Effects of EBV infection on growth and apoptosis of nasopharyngeal carcinoma cell line

AIM: To explore the effect of EBV infection on growth and apoptosis of nasopharyngeal carcinoma(NPC) cell line.METHODS: NPC cell line CNE1 was directly infected by Epstein Barr virus (EBV). The expression of EBV-latent membrane protein 1 (EBV-LMP1) and bcl-2 were detected by immunohistochemistry method (LSAB). The growth of NPC cells was identified by MTT method. Apoptotic carcinoma cells were detected by flow cytometry analysis and the terminal deoxynucletidyl transferase-medicated dUTP-biotin nick end labeling (TUNEL) methods. RESULTS: EBV-LMP1 was positive in CNE1 infected by EBV(E-CNE1). Compared with CEN1, the growth of E-CNE1 apparently increased (P＜0.01). No apoptotic carcinoma cells were detected and bcl-2 postive cells were 2%~3% respectively in 2 kinds of NPC cells.CONCLUSION: Growth of NPC cells is enhanced by EBV infection and EBV-LMP1 expression, but no influence on expression of bcl-2 and apoptosis of NPC cells.     

Effects of EBV infection on growth and apoptosis of nasopharyngeal carcinoma cell line

AIM:To explore the effect of EBV infection on growth and apoptosis of nasopharyngeal carcinoma(NPC)cell line.METHODS:NPC cell line CNE1 was directly infected by Epstein Barr virus(EBV).The expression of EBV-latent membrane protein 1(EBV-LMP1)and bcl-2 were detected by immunohistochemistry method(LSAB).The growth of NPC cells was identified by MTT method.Apoptotic carcinoma cells were detected by flowcytometry analysis and the terminal deoxynucletidyl transferase-medicated dUTP-biotin nick end labeling(TUNEL)methods.RESULTS:EBV-LMP1 was positive in CNE1 infected by EBV(E-CNE1).Compared with CEN1, the growth of E-CNE1 apparently increased(P<0.01).No apoptotic carcinoma cel s were detected and bcl-2 postive cells were 2%~3%respectively in 2 kinds of NPC cells.CONCLUSION:Growth of NPC cells is enhanced by EBV infect ion and EBV-LMP1 expression, but no influence on expression of bcl-2 and apoptosis of NPC cells.     

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.     

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.     

The latent gene expression of Epstein-Barr virus in nasopharyngeal carcinoma

AIM: To examine the latent membrane protein 1(LMP1)-DNA sequence in nasopharyngeal carcinoma(NPC) and detect mRNA expression of LMP1,EBNA1,EBNA2,and to explore the relationship between EBV infectious status,expression products and NPC carcinogenesis.METHODS: LMP1 DNA was detected in NPC by PCR.Direct sequence was applied to analyze the difference between NPC-LMP1-DNA and B95-8- LMP1-DNA.mRNA expressions of LMP1,EBNA1,EBNA2 in NPC were detected by nested RT-PCR.RESULTS: LMP1 DNA existed in all 47 NPC tissues.Several single nucleotide variations were found between NPC-LMP1-DNA and B95-8- LMP1-DNA.The notable variation was the lost of XhoⅠrestriction site in NPC.Direct sequence showed 30 bp deletion in NPC.The mRNA expressions of LMP1,EBNA1 and EBNA2 in NPC were 76.6%,80.0% and 74.5% respectively by nested RT-PCR.The expression of EBNA1 in NPC was promoted by Q promoter while the expression of EBNA1 in B95-8 was promoted by C promoter.CONCLUSION: The way of EBV involved in NPC is complex.Latent genes such as LMP1,EBNA1 and EBNA2 as well as early lytic gene BARF1 may all play certain roles in NPC carcinogenesis.     

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.     

Deletion of the LMP-1 gene integrated in the nasopharyngeal carcinoma cell line SUNE-1

AIM: To study the EBV LMP-1 gene integrated in the chromosome of poorly differentiated nasopharyngeal carcinoma cell line SUNE-1. METHODS: The LMP-1 gene of SUNE-1 was detected with PCR; Deletion of LMP-1 was examined by restriction endonuclease analysis and PCR. The deletion was precisely localized by DNA sequencing. RESULTS: The LMP-1 gene integrated in the chromosome of SUNE-1 could be deleted or non-deleted. The two introns of LMP-1 gene were shown being lost in SUNE-1 cell line. CONCLUSION: Deletion of intron 1 and intron 2 happen in some of the LMP-1 gene integrated in the chromosome of SUNE-1.     

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.     

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.     

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.     

Inhibition of miR-9 expression suppresses proliferation,invasion and migration of nasopharyngeal carcinoma cells

AIM: To investigate the effects of down-regulated miR-9 expression on the proliferation, invasion and migration of nasopharyngeal carcinoma (NPC) cells. METHODS: Human NPC CNE1 and CNE2 cells were transfected with the inhibitor of miR-9 by Lipofectamine to down-regulate the expression of miR-9, and the cells transfected with an inhibitor control were also set up. The cell proliferation and cell cycle were evaluated by CCK-8 assay and flow cytometry. The cell invasion and migration abilities were detected by Transwell invasion and wound-healing assays. Immunoblotting was applied to analyze the levels of the proteins. RESULTS: Compared with control group, inhibition of miR-9 expression in the NPC cells by transfection of the miR-9 inhibitor significantly decreased the proliferation ability (P<0.05). The percentages of the cells in G0/G1 phase ［CNE2: (57.96±1.39)% vs (47.93±1.76)%, P<0.05; CNE1: (51.24±0.88)% vs (48.29±0.39)%, P<0.05］ were significantly increased. The migration distances ［CNE2: (186.50±7.94)μm vs (247.56±15.56)μm, P<0.05; CNE1: (139.06±16.73)μm vs (230.66±14.27)μm, P<0.01］ and the invasion ability of the CNE2 cells (43.00±3.17 vs 65.80±5.20, P<0.01) were also significantly inhibited. Moreover, the tumor cells transfected with the inhibitors produced lower β-catenin. CONCLUSION: Inhibition of miR-9 expression suppresses the proliferation, invasion and migration of nasopharyngeal carcinoma cells.     

Effect of tanshinone IIA on expression of cell cycle regulators and proliferation of pancreatic cancer cells

AIM：To observe the effect of tanshinone IIA on the expression of cell cycle regulators and the proliferation of pancreatic cancer cell line BX-PC-3. METHODS： The pancreatic cancer cell line BX-PC-3 was treated with tanshinone ⅡA at various concentrations for 48 h. The inhibition of proliferation was measured by MTT method. The change of the cell cycle was detected by flow cytometry. The protein levels of cyclin A and cyclin D2 were determined by Western blotting. RESULTS：Tanshinoone IIA significantly inhibited the proliferation of BX-PC-3 cells in a dose-dependent manner. The cancer cells were arrested in stage G0/G1 after treated with tanshinone IIA at low dose. The protein levels of cyclin A and cyclin D2 were decreased after drug intervention. CONCLUSION：Tanshinone IIA inhibits the proliferation of human pancreatic cancer cell line BX-PC-3 and the expression of cell cycle-promoting factors (cyclin A and cyclin D2), which may be the mechanism of attenuating the proliferation of pancreatic cancer 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.     

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.     

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 microtubule-destabilizing protein stathmin gene silencing on proliferation and apoptosis of nasopharyngeal carcinoma cell line 5-8F

AIM: To investigate the effects of stathmin gene silencing on nasopharyngeal carcinoma cell line 5-8F. METHODS: Double-strand siRNA targeting to stathmin gene was obtained by chemical synthesis and annealing, and was sub-cloned into the vector pGenesil-1.1. The plasmid was introduced into 5-8F cells by liposome-mediated transfection. The gene expression of stathmin, and the proliferation, morphology and apoptosis of the cells were analyzed by Western blotting, MTT assay and flow cytometry. RESULTS: The cell suppression rate in stathmin gene silencing group was (53.01?1.12)%, significantly higher than that in transfection reagent group and in negative control group. The cell apoptotic rate in stathmin gene silencing group was (8.75?0.67)%, also significantly higher than that in transfection reagent group and in negative control group (P<0.05). CONCLUSION: Silencing of stathmin gene in nasopharyngeal carcinoma cells inhibits the cell proliferation and induces cell apoptosis.     

Latent membrane protein 1 regulates expression of p27 and RPLP0 in nasopharyngeal epithelial and nasopharyngeal carcinoma tissues

AIM: To observe the protein expression of p27 and ribosomal phosphoprotein large P0(RPLP0) regulated by latent membrane protein 1 (LMP1) in nasopharyngeal epithelial and nasopharyngeal carcinoma tissues. METHODS: The protein levels of p27 and RPLP0 and the relationship with LMP1 were analyzed by Western blotting. The protein expression of LMP1, p27 and RPLP0 was also detected by the method of immunohistochemistry in 30 nasopharyngeal epithelial and 60 nasopharyngeal poorly-differentiated squamous-cell carcinoma tissues. Meanwhile, the significance of clinical pathology was evaluated. RESULTS: The positive rate of LMP1 protein was 73.3% and 90.0% in nasopharyngeal epithelial and nasopharyngeal poorly-differentiated squamous-cell carcinoma tissues, respectively. Compared with the LMP1-negative tissues, the protein levels of RPLP0 were low in the nasopharyngeal epithelial and nasopharyngeal poorly-differentiated squamous-cell carcinoma tissues with LMP1-positive expression, but the levels of RPLP0 protein were overexpressed. The protein expression of RPLP0 and RPLP0 was related to the age of nasopharyngeal carcinoma patients, the protein level of LMP1, the metastasis of lymph nodes and the TNM classification. The positive expression of p27 protein at high level was usually observed in the patients with young age, or had the characteristics of LMP1 (-), non-metastasis of lymph nodes, and in I or II stage of TNM classification. However, the protein expression of RPLP0 was low (P<0.05). CONCLUSION: LMP1 down-regulates p27 and up-regulates RPLP0 in nasopharyngeal epithelial and nasopharyngeal poorly-differentiated squamous-cell carcinoma tissues.     

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.     

Tetrandrine enhances radiosensitivity of human nasophayrngeal carcinoma cell lines

AIM: To study whether tetrandrine (Tet) enhances the radiosensitivity of human nasopharyngeal carcinoma cell lines in vitro and its mechanism.METHODS: The inhibitory effect on proliferation was evaluated by MTT assay. The radiosensitivity of the cells was compared by colony formation assay. The cell cycle was analyzed by flow cytometry. RESULTS: The maximum non-cytotoxic doses of Tet for CNE1 and CNE2 cells were 1.5 and 1.8 μmol/L, respectively. Compared with radiation group, the cell proliferation in Tet plus radiation group was significantly inhibited on the 4th to 6th days (P<0.01). The mean lethal doses for CNE1 and CNE2 cells in radiation group were (1.26±0.02) Gy and (2.27±0.04) Gy, respectively,and the values changed to (0.73±0.05) Gy and (1.61±0.08) Gy in Tet plus radiation group, respectively, resulting in the sensitivity enhancement ratio of 1.73 and 1.40, respectively (P<0.05). The CNE1 and CNE2 cells in G₂ phase of the cell cycle in radiation group were (42.62±2.07)% and (34.82±2.74)%, respectively, while those in Tet plus radiation group were (17.02±1.87)% and (19.64±4.82)%, respectively. CONCLUSION: Tetrandrine enhances the radiosensitivity of human nasopharyngeal carcinoma cell lines and the mechanism may be related to the abrogation of radiation-induced G₂ phase arrest.