Nasopharyngeal Carcinoma Cell CNE2 Research Articles

Hypoxia increases chemotherapy resistance in nasopharyngeal carcinoma via inducing CDK6 deSUMOylation

Objective: To understand the mechanism of chemotherapy resistance in nasopharyngeal carcinoma under hypoxic conditions through the perspective of protein SUMOylation modification. Methods: Cobalt chloride (CoCl(2)) was used to establish the hypoxic model of human nasopharyngeal carcinoma CNE1 cells. Then, the cell cycle was detected by flow cytometry, and the expression level of small ubiquitin-related modifier(SUMO) and cyclin-dependent kinase 6 (CDK6) proteins were detected by western blotting. MTT assay was used to determine the median lethal dose (IC(50)) of cancer cells against cisplatin, and enzyme-linked immunosorbent assay (ELISA) was used to determine lactate dehydrogenase (LDH) level. Results: The cell cycle of CNE1 induced by hypoxia was arrested in G0/G1 phase.The results of Western blot showed that the protein expression level of CDK6 in CNE1 cells was lower than that in the control group (0.83±0.25 vs. 0.43±0.21, t=14.67, P=0.003). The protein level of conjugated SUMO1 was significantly lower than that in the control group (2.69±0.48 vs. 1.38±0.31, t=17.22, P=0.001), while the level of free SUMO1 protein was significantly higher than that in the control group (2.01±0.43 vs. 2.60±0.59, t=15.45, P=0.002).The LC50 of CNE1 cells in the control group was significantly lower than that in the hypoxic group (29.44 μg/ml vs. 97.72 μg/ml, t=12.79, P=0.001). After CNE1 cells received 50 μg/ml cisplatin for 48 h, the LDH content in the supernatant of the control group was significantly higher than that in the hypoxic group ((541.49±64.59) ng/ml vs. (234.67±41.03) ng/ml, t=11.94, P=0.007)). The apoptosis rate of CNE1 cells in the control group was significantly higher than that in the hypoxic group ((76.64±5.37)% vs. (32.84±4.77) ng/ml, t=8.49, P=0.003)). Conclusion: Hypoxia can dissociate the covalent modification of CDK6 and SUMO1, inhibit cell cycle and increase the chemotherapy resistance of nasopharyngeal carcinoma.

Bioinformatics Study of RNA Interference on the Effect of HIF-1α on Apelin Expression in Nasopharyngeal Carcinoma Cells

Background: HIF-1α can affect the apelin expression and participates in the developments in cancers but the mechanism need to be explored further. Objective: This paper investigates apelin expression in nasopharyngeal carcinoma CNE-2 cells and its regulation by hypoxia inducible factor-1α (HIF-1α) under hypoxic conditions. Methods: CoCl2 was used to induce hypoxia in CNE-2 cells for 12h, 24h and 48h. HIF-1α small interference RNA (siRNA) was transfected into CNE-2 cells using a transient transfection method. HIF-1α and apelin mRNA levels were detected by real time PCR. Western blot was used to measure HIF-1α protein expression. The concentration of apelin in cell culture supernatant was determined by enzyme linked immunosorbent assay (ELISA). Results: HIF-1α and apelin mRNA levels and protein expression in CNE-2 cells increased gradually with increased duration of hypoxic exposure and were significantly reduced in HIF-1α siRNA transfected cells exposed to the same hypoxic conditions. Conclusion: Apelin expression is induced by hypoxia and regulated by HIF-1α in CNE-2 cells.

Effects of DACT1 methylation status on invasion and metastasis of nasopharyngeal carcinoma

BackgroundThe purpose of the present study was to investigate the role of the methylation status of the DACT1 gene on the invasion and metastasis of nasopharyngeal carcinoma cells.MethodsThe levels of methylation and expression of the DACT1 gene in nasopharyngeal carcinoma tissues and CNE2 cells were determined by methylation-specific PCR and RT-PCR, respectively. CNE2 cells were treated with 5-aza-2-deoxycytidine, and the variation in the methylation status of the DACT1 gene was detected, as well as the influence of methylation on invasiveness of nasopharyngeal carcinoma cells.ResultsThe DACT1 gene was hyper-methylated in 44 of 62 cases of nasopharyngeal carcinoma. The DACT1 gene was hyper-methylated in 32 of 38 cases of nasopharyngeal carcinoma with lymph node metastasis, and the DACT1 gene was hyper-methylated in 7 of 24 cases of nasopharyngeal carcinoma without lymph node metastasis. The DACT1 mRNA level was weakly expressed or not expressed in all nasopharyngeal carcinoma tissues with hyper-methylated DACT1 genes; however, the DACT1 mRNA level was highly expressed in nasopharyngeal carcinoma tissues with low expression of the methylated DACT1 gene. The DACT1 gene was hyper-methylated and not expressed in CNE2 cells that did not have 5-aza-2-deoxycytidine treatment. After 5-aza-2-deoxycytidine treatment, the DACT1 gene was demethylated and the expression of DACT1 was restored. Moreover, the invasion ability was inhibited in CNE2 cells treated with 5-aza-2-deoxycytidine.ConclusionThe expression of DACT1 was related to the methylation status. High expression of DACT1 may inhibit the invasion and metastasis of nasopharyngeal carcinoma cells.

A Study on the Correlations of the miR-31 Expression with the Pathogenesis and Prognosis of Head and Neck Squamous Cell Carcinoma.

To investigate the correlations of miR-31 expression with cell proliferation, invasion, and prognosis of patients with head and neck squamous cell carcinoma (HNSCC). The expression of miR-31 in human laryngeal cancer TU686 cells, human nasopharyngeal carcinoma CNE-2 cells, and normal human oral keratinocyte (NHOK) epithelial cells was detected via quantitative real-time polymerase chain reaction (qRT-PCR). The effects of miR-31 on the proliferation and invasion of HNSCC cells were explored through transfecting miR-31 analogs (miR-31 mimics) and miR-31 inhibitors (anti-miR-31). qRT-PCR was applied to detect the expressions of miR-31 in 56 cases of HNSCC tumor tissues and tumor-adjacent normal tissues. The correlation of miR-31 expression with pathological parameters and survival prognosis of HNSCC patients was also analyzed. The expressions of miR-31 in TU686 and CNE-2 cell lines were significantly higher than that in NHOK cells (p < 0.01). Compared with those in the negative control group, the proliferation and invasion abilities of cells transfected with miR-31 mimics were notably enhanced (p < 0.01), and those of cells transfected with anti-miR-31 were significantly reduced (p < 0.01). In addition, miR-31 mimics significantly reduced ARID1A expression (p < 0.01) and anti-miR-31 increased its expression (p < 0.05). The expression of miR-31 in tumor tissues of HNSCC patients was remarkably higher than that in tumor-adjacent normal tissues (p < 0.01). This, together with clinical data analysis, revealed that the expression of miR-31 was associated with tumor differentiation, metastasis, and staging of patients, and the survival period of patients with lowly expressed miR-31 was longer. The highly expressed miR-31 can stimulate the proliferation and invasion of HNSCC cells, closely correlated with tumor differentiation, metastasis, and staging of patients. Patients with lowly expressed miR-31 have a longer survival period. Therefore, miR-31 expression can be taken as a crucial reference indicator for the prognosis of HNSCC patients.

Synergistic Effects of Targeting Survivin and CDK1 on Nasopharyngeal Carcinoma &amp;lt;i&amp;gt;in Vitro&amp;lt;/i&amp;gt; and &amp;lt;i&amp;gt;in Vivo&amp;lt;/i&amp;gt;

Background: To explore the impact of pU6-based tandem survivin and CDK1-specific short hairpin RNA on the biological behaviors of CNE-2 nasopharyngeal carcinoma cells in vitro and in vivo. Patients and Methods: The vectors of pU6-survivinshRNA, pU6-CDK1shRNA and pU6-survivinshRNA-CDK1shRNA were constructed and transfected into CNE-2 cells with Lipofectamine TM 2000, respectively. The mRNAs and proteins of CDK1 and survivin were determined by RT-PCR and Western blotting, accordingly. MTT assay was employed to evaluate the proliferation of CNE-2 cells, and flow cytometry was performed to determine the apoptosis of CNE-2 cells. The effects of interfering survivin and CDK1 on tumorigenesis were evaluated by tumor xenografts experiments. Results: Effective plasmids were successfully constructed knocking down survivin and/or CDK1. The proliferation inhibition of CNE-2 cells by pU6-survivinshRNA-CDK1shRNA (32.5%) was higher than that of by pU6-survivinshRNA (25.6%) and pU6-CDK1shRNA (15.6%), and apoptosis in CNE-2 cells simultaneously interfering survivin and CDK1 (15.2%) dramatically increased when compared to those of interfering survivin (5.4%) or CDK1 (4.7%) alone. Furthermore, simultaneously interfering survivin and CDK1 is more effective than interfering alone component in inhibiting tumor growth of fBalb/C nude mice xenografted with CNE-2 cells. Conclusion: The results altogether indicate that interfering survivin and CDK1simutaneously can produce synergistic effects of anti-nasopharyngeal carcinoma, which could be a potential therapeutic method.

Extraction of matrine from Sophora flavescens Ait. and evaluation of its inhibitory effects on human nasopharyngeal carcinoma CNE-2 cells

Abstract The present study investigated the extraction and purification technology of matrine from Sophora flavescens Ait. and the inhibitory effects of matrine on human nasopharyngeal carcinoma (NPC) CNE-2 cells. The matrine was extracted from Sophora flavescens Ait. by decocting using ethanol-aqueous solution as the solvent, followed by purification using ion exchange resin adsorption. The extraction and purification conditions were investigated. The effects of matrine on growth, cell apoptosis and cycle of CNE-2 cells and expressions of Bac-2 and caspase-3 protein in cells were detected. Results showed that, the optimal extraction and purification technologies of matrine from Sophora flavescens Ait. were obtained, under which the purity of matrine was 81.56%. Matrine could obtain inhibit the growth of CNE-2 cells, promote the cell apoptosis, and arrest most cells in G0/G1 phase. In addition, after treated matrine, the relative expression level of Bcl-2 protein in CNE-2 cells was decreased, and the expression level of Caspase-3 protein was increased. In conclusion, matrine has inhibitory effects on human NPC CNE-2 cells. The mechanism may be related to its down-regulation of Bcl-2 protein and up-regulation of Caspase-3 protein expression.

PP-22 promotes autophagy and apoptosis in the nasopharyngeal carcinoma cell line CNE-2 by inducing endoplasmic reticulum stress, downregulating STAT3 signaling, and modulating the MAPK pathway.

Paris polyphylla var. yunnanensis, named Chong Lou, is considered an antitumor substance. In this study, we investigated the effect of PP-22, a monomer purified from P. polyphylla var. yunnanensis, on the nasopharyngeal carcinoma cell line CNE-2 in vitro. The results showed that PP-22 could inhibit the proliferation of CNE-2 cells via the induction of apoptosis, with evidence of the characteristic morphological changes in the apoptosis in the nucleus and an increase in Annexin V-positive cells. In addition, we found that PP-22 could activate the p38 mitogen-activated protein kinase (MAPK) pathway and that this activation was reversed by SB203580, a specific inhibitor of the p38 MAPK pathway. In contrast, PP-22 promoted apoptosis via an intrinsic pathway, including the endoplasmic reticulum stress pathway, in a caspase-dependent manner. A further study showed that PP-22 also induced apoptosis by downregulating the signal transducers and activators of transcription 3 (STAT3) pathway, and the inhibitory effect was also confirmed by STAT3 small interfering RNA. In addition, PP-22 could promote autophagy by inhibiting the extracellular regulated protein kinases (ERK) pathway. And autophagy plays a protective role against apoptosis. Together, these data show that PP-22 promotes autophagy and apoptosis in the nasopharyngeal carcinoma CNE-2 cell line.

Targeted regulationof STAT3 by miR-29a in mediating Taxol resistance of nasopharyngeal carcinoma cell line CNE-1.

STAT3 is an important molecule in Janus kinase (JAK) signal transducer and activator of transcription (STAT) signal pathway, and facilitates expression of various oncogenic genes such as Bcl-2, thus is correlated with tumor onset, progression and drug resistance. MiR-29a down-regulation is associated with the pathogenesis of nasopharyngeal carcinoma. Bioinformatics analysis demonstrated a complementary binding between miR-29a and 3'-UTR of STAT3. This study aims to investigate the role of miR-29a in regulating STAT3, as well as in Taxol resistance of nasopharyngeal carcinoma CNE-1 cells. Dual luciferase reporter gene assay showed a regulatory relationship between miR-29a and STAT3. Rhodamine 123 repository in CNE-1 and CNE1/Taxol drug resistant cells was measured together with the expression of miR-29a, STAT3, and p-STAT3. Flow cytometry was used to measure cell apoptosis and PCNA expression under Taxol treatment. CNE-1/Taxol cells were treated with miR-29a mimic and or si-STAT3, followed by measuring the expression of miR-29a, STAT3, and p-STAT3 and cell apoptosis. CCK-8 assay was performed to evaluate cell proliferation. MiR-29a inhibited STAT3 expression. Significantly lower Rhodamine 123 repository, miR-29a expression and apoptosis and higher expression of STAT3, p-STAT3 and PCNA were observed in CNE-2/ Taxol cells than those in CNE-1 cells. Transfection of miR-29a mimic and/or si-STAT3 decreased STAT3, p-STAT3 and PCNA expression, inhibited proliferation and promoted cell apoptosis. MiR-29a down-regulation is correlated with drug resistance of nasopharyngeal carcinoma cell line CNE-1 and MiR-29a up-regulation decreases Taxol resistance of nasopharyngeal carcinoma CNE-1 cells possibly via inhibiting STAT3 and Bcl-2 expression.

Effects of osthole on the proliferation, invasion and migration of nasopharyngeal carcinoma cells CNE2

Objective To study the effects of osthole on the proliferation, invasion and migration of nasopharyngeal carcinoma cells CNE2, and to investigate the possible molecular mechanism involved in epithelial to mesenchymal transition (EMT) of CNE2. Methods CNE2 cells were cultured in vitro and were treated with 0, 20, 40 and 80 μg/ml osthole for 24 or 48 hours, and then methyl thiazolyl tetrazolium (MTT) assay and Transwell assay were used to explore their effects on the cell proliferation, invasion and migration while cells treated with 0 μg/ml osthole were used as the control group. Meanwhile, the mRNA and protein levels of markers of EMT (E-cadherin and vimentin ) and Wnt/β-catenin signaling (β-catenin and cyclin D1) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting respectively. Results After treatment for 24 and 48 hours, the inhibitory rates of treatment with various concentration of osthole (0, 20, 40, 80 μg/ml) were 0.00%±0.00%, 7.45%±0.87%, 14.12%±2.29%, 27.26%±0.43% and 0.00%±0.00%, 13.44%±0.84%, 29.03%±0.78%, 57.49%±1.70%, with significant differences (F=174.33, P<0.001; F=1 041.40, P<0.001), and the following contrast between each two groups met the statistical significance (all P<0.01). The migration cells per field of CNE2 cells treated with 0, 20, 40, 80 μg/ml osthole for 48 hours were 52.13±4.49, 29.00±4.49, 18.50±1.93, 13.75±2.77, which exhibited a significant difference (F=200.37, P<0.001), and the following contrast between each two groups met the statistical significance (all P<0.01). The invasion cells per field of CNE2 cells treated with 0, 20, 40, 80 μg/ml osthole for 48 hours were 46.63±2.87, 24.13±2.87, 16.75±5.29, 11.00±1.77 respectively, which exhibited a significant difference (F=131.92, P<0.001), and the following contrast between each two groups met the statistical significance (all P<0.01). Meanwhile, the relative mRNA and protein expressions of E-cadherin in 0, 20, 40 and 80 μg/ml osthole treated-cells (exposure for 48 hours) were 1.00±0.13, 2.61±0.03, 3.12±0.09, 3.60±0.06 (F=20.92, P<0.001) and 0.22±0.03, 0.35±0.01, 0.60±0.04, 0.82±0.03 (F=178.63, P<0.001) respectively, and the differences were statistically significant, and further pairwise comparison showed the differences were statistically significant (all P<0.05). Furthermore, the relative mRNA and protein levels of vimentin, β-catenin, cyclin D1 in 0, 20, 40 and 80 μg/ml osthole treatment for 48 hours were statistically significant difference (mRNA level of vimentin: 1.00±0.12, 0.68±0.03, 0.56±0.01, 0.40±0.09, F=9.48, P<0.010; mRNA level of β-catenin: 1.00±0.14, 0.78±0.04, 0.69±0.07, 0.46±0.12, F=4.84, P<0.050; mRNA level of cyclin D1: 1.00±0.09, 0.82±0.03, 0.58±0.09, 0.40±0.03, F=9.49, P<0.010; protein level of vimentin: 0.85±0.02, 0.74±0.01, 0.34±0.01, 0.27±0.01, F=610.58, P<0.001; protein level of β-catenin: 0.83±0.00, 0.44±0.02, 0.39±0.00, 0.23±0.03, F=985.74, P<0.001; protein level of cyclin D1: 0.86±0.02, 0.67±0.00, 0.35±0.01, 0.25±0.01, F=910.57, P<0.001), and further pairwise comparison showed the differences were statistically significant (all P<0.05). Conclusion Osthole can inhibit the proliferation, invasion and migration of CNE2 cells, which is related to the regulation of Wnt/β-catenin signal pathway and then suppressing of EMT. Key words: Osthole; Nasopharyngeal neoplasms; Cell proliferation; Neoplasm invasiveness; Cell movement

Berberine Hydrochloride-Loaded Chitosan Nanoparticles Effectively Targets and Suppresses Human Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is a common epithelial malignancy that occurs in the nasopharynx and it is one of the high incidences of malignant tumors in China. As previous reports, berberine hydrochloride (BH) possesses a repressive effect on the proliferation of various cancer types. But the application of BH was hampered for a long time due to its hydrophobic properties, along with poor stability and bioavailability. In this study, folate acid modified chitosan nanoparticles loaded berberine hydrochloride (BH/FA-CTS NPs) were prepared by the ionic cross-linking technique, the physicochemical properties and the influence of BH/FA-CTS NPs on proliferation, migration and apoptosis in human nasopharyngeal carcinoma CNE-1 cells were studied in vitro and in vivo. Results of scanning electron microscopy (SEM) images showed that BH/FA-CTS NPs were sphere and the diameter was 258.2±9.1 nm. The drug loading and encapsulation efficiency were 8.17±1.12% and 87.73±4.21%, respectively. The drug release kinetics exhibited a slower and sustained release over a period of 48 h. Moreover, results of MTT assay and scratch assay displayed that BH/FA-CTS NPs inhibited proliferation and migration of CNE-1 cells remarkably. BH/FA-CTS NPs promoted apoptosis and necrosis of CNE-1 cells. And BH/FA-CTS NPs displayed distinguished higher tumor inhibition than control group, free BH and BH/CTS NPs in vivo. Therefore, as a nanocomposite, BH/FA-CTS NPs provide a new method and option for the treatment of nasopharyngeal carcinoma patients.

X线诱导人鼻咽鳞状细胞癌CNE1细胞株MDR-1及P-gp的表达研究

Objective To examine the expressions of multidrug resistance gene (MDR)-1 and P-glycoprotein (P-gp) in nasopharyngeal squamous cell carcinoma CNE1 cell line before and after X-ray exposure. Methods CNE1 cells were exposed to X-ray. After the irradiation, the CNE1 cells were cultured for 24 hours and tested. The mRNA expressions of MDR-1 in CNE1 cells were measured by semi-quantitative real-time polymerase chain reaction (RT-PCR) before and after X-ray exposure, and the protein expressions of P-gp in CNE1 cells were detected by Western blotting. The protein expressions of P-gp in CNE1 cells were observed by confocal microscope before and after X-ray exposure. Results The results of RT-PCR showed that the absorbance (A) values of MDR-1 mRNA in CNE1 cells were 0.17±0.01 and 0.34±0.03 before and after irradiation, and the difference was statistically significant (t=16.541, P<0.001). The results of Western blotting showed that the A values of P-gp protein in CNE1 cells were 0.02±0.01 and 0.04±0.01, and the difference was statistically significant (t=4.612, P=0.016). The green fluorescence intensity of P-gp protein in CNE1 cells after X-ray irradiation was higher than that before X-ray irradiation by confocal microscope. Conclusion X-ray irradiation can cause the high expressions of MDR-1 and P-gp in CNE1 cells, suggesting that X-ray irradiation can induce the occurrence of multidrug resistance in CNE1 cells. Key words: Nasopharyngeal neoplasms; CNE1 cells; Drug resistance, multiple; X-ray irradiation

Characterization of CSF2A fusion gene and its effect on Epstein-Barr virus-positive tumor cells.

We build the latent membrane protein gene latent membrane protein 2A (LMP2A) and the granulocyte-macrophase colony-stimulating factor (GM-CSF) gene fusion gene (CSF2A) and discuss how the CSF2A fusion protein influenced the proliferation and apoptosis of Epstein-Barr virus-positive (EBV+ ) tumor cells. Reverse-transcription polymerase chain reaction (RT-PCR) method was used to amplify the LMP2A gene and GM-CSF gene fragments, respectively, according to the principle of overlap extension in the coding (Gly4Ser)3 polypeptide gene fragments of DNA restructured under the connection. The CSF2A gene could be connected with the pIRES2-enhanced green fluorescent protein vector by recombinant DNA technology and identified by enzyme electrophoresis analysis and DNA sequencing. Then, the recombinant vector was transfected into dendritic cells (DCs); RT-PCR and Western blot analysis were used for testing the CSF2A gene messenger RNA and protein expression. The impacts of CSF2A on the proliferation and apoptosis of EBV+ tumor cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Hochest 33342 staining. We successfully obtained the recombinant vector named pIRES2-CSF2A. The expression of CSF2A could be detected by transfecting pIRES2-CSF2A into DCs. The DCs were cocultured with T lymphocytes and then acted on the EBV+ CNE2 nasopharyngeal carcinoma cells. MTT assay showed that the inhibiting effect of CSF2A obviously increased and the time dependency (**P < 0.01, *P < 0.05) also existed. Hochest 33342 staining showedapoptosis morphological changes of cells in nucleus staining and generated the apoptotic body. Apoptosis cells of the pIRES2-CSF2A group increased significantly at 48 hours. The results showed that the pIRES2-CSF2A recombinant vector was effectively transfected into DCs and the fusion gene CSF2A could promote EBV+ CNE2 cell apoptosis, laying the foundation for the specificity of EBV+ tumor targeting immune gene therapy in the future.

Overexpression of β-Catenin Decreases the Radiosensitivity of Human Nasopharyngeal Carcinoma CNE-2 Cells

Background/Aims: Nasopharyngeal carcinoma (NPC) is rare worldwide but remains highly prevalent in endemic regions, notably in southern China. Radiotherapy remains the treatment of choice for NPC, but radioresistance has been identified as a major cause of therapeutic failure. The Wnt/β-catenin signaling has been found to be involved in NPC radioresistance; however, the effect of β-catenin overexpression on radioresistance remains unknown in NPC until now. This study aimed to examine the impact of β-catenin overexpression on the radiosensitivity of human NPC CNE-2 cells. Methods: Immunohistochemistry was performed to detect the β-catenin expression in normal nasopharyngeal specimens and NPC specimens. The human NPC CNE-2 cell line overexpressing β-catenin was modeled by transfection with the pcDNA3.1/Hygro(+)/β-catenin recombinant vector (transfection group), while cells transfected with the pcDNA3.1/Hygro(+) vector served as negative controls and non-transfected cells served as blank controls. The expression of key molecules of the Wnt/β-catenin signaling pathway was determined using Western blotting and qPCR assays, and the changes of radiation sensitivity were measured with a colony-formation assay. Cell viability was measured by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide) assay. In addition, the cell cycle and apoptosis was detected using flow cytometry and the TCF/LEF transcriptional activity was measured with a Dual Luciferase Reporter Assay System. Results: Immunohistochemical staining showed high β-catenin expression in radioresistant NPC specimens, and low expression in radiosensitive NPC specimens and normal nasopharyngeal specimens. Western blotting and qPCR assays detected higher β-catenin expression in the transfection group than in the negative and blank controls (P < 0.01). Down-regulation of GSK-3β expression (P < 0.05) and up-regulation of Cyclin D1 expression (P < 0.01) was detected in β-catenin overexpressing NPC cells exposed to X-ray radiation relative to negative and blank controls. Colony-formation assay revealed higher D₀, D_q and SF in the transfection group than in the negative and blank control groups post-radiation, and the SER in the transfection group was 0.75-fold and 0.68-fold greater than that in the blank and negative control groups, respectively. MTT assay revealed that the viability of CNE-2 cells was significantly higher in the transfection group (96% ± 8.72%) than in the negative control group (74.67 ± 7.05%) and the blank control group (75.33% ± 7.02%) 24 h post-exposure to 6 Gy X-ray radiation (P < 0.05). X-ray radiation led to a lower proportion of CNE-2 cells at the G2/M phase and a lower apoptotic rate in the transfection group than in the negative and blank control groups (P < 0.05). In addition, the TCF/LEF transcriptional activity was higher in the transfection group than in the negative and blank control groups (P < 0.01), and 6 Gy X-ray radiation elevated the TCF/LEF transcriptional activity relative to 0 Gy radiation in the transfection group (P < 0.01). Conclusion: β-catenin overexpression may decrease the radiation sensitivity in NPC CNE-2 cells through activating the downstream transcriptional factors of β-catenin, and reducing G2/M arrest and cell apoptosis.

Prodigiosin Inhibits Proliferation, Migration, and Invasion of Nasopharyngeal Cancer Cells

Background/Aims: Nasopharyngeal carcinoma remains a devastating and difficult disease to treat. This study explores the antineoplastic effect of prodigiosin on nasopharyngeal cancer cells. Methods: Human nasopharyngeal carcinoma CNE2 cells and human normal nasopharyngeal epithelial NP69 cells were obtained and treated with prodigiosin or fluorouracil (5-FU). Colony formation assay was performed to screen for the optimal experimental concentrations of prodigiosin and 5-FU, and MTT assay was used to examine cell proliferative ability. Flow cytometry was used to examine cell cycle distribution, the scratch test was employed to examine cell migration, and Transwell migration assay (Boyden chamber) was used to study cell invasion. Results: The optimal concentrations of prodigiosin and 5-FU for treatment were 4 mg/L and 0.35 mg/L, respectively. Both prodigiosin and 5-FU inhibited tumor cell proliferation. The percentage of cells in G0/G1 phase was higher and the percentage of cells in S phase was lower in the prodigiosin and 5-FU groups than in the untreated groups. Both prodigiosin and 5-FU inhibited tumor cell migration and tumor cell invasion. Conclusions: Our results suggest that prodigiosin can inhibit proliferation, migration, and invasion of nasopharyngeal carcinoma cells.

Snail-mediated cancer stem cell-like phenotype in human CNE2 nasopharyngeal carcinoma cell.

Cancer stem cell (CSC)-like phenotype, which has been proven to play a critical role in invasion and metastasis of many kinds of cancers, has also been reported to be associated with epithelial-mesenchymal transition. Snail, a potent repressor of E-cadherin expression, was found to have a function to regulate the aforementioned processes. In the current study, expression of putative CSCs biomarkers and the ratio of CSC-like CNE2 (cancer cell line) in total CNE2 were measured, and CSC-like characteristics were analyzed with tumor-sphere self-renewal and colony-forming assays. Migration and invasion properties were determined by using transwell and wound healing assays. Xenograft tumor assays in vivo were done to evaluate the function of Snail and radiation in the tumor forming ability. In human nasopharyngeal carcinoma (NPC) cells, overexpression of Snail mediates a CSC-like phenotype, which enhances the initiation, invasion, and migration ability of cancer cells. Thus, Snail is a potential therapeutic target in NPC.

Radio-sensitizing effect of ethyl caffeate on nasopharyngeal carcinoma CNE-2 cell line

Purpose: To investigate the radio-sensitizing effect of ethyl caffeate (ETF) on naso-pharyngeal carcinoma. Methods: MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay was used to evaluate the cell viability of CNE-2 cells, while their levels of caspase-3 and caspase-9 were determined by enzyme-linked immunosorbent assay (ELISA). In addition, a xenograft model was established in nude mice. The model was treated with ETF (40 mg/kg) and subjected to β-irradiation (10 Gy) for 28 days, during which tumor volume was determined at 4-day intervals. Expressions of caspase-3, caspase-9 and Bcl-2 were determined by western blotting assay. Results: β-irradiation (10 Gy) did not produce any obvious inhibitory effect on the proliferation of CNE-2 cells. However, ETF (10, 20 and 40 μg/mL) significantly enhanced the radiosensitivity of the cells to β- irradiation ( p < 0.01) and significantly increased their levels of caspase-3 and caspase-9 ( p < 0.01). The combination of ETF (40 mg/kg) with β-irradiation resulted in significant inhibition of tumor growth in mice xenograft model ( p < 0.01). The combined treatment also resulted in significant up-regulation of expressions of caspase-3 and casepase-9 and significant down-regulation of Bcl-2 in the tumor tissues when compared with corresponding tissues from the control mice ( p < 0.01). Conclusion: ETF significantly enhances the sensitivity of naso-pharyngeal carcinoma CNE-2 cells to β- irradiation, probably through induction of mitochondria-mediated apoptosis. ETF may be useful for treating naso-pharyngeal carcinoma in combination with radiation therapy. Keywords: Ethyl caffeate, Radio-sensitizing effects, Caspase, Nasopharyngeal carcinoma, CNE-2 cell line, β-irradiation

Investigation of the effects induced by 1 400 W on nasopharyngeal carcinoma cell and the mechanism

Objective To investigate the effect induced by specific inducible nitric oxide synthase (iNOS) inhibitor 1 400 W on nasopharyngeal carcinoma CNE-2 cells lines and its mechanism. Methods CNE-2 cells were treated by different concentrations of 1 400 W, diamminedichloroplatinum (DDP), and both chemicals. Cell counting kit-8 (CCK-8) was used to examine the viability of cells. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect the iNOS mRNA expression. Results The expression of iNOS mRNA was down-regulated by 1400W and was positively correlated with inhibition rate of cell proliferation. 1 400 W inhibits proliferation of CNE-2 cell in a concentration-dependent manner. The proliferation inhibition rate of CNE-2 cells treated by 1 400 W combined with DDP was not enhanced. Conclusions Specific inducible nitric oxide synthase inhibitor 1 400 W can exerts anti-tumor effect though inhibiting the expression of iNOS mRNA; The mechanism of chemosensitization induced by iNOS inhibitor on CNE-2 cells may be closely related level of down-regulation of iNOS expression. Key words: Nitric oxide synthase/AI/ME; Nasopharyngeal neoplasms/DT/ME

Radiotherapy induces cell cycle arrest and cell apoptosis in nasopharyngeal carcinoma via the ATM and Smad pathways

ABSTRACTNasopharyngeal carcinoma (NPC) is a common malignant neoplasm of the head and neck which is harmful to human's health. Radiotherapy is commonly used in the treatment of NPC and it induces immediate cell cycle arrest and cell apoptosis. However, the mechanism remains unknown. Evidences suggested the activation of Ataxia telangiectasia mutated (ATM) pathway and Smad pathway are 2 of the important crucial mediators in the function of radiotherapy. In this study, we performed in vitro assays with human nasopharyngeal carcinoma CNE-2 cells and in vivo assays with nude mice to investigate the role of the ATM and Smad pathways in the treatment of nasopharyngeal carcinoma with radiotherapy. The results suggested that radiation induced activation of ATM pathway by inducing expression of p-ATM, p-CHK1, p-CHK2, p15 and inhibiting expression of p-Smad3. In addition, Caspase3 expression was increased while CDC25A was decreased, leading to cell cycle arrest and cell apoptosis. On the other hand, activation of Smad3 can inhibited the ATM pathway and attenuated the efficacy of radiation. In summary, we suggest that both ATM and Smad pathways contribute to the cell cycle arrest and cell apoptosis during nasopharyngeal carcinoma cells treated with radiation.

In Vivo and In Vitro Effects of ATM/ATR Signaling Pathway on Proliferation, Apoptosis, and Radiosensitivity of Nasopharyngeal Carcinoma Cells.

The study investigated the ability of ataxia-telangiectasia mutated (ATM)/Rad3-related (ATR) signaling pathway to influence the proliferation, apoptosis, and radiosensitivity of nasopharyngeal carcinoma (NPC) cells. NPC tissues and corresponding adjacent normal tissues were collected from 143 NPC patients. The NPC CNE2 cells were assigned into a control group, X-ray group, CGK-733 group, and X-ray+CGK-733 group. The mRNA levels of ATM and ATR were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR) and the protein levels of ATM and ATR using western blotting. The positive expression of ATM and ATR in tissues and nude mouse tumor tissues was determined by immunohistochemistry. Cell proliferation, migration, invasion, and apoptosis rates were analyzed by the 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay, scratch test, transwell assay, and flow cytometry, respectively. A nude mouse model of NPC was established to observe tumor volume and growth. The mRNA levels of ATR and ATM and the expression of ATR and ATM protein in NPC tissues were significantly higher than those in adjacent normal tissues. The colony formation assay showed that the colony-forming rate decreased, showing radiation dose-dependent and CGK-733 concentration-dependent manners. Expression of ATM, ATR, Chk1, and Chk2 was evidently increased in the X-ray, CGK-733, and X-ray+CGK-733groups compared with the control group, and the aforementioned expression was highest in the X-ray+CGK-733 group among the four groups. The cell proliferation, invasion, and migration were decreased, tumor volume decreased and cell apoptosis increased in the X-ray, CGK-733, and X-ray+CGK-733 groups compared with the control group; the X-ray+CGK-733 group exhibited lowest cell proliferation, invasion and migration, smallest tumor volume, and highest cell apoptosis among the four groups. Inhibition of ATM/ATR signaling pathway reduces proliferation and enhances apoptosis and radiosensitivity of NPC cells.

Vimentin is a crucial target for anti-metastasis therapy of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a unique subtype of head and neck cancer, with tendency to spread to regional lymph nodes and distant organs at early stage. Vimentin, a major cytoskeletal protein constituent of the intermediate filament, plays a critical role in the epithelial-mesenchymal transition. Overexpression of vimentin is considered to be a critical prerequisite for metastasis in numerous human cancers. Therefore, targeting vimentin for cancer therapy has gained a lot of interest. In the present study, we detected vimentin expression in NPC tissues and found that overexpression of vimentin is associated with poor prognosis of NPC patients. Silencing of vimentin in NPC CNE2 cells by RNAi suppresses cells migration and invasion in vitro. However, blocking vimentin did not affect cell proliferation of CNE2 cells. In addition, the in vivo metastatic potential of CNE2 cells transfected with Vimentin shRNA was suppressed in a nude mouse model of pulmonary metastasis. Silencing of Vimentin in CNE2 cells leads to a decrease of microvessel density and VEGF, CD31, MMP2, and MMP9 expressions in pulmonary metastatic tumors. Importantly, we found that it is easier for the tumor cells from the high vimentin-expressing pulmonary metastatic tumors to reinvade the microvessel and to form stable tumor plaques attached to the endothelial cells, which resemble the resource of circulating tumor cells and are very hard to eliminate. However, depletion of vimentin inhibits the formation of vascular tumor plaques. Our findings suggest that RNAi-based vimentin silencing may be a potential and promising anti-metastatic therapeutic strategy for NPC.