Synergistic Effects Of Astragaloside Ⅳ And Cisplatin On Sensitizing In Human Non-small Lung Cancer Cell Lines

Background and Specific Aims:Lung cancer is the world’s highest rates of malignant tumor, chemotherapy is one of the main means of treatment of lung cancer, but over the past 25 years the 5-year survival rate still not see increased significantly, about 15%. The multi-drug resistance (MDR) is one of the main causes of lung cancer chemotherapy failure. Cisplatin(DDP) in lung cancer chemotherapy is an effective and widely used first-line drugs, is a kind of cell cycle non-specific cytotoxic drugs. Now think cancer cell apoptosis. DNA damage repair process of shear increases, and regulate the regulation of signaling pathways related protein expression changes, drug intake reduce lead to reduced CDDP concentration in cells, etc. May be associated with cisplatin resistance, lung cancer cells, however, the drug resistance mechanism has not been fully elucidated.Ras genes located in the upstream of PI3K/AKT pathway, and PI3K/AKT pathway is the main resistance to apoptosis pathway. The study found that the AKT1 expression and gene amplification is closely related to the lung cancer cells to cisplatin resistance, can inhibit the expression of AKT1 reverse cisplatin resistance.Study found B7-H3 is highly expressed in a variety of malignant tumors, and thus is more and more attention, the study found that B7-H3 is by some mechanisms involved in regulating the tumor promoting apoptosis or antiapoptotic genes, that may be some new diagnosis of tumor markers and potential therapeutic targets.Astragalus membranaceus methyl glucoside (Astragaloside IV. the AS-IV) is one of the major components of radix astragali. Studies have shown that the AS-IV by regulatory T cells and cytotoxic T lymphocytes and PKC alpha ERK1/2-the nf-kappa B signaling pathway inhibition of lung cancer cell migration and invasion.Research purpose is to (1) by comparing the radix astragali oral liquid of adjuvant chemotherapy and the curative effect of conventional chemotherapy in treatment of elderly patients with lung cancer and the influence on the immune function, so as to radix astragali oral liquid of adjuvant chemotherapy in the treatment of elderly lung cancer to provide a certain basis; (2) the research through the detection of B7-H3 expression in lung cancer cell line, analysis of B7-H3 effect on lung cancer cell line; (3) the CCK 8 evaluate different concentration astragalus methyl glucoside in combination with cisplatin ACTS on the A549/DDP cells, observe the A549/DDP cell proliferation; (4) after CCK siRNAB7-H3-8 method, astragalus membranaceus methyl glucoside in combination with cisplatin effects on cell proliferation; (5) to investigate the drug resistance of lung cancer cells in B7-H3 and interaction between Ras/AKT signal pathway.Research methods:1, through comparing the radix astragali oral liquid adjuvant chemotherapy and conventional chemotherapy in elderly patients with lung cancer RR, the comparison of DCR and productivity, and peripheral blood of CD3+, CD4+, CD8+ and NK lymphocyte change;2, by using immunohistochemical method to detect NSCLC tumor tissue, the tissue adjacent to carcinoma and benign lesion in the expression of B7-H3, and collect the patients clinical data, analysis of B7-H3 its relationship with clinical parameters; QRT PCR and protein imprinting method determination of B7-H3 changes in different lung cancer cell line;3, CCK 8 method to evaluate the AS-IV of A549 and A549/DDP proliferation inhibition condition; Evaluation of cisplatin resistance ratio and toxic dose; And AS to IV associated with cisplatin, one of the best dose;4, after siRNAB7-H3, the AS-IV, cisplatin and co-ordinated intervention A549/ DDP cell proliferation inhibition;5, After drug intervention in different cell group, protein imprinting method B7-H3, Ras, pAKT1 protein changes.Results:1, according to the results, compared two groups of patients with RR, DCR, there was no significant difference (P>0.05). The experiment Group of patients 6 months and 12 months and 18 months of survival rates were significantly higher than that of control group, with significant difference (P< 0.05). Experimental CD3+, CD4+’ CD8+ and NK cells activity significantly higher than the control group, with significant difference (P< 0.05).2, the study found that B7-H3 expressed in A549 and SPAC-1 is low, expressed in H466, H1299 and H460 is higher. Immunohistochemical detection of patients with NSCLC group found that B7-H3 expression within the cell membrane or within the cytoplasm or both are expressed. B7-H3 positive expression rate higher than that of benign lesions in the lung cancer tissues and tissue adjacent to carcinoma, benign lesion are tissue adjacent to carcinoma express high quantity. The expression of B7-H3 intensity is closely related to the degree of differentiation, lymph node metastasis, and may be associated with progress in non-small cell lung cancer;3, this experiment human lung adenocarcinoma A549 by different concentration of the AS-IV intervention after 48 h, cell proliferation inhibition rate increased with the concentration of the AS-IV. A549 cell proliferation inhibition rate increases; A549/DDP by different concentration of the AS-IV intervention after 48 h, cell proliferation inhibition rate increased with the concentration of the AS-IV,12 ng/ml. 24 ng/ml compared with control group, the statistical significance, P<0.01; DDP resistance ratio of 14.14 times, highly resistant cells:12 ng/joint DDP0 mlAS to IV. 625 ug/mL. synergy effect is best;4, B7-H3 expression by inhibiting the A549/DDP cell proliferation; 4, B7-H3 expression by joint AS-IV. DDP very significantly inhibited A549/DDP cell proliferation;5, after the inhibitory effect of B7-H3 joint AS-IV B7-H3, Ras, pAKT1 expression had the greatest reduction, hint AS to IV may inhibit B7-H3 and through B7-H3/Ras/pAKT1, inhibiting cell antiapoptotic, make its produce sensitization effect.Conclution:Research shows that 1, B7-H3 in NSCLC tissues and the expression of lung cancer cells; 2, the AS-IV has inhibitory effect to the lung cancer cells, showed a trend of concentration; 3, the AS-IV combined cisplatin can increase the rate of tumor cell inhibition; 4, the inhibition of B7-H3 can inhibit the activation of the Ras/pAKT1; 5, the AS-IV can be adjusted by B7-H3, inhibition of the Ras/pAKT1 activation, promoted the cisplatin chemotherapy sensitivity.