| It was an important advancement in the field of cell signal transduction that phospholipids were demonstrated participating in cell signal transduction. Phosphosphingolipids were considered important in maintaining normal structure of cell membrane. Moreover some metabolites of phosphosphingolipids were found taking part in the regulation of many physiological functions such as cell growth, differentiation and apoptosis. Ceramide, as the core metabolites of phosphosphingolipids, plays a crucial role during the process of apoptosis and so it was regarded as one of the distinctive markers in the determination of cell apoptosis. Scientific researches these years revealed that endogenous ceramide, as an intracellular second messenger, could transmit apoptotic signals in the process of tumor cell apoptosis induced by stimulating factors such as radiation, heat shock and chemotherapeutics. Meanwhile several researches in vitro showed exogenous application of ceramide alone could induce lung cancer cells, endometrial cancer cells and colon cancer cells apoptosis through a number of different mechanisms. Up to now, however, little is known about the apoptosis inducing effect and its mechanism of ceramide to human bladder cancer cells.Researches indicated that mitochondria, as power manufactory of cells, play a critical role in the process of cell apoptosis. There are many important cell factors such as cytochrome C, apoptosis inducing factor, Smac/Diablo, sulfuric acid oxidase, et al that are closely relevant to apoptosis. These cell factors will be released to cytosol under stimulating factor by involucrum penetrability changes or involucrum breaking. This will render amplifying of apoptosis signals. So some researchers called the apoptosis pathway mediated by mitochondria "mitochondria pathway". However, it is also a pendent problem whether the apoptosis of human bladder cancer cells induced by ceramide is through "mitochondria pathway".Chemotherapy is an important way to reduce the recurrence rate of bladder cancer, but both toxic or side effects of chemotherapeutic agents and the drug resistance of cancer cells could interfere clinical treatments. Up to now, the effect of chemotherapy to bladder cancer is still unsatisfactory. Reasonable combination of anticancer agents may inhibite the growth of cancer cells synergistically, promote the treatment effect and decrease the concentrations of anticancer agents, so as to prevent toxic or side effects of anticancer agents and the drug resistance of cancer cells. A new therapy way will be provided if traditional chemotherapeutics can be synergetic with ceramide in inhibiting the growth of human bladder cancer cells.In this research, we will study the problem from three aspects. First, the apoptosis inducing effect and its mechanism of ceramide to human bladder cancer cells will be studied; Second, the synergy between mitomycin C (MMC) and ceramide will be analysis; Third, the mechanism of synergy between MMC and ceramide in inhibiting the growth of human bladder cancer cells will be studied.Materimals and Methods1. The inhibiting effect of C2-Cer to the growth of BIu-87 cellsBIU-87 cells were treated with different concentrations of C2-Cer, and then method of MTT was used to detect the inhibition rate. Inhibition rate=[(A490 of control group-A490 of treated group)/ A490 of control group]×100%.2. Detection of the synergetic effect The cells were divided into four groups: (1) Group of MMC: final concentrations of MMC were 24, 48, 96, 192μmol/l individually; (2) Group of C2-ceramide (C2-Cer): final concentrations of C2-Cer were 5, 10, 20, 40μmol/l individually; (3) Group of combination: final concentrations of MMC and C2-Cer were 24 and 5,48 and 10,96 and 20,192 and 40μmol/l; (4) Untreated cells served as the control group. The inhibition rate was detected by method of MTT. Inhibition rate=[(A490 of control group-A490 of treated group)/ A490 of control group]×100%. The effect of combining MMC and C2-Cer was analyzed by the combination index method of Chou and Talalay.3. Detection of morphology changes of apoptoticCells were collected after exposed to different concentrations of MMC and C2-Cer. Then suspend the cells with 1×PBS for 2 times.. Regulate the concentration of cells to 1×107/mL. After cells were stained by AO, observe cells' appearance through a fluorescence microscope.4. Detection of the apoptosis rateCells were collected after exposed to different concentrations of MMC and C2-Cer. Cell apoptosis was detected by FCM after PI staining. Collecting software and analysis software were CellQuest 3.0 and ModFit, respectively. The apoptosis rate was expressed as percentage.5. Detection of Caspase-3 activityCaspase-3 activity is determined according to that the substrate DEVD-pNA could be disassociated by activated Caspase-3 and the absorbance of monomer pNA at 405nm (A405) is positive correlated to Caspase-3 activity. Cells were collected after exposed to different concentrations of MMC and C2-Cer and then did the experiment according to the instruction. Finally read samples at 405nm in an ultraviolet spectrophotometer. The Caspase-3 activity was expressed as the value of A405.6. Detection of intracellular distribution change of cytochrome C and the amount of Bcl-2 protein Then mitochondria protein and cytosol protein were extracted according to the instruction of mitochondria/cytosol fractionation kit. Total cell protein was extracted according to the reference. Protein concentration was determined by the method of coomassie brilliant blue. Total cell protein was used to analysis the amount of intracellular Bcl-2 protein. Mitochondria protein and cytosol protein were used to analysis the distribution change of cytochrome C. 40μg of Protein was fractioned by sodium dodecylsulphate polyacrylamide (SDS-PAGE) and transferred to nitrocellulose membrane. Then the membranes were incubated with antibodies. The bands were detected by Coloring reagent. Measure the gray scale value of each band by Fluorchem v2.0 software. Protein contents were quantitated as the ratio of gray scale values of purpose bands to internal standards.7. Statistical analysist test or q test.ResultsWith increasing concentration of C2-ceramide, A490 values decreased and inhibition rates increased (Table 1-1). The inhibition rate was positively correlated with the concentration of C2-Cer (P＜0.05). Apoptotic cells appeared after cells were exposed to C2-ceramide (Figure 1-1). Apoptotic cells determined by FCM increased in a dose-dependent manner after treatment with C2-Cer (P＜0.05) (Figure 1-2). With the increasing concentration of C2-Cer, the Caspase-3 activity increased. In control group, cytochrome C resided only in mitochondria. The expression was negative in cytosol. While after BIU-87 cells were exposed to C2-Cer, cytochrome C was found in cytosol and the amount of cytochrome C in mitochondria decreased significantly compared to control group (P＜0.05) (Figure 1-3). Bcl-2 protein was found whether BIU-87 cells were exposed to C2-Cer or not. But it was obvious that the expression level, decreased in cells exposed to C2-Cer compared to control group (P＜0.05) (Figure 1-4).Inhibition effects of MMC and C2-Cer to the growth of BIU-87 cells when they were applied individually or simultaneously were shown in Table 2-1. When they were combined, IC50 of MMC and C2-Cer reduced to 35.0% and 40.6% of that when they were alone. The median effect equations of MMC and C2-Cer when they were used alone were D=153.003(fa/fu)0.771 and D-26.644 (fa/fu)0.649, respectively. The median effect equations of MMC and C2-Cer when they were combined were D=54.325(fa/fu)0.665 and D=11.350(fa/fu)0.665. These data indicated synergy between the two drags (CI＜1) (Table 2-2, Figure 2-1).Apoptosis rate when the two drugs were combined at any concentration was higher than that when the two drags were applied alone. The differences were statistically significant (P＜0.05) (Table 3-1, Figure 3-1 and 3-2). Apoptotic cells, the morphology characters of which are shrinkage of the contour, chromatin margination, pyknosis, or broken into pieces, appeared after BIU-87 cells were exposed to MMC and C2-Cer individually or simultaneously. The phenomenon was much more obvious when MMC and C2-Cer were combined than that when they were applied alone (Figure 3-3). The Caspase-3 activities of groups of MMC and C2-Cer increased obviously compared with control group (P＜0.05). The Caspase-3 activity of group of combination was the highest among the four groups. The differences were statistically significant (P＜0.05).Cytochrome C was released from mitochondria to cytosol whether BIU-87 cells were exposed to MMC and C2-Cer individually or simultaneously, which result in the decreasing of the amount of cytochrome C in mitochondria. The amount of cytochrome C in mitochondria was less in groups of MMC, C2-Cer and combination than in control group (P＜0.05). The amount of cytochrome C in mitochondria was less in group of combination than in groups of MMC and C2-Cer (P＜0.05) (Figure 3-4). The expression of cytochrome C was negative in cytosol of control group, while it was positive in any other three groups. The amount of cytochrome C in cytosol was more in group of combination compared with groups of MMC and C2-Cer (P＜0.05) (figure 3-5).Conclusions1. C2-Cer induces apoptosis of human bladder cancer BIU-87 cells by decreasing intracellular amount of Bcl-2 protein, promoting the release of cytochrome C and activating Caspase-3.2. The combination of MMC and C2-Cer may promote cell apoptosis, inhibit the growth of bladder cancer cells synergistically.3. The release of cytochrome C from mitochondria to cytosol and the changes of Caspase-3 activity may play a critical role in the synergy between MMC and C2-Cer in inhibiting the growth of human bladder cancer cells.
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