The Inhibitory Effect Of Triptolide On Human Lung Adenocarcinoma Nude Mice Transplantation Tumor Growth And Autophagy Mechanism

Tripterygium wilfordii is a traditional Chinese herb,which has an anti-cancer effect.Under comparison,Tripoclide(T4)is a substance with lower toxicity and stronger anti-cancer activity extracted from Tripterygium wilfordii.In our study,we established a nude mouse xenograft model using non-small cell lung cancer cell A549 and A549/DDP,then different concentrations of T4 were injected into the mice enterocoelia,and afterwards,we observed the growth of xenografts,detected the expression levels of LC3 protein and PI3K/AKT/m TOR signaling pathway.Our study was to confirm the effect of triptolide in inhibiting the growth and inducing autophagy on lung cancer xenografts and to explore their possible molecular mechanism,providing a theoretical basis of clinical anti-cancer applications for T4.Methods: 1.We established a nude mice subcutaneous xenograft model of A549 and A549/DDP,then they were randomly divided into control group as below,T4 low dose group(0.05mg/kg),T4 medium dose group(0.2mg/kg)and T4 high dose group(0.4mg/ Kg).Then long diameter(a)and short diameter(b)of subcutaneous xenografts in nude mice were measured with vernier calipers,the volume of transplanted tumors calculated by the formula V=0.5a*b2,and then the growth curve of subcutaneous transplanted tumors was plotted.After the experiment,we weighed the tumors and calculated the tumor inhibition rate of each group.2.The pathological morphology of subcutaneous xenografts in nude mice was observed by HE staining.The expression of ki-67 in subcutaneous xenografts was detected by immunohistochemistry.3.The expression levels of LC3 protein and p62 protein in xenografts were detected by Western blot,and autophagosomes in xenografts were observed by transmission electron microscopy.4.The expression levels of PI3 K,AKT,TSC2,m TOR,p70S6 K,4E-BP1 and phosphoprotein p-PI3 K,p-AKT,p-TSC2,p-m TOR,p-p70S6 K,p-4E-BP1 protein in xenografts were detected by Western blot.5.Western blot was used to detect the expression levels of MDR-1 in subcutaneous xenografts of nude mice,and q RT-PCR was employed to detect the m-RNA levels of MDR-1 in subcutaneous xenografts of nude mice.6.The primary cells in each group of subcutaneous xenografts were isolated and cultured by tissue culture method.Immunohistochemistry was used to detect the expression of CK7,TTF-1,and Napsin A in primary cells.7.We detected the sensitivity of primary drug-resistant lung cancer cells to cisplatin by CCK8.Results:1.There was no statistically significant difference in the effect of T4 on albumin,alanine aminotransferase,aspartate aminotransferase,urea nitrogen,creatinine and glucose in nude mice.2.The growth rate of xenografts in the 0.4 mg/kg concentration group was significantly lower than the control group;the tumor inhibition rate in the 0.4 mg/kg concentration group was significantly higher than the control group.3.The expression of Ki-67 in xenografts was significantly reduced in nude mice after T4 intervention.4.Compared with the control group,the ratio of LC3II/LC3 I in the subcutaneous xenografts was significantly increased,and the expression level of p62 protein was decreased in the 0.2 mg/kg concentration group and the 0.4 mg/kg concentration group.The number of autophagosomes by transmission electron microscopy in the transplanted tumor,revealed that T4 treated nude mice were increased.5.Compared with the control group,the expression levels of p-PI3 K,p-AKT,p-m TOR,p-p70S6 K and p-4E-BP1 protein were significantly decreased in the 0.2 mg/kg concentration group and the 0.4 mg/kg concentration group,and the expression level of p-TSC2 protein was significantly increased.6.The protein levels and m RNA levels of MDR-1 were significantly lower than the control group with application of T4 at the concentrations of 0.2 mg/kg and 0.4 mg/kg,.7.Immunohistochemistry results showed that the expression of CK7,TTF-1 and Napsin A in primary cells and A549/DDP were same.8.The viability of primary lung cancer cells in the 0.05 mg/kg group and the 0.2 mg/kg group showed no differences with the control group under different concentrations of cisplatin;Compared with the control group,the activity of the cells was decreased with a difference.Conclusions:1.Triptolide inhibits the growth of non-small cell lung cancer in subcutaneous xenografts.2.Triptolide induces autophagy in lung cancer cells by inhibiting the PI3K/AKT/m TOR signaling pathway.3.Triptolide reduces the expression of MDR1 in subcutaneous xenografts in nude mice.4.Triptolide enhances the sensitivity of primary drug-resistant lung cancer cells to cisplatin.