OVERCOMING TUMOR-NECROSIS-FACTOR-ALPHA RESISTANCE OF HUMAN RENAL AND OVARIAN-CARCINOMA CELLS BY COMBINATION TREATMENT WITH BUTHIONINE SULFOXIMINE AND TUMOR-NECROSIS-FACTOR-ALPHA - ROLE OF TUMOR-NECROSIS-FACTOR-ALPHA MESSENGER-RNA DOWN-REGULATION IN TUMOR-CELL SENSITIZATION

Background. Previous studies have reported that glutathione plays a central role in a wide range of cellular functions, including protection, detoxification, transport, and metabolism. Buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamyl-cysteine synthetase, depletes intracellular glutathione. The study investigates the cytotoxic effect of BSO and tumor necrosis factor-alpha (TNF-alpha) used in combination on TNF-alpha-resistant human renal and ovarian cancer cells. Methods. Cytotoxicity was determined by a 1-day microculture tetrazolium dye assay. TNF-alpha mRNA was examined by Northern blot analysis. Results. Combination treatment of TNF-alpha-resistant R4 and R11 human renal cell carcinoma cells with BSO and TNF-alpha overcame their resistance to TNF-alpha. In addition, the combination of BSO and TNF-alpha resulted in a synergistic cytotoxic effect on TNF-alpha-resistant OVC-8 and C30 human ovarian cancer cells. Treatment of R4, R11, and OVC-8 cells with TNF-alpha in combination with glutathione or N-acetyl-cysteine(NAC) showed an antagonistic cytotoxic effect. A possible mechanism of resistance to TNF-alpha in tumor cells is the expression of TNF-alpha mRNA or protein. R4 cells and OVC-8 cell constitutively expressed mRNA for TNF-alpha. Treatment of R4 cells or OVC-8 cells with BSO down-regulated the expression of TNF-alpha mRNA; however, treatment with TNF-alpha up-regulated the expression of TNF-alpha mRNA. When BSO was used in combination with TNF-alpha, the level of TNF-alpha mRNA enhanced by TNF-alpha was markedly reduced. Incubation of R4 cells with glutathione or NAC also down-regulated the expression of TNF-alpha mRNA. R11 and C30 cells did not constitutively express mRNA for TNF-alpha, and the BSO treatment had no effect on the TNF-alpha mRNA level. Conclusions. This study demonstrates that the combination of BSO and TNF-alpha can overcome the TNF-alpha resistance of tumor cells and that depletion of intracellular glutathione and down-regulation of TNF-alpha mRNA by BSO may play a role in the enhanced cytotoxicity seen with the combination of BSO and TNF-alpha. There may not be always a correlation between the expression of TNF-alpha mRNA in tumor cells and their resistance to TNF-alpha. The synergistic effect obtained with established renal cell carcinoma cells and ovarian cancer cells suggests that combination treatment with TNF-alpha and BSO could have clinical application in the therapy of TNF-alpha-resistant tumors.