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.     

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.     

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.     

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.     

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.     

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.     

Molecular mechanism underlining ethanol-induced chloride currents in nasopharyngeal carcinoma cells

AIM：To study the effects and mechanisms of ethanol on chloride channels in poorly differentiated nasopharyngeal carcinoma CNE-2Z cells. METHODS：The effect of ethanol on the cell growth was analyzed by MTT assay. The technique of whole-cell patch-clamp was used to detect the chloride current. The characteristics of the chloride current were analyzed by using the chloride channel blockers. The siRNA technique was used to analyze the molecular basis of the ethanol-sensitive chloride channels. RESULTS：Under isotonic conditions, the background current was weak and stable. Ethanol at concentrations of 0.17~170 mmol/L activated a chloride current in a concentration-dependent manner (an inverted U-shape), with a maximum effect at the concentration of 17 mmol/L. The currents showed obviously outward rectification and were susceptible to extracellular hypertonicity and the chloride channel blocker, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). ClC-3 siRNA obviously decreased the currents activated by ethanol. CONCLUSION：Extracellular ethanol induces chloride currents through activating the ClC-3 chloride channels.     

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.     

Multidrug-resistance antisense inhibits volume-activated chloride current in non-pigmented ciliary epithelial cells

AIM: To study the relationship between multidrug-resistance (MDR1) gene product P-glycoprotein (P-gp) and the volume-activated chloride current. METHODS:The volume-activated chloride current in bovine non-pigmented ciliary epithelial cells was recorded using a whole cell recording technique. An antisense technique was used to inhibit the expression of MDR1 gene. The immunofluorescence of P-gp was monitored with a real-time laser confocal microscope.RESULTS:P-gp immunofluorescence correlated negatively with the concentration of the human MDR1 antisense oligonucleotide. The antisense oligonucleotide inhibited the volume-activated chloride current specifically and partially. The latency of activation of the current increased and the peak current decreased. The percentage of inhibition of peak current correlated positively to the concentration of the antisense oligonucleotide(r＝0.99, P＜0.01) and to the reduction(%) of P-gp immunofluorescence(r=0.99,P＜0.01).CONCLUSION:P-gp, the product of MDR1 gene, plays an important role in the activation pathway of volume-activated chloride current in non-pigmented ciliary epithelial 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.     

Role of Cl- in regulatory volume decrease of nasopharyngeal carcinoma cells

AIM:To clarify the role of Cl^- in regulatory volume decrease (RVD) of nasopharyngeal carcinoma cells (CNE-2Z).METHODS:Analysis of living cell images was used to detect the volume changes following exposure to hypotonic solution. Iron replacement and block of iron channels were also applied in the present study. RESULTS:Extracelluar hypotonic treatment made the cells swell and induced RVD. The RVD was correlated positively to the swelling in the range of 160-230 mOsmol/L. Substitution of gluconate for Cl^- in perfusing solutions markedly increased RVD. Depletion of cellular Cl^- abolished, and chloride channel blockers inhibited RVD. CONCLUSION:Cl^- is the key iron to establish the RVD in CNE-2Z cells. Activation of Cl^- channels and Cl^- efflux are the major mechanisms of RVD.     

Volume-activated chloride currents in highly differentiated nasophyaryngeal carcinoma CNE-1 cells

AIM: To characterize the induction of volume-activated chloride currents by extracellular hypotonic stress. METHODS: Whole cell recording was used to detect the Cl- currents. The properties of the currents were clarified by applying the blockers of chloride channel, substitution of anions and changing cell volume. RESULTS: Under isotonic conditions, background currents were tiny and stable. However, large currents were induced rapidly by perfusing the cells with 47% hypotonic solution. The currents showed weak outward rectification. The permeability sequence of the 4 anions was I->Br- >Cl->gluconate. Chloride channel blockers, NPPB and ATP, inhibited the currents reversibly. Moreover, the inhibition of ATP on the outward currents was stronger than that on the inward currents. The currents were sensitive to the changing of cell volume, which can be activated by cell swelling and be inhibited by cell shrinkage. CONCLUSION: Extracellular hypotonicity induces chloride currents, which are sensitive to the changing of cell volume and play an important role in cell volume regulation.     

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.     

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.     

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 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 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.     

Volume-sensitive chloride current of breast cancer cells

AIM: To investigate the volume-sensitive chloride current of breast cancer cells MCF-7 and MDA-MB-231. METHODS: The technique of whole-cell patch clamp was used to record the chloride current. The background chloride current of MCF-7 cells and MDA-MB-231 cells was recorded in isosmotic solution. The changes of chloride current were observed when the cells were perfused by 47% hypotonic or 47% hypertonic solutions. The changes of chloride current were observed after adding the chloride channel blocker NPPB (100 μmol/L) or tamoxifen (20 μmol/L). RESULTS: The background currents in estrogen receptor (ER) positive breast cancer MCF-7 cells and ER negative breast cancer MDA-MB-231 cells were statistically different under isotonic conditions. Perfusion of 47% hypotonic solution induced cellular swelling and activated volume-sensitive chloride current. Perfusion of 47% hypertonic solution induced cell shrinkage and inhibited the volume-sensitive chloride current. NPPB and Tamoxifen inhibited the hypotonicity-activated chloride current. CONCLUSION: The volume-sensitive chloride current was recorded in the breast cancer cells MCF-7 and MDA-MB-231, which was inhibited by hypertonic solution and chloride channel blockers.     

Inhibitory effect of extract of Oratosquilla on migration and vasculogenic mimicry in human nasopharyngeal carcinoma cell line in vitro

AIM: To study the inhibitory effect of the extract of Oratosquilla (EOS) on the migration and vasculogenic mimicry in human poorly differentiated nasopharyngeal carcinoma cell line CNE-2.METHODS: CNE-2 cells were cultured in the medium with different concentrations of EOS (0 mg/L, 125 mg/L, 250 mg/L and 500 mg/L). The migration of CNE-2 cells and the formation of tube-like structures (TLSs) by CNE-2 cells were determined with wound healing assay and in vitro anti-angiogenesis test, respectively. The formation of TLSs by CNE-2 cells and their structural characteristics were observed by anti-angiogenesis test on the Matrigel. The protein expression of fascin 1 and vascular endothelial growth factor (VEGF) was detected by Western blotting.RESULTS: Compared with control group, EOS significantly decreased the migration velocity of CNE-2 cells in a dose-dependent manner. CNE-2 cells formed TLSs on the Matrigel, and the formation of TLSs by CNE-2 cells was inhibited by EOS in a dose-dependent manner. The expression of fascin 1 and VEGF in CNE-2 cells was also decreased after treatment with EOS. A positive correlation between the expression of fascin 1/VEGF and the formation of TLSs by CNE-2 cells was observed.CONCLUSION: CNE-2 cells form TLSs on the Matrigel, and EOS inhibits the migration and vasculogenic mimicry of CNE-2 cells, which are related with down-regulating the expression of fascin 1 and VEGF in CNE-2 cells.     

Changes of K+ channels of outer hair cells in guinea pig cochlea with streptomycin ototoxicity

AIM:To study the changes of K⁺ channels of outer hair cells in guinea pig cochlea with streptomycin ototoxicity. METHODS:Auditory brainstem responses (ABR) and whole-cell patch clamp techniques were used.RESULTS:(1) The body weight of guinea pigs with streptomycin ototoxicity decreased significantly; (2) The ABR threshold markedly increased in streptomycin group (Ⅱ,Ⅲ);(3)The number of dissociated outer hair cells of guinea pigs (Ⅱ,Ⅲ) was lower than that of control (Ⅰ); (4) Streptomycin decreased the Ca²⁺-sensitive K⁺ currents and delayed outward K⁺ currents distinctly; (5) There was no significant difference of K⁺ currents between Ⅰ and Ⅱ/Ⅲ. CONCLUSION:These results suggest that the inhibition of K⁺ channels is the basis of streptomycin ototoxicity, but not the direct reason for cell death.