Human Nasopharyngeal Carcinoma CNE-2 Cells Research Articles

Salinomycin enhances radiotherapy sensitivity and reduces expressions of BIRC5 and NEIL2 in nasopharyngeal carcinoma.

The aim of this study was to investigate the effects of salinomycin (Sal) on expressions of baculoviral IAP repeat-containing 5 (BIRC5) and Nei endonuclease VIII-like 2 (NEIL2) and radiotherapy sensitivity of nasopharyngeal carcinoma (NPC). Human NPC CNE-2 cell lines were used as research objects in this study. Subsequently, the cells received intervention with Sal at different concentrations, radioactive rays at different doses and Sal combined with radioactive rays. The growth inhibition rate of CNE-2 cells was detected via methyl thiazolyl tetrazolium (MTT) assay. The dose-effect relations of Sal, radioactive rays and combination therapy with the inhibitory effect on CNE-2 cells were obtained. CNE-2 cells receiving intervention with Sal at an appropriate concentration or radioactive rays at an appropriate dose alone and Sal combined with radioactive rays were used as intervention groups (Sal group, Radiation group and Combination group). However, those added with an equal amount of DMSO were set as Control group. Next, the cycle, apoptosis and apoptotic morphology of CNE-2 cells were observed via flow cytometry and Hoechst assay, respectively. Moreover, the expressions of apoptosis-related proteins Caspase-3, B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax), as well as BIRC5 and NEIL2 proteins in CNE-2 cells were determined using Western blotting. Under the intervention with Sal or radioactive rays alone, the growth inhibition rate of CNE-2 cells rose in a concentration/dose-dependent manner. With the increase in Sal concentration in combination therapy, the growth inhibition rate of CNE-2 cells significantly increased (p<0.05). Compared with Control group, Sal group, Radiation group, and Combination group exhibited remarkably lower colony formation rate, higher proportion of CNE-2 cells in the G2/M phase, enhanced apoptosis of CNE-2 cells with nuclear fragmentation, increased expressions of pro-apoptotic proteins Caspase-3 and Bax, decreased expression of anti-apoptotic protein Bcl-2, and lower protein expressions of BIRC5 and NEIL2 in cells (p<0.05). Compared with Radiation group, the Combination group had significantly decreased colony formation rate, increased proportion of CNE-2 cells in the G2/M phase, enhanced apoptosis of CNE-2 cells with more nuclear fragmentation and other apoptosis characteristics, increased expressions of pro-apoptotic proteins Caspase-3 and Bax, decreased expression of anti-apoptotic protein Bcl-2, and decreased protein expressions of BIRC5 and NEIL2 in cells (p<0.05). Sal enhances the radiotherapy sensitivity of NPC and reduces the protein expressions of BIRC5 and NEIL2 in cells.

Spectomycin B1 induces VEGFR2 de-SUMO modification to inhibit angiogenesis in nasopharyngeal carcinoma

Objective:To explore the new mechanism of spectomycin B1 in inhibiting angiogenesis of nasopharyngeal carcinoma and to provide a theoretical basis for targeted gene therapy of nasopharyngeal carcinoma. Method:Human nasopharyngeal carcinoma CNE1 cells were divided into two groups, the control group and spectomycin B1 group. Western blot was used to detect the expression levels of small ubiquitin-related modified protein（SUMO） 1 and vascular endothelial growth factor receptor 2（VEGFR2）. The angiogenesis assay was used to detect the angiogenic ability of CNE1 cells, and the apoptosis was detected by flow cytometry. The model of nasopharyngeal carcinoma-bearing mice was established, spectomycin B1 was administered, tumor volume and weight were measured, and protein expression of CD31 was detected by immunohistochemistry and microvessel density was compared. Result:Spectomycin B1 could reduce deSUMOylation of VEGFR2 protein by 4.05 times, significantly reduce the angiogenic ability of CNE1 cells, and increase the apoptosis rate by 20.68%. In the tumor-bearing mouse model, spectomycin B1 treatment could inhibit subcutaneous tumor growth rate and weight, and the blood vessel density decreased by 40.04%. Conclusion:Spectomycin B1 can inhibit neovascularization of nasopharyngeal carcinoma by inducing deSUMOylation of VEGFR2 protein.

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.

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.

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.

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.

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.

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.

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.

Inhibition of Anaphase-Promoting Complex by Silence APC/CCdh1 to Enhance Radiosensitivity of Nasopharyngeal Carcinoma Cells.

The aim of this study was to investigate the possibility of APC/CCdh1 as a potential therapeutic target in the radiosensitivity of nasopharyngeal carcinoma (NPC) cell CNE-1, and explain the role of APC subunits after silence of Cdh1 combined with radiotherapy. Transfection with Cdh1 shRNA significantly increased the radiosensitivity of CNE-1 cells and the radiation enhancement ratio (RER) of sh-Cdh1 cells was 1.76. Knockdown of Cdh1 in CNE-1 cells increased irradiation induced apoptosis and G2/M phase cell cycle arrest. The levels of CDC20 and CylinB1 increased and the levels of Ku70 and APC3 decreased after irradiation. APC/CCdh1 is involved in regulation of radiosensitivity in human NPC CNE-1 cells. Our study may provide a promising therapeutic strategy for NPC by targeting Cdh1. J. Cell. Biochem. 118: 3150-3157, 2017. © 2016 Wiley Periodicals, Inc.

Study of gonadotropin releasing hormone on suppressing migrationg and invationg of human nasopharyngeal carcinoma cells CNE2

Objective:The aim of this study is to investigate the effect of gonadotropin releasing hormone (GnRH) on suppressing cell viability, apoptosis, migrationg and invationg of human nasopharyngeal carcinoma cells CNE2. Method:Nasopharyngeal carcinoma tissues and postnasal catarrh tissues were collected, the expression of GnRH positive cells and GnRH mRNA were detected by immunohistochemical staining and qRT-PCR. The human nasopharyngeal carcinoma CNE2 cells and immortalized nasopharyngeal epithelial cell line NP69 were cultured in vitro, and the expression of GnRH positive cells and GnRH mRNA were detected by immunohistochemical staining and qRT-PCR. The CNE2 cells were treated with GnRH with various concentrations 0 (Blank group), 10⁻², 10⁻¹, 10⁰ nmol/L. The effects of GnRH on the viability, apoptosis, migration and invasion of CNE2 cells were detected by cell Counting Kit (CCK-8), flow cytometry, wound healing assay and transwell chamber assay in vitro. Result:The expression of GnRH positive cells and GnRH mRNA in nasopharyngeal carcinoma tissues were markedly down regulated than postnasal catarrh tissues (P<0.05). The expression of GnRH positive cells and GnRH mRNA in CNE2 cells were markedly down regulated than NP69 cells (P<0.05). Compared with blank group, GnRH can significantly inhibite the cell viability cells, apoptosis, migration and invasive ability (P<0.05 or P<0.01). Conclusion:GnRH significantly inhibited the cell viability, apoptosis, migration and invasive ability of CNE2 cells.

Apogossypolone induces apoptosis and autophagy in nasopharyngeal carcinoma cells in an in vitro and in vivo study.

Nasopharyngeal carcinoma (NPC) has a high incidence and mortality rate, particularly in Southern China. Apogossypolone (ApoG2) is a novel derivative of gossypol with antitumor activity and less toxicity. The human NPC CNE-2 cell line was studied in the in vitro model; whilst 4 week-old male nude mice (BALB/c-nu) were inoculated subcutaneously with CNE-2 cells, and xenograft tumors were studied in the in vivo model. Graded concentrations of ApoG2 were used in treatment studies. In ApoG2-treated and control in vitro and in vivo tumor cells, cell apoptosis, and autophagy were evaluated and quantified using fluorescent and transmission electron microscopy and flow cytometry. Hoechst-33258 fluorescence staining was used to evaluate apoptosis in treated and non-treated cell culture and xenograft NPC cells. Western blotting was performed on lysed tumor cells using primary antibodies to B-cell lymphoma-2 (Bcl-2), beclin-1, and β-actin, and flow cytometry results indicated cell apoptosis rates of 3.90±0.34 and 19.52±1.18% in the control and ApoG2-treated cells, respectively (F=485.294, P<0.001). Western blot analysis showed that ApoG2 significantly decreased expression of the Bcl-2 protein in CNE-2 cells, when compared with control cells (F=68.909, P=0.001) and flow cytometry showed cell autophagy rates of 0.92±3.10% of control cells compared with 28.24±7.35% of ApoG2-treated cells (F=31.035, P=0.003). ApoG2 treatment significantly increased beclin-1 protein expression in CNE-2 cells (F=497.906, P<0.001). ApoG2 treatment inhibited NPC xenograft tumor growth by 65.49% (P<0.05). In conclusion, these results support a role for ApoG2 in inhibiting the growth of human NPC cells by inducing apoptosis and autophagy. Future controlled clinical studies could be planned, to define safety, efficacy and dosing regimens for ApoG2 as a potential treatment for patients with NPC.

Anticancer and anti-angiogenic activities of extract from Actinidia eriantha Benth root

Ethnopharmacological relevanceThe roots of Actinidia eriantha Benth (AER) are commonly used traditional folk medicine for the treatment of gastric carcinoma, nasopharyngeal carcinoma, and breast carcinoma. Besides, the anti-proliferative and immunomodulatory effects of AER polysaccharides on tumor-bearing mice have been reported previously. Aim of the studyThis work was carried out to investigate the anticancer and anti-angiogenic activities of AER. Materials and methodsThe growth inhibitory effects of ethanol extracts from the leaves (EEL), stems (EES) and roots (EER) of A. eriantha on human gastric carcinoma SGC7901 cells, human nasopharyngeal carcinoma CNE2 cells, human breast carcinoma MCF7 cells and human umbilical vein endothelial cells (HUVECs) were evaluated by MTT assay. The ethyl acetate fraction from EER (EA-EER) was further investigated for the anticancer activity against SGC7901 cells and the anti-angiogenic activity in HUVECs in vitro. The apoptosis in SGC7901 cells and HUVECs was confirmed by DAPI nuclear staining and flow cytometry analysis, the effect on cellular DNA fragmentation was detected in SGC7901 cells. And the cell cycle-arresting activity in HUVECs was determined by flow cytometry. Moreover, the inhibitory effect of EA-EER on cell migration in HUVECs was observed by both wound-healing and Transwell migration assays. RT-PCR and Western-blotting were performed to determine the mRNA and protein expression levels, respectively, including Bax, Bcl-2 and caspase-3 in SGC7901 cells, as well as VEGF-A and VEGFR-2 in HUVECs. Furthermore, the in vivo anti-angiogenic activity of EA-EER was evaluated by using chick embryo chorioallantoic membrane (CAM) assay. Ultimately, the chemical components in EA-EER were isolated and purified by repeated column chromatography followed by structure characterization using 1H and 13C nuclear magnetic resonance and mass spectroscopy. ResultsCompared with EEL and EES, EER displayed the strongest growth inhibitory effect on SGC7901 cells, CNE2 cells and HUVECs. Among the EER fractions, EA-EER exhibited the most potent growth inhibitory activity against SGC7901 cells, CNE2 cells and HUVECs. Moreover, EA-EER induced obvious apoptosis in SGC7901 and HUVECs, and significantly inhibited the proliferation of HUVECs via blockade of cell cycle G1 to S progression. Furthermore, EA-EER suppressed the expression of Bcl-2 and improved the expression Bax and caspase-3 in SGC7901 cells. EA-EER not only inhibited migration of HUVECs, but also down-regulated the expression of VEGF-A and VEGFR-2 in HUVECs. In vivo, EA-EER exposure reduced the formation of blood vessels in chick embryos. A bio-guided isolation of EA-EER led to the isolation of three compounds for the first time, namely (6R, 7E, 9S)-6, 9-hydroxy-megastigman-4, 7-dien-3-one-9-O-β-D-glucopyranoside, Oleanolic acid-23-O-β-D-glucopyranoside, 3β, 23, 24-trihydroxyl-12-oleanen-28-oic acid. ConclusionThe present research demonstrated that the significant anticancer and anti-angiogenic effects of AER, providing the supportive evidence for its traditional use in the treatment for cancer. It was suggested that AER could be use as a potential source of cancer therapeutic drug.

Anticancer activities of alkaloids extracted from the Ba lotus seed in human nasopharyngeal carcinoma CNE-1 cells.

To investigate the anticancer activities of alkaloids from the Ba lotus seed (BLSA) in human nasopharyngeal carcinoma (NPC) CNE-1 cells, an MTT assay, flow cytometry, reverse transcription-polymerase chain reaction and western blotting were performed. BLSA was found to significantly reduce CNE-1 cell proliferation in a dose-dependent manner at all concentrations compared with the control (P<0.05). In addition, flow cytometry analysis identified that BLSA treatment significantly increased the sub-G1 content in CNE-1 cells (P<0.05). Following BLSA treatment, the mRNA and protein levels of a number of apoptosis-related factors, such as caspase family members (caspase-3, -8 and -9), B-cell lymphoma (Bcl)-2-associated X protein, Fas and Fas ligand were significantly increased compared with the control (P<0.05). This was accompanied by a significant decrease in anti-apoptotic Bcl-2 and Bcl-extra large protein expression compared with the control (P<0.05). Furthermore, BLSA treatment was determined to modulate CNE-1 cell expression of nuclear factor (NF)-κB and NF-κB inhibitor α. The results of the present study indicate that BLSA has anticancer activity through inducing cellular apoptosis. In addition, these results suggest that BLSA can be used as a therapeutic agent in NPC.

Proteomic analysis of docetaxel resistance in human nasopharyngeal carcinoma cells using the two-dimensional gel electrophoresis method.

Docetaxel-based chemotherapy has been recommended for advanced nasopharyngeal carcinoma (NPC). However, treatment failure often occurs because of acquired drug resistance. In this study, a docetaxel-resistant NPC cell line CNE-2R was established with increasing doses of docetaxel for more than 6 months. Two-dimensional gel electrophoresis and ESI-Q-TOF-MS were used to compare the differential expression of docetaxel-resistance-associated proteins between human NPC CNE-2 cells and docetaxel-resistant CNE-2R cells. As a result, 24 differentially expressed proteins were identified, including 11 proteins with increased expression and 13 proteins with decreased expression. These proteins function in diverse biological processes such as metabolism, signal transduction, calcium ion binding, immune response, proteolysis, and so on. Among these, α-enolase (ENO1), significantly upregulated in CNE-2R, was selected for detailed analysis. Inhibition of ENO1 by shRNA restored CNE-2R cells' sensitivity to docetaxel. Moreover, overexpression of ENO1 could facilitate the development of acquired resistance of docetaxel in CNE-2 cells. Western blot and reverse-transcription PCR data of clinical samples confirmed that α-enolase was upregulated in docetaxel-resistant human NPC tissues. Finding such proteins might improve interpretation of the molecular mechanisms leading to the acquisition of docetaxel chemoresistance.

MicroRNA-15a Inhibits Proliferation and Induces Apoptosis in CNE1 Nasopharyngeal Carcinoma Cells.

Nasopharyngeal carcinoma (NPC) is a highly metastatic cancer, frequently occurring in Southeast Asia and Southern China. Several microRNAs (miRNAs) have been shown to have an inhibitive effect on NPC, while the effect of miR-15a on NPC remains unclear. Thus, our study aimed to investigate the potential effect of miR-15a on NPC cell proliferation, apoptosis, and possible functional mechanism. Human NPC CNE1 cells were transfected with miR-15a mimics, miR-15a inhibitors, or a control. Afterward, cell viability and apoptosis were assayed by using CCK-8, BrdU assay, and flow cytometry. Moreover, Western blot was used to detect the expression changes of proliferation and apoptosis of related proteins. As a result, miR-15a overexpression significantly reduced cell proliferation (p < 0.01 or p < 0.001) and induced cell apoptosis (p < 0.001), while miR-15a suppression got the opposite result for cell proliferation and apoptosis. In addition, miR-15a overexpression upregulated the protein levels of p27, GSK-3β, Bax, procaspase 3, and active caspase 3, whereas miR-15a suppression downregulated these proteins. The protein level of p21 was not significantly regulated by miR-15a overexpression or suppression. These results indicated that miR-15a played a role for inhibition of proliferation and induction of apoptosis in CNE1 cells.

Grape seed proanthocyanidins induce apoptosis through the mitochondrial pathway in nasopharyngeal carcinoma CNE-2 cells

Although modern radiotherapy offers excellent local control in the treatment of nasopharyngeal carcinoma (NPC), current therapeutic decisions remain burdensome due to the frequency of local recurrence and treatment failure at distant sites. One potential and promising strategy for the prevention or treatment of cancers is the use of bioactive components of plant origin, including dietary plant products. Herein, we studied one class of these bioactive compounds, grape seed proanthocyanidins (GSPs), and explored their effect on NPC CNE-2 cells, as well as the primary mechanism underlying this effect. Our results revealed that treatment of human NPC CNE-2 cells with GSPs reduced cell viability in a dose-and time-dependent manner, and moreover, markedly induced cell cycle arrest at the G2/Mphase, leading to induction of apoptosis. In addition, we found that the underlying mechanism was associated with increased expression of the pro-apoptotic protein Bax, decreased expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL, upregulation of cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PRAP) protein, and the loss of mitochondrial membrane potential (MMP) (Δψm). Furthermore, GSPs upregulated the Bcl-2 homology3 (BH3)-only proteins, Bim and Bad, in a concentration-dependent manner. Taken together, these data supported our hypothesis that, in human NPCCNE-2 cells, GSPs could induce apoptosis through the mitochondrial pathway and ultimately reduce cell viability. Collectively, the results discussed above provide substantive evidence for the potential of GSPs as an effective bioactive phytochemical for the treatment of NPC.

Morphine, a potential antagonist of cisplatin cytotoxicity, inhibits cisplatin-induced apoptosis and suppression of tumor growth in nasopharyngeal carcinoma xenografts.

Morphine is an opioid analgesic drug often used for pain relief in cancer patients. However, there is growing evidence that morphine may modulate tumor growth, progression and metastasis. In this study, we evaluated whether morphine modulates cisplatin-induced apoptosis in human nasopharyngeal carcinoma CNE-2 cells and whether morphine affects the antitumor activity of cisplatin on tumor growth in human nasopharyngeal carcinoma CNE-2 xenografts in nude mice. We showed that a pretreatment with morphine (1 μg/ml) inhibited the sensitivity of CNE-2 cells to cisplatin by inhibiting cisplatin-induced CNE-2 cell apoptosis, decreasing caspase-3 activity and increasing the Bcl-2/Bax ratio. However, a high dose of morphine (1000 μg/ml) had the opposite effect. We also showed that at a low dose, morphine enhances chemoresistance in an in vivo nasopharyngeal carcinoma (NPC) model by inhibiting cisplatin-induced apoptosis and decreasing neovascularization. Taken together, our results indicate that a low dose of morphine may lead to chemoresistance of cisplatin in NPC models in vitro and in vivo by inhibiting cisplatin-induced apoptosis and decreasing neovascularization.

Effect of silencing HIF-1α by RNA interference on adhesion and invasion of the human nasopharyngeal carcinoma cell line CNE-1

To investigate the effects of HIF-1α on adhesion and invasion of human nasopharyngeal carcinoma CNE-1 cells under hypoxia and underlying molecular mechanisms. CoCl₂was used to mimic tumor hypoxic microenvironment. mRNA and protein expressions of HIF-1α, E-cadherin and CXCR4 in CNE-1 cells at different hypoxic time phases were detected by RT-PCR and ELISA respectively. The influences of silencing HIF-1α using RNA interference on E-cadherin and CXCR4 expressions were evaluated. Adhesion test Transwell invasion test were used to evaluate the effects of HIF-1α gene silencing on cell adhesion and invasion. Under hypoxia, HIF-1α mRNA expression in CNE-1 cells was stable, but its protein expression increased obviously (P<0.05). Both mRNA and protein expressions of E-cadherin were decreased significantly with prolonged hypoxia, while mRNA and protein expressions of CXCR4 increased significantly (P<0.05). After silencing HIF-1α gene, expression of E-cadherin protein was up-regulated, but with down-regulated expression of CXCR4 protein, with a decrease significantly in adhesion rate or invasive cell number of CNE-1 cells (P<0.05). Hypoxia can increase HIF-1α protein expression in nasopharyngeal carcinoma cell line CNE-1. Silencing HIF-1α by RNA interference can reduce inhesion and invasion abilities of CNE-1 cells, which may be mediated by down-regulating E-cadherin expression and up-regulating CXCR4 expression.

A dimeric Phaseolus coccineus lectin with anti-oxidative, anti-proliferative and cytokine-inducing activities

In the present study, a dimeric glucosamine binding lectin, designated as CHL, was purified from Phaseolus coccineus L. var. albonanus Bailey through three chromatographic steps. The molecular weight of CHL was approximately 66kDa. Its hemagglutinating activity toward rabbit erythrocytes was dependent on carbohydrates, especially glucosamine, and was stable at temperatures between 20 and 70°C, and at pH between 1 and 13. Intriguingly, further characterization showed that CHL served as a potent antioxidant to prevent erythrocytes from haemolysis induced by 2′-azobis (2-amidinopropane) dihydrochloride (AAPH) in a dose-dependent manner. Moreover, it exerted antitumor activity against human nasopharyngeal carcinoma CNE1 cells, hepatoma HepG2 cells, and breast cancer MCF7 cells but was devoid of antifungal activity. In addition, the CHL could bring about a significant dose-dependent increase in the production of mRNAs of pro-inflammatory cytokines including interferon-gamma and interleukin-2. These results suggest the potential therapeutic utility of CHL.