The Inhibitory Effects Of Adinbitor, A Recombinant Disintegrin From Gloydius Blomhoffi Brevicaudus On Human Nonsmall Cell Lung Cancer A549 Cell

Objectives: The nonsmall cell lung cancer, a highest malignant cancer, is one of the major causes of morbidity and mortality with poor sensitivities to radiation and chemotherapy. Therefore, searching antitumor drugs with high efficiency and low toxicity from organisms is a very important part of new drug development.Disintegrins are a group of polypeptides found in snake venom of viperine family and Agkistrodon family maily and leeches toxins [1], Rich of cysteines and hydrophilic Arg -Gly - Asp (RGD) or Lys - Gly– Asp (KGD) motif are the typical characteristics of disintegrins. Most disintegrins are single peptides with molecular weight of 5 ~ 9kD [2]. Because integrins in most cells can recognize RGD or KGD sequence, disintegrins based on their RGD or KGD motif can be strong inhibitors for competitively blocking the interaction between extracellular matrix (ECM) and target cells. This mechanism indicates a direction for developing new anti-cancer drugs. In this paper, the in vitro effects of recombinant disintegrin Adinbitor on the proliferation and apoptosis of nonsmall cell lung cancer cell A549 and its molecular mechanisms were investigated.Methods: The 9KD recombinant Adinbitor used in this study was purified with His-affinity chromatography and stored in -20°C freezer. 1. the nonsmall cell lung cancer A549 cells were incubated with different concentrations of 0μmol/L,1μmol/L, 2μmol/L,3μmol/L,4μmol/L,5μmol/L,and 6μmol/L Adinbitor, or 10ng/L TNF-α, or TNF-α/Adinbitor. The effect of Adinbitor on the cell proliferation was detected by using MTT. The A549 cells were incubated with 3μmol/L Adinbitor and 10ng/L TNF-α, or T 10ng/L TNF-α/3μmol/L Adinbitor , the apoptonic effects of Adinbitor and TNF-αon A549 cells were morphologically detected by Using hematoxylin-eosin red (HE) staining and combined with phase-contrast microscope, further using Hoechst staining combined with fluorescence microscope.3. The Western blotting was emplied to determine the influence of Adinbitor and TNF-αon expression of IL-8 and NF-κB p65 in A549 cells.Results: 1. By MTT detection, Adinbitor could inhibit the proliferation of A549 cells in the dose-dependent manner, the IC50 was 3.28μmol/L; 2. TNF-αcould promote the cell proliferation, and that could be inhibited by adding Adinbitor to the culture medium; 3. Through the phase-contrast microscope, HE staining and Hoechst staining, it was found that Adinbitor could promote the apoptosis of TNF-αactivated A549 cells; 4. Western blotting showed that TNF-αcould increase IL - 8 and NF-κB p65 expression in the cells, in contrast, Adinbitor could down regulate IL-8 and NF-κB p65 expression.Conclusion: In this study, the effects of purified recombinant disintegrin Adinbitor on human nonsmall cell lung cancer A549 cells were detected. The results indicated that Adinbitor could inhibit the proliferation of A549 cells in a dose-dependent manner. Adinbitor also inhibited TNF-αinduced cell proliferation. TNF-αcould induce IL-8 expression, and Adinbitor could down regulate TNF-αinduced this expression and promote apoptosis. TNF-αwas able to increase NF-κB p65 expression, in contrast, Adinbitor could inhibit this expression. These results suggested that the inhibitory effects of Adinbitor on TNF-αindeced A549 cell proliferation might be practiced by repressing IL-8 expression. The inhibitory effect of TNF-αmight be carried out by inhibiting NF-κB p65 expression. These results provided a basic data for further studying the regulatory effects of Adinbitor on signaling pathway, and for further opening the thought to investigate the molecular mechanism of anti-tumor of Adinbitor.