Resistance to Fas-mediated apoptosis in malignant tumours is rescued by KN-93 and cisplatin via downregulation of c-FLIP expression and phosphorylation

Abstract

1. The purpose of the present study was to investigate the molecular mechanisms that control tumour cell resistance and to search for molecules that could overcome Fas ligand (FasL) or CH-11 resistance in certain tumours, including glioma and melanoma. 2. Twelve tumour cell lines were examined for their sensitivity to CH-11-induced apoptosis and then two of each of the CH-11-sensitive and -resistant tumour cell lines were analysed for Fas-mediated death-inducing signalling complex (DISC). The calmodulin kinase II (CaMKII) inhibitor KN-93 and the chemotherapeutic drug cisplatin were used to treat resistant cells; the effects of these two drugs on CH-11-resistant tumour cells were investigated. 3. In CH-11-sensitive tumour cells, apoptosis-initiating caspase 8 and caspase 10 were recruited to the DISC, where they became activated through autocatalytic cleavage, leading to apoptosis through cleavage of downstream substrates, such as caspase 3 and DNA fragmentation factor 45. 4. In CH-11-resistant cells, cellular Fas-associated death domain-like interleukin-1b-converting enzyme inhibitory protein (c-FLIP) proteins were recruited to the DISC, resulting in inhibition of caspase 8 and caspase 10 cleavage. The c-protein expression and phosphorylation of FLIP and CaMKII protein and enzyme activity were upregulated in resistant cells. Treatment of resistant cells with 100 micromol/L KN-93 and 10 microg/mL cisplatin downregulated c-FLIP expression, inhibited c-FLIP phosphorylation and rescued CH-11 sensitivity. 5. In conclusion, KN-93 and cisplatin inhibit c-FLIP protein expression and phosphorylation restores CH-11-induced apoptosis in tumour cells. tHe present study provides evidence for the use of a new combination therapeutic strategy in the treatment of malignant tumours.