simply from the research: KN-93 and cisplatin inhibit c-FLIP protein expression and phosphorylation restores CH11-induced apoptosis in tumour cells. This study provided a new combined therapeutic strategy for malignant tumours.