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 mRNA down-regulation in tumor cell sensitization.
BACKGROUND
Previous studies have reported the 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 cells 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.
Mizutani
Y
Y
Department of Urology, Faculty of Medicine, Kyoto University, Japan.
Yoshida
O
O
D000111:Acetylcysteine / Q000494:pharmacology; D000964:Antimetabolites, Antineoplastic / Q000008:administration & dosage* / Q000494:pharmacology; D015152:Blotting, Northern; D019328:Buthionine Sulfoximine; D002292:Carcinoma, Renal Cell / Q000473:pathology*; D015536:Down-Regulation; D004351:Drug Resistance; D004357:Drug Synergism; D005260:Female; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D007680:Kidney Neoplasms / Q000473:pathology*; D008717:Methionine Sulfoximine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology; D010051:Ovarian Neoplasms / Q000473:pathology*; D012333:RNA, Messenger / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000008:administration & dosage* / Q000235:genetics* / Q000494:pharmacology
D000964:Antimetabolites, Antineoplastic; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D008717:Methionine Sulfoximine; D019328:Buthionine Sulfoximine; D005978:Glutathione; D000111:Acetylcysteine