Erlotinib enhanced chemoradiotherapy sensitivity via inhibiting DNA damage repair in nasopharyngeal carcinoma CNE2 cells.

BACKGROUND: Nasopharyngeal carcinoma (NPC), arising from nasopharynx epithelium, is a rare type of malignant carcinoma that has a specific geographical distribution and a high risk of distant metastases. For most of the diagnosed NPC patients, the total survival rate decreased significantly due to the high local recurrence rate and metastasis rate. Concurrent chemoradiotherapy (CCRT), as routine therapy strategy of NPC, usually accompanies with high-dosage cytotoxic agents and serious toxic side reaction. Therefore, there is an urgent need for update the existing therapy strategies. In this study, we sought to investigate the effects of a combined therapy strategy, erlotinib combined with cisplatin and radiotherapy, on biological characteristics of NPC CNE2 cells and the potential reasons. METHODS: CNE2 cells in logarithmic phase seeding in 96-well plates received concentration gradients of erlotinib (at 0, 10, 20, 40, 80, 160, 320 mmol/L) or cisplatin (at 0, 0.25, 0.5, 1, 2, 4, 8 mg/L), in order to obtain the optimal working concentration of erlotinib and cisplatin via 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Then, cells were divided into control group and four treatment groups (Group I-IV). All treatment groups received irradiation of 4 Gy, moreover, Group II-IV respectively received erlotinib, cisplatin and erlotinib plus cisplatin at optimal working concentration. After 24 and 48 h of irradiation, growth inhibition rate was determined; invasion ability and migration ability was respectively detected by Boyden's chamber assay and cell scratch test; flow cytometry was performed for determining apoptosis rate and cell cycle distribution; the expression levels of epidermal growth factor receptor (EGFR) signal pathway proteins were semi-quantitatively analyzed by Western-blot. RESULTS: Compared with Group I, all the other treatment groups showed better inhibition effect on cell viability, invasion and migration ability and higher apoptosis rate, while Group IV showed the strongest growth inhibition effect and highest apoptosis rate. In addition, EGFR signal pathway proteins of Group IV showed the lowest expression level. CONCLUSIONS: In combined therapy with radiotherapy/chemotherapy, erlotinib could enhance radiotherapy/chemotherapy sensitivity, probably because it could suppress DNA damage repair after radiotherapy/chemotherapy, thus weakening radiotherapy/chemotherapy resistance of tumor cells.

AMIGO2 promotes proliferation of nasopharyngeal carcinoma cells by activating the PI3K/AKT/mTOR signaling pathway / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To explore the role of adhesion molecule with Ig like domain 2 (AMIGO2) in the proliferation of nasopharyn‐ geal carcinoma (NPC) cells and its mechanisms. Methods: A total of 10 NPC tissue samples and 10 normal nasopharyngeal epithelial tissue samples collected at Fujian Cancer Hospital during September 2017 and November 2017 were used for this study; in addition, NPC cell lines (CNE-1, CNE-2, SUNE-1, 6-10B, C666-1) and human immobilized nasopharyngeal epithelial cell line NP69 were also collected. The relative expression of AMIGO2 mRNAin above mentioned tissues and cell lines was detected by qPCR. Lentivirus vectors were constructed to interfere AMIGO2 mRNA expression, and qPCR was used to verify its interference efficiency. CCK-8 method, Clonal formation and Flow cytometry were performed to evaluate the effect of AMIGO2 interference on proliferation, clone formation and apoptosis of NPC cells. The influence of AMIGO2 interference on PI3K/AKT/mTOR signaling pathway and proliferation related molecular markers in NPC cells was assessed by Western blotting. Results: The results of qPCR showed that AMIGO2 was highly expressed in NPC tissues, CNE-2, and SUNE-1 cells (all P<0.01). The interference efficiency of AMIGO2 in CNE-2 and SUNE-1 cells could reach over 50%. The interfering of AMIGO2 expression significantly inhibited the proliferation and clone formation of CNE-2 and SUNE-1 cells (all P<0.01), promoted cell apoptosis (all P<0.01), reduced the phosphorylated protein expression levels of PI3K, AKT and mTOR in SUNE-1 cells (all P<0.01), as well as down-regulated the protein expressions of survivin and PCNA (all P<0.01). Conclusion: AMIGO2 may promote the proliferation as well as inhibit apoptosis of NPC cells by activating the PI3K/AKT/mTOR signaling pathway, suggesting that AMIGO2 may be a potential target for NPC therapy.

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

BACKGROUND: The 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. METHODS: The 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. RESULTS: The 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. CONCLUSION: The expression of DACT1 was related to the methylation status. High expression of DACT1 may inhibit the invasion and metastasis of nasopharyngeal carcinoma cells.

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

BACKGROUND: The 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. METHODS: The 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. RESULTS: The 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. CONCLUSION: The expression of DACT1 was related to the methylation status. High expression of DACT1 may inhibit the invasion and metastasis of nasopharyngeal carcinoma cells.

miR-17-5p regulates proliferation, invasion, migration and apoptosis of nasopharyngeal carcinoma CNE2 cells by down-regulating BRMS1Lexpression / 中国肿瘤生物治疗杂志

@# Objective: To explore the molecular mechanism of miR-17-5p regulating the proliferation and invasion of nasopharyngeal carcinoma cells by regulating the expression of breast cancer metastasis suppressor 1 like (BRMS1-like or BRMS1L) gene. Methods:A total of 40 cases of nasopharyngeal carcinoma tissues and corresponding paracancerous tissues resected from nasopharyngeal carcinoma patients, who were admitted to the General Hospital of Pingdingshan Shenma Medical Group during January 2014 to December 2017, were included in this study; in addition, nasopharyngeal carcinoma cell lines CNE 2, HONE 1, C666-1 and nasopharyngeal immortalized epithelial cell line NP69 were also collected for this study. The expression of miR-17-5p in nasopharyngeal carcinoma tissues and cell lines was detected by qPCR. The targeted relationship between BRMS1L and miR-17-5p was predicted by the StarBase and verified by the Dual luciferase reporter gene assay. Effects of transfection of miR-17-5p mimics and inhibitors on the expression of BRMS1Lin CNE2 cells were detected by WB assay. CCK-8, Transwell and Flow cytometry were used to detect the effects of miR-17-5p/BRMS1L axis on the proliferation, migration, invasion and apoptosis of CNE 2 cells. Results: miR-17-5p was highly expressed in nasopharyngeal carcinoma tissues and cell lines (P<0.05 or P<0.01). Knockdown of miR-17-5p significantly inhibited proliferation, invasion and migration of CNE2 cells but promoted apoptosis (P<0.05 or P<0.01); miR-17-5p targeted BRMS1Land down-regulated its expression. Over-expression of BRMS1Lsignificantly inhibited the proliferation, invasion and migration of CNE2 cells but promoted apoptosis (all P<0.01); while simultaneous over-expression of miR-17-5p and BRMS1L reversed the above effects (all P<0.01). Conclusion: miR-17-5p promoted proliferation, invasion, migration and inhibited apoptosis of CNE 2 cells by down-regulating the expression of BRMS1L.

ß-Catenin is important for cancer stem cell generation and tumorigenic activity in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors with poor prognosis and recurrence in South China. The hard eradication of NPC in clinic is predominantly due to cancer stem cells (CSCs). Increasing evidence revealed that the aberrant activation of Wnt/ß-catenin was positively correlated with the produce of CSCs. To further investigate the effect of ß-catenin on CSCs and tumorigenesis in NPC, a CNE2 cell line (pLKO.1-sh-ß-catenin-CNE2) with stably suppressed expression of ß-catenin was used in this study. The expressions of biomarkers in CSCs including c-myc, Nanog, Oct3/4, Sox2, EpCAM as well as adhesion-related proteins like E-cadherin and vimentin were analyzed by western blot analysis and immunofluorescent staining. The proliferation and migration abilities were investigated by MTT assay and Transwell assay, respectively. Cell cycle was analyzed by flow cytometry. Finally, xenograft was performed to determine the effect of ß-catenin on oncogenesis in vivo. Results showed that the expressions of c-myc, Nanog, Oct3/4, Sox2, and EpCAM were all decreased in pLKO.1-sh-ß-catenin-CNE2 cells. It was also found that vimentin was downregulated, while E-cadherin was upregulated. Results of MTT and Transwell assays suggested that the proliferation and migration abilities were impaired by silencing of ß-catenin, and more cells were arrested in G1 phase when compared with the control. In vivo study indicated that the tumor growth was markedly suppressed in experimental group. Based on current findings, ß-catenin may function as an essential protein for the maintenance of migration and proliferation abilities of NPC cells, and a complicated network consisting of c-myc, Nanog, Oct3/4, Sox2, EpCAM, E-cadherin, vimentin, and ß-catenin may be involved in the inherent regulation mechanisms.

Promotion of combination of chrysin and camptothecin on apoptosis of CNE2 cells / 中草药

objective To research whether the combination of chrysin and camptothecin can promote the apoptosis of human nasopharyngeal carcinoma cell line CNE2 and to explore the molecular mechanism of the combinative effect. Methods: CNE2 cells were pretreated with designed dose of chrysin (10|, 20, and 40 μmol/L) for 2 h, then treated with camptothecin (1 μg/mL) for 24 h. The morphologic changes were observed under inversed microscope and the cell viability was measured using MTT. The activity of caspase-3 and PARP, which was regarded as the protein marks of apoptosis, was determined by Western blotting. Then the cells were treated with chrysin for different time and the time course of apoptosis inhibitory protein, Bcl-xL was also detected using Western blotting. Results: Increases of cell death were observed in the group with combined chrysin and camptothecin, but no obvious cell death could be found in chrysin, camptothecin alone, and control groups; The data of cell viability supported this results; With the enhance of pretreatment dose of chrysin, the cell viability decreased. There were the significant differences between the combined groups and the control one (P<0.05), and between the combined groups and both the chrysin and camptothecin groups separately (P<0.05). Chromatin condensation, which was the indication of apoptosis, could be observed when the cells were stained with Hochest 33342; The proprotein of caspase-3 and PARP degraded and there were the dose-dependent and time-dependent effect. The pan-caspase inhibitor Z-VAD-fmk could inhibit the apoptosis of CNE2 cells which were treated with the combination of chrysin and camptothecin, according to the cell viability and the activation of caspase-3 and PARP; The time-dependent down-regulation in the apoptosis inhibitory protein Bcl-xL could be observed. Conclusion: The cotreatment of chrysin and camptothecin could promote the apoptosis of CNE2 and the down-regulation of apoptosis inhibitory protein Bcl-xL played an important role in the combinative effect.

Growth Difference between CNE-2 and S-18 Cell Lines after Subcutaneous Xenograft / 昆明医科大学学报

Objective To study the growth difference and possible mechanism between nasopharyngeal carcinoma (NPC) cell line CNE-2 and its subclone S-18.Methods CNE-2 and S-18 cells were cultured in vitro.6 x 105 cells/mouse were xenografted subcutaneously in the back of nude mice.The volumes of rumors were measured on the 3 rd,7 th,10 th,14 th day after grafting.Mice were sacrificed on the 14 th day and tumors were isolated and weighed.RNA from tumor tissues were extracted and transcriptional levels of HSP27 and NF-K B were detected.Results (1) S-18,instead of CNE-2,grew to form tumor mass 7 days after xenografting subcutaneously;both cell lines formed tumor mass 10 days after xenografting,however,the volumes of S-18 tumors [(223.13 ± 21.32) mm3,10 th day;(420.25 ± 24.52) mm3,14 th day] were significant bigger than CNE-2tumors [(113.70±11.70) mm3,10thday;(279.86±25.78) mm3,14thday];The weights of S-18 umors were significantly higher than CNE-2 tumors on the 14 th day after xenografting;(2) The transcriptional levels of HSP27 and NF-KB in S-18 tumor were significantly higher than in CNE-2 tumor.Conclusion Xenografted S-18 NPC grows faster than Xenografted CNE-2 NPC.HSP27 and NF-κ B are probably involved in the regulation of growth in NPC.

Inhibitory effect and its molecular mechanisms of MicroRNA-34a on human na-sopharyngeal carcinoma CNE-2 cell line subcutaneous xenograft tumor in nude mice / 中国免疫学杂志

Objective:To investigate the inhibitory effect and its possible molecular mechanisms of MicroRNA-34a(miR-34a) on the human nasopharyngeal carcinoma CNE-2 cell line subcutaneous xenograft tumor in nude mice.Methods: The human nasopharyngeal carcinoma CNE-2 cell line was cultured in vitro.miR-34a and Scrambled miRNA recombinant plasmids were successfully established and stably transfected into CNE-2 cells.Fifteen six-week-old male nude mice were divided randomly into three groups:miR-34a group(5 mice) ,Scrambled miRNA group(5 mice) ,Blank control group(5 mice).Different CNE-2 cells were subcuta-neously injected on the back near right lower limb.Tumor volumes were examined every 7 days.Mice were executed on the 35 days,and the eventual average tumor volumes and weights were examined.Total RNA and protein were isolated from tumors,and the expression of miR-34a,CDK6,and Bcl-2 mRNA and protein were determined by qRT-PCR and western blot,respectively.Results: The relative expressions of miR-34a was significantly up-regulated in miR-34a transfected group compared to Scrambled miRNA transfected group (P (849.62±101.32) mm3 ,respectively,and the eventual average tumor weights in miR-34a group,Scrambled miRNA group and blank control group were(0.81±0.13)g,(1.47±0.21)g and(1.58±0.37)g,respectively.Both the eventual average tumor volumes and weights in miR-34a group were lower compared to the other two groups(P<0.05).qRT-PCR results revealed that the expression of miR-34a in miR-34a transfected group was significantly higher than in the other two groups,while the mRNA and protein expression of CDK6 and Bcl-2 were lower than the other two groups ( P<0.05 ) .Conclusion: miR-34a may inhibit the growth of human nasopharyngeal carcinoma CNE-2 cell line subcutaneous xenograft tumor in nude mice by down-regulating CDK6 and Bcl-2.

Effects of SAA protein on biological behavior of nasopharyngeal carcinoma CNE2 cells / 重庆医学

Objective To observe the effects of over expression and inhibition expression of SAA protein on biological behavior of nasopharyngeal carcinoma CNE2 cells.Methods pcDNA3.1 (+)-SAA-CNE2 cell lines of high expression and pGPU6/GFP/Neo-SAA-CNE2 cell lines of interference expression of SAA protein in vitro.These two cells constructed by transfection of pcD-NA3.1(+)-SAA plasmid of SAA high expression and pGPU6/GFP/Neo-SAA plasmids of SAA inhibition expression respectively, plasmids of which were previously successfully reconstructed by the research group.Cell cycle of these two cells was analyzed by flow cytometry with PI staining.The ability of cell proliferation was inspected by plate cloning-forming test.Results Flow cytome-try showed that with the increase of expression of SAA protein,it had effect on promoting CNE2 cell division.Plate cloning-forming test showed that SAA protein can improve proliferation of the CNE2 cells.Conclusion SAA protein has the effect on promoting proliferation of human nasopharyngeal carcinoma CEN2 cell and migration in vitro.

Radiosensitization of lobaplatin on human nasopharyngeal cancer cell line CNE2 in vitro / 中华放射医学与防护杂志

Objective To investigate the effect of lobaplatin combined with irradiation on human nasopharyngeal cancer cell line CNE2,and to illuminate its mechanism of radiosensitization.Methods MTT assay was used to detect the outcome of lobaplatin and irradiation on CNE2 cell proliferation.Clonogenic assay was applied to testify the radiosensitization effect of lobaplatin on the cells.Flow cytometry was used to check the cell cycle distribution and cell apoptosis.Western was used to detect the expression of Bcl-2,Bax and cleaved Caspase-3.Results The proliferation of CNE2 cells was reduced by lobaplatin in a dose-dependent manner.50IC of lobaplatin on CNE2 cells and lobaplatin combined with 4 Gy irradiation was 1.610 μmol/L and 0.077 μmol/L,respectively.The radiosensitization ratio of the combination group was over 3.Within 24 h of drug treatment,the percent of cells in G2/M phase increased with the concentration of lobaplatin.When the concentration of lobaplatin increased to 6 μmol/L,the cells of combination group were arrested at S phase.The apoptosis rate of lobaplatin (5 μmol/L) group,radiotherapy(4 Gy)group and combination group was 15.6％,11.3％ and 61.8％,respectively.Western blot showed that the expressions of Bax and cleaved Caspase-3 increased but Bcl-2 decreased in the combination group.Conclusion Lobaplatin could increase radiosensitization of human nasopharyngeal cancer cell line CNE2,probably by depressing Bcl-2 but enhancing Bax expression and hence activating Bcl-2/Bax-Caspase signaling pathway.

STGC3 inhibits xenograft tumor growth of nasopharyngeal carcinoma cells by altering the expression of proteins associated with apoptosis.

STGC3 is a potential tumor suppressor that inhibits the growth of the nasopharyngeal carcinoma cell line CNE2; the expression of this protein is reduced in nasopharyngeal carcinoma compared with normal nasopharyngeal tissue. In this study, we investigated the tumor-suppressing activity of STGC3 in nude mice injected subcutaneously with Tet/pTRE-STGC3/CNE2 cells. STGC3 expression was induced by the intraperitoneal injection of doxycycline (Dox). The volume mean of Tet/pTRE-STGC3/CNE2+Dox xenografts was smaller than that of Tet/pTRE/CNE2+Dox xenografts. In addition, Tet/pTRE-STGC3/CNE2+Dox xenografts showed an increase in the percentage of apoptotic cells, a decrease in Bcl-2 protein expression and an increase in Bax protein expression. A proteomic approach was used to assess the protein expression profile associated with STGC3-mediated apoptosis. Western blotting confirmed the differential up-regulation of prohibitin seen in proteomic analysis. These results indicate that overexpression of STGC3 inhibits xenograft growth in nude mice by enhancing apoptotic cell death through altered expression of apoptosis-related proteins such as Bcl-2, Bax and prohibitin. These data contribute to our understanding of the function of STGC3 in human nasopharyngeal carcinoma and provide new clues for investigating other STGC3-associated tumors.

STGC3 inhibits xenograft tumor growth of nasopharyngeal carcinoma cells by altering the expression of proteins associated with apoptosis

STGC3 is a potential tumor suppressor that inhibits the growth of the nasopharyngeal carcinoma cell line CNE2; the expression of this protein is reduced in nasopharyngeal carcinoma compared with normal nasopharyngeal tissue. In this study, we investigated the tumor-suppressing activity of STGC3 in nude mice injected subcutaneously with Tet/pTRE-STGC3/CNE2 cells. STGC3 expression was induced by the intraperitoneal injection of doxycycline (Dox). The volume mean of Tet/pTRE-STGC3/CNE2+Dox xenografts was smaller than that of Tet/pTRE/CNE2+Dox xenografts. In addition, Tet/pTRE-STGC3/CNE2+Dox xenografts showed an increase in the percentage of apoptotic cells, a decrease in Bcl-2 protein expression and an increase in Bax protein expression. A proteomic approach was used to assess the protein expression profile associated with STGC3-mediated apoptosis. Western blotting confirmed the differential up-regulation of prohibitin seen in proteomic analysis. These results indicate that overexpression of STGC3 inhibits xenograft growth in nude mice by enhancing apoptotic cell death through altered expression of apoptosis-related proteins such as Bcl-2, Bax and prohibitin. These data contribute to our understanding of the function of STGC3 in human nasopharyngeal carcinoma and provide new clues for investigating other STGC3-associated tumors.