Tumoricidal Effect of Taxol on Murine Bladder Tumor-2 (MBT-2) cells via Nitric Oxide Production

Oh, Sang Jin; Rim, Joung Sik; Chung, Hun Taeg

Abstract

PURPOSE: Taxol, an anticancer drug, blocks cell division by stabilizing microtubules. However, taxol has distinct cell-cycle-independent effects. For example, taxol and interferon gamma(IFN-gamma) induce tumoricidal activity of murine peritoneal macrophages. This study was designed to know whether taxol has indirect tumoricidal effect on murine bladder tumor-2(MBT-2) cells besides its direct cytotoxicity.
MATERIALS AND METHODS
The original stock of C57BL/6 mice were used at 8 to 12 weeks of age. Macrophages were obtained by peritoneal lavage from the mice which had been treated with thioglycollate. The tumor target cells were MBT-2 cell line. MBT-2 cells were cultivated in different concentration of taxol for various times and the growth of MBT-2 cells were tested. Tumoricidal activitiy was measured by indirect methylthiazol-2-yl-2,5-diphenyl tetrazolium bromide(MTT) assay after co-cultures of stimulated macrophage and MBT-2 cells with taxol, INF-gamma, lipopolysaccharide(LPS) or with combination of taxol and INF-gamma or LPS and INF-gamma. Nitric oxide(NO) formation was measured by Griess method under the same conditions. Effect of NG -monomethyl-L-arginine(NGMMA) on nitrite formation and cytotoxicity toward MBT-2 cells were also evaluated.
RESULTS
Significant retardation of cell growth was observed after treatment of tumor cells with taxol in a dose dependent manner but does not affect cell viability. Taxol(19+/-2%) or LPS(19+/-4%) alone weakly activated macrophages to kill MBT-2 cell lines, whereas combinations of taxol(77+/-3%) or LPS(75+/-4%) with IFN-gamma(control: 2%, IFN-gamma:18+/-3%) synergized to activate macrophages to kill tumor cells in a dose dependent manner. Taxol(20+/-5microM), LPS(15+/-5microM) or IFN-gamma(25+/-3microM) alone induced small amounts of NO secretion but the combinations of either taxol and INF-gamma(73+/-5microM) or LPS and IFN-gamma(77+/-5microM) synergistically induced large amounts of NO secretion. The production of NO(control:<5, IFN-gamma+ taxol: 73+/-5microM, IFN-gamma+ taxol + NGMMA: 15+/-3microM) and tumor cell killing(control: 2%, IFN-gamma+ taxol: 77+/-3%, IFN-gamma+ taxol + NGMMA: 20+/-3%) were blocked in the presence of NGMMA, a competitive inhibitor of NO synthase.
CONCLUSIONS
Collectively, the data indicate that taxol is directly non-cytotoxic for MBT-2 cells via its effect on micrtubules but indirectly activates macrophages to kill MBT-2 cells probably via NO secertion.