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. 2014 Sep 11;2(9):e12137.
doi: 10.14814/phy2.12137. Print 2014 Sep 1.

Functional expression of chloride channels and their roles in the cell cycle and cell proliferation in highly differentiated nasopharyngeal carcinoma cells

Weiyuan Huang  1 , Mei Liu  2 , Linyan Zhu  2 , Shanwen Liu  1 , Hai Luo  1 , Lianshun Ma  2 , Haibo Wang  2 , Ruiling Lu  1 , Xiaoxue Sun  1 , Lixin Chen  2 , Liwei Wang  1
Affiliations

Functional expression of chloride channels and their roles in the cell cycle and cell proliferation in highly differentiated nasopharyngeal carcinoma cells

Weiyuan Huang et al. Physiol Rep. .

Abstract

We previously demonstrated that the growth of the poorly differentiated nasopharyngeal carcinoma cells (CNE-2Z) was more dependent on the activities of volume-activated chloride channels than that of the normal nasopharyngeal epithelial cells (NP69-SV40T). However, the activities and roles of such volume-activated chloride channels in highly differentiated nasopharyngeal carcinoma cells (CNE-1) are not clarified. In this study, it was found that a volume-activated chloride current and a regulatory volume decrease (RVD) were induced by 47% hypotonic challenges. The current density and the capacity of RVD in the highly differentiated CNE-1 cells were lower than those in the poorly differentiated CNE-2Z cells, and higher than those in the normal cells (NP69-SV40T). The chloride channel blockers, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and tamoxifen inhibited the current and RVD. Depletion of intracellular Cl(-) abolished the RVD. The chloride channel blockers reversibly inhibited cell proliferation in a concentration- and time-dependent manner, and arrested cells at the G0/G1 phases, but did not change cell viability. The sensitivity of the three cell lines to the chloride channel blockers was different, with the highest in poorly differentiated cells (CNE-2Z) and the lowest in the normal cells (NP69-SV40T). ClC-3 proteins were expressed in the three cells and distributed inside the cells as well as on the cell membrane. In conclusion, the highly differentiated nasopharyngeal carcinoma CNE-1 cells functionally expressed the volume-activated chloride channels, which may play important roles in controlling cell proliferation through modulating the cell cycle, and may be associated with cell differentiation. Chloride channels may be a potential target of anticancer therapy.

Keywords: Cell cycle; cell proliferation; chloride channels; nasopharyngeal carcinoma; regulatory volume decrease.

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Figures

Figure 1.
Figure 1.
Activation of hypotonicity‐induced chloride currents and inhibition of the currents by the chloride channel blockers NPPB and tamoxifen in CNE‐1 cells. Cells were held at 0 mV and then stepped in sequence to ±80, ±40 and 0 mV repeatedly. Forty‐seven percent hypotonic challenges (Hypo) activated a chloride current which was inhibited by 100 μmol/L NPPB (A & B) and 20 μmol/L tamoxifen (C & D). (E) shows the inhibitory effect of NPPB (100 μM) on the tamoxifen (20 μmol/L)‐insensitive current. (F) presents the comparison of the hypotonicity‐activated currents between CNE‐2Z, CNE‐1 and NP‐69‐SV40T cells (n = 15, 18,16 respectively). Data in B, D and F are mean ± SE of 8–18 cells. *P <0.05, **P <0.01.
Figure 2.
Figure 2.
Hypotonicity‐induced RVD and the effects of depletion of intracellular Cl− and extracellular application of the chloride channel blockers NPPB and tamoxifen on RVD in CNE‐1 cells. Exposure to a 47% hypotonic solution swelled CNE‐1 cells and induced a regulatory volume decrease (RVD) (A). RVD in CNE‐2Z cells was the largest and that in NP‐69‐SV40T cells was the smallest with that in CNE‐1 in the middle (B, five experiments). Depletion of intracellular Cl− by incubation the cells in the Cl−‐free solution (substitution of NaCl with equimolar sodium gluconate) for 2 h (C), or extracellular application of 100 μmol/L NPPB (D) or 20 μmol/L tamoxifen (E) abolished the hypotonicity‐induced RVD. Data in the figures are mean ± SE of 16–39 cells in 3–5 experiments. *P <0.05, **P <0.01.
Figure 3.
Figure 3.
Inhibitory effects of the chloride channel blockers NPPB and tamoxifen on CNE‐1 cell proliferation. Relative cell number was detected by the MTT assay and expressed as the optical density (OD value). (A) and (B) present the inhibitory effects of 100 μmol/L NPPB and 20 μmol/L tamoxifen on cell proliferation. (C) presents the comparison of the inhibitory effects of NPPB and tamoxifen between CNE‐2Z, CNE‐1, and NP‐69‐SV40T cells (D) and (E) shows the release of cells from the inhibitory effects of NPPB and tamoxifen by the washout of the inhibitors. Data in the figures are mean ± SE of four experiments.
Figure 4.
Figure 4.
Effects of the chloride channel blockers, NPPB, and tamoxifen, on CNE‐1 cell cycle detected by the flow cytometry. (A–F) The typical cell cycle distribution of cells incubated in the medium with or without chloride channel blockers (100 μmol/L NPPB or 20 μmol/L tamoxifen) for 24 and 48 h. (G–I) The quantitative distribution of cells in different phases received different treatments (mean ± SE of four experiments). *P <0.05, **P <0.01 (vs. Control).
Figure 5.
Figure 5.
Expression of ClC‐3 chloride channel proteins detected by immunofluorescence in CNE‐1 cells. (A) ClC‐3 immunofluorescence (green). (B) The nuclei labeled by DAPI staining (blue). (C) Presents the transmitted light images of the cells.
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