Antitumor Activity And Related Mechanisms Of Deguelin And Its Carbamate Derivative In Vitro And In Vivo

ObjectiveDeguelin,a rotenone?flavonoid?compound,is a potential natural broad-spectrum anti-tumor drug.It has been demonstrated that deguelin restrains tumors proliferation mainly through arresting cell cycle,inducing cell apoptosis,inhibiting angiogenesis,inhibiting cell migration and regulating metabolism.However,due to long-term or high-dose use of deguelin may cause damage to the nervous system,in order to reduce the side effects,our group has synthesized a variety of deguelin derivatives.Based on previous studies,we chose deguelin carbamate derivative to do further research.CCK-8 test,Cell scratch test,Flow cytometry with PI single staining and Annexin V-FITC/PI double staining experiment were used to evaluate and compare the effects of deguelin carbamate derivative and deguelin on cell proliferation,cell migration,cell cycle and apoptosis.RT-qPCR and Western Blot experiments were used to detect the regulatory effects of deguelin and its carbamate derivative on the Cyclin D-CDK4/6complex at the gene and protein level,respectively.Tumor xenograft experiment and HE staining were conducted to investigate the effect of deguelin and its carbamate derivative on the growth of transplanted tumor in nude mice.These studies are to provide new scientific basis for the anti-tumor activity and mechanism of deguelin and its carbamate derivatives.Results:?1?1.5625?M-50?M of deguelin and its carbamate derivative all had significant inhibitory effects on the proliferation of H1299,A549,MCF-7,Hela and PC-3 cells?P<0.05?.Deguelin and its carbamate derivative had the strongest inhibitory effect on the proliferation of H1299cells.?2?3.125?M-50?M deguelin and its carbamate derivative inhibited the healing ability of H1299cells and inhibited the migration of H1299 cells significantly?P<0.05?.?3?1?M,2?M,4?M of deguelin blocked H1299 cell cycle in G₁ phase,while 3?M,6?M,12?M of deguelin carbamate derivative not only increased cell ratio in G₁ phase significantly?P<0.05?,but also decreased the percentage of cells in S phase significantly at the same time?P<0.05?,but there was no significant change in cells in G₂/M phase?P>0.05?.That is,it could arrest cells in G₁/S phase.?4?Flow cytometry combined with Annexin V-FITC/PI double staining showed that deguelin and its carbamate derivative could induce apoptosis of H1299 cells.?5?RT-qPCR experiments showed that deguelin and its carbamate derivative can down-regulate the expression of CDK4,CDK6 and CCND1 genes significantly?P<0.05?.Western Blot experiments verified the RT-qPCR experimental results from the protein level,and preliminary indicated deguelin and its carbamate derivative may modulate the cell cycle of H1299 through the Cyclin D-CDK4/6 complex.?6?H1299 xenograft tumor model and HE staining experiments showed that deguelin and its carbamate derivative significantly inhibited the growth of transplanted tumors in H1299 nude mice?P<0.05?.Conclusions:?1?Deguelin and its carbamate derivative inhibited the proliferation of H1299,A549,MCF-7,Hela and PC-3 cells.?2?Deguelin and its carbamate derivative inhibited the migration,arrested the cell cycle and induced apoptosis in H1299 cells.?3?Deguelin and its carbamate derivatives may regulate cell cycle by down-regulating the expression of CDK4,CDK6 and Cyclin D1 genes and proteins in the cell cycle regulatory system,so as to achieve anti-tumor effects.?4?Deguelin and its carbamate derivative inhibited the growth of transplanted tumors in H1299nude mice.