| Bladder cancer is the most common cancer of genitourinary tract in our country. The majority is the superficial bladder cancer. The treatment by surgery is the transurethral resection or laser vaporization, or partial cystectomy. After the surgery, intravesical chemotherapy is often undertaken. The most common agents are bacillus Calmette- Guerin vaccine, mitomycin C, hydroxycamptothecin etc.Bruceajavanica oil emulsion (BJOE) is a new agent of the traditional: Chinese medicine developed by Shenyang Pharmacy College. It has been put into clinical practice from the 1970s and showed satisfactory anti-cancer effect. Irrigation of bladder with Bruceajavanica oil emulsion has some advantages, such as low recurrence rate, few side effects and well tolerance etc.So it has great significance to investigate the mechanism of BJOE for the therapy of bladder cancer. This study observed the effect of different concentration BJOE on proliferation of BIU-87 cell and the possible mechanism involved. The effect of BJOE on the motor capacity and invasiveness were also examined.The inhibitory effect of BJOE on BIU-87 cell proliferation was measured by MTT assay, and IC50 was estimated. Stained by PI and Hoechst 33342 treated BIU-87 cells were observed by fluorescence microscope. And the ratio of necrosis/ apoptosis cells was detected by Annexin V-FITC apoptosis detecting kit after the cell were exposed to different concentration of BJOE and different time. The mitochondrial membrane potentials were also measured by fluorescence probe Rhodamine123. After labeling fluorescence antibodies (anti-cyt c and anti-OMP25, a protein located at mitochondrion), the distribution of cytochrome c were examined by laser confocal microscopy. Treated with 5mL/L BJOE, the whole RNA and protein were abstracted in different ways. The activation of caspase-3 was detected by western blot analysis with anti-procaspase-3 antibody which could bind to both procaspase-3 and the large subunit of activate Ca$pase-3. RT-PCR assay were used to detect the transcription level of procaspase-3 mRNA. At last the in vitro motor capacity and invasiveness were assessed by Boyden chamber and Millicell chamber.Our MTT results showed that BJOE inhibited the proliferation of BIU-87 cells in a concentration and time dependent way. According to the diagram of 48h the IC50 was about 6mL/L. The Annexin V-FITC/PI apoptosis detecting kit results indicated that at 24h and 36h, low concentration of BJ0E(5mL/L) induced more apoptosis cells comparing to the control and the high concentration of BJOE (20mL/L) induced cell death most likely in a necrosis way. Decrease of the mitochondrial membrane potential had been detected by FLC, and the high concentration of BJOE is more effective. The results of laser confocal microscopy showed that the cytochrome c released from mitochondrition to cytoplasm after the cell treated with low concentration BJOE. In control group the mRNA of procaspase-3 didn't observed by semi-quantitive RT-PCR, while the faint strip was observed in 5mL/L BJOE group(12h). The total protein of 5mL/L BJOE treated cells were abstracted and then the expression of procaspase-3 and large subunit were analysed by western blotting assay which indicated the activation of Caspase-3. After treated with lmL/L BJOE, the chamber membrane were stained with HE, and the cells which got through the membrane were counted. In this experiment the number of cells had got through the membrane in BJOE group is smaller than control group. The invasiveness test got the similar result. These indicated that BJOE possibly could inhibit the motor capacity and invasiveness of BIU-87 in vitro.The results indicate that BJOE can inhibit the proliferation of BIU-87 cell in vitro,' which could induce both necrosis and apoptosis. In low concentration BJOE group the apoptosis is the more popular death style, and in high concentration BJOE group the necrosis is more common. Lowconcentration BJOE could decrease the MMP, induce cytochrome c release and procaspase-3 mRNA transcription, and Caspase-...
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