Influence Of Hdm2 E3ligase Inhibitor On Protein Expression Of ASPPs Of Non-small Cell Lung Cancer

Objective:Tumor suppressor gene p53 plays an important role in the regulation of cells response to chemotherapeutic drug-induced DNA damage. ASPPs (apoptosis-stimulating protein of p53) is a new family of proteins which regulated proapoptotic function of p53, ASPP1 and ASPP2 can specifically stimulate proapoptosis function, iASPP exhibit inhibitory effect on the function. The oncoprotein Hdm2 E3ubiquitinates P53, resulting in the rapid degradation of p53 through the ubiquitin-proteasome pathway, hdm2-mediated p53 inactivation and degradation are considered resistance to chemotherapeutic agents of tumors. In the experiment non-small lung cancer cell lines A549 (p53 wild type) and NCI-157 (p53 mutation) were treated with proteasome inhibitors (Hdm2 E31igase inhibitor) to observe the influence of ubiquitination on expression of ASPPs and to discuss the relationship between ASPPS and the sensitivity of tumor cells on chemotherapy, especially DNA injuring chemotherapeutic agents-induced apoptosis.Methods:A549 and NCI-157 were treated with cisplatin (CDDP), pirarubicin (THP), Hdm2 E31igase inhibitor and combined treatment (cisplatin+protease inhibitor; THP+proteasome inhibitor). The effective concentrations of different DNA-damaging agents and the growth arrest of cell lines A549 and NCI-157 were detected by MTT assay and then calculation the proliferation inhibition rate of A549 and NCI-157 under three medicines after 24h; Western Blot are adopted to manifest the expression level of ASPPs in A549 and NCI-157 cell line treated for 24h; Immunoprecipitation assay were used to measure JNK/SAPK activity of A549 treated with CDDP or combined CDDP and Hdm2 E31igase inhibitor. Finally all the data were processed in SPSS software. Means was denoted with means±SD, and analyzed by variance analysis (ANOVA) statistical methods according to the features of different data. P<0.05 has statistical significance.Results:(1) Inhibitory effects on cell proliferation of Hdm2 E31igase inhibitor:The results of MTT assay showed that the proliferation of two types of cells was significantly inhibited by CDDP, THP (P<0.05). The inhibitory effect of Hdm2 E31igase inhibitor on proliferation of both cell lines was not significant (P>0.05), inhibition rate of proliferation is<11% the rate was not increased combined treatment with CDDP or THP (p> 0.05). (2) Influence of Hdm2 E31igase inhibitor on the protein expression of ASPPs family:The results of Western Blot showed that after the drug treatment for 24 hours expression of ASPP1 and ASSP2 of two cell lines was upregulated. The relative expression of ASPP1 of A549 cells treated with Hdm2 E31igase inhibitor was 0.887±0.970, higher than 0.760±0.80 of untreated A549 cells; the relative expression of ASPP2 of A549 cells treated with Hdm2 E31igase inhibitor was 0.968±0.654, higher than 0.544±0.67 of untreated A549 cells. The relative expression of ASPP1 of NCI-157 cells treated with Hdm2 E31igase inhibitor was 0.334±0.133, higher than 0.157±0.05 of untreated NCI-157; the relative expression of ASPP2 of NCI-157 cells treated with Hdm2 E31igase inhibitor was 0.391±0.091, higher than 0.239±0.09 of untreated NCI-157 cells.The expression of iASPP treated with Hdm2 E31igase inhibitor was different. The relative expression of iASPP of A549 cells treated with Hdm2 E31igase inhibitor was 0.55±0.211, lower than 0.157±0.05 of untreated A549; The relative expression of iASPP of NCI-157 cells treated with Hdm2 E31igase inhibitor was 1.459±0.9, higher than 1.188±0.755 of untreated NCI-157. (3) Influence of Hdm2 E31igase inhibitor on JNK/SPAK activity of A549 treated with CDDP:A549 cells treated with CDDP and CDDP+Hdm2 E3ligase inhibitor for 24 hours has higher expression of phosphorylated c-Jun than that of untreated cells, indicating higher JNK/SAPK activity of treated cells than untreated cells.Conclusion:Hdm2 E31igase inhibitor which inhibited proteosome mediated degradation of p53 increased the protein expression of ASPP1 and ASPP2 of A549 and NCI-157 cells. Changes of iASPP was different, in A549 with wild-type p53 its expression was downregulated, in NCI-157 with mutant-type p53 its expression was upregulated. Hdm2 E31igase inhibitor enhanced the activity of JNK/SAPK.