Restructuring Agkistrodon disintegrin Adinbitor human non-small cell lung cancer cell line A549
|School||Dalian Medical University|
|Course||Biochemistry and Molecular Biology|
|Keywords||Disintegrin Non-small cell lung cancer Tumor Necrosis Factor Nuclear factor NF-κB p65 subunit Interleukin-8|
Purpose: Lung cancer is currently the highest incidence and mortality rates of cancer, to radiotherapy and chemotherapy sensitivity are poor. Therefore, in vivo Looking efficiency, low toxicity of anticancer drugs have become resistant to cancer research and drug development an important part. Because most cell integrins (integrins) can identify RGD or KGD sequence, and this venom protein family by virtue of its molecular sequence RGD become extracellular matrix (extracellular matrix, ECM) and cell integrin binding strong competition between antagonist, inhibits platelet aggregation and angiogenesis, tumor metastasis and other functions. Adinbitor is our laboratory from China Lushun browed viper venom gland obtained by cloning of an RGD motif containing disintegrin, the experiment confirmed that it inhibits platelet aggregation and angiogenesis function, and has significantly inhibited The role of tumor formation. This study aimed to investigate the Adinbitor on human non-small cell lung cancer A549 cell proliferation, apoptosis, its research and development into new anticancer drugs provide a theoretical basis. Methods: The molecular weight used in the study of the disintegrin 9KD Adinbitor, histidine is the laboratory were purified by affinity chromatography high concentration of proteins. 1 using in vitro culture methods, using different concentrations of Adinbitor (0,1,2,3,4,5,6 μmol / L), and 10ng / L TNF-α, TNF-α (10ng / L) / Adinbitor (3μmol / L) the role of human non-small cell lung cancer A549 cells, the application of MTT (MTT) method for the determination Adinbitor and TNF-α on cell proliferation; 2. adopt 3μmol / L Adinbitor and 10ng / L TNF-α, TNF-α ( 10ng / L) / Adinbitor (3μmol / L) treated A549 cells, the application of hematoxylin - eosin (HE) staining method combines phase contrast microscope observation of morphological changes of apoptosis, and further by Hoechst staining and fluorescence microscopy Adinbitor and TNF -α on apoptosis of A549 cells; 3 detected by Western blot Adinbitor and TNF-α in A549 cells, IL-8, NF-κB p65 expression. Results: 1. By MTT assay, Adinbitor in a dose dependent manner, the proliferation of A549 cells, the inhibition of cell proliferation IC50 of 3.28μmol / L; 2. TNF-α can promote cell proliferation, and the TNF-α Adinbitor induced proliferation inhibition; 3 by phase contrast microscopy HE staining and Hoechst staining to Adinbitor promote the role of TNF-α-producing cells apoptosis; 4.Western blot method to detect the role of TNF-α and IL-8 cells NF-κB p65 expression was increased, while Adinbitor be reduced IL-8 and NF-κB p65 expression. Conclusion: In summary, the present study obtained by inducing the expression and purification of recombinant proteins that disintegrin Adinbitor, in vitro study its effect on human non-small cell lung cancer A549 role. Experimental results show that: Adinbitor A549 non-small cell lung cancer growth inhibition in a dose-dependent manner, TNF-α can induce proliferation of A549 cells, while Adinbitor against TNF-α-induced cell proliferation inhibition; TNF-α, IL role in cellular -8 expression, Adinbitor reduced TNF-α cells, the role of expression of IL-8; Adinbitor containing TNF-α promote apoptosis; TNF-α cells can increase the expression of NF-κB p65 and inhibit Adinbitor its expression. These results suggest that, Adinbitor inhibited TNF-α-induced proliferation of A549 cells probably by inhibition of IL-8 expression as may be the inhibition of apoptosis by inhibiting the expression of NF-κB p65 and biological effect. This is discussed Adinbitor on the regulation of signal transduction pathways provide an experimental basis, and will further explore the molecular mechanism of antitumor open up new ideas.