Effects Of Benzalkonium Bromide On Bladder Tumor Cell&Mitochondria Mediated Apoptotic Signaling Pathway

Objective:Although a variety of intravesical measures are taken after the non-muscle invasive bladder urothelial carcinoma treatment, the recurrence rate is kept high. To seek more effective intravesical drug and method is a new focus of urological surgeons. Benzalkonium bromide is used clinically and hygienically for the control of bacterial growth. There have reports that benzalkonium bromide induce apoptosis in normal and in cancerous cells; and we found in the clinical work benzalkonium bromide has the ability to destroy bladder mucosa, so we speculate that benzalkonium bromide maybe a novel agent for use in post-operative administration. In this paper, we chose human bladder cancer cell line5637as the research object, and explored the biological characteristics (proliferation, apoptosis, migration and immunostimulation) of bladder cancer after benzalkonium bromide administration, normal bladder mucosa permanent biochemical SV-HUC-1cell line as control cell, mitomycin and BCG as control drug. We also detected the gene expression of caspase-dependent and non caspase-dependent cell apoptosis signaling pathways mediated by mitochondria.Methods:1) Using MTT, FCM method for detection of benzalkonium bromide in5637cells proliferation, apoptosis and cell cycle effects.2) Using wound healing assay and transwell assay for detect the migration of5637cells after benzalkonium bromide administration.3) Detection secretion of level of IL-2, IL-6, IL-8, TNF-a on5637cells by double antibody sandwich ELISA and real time quantative PCR.4) Real time quantative PCR was performed to detect the expression level of cyt c, apaf-1, caspase-9, caspase-3, PARP-1, AIF and Endo G.Results:1) According to the different concentration, benzalkonium bromide can directly cause cell necrosis or apoptosis. Low concentration effects of benzalkonium bromide on cells include inhibition of proliferation, apoptosis and cell cycle arrest in a time and dose dependent. The effects of benzalkonium bromide on SV-HUC-1cells is weaker than on5637cells. Inhibitory effect of5-10μg/ml benzalkonium bromide was similar with mitomycin actually.1.25-2.5ug/ml benzalkonium bromide and100μg/ml mitomycin C had similar G0/G1arrest after administration for24h; arrest effect of benzalkonium bromide is stronger in mitomycin C after48h.2) Benzalkonium bromide can induce IL-2, IL-6, IL-8and TNF-a secretion, cytokine concentration increased with longer time and higher drug concentration.3) Benzalkonium bromide reduce reduced capacity of migration on5637cells and with the higher concentration, the inhibition effect was stronger trend. Compared with the100ug/ml mitomycin C, migration of2.5ug/ml benzalkonium bromide had a stronger inhibition trend.4) Expression of cyt c, apaf-1, caspase-9, caspase-3gene was up-regulated after benzalkonium bromide administration in5637cells, but the expression of PARP, AIF, Endo G gene had no obvious regularity.Conclusion:Benzalkonium bromide on bladdder cancer cells can play against proliferation, induce apoptosis and cell cycle arrest effect, also reduced the migration ability and had immunostimulating ability of bladder cancer cells in vitro. Benzalkonium bromide exerts its apoptosis effects on5637cells via the caspase-dependent signaling pathways.