Inhibitory Effect And Mechanism Of Metformin On Thyroid Papillary Carcinoma Graft

Part 1.Effects of metformin on the apoptosis and reactive oxygen species of human papillary thyroid carcinoma xenografted in nude miceBackgroundthe Rate of Growth of human papillary thyroid carcinoma xenografted in nude mice was analyzed after different treatments.The effects of metformin on the apoptosis and oxidative stress of human papillary thyroid carcinoma(PTC)xenografted in nude mice were detected by TUNEL assay and biochemical assay,respectively.MethodsThe human PTC-K1 cell line was used to establish a subcutaneous xenograft mouse model in nude mice,when subcutaneous tumor xenograft models were developed,The nude mice were randomly assigned to four groups:control group(Con group),metformin(Met group),Nrf2-shRNA group(Nrf2-shRNA group),metformin+Nrf2shRNA group(combination group),The nude mice were killed on the 14th day,and the xenografted tumors were taken out for test:(1)TUNEL assay was used to detect apoptosis of xenograft tumor tissues.;(2)Immunohistochemistry assay was used to evaluate the levels of Bcl-2 and activated caspase-3 protein.(3)ROS,GSH-Px,and SOD were measured in xenograft tumor tissues by Biochemical analysis.ResultsCompared with the Con group,the Rate of Growth of human papillary thyroid carcinoma xenografted in nude mice was slower in the Met group and the Nrf2-shRNA group,and the Rate of Growth in the combination group was the slowest.The apoptosis rates in the Control group,Met group,Nrf2-shRNA group,and combination group was(8±4)%,(19±21)%,(14±9)%,and(73±13)%,respectively.Compared with the control group,the apoptosis of xenografted tumor tissues in the other three groups was significantly different(F apoptosis=19.12,P<0.001).Compared with the Met group and Nrf2-shRNA group,the apoptosis rate of xenografted tumor tissues in the combination group was significantly increased(all P<0.05);Compared with the control group,the levels of ROS and Caspase-3 were significantly increased and the levels of GSH-Px,SOD and Bcl-2 were significantly decreased in the metformin/Nrf2-shRNA groups alone or combination with metformin/Nrf2-shRNA groups(all P<0.05).ConclusionsMetformin plays a pro-apoptotic role by decreasing the expression of Nrf2 and increasing the level of intracellular reactive oxygen species.Metformin may decrease Nrf2 expression through activation of the AMPK/mTOR signaling pathway.Part 2.Effects of metformin on AMPK/mTOR-Nrf2 signaling pathway of human papillary thyroid carcinoma xenografted in nude miceBackgroundThe expression of the AMPK/mTOR pathway and Nrf2 and its downstream proteins of human papillary thyroid carcinoma xenografted in nude mice were detected to further analyze the molecular mechanism about metformin promoting apoptosis in the xenograft tumor tissues of nude mice.MethodsWestern blot and qRT-PCR were used to detect the expression of Nrf2 protein in the xenografted tumor tissues of nude mice treated with metformin.Immunohistochemical was used to analyze the expression of AMPK/mTOR pathway,Nrf2,and downstream proteins in human thyroid papillary carcinoma xenografted in nude mice.Resultsthe expression of the total Nrf2,nucleus Nrf2 protein,and Nrf2mRNA levels were attenuated compared to Con group,the plasma Nrf2 displayed an enhanced in Met group,compared with Con group,p-AMPK expression levels of Met,combination group were increased,the expression of p-mTOR level decreased,with Nrf2,NOQ1,HO-1 protein expression levels were decreased,the difference was statistically significant(P<0.05).ConclusionsMetformin plays a pro-apoptotic role by decreasing the expression of Nrf2 and increasing the level of intracellular reactive oxygen species.Metformin may decrease Nrf2 expression through activation of the AMPK/mTOR signaling pathway.