kn-93 and Glioma

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.

Topics: Antibodies; Antineoplastic Agents; Apoptosis; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 10; Caspase 8; Cell Line, Tumor; Cell Survival; Cisplatin; Down-Regulation; Drug Resistance, Neoplasm; Fas Ligand Protein; Glioma; Humans; Melanoma; Phosphorylation; Protein Kinase Inhibitors; Sulfonamides

Fas, upon cross-linking with Fas ligand (FasL) or Fas agonistic antibody, transduces apoptotic yet also proliferative signals, which have been implicated in tumor pathogenesis. In this study, we investigated the molecular mechanisms that control Fas-mediated signaling in glioma cells. Fas agonistic antibody, CH-11, induced apoptosis in sensitive glioma cells through caspase-8 recruitment to the Fas-mediated death-inducing signaling complex (DISC) where caspase-8 was cleaved to initiate apoptosis through a systematic cleavage of downstream substrates. In contrast, CH-11 stimulated cell growth in resistant glioma cells through recruitment of c-FLIP (cellular Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme (FLICE)-inhibitory protein) to the Fas-mediated DISC. Three isoforms of long form c-FLIP were detected in glioma cells, but only the phosphorylated isoform was recruited to and cleaved into a p43 intermediate form in the Fas-mediated DISC in resistant cells. Calcium/calmodulin-dependent protein kinase II (CaMK II) activity was up-regulated in resistant cells. Treatment of resistant cells with the CaMK II inhibitor KN-93 inhibited CaMK II activity, reduced c-FLIP expression, inhibited c-FLIP phosphorylation, and rescued CH-11 sensitivity. Transfection of CaMK II cDNA in sensitive cells rendered them resistant to CH-11. These results indicated that CaMK II regulates c-FLIP expression and phosphorylation, thus modulating Fas-mediated signaling in glioma cells.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Benzylamines; Blotting, Western; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Carrier Proteins; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 8; Caspase 9; Caspases; Cell Death; Cell Division; Cross-Linking Reagents; DNA, Complementary; Electrophoresis, Gel, Two-Dimensional; Enzyme Inhibitors; Fas Ligand Protein; Glioma; Humans; Immunoblotting; Intracellular Signaling Peptides and Proteins; Membrane Glycoproteins; Phosphorylation; Precipitin Tests; Protein Isoforms; Proteins; Signal Transduction; Sulfonamides; Time Factors; Transfection; Tumor Cells, Cultured; Up-Regulation