Reduction of mitomycin C resistance in human bladder cancer T24 cells by knocking-down ras oncogene

Abstract

Aim: Mitomycin C (MMC) is a commonly used as intravesical treatment for superficial bladder cancer. However, its role in combination with ras inhibition has not been investigated. The aim of this study was to explore the role of ras in MMC-induced apoptosis in T24 bladder cancer cells and to determine the efficacy of combination therapy in vitro.

Methods: We measured the effects of various doses of MMC on apoptosis induction as well as on ras, ERK and Ki-67 protein expression by T24 cell line using immunocytochemistry, flow cytometry and Western blotting. We also tested the effect of siRNA on ras employed singly or in combination with MMC.

Results: T24 cells expressed high level of ras protein. MMC treatment increased the level of ras and ERK protein expression after 24 h, and decreased these levels after 72 h. Ras siRNA (100 nmol/L) caused massive apoptosis associated with a marked decrease in ras expression in T24 cells. When combined with low doses of MMC, ras siRNA (50 nmol/L) sensitized T24 cells to apoptosis and decreased their expression of ras. The effect of combined therapy was higher than that of either compound used alone. Expression levels of ERK, a downstream target of ras, declined following combination therapy.

Conclusion: Ras siRNA in combination with low dose MMC is a possible treatment strategy for patients with ras-positive bladder tumors.

Keywords

Mitomycin C / ras oncogene / T24 cells / bladder cancer / siRNA

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