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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Establishment of nasopharyngeal carcinoma (NPC) cell lines stable expressing NPC-derived LMP1

AIM: To establish nasopharyngeal carcinoma(NPC) cell lines stable expressing NPC-derived latent membrane protein 1(LMP1) gene.METHODS:General expression vector and epithelium-specific expression vector of NPC-derived LMP1 gene were constructed by using recombinant techniques, then transfected these vectors into a poor differentiated NPC cell line named CNE-2 ,integration and expression of N-LMP1 in CNE-2 cells were detected by PCR,RT-PCR and Western blot. RESULTS:(1) General expression vector and epithelium-specific expression vector of NPC-derived LMP1 gene were constructed successfully.(2) It showed that N-LMP1 gene expressed in CNE-2 cells correctly.CONCLUSION: The first NPC cell lines which stable express NPC-LMP1 were established. The cell lines obtained will provide important basis for exploring the role of NPC-LMP1 in nasopharynx carcinogenesis.     

The expression of 53BP1 and 53BP2 gene in nasopharyngeal carcinoma

AIM:To investigate the expression map of two p53 binding proteins 53BP1 and 53BP2 in nasopharyngeal carcinoma (NPC) tissue. METHODS:The expression of 53BP1 and 53BP2 mRNA in NPC biopsy and control group are tested by RT-PCR. The expression of two mRNA in NPC paraffin section are examined by in situ hybridization. RESULTS:No expression of 53BP1 mRNA was found in NPC tissue and control group. However, expression of 53BP2 was detected in NPC biopsy and control group by RT-PCR, specific expressoin found cancerous nest in NPC paraffin section by in situ hybridization. CONCLUSION:The high expression of 53BP2 may be related to the development of NPC.     

Effects of curcumin analogue T83 on apoptosis of homologous nasopharyngeal carcinoma cells with different radioresistance

AIM：To compare the effect of T83 (a 4-arylidene curcumin analogue) on the apoptosis of homologous nasopharyngeal carcinoma cells with different radioresistance. METHODS：The effects of T83 on the viability, apoptosis, mitochondrial membrane potential (MMP), expression of procaspase-3/procaspase-9/Cyt-C proteins and relative PTEN/Akt/p27 mRNA expression in CNE-2R cells and CNE-2 cells were detected and compared by the methods of MTT assay, Hoechst staining, flow cytometry, Western blotting and qRT-PCR. RESULTS：T83 inhibited the viability of CNE-2R cells with the IC50 of 0.9,0.4 and 0.2 μmol/L for 24 h, 48 h and 72 h,respectively, which was more effective than that inhibiting the viability of CNE-2 cells with the IC50 of 1.8,0.5 and 0.4 μmol/L, respectively. After treated with T83 for 48 h, chromatin condensation, margination and splitting into a massive structure were observed in CNE-2R cells and CNE-2 cells,and the late apoptotic rate of CNE-2R cells was increased from 1.57% to 27.26%, which was higher than that of CNE-2 cells (1.74% to 8.15%). After treated with T83 for 36 h, the MMP in CNE-2R cells decreased by 87.71% and that decreased by 50.47% in CNE-2 cells. After treated with T83 for 48 h, the protein levels of procaspase-3 and procaspase-9 were decreased, and the protein level of Cyt-C was increased, which were more susceptible in CNE-2R cells than those in CNE-2 cells. After treated with T83 for 24 h, the relative mRNA expression of PTEN and p27 was significantly up-regulated, and the mRNA expression of Akt was down-regulated, which were more susceptible in CNE-2R cells than those in CNE-2 cells. CONCLUSION：Compared with CNE-2 cells, the inhibitory effect of T83 on the viability of CNE-2R cells is more specific by starting the mitochondrial apoptotic pathway, which is due to the inhibition of PTEN/Akt/p27 signaling pathway.     

Effects of aspirin on apoptosis of homologous nasopharyngeal carcinoma cells with different radioresistance

AIM: To investigate the effects of aspirin on the apoptosis of nasopharyngeal carcinoma cell lines CNE2R and CNE2 with different radioresistance and its potential mechanism. METHODS: The effects of aspirin on the cell viability, apoptosis, and protein levels of procaspase-3, cleaved caspase-3, procaspase-9, procaspase-12, PARP and cleaved PARP, PI3K p110α, Akt, Bcl-2, Bax and p27 in the CNE2R cells and CNE2 cells were detected by the methods of MTT assay, flow cytometry and Western blot. RESULTS: Aspirin inhibited the viability of homologous nasopharyngeal carcinoma cell lines CNE2 and CNE2R (with the IC₅₀ to CNE2 cells of 6.18, 3.92 and 3.06 mmol/L for 24 h, 48 h and 72 h, respectively; and with the IC₅₀ to CNE2R cells of 7.05, 3.90 and 2.20 mmol/L for 24 h, 48 h and 72 h, respectively). After treated with aspirin for 48 h, the apoptotic rate of CNE2R cells was higher than that of CNE2 cells (P<0.05). After treated with aspirin for 48 h, the protein levels of procaspase-3, procaspase-9, procaspase-12 and PARP were decreased, the protein levels of cleaved caspase-3, cleaved PARP and p27 were increased, and the protein levels of PI3K p110α, Akt and Bcl-2/Bax were decreased. CONCLUSION: Aspirin inhibits the viability of homologous nasopharyngeal carcinoma cell lines CNE2R and CNE2 with different radioresistance. Aspirin also induces the apoptosis of CNE2 and CNE2R cells, which is more effective in CNE2R cells. The underlying mechanisms may be involved in affecting PI3K/Akt signaling pathway, Bcl-2/Bax and p27 expression.     

Inhibitory effect of JIP on AP-1 activity induced by LMP1 in nasopharyngeal carcinoma cells and its mechanism

AIM: To investigate the mechanism of the AP-1 signal transduction pathway inhibited by JIP in nasopharyngeal carcinoma cells. METHODS: AP-1 activity was triggered by Dox-induced LMP1 expression in Tet-on-LMP1-HNE₂ cells (L7). The retention of phospho-JNK in the cytoplasm caused by JIP was examined with immunofluroscence assay. RESULTS: 24 h after transfection of L7 cells with the JIP expression plasmid, the translocation of activated JNK was inhibited, which resulted in the retention of phospho-JNK in the cytoplasm and down-regulation of the AP-1 activity. CONCLUSION: JIP down-regulates the activity of AP-1 through the inhibition of the translocation of JNK.     

Effects of curcumin analogue B50 on proliferation and apoptosis of homologous nasopharyngeal carcinoma cells with different radioresistance

AIM: To compare the effects of B50, a mono-carbonyl analogue of curcumin, on the proliferation and apoptosis between homologous nasopharyngeal carcinoma cells CNE-2R and CNE-2 with different radioresistance.METHODS: The effects of B50 on cell viability and cell growth were detected by MTT assay and colony-forming experiment, respectively. The changes of cell cycle, apoptosis and mitochondrial membrane potential (MMP) were determined by flow cytometry.RESULTS: B50 inhibited the cell viability of CNE-2R cells in a time-and dose-dependent manner with the IC₅₀ of (8.06±0.14) μmol/L (24 h), (2.49±0.02)μmol/L (48 h) and (1.42±0.02) μmol/L (72 h), which was more effective than that in CNE-2 cells . The inhibitory effect of B50 on CNE-2R cell growth was more effective than that on CNE-2 cells . After treated with B50 for 48 h, the proportion of CNE-2R cells in G₂/M stage was increased from 7.1% to 34.9%, which was better than that of CNE-2 cells (from 12.4% to 35.7%). After treated with B50 for 24 h, the early apoptotic rate in CNE-2R cells was increased from 3.7% to 19.5%, which was better than that in CNE-2 cells (from 4.4% to 14.8%), and the MMP in CNE-2R cells was decreased by (43.17±3.11)%, which was better than that in CNE-2 cells .CONCLUSION: B50 is more effective on inhibiting the cell viability and cell growth, blocking the cell cycle at G₂/M stage, inducing apoptosis and decreasing MMP in CNE-2R cells than those in CNE-2 cells, indicating that B50 may enhance the radio-sensitivity of CNE-2R cells by blocking the cell cycle and inducing apoptosis through mitochondrial pathway.     

T63 enhances radiotherapeutic sensitivity of nasopharyngeal carcinoma cells through apoptosis and G2/M arrest

AIM: To explore the antitumor effect of a curcumin analogue T63 in human nasopharyngeal carcinoma (NPC) cell lines CNE-2 and CNE-2R. METHODS: Cell viability was monitored by the methods of MTT and colony formation assay. Cell cycle distribution was detected by flow cytometry. Apoptosis was examined using the annexin V-FITC/PI staining assay. RESULTS: A growth inhibitory effect was observed with T63 treatment in a dose-dependent manner. Either T63 or ionizing radiation (IR) significantly induced G2/M arrest and apoptosis in NPC cells. In addition, T63 treatment combined with IR induced significantly higher apoptosis and G2/M arrest in NPC cells. CONCLUSION: T63 exhibits potent inhibitory activity on NPC cells and induces the radiotherapeutic sensitivity. Therefore, T63 has a potential as a preventive or therapeutic agent for treating NPC.     

Effect of transketolase-like protein 1 on proliferation of human nasopharyngeal carcinoma cells

AIM: To investigate the effect of transketolase-like protein 1 (TKTL1) on proliferation of human nasopharyngeal carcinoma cells in vitro. METHODS: The siRNA against TKTL1 mRNA was constructed and transfected into human nasopharyngeal carcinoma cells (CNE cell line). The activity of transketolase was detected before and after RNA interference.Real-time PCR was used to determine the mRNA expression of transketolase (TKT) gene family in the CNE cells.Flow cytometry and MTT test were used to detect the effect of anti-TKTL1 siRNA on cell proliferation and cell cycle in the CNE cells. RESULTS: The total transketolase activity was significantly decreased in the CNE cells transfected with siRNA TKTL1 construct compared with the cells transfected with control vector or untransfected CNE cells. No significant difference in the expression level of TKT and TKTL2 gene between the CNE cells transfected with siRNA TKTL1 construct and the cells transfected with control vector or untransfected CNE cells was observed (P>0.05). However, the expression level of TKTL1 gene was significantly downregulated in the CNE cells transfected with siRNA TKTL1 construct compared with the cells transfected with control vector.Cancer cells were arrested in G0/G1 phase, and cancer cell proliferation was significantly inhibited in the CNE cells transfected with siRNA TKTL1 construct. CONCLUSION: TKTL1 plays an important role in the total transketolase activity and cell proliferation of human nasopharyngeal carcinoma. TKTL1 may be considered as a potential target for novel anti-cancer therapy.     

Curcumin analogue B67 induces apoptosis and G2/M arrest to suppress radioresistant nasopharyngeal carcinoma cells

AIM: To study the effect of curcumin analogues B67 on radioresistant nasopharyngeal carcinoma cells (CNE-2R). METHODS: The effects of B67 on the cell viability and proliferation of CNE-2R and the parent cells CNE-2 were detected by MTT assay and colony formation assay, respectively. The changes of cell cycle, apoptosis and mitochondrial membrane potential were determined by flow cytometry. The morphological changes of the cells were observed under fluorescence microscope. Node mice were subcutaneously inoculated with the cells to determine the tumorigenic ability. RESULTS: The IC₅₀ of B67 on the viability of CNE-2R cells after treatment for 24 h, 48 h and 72 h were 3.96,2.59 and 0.89 μmol/L, respectively, and those of CNE-2 cells were 8.84, 3.55 and 1.10 μmol/L,respectively. The IC₅₀ of B67 on the proliferation of CNE-2R cells after treatment for 48 h was 0.55 μmol/L, and that of CNE-2 cells was 0.73 μmol/L. After treated with B67 for 24 h, CNE-2R and CNE-2 cells at G₂/M stage increased from 5.32% to 40.01% and from 9.07% to 15.73%,respectively. After treated with B67 for 48 h, the apoptosis of CNE-2R and CNE-2 cells increased from 5.49% to 38.06% and from 4.99% to 35.74%, respectively. The mitochondrial membrane potential in CNE-2R and CNE-2 cells was decreased by 66.76% and 72.09%, respectively. After treated with B67 for 24 h, the tumorigenic rate of CNE-2R cells was 0%, while the rates of CNE-2 cells in low- and high-concentration groups were 100% and 0%, respectively.CONCLUSION: Curcumin analogue B67 exhibits enhanced suppressive activity on radioresistant nasopharyngeal carcinoma cells by inducing G₂/M-phase arrest, promoting cell apoptosis and changing mitochondrial membrane potential.     

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.     

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.