The Study Of The Influence Of Withaferin A And Its Anologs On The Biological Effects Of Human Pancreatic Cancer Cells And The Preliminary Study Of Its Pharmacological Mechanisms

Part I the influnce of Withaferin A on the biological effects of pancreatic cancer cellsObjective:To study the influnce of withaferin A (WA), the effective component extracted isolated from Withania somnifera on the biological effects of human pancreatic cancer cellsMethods:To observe the influence of WA for pancreatic cancer cell morphology by inverted microscope; We used Annexin V/PI double staining flow cytometry to detected Cell apoptosis. CCK-8 assay was performed to detect cell viability and drug fast; Scratch test and transwell assay were used to detect cell migration and invation; CM-H2DCFDA fluorescent probe were used to confirm the reactive oxygen species (ROS) production; Using westren blot to detect the expression of apoptosis related proteins; Finally, subcutaneous implanted tumor in nude mice was established to observe proliferation in vivo using WA or blank.Result:CCK-8 assay and subcutaneous implanted tumor in nude mice assay indicated that WA can on proliferation inhibition of pancreatic cancer cells in vivo and in vitro, Scratch test and transwell assay indicated that WA can significantly inhibit the invasion and migration of pancreatic cancer cells in vitro, Western Blot showed that WA can cause the Caspase family protein increases, through the mitochondrial membrane potential decreased to activated ROS high levels to induce cell apoptosis; WA block the cell cycle in G2/M phase. In vivo experiment found that WA can result in the nude mouse model with hepatic and renal fibrosis.Conclusion:WA can on proliferation inhibition of pancreatic cancer cells in vivo and in vitro, and can significantly inhibit the invasion and metastasis of pancreatic cancer cells in vitro, WA can activate the Caspase family proteins induce cell apoptosis; WA block the cell cycle in G2/M phase. In vivo experiment found that WA the nude mouse model has obvious adverse reactions such as liver toxicity and kidney toxicity.Part ? the influnce of WA and its chemical formula space structure modification analogs on the biological effects of pancreatic cancer cellsObjective:To study the influnce on the biological effects of human pancreatic cancer cells of WA and its analogs, the lipidosome of chemical formula space structure modification.Methods:Cooperation with pharmaceutical college of HUST to develop WA with spatial structure modification, using hydrogen pectrum to examine chemical formula. To observe the influence of WA and analogs for pancreatic cancer cell morphology by inverted microscope; We used Annexin V/PI double staining flow cytometry to detected Cell apoptosis.CCK-8 assay was performed to detect cell viability and drug fast; Scratch test and transwell assay were used to detect cell migration and invation; CM-H2DCFDA fluorescent probe were used to confirm the reactive oxygen species (ROS) production; Using westren blot to detect the expression of apoptosis related proteins; Finally, subcutaneous implanted tumor in nude mice was established to observe proliferation and toxicity in liver and kidney in vivo using WA or anologs. Using western blot to detect extract protein detecting the change of the key protein from the tumor in vivo.Result:Compare with analogs, WA has a stronger inhibition on proliferation of pancreatic cancer cells in vivo and in vitro,at the same time scratch test and transwell assay indicated that analogs can a stronger inhibition on the invasion and migration of pancreatic cancer cells in vitro, Western Blot showed that both can cause the Caspase family protein increases, flow cytometry showed to have effects on cell apoptosis; In vivo experiment found that anologs can result in the nude mouse model with weaker hepatic and renal fibrosis, compairing with a lower inhibition on proliferation. Both of them can activate autophagy pathway to inhibit of tumor on the biological effects.Conclusion:WA compared its analogs in a stronger inhibitionon proliferation of pancreatic cancer cells in vivo and in vitro,at the same time scratch test and transwell assay indicated that analogs can a stronger inhibition on the invasion and migration of pancreatic cancer cells in vitro. Both of them can activate autophagy pathway to inhibit of tumor on the biological effects. In vivo experiment found that anologs can result in the nude mouse model with hepatic and renal fibrosis weaker.Part III the preliminary study of pharmacological mechanisms of WA and its anologs by inducing activated autophagy pathwayObjective:the preliminary study of WA and its anologs inducing activated autophagy pathway by pharmacological mechanismMethods:The super-microstructural changes by activated autophagy pathway in pancreatic cancer cells were observed by transmission electron microscopy after WA and its analogs treatment. Using western blot to detect autophagy protein expression in pancreatic cancer cells WA and its analogs treatment. C57 mice model was used to simulate an intraperitoneal injection of chemotherapy through WA and its anologs, to observe the changes in liver and kidney, using immunohistochemical and Western bolt to verification. Using the down-regulated gene lentivirus and overexpression plasmid to obtain stable transfection of pancreatic cancer cells, Using western bolt to detect the changes of autophagy pathway.Result:WA and its anologs activate autophagy pathway to inhibit pancreatic cancer proliferation in vitro. Using western blot to find out the down-regulation SNARE complexes expression. In vivo experiment found that anologs can result in the C57 mice model with weaker hepatic and renal fibrosis, and increased the expression of SNARE complexes in the liver and kidney.Using the down-regulated STX17 lentivirus and STX17 overexpression plasmid to obtain stable transfection of pancreatic cancer cells, using western bolt to confirm SNARE complexes is one of the key protein in autophagy pathway.Conclusion:WA and its anologs activate autophagy pathway to inhibit pancreatic cancer proliferation in vitro. The preliminary study suggested that increased SNARE complexes expression in liver and kidney might be associated with its fibrosis performance in vivo. Preliminary evident that in vitro SNARE complex is one of the key protein that affect autophagy pathways in pancreatic cancer. We also suggested that confirm SNARE complexes is one of the key protein in autophagy pathway.