| Charpter1The correlation of LMP1expression and TRAIL sensitivity in nasopharyngeal carcinoma cellsObjective:To evaluate the expression of LMP1and TRAIL sensitivity in nasopharyngeal carcinoma (NPC) cells, and correlate LMP1expression with TRAIL resistance in NPC cells.Methods:LMP1protein and mRNA expression in3NPC cell lines (CNE-1, CNE-2, C666-1) was analyzed by RT-PCR and western blot. The TRAIL sensitivity of3NPC cell lines was analyze by MTT and flow cytometric analysis.Results:TRAIL inhibited cell growth and induced cell apoptosis in time-dependent and does-dependent manner. CNE-1was the most TRAIL-sensitive cell and C666-1was TRAIL-resistant cell. The relative mRNA expressions of CNE-1, CNE-2and C666-1were0.27±0.02,0.52±0.02,1.44±0.12, respectively. The relative protein expressions of CNE-1, CNE-2and C666-1were0.29±0.02.0.49±0.02.0.95±0.06, respectively. The LMP1expression in C666-1was highest, while which in CNE-1was lowest.Conclusion:TRAIL could induce NPC cell apoptosis. TRAIL-sensitivity in NPC cells may negative correlative with LMP1expression. The higher LMP1expresses, the more resistant to TRAIL NPC cell shows. This study demonstrated that LMP1may be the anti-apoptosis factor which effects the TRAIL-sensitivity.Charpter2Investigation of the regulation of LMP1on TRAIL resistance of NPC cells in vitroObjective:Some researches showed that LMP1could regulate apoptosis of cells. Our previous study has demonstrated that TRAIL-sensitivity in NPC cells may negative correlative with LMP1expression. In this part of study, we up-regulated the expression of LMP1in NPC cells. Then we observed the effect of LMP1over-expression on TRAIL-induced apoptosis and TRAIL-activated apoptotic pathway.Methods:We up-regulated LMP1expression in CNE-1which was TRAIL-resistant LMP1low expressive NPC cell by liposome. The efficiency of transfection was observed by fluorescence microscope. G418screened the stable transfected cells CNE-1-LMP1, and the expression of LMP1was analyzed by RT-PCR and western blot. The TRAIL sensitivity of CNE-1-LMP1and CNE-1was analyze by MTT and flow cytometric analysis. The expression of death receptor (DR), activation of apoptotic pathway and mitochondrial transmembrane potential were analyzed by flow cytometric analysis, western blot and JC-1assessment, respectively.Results:The pGL6-LMP1transfection up-regulated the expression of LMP1in CNE-1, and the stable transfective cell CNE-1-LMP1was constructed. The cytotoxicity and apoptosis-induced effect of TRAIL to CNE-1-LMP1were weaker than CNE-1and the empty vector control cell CNE-1-pGL6. However, there was no significant difference in expression of DR4and DR5among three cell lines (P>0.05). Moreover, after TRAIL (100ng/ml) administration for2,4,6,12hours, the expressions of caspase-8p43/p41, p18and caspase-3p17, p10in CNE-1were higher than CNE-1-LMP1. However, there was no significant difference in expression of tBid and caspase-9p35between this two cell lines. JC-1assessment showed that TRAIL administration could not affect the mitochondrial transmembrane potential and there was no significant difference in mitochondrial transmembrane potential between CNE-1and CNE-1-LMP1.Conclusion:LMP1induced TRAIL-resistance in NPC cell through inhibiting cell extrinsic apoptotic pathway.Charpter3LMP1induced TRAIL-resistance in NPC cell through activating PI3K/Akt pathwayObjective:Some researches demonstrate that activation of PI3K/Akt pathway is an important mechanism of TRAIL resistance. In this part, we explored that over-expression of LMP1could activate PI3K/Akt pathway, and we further confirmed that LMP1induced TRAIL-resistance in NPC cell through activating PI3K/Akt pathwayMethods:After the LMP1was up-regulated, the Akt and p-Akt (?)pressions were detected by western blot. The co-location of LMP1and p-Akt was detected by double immunofluorescence. We inhibited PI3K/Akt pathway in CNE-1and CNE-1-LMP1. Then the TRAIL sensitivity of CNE-1-LMP1and CNE-1was analyze by MTT and flow cytometric analysis. Activation of cell extrinsic apoptotic pathway was analyzed by western blot.Results:The expression of p-Akt in CNE-1-LMP1was higher than CNE-1. Double immunofluorescence showed that after LMP1up-regulated, p-Akt was trans-located from cytoplasm to membrane. With1μmol/ml LY294002pre-treatment for8hours, p-Akt expression was down-regulated, and there was no significant difference between CNE-1and CNE-1-LMP1(P>0.05). Moreover, the cytotoxicity and apoptosis-induced effect of TRAIL were enhanced and there was no significant difference between CNE-1and CNE-1-LMP1(P>0.05). After TRAIL (100ng/ml) administration for2,4,6,12hours, the expressions of caspase-8p43/p41, p18and caspase-3p17, p10in CNE-1-LMP1pre-treated by LY294002were higher than CNE-1-LMP1without LY294002pre-treatment.Conclusion:LMP1induced TRAIL-resistance in NPC cell through activating PI3K/Akt pathway, suggesting that therapy targeting PI3K/Akt pathway could reverse TRAIL-resistance in NPCCharpter4Investigation of the role of LMP1on TRAIL resistance of NPC cells in vivoObjective:To further confirm the above results we obtained in vivo, and initially explore the mechanisms of LMP1-induced TRAIL resistance in NPC cells by analysis of xenografted tumor tissues from nude mouse.Methods:CNE-1, CNE-1-LMP1were used to establish xenograft mouse model with different LMP1expression level. The xenograft mouse model was treated with TRAIL. The tumor size measurement and hematoxylin-eosin staining were applied to evaluate xenografted tumor formation and growth. Western blot was used to investigate the protein expression of LMP1. TUNEL assessment was used to detect the apoptotic index. At last, rumor growth inhibition rate of TRAIL was analyzed.Results:CNE-1, CNE-1-LMP1were successfully utilized to establish LMP1negative expressive and LMP1over-expressive xenograft mouse model. Hematoxylin-eosin staining confirmed that the tumor formation rate was100%. Western blot showed that LMP1protein expression in CNE-1-LMP1xenografted tumor was higher than CNE-1xenografted tumors (P<0.05). There was no significant difference in tumor volume, tumor weight and apoptotic index between two control group (P>0.05). The tumor volume, tumor weight in TRAIL treatment group were higher than the relative control group. However, the apoptotic index in TRAIL treatment group were lower than the relative control group(P<0.05). The tumor volume, tumor weight in CNE-1TRAIL treatment group were lower than CNE-1-LMP1TRAIL treatment group. However, the apoptotic index in CNE-1TRAIL treatment group were lower than CNE-1-LMP1TRAIL treatment group (P<0.05).Conclusion:TRAIL can induce NPC cell apoptosis in vivo, and inhibit the tumor growth. However, LMP1can induce NPC cell TRAIL resistance in vivo. |
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