Roles Of ClC-3 Chloride Channels In Highly Selective Anti-tumor Of Disulfiram/Copper In Nasopharyngeal Carcinoma

Objective:Disulfiram had sixty years of clinical application background as an anti- alcoholism drug. In recent years, disulfiram chelated with bivalent metal cation show a highly selective anti-tumor effect. The underlying mechanisms, however, are unclear. Chloride channels were the most important anion channel that involved in physiological and pathophysiological processes of cells such as cell proliferation, apoptosis, cycle regulation. Our previous studies found that, compared those in normal nasopharyngeal epithelial cells, the functional activities and protein expression of Cl C-3 chloride channel were up-regulated in nasopharyngeal carcinoma cells. These results showed that chloride channel may be considered as a potential tumor marker and therapeutic target for human carcinoma. This study based on higher effective and lower toxic anti-tumor effect of disulfiram/copper and determined the roles of the Cl C-3 chloride channel in cell apoptosis induced by DSF/Cu. So as to provide evidence for the development of the anti-tumor drugs which target on Cl C-3 chloride channel.Methods:Experiments were performed on poorly-differentiated human nasopharyngeal carcinoma cells(CNE-2Z)and normal human nasopharyngeal epithelial cells(NP69-SV40T).1) DSF/Cu induced apoptosis of CNE-2Z cells and NP69-SV40 T cells were monitored by flow cytometry.2) DSF/Cu induced the ultra-microstructure changes of CNE-2Z cells and NP69-SV40 T cells observed by AFM.3) Specific si RNA was employed to down-regulate the expression of Cl C-3 chloride channel protein in CNE-2Z cells.4) Transfection efficiency of Cl C-3 si RNA in CNE-2Z cells was detected by the confocal microscopy and flow cytometry.5) Western blot technique was used to analyze the expression of Cl C-3 protein.6) Chloride currents in different conditions were recorded by the whole cell patch-clamp technique. Results:1. DSF/Cu induced apoptosis of CNE-2Z cells in dose-dependent and time-dependent manner.DSF/Cu had less influence on NP69-SV40 T cells. 2. Height, width, Ra and Rq were compared between stimulated and non-stimulated cellswhich detected by AFM. DSF/Cu induced the ultra-microstructure changes of CNE-2Z cellsmore than that in NP69-SV40 T cells. The volume of CNE-2Z cells shrunk or rounded andthe membrane surface of CNE-2Z cells were smooth. 3. The apoptosis of CNE-2Z cells induced by DSF/Cu were inhibited by chloride channelblocker NPPB. 4. Both CNE-2Z cells and NP69-SV40 T cells expressed Cl C-3 chloride channel proteins.Compared those in NP69-SV40 T cells, the protein expression of Cl C-3 chloride channelwere up-regulated in CNE-2Z cells. 5. The protein expression of Cl C-3 chloride channel was up-regulated with increase ofadministration time and correlated with apoptosis induced by DSF/Cu.6. The whole cell patch-clamp results showed that the chloride currents induced by DSF/Cu inCNE-2Z cells were larger than that in NP69-SV40 T cells. DSF/Cu-activated chloridecurrents were inhibited by 47% hypertonic solution and the chloride channel inhibitor NPPBin CNE-2Z cells. 7. Down-regulation of Cl C-3 chloride channel protein expression by si RNA attenuated theapoptosis and current of CNE-2Z induced by DSF/Cu. Conclusion: 1) DSF/Cu had a highly selective activity that induced apoptosis effect on nasopharyngealcarcinoma CNE-2Z cells. 2) Cl C-3 chloride channel might be involved in the process that apoptosis of CNE-2Z cellsinduced by DSF/Cu. 3) The highly selective anti-tumor effect of DSF/Cu was related to the different expression ofCl C-3 chloride channel protein in normal and carcinoma cells.