Studies On Inhibitory Effect Of Benzalkonium Bromide On Human Bladder Carcinoma T24Cells In Vitro

Object To investigate the effects of Benzalkonium Bromide(BB) on growth inhibition,cell cycle and apoptosis of houman bladder carcinoma T24cells in vitro, and provide some theoretical evidences for further investigating the mechanism of anti-tumor of BB and for developing it as a new intravesical therapy drug.Methods1.The cytotoxic effect of BB on human bladder carcinoma T24cells was determined with varying concentration of BB (0,0.1,0.5,1,5,10,100,1000μg/ml) treatment for24,48and72hours by MTT assay taking20μg/ml Mitomycin c (MMC) as a positive control.2.The cell cycle of T24cells treated by BB(0,0.5,1,5,10μg/ml) for24hours were detected by flow cytometry(FCM) with PI staining method.3. Morphologic changes of T24cells were studied with inverted phase contrast microscope with different concentration of BB at different time.4. The extent of apoptosis of T24cells treated by BB (0,0.5,1,5,10μg/ml) for24hours were detected by (FCM) with annexin V and PI staining method.Results1.Using the human bladder carcinoma T24cell line, we found that BB treatment resulted in dose-dependent(0.1,0.5,1,5,10,100,1000μg/ml) inhibition of cellular proliferation and cell viability. The inhibition rate of cellular proliferation with BB treatment at concentrations of0.1～1000μg/ml treatment after24hours ranged from 5.30%to97.17%respectively. Except the concentration of0.1μg/ml BB group, the inhibition rate of cellular proliferation with BB treatment was significantly different compared with negative control group(P<0.01). The inhibition rate of each concentration of BB group was significantly different compared with MMC(22.84%), but the inhibitory effect of5μg/ml BB group with27.7%inhibition was similar with MMC group actually. The inhibition of cellular proliferation with BB treatment at concentrations of0.1～1000μg/ml after48and72hours ranged from10.86%to97%,29.27%to99%respectively in a time-dependent manner.2. As shown by FCM with PI staining method, BB treatment (0.1,0.5,1,5,10,100,1000μg/ml) of the T24cells resulted in significant G2/M-phase cell arrest ranging from12.97%to44.64%, and each concentration of BB group(except0.5μg/ml concentration) was significantly different compared with negative control group8.81%and MMC group12.03%(P<0.05).3. In lower concentrations(≤10μg/ml) treatment of BB for24hours, it shows a moderate apoptotic effect, while in higher concentrations(≥100μg/ml) it showed a necrotic effect on T24cells observed with inverted phase contrast microscope.4. As shown by FCM with PI and Annexin V staining method, we found that BB caused a dose-dependent increase in T24cell apoptosis. It was observed that treatment of T24cells with varying concentration of BB (0.5～10μg/ml) for24hours increased the number of early apoptotic cells from6.25%to 24.43%, and total apoptotic cells from12.12%to50.72%in a dose-dependent manner, and significantly different compared with negative control group1.63%and2.70%respectively (P<0.05). The rate of apoptotic cells induced by BB was also significantly different compared with MMC group18.4%and24.95%(P<0.05), except5μg/ml BB group.Conclusion1.Benzalkonium Bromide(BB) can inhibit the proliferation of human bladder carcinoma T24cells effectively in a dose and time-dependent manner in vitro.2. BB can arrest the T24cells in G2/M phase at a concentration(0.5～10μg/ml)in a dose-dependent manner.3. BB induces human bladder carcinoma T24cells necrosis at a high concentration(≥100μg/ml), but induces cell apoptosis at a lower concentration(0.5～10μg/ml)in a dose and time-dependent manner.4. The effect of proliferation inhibition and apoptosis induction to human bladder carcinoma T24cells caused by5μg/ml BB is considerable with that by20μg/ml MMC in vitro.