Study On The Effect Of Oridonin Analogue 2y On Esophageal Cancer In Vivo And In Vitro And Its Molecular Mechanism

Aims1.To explore the effect of oridonin analogues on the proliferation of tumor cells,to screen compounds with anti-tumor activity,to investigate their effects on the proliferation,apoptosis,and reactive oxygen species of esophageal cancer cell EC 109 and their mechanism of action.2.To explore the effect of compound 2y on the growth of transplanted tumors in nude mice and the related molecular mechanism in vivo.Methods1.Using SRB staining method to investigate the effects of oridonin analogue compounds on the proliferation of four tumor cells(MGC-803,TE-1,EC109,MCF-7)after 72 hours.2.The SRB staining method was used to investigate the effect of the compound on the proliferation of esophageal cancer cell EC 109 at 24,48,and 72 h;the cell clone formation experiment was used to examine the effect of the compound on esophageal cancer cell EC 109;And late apoptosis;simultaneously investigate the effect of compound 2y on the reactive oxygen level of EC 109;and the effect of compound 2y on the cell mitochondrial membrane potential.Western Blotting experiment was used to detect apoptosis and ROS related proteins,and to explore the anti-tumor mechanism of compound 2y on esophageal cancer cell EC 109.The intervention by adding active oxygen scavenger N-acetylcysteine(NAC)further confirmed the effect of compound 2y on intracellular reactive oxygen levels.3.Using the nude mouse human cancer EC 109 cell transplantation tumor model,investigate the antitumor effect of compound 2y in animals.The HE staining experiment was used to investigate the effect of compound 2y on tumor cells in vivo.Observation of the changes in the body weight of nude mice during medication,and further through the HE staining experiment and blood biochemical index results,the safety of compound 2y in animals was preliminarily evaluated.Western Blotting was used to detect the related proteins of apoptosis pathway and reactive oxygen pathway,and to explore the anti-tumor mechanism of compound 2y in vivo.Results1.The SRB test results show that most compounds in the oridonin analogue have IC50 of less than 1 ?M for the four cell lines,which shows good antitumor activity.We selected compound 2y from this series of compounds,and their IC50 were:MGC-803 about 0.20 ± 0.01 ?M,EC109 about 0.13 ± 0.01 ?m,TE-1 about 0.27 ± 0.02 ?M,MCF-7 about 0.35± 0.03 ?M.2.SRB experiment results show that compound 2y has a significant inhibitory effect on the proliferation ability of EC 109 cells,and shows a concentration-dependent.The results of clone formation experiments showed that compound 2y had an inhibitory effect on the cloning ability of EC 109 cells.Experimental results related to flow cytometry showed that compound 2y reduced the mitochondrial membrane potential of esophageal cancer cell EC 109,which in turn led to an increase in the proportion of apoptosis.Western Blotting results showed that the expression of Bax,Bad,Bim,Cyc C,Cleaved-Caspase 3,Cleaved-Caspase 7,Cleaved-Caspase 9,and Cleaved-PARP in compound 2y group were up-regulated,and Bcl-2 Expression is down-regulated.3.Compound 2y can significantly increase the ROS level of EC 109 cells in a dose-dependent manner;Western Blotting experiment results show changes in ROS/JNK pathway-related proteins,p-ASK1,p-MKK4,p-JNK,pc-jun(Ser 63)Are showing an upward trend.Antioxidant NAC can reverse the effect of compound 2y on EC 109.After intervention with antioxidant NAC,it almost completely reversed the inhibitory effect of compound 2y on the proliferation and cloning ability of EC 109.Cell morphology also changed from normal shrinkage after drug addition.Mitochondrial membrane potential returned to nonnal,and the proportion of apoptosis decreased.At the same time,it reversed the expression changes of apoptosis-related and reactive oxygen pathway related proteins.4.Nude mouse human cancer xenograft model experiment results show that compound 2y can inhibit tumor growth in nude mice.The mechanism is to increase the protein expression in the reactive oxygen pathway,leading to changes in apoptosis-related proteins and ultimately to apoptosis.The in vivo and in vitro mechanisms are the same.The HE staining results of the tumor tissue further confirmed that compound 2y can promote the apoptosis of tumor cells in the tumor tissue.The body weight gain curve and blood biochemical index results of nude mice showed that the effect of compound 2y on the body weight of nude mice was not significantly different from that of the control group(NC).At the same time,compound 2y had no obvious toxicity to the heart,liver,spleen,lung and kidney of nude mice There is no obvious influence on blood routine indexes and blood biochemical indexes.Conclusions1.Compound 2y can inhibit the proliferation of esophageal cancer cells in vitro,and its mechanism is to induce apoptosis by activating the ROS/JNK pathway.2.Compound 2y also inhibits the growth of tumor tissues in vivo,and its molecular mechanism is also through the activation of ROS/JNK pathway to promote tumor cell apoptosis,which is consistent with the mechanism in vitro.At the same time,Compound 2y did not show significant toxicity in vivo.