Vascular Endothelial Growth Factorl65-regulated Nasopharyngeal Carcinoma Cell Lines Invasion and Migration Involve Expression and Activation of Matrix Metalloproteinase-2

The effect of vascular endothelial growth factor （VEGF） overexpression on matrix metalloproteinase-2 （MMP-2） in nasopharyngeal carcinoma （NPC） cells in vitro and the possible mechanism involved were investigated, and the correlation between the expression of VEGF and MMP-2 in NPC evaluated. The NPC cells were transfected with PAd-trackVEGF165 plasmid. The expression levels of VEGF and MMP-2 mRNA and protein in NPC cells were detected by semi-quantitative RT-PCR and Western blot respectively. It was found that the expression of VEGF and MMP-2 mRNA and protein was significantly increased in NPC cells after transfection of VEGF 165. It was concluded that the expression of VEGF was correlated to the in vitro invasion of NPC cells, and the induction of MMP-2 by VEGF was a key process of NPC cell invasion.

Research on cell cycle retardation, apoptosis and the expression of antioncogene p57^(Kip2) by radioactive rays in nasopharyngeal carcinoma cell line in vitro

Objective: To study cell cycle retardation, apoptosis and the expression of antioncogene p57kip2 by radioactive rays in nasopharyngeal carcinoma cells. Methods: Cell cycle retardation, apoptosis and cell survival rate induced by radioac- tive rays were tested by the methods of flow cytometry and MTT method. The expression of antioncogene p57kip2 was detected by immunohistochemistry and Western blot. Results: After irradiation, G1 phase had no obvious retardation, S phase showed transient delay. There was a positive correlation between irradiation dosage and retardation strength in G2/M phase (P < 0.01). Peak value appeared at 24 h after 12 Gy irradiation, then decreased. There was a positive correlation between apop- tosis incidence and irradiation dosage or after-irradiation time extention (P < 0.01). There was a negative correlation between cell survival rate and irradiation dosage or apoptosis incidence (P < 0.01). The expression of p57kip2 protein was up-regulated along with the prolongation of time and dosage after irradiation (P < 0.01). Conclusion: G2/M phase arrest, apoptosis and the up-regulation of the expression of p57kip2 protein all can reflect predict the radiosensitivity of nasopharyngeal carcinoma cells.

c-myc gene inactivation during induction of nasopharyngeal carcinoma cells with retinoic acid

To investigate the effects of retinoic acid (RA) on the growth, morphology, oncogene expression and regulation of nasopharyngeal carcinoma cells Methods Nasopharyngeal carcinoma cell line (HNE 1) was induced by RA The RA-treated and control cells were established and cellular morphology and growth patterns were defined Oncogene expression and regulation were detected by Northern hybridization and DNase-Ⅰ hypersensitive site analysis Results RA markedly inhibited cell growth The growth of HNE 1 cells was reduced to 50% of the control level on the 4th day of RA (10 -4 mol/L) treatment After 4 days of treatment, the rapidly growing polygonal cells were reversed into a slow growing phenotype, with flattened morphology similar to fibroblast-like cells Northern hybridization showed that c-myc and c-Ha-ras expression was high in HNE 1 cells and undetectable in normal blood cells c-myc was down-regulated at 48*!h of RA treatment In contrast, the c-Ha-ras was not affected DNase Ⅰ hypersensitive site analysis detected changes in the regulatory elements of c-myc and c-Ha-ras genes 5 hypersensitive sites were found in the c-myc of HNE 1 cells, while 3 hypersensitive sites disappeared upon HNE 1 induction However, only 1 hypersensitive site was found in c-Ha-ras of RA treated cells and controls In normal peripheral white blood cells, no DNase Ⅰ hypersensitive sites were found in the inactive c-myc and c-Ha-ras gene Conclusion RA can induce differentiation in a nasopharyngeal carcinoma cell line at high concentration of RA; HNE 1 shows some similar patterns of DNase Ⅰ hypersensitive sites with the common one in other types of cells expressing c-myc The repression of c-myc expression with induction is accompanied by the loss of 3 DNase-Ⅰ hypersensitive sites; c-myc has more than one inactive conformation

Yi Qi Jie Du Formula and Salinomycin Combination Treatment Mediates Nasopharyngeal Carcinoma Stem Cell Proliferation,Migration and Apoptosis via CD44/Ras Signaling Pathway

Objective To assess the effects of Yi Qi Jie Du Formula(YQJDF)combined with salinomycin(SAL)on nasopharyngeal carcinoma stem cells(NPC-SCs)and investigate the underlying molecular mechanisms.Methods Cell counting methods,the CCK-8 assay,transwell migration assay and JC-1 staining,were used to observe the effects of the combination on the proliferation,migration and apoptosis of NPC stem cells,respectively.Western blot was used to detect the levels of protein in NPC-SCs.Results YQJDF combined with SAL had a synergistic effect on the inhibition of proliferation and migration and induction of NPCSC apoptosis.Mechanistically,YQJDF combined with SAL synergistically upregulated the levels of apoptotic proteins,including cleaved Caspase-3,cleaved Caspase-7 and cleaved Caspase-9.Moreover,YQJDF combined with SAL synergistically decreased the levels of CD44,p-c-Src,Ras,p-PKCδ,p-MEK,p-c-Raf,p-ERK1/2 and p-AKT proteins.Conclusions The combination of YQJDF and SAL has a synergistic effect on the inhibition of NPC-SC proliferation and migration and induction of apoptosis,which may be closely related to the downregulation of the CD44/Ras signaling pathway.

Induction of cytotoxic T lymphocyte respones in vivo after immunotherapy with dendritic cells in patients with nasopharyngeal carcinoma

The aim of the present study was to determine the efficacy of immunotherapy with dendritic cells to elicit EBV-specific CTL-immunity in advanced cases of EBV-positive patients with nasopharyngeal carcinoma （NPC） and to determine the safety and toxicity of this preparation. Nine cases of histologically confirmed patients with NPC undergoing treatment with radiological therapy were enrolled in this study. Dendritic cells, generated in vitro from blood monocytes of patients were cultured and matured with cytokines and then infected with recombinant adenovims vaccine containing EBV-latent membrane protein-2 （Ad-LMP2）. On 9 days＇ cultivation of cells, the matured DCs were harvested, irradiated with 60^Co and then injected intradermally to patients with NPC. The injections were performed 3 times totally. After immunization, the CTL responses were assayed by means of cytotoxicity and epitope-specific IFN-γ production. The results of this trial showed that all patients could tolerate this kind of treatment without any side effect, during which marked increase of LMP2-specific CIL-responses could be demonstrated in 5 patients of this group. And the level of IgA/VCA antibody decreased in 8 of 9 patients, thus accounting for a better prognosis for these patients. All patients will be followed up for another one year. At least, the present work shows that intradermal vaccination with autologons DCs infected with recombinant Ad-LMP2 adenovirus is a safe procedure in NPC patients, in which this procedure can enhance the LMP2-specific CTL responses in patients. These data are encouraging to develop more effective vaccine strategies for the treatment of nasopharyngeal carcinoma.

CONDITIONED MEDIUM OF HUMAN NASOPHARYNGEAL CARCINOMA EPITHELIOID CELL LINE CNE_(1) CONTAINED THE ACTIVITIES OF TRANSFORMED GROWTH-INHIBITING FACTORS

The hormone defined serum free conditioned medium (SFCM) of human nasopharyngeal carcinoma epithelioid cell line (CNE1) was assayed by both the 3H-thymidine incorporation test and the soft agar test. It was found that the SFCM stimulated the growth of long-term serum-free cultured CNE4 cells in ac-cordence with the fact that the growth rate of long-term serum-free cultured CNE1 cells was directly proportional to the plating density. Alternatively 5% SFCM inhibited the growth of short-term serum-free cultured CNE4 cells by 51% in which the indicator cell remained the responsiveness state of growing in the serum-supplemented medium to the effector of interest. Furthermore, SFCM resulted in the inhibition of anchorage-independent growth of CNE4 cells and A431 cells. Also in soft agar test. SFCM reduced the colony formation of NRK(?),9F cells in the presence of EGF or EGF plus TGF-β. These finding suggested that CNE4 secreted autocrine growth stimulating factor(s) and growth inhibiting factor(s) in the serum-free medium, the latter strongly reverse malignant phenotypes of CNE4 and A431 cells in serum-supplemented surrounding.

Role of JNK signaling pathway in sensitivity to radiotherapy of nasopharyngeal carcinoma

Objective:The aim of the study was to investigate the effect of c-Jun N-terminal protein kinase(JNK) signaling pathway on influencing the sensitivity to radiotherapy of human nasopharyngeal carcinoma CNE cells.Methods:Human nasopharyngeal carcinoma CNE multicellular spheroids(MCS) were constructed with three dimensional cell culture methods.Western blot was employed to analyze the activity of JNK signaling pathway in MCS after X-ray irradiation,and the expression of caspase-3 protein before and after using SP600125(a special inhibitor of JNK).X-ray induced cell apoptosis in MCS before and after treated with SP600125 were detected by TUNEL.Results:The level of JNK phosphorylation in MCS was a dynamic course after radiation,and there was a phosphorylation peaks at 2 h later,the apoptotic rate of MCS(P < 0.05) and the expression of caspase-3 protein(P < 0.05) were significantly increased after treated with SP600125.Conclusion:The transient activation of JNK played a important role in sensitivity to radiotherapy of CNE MCS via mediating survival signals,blocking this pathway accelerate cell apoptosis,which may be related to the increased expression of caspase-3.