Antitumor Of RAdinbitor, A Novel Disintegrin From The Snake Venom Of Gloydius Blomhoffi Brevicaudus

Objective: to research anti-tumor mechanism of rAdinbitor, so as to provide a theoritc basis of laboratory for clinical application in the future.Methods: The experiments included two parts. In the first part, the genetic engineering was employed to transform the gene of rAdinbitor derived from Gloydius blomhoffi brevicaudus in the Escherichia coli BL21 cells. The gene was induced and expressed. Then the cells were harvested and sonicated to get the supernatant. The purified rAdinbitor was obtained by His·Tag affinity chromatography. After getting the samples, in vivo experiments were performed to reveal the anti-tumor effects of rAdinbitor on melanoma B16 and pulmonary carcinoma LLC. The mice bearing cancers were employed to prove the anti-tumor mechanism of rAdinbitor. The models of mice bearing cancers were established by inoculating the tumor cell B16 and LL, respectively, and then the three doses of rAdinbitor were injected into subaxilla, and sodium chloride wase used as controlls. The experimental mice were sacrificed after 15 days and pick out of tumors, and weighed. The pathological sections were made to calculate the numbers of blood vessels in the tumor. In the second part of the experiments, the effect of rAdinbitor mutant RI(49)A(50)RGD→RPTRGD on FAK-Ras-MEK signaling pathway was examed.Results: The method of genetic engineering was utilized sucessfully to obtain a great quantity purified rAdinbitor, 3.42mg purified samples per liter of medium were obtained. In vivo experiments of mice proved that rAdinbitor could inhibit the growth and metastasis of B16 and LLC tumors. The three doses of rAdinbitor, 0.5mg, 1.25mg, and 5mg/ kg body weigh of mouse, could inhibit the growth of melanoma B16 tumors up to 40.0%, 78.8%, and 94.4%, respectively; pulmonary carcitoma LLC tumors up to 39.6%, 71.7%, and 94.3%, respectively. The pathological section displayed that rAdinbitor could inhibit the angiogenesis of the two groups of tumors, 45.9%, 71.6%, and 91.7% respectively for B16 tumor, and 44.0%, 70.3%, and 90.7% respectively for LLC tumor. The results of Western blotting showed that the mutant of rAdinbitor RPTRGD exhibited the characteristics of disintegrins, and could negatively regulate the activities of FAK-Ras-MEK signaling pathway by repressing the expression of FAK, Ras, MEK1/2, and ERK1/2. Meanwhile, the mutant could induce apoptosis of the neuroglioma C6 cells by induce the expression of caspase-3, the key enzyme of apoptosis.Conclusion: (1) rAdinbitor with character of disintegrins could inhibit angiogenesis of tumors B16 and LLC, and further inhibited the growth of these tumors. (2) The RPTRGD mutant of rAdinbitor with the characteristics of disintegrins could negatively regulate FAK-Ras-MEK signaling pathway, and induce apoptosis of neuroglioma C6 cells.