Apoptosis Of CNE-2 Cells Research Articles

Effect of capilliposide for induction apoptosis in human nasopharyngeal cancer CNE-2 cells through up-regulating PUMA expression.

To observe the apoptosis of capilliposide against human nasopharyngeal cancer CNE-2 cells and to study its primary mechanisms. Vectors pSilencer-PUMA-small interfering RNA (siRNA) were constructed to transcribe functional siRNA specially targeting PUMA. The interfering plasmids were used to transfect CNE-2 cells with lipofectamine 2000 transfection reagent. PUMA messenger RNA (mRNA) expression levels were analyzed by polymerase chain reaction. The proliferation of CNE-2 cells was detected using MTT colorimetry. Annexin V/propidium iodide double staining was applied to detect the apoptosis rate of CNE-2 cells. The protein levels of p53, PUMA, and Bax were detected using Western blot analysis. Recombinant siRNA expression vector targeting PUMA was constructed. MTT assays showed capilliposide inhibited the proliferation of CNE-2 cells in a concentration-dependent manner. The inhibition was strengthened along with increased concentrations. Apoptosis detected by flow cytometry in control group, drug group, siRNA group, and drug combined siRNA group was 9.3 ± 2.3%, 31.4 ± 5.6%, 12.3 ± 4.1%, and 13.2 ± 3.7%, respectively. After pretreated by capilliposide, PUMA protein was upregulated, and BAX was distributed to mitochondria in CNE-2 cells using Western blot analysis, but this effect can be interrupted by PUMA-siRNA. Capilliposide could induce the apoptosis of CNE-2 cells, which might be related with the increasing in PUMA-Bax pathway.

Effects of epigallocatechin gallate on the proliferation and apoptosis of the nasopharyngeal carcinoma cell line CNE2.

The present study explored the effects of epigallocatechin gallate (EGCG) on the cell cycle, proliferation and apoptosis of the nasopharyngeal carcinoma cell line CNE2 in vitro. The proliferation of CNE2 cells was detected using the cell counting kit-8 method. Cell cycle distribution and apoptosis were detected using flow cytometry. The human telomerase reverse transcriptase (hTERT) mRNA expression was determined using reverse transcription polymerase chain reactions. The protein expression of hTERT and Myc proto-oncogene protein (c-Myc) was observed using western blot analysis. EGCG inhibited the proliferation of CNE2 cells in a concentration-dependent manner (P<0.05) and blocked the cell cycle progression of the cells. In the low concentration (100 μg/ml) group, the cell cycle arrest showed a time-dependent manner. However, as the concentration increased and action time was prolonged, this time dependency became less marked. EGCG promoted the apoptosis of CNE2 cells in a time-dependent manner. In addition, EGCG downregulated the mRNA and protein expression of hTERT and downregulated the expression of c-Myc protein. Downregulation of the expression of hTERT and c-Myc was more evident in the high-dose group (200 μg/mL). In conclusion, EGCG has proliferation-inhibiting, cell cycle-blocking and apoptosis-promoting effects on CNE2 cells. EGCG may be developed into an auxiliary therapeutic agent for the treatment of nasopharyngeal carcinoma.

The 656C and 725C are two important sites in gene STGC3 for its negative regulation on cell growth

The aim of this study was to analyze the functional sites of the nasopharyngeal candidate tumour suppressor gene STGC3. Recombinant plasmid pcDNA3.1TM/myc-His B-STGC3 was constructed. Site-directed mutagenesis of pcDNA3.1TM/myc-His B-STGC3 plasmid at sites of C656G, C725T and T913G was induced by the Stratagene mutagenesis method. Recombinant plasmids with point mutations at C656G, C725T and T913G of gene STGC3 were named as STGC3-C656G, STGC3-C725T and STGC3-T913G, respectively. CNE2 cell lines stably expressing wild and mutant STGC3 genes were established. STGC3 expression was detected by Western Blotting and immunocytochemistry. Cell proliferation was analyzed by 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue staining. Flow cytometry analysis was used to assess apoptosis of CNE2 cells. Bax protein expression was detected by Western Blotting.Proteins of wild-type and mutant STGC3 genes were expressed in the cytoplasm and nucleus of CNE2 cells. Compared with the control groups, in cells stably expressing wild-type STGC3 and STGC3-T913G genes, cell proliferation was significantly inhibited, whereas levels of apoptosis and Bax protein expression were significantly increased. However, the cell proliferation, apoptosis and Bax protein expression in cells stably expressing STGC3-C656G and STGC3-C725T genes were not significantly different from those in the control groups. Our results suggest that mutations at 656C and 725C, but not 913T, abolished the effects of the wild-type STGC3 gene on CNE2 cells and that the 656C and 725C were important sites in gene STGC3 for its negative regulation on cell growth.

Berberine Hydrochloride Impact on Physiological Processes and Modulation of Twist Levels in Nasopharyngeal Carcinoma CNE-1 Cells

The main purpose of this work was to investigate the effect of berberine hydrochloride (BH) on the proliferation, apoptosis, migration, and invasion of CNE-1 nasopharyngeal carcinoma cells. Our results shed light on the functional components of traditional Chinese herbs for potential use in modern medicine. The CNE-1 cell line was treated with different concentrations of BH and effects on cell viability and proliferation were evaluated using the Cell Counting Kit-8 (CCK-8) assay. Anti-migratory and anti-invasive actions of BH were investigated using wound healing assays and the Millicell Hanging cell culture insert system, respectively. Expression of the epithelial-mesenchymal transition (EMT)-related gene twist (Twist) was analyzed by real-time PCR and Western blotting. Apoptosis was estimated with an annexin-V fluorescein (FITC) apoptosis detection kit, as well as with reference to levels of activated caspase-3 of CNE-1 cells before and after treatment with BH utilizing fluorescence spectroscopy. BH was capable of reducing proliferation and viability of CNE-1 cells in a dose- and time-dependent manner, also demonstrating anti-migratory and anti-invasive capacities which correlated with reduction in expression of Twist. Finally, BH was able to induce significant amounts of apoptosis in CNE-1 cells, as demonstrated by an increase in the activity of caspase-3 and in annexin-V staining following treatment. BH extracted from rhizoma coptidis demonstrated an ability to block proliferation, induce apoptosis, and impair the migration and invasion of the CNE-1 cell line Considering these properties, our results suggest that BH could be an important compound for consideration in the treatment of nasopharyngeal carcinoma.

Anticancer effects of 3,3′-diindolylmethane are associated with G1 arrest and mitochondria-dependent apoptosis in human nasopharyngeal carcinoma cells

The antitumor effects of 3,3′-diindolylmethane (DIM) are exhibited in a number of human cancer cells. However, there have been few studies performed concerning the effect of DIM on nasopharyngeal cancer (NPC) cells. In the present study, we examined the in vitro antitumor activity of DIM on the poorly differentiated NPC cell line CNE-2. The potential molecular mechanisms of the activity were also explored. CNE-2 cells were treated with varying concentrations of DIM for different times. Cell proliferation and apoptosis were detected and the molecular mechanisms involved in these effects were characterized. The results demonstrated that DIM at concentrations of 15–100 μM caused dose- and time-dependent inhibition of CNE-2 cell proliferation. Flow cytometry analysis revealed a high sub-G1 cell peak following treatment with DIM, and the rate of apoptosis increased. DIM may elevate the levels of cleaved Bid and Bax and enhance mitochondrial membrane depolarization, allowing the efflux of cytochrome c, Smac and Omi into the cytosol. The levels of caspases-3, -8 and -9 and cleaved poly (ADP-ribose) polymerase (PARP) were upregulated following DIM treatment in a dose-dependent manner. DIM also inhibits the phosphorylation of IκB-α, and showed dose-dependent inhibition of Bcl-2, XIAP and NF-κB in CNE-2 cells in vitro. These results indicate that DIM inhibits cell proliferation by inducing cell cycle arrest at G0/G1 phase and induces the apoptosis of CNE-2 cells by regulating multiple molecules in a mitochondria-dependent pathway. DIM may be a preventive and therapeutic agent against NPC.

Trichosanthin down-regulates Notch signaling and inhibits proliferation of the nasopharyngeal carcinoma cell line CNE2 in vitro

The prognosis of nasopharyngeal carcinoma (NPC) is still poor. Trichosanthin (TCS) has abortifacient, anti-virus, immunoregulation and various anti-tumor pharmacological activities, but there are no reports about its effect on NPC and the exact mechanisms that TCS inhibits tumor are not well known. In this study, the proliferation, apoptosis and soft agar colony formation abilities of CNE2 cells were examined with various assays in vitro followed by treatment with TCS. Furthermore, the activation status of Notch signaling pathway in TCS and control cells also was examined. The results revealed that TCS could inhibit NPC cell line CNE2 in vitro, reduce clone formation ability and induce apoptosis of CNE2 cells. Down-regulation of Notch signaling may be one of the mechanisms that TCS inhibits NPC.

Effect of Adv Vector-mediated shRNA Targeting hTERT on Proliferation and Apoptosis of Nasopharyngeal Carcinoma Cells

Objective To evaluate the effect of targeting hTERT gene on the proliferation and apoptosis of CNE-2 cells by applying RNA interference to restrain the expression of hTERT in nasopharyngeal carcinoma CNE-2 cells.Methods Recombinant adenovirus vectors expressing EGFP and human TERT shRNA were construted and transfected in human nasopharyngeal carcinoma CNE-2 cells.The expression levels of hTERT mRNA and protein were detected respectively by RT-PCR and Western blot method.Cell proliferation was determined by CCK-8 assay and cell apoptosis was observed by FCM(Flow cytometric).Results The transfection rate of Adv-EGFP-shTERT recombinant adenovirus plasmid in CNE-2 cell line was more than 90 %.The expression levels of the hTERT mRNA and protein were dramatic declining respectively at 24h and 48h after transfection.Cell proliferation activity was significantly inhibited and the cell apoptosis reached 23.0%.Conclusion Adv vector-mediated RNAi targeting hTERT can inhibit the activity of telomerase reverse transcriptase by down-regulating the expression of the hTERT mRNA and its protein significantly,therefore can inhibit the growth of the cells and induce their apoptosis.Future application of RNAi to the gene therapy of nasopharyngeal carcinoma might be expected.

γ-secretase inhibition combined with cisplatin enhances apoptosis of nasopharyngeal carcinoma cells

The Notch signaling pathway plays an important role in the proliferation and differentiation of cells. Although recent studies have shown that Notch plays a role in the mechanisms of cisplatin resistance, the mechanism by which Notch plays roles in intrinsic or acquired cisplatin resistance remains unclear. In the present study, poorly differentiated nasopharyngeal carcinoma cells were treated with a γ-secretase inhibitor (DAPT), which led to a decrease in the Notch intracellular domain and inhibition of Notch signaling. Treatment was not sufficient to induce pronounced apoptosis of CNE-2 cells, but did result in the down-regulation of the P-glycoprotein and ERCC1 protein. In contrast, the combined treatment of DAPT and cisplatin induced substantial cell apoptosis compared to cisplatin treatment alone.

Silencing of c-Met by RNA interference inhibits the survival, proliferation, and invasion of nasopharyngeal carcinoma cells

c-Met is a tyrosine kinase receptor that mediates pleiotropic cellular responses following its activation by hepatocyte growth factor. The overexpression of c-Met in nasopharyngeal carcinoma (NPC) has been described recently, but the functional role of c-Met in NPC remains incompletely understood. This study aimed to investigate the potential mechanism by which c-Met contributes to the tumorigenesis of NPC. In the present study, by using RNA interference we silenced the expression of c-Met in CNE-2 cells, a poorly differentiated NPC cell line. Our in vitro studies showed that shRNA-mediated depletion of c-Met resulted in the suppression of proliferation, migration, and invasion, as well as an increase in the apoptosis of CNE-2 cells. Moreover, in xenograft nude mice we demonstrated that the depletion of c-Met resulted in reduced tumor growth and increased apoptosis in xenografts. Taken together, these results suggest that c-Met plays an oncogenic role in the development of NPC and reveal it as a potential novel therapeutic target for NPC.

Purified alkaloid extract of Scutellaria barbata inhibits proliferation of nasopharyngeal carcinoma CNE-1 cells by inducing apoptosis and cell cycle arrest at S phase

Scutellaria barbata was widely used as an antitumor agent in traditional Chinese Medicine. However, its antitumor components and mechanism remained unclear. In the present study, the effects of purified alkaloid extract of S. barbata (PAESB) on the cultured nasopharyngeal carcinoma CNE-1 cells were investigated. MTT assay showed that PAESB could inhibit the proliferation of CNE-1 cells in a dose- and time-dependent manner. Annexin V-FITC and propidium iodide (PI) staining showed that PAESB had a positive effect on apoptosis of CNE-1 cells. After treated by PAESB (0.75 mg/ml) for 48 h, the CNE-1 cells apoptosis rate (5.13 ± 0.70%) had statistical significance as compared with the negative group (1.42 ± 0.26%) (P < 0.05). Among the various phases of cell cycle, significant increase in the percentage of S phase occurred after treated with PAESB (0.75 mg/ml) for 48 h while the percentage of cells at G0/G1 and G2/M phases were decreased by comparison with the corresponding values for CNE-1 cells without PAESB (P < 0.05). These results indicated that PAESB exhibited potential anticancer activity against nasopharyngeal carcinoma CNE-1 cells through induction of apoptosis and S phase cell cycle arrest. Key words: Scutellaria barbata, purified alkaloid extract, apoptosis, cell cycle arrest, nasopharyngeal carcinoma CNE-1 cells.

Expression of mismatch repair gene PMS2 in nasopharyngeal carcinoma and regulation by glycogen synthase kinase-3β in vivo and in vitro

To evaluate the expression of mismatch repair gene PMS2 in human nasopharyngeal carcinoma (NPC) tissues and evaluate the effect of glycogen synthase kinase (GSK)-3β on PMS2 production in vivo and in vitro. The expression of PMS2 and inactivated phosphorylated GSK-3β(s9) was examined by immunohistochemical staining in 25 NPC tissues and the relation was determined by correlation analysis. The effect of GSK-3β transfection in CNE-2 cells on PMS2 production as well as cell apoptosis and chemosensitization were evaluated using small interference RNA (siRNA), immunoblotting and flow cytometric analysis in vitro. The expression of inactivated phosphorylated GSK-3β(s9) was found to negative correlated with PMS2 in vivo. And transfected GSK-3β was found to be able to enhance PMS2 production, and increase cell apoptosis in CNE-2 cells in combination with cisplatin administration in vitro. Inactivation of GSK-3β might be important for NPC tumorgenesis through negatively regulating PMS2 production, and enhanced PMS2 production by GSK-3β is beneficial for understanding the NPC tumorgenesis and developing potential strategy for future therapy.

Antitumor and immunoregulatory effects of astragalus on nasopharyngeal carcinoma In Vivo and In Vitro

This study was carried out to evaluate the effects of Astragalus on human nasopharyngeal carcinoma (NPC) viability and apoptosis and to investigate the mechanism of Astragalus in a NPC cell line (CNE2). Cell viability was measured using the MTT assay. CNE2 cells treated with Astragalus were stained with acridine orange/ethidium bromide and subjected to fluorescence microscopy. Bcl-2, Bax, caspase-3 and -8 were measured by western blotting. Rat NPC cells were used to establish a NPC model. Tumor weight, immune organ index and T lymphocyte subsets were employed to detect the immunoregulatory and antitumor effects of Astragalus after administration. Astragalus was effective in inducing apoptosis in CNE2 cells. Morphological changes associated with cell injury were found. Western analysis showed caspase-3, -8, and Bax protein levels were increased after Astragalus treatment, while the bcl-2 protein level was decreased. Astragalus increased the percentage of CD3(+) , CD4(+) T-lymphocytes, and the ratio of CD4(+) /CD8(+) . Astragalus also restored the immunological effects of DDP-induced immunosuppression. These findings suggest that the immunomodulatory and anticancer effects of DDP + Astragalus were better than those of DDP alone, and Astragalus could inhibit immunosuppression induced by DDP. The combination of CDDP + Astragalus could be developed as an effective chemotherapeutic regimen in the treatment of nasopharyngeal carcinoma.

Gambogenic acid mediated apoptosis through the mitochondrial oxidative stress and inactivation of Akt signaling pathway in human nasopharyngeal carcinoma CNE-1 cells

In the present study, Gambogenic acid exhibits potential anti-tumor activity in several cancer cell lines. However, Gambogenic acid-induced apoptosis mechanism is not well understood. Here, we report that Gambogenic acid was capable to induce CNE-1 cells apoptosis and caused mitochondrial and endoplasmic reticulum injury, analyzed via transmission electron microscopy and acridine orange/ethidium bromide (AO/EB) double staining. To quantitatively analyze apoptosis, through the propidium iodide (PI)/Annexin V-FITC double staining to detect cell apoptosis, PI staining of the cell cycle distribution. To further explore the potential mechanism of Gambogenic acid mediated apoptosis in CNE-1 cells, we also examined mitochondrial oxidative stress in the levels of reactive oxygen species, the release of cytochrome c, intracellular Ca 2+ concentration and mitochondrial membrane potential by flow cytometry. Moreover, Gambogenic acid could result in a time and concentration-dependent decrease in Phospho-Akt expression, basal expression levels of Akt change was not obvious, In addition, we detected Bcl-2 family including Bcl-2, Bax and Bad expression in mRNA level. This resulted in a decrease of Bcl-2 and Bad increased in CNE-1 cells after Gambogenic acid treatment. Overall, our results indicated that Gambogenic acid mediated apoptosis through inactivation of Akt, accompanied with mitochondrial oxidative stress and cross-talk with Bcl-2 family in the process of apoptosis.

Synthesis, Identification and Anti-Cancer Activity of 1-(4-Methylpent-2-enyl)-2-(4-phenylbut-2-enyl)disulfane

In this study, we synthesized 1-(4-methylpent-2-enyl)-2-(4-phenylbut-2- enyl)disulfane using sodium sulfide, 1-bromine-4-methyl-2-amylene and 1-(4-bromine-2- butylene)benzene as raw materials. The yield rate of target product was 84%. The structure of the target product was confirmed by GC-MS, 1H-NMR and elemental analysis. The results of anti-cancer activity experiments showed that 1-(4-methylpent-2-enyl)-2-(4- phenylbut-2-enyl)disulfane could significantly inhibit the proliferation, induce the apoptosis of CNE2 cells in a dose dependent manner, and could significantly enhance the activity of XIAP.

Indole-3-carbinol (I3C)-induced apoptosis in nasopharyngeal cancer cells through Fas/FasL and MAPK pathway

The apoptotic effects of indole-3-carbinol (I3C) were exhibited in many human cancer cells. However, the apoptotic effects of I3C on nasopharyngeal cancer cells were still unknown. In this study, we first examined the potential effects of I3C on the induction of apoptosis in human nasopharyngeal cancer cells CNE-2 and further characterized the mechanism(s) involved in the effects. Apoptotic cells after treatment with I3C were analyzed by flow cytometry. The change in relative mitochondrial transmembrane potential in the CNE-2 cells was analyzed with rhodamine 123 staining. The protein levels of MAPK family and Fas/FasL were examined by Western blot. Our results indicated that I3C could induce apoptosis of CNE-2 cells in a dose- and time-dependent manner accompanied with increased mitochondrial membrane potential. Treatment with I3C significantly suppressed XIAP, c-IAP1 and Survivin protein, while elevated the expression of Omi, Smac and Cyto-c. Fas/FasL and MAPK pathway were involved in the induction of apoptosis. Taken together, these results demonstrated that I3C may induce mitochondria-mediated apoptosis via the Fas death receptor in CNE-2 cells. This molecular mechanism for apoptotic effect of I3C on nasopharyngeal cancer cells suggested that I3C might become a preventive and therapeutic agent against nasopharyngeal cancer.

Effect of heparin on apoptosis in human nasopharyngeal carcinoma CNE2 cells.

In order to study the mechanism of the effect of heparin on apoptosis in carcinoma cells, the nasopharyngeal carcinoma cell line CNE2 was used to identify the effect of heparin on apoptosis associated with the expression of c-myc, bax, bcl-2 proteins by use of Hoechst 33258 staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), agarose gel electrophoresis, and flow cytometry, as well as Western blot analysis. The results showed that heparin induced apoptosis of CNE2 cells including the morphologic changes such as reduction in the volume, and the nuclear chromatin condensation, as well as the "ladder pattern" revealed by agarose gel electrophoresis of DNA in a concentration-dependent manner. The number of TUNEL-positive cells was dramatically increased to 33.6+/-1.2% from 2.8+/-0.3% by treatment with heparin in different concentrations (10 to approximately 40 kU/L). The apoptotic index was increased to 32.5% from 3.5% by detecting SubG1 peaks on flow cytometry. Western blot analysis showed that levels of bcl-2, bax and c-myc were significantly overexpressed by treatment with the increase of heparin concentrations. These results suggest that heparin induces apoptosis of CNE2 cells, which may be regulated by differential expression of apoptosis-related genes.