Research about Synergetic Effect and Its Mechanism of Combining Mitomycin C with C(subscript 2-) Ceramide in Inhibiting Growth of Human Bladder Cancer Cells

[Objective] To evaluate the therapeutic efficacy and its mechanism of combining mitomycin C with C(subscript 2-) ceramide in human bladder cancer cells. [Methods] Different concentrations of mitomycin C and C(subscript 2-) cer were applied, individually or simultaneously, to human bladder cancer BIU-87 cells. MIT method was used to detect the cytotoxicity and then assayed the synergetic effect by calculating combination index (CI). Flow cytometry (FCM) and fluorescent staining with acridine orange (AO) were used to detect cell apoptosis. Western blot was used to detect the distribution changes of intracellular cytochrome C. At the same time, the changes of Caspase-3 activity were detected. [Results] The IC50 of mitomycin C and C (subscript 2-) ceramide were 159 μmol/L and 28 μmol/L when they were applied alone, while the numbers were 55 μmol/L and 11 μmoL/L when they were combined. Combination index (CI) was 0.74. Cell apoptosis may be induced no matter mitomycin C and C(subscript 2-) cerarnide were applied alone or together, while the apoptosis rates when these two drugs were combined were higher than that when they were alone (P＜0.05). The amounts of cytochrome C in mitochondria were decreased whether or not mitomycin C and C(subscript 2-) ceramide were applied together, while the phenomenon was the most obvious when they were combined. The amounts of cytochrome C in cytosol were also increased most obviously when two drugs were combined together (P＜0.05). The Caspase-3 activity of BIU-87 cells was the highest when mitomycin C and C(subscript 2-) ceramide were used together although Caspase-3 activities increased either these two drugs were applied alone or together. [Conclusions] Combination of mitomycin C with C(subscript 2-) ceramide can promote cell apoptosis, inhibit the growth of bladder cancer cells synergistically. The release of cytochrome C from mitochondria to cytosol and the changes of Caspase-3 activity play critical roles during this process.