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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Difference of ClC-3 protein expression and channel function between cisplatin-sensitive and -resistant ovarian cancer cells

AIM: To study the difference of ClC-3 chloride channel protein expression and channel function between cisplatin-sensitive (a2780) and -resistant (a2780cp) ovarian cancer cells. METHODS: The inhibition of a2780 and a2780cp cell proliferation induced by cisplatin were detected by MTT assay. The mRNA and protein expressions of ClC chloride channel families in a2780 cells and a2780cp cells were detected by real-time PCR and Western blot, respectively. The distribution of ClC-3 protein in a2780 cells and a2780cp cells were analyzed by immunofluorescence staining. The whole cell patch-clamp technique was used to record the chloride current in the cells. RESULTS: The sensitivities of a2780 cells and a2780cp cells to cisplatin were different. The IC₅₀ values of a2780 cells and a2780cp cells to cisplatin were 5 μmol/L and 20 μmol/L, respectively (P<0.01). The a2780 cells and a2780cp cells mainly expressed ClC-3 in ClC families. However, the mRNA expression of ClC-3 was much lower in a2780cp cells than that in a2780 cells (P<0.01). Compared with a2780 cells, the protein expression of ClC-3 in a2780cp cells was also significantly decreased (P<0.01). ClC-3 protein was mainly distributed on the membrane in a2780 cells, while was in cytoplasma in a2780cp cells. Cisplatin activated the chloride channel and induced the chloride current in the a2780 cells, but not in the a2780cp cells. Cisplatin did not induced the chloride current in a2780 cells treated with ClC-3 siRNA. CONCLUSION: The differences in protein distribution, expression and function of ClC-3 chloride channel were observed in cisplatin-sensitive and -resistant ovarian cancer cells, which may be one of the underlying mechanisms of cisplatin resistance.     

Relationship between calcium-activated chloride channels and mucus overproduction,allergic airway inflammation in asthma

Calcium-activated chloride channels play important roles in the pathological processes in asthma with mucus overproduction and a series of airway inflammation. The function of calcium-activated chloride channels depends on their structure and characterization. The members of chloride channels, calcium activated (CLCA) family of proteins and in particular murine mCLCA3 (alias Gob-5) are possible initial factors of mucus overproduction in asthma. Regulation of mCLCA3 is relevant with cytokines secreted by Th2 cells. Over-expression of Gob-5 and hCLCA1 increase the translation of MUC5AC gene, which upregulates the secretion of goblet cells. Further study on the function and structure of calcium activated chloride channels may provide new evidence for understanding the pathogenesis of asthma.     

Effect of lipopolysaccharide on L-type voltage-gated calcium channel currents in primarily cultured rat caudate nucleus neurons and its modulation by 2-arachidonoylglycerol

AIM: To explore whether L-type voltage-gated calcium channel (L-VGCC) currents are involved in neurotoxicity of lipopolysaccharide (LPS) and the neuroprotective mechanism of 2-arachidonoylglycerol (2-AG) in caudate nucleus (CN) neurons. METHODS: On the 7th day, the primarily cultured CN neurons were treated with 2-AG, LPS, SR141716A (a CB1 receptor inverse agonist) and AM630 (a CB2 receptor inverse agonist) for 12 h. The L-VGCC currents in the CN neurons were recorded using the whole-cell patch-clamp technique. Hoechst staining was used to detect the effect of 2-AG on neurotoxicity of LPS. Caspase-3 assay kit was used to determine the activity of caspase-3 induced by LPS alone or with 2-AG. RESULTS: LPS enhanced the current density of L-VGCC, but did not influence the activation and deactivation of L-VGCC. 2-AG inhibited the enhancement of L-VGCC currents induced by LPS. The effect of 2-AG on the suppression of L-VGCC currents induced by LPS was not mediated through CB1 or CB2 receptor. 2-AG alone did not affect the L-VGCC currents, activation and deactivation in the CN neurons. 2-AG inhibited LPS-induced elevated activity of caspase-3 in the CN neurons. This effect was inhibited by SR141716A. 2-AG inhibited the cell apoptosis induced by LPS, and this effect was mediated via CB1 receptor. CONCLUSION: Endocannabinoid 2-AG may exert the anti-inflammatory and neuroprotective effects through the modulation of L-VGCC currents in primarily cultured rat CN neurons.     

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.     

Expression of TMEM16A as a calcium-activated chloride channel in Fischer rat thyroid follicular epithelial cells and its electrophysiologic pro-perties

AIM：To investigate the expression of transmembrane protein 16A(TMEM16A) in Fischer rat thyroid follicular epithelial (FRT) cells and its electrophysiologic properties. METHODS：The eukaryotic expression vector of pUB6/V5-TMEM16A was constructed and transfected into FRT cells by liposome-mediated transfection. In order to obtain the high efficiency of gene transfection and expression, the quantity and ratio of lipid/DNA complexes were optimized. The FRT cells stably expressing TMEM16A were gained by the selection with blasticidin and confirmed by the techniques of RT-PCR and immunofluorescence. The expression and location of TMEM16A in the FRT cells were observed under an inverted fluorescence microscope. TMEM16A protein was associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent protein and patch-clamp technique. RESULTS：The results of double digestion and sequencing indicated that TMEM16A was cloned into pUB6/V5. The results of RT-PCR and immunofluorescence confirmed that TMEM16A was expressed in the FRT cells after transfection with TMEM16A. The classical calcium-activated chloride channel currents were recorded in the FRT cells stably expressing TMEM16A by the technique of patch-clamp and halide-sensitive fluorescent protein YFP-H148Q/I152L. CONCLUSION：The protein expression of TMEM16A in the FRT cells was observed. TMEM16A is the molecular identity of calcium-activated chloride channels.     

Effects of apelin-13 on transient sodium currents in rat ischemic ventricular myocardial cells

AIM：To investigate the effects of apelin on ventricular arrythmias and cardiac functions in rat Langendorff perfusion-simulated myocardial ischemia model by observing the changes of transient sodium currents (INa) in normal cells and the simulated ischemic cells in rat left ventricle. METHODS：Ventricular cells were enzymatically isolated by the Langendorff perfusion system. INa was recorded by the technique of whole-cell patch-clamp. Some elements in the extracellular fluid were changed to simulate the normal or ischemic status. Forty Wistar rats were divided into 4 groups：normal group, ischemic group, normal with apelin group and ischemic with apelin group. The effect of apelin-13 on INa was observed. The method of rat Langendorff perfusion was used to simulate the ischemic heart model. The ventricular arrhythmia scores and heart functional parameters were compared. The expression level of sodium channel protein,type V,alpha subunit (SCN5A) in ventricular ischemic cells was measured by Western blotting. RESULTS： Apelin-13 increased INa amplitude in both normal myocardial cells ［(-86±13） pA/pF］ and ischemic myocardial cells ［(-52±15） pA/pF］. The results of current-voltage curve analysis indicated that apelin-13 did not change the conduction velocity of INa in the 4 groups ［(3.2±0.2） pS/pF, （3.1±0.3） pS/pF,（2.9±0.1）pS/pF and （2.8±0.4） pS/pF,respectively, P>0.05］. The membrane potentials at 50% maximal activation in the 4 groups were （-21.9±0.6） mV, （-28.7±0.3） mV, （-30.5±0.7） mV and （-36.8±0.2） mV, respectively, and the slope of activation curves was 5.6±0.3, 5.1±0.4, 4.3±0.3 and 4.9±0.6 (P>0.05), respectively. No difference of ventricular arrhythmia scores between normal group and normal with apelin group, as well as between ischemic group and ischemic with apelin group was observed. LVEDP in normal with apelin group was lower than that in normal group.The dp/dtmax and dp/dtmin in normal with apelin group were higher than those in normal group. Apelin improved cardiac function parameters in the ischemic hearts. The expression of SCN5A was not affected by apelin (28.8±3.6, 29.4±4.1, 30.1±2.9 and 31.3±3.8,respectively,P>0.05). CONCLUSION：Apelin-13 changes the gating properties of sodium channel, enhances the peak INa and facilitates the opening of sodium channel without inducing ventricular arrhythmias. Apelin-13 has a positive inotropic effect on both normal and ischemic hearts.     

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.