The Predictive Value Of RCC1 And P53 For The Effect Of Panobinostat And Cisplatin Combination Treatment In NSCLC

(Chapter 1)Cell Sensitivity to Cisplatin and the Combination of Cisplatin and Panobinostat in NSCLC cellsObjective:Determine cell sensitivity to Cispaltin and the combination of cisplatin and Panobinostat in 8 NSCLC cell lines.Methods:Eight NSCLC cell lines with different ERCC1 expression level and p53 status were treated with Cisplatin alone or Cisplatin combined with Panobinostat. The sensitivity of all cell lines to the drugs was analyzed by cell growth assay.Results:In the 8 NSCLC cell lines, we found ERCC1 low expression in cell line: A549, HCC827, NCI-H23, NCI-H441 andNCI-H1975 and high expression in the other three cell lines:NCI-H1299, NCI-H2172 and PC-14. In the 8 NSCLC cell lines,7 cell llines but not A549 showed resistance to cisplatin. When treat with the combination of cisplatin and panobinostat, an HDAC inhibitor, four out of 5 ERCC1 low expression cell lines showed increased sensitivity. While the other 3 cell lines with high ERCC1 expression are still resistant to the combination treatment.Conclusion:ERCC1 expression level alone cannot predict cisplatin effeicient in NSCLC cell lines. While Panobinostat can increase cell sensitivity in ERCCllow expression NSCLC cell lines.(Chapter 2)The predictive value of ERCC1 and p53 for the effect of panobinostat and cisplatin combination treatment in NSCLCObjective:To evaluate ERCC1 and p53 as the biomarkers to predict the effect of HDACis and Cisplatin combination in NSCLC treatment.Methods:We knockdown ERCC1 in 3 ERCC1 high expression cell lines and overexpression ERCC1 in ERCC1 low expression cells to investigate the predictive value of ERCC1 for cell sensitivity to Cisplatin and Panobinostat combined treatment. Confirm the importance of p53 satusin predicting the effect of the combination treatment through GOF mutant p53 KD and WT or GOF mutant p53 overexpress. In PC-14 cell line, double knockdown ERCC1 and GOF mutant p53can increase cell sensitivity to the combination treatment.Results:In the 3 ERCC1 high expression cell lines:NCI-H1299, NCI-H2172 and PC-14, knockdown ERCC1 can increase cell sensitivity to ciaplstin and panobinostat combination in NCI-H1299 and NCI-H2172cell lines.While in ERCC1 low expression cell lines HCC827 and NCI-H23, overexpress ERCC1 can decrease cell sensitivity to the combination treatment. Knockdown p53 in NCI-H1975, which habors GOF mutant p53 and double knockdown ERCC1 and p53 in PC-14+, which both have high ERCC1 and GOF mutant p53 can increase the sensitivity. Overexpress WT p53 in NCI-H2172, a p53 null cell line, can increase the sensitivity, while overexoress GOF mutant p53 can reverse the sensitization effect.Conclusion:ERCC1 and p53 could act as the biomarkers to predict the effect of HDACis and Cisplatin combination in NSCLC treatment.(Chapter 3)Panobinostat can increase cisplatin induced cell apoptosisObjective:Further investigate the mechanism of panobinostat sensitize cisplatin and deeply understand the molecular biology of the predictive effect of ERCC1 and p53.Methods:Finally, Apoptosis was analyzed by flow cytometry and apoptosis related genes expression was examined by western blot for p-p53 and cleaved-PARP and q-PCR for p21.Results:In the three ERCC1 low expression cell lines A549, HCC827 and NCI-H23, which also have WT or non-GOF mutant p53, the combination treatment of cisplatin and panobinostat can significantly increase p-p53, p21 and cleaved-PARP expression. Cell apoptosis analyzed by flow cytometry also showed increased AnnexinV positive/PI negative cells. In cell line而NCI-H1975 and PC-14, both habors GOF mutant p53, we can only detect increased p-p53 but not p21 and cleaved-PARP. In NCI-H1975cell line, knockdown GOF mutant p53 can increased cleaved-PARP expression and cell apoptosis. In PC-14 cell line, which both have high ERCC1 expression level and GOF mutant p53, double knockdown ERCC1 and p53 can increased cleaved-PARP expression and cell apoptosis.Results:Panobinostat can increase cell sensitivity to cisplatin in NSCLC cell lines through inducing cell apoptosis.Objective:To construct plasmid pT28a-HBVcAg-Mage A3 and optimize the condition of it’s expression and purification.Materials & Methods:Construct plasmid pT28a-HBVcAg-Mage A3 and explore the condition of it’s expression and purification by Nickel-affinity chromatography column and ultrafiltration.Results:We confirmed the success of construction of plasmid pT28a-HBVcAg-Mage A3 and optimization of it’s expression and purification. After the transformation of the plasmid into BL21 competent cell, we confirmed correct plasmid construction by plasmid extraction and DNA electrophoresis. Next we find that HBVcAg-Mage A3 fusion protein can efficiently expressed under the condition of an initial BL21 ODλ600 1.3-1.5,0.8-1.0mM IPTG, lOmM glucose and 100μg/ml kanamycin and following incubation on shaker (140 rpm) for 17 hours. SDS-page gel analysis confirmed the expressed HBVcAg-Mage A3 fusion protein was inclusion body. Then we purified HBVcAg-Mage A3 fusion protein and performed desalination by ultrafiltration and finally we test the concentration of target protein by SDS-page gel electrophoresis and BCA method.