Effects Of Chloride Channel Blockers On The Proliferation Of Human Hepatoma Cell HHCC

There are many types of ion channels on mammal cells that take part in electric activity, and have a close relation with cell proliferation, differentiation and apoptosis. Chloride channels are a family of putative cell membrane protein that allow the passive diffusion of negatively charged ions according to their electrochemical gradient. Under physiological condition, these channels mainly mediate the transport of chloride ions, they are usually called chloride channels. Recently, many laboratories have found that chloride channels play a very important role in cell proliferation. Wondergem et al. reported that chloride channel blockers DIDS, NPPB, tamoxifen, and mibefradil inhibited the growth of mouse liver cell AML12, and concluded that the inhibitory effect was due to blocking volume-regulated anionic current (VRAC, Icl, vol)- Shen et al. demonstrated that cell cycle progression correlates with the expression of VRAC activity. Arrest of cell growth in the G0/G1 phase was accompanied by a significant decrease of VRAC activity. When cell reentry into cell cycle, and progress through S and G2/M, VRAC activity increased. The molecular identification of the protein mediating Icl, volremains uncertain so far. Progress in this area is severely hampered by the absence of specific high-affinity inhibitor and the presence of ICl,vol in most, if not all, cells that are used as expression system. The subtype of chloride channel that contribute to cell proliferation could not be identified, and there's no report on the mechanisms underlying the mediating of cell proliferation.We aimed to determine the effects of chloride channel blockers niflumic acid (NFA), 5-nitro-2-(3-phenylpropyIamino)-benzoic acid (NPPB) on the proliferation of hepatoma cell HHCC, and analyze which chloride channel may contribute to tumor proliferation, and discuss the probability of controlling of tumor proliferation by inhibiting chloride channel.hi this study we focus on (1) the effects of chloride channel blockers NFA and NPPB on the proliferation of hepatoma cells; (2) the role of L-type calcium channel in the proliferation of hepatoma cells; (3) the effect of environmental osmolarity on cell proliferation. The following methods were adopted in the experiments:1. The culture of HHCC hepatoma cell line.2. We adopted cell counting and MTT assay to determine the effects of different treatments on cell proliferation.3. Cell cycle analysis and apoptosis detecting were carried out by flow cytometry.4. Laser scanning confocal cytometry was used to investigate the influence of different treatments on intracellular Ca2+.The results were as follows:1. Chloride channel blockers NFA, NPPB, and calcium channel blockers Verapamil, Nifedipine inhibited the proliferation of HHCC reversibly in a dose-dependent manner.2. After treated with chloride channel blockers NFA, NPPB and calcium channel blockers verapamil, nifedipine for 2 days, the cells were arrested in G1 phase accumulated and the number of cells in S phase decreased significantly. These blockers block cell cycle progression at G1 phase.3. High dose of chloride channel blockers NFA, NPPB and calcium channel blockers verapamil, nifedipine did not induce cell apoptosis.4. [Ca2+]i decreased significantly after treating the cells with 100 μmol/L NFA or 100 μmol/L NPPB for 2 min, and recovered about 3 min after removing the blockers.5. HHCC grew fast in hypotonic environment, but significantly slowly in hypertonicity compared with those in isotonic condition. It suggests that VRAC may play an important role in cell proliferation.The present study indicates that the activity of both VRAC and L-type calcium channel is crucial for d/S checkpoint progression of hepatoma cell HHCC. Pharmacological blockade of the growth of HHCC cells by NFA or NPPB perhaps is via inhibiting Ca2+/CaM pathway.