Influence Of NF-κB Inhibitor (PDTC) On Protein Expression IASPP And JNK Of Non-small Cell Lung Cancer Cell

The lung cancer is one of most common malignant , lung cancer is currently one of the world's highest incidence and mortality of malignant tumors. Because of its insidious onset, highly malignant, approximately 80% of is at the time of the late stage patients in their treatment, due to tumor metastasis and multi-drug resistance, even with surgery, radiotherapy, chemotherapy and other comprehensive treatment, 5-year survival rate is less than 15%。Now research shows that the incidence, development, prognosis of NSCLC are closely related to many molecular events, nuclear factor NF-KB activation and the composition of the P53 tumor suppressor gene inactivation is the mutations and the development of lung cancer the most common molecular changes. Particularly, p53 is an important tumor suppressor gene, but in the wild-type p53 state, why would a tumor? ASPP (Apoptosis Stimulating Protein of p53) family, especially iASPP, induced apoptosis by p53 functional regulation to explain the problem. There are three members of the ASPP family: ASPPl, ASPP2 and iASPP, ASPPs contains C-terminal ankyrin repeats, SH3domain, proline-rich domain (Proline-rich region) . The ASPPl and ASPP2 combine with the P53 can activate the tumor suppressor function of P53, while P53 occurred combined with iASPP the effect is inhibition of P53 tumor suppressor function, it can inhibit apoptosis, because iASPP, ASPP1 and ASPP2 have the same regions, this regions can be combined with p53, so it can be competitive with ASPP1 and ASPP2 with the p53 binding, thereby inhibiting the p53 tumor suppressor function.NF-κB and inflammatory, immune response, trauma, cell proliferation, apoptosis, and embryonic development and other important events are closely linked, are now known, including five members of his family, namely RelA (p65), RelB, c-Rel, p50 (NF-κB1) and p52 (NF-κB2), were expressed in all cells. NF-κB is a more dominant gene transcription factor, and transcription of multiple genes related to, including participation in the development of tumor genes and factors. Expression of the activation of NF-κB exist in a variety of tumors, NF-κB p65 is the activation of NF-κB components. So far, NF-κB is activated througth 3 species pathway, mostly by the main process of phosphorylation, ubiquitination and so on, will be resting state NF-κkB/κB complex processing or separation, NLS of NF-κB can be exposured, then the activation of NF-κB making it located in the nucleus, with its combination of corresponding target genes play a regulatory function of gene transcription.Studies have shown that iASPP (RAl) can unit with NF2kappaB p65 (RelA) and inhibit the transcriptional activity of NFkappaB,highly expressed in the heart, placenta, prostate and other tissues, lowly expressed in liver tissue, testis tissue and leukocytes . However, the studies on protein expression of iASPP of non-small cell lung cancer (NSCLC) have been rarely reported.Objective: To observe the influence of NF-κB inhibitor (PDTC) on protein expression of ASPPs (iASPP)﹑JNK of Non-small cell lung cancer cell lines A549 (wild-type p53)and NCI-157(mutation p53) and its synergistic anticancer effect with chemotherapeutic drugs (CDDP).Methods: Human non-small cell lung cancer A549 and NCI-157 were treated with drugs (cisplatin, CDDP; Ammonium Pyrrolidine dithiocarbam ate PDTC) and combined treatment (PDTC+CDDP).the minimum effective concentration of different 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 two drugs after 24h; the initial detection of the drugs on the A549 and NCI157 cell growth inhibition; Western blot was used to detect the expression of iASPP of single agent and combination treatment after 24; immunoprecipitation assay were used to measure JNK/SAPK activity of A549 and NCI157 treated with PDTC or combined CDDP. Experimental data using SPSS statistical software, mean was denoted with mean±SD and analyzed by variance (ANOVA), P <0.05 was considered statistically significant.Results:1 The results of MTT showed that the proliferation of two types of cells was significantly inhibited by CDDP (P <0.05).the inhibitory effect of PDTC on proliferation of A549 cell lines was not significant (P> 0.05), the inhibitory effect of PDTC on proliferation of NCI-157 cell lines was significant (P <0.05), but Inhibition of PDTC to NCI-157 is useless good dose dependent manner.the inhibitory effect of PDTC(100μmol/L, 50μmol/L, 25μmol/L, 12.5μmol/L) combined CDDP (6μg/ml) for two A549 and NCI-157 of cell lines was not significant (P <0.05).2 The results of Western blot were treated with PDTC for 24 hours, the protein expression of iASPP of two cell lines A549 and NCI-157 in the treatment group than in control group, protein expression of iASPP in A549 was 0.299±0.093, control group was 0.208±0.082, and protein expression of iASPP in the NCI-157 was 4.078±0.864, control group was 1.685±0.19.3. Immunoprecipitation results show that, A549 and NCI-157 cells was treated with PDTC, CDDP, and PDTC + CDDP, these three drugs can activate the activity of JNK / SAPK in A549, the expression of phosphorylated c-Jun was in higher, but in the NCI-157 JNK / SAPK activity can be activated only by CDDP, PDTC and PDTC + CDDP can reduce activation JNK / SAPK to some extent.Conclusion:1. In our study, we select two cell lines A549 and NCI-157, MTT shows with PDTC, CDDP, and after combination therapy (cisplatin concentration fixed), PDTC and CDDP have synergy can increase chemotherapy (CDDP) sensitivity of non-small cell lung cancer .2.Nuclear factor kappaB(NF-κB) inhibitor (pyrrolidine dithiocarbamate, PDTC) can increase the expression of protein iASPP A549 and NCI-157.3. The experimental drugs(PDTC)was used in the two cell A549 and NCI-157 ,the activity of JNK are different.