Cell Line CNE2 Research Articles

Stable knockdown of ZBTB7A promotes cell proliferation and progression in nasopharyngeal carcinoma

Although high expression of ZBTB7A is positively relative to metastasis in nasopharyngeal carcinoma (NPC) patients, the association between its low expression and metastasis of NPC remains unclear. The present study aimed to definitely identify the association. The level of ZBTB7A was effectively knocked down by stable transfection of short hair RNA plasmid in NPC cell lines CNE2 and 5-8F (shRNA-CNE2 and shRNA-5-8F), compared with the cells that stably transfected empty plasmid (NC-CNE2 and NC-5-8F). The levels of ZBTB7A were assessed by real-time polymerase chain reaction and Western blot in the cell lines. MTT assay, colorimetric focus-formation assay, flow cytometry, wound healing assay, transwell assays, and xenograft model were performed to analyze cell vitality, proliferation, cell cycle, migration, invasion, and tumorigenicity. The levels of ZBTB7A were effectively reduced in shRNA-CNE2 and shRNA-5-8F. Their carcinogenicity was stronger separately than the abilities of NC-CNE2 and NC-5-8F. NC-CNE2 and shRNA-CNE2 were selected to establish the xenograft model because of their stronger tumorigenicity than NC-6-10B and shRNA-5-8F. The assay showed that shRNA-CNE2 had stronger tumorigenicity than NC-CNE2. The results demonstrated the reverse association between the expression of ZBTB7A and the tumorigenicity of NPC. We postulate that some oncogenic pathways, which are suppressed by ZBTB7A, will vicariously promote the proliferation and progression of NPC when ZBTB7A is decreased.

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.

Sers Imaging of Nasopharyngeal Carcinoma Markers Using an Antibody-Conjugated Gold Nanoparticles Probe

Surface-enhanced Raman scattering (SERS) nanotags as an ultrasensitive nanoprobe is becoming popular for the detection of biomarkers. Herein, antibody-conjugated gold nanoparticles (AuNPs) were used to target LMP2A in an LMP2A-infected CNE2 cell line. SERS maps showed that the LMP2A was distributed around the cell, which was consistent with the results of immunofl uorescence staining in the previous report. This location could be due to the specific binding of the bioconjugated nanotags to the receptors on the cell surface. However, the CNE2 cell line without LMP2A-infected showed no detectable signal at 1044 cm–1. The results demonstrated the potential feasibility of AuNPs nanotags as highly sensitive probes conjugated at the subcellular level for detection and localization of cancer markers in nasopharyngeal carcinoma (NPC).

Toll-like receptor-9 in hypoxic nasopharyngeal carcinoma cells and its correlation with cell proliferation and apoptosis.

The purpose of this study was to investigate the correlation between the expression of Toll-like receptor-9 (TLR-9) and cell proliferation and apoptosis in hypoxic nasopharyngeal carcinoma cells. Human nasopharyngeal carcinoma cell line HNE-1 (EBV positive) and CNE-1 (EBV negative) were used. Cells were divided into normal control group, hypoxia group and hyperoxia group. Hypoxic conditions were 5% CO2 and 0.01% partial pressure of oxygen, hyperoxia conditions were 5% CO2 and 10% partial pressure of oxygen. Reverse transcription-PCR (RT-PCR) and western blot analysis were used to detect the expression of TLR-9 mRNA and protein at 6, 12 and 24 h after the beginning of cell culture. MTT assay was used to detect the cell proliferation rate and flow cytometry was used to detect cell apoptosis rate. Expression levels of TLR-9 mRNA and protein in hypoxia group reached the peak at 12 h after the beginning of cell culture, and were significantly higher than those of hyperoxia group at all time-points, expression levels of TLR-9 mRNA and protein of control group were the lowest, difference between groups were all statistically significant (P<0.05). No significant changes in expression levels of TLR-9 mRNA and protein were found in control group and hyperoxia group between different time-points (P>0.05). Compared with the other two groups, cell proliferation rate was gradually decreased and apoptotic rate was gradually decreased in hypoxia group, significant differences were found between hypoxia group, and control group and hyperoxia group (P<0.05), no significant differences were found between control group and hyperoxia group (P>0.05). In conclusion, TLR-9 was highly expressed in hypoxic nasopharyngeal carcinoma cells regulating cell proliferation and apoptosis, which may be an important mechanism of tumorigenesis and a potential target for intervention therapy.

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

Design and synthesis of 2-phenylpyrimidine coumarin derivatives as anticancer agents

A series of 2-phenylpyrimidine coumarin derivatives with potential telomerase-inhibiting activity was designed and synthesized. All of the compounds were screened for antiproliferative activity against CNE2, KB, and Cal27 cell lines in vitro. The results showed that most of the derivatives had a favorable effect on resisting tumor cell proliferation; compound 13, 3-(4-amino-5-oxo-5H-chromeno[4,3-d]pyrimidin-2-yl)phenyl 4-(dimethylamino)benzenesulfonate, exhibited the best activity. Flow cytometry revealed that compound 13 can inhibit CNE2 proliferation. Telomerase inhibition and in vitro antitumor activity were consistent among the compounds, but compound 13 showed the best telomerase-inhibiting activity and could inhibit telomere extension. Molecular docking results indicated that compound 13 bonded with telomerase reverse transcriptase (TERT) through multiple interactions, including hydrogen bonding and hydrophobic interactions. The results of the study provide further information on 2-phenylpyrimidine coumarins, expanding the types of telomerase inhibitors as the parent structures.

Abstract 14: Anticancer activities of terpenoid compounds isolated from the plant centipeda minma against nasopharyngeal carcinoma cells

Abstract Nasopharyngeal carcinoma (NPC) is one of common cancers throughout south-east Asia, southern China and other regions. Radiotherapy and chemotherapy are the standard treatments for NPC with severe adverse effects, multi-drug resistance, and insensitivity to advanced NPC. Complementary and novel treatments for NPC gain considerable interest around the world. The medicinal plant Centipeda minma is traditionally used in China for the treatment of various nasal diseases such as allergy, rhinitis, sinusitis and nasopharyngeal carcinoma. In the current study, we studied the anticancer potential of an ethanol extract (CME) and 3 pure sesquiterpenoids (A1, A2, A3) extracts from Centipeda minma against NPC cells in vitro and some underlying mechanisms of actions. The NPC cell lines CNE-1 and CNE-2 cells were treated with either CME or 3 pure compounds (A1, A2, A3). Proliferation of NPC cells was inhibited in a concentration- and time-dependent manner. For 48 h treatment, the IC50 values were 41.57 to 24.98 μg/mL for CME, and 2.3 to 7.5 μM for A1 to A3. While these extracts showed no appreciable effect on normal human LO2 hepatocytes, significant numbers of NPC cells underwent apoptotic morphological changes following treatments. Cytometric analysis demonstrated that treatments with CME and compounds results in NPC cell cycle arrest at the G2/M phase and eventually apoptosis in a concentration and time-dependent manner. Western blot analysis showed that these extracts could down-regulate Bcl-2 expression, up-regulate Bax expression and caspase-8, 9, 3 activities. In addition, the potential anti-NPC actions might be also associated with the marked inhibition of multiple signal transduction pathways including EGFR, VEGFR, HER2, FGFR, IGF-1R, PI3K/AKT/mTOR, MAP/ERK and Jan/Stat3 pathways. In summary, the Centipeda minma extract and terpenoids are potential apoptosis inducing agents to kill NPC cells and may be promising anti-cancer drug leads against nasopharyngeal carcinoma. The exact mechanism of actions remains further study. Note: This abstract was not presented at the meeting. Citation Format: Bei-bei Liu, Kui-yuan Zhu, Chi-on Chan, Ling-Hua Zhang, Daniel Kam-Wah Mok, Si-bao Chen. Anticancer activities of terpenoid compounds isolated from the plant centipeda minma against nasopharyngeal carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 14. doi:10.1158/1538-7445.AM2017-14

Synthesis and In Vitro Anticancer Activity of Novel Dehydroabietic Acid-Based Acylhydrazones.

In order to develop novel chemotherapeutic agents with potent anticancer activities, a series of dehydroabietic acid (DHA) derivatives bearing an acylhydrazone moiety were designed and synthesized by the condensation between dehydroabietic acylhydrazide (3) and a variety of substituted arylaldehydes. The inhibitory activities of these compounds against CNE-2 (nasopharynx), HepG2 (liver), HeLa (epithelial cervical), and BEL-7402 (liver) human carcinoma cell lines were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay in vitro. The screening results revealed that many of the compounds showed moderate to high levels of anticancer activities against the tested cancer cell lines and some displayed similar potent inhibitory activities to the commercial anticancer drug cisplatin, while they exhibited lower cytotoxicity against normal human liver cell (HL-7702). Particularly, compound 4w, N’-(3,5-difluorobenzylidene)-2-(dehydroabietyloxy)acetohydrazide, with an IC50 (50% inhibitory concentration) value of 2.21 μM against HeLa cell, was about 17-fold more active than that of the parent compound, and showed remarkable cytotoxicity with an IC50 value of 14.46 μM against BEL-7402 cell. These results provide an encouraging framework that could lead to the development of potent novel anticancer agents.

Study of signal transduction mechanisms of X-ray induced multidrug resistance in nasopharyngeal squamous cell carcinoma CNE1 cells

Objective To investigate the relationship between multidrug resistance of X-ray irradiated human nasopharyngeal squamous carcinoma cell line CNE1 and platelet endothelial cell adhesion molecule-1 (PECMA-1)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/Bcl-2 signaling pathway. Methods ①The CNE1 cells were cultured in incubator. Exponentially growing CNE1 cells were exposed to 50 Gy ionizing radiation administered in 25 fractions, 2 Gy per fraction, once every two days, and the CNE1/R cells were collected. ②CNE1/R cells were cultured for 24 h, and divided into three groups: the control group, experimental group one (CNE1/R cells were processed through 1∶600 PECAM-1 antibody for 24 h) and experimental group two (CNE1/R cells were processed through 10 μmol/L PI3K inhibitor LY294002 for 24 h). ③The half maximal inhibitory concentration (IC50) values of the control group and two experimental groups of CNE1/R cells were detected by methyl thiazolyl tetrazolium (MTT). ④The mRNA expressions of PI3K and Bcl-2 in the control group and experimental group one were measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). ⑤The mRNA expressions of AKT and Bcl-2 in the control group and experimental group two were measured by semi-quantitative RT-PCR. Results ①The IC50 of the control group, experimental group one and two were (2.99±0.02)μg/ml, (1.50±0.03)μg/ml and (1.60±0.05)μg/ml, and the difference among the three groups was statiatically signficant (F=4 381.263, P=0.000). The IC50 of the two experimental groups were obviously lower than that of the control group (t=116.885, P=0.000; t=73.006, P=0.000). ②The semi-quantitative values of PI3K in the control group and experimental group one were 1.66±0.01 and 0.90±0.05 (t=50.394, P=0.000), and the semi-quantitative values of Bcl-2 were 1.66±0.02 and 0.71±0.05 (t=183.165, P=0.000), and the differences were statistically significant. ③The semi-quantitative values of AKT of control group and experimental group two were 1.53±0.01 and 0.72±0.01 (t=135.281, P=0.000), and the semi-quantitative values of Bcl-2 were 1.66±0.02 and 0.63±0.02 (t=128.814, P=0.000), and the differences were statistically significant. Conclusion PECAM-1 induced multidrug resistance of human nasopharyngeal squamous cell carcinoma CNE1 cells may be related to the activity of Bcl-2 through the signal pathway of PI3K/AKT. Key words: Nasopharyngeal neoplasms; Drug resistance, neoplasm; Antigens, CD31; X-rays

Expression of C2orf40 protein and its clinical significance in human nasopharyngeal carcinoma

Objective To evaluate the protein expression of chromosome 2 open reading frame 40 (C2orf40) in nasopharyngeal carcinoma (NPC) tissues and cells, and to explore its association with cell diffe-rentiation and clinicopathological features. Methods A total of 122 patients with NPC between January 2001 and December 2003 were enrolled in Cancer Hospital of Shantou University Medical College. The paraffin-embedded tissue sections and medical records were collected. Twenty-five samples with chronic nasopharyngitis were used as controls. Tumor and control tissues from biopsies underwent immunohistochemical staining for C2orf40. C2orf40 expression was analyzed with clinicopathological variables. Besides, the protein expressions of C2orf40 were measured by Western blotting in three NPC cell lines, including CNE1, CNE2 and C666-1. Results Ninety-six percent (24/25) of control tissues showed positive expression, among which 88.0% showed dense staining. Otherwise, only 58.3% (71/122) of NPC samples were positive for C2orf40 protein and 82.0% showed weak staining. There was significantly difference between the two groups (U=255.500, P<0.001). It was inversely related to lymph nodes status (r=-0.058, P<0.001) and clinical stage (r=-0.202, P=0.026) by Spearman rank correlation test. In vitro, higher level of C2orf40 protein was found in well differentiated CNE1 cells, while lower levels were found in poorly differentiated cell lines CNE2 and C666-1. Conclusion Down-regulated C2orf40 expression is correlated with tumor cell differentiation, lymph nodes metastasis and clinical stage, and may be a molecular event in the occurrence and development of NPC. C2orf40 is likely to be a potential target of anticancer therapy. Key words: Nasopharyngeal neoplasms; Pathology, clinical; C2orf40

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.

Laminaria Japonica Polysaccharides effectively inhibited the growth of nasopharyngeal carcinoma cells in vivo and in vitro study

AimsLaminaria Japonica Polysaccharides (LJP) is a kind of plant polysaccharide isolated from Laminaria Japonica Aresch. LJP has a variety of biological activity, including anti-tumor, improving immune function, anti-radiation and others. This study observed the biological activity of LJP in vitro and in vivo on human nasopharyngeal carcinoma(NPC), and the possible anticancer mechanism was explored. MethodsNasopharyngeal poorly differentiated squamous cell carcinoma cell lines CNE2 and HONE1 were used for the study. MTT method was used to detect the proliferation of HONE1 and CNE2 treated with gradient concentrations of LJP. The apoptosis of HONE1 treated with LJP was detected by annexin V-FITC/PI double staining method. HONE1 was used to establish subcutaneous implanted tumor model in nude mice. The changes of transplanted tumor volume and body weight of nude mice in each group were observed and recorded. The changes of the ultrastructure of transplanted tumor were observed by transmission electron microscope (TEM). ResultsMTT results showed that LJP has inhibitory effect on proliferation of both HONE1 and CNE2, and the effects were dosage-dependent; results of flow cytometry (FCM) analysis showed that, LJP could efficient induce apoptosis in HONE1, and apoptosis rate increased with the increase of LJP concentration. In vivo experiments, the inhibition rate was 33.7% (P<0.05) and 47% (P<0.01) in middle and high dose LJP group, respectively. TEM results suggested that the cancer cells in the transplanted tumor tissue treated with middle and high dose LJP presented unique apoptosis changes. ConclusionsLJP can effectively inhibit the growth of NPC cells. And it may be achieved by inducing apoptosis of NPC cells.

Curcumin exerts its antitumor activity through regulation of miR-7/Skp2/p21 in nasopharyngeal carcinoma cells.

Curcumin, a natural polyphenol compound, exhibits tumor suppressive activity in a wide spectrum of cancers, including nasopharyngeal carcinoma cells. However, the exact molecular mechanisms governing this tumor suppressive activity remain elusive. Multiple studies have revealed that miRNAs are critically involved in tumorigenesis, indicating that targeting miRNAs could be a therapeutic strategy for treating human cancer. In the current study, we set out to determine whether curcumin regulates miR-7 expression in nasopharyngeal carcinoma cells. We found that curcumin inhibited cell growth, induced apoptosis, retarded cell migration and invasion, and triggered cell cycle arrest in the human nasopharyngeal carcinoma cell lines CNE1 and CNE2. Importantly, we observed that curcumin upregulated the expression of miR-7 and subsequently inhibited Skp2, a direct miR-7 target. Our results identified that upregulation of miR-7 by curcumin could benefit nasopharyngeal carcinoma patients.

Antitumor activity of the pachymic acid in nasopharyngeal carcinoma cells

ABSTRACTObjective: To investigate the antitumour efficacy of pachymic acid (PA), which is a fungal extract component, on nasopharyngeal carcinoma (NPC) cells CNE-1, CNE-2. Methods: We have chosen NPC cell line CNE-2 for the study, and the cells were treated with PA before the detection. CCK-8 assay was used to detect the proliferative ability, and Annexin V-PI double staining was used for the detection of apoptosis rate; and the nucleus damage was detected by transmission electron microscope, the protein expression of the DNA damage pathway were detected by Western blot. Results: PA can significantly inhibited proliferation of CNE-1, CNE-2 cells. The proportion of apoptotic cells of all cell lines gradually increased in a dose-dependent manner induced by PA, P < 0.05. Meanwhile, the nucleus could be caused morphological changes and the expression of DNA damage-related proteins was upregulated by PA in CNE-2. Conclusions: PA can significantly inhibit cell proliferation and increase the apoptosis rates and may induce the apoptosis of the human NPC cells.

Chemosensitizing Effect of Astragalus Polysaccharides on Nasopharyngeal Carcinoma Cells by Inducing Apoptosis and Modulating Expression of Bax/Bcl-2 Ratio and Caspases.

BackgroundPlatinum-based chemotherapy is the most effective regimen for nasopharyngeal carcinoma, which presents highly invasive and metastatic activity. However, the dose-related toxicity of chemotherapy agents limits the dose administration. Astragalus polysaccharide (APS) is the major active ingredient extracted from Chinese herb Radix Astragali and is proven to be active against carcinomas. We aimed to assess the chemosensitizing effects of Astragalus polysaccharides on nasopharyngeal carcinoma in vitro and in vivo and to explore the underlying mechanism.Material/MethodsWe used BALB/c nu/nu mice and human nasopharyngeal carcinoma cell lines CNE-1, CNE-2, and SUNE-1. MTT, Annexin V/PI, Western blot analysis, and TUNEL assay were carried out.ResultsAPS significantly promoted anti-proliferative and apoptotic effects of cisplatin on nasopharyngeal carcinoma cells. APS also enhanced the anti-tumor effects and cisplatin-induced apoptosis in the xenograft model. The level of Bcl-2 decreased, while the levels of Bax, caspase-3, and caspase-9 increased in cisplatin combined with APS treatment compared to cisplatin only treatment. The ratio of Bax to Bcl-2 was significantly enhanced by the APS to cisplatin.ConclusionsAPS enhanced the anti-proliferative and apoptotic effect of cisplatin by modulating expression of Bax/Bcl-2 ratio and caspases on nasopharyngeal carcinoma cells and in the xenograft model.

Promoter Methylation of Dual Specificity Phosphatase-6 Gene effect on Invasion and Metastasis of Nasopharyngeal Carcinoma

Objective: The dual-specificity phosphatase-6 (DUSP-6) is a member of dual-specificity protein phosphatase (DUSP) family, it can make residues of threonine and tyrosine dephosphorylate. In recent years, it was found that DUSP-6 is correlated with tumour generation and progress. In this study we have detected the effect of DUSP-6 gene methylation on invasion and metastasis of nasopharyngeal carcinoma. Methods: RT-PCR and methylation-specific PCR are used to detect mRNA expression and methylation state of DUSP-6 gene in nasopharyngeal carcinoma tissues and CNE2 cell line. The changes of mRNA expression and methylation state of DUSP-6 gene, proliferation and invasion of cells were detected after 5-aza- 2’-deoxycytidine (5-aza-cdR) treated CNE2 cell line. Results: These results showed that DUSP-6 gene expression is related to its promoter methylation state, cervical lymph node metastasis is related to DUSP-6 gene promoter methylation state in patients with nasopharyngeal carcinoma, 5-aza-cdR can inhibit invasion of CNE2 cells, and make re-expression of DUSP-6 gene by promoter demethylation in CNE2 cells. Conclusion: DUSP-6 gene promoter methylation state is possibly an important factor that effected invasion and metastasis of nasopharyngeal carcinoma. DUSP-6 gene may make a novel marker for prognostic evaluation in nasopharyngeal carcinoma.

Role of the TGFβ/PDCD4/AP-1 Signaling Pathway in Nasopharyngeal Carcinoma and Its Relationship to Prognosis

The objective of the present study was to evaluate the role of the TGFβ/PDCD4/AP-1 pathway in nasopharyngeal carcinoma (NPC) and its relationship to NPC prognosis. NPC tissues collected from 66 NPC patients were compared to 17 nasopharyngeal mucosa biopsy specimens collected as normal tissues. Immunohistochemical staining was performed to assess expression of transforming growth factor-β receptor I (TGFβRI), programmed cell death 4 (PDCD4) and activator protein-1 (AP-1). The Kaplan-Meier method was applied to evaluate NPC patient overall survival (OS) and progression-free-survival (PFS). Cox regression analysis was used to estimate independent prognostic factors for NPC. The human NPC cell line CNE2 was selected and treated with SB431542, an inhibitor of TGFβRI; expression of TGFβRI and PDCD4 in CNE2 cells was determined by western blotting. NPC tissues showed higher expression of TGFβRI and AP-1 but lower expression of PDCD4 than normal tissues (all P < 0.05). The results of Kaplan-Meier analysis showed that TGFβRI-positive patients and AP-1-positive patients had shorter OS and PFS than TGFβRI-negative patients and AP-1-negative patients; additionally, PDCD4-positive patients had higher OS and PFS than PDCD4-negative patients. Cox regression analysis revealed that advanced tumor stage, overexpression of TGFβRI and AP-1, and low expression of PDCD4 were unfavorable factors influencing OS and PFS in NPC patients. Compared with the control group, expression of TGFβRI decreased and that of PDCD4 increased significantly in CNE2 cells treated with the inhibitor (all P < 0.05). These findings indicate that the TGFβ/PDCD4/AP-1 pathway may be associated with NPC development and progression. High expression of TGFβRI and AP-1 and low expression of PDCD4 may be unfavorable prognostic factors for NPC.

Mutant Rad50 enhances killing effects of radiation on nasopharyngeal carcinoma cell line CNE1

Objective: To investigate the killing effects of radiation and mutant Rad50 transfection on human nasopharyngeal carcinoma cell line CNE1. Methods: The experimental groups included: control group, Ad-Rad50-GFP group, Ad-EGFP group, irradiation group, Ad-Rad50-GFP combined with irradiation group, and Ad-EGFP combined with irradiation group. CNE1 cells were transfected with recombinant adenoviral vector Ad-Rad50-GFP carrying mutant Rad50 gene. The expressions of Mre11, Rad50, Nbs1, and relevant constituents composing MRN complex were detected by Western Blot. Neutral comet assay was used to detect the effect of mutant Rad50 on restoration process of DNA damage. Cell growth curve was used to evaluate the growth inhibition of CNE1 by mutant Rad50 and radiation. Results: Expressions of Mre11, Rad50, and Nbs1 in cells of Ad-Rad50-GFP group were less significantly than those in control group when irradiation was completed (0.48 vs 0.62, 0.42 vs 0.5, and 0.53 vs 0.69, respectively, P<0.05) and 24 hours after irradiation (0.41 vs 0.69, 0.46 vs 0.58, and 0.34 vs 0.78, respectively, P<0.05). The mean tail moment (MTM) in Ad-Rad50-GFP plus irradiation group was higher than that in irradiation group when irradiation was completed (16.06 vs 14.8, P<0.05), 24 hours after irradiation (58.23 vs 15.89, P<0.05) and 48 hours after irradiation: (45.12 vs 11.42, P<0.05). Seven days after irradiation, the cells in Ad-Rad50-GFP plus irradiation group was less than those in control group or irradiation group (both P<0.05). Conclusion: Mutant Rad50 enhances killing effects of radiation on nasopharyngeal carcinoma cell line CNE1.

Detection of Genomic DNA Damage from Radiated Nasopharyngeal Carcinoma Cells Using Surface-Enhanced Raman Spectroscopy (SERS).

Structural changes and chemical modifications in DNA during interactions with X-ray radiation are still not clear within 48 h of incubation. We investigate genomic DNA from the radiated CNE2 cell line within 48 h of incubation using surface-enhanced Raman spectroscopy (SERS). Multivariate methods including principal component analysis (PCA) and random forest are proposed to explore the statistical significance before and after radiation. Our results show that intensities of several bands change after radiation, which indicates backbone damage and base-unstacking. Biological effects from DNA damage repairing process may be simultaneously stimulated and different from incubation time. Under doses of 10 Gy (with 24 and 48 h of incubation) and 20 Gy (with 48 h of incubation), the relative contents of C against T and A against G deviate obviously from the control level. Statistical results strengthen significantly the idea that modification in DNA bases is associated with the disruption of base-stacking in the DNA duplex. Our findings provide vital information for radiation-induced the DNA damage at the molecular level, which may provide insight into the effect and mechanism of anticarcinogens in tumor therapy.

Physcion, a naturally occurring anthraquinone derivative, induces apoptosis and autophagy in human nasopharyngeal carcinoma.

Physcion is a major bioactive ingredient in the traditional Chinese medicine Radix et Rhizoma Rhei, which has an anthraquinone chemical structure and exhibits a variety of pharmacological activities including laxative, hepatoprotective, anti-inflammatory, anti-microbial and anti-proliferative effects. In this study we investigated the effect of physcion on human nasopharyngeal carcinoma in vitro and in vivo, as well as the mechanisms underlying the anti-tumor action. The nasopharyngeal carcinoma cell line CNE2 was treated with physcion, and cell viability was detected using MTT and colony formation assays. Flow cytometry was used to assess the cell cycle arrest, mitochondrial membrane potential loss, apoptosis, autophagy and intracellular ROS generation. Apoptotic cell death was also confirmed by a TUNEL assay. The expression of target or marker molecules was determined using Western blotting. The activity of caspase-3, 8, and 9 was detected with an ELISA kit. A xenograft murine model was used to evaluate the in vivo anti-tumor action of physcion, the mice were administered physcion (10, 20 mg·kg-1·d-1, ip) for 30 d. Treatment with physcion (5, 10, and 20 μmol/L) dose-dependently suppressed the cell viability and colony formation in CNE2 cells. Physcion (10 and 20 μmol/L) dose-dependently blocked cell cycle progression at G1 phase and induced both caspase-dependent apoptosis and autophagy in CNE2 cells. Furthermore, physcion treatment induced excessive ROS generation in CNE2 cells, and subsequently disrupted the miR-27a/ZBTB10 axis, resulting in repression of the transcription factor Sp1 that was involved in physcion-induced apoptosis and autophagy. Moreover, physcion-induced autophagy acted as a pro-apoptotic factor, and possibly contributed to physcion-induced apoptosis. In the xenograft murine model, administration of physcion dose-dependently suppressed the tumor growth without affecting the body weight. Furthermore, the anti-tumor effects of physcion were correlated with downregulation of Sp1 and suppression of miR-27a in the tumor tissues. Physcion induces apoptosis and autophagy in human nasopharyngeal carcinoma by targeting Sp1, which was mediated by ROS/miR-27a/ZBTB10 signaling. The results suggest that physcion is a promising candidate for the treatment of human nasopharyngeal carcinoma.