EM-2,an Elephantopus Mollis H.B.K.monomer,induces Apoptosis In Human Colon Cancer Cells By Reactive Oxygen Species(ROS)-mediated PI3K/Akt Signaling Pathways

Objective: This study explored the effect and mechanism of the the EM-2 of an Elephantopus mollis H.B.K.monomer in inducing human colon cancer,and provided experimental evidence for the EM-2 of an Elephantopus mollis H.B.K.monomer to replace clinical practice in the future.Methods: MTT method and cell clone formation were used to detect the effect of EM-2 on the viability and proliferation of human hepatocellular carcinoma cells;after EM-2 was treated with SW620 cells,flow cytometry was used to detect the changes in the level of reactive oxygen species and the rate of apoptosis in the cells;Western blotting method Autophagy and apoptosis-related protein levels;the effect of ROS scavenger(NAC)pretreatment on the apoptosis of human colon cancer cells induced by EM-2 and the detection of related signal pathways;immunofluorescence detection of autophagy-related proteins Variety..Result: MTT results show that EM-2 can inhibit the activity of human colon cancer cells in a dose-dependent manner,and is less toxic to normal human epithelial cells LO2 and 293 T.At the same time,the clone formation experiment proves that EM-2can proliferate colon cancer cells.Has an inhibitory effect.Next,through flow cytometry detection,the results showed that in SW620 cells,EM-2 treatment can significantly induce apoptosis in a dose-dependent manner;consistent with this,Western blot detection showed that EM-2 It can dose-dependently down-regulate apoptosis-related proteins(Bcl-2,Bcl-xl)and up-regulate activated Caspase-3/9 and activated PARP.Further detection by the DCFH-DA probe revealed that EM-2treatment can increase the level of ROS in cells;NAC pretreatment can reduce the increase in ROS levels caused by EM-2 and inhibit the effect of EM-2 on inducing cell apoptosis,indicating that EM-2 may induce cell apoptosis by increasing the production of ROS in the cell leading to the destruction of mitochondria.Mechanism studies have shown that EM-2 treatment can reduce the phosphorylation level of AKT and m TOR,while inhibiting the expression of PI3 K,AKT and the total protein of m-TOR.On the contrary,NAC pretreatment reversed the inhibitory effect of EM-2 on AKT and m TOR phosphorylation,thereby down-regulating bik and up-regulating the expression of Bcl-xl and Mcl-1,further inhibiting the activation of Caspase-3/9 and PARP.Finally,it reversed the apoptosis induced by EM-2,indicating that EM-2 may induce apoptosis by regulating the PI3K/AKT-m TOR pathway.On the other hand,this study found that EM-2 can promote the expression of LC3 B and p62 protein and inhibit the expression of beclin1;consistent with this,immunofluorescence detection also showed that the expression of LC3 B and p62 increased.At the same time,we found that EM-2 significantly inhibited the expression of lysosomal-associated membrane protein 2,and the expression of mitochondrial outer membrane receptor Tom20 increased significantly.It shows that EM-2 can impair lysosome function and inhibit the occurrence of cell mitochondrial autophagy.Conclusion:1.EM-2 can reduce the activity of human colon cancer cells and inhibit their proliferation.2.EM-2 induces apoptosis by up-regulating the level of ROS and destroying mitochondria.3.EM-2 may play an anti-tumor effect by regulating the PI3K/AKT-m TOR pathway.4.EM-2 can destroy lysosomes and block autophagy.Overall,the monomer EM-2 of B.radiata can induce apoptosis of human colon cancer cells and exert anti-tumor effects.The mechanism may be through destroying lysosomes and blocking the occurrence of cell mitochondrial autophagy,thereby inducing ROS Increase,inhibit PI3K/AKT-m TOR pathway,cause mitochondrial destruction,and then induce cell apoptosis.These results indicate that the the EM-2 of an Elephantopus mollis H.B.K.monomer has certain anti-tumor activity and potential clinical application value.