Roles of volume-activated chloride channels in control of growth of normal and cancerous nasopharyngeal epithelial cells

Linyan Zhu1, Zhenfeng Liu1,2, Bingxue Li1, Shuangfeng Teng1, Haifeng Zhang1,2, Linjie Yang1, Liwei Wang1,2, Lixin Chen1. 1Department of Pharmacology, Medical College, Jinan University, Guangzhou, China, 2Department of Physiology, Medical College, Jinan University, Guangzhou, China.

It was proved by us previously that chloride channel activities were cell cycle-dependent and were involved in cell proliferation in nasopharyngeal carcinoma cells (Chen et al. 2002; Chen et al. 2007). In this study, the expression and roles of volume-sensitive chloride channels in cell growth were investigated in poorly-differentiated human nasopharyngeal carcinoma cells (CNE-2Z, kindly provided by Professor Weiping Tang, Department of Pathology, Guangdong Medical College, China) and its counterpart, the normal human nasopharyngeal epithelial cells (NP69-SV40T, obtained from Hunan Xiangya Type Culture Collection, Hunan, China) using the patch clamp technique, the MTT essay and the siRNA technology described by us previously (Chen et al. 2002; Chen et al. 2007; Yang et al. 2011). The results are expressed as mean ± standard error and, where appropriate, have been analyzed using ANOVA. Consistent with growth ability, the activities and expression of the volume-sensitive chloride channels were different between the cancerous and normal cells. The background chloride currents recorded under isotonic condition, the volume-activated chloride currents induced by 47% hypotonic challenges and the hyponinicity-induced regulatory volume decrease (RVD) were much larger in cancerous cells (12.1±1.3 pA/pF, 78.3±9.5 pA/pF, and 56.1±4.3%, respectively; n = 11-39) than those in NP69-SV40T cells (5.5±1.1 pA/pF, 42.3±5.3 pA/pF and 22.1±5.3%, respectively; n = 10-62; P < 0.01), suggesting the up-regulation of expression of volume-activated chloride channels. This was proved by the up-regulation of ClC-3 proteins (by 70.3±10.5%, P<0.01, n = 4), a candidate of volume-activated chloride channels, in the cancerous cells. Functional inhibition of chloride channel activities by the chloride channel blocker, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and knock-down of ClC-3 expression by specific ClC-3 siRNA attenuated the background currents, suppressed the activation of the volume-activated chloride currents and inhibited cell growth in the cancerous and normal cells. However, the sensitivities of the cancerous cells were much higher than that of the normal cells. The inhibition of proliferation by 48 h treatment with ClC-3 siRNA (100 nM) was higher in CNE-2Z cells (by 50.3±6.3%, n = 5) than in NP69-SV40T cells (by 22.5±5.3%, n = 5, P < 0.01). Our data suggest that volume-activated chloride channels play a more important role in control cell proliferation in the cancerous cells than in the normal cells; the growth of cancerous cells is more dependent on the activities of volume-activated chloride channels than that of the normal cells. ClC-3 protein may be considered as a potential tumor marker and therapeutic target for human nasopharyngeal carcinoma.

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