Inhibition of cell proliferation and arrest of cell cycle progression by blocking Cl- channels in nasopharyngeal carcinoma cells

AIM: The roles of Cl^-channels in regulatory volume decrease (RVD), cell proliferation and cell cycle progression in nasopharyngeal carcinoma cells (CNE-2Z) were investigated. METHODS: Image analysis of living cells was used to detect the volume changes following exposure to hypotonic solutions. Cell viability was determined by the trypan blue assay. MTT method was applied to detected cell proliferation. The effect of the blocker on the cell cycle distribution was monitored by the flow cytometry. RESULTS: 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) inhibited RVD and cell proliferation in a dose-dependent manner. NPPB at the concentration of 100 μmol/L arrested cells in G₁ phase (G₁ population increased from 54% to 71% at 48 h after treatments), but did not significantly alter cell viability. CONCLUSION: Block of chloride channels suppressed cell proliferation by arresting cells in G₁ phase. The results suggest that activation of Cl^-channels and RVD is necessary for facilitating cells to proceed to the S phase from G₁ phase and maintaining cell proliferation.     

Hypotonic challenges induce volume-activated chloride currents and regulatory volume decrease in rat embryonic myocardial H9c2 cells

AIM:To investigate the volume-activated chloride currents and regulatory volume decrease(RVD) induced by hypotonic challenges in rat embryonic myocardial cell line H9c2. METHODS:The technique of whole-cell patch-clamp was used to record the chloride currents induced by hypotonic challenges and to clarify the properties of the currents in H9c2 cells. The changes of cell volume were observed by the technique of real-time living cell imaging, and the roles of chloride channels in RVD were analyzed. RESULTS:A weak background current was recorded in H9c2 cells under isotonic condition. Extracellular application of 47% hypotonic solution rapidly activated an outward rectified current, which did not exhibit time-and voltage-dependent inactivation with the current density of(47.77±3.80) pA/pF at +80 mV and(-33.36±2.80) pA/pF at-80 mV. The reversal potential was(-9.02±0.61) mV, closed to the calculated equilibrium potential for Cl^-(-0.9 mV). The current was volume-sensitive and was completely suppressed by 47% hypertonic solution. In addition, chloride channel blockers tamoxifen(20 μmol/L), 5-nitro-2-(3-phenylpropylamino) benzoic acid(NPPB,100 μmol/L) and ATP(10 mmol/L) significantly inhibited the current with different inhibitory ratios. The phenomenon of RVD was also observed in H9c2 cells under the condition of perfusion with 47% hypotonic solution. The chloride channel blocker NPPB at concentration of 100 μmol/L completely inhibited the RVD process. CONCLUSION:The volume-activated chloride channels, which are activated by extracellular hypotonic challenges, play an important role in the process of regulatory volume decrease in H9c2 cells.     

Bufalin activates chloride channels in poorly differentiated nasopharyngeal carcinoma cells

AIM: To investigate the activation of chloride channels induced by bufalin and the properties of the channels in poorly differentiated nasopharyngeal carcinoma (CNE-2Z) cells. METHODS: The technique of whole-cell patch clamp was used to record the chloride currents and to analyze the characteristics of the currents in CNE-2Z cells.RESULTS: A chloride current was slowly activated by extracellular application of bufalin (1 μmol/L). The activation of the current was slower than that of the volume-activated chloride current, with an activation latency of(12.1±6.4) min. The reversal potential of the current was close to the calculated Cl^- equilibrium potential (E_Cl =-0.9 mV). The chloride current was outward-rectified and did not show significant time-dependent or voltage-dependent inactivation. The chloride channel blocker tamoxifen completely inhibited the outward and inward currents. The current was also completely inhibited by extra-cellular application of 47% hypertonic solution. CONCLUSION: Bufalin activates chloride channels and induces a chloride current in CNE-2Z cells. Compared with the volume-activated chloride current in CNE-2Z cells, the activation latency of the bufalin-induced current is longer and the outward rectification is more obvious.     

Volume-activated chloride currents in human acute lymphoblastic leukemia Molt4 cells

AIM: To characterize the chloride current activated by extracellular hypotonic stress in human acute lymphoblastic leukemia Molt4 cells.METHODS: The technique of whole-cell patch clamp recording was used to detect the volume-activated Cl^- current in Molt4 cells. The characteristics of the current were investigated.RESULTS: The background Cl^- current was weak and stable under isotonic condition. However, a large Cl^- current was induced by exposure of the cells to 47% hypotonic solution. The current showed a characteristic of outward rectification. No voltage-dependent inactivation and time-dependent inactivation were observed. The current was sensitive to the change of cell volume and was inhibited by extracellular hypertonic solution. Extracellular tamoxifen, which is one of the chloride channel blockers, significantly inhibited the current. The effects of tamoxifen were almost equal for both inward and outward currents (P>0.05).CONCLUSION: There are volume-activated chloride channels on the cell membrane of Molt4 cells. Exposure of the cells to a hypotonic solution activates the chloride channels and induces a volume-activated chloride current. The volume-activated Cl^- channels are sensitive to tamoxifen in Molt4 cells.     

Role of ClC-3 chloride channel in inhibition of nasopharyngeal carcinoma cell cycle by metformin

AIM: To study the roles of ClC-3 chloride channel in the inhibition of nasopharyngeal carcinoma cell cycle by metformin. METHODS: The CNE-2Z cells were treated with metformin at different concentrations. The viability of CNE-2Z cells was measured by CCK-8 assay. The cell cycle distribution was detected by flow cytometry. The protein expression of ClC-3 was determined by Western blot. The Cl^- currents was record by the patch-clamp technique. In addition, the cell cycle distribution was analyzed in the nasopharyngeal carcinoma CNE-2Z cells which over-expressed ClC-3 by pEZ-M03-ClC-3 plasmid transfection. RESULTS: Metformin inhibited the viability of CNE-2Z cells at 5, 10 and 20 mmol/L. Metformin at 10 mmol/L prevented the activation of chloride currents induced by hypotonicity, inhibited the protein expression of ClC-3 chloride channel and arrested the nasopharyngeal carcinoma CNE-2Z cells at G₀/G₁ phases. ClC-3 chloride channel protein over-expression reversed the effect of metformin on the cell cycle distribution of CNE-2Z cells. CONCLUSION: Metformin inhibits the CNE-2Z cell cycle, which may be related to the inhibition of ClC-3 chloride channel function and protein expression.     

Roles of chloride channels in the migration of nasopharyngeal carcinoma cells at different stages of the cell cycle

AIM: To clarify the migration capability of nasopharyngeal carcinoma cells (CNE-2Z) at different stages of the cell cycle and the roles of chloride channels in cell migration. METHODS: Synchronous cells were obtained by the serum deprivation，double chemical-block, mitotic arrest and shake-off techniques. Cell cycle distribution of CNE-2Z cells was analyzed by the flow cytometry. Migration rate was assayed by transwell chambers and by image analysis. The cytotoxicity of chemicals on cells was tested by MTT assay. RESULTS: CNE-2Z cells at different stages of the cell cycle exhibited different migratory ability. The migration rate of the three stages was G1>M> S. The migration of CNE-2Z cells was inhibited by chloride channel blockers (ATP, NPPB and tamoxifen), but the inhibitory effect of the blockers varied with cells at different stages. CONCLUSIONS: The migratory ability is associated with the cell cycle in CNE-2Z cells. Chloride channels play an important role in cell migration of CNE-2Z cells.     

Knockdown of IK1 potassium channel inhibits expression and function of ClC-3 chloride channel

AIM: To investigate whether the ClC-3 chloride channel is an acting target of the IK1 potassium channel, and to study the action of IK1 potassium channel on the functional activities and expression of ClC-3 chloride channels. METHODS: IK1 gene was silenced by IK1 siRNA in poorly-differentiated nasopharyngeal carcinoma cells (CNE-2Z). Real-time PCR and Western blot were used to detect the expression of ClC-3 at mRNA and protein levels. The distribution of ClC-3 protein in the cells was observed under confocal immunofluorescence microscope. The chloride current was recorded by the patch-clamp technique. RESULTS: IK1 siRNA was successfully transfected into the CNE-2Z cells and knocked down the expression of IK1 potassium. The mRNA expression of ClC-3 was increased by the IK1 siRNA. IK1 siRNA inhibited the expression of ClC-3 protein. A chloride current was activated by hypotonic challenges, and the hypotonicity-induced current was reduced in the cells which successfully transfected with IK1 siRNA. CONCLUSION: The knockdown of IK1 potassium channels inhibits the expression and function of ClC-3 chloride channel.     

Osmolarity,cell volume and proliferation in nasopharyngeal carcinoma cells

AIM: To investigate the relationship between osmolarity, cell volume and cell proliferation in nasopharyngeal carcinoma cells. METHODS: MTT method was applied to detect the proliferation ability of the poorly-differentiated nasopharyngeal carcinoma cell (CNE-2Z) under various osmolarity conditions. The flow cytometry was used to analyse cell cycle distribution. Cell volume was obtained by the image analysis of living cells and cell viability was determined by the trypan blue assay. RESULTS: Cultivation of cells under the hypertonic conditions of 370 and 440 mOsmol/L increased cell volume by 8.7% and 27.8% and facilitated cell proliferation by 22.2% and 33.9%, respectively. However, hypotonic incubation of cells with osmolarity of 160 and 230 mOsmol/L decreased cell volume by 12.8% and 4.1% and inhibited cell proliferation by 34.0% and 15.6%, respectively. Cell volume was positively correlated with cell proliferation rate. Long-term cultivation of cells under anisotonic conditions did not significantly alter cell cycle distribution, but hypotonic cultivation decreased cell viability. CONCLUSION: Proliferation of nasopharyngeal carcinoma cells was closely correlated with the osmolarity of culture medium and cell volume. Hypotonic cultivation may inhibit cell proliferation by decreasing cell volume to facilitate cell death mechanisms.     

Effects of tamoxifen on volume-activated Cl- currents of nasopharyngeal carcinoma cells at different stages of the cell cycle

AIM:To investigate the inhibitory effects of Cl- channel blocker, tamoxifen, on volume-activated Cl- currents of nasopharyngeal carcinoma cells (CNE-2Z cells) in G₁ and S phases. METHODS:Highly synchronous cells in G₁ phase and S phase were obtained by the serum starvation and the double-block techniques. The whole-cell patch clamp technique was used to observe the effects of tamoxifen on volume-activated Cl- currents and to analyze the anion permeability of volume-activated Cl- channels. RESULTS:47% hypotonic stimulation activated a Cl- current in the nasopharngeal carcinoma cells at the cell cycle stage G₁ phase and S phase. Tamoxifen at concentration of 10 to 30 μmol/L completely inhibited the current. However, the time needed to completely inhibit the current was dose-dependent and was different between G₁ phase and S phase. The time needed to completely inhibit the current was shorter in G₁ cells than that in S phase cells. The anion permeability sequence of the volume-activated Cl- channel was I->Cl->gluconate in both G₁ phase and S phase cells. The permeability of G₁ phase cells to I- was higher than that in S phase cells, but to gluconate was lower than that in S phase cells. CONCLUSIONS:The density of the volume-activated Cl- current, the anion permeability of the channel and the sensitivity of the current to tamoxifen were different between the CNE-2Z cells in G₁ phase and those in S phase. The results suggest that the expression of tamoxifen-sensitive, volume-activated chloride channels is differentiated at different stages of the cell cycle.     

Ca2+-activated Cl- channels and pulmonary hypertension induced by high pulmonary blood flow

Pulmonary hypertension induced by high pulmonary blood flow involves a variety of complex mechanisms, including endothelial damage, pulmonary artery smooth muscle relaxation-contraction disorder and vascular remodeling. Besides, the factor of ion channels in pulmonary artery smooth muscle cells is also highly correlated to vasoconstriction. In recent years, many studies have shown that activation of Ca²⁺-activated Cl^- channels is responsible for the membrane depolarization of pulmonary artery smooth muscle cells, and plays an important role in the regulation of vascular tone and vasoconstriction. This article reviews the biophysical and pharmacological characteristics of Ca²⁺-activated Cl^- channels as well as the influence of Ca²⁺-activated Cl^- channels in high pulmonary blood flow-induced pulmonary hypertension.     

Modulation of K+ channels by Ca2+ and pH in regulatory volume decrease

It has been widely appreciated that animal cells rely on the mechanism of regulatory volume decrease(RVD) after swell in a hypotonic environment. Activation of K channels is crucial in the process of self-protection. There are a characterized increase in cytoplasmic Ca and a decrease in pH in the process of RVD in many cell types. Ca entry via transient receptor potential(TRP) channel is crucial for the cytoplasmic Ca increase, which in turn induces the decrease in pH.The increase in cytoplasmic Ca and decrease in pH activate or inhibit the activity of K channel, respectively. In this review, the regulatory network at cellular level between cytoplasmic Ca and pH, and the modulation of K channels by Ca and pH in the process of RVD are discussed.     

Effects of obstructive jaundice on enterocyte chloridion secretion in rats

AIM: To investigate the effects of obstructive jaundice (OJ) on enterocyte chloridion secretion in rats. METHODS: The OJ model of rats was set up. The rats were divided into OJ1 group and OJ2 group randomly. The animals in OJ1 group were sacrificed by exsanguination 7 days after operation and the rats in OJ2 group were sacrificed 14 days after operation. The Cl^- concentration in peripheral blood was detected. The epithelial cells in the terminal ileum mucosas were cultured in vitro. The concentration of intracellular Cl^- was detected by fluorospectrophotometry. The Cl^- current was measured by whole-cell patch clamp experiments. Western blotting was used to examine the change of voltage-gated Cl^- channel 2 protein (ClC-2) and the images were analyzed by image analysis system and statistical software quantitatively. RESULTS: The serum Cl^- concentration in OJ1 group and OJ2 group were obviously lower than that in control group . The intracellular Cl^- relative concentrations in OJ1 group and OJ2 group were higher than that in control group (3.14±0.38 and 3.55±0.47 vs 2.69±0.41,both P<0.05). The Cl^- current of normal epithelial cells displayed outward rectification, and transformed negative value into positive value following cell membrane depolarization. The Cl^- current was (-15.45±7.56) pA/pF and (5.85±0.81) pA/pF when voltage was -80 mV and 80 mV. The ranges of upgrade of absolute values of average electric current density in OJ groups were lower than those in control group (all P<0.05). Western blotting analysis showed that the ClC-2 protein generated a broad band about 90 kD. The average gradations of Western blotting images were lower in OJ groups than that in control group (0.20±0.04 and 0.19±0.06 vs 0.27±0.06, both P<0.05). However, no difference between the 2 OJ groups was observed (P>0.05). CONCLUSION: The chloridion secretion of intestinal epithelium is restrained in rats with OJ. The concentration gradient between exterior and interior of epithelial cells is decreased, and the Cl^- outward current is reduced. All of above are concerned with downregulation of ClC-2 protein in cell membrane.     

Effect of extract of Oratosquilla oratoria on telomerase activity in human nasopharyngeal carcinoma cell line

AIM: To study the inhibitory effect and its mechanisms of the extract of Oratosquilla oratoria (EOS) on the activity of telomerase in human nasopharyngeal carcinoma cell line CNE-2Z. METHODS: MTT assay was used to determine the effect of different doses of EOS on the proliferation of CNE-2Z cells. The activity of telomerase was analyzed by TRAP-ELISA. The mRNA expression of hTERT was determined by RT-PCR, and the protein expression of c-Myc was detected by Western blotting. RESULTS: EOS inhibited the proliferation of CNE-2Z cells in a dose-dependent manner (P<0.01). Telomerase activity was decreased, the mRNA expression of hTERT and c-Myc in CNE-2Z cells was also decreased (P<0.01) by the treatment of EOS. The correlation between the down-regulatory expression of hTERT mRNA and inhibitory expression of c-Myc protein (P<0.05) under the condition of EOS exposure was observed. CONCLUSION: EOS inhibits the proliferation of CNE-2Z cells by reducing the activity of telomerase, which is related with the inhibitory expression of hTERT mRNA caused by the decrease in c-Myc production.     

Chloride currents activated by cisplatin in nasopharyngeal carcinoma cells

AIM: To study the activation and the properties of chloride channels activated by the antineoplastic agent cisplatin (cDDP) in nasopharyngeal carcinoma (CNE-2Z) cells. METHODS: The whole-cell patch clamp technique was used to record chloride currents. The characteristics of the channel were investigated using ion-exchange and pharmacological methods. RESULTS: A chloride current was activated by extracellular application of cDDP (5 μmol/L). The current showed significant outward rectification. The reversal potential of the current was close to the calculated equilibrium potential for Cl-(ECl=-0.9 mV). The activation of the chloride channel was dependent on the existence of the intracellular ATP. The permeability sequence of the four anions was I-≥Br->Cl->gluconate. The current was almost completely inhibited by extracellular application of chloride channel blocker tamoxifen (30 μmol/L). CONCLUSION: Antineoplastic agent cDDP can activate a chloride channel with characteristics similar to the volume-activated chloride channel in CNE-2Z cells.     

Chloride currents activated by cisplatin in poorly differentiated naso-pharyngeal carcinoma cells are not Ca2+-activated chloride currents

AIM：To investigate the type of chloride channel activated by cisplatin in poorly differentiated nasopharyngeal carcinoma cells (CNE-2Z cells). METHODS：The technique of whole-cell patch-clamp was used to investigate the role of Ca2+ in the activation of cisplatin-activated chloride currents and to analyze the effect of hypertonic stress on these currents in CNE-2Z cells. RESULTS：Chloride currents were induced when the cells were exposed to the calcium-free cisplatin solution, showing the similar density to the currents induced by cisplatin with the presence of extracellular calcium. However, the latency and the peak time of cisplatin-activated currents in the absence of extracellular calcium were prolonged. The activation of cisplatin-activated chloride currents was insensitive to the depletion of intra- and extracellular calcium. Calcium channel antagonist nifedipine had no effect on the cisplatin-activated chloride currents, while hypertonic solution completely inhibited those currents. CONCLUSION：The cisplatin-activated chloride currents are independent on intra/extracellular calcium. The chloride channels activated by cisplatin are not calcium-activated chloride channels, but are probably volume-sensitive chloride channels.     

Effects of PKC-α asODN and PKA-ⅠasODN on growth and proliferation of the CNE-2Z cells of nasopharyngeal carcinoma

AIM:To investigate the effects of antisense oligonucleotides (asODN) of PKC-α and PKA-Ⅰon growth and proliferation of the CNE-2Z cells.METHODS:The expression of PKC-α and PKA-Ⅰ was observed with immunohistochemistry method. The asODNs of (1)PKC-α, (2)PKA-Ⅰ, (3)PKC-α and PKA-Ⅰ, were transfected into CNE-2Z cells by lipofectin (LP), and a random sequence as a control was used. The cell growth index (GI) and the clone formation rate of CNE-2Z were detected by MTT colorimetric assay and soft agar assy, respectively.RESULTS:The expression of PKC-α or PKA-Ⅰin CNE-2Z in experimental group were both significantly lower than that of control group(P<0.05). The GI and clone formation rates of CNE-2Z cells transfected by PKC-α and PKA-ⅠasODN with concentrations ranging from 0.05 μM to 1.00 μM were lower significantly than that of control groups(P<0.05), and there was a dose-dependent relationship among them. The inhibitory effects of PKC-α and PKA-ⅠasODNs both on the cell growth index (GI) and clone formation rates were more significant than that of control group(P<0.01),and the GI were significantly lower than that of the other experimental groups(P<0.05).CONCLUSION:PKC-α asODN and PKA-ⅠasODN inhibited CNE-2Z growth and proliferation in vitro, and a synergetic inhibitory effect of PKC-α asODN and PKA-ⅠasODN was also observed.     

Inhibitory role of epigallocatechin-3-gallate in proliferation of human nasopharyngeal carcinoma cells by targeting P53/miR-34a

AIM: To study the effect of epigallocatechin-3-gallate(EGCG) on the proliferation of human nasopharyngeal carcinoma(NPC) cells, and to explore its mechanism by targeting miR-34a.METHODS: Nasopharyngeal carcinoma CNE-2Z cells were treated with various concentrations of EGCG. The ability of cell proliferation was detected by CCK-8 assay, 5-ethynyl-2-deoxyuridine(EdU) incorporation assay and colony-forming assay. The cell cycle distributions were analyzed by flow cytometry. The protein levels of P53 and Notch1 were detected by Western blot. The expression of miR-34a and Notch1 mRNA was measured by real-time PCR.RESULTS: EGCG effectively inhibited the proliferation and colony formation of CNE-2Z cells in a dose-dependent manner, which was related to its induction of cell cycle arrest at G₀/G₁ phase. The expression of P53 and miR-34a in CNE-2Z cells was significantly increased after treated with EGCG, while the expression of Notch1 at mRNA and protein levels was markedly suppressed.CONCLUSION: EGCG induces cell cycle arrest and suppresses cell proliferation by regulating the P53/miR-34a/Notch1 pathway in NPC cells.     

Effects of extract of Oratosquilla on expression of P53, COX-2 and VEGF in human nasopharyngeal carcinoma cell line

AIM: To study the inhibitory effect of the extract of Oratosquilla(EOS) on the expression of P53, cyclooxygenase-2(COX-2) and vascular endothelial growth factor(VEGF) in nasopharyngeal carcinoma cell line CNE-2Z.METHODS: CNE-2Z cells were treated with different concentrations of EOS for 24 h. The methods of RT-PCR and immunocytochemistry were used to detect the expression of COX-2 and VEGF at mRNA and protein levels, respectively. The protein expression of P53 was determined by Western blotting.RESULTS: After treated with EOS, the protein expression of P53 in CNE-2Z cells was decreased(P<0.01), and the expression of COX-2 and VEGF at mRNA and protein levels was also significantly decreased in a dose-dependent manner(P<0.01). The expression of COX-2 was positively correlated with that of VEGF(P<0.05), and a positive correlation between the expression of COX-2 and P53 protein(P<0.05) was also observed.CONCLUSION: EOS may play its antitumor effect by inhibiting the expression of P53, COX-2 and VEGF in nasopharyngeal carcinoma cell line CNE-2Z.     

Effect of protein kinase C-α antisense oligonucleotide on cell cycle of CNE-2Z cells

AIM:To observe the effect of protein kinase C-α(PKCα)antisense oligonucleotide on cell growth, cell cycle and the expression of cyclin E in human poor-differentiated nasopharyngeal carcinoma(NPC) cell line CNE-2Z. METHODS:Antisense PKCα was transfected by cationic liposome(LP) in CNE-2Z cells to analyze the cell growth and cell cycle by MTT colorimetric assay and flow cytometry, respectively. Moreover, the expression of cyclin E was determined by immunocellularchemistry and scanning the result of dot-blotting. RESULTS:①With the concentration of antisense PKCα increasing, the relative cell growth index was decreased gradually(P＜0.01). ②After treated with antisense PKCα, the percentage of cells in G₁ phase enhanced(P＜0.05). ③Compared with the control group, the expressing intensity of cyclin E reduced in antisense PKCα group, and the expression of cyclin E decreased to 66.5%±18.4％(P＜0.05) of the control by scanning quantitative analysis. CONCLUSION:These results indicated that antisense PKCα may inhibit cell growth in CNE-2Z via suppressing the expression of cyclin E and hindering cell process in G₁ phase.     

Inhibitory effect of polypeptide from Buthus martensii Karsch on neoplasm cells

AIM: To observe the inhibitory effects of polypeptide from Buthus martensii venom (PBMV) on human tumor cell lines and the influence of PBMV on cell cycle migration, expression of tumor-suppressor gene p53 and the membrane potential of CNE-2Z cells. METHODS: MTT colorimetric method, the colony formation and mitosis index, flow cytometry assay and intracellular recording with glass microelectrodes were used. RESULTS: PBMV had obvious cytotoxicity on several tumor cell lines. The cells grow was inhibited by PBMV, colony formation rate and mitotic index of tumor cells were reduced and the number of polykaryocyte was decreased. CNE-2Z cells in S phase were reduced evidently after they were treated with PBMV (P<0.01). After CNE-2Z cells were incubated with PBMV, P53 protein expression in the cells decreased obviously. Differences were very distinct (P<0.01). The negative potential of cell membrane reduced evidently and cell membrane was in depolarization state. CONCLUSION: PBMV probably possesses the effects of the "membrane toxin". By decreasing the membrane potential, the growth and proliferation of tumor cells were interfered by PBMV.