Study On The Dihydrotanshinone Ⅰ Induced Oncosis Of Non-small Cell Lung Cancer And Its’ Mechanism

Objective:The purpose of this study was to investigate the oncosis of A549 cells induced by DHT in vitro and its mechanism.DHT could inhibit the growth of LLC tumor-bearing mice in vivo,and provide theoretical support for the clinical application of DHT.In order to provide theoretical support for the clinical application of DHT.Methods:In vitro:(1)A549 cells were treated with DHT(2,4,8 μM),then Cell Viability,Lactate Dehydrogenase(LDH)level and Cell Death Rate were measured to observe whether DHT could kill A549 cells in vitro.(2)A549 cells were pre-incubated with Z-VAD-FMK(20 μM)and VX765(60 μM)for 1 h,followed by intervention with DHT(8 μM),then Cell Viability,Apoptosis Rate,Cell Death Rate.(3)The A549 cells were pre-treated with DHT(2,4,8 μM),then cellular reactive oxygen species(Ros),calcium ions,porimin protein expression,porimin mRNA expression,mitochondrial membrane potential and ATP level were detected.(4)ROS inhibitor NAC(10 mM)was used to pre-incubate A549 cells for 1h,then DHT(8 μM)was used to interfere A549 cells,and the cellular reactive oxygen species(Ros),calcium and ATP levels were measured.In vivo:(1)LLC cells were implanted to establish mouse tumor model in vitro.(2)After tumor formation,animals were randomly divided into groups.The weight of tumor mice was recorded by weight scale,and the volume of tumor mice was recorded by vernier caliper.The drug was given continuously for 14 days.(3)The expression of Porimin protein in tumor tissues was detected by WB.The expression of porimin mRNA in tumor tissues was detected by qPCR.The pathological changes of organs and tumor tissues were observed by H&E stain.Results:In vitro:(1)Compared with the control group,DHT(2,4,8 μM)could decrease the activity of A549 cells and increase the level of LDH in a dose-dependent manner.Compared with the control group,DHT(2,4,8 μM)could increase the cell death rate,the results were statistically significant.(2)Compared with DHT(8 μM)group,VX765(60 μM)+DHT(8 μM)group could not reverse the DHT-induced decrease in cell activity.Compared with DHT(8 μM)group,z-VAD-FMK(20 μM)+DHT(8 μM)group reversed the DHT-induced decrease in cell activity,and the results were statistically significant.Compared with DHT(8 μM)group,z-VAD-FMK(20μM)+DHT(8 μM)group increased apoptosis rate but decreased cell death rate.Apoptosis can not explain the decline in cell death.(3)Compared with the control group,DHT(2,4,8 μM)could up-regulate ROS level,Ca2+level,Porimin protein expression level,porimin mRNA expression level.Compared with the control group,DHT(2,4,8 μM)could reduce mitochondrial membrane potential and ATP level in A549 cells.DHT induced oncosis in A549 cells.(4)Compared with DHT(8 μM)group,NAC(10 mM)+DHT(8 μM)group could increase cell activity,ATP level,inhibit ROS level.But it could not inhibit calcium ion level.After DHT administration,ROS mediated mitochondrial dysfunction and participated in oncosis of A549 cells.In vivo:(1)LLC tumor mouse model was successfully constructed.Compared with the control group,the weight of LLC tumor-bearing mice decreased.Compared with the model group,the tumor volume of the treated group was decreased.DHT administration can inhibit tumor growth in vivo.(2)The results of HE staining in mice spleen showed that the structure of spleen was improved and the splenic nodules were more clear in the treatment group than in the model group.(3)Compared with the model group,western blotting showed that DHT up-regulated the expression of porimin protein in tumor tissues of mice.DHT administration may induce tumor cell oncosis in vivo.Conclusion(s):DHT could induce cell death of A549 cells by oncosis.The mechanism may be through the porimin protein/ROS/mitochondria pathway to exert anti-tumor effects.DHT can inhibit the growth of tumor and up-regulate the expression of porimin protein in tumor tissue.DHT administration may induce the oncosis of tumor cells in mice.