Impairment of both apoptotic and cytoprotective signalings in glioma cells resistant to the combined use of cisplatin and tumor necrosis factor α

Purpose: Tumor necrosis factor (TNF)-alpha elicits two opposing effects, the induction of apoptosis and the transcription of antiapoptotic genes. We have recently shown that cisplatin sensitizes glioma cells to TNF-induced apoptosis, but only in some cell lines. To understand the mechanism involved in the different susceptibilities, we examined both the activation of caspases and cytoprotective signaling by TNF-alpha. Experimental Design: Caspase activation was examined by estimating the cleavage of substrate peptides and by immunoblot to identify the cleavage of procaspases. Peptide inhibitors of caspases were used to reverse the cytotoxicity. The binding of TNF-alpha to the receptor was analyzed by flow cytometry. Nuclear factor (NF)-KB activation was assayed by the binding of NF-KB to oligonucleotides containing the consensus binding site. Interleukin (IL)-1beta, IL-6, IL-8, and manganous superoxide dismutase (MnSOD) were measured by enzyme-linked immunoassays. Results: T98G and U87MG underwent apoptosis on treatment with cisplatin and TNF-alpha, but U373MG and A172 were resistant. Caspases 2, 3, and 6-10, but not caspases 1, 4, and 5, were activated in sensitive cells, and none were activated in resistant cells. The binding of TNF-alpha to the receptor was the same in all four of the cell lines. In the sensitive cells, NF-KB activation and the production of IL-1beta, IL-6, IL-8, and MnSOD were significantly elevated by TNF-alpha. However, in the resistant cells, the production of IL-1beta and IL-6 were specifically impaired in response to TNF-alpha. Conclusions: Our results indicate that both apoptotic and cytoprotective pathways are impaired in glioma cells that are resistant to treatment with cisplatin and TNF-alpha.