The Effect Of Rosmarinus Acid Extract RAA9 On Autophagy Mediated By PI3K/Akt/mTOR Signaling Pathway

Objective:(1)To explore the effect of rosmarinic acid derivative RAA9 on the proliferation and migration of human colon cancer HCT116 cells.(2)To investigate whether RAA9 induces autophagy in human colon cancer HCT116 cells.(3)To explore the possible mechanism of RAA9-induced HCT116 autophagy.Methods:(1)MTT method was used to detect the effect of RAA9 on the proliferation of human colon cancer HCT116 cells.(2)Wound healing experiment to observe the effect of RAA9 on migration of HCT116 cells.(3)Acridine orange(AO)staining to observe autophagic vesicles(4)Cellular immunofluorescence observation of LC3 expression in HCT116 cells.(5)PCR and Western blot to detect the expression of autophagy-related genes and proteins LC3 and Beclin-1 at different RAA9 concentrations.(6)Western blot to detect the effect of RAA9 on the autophagy-related pathway extract RAA9 on the expression of Raptor,AKT,P-AKT,S6K1,P-S6K1,4ebp1 protein in PI3 K /Akt / mTOR signaling pathway.Results:(1)The MTT results show that RAA9 has a proliferation-inhibiting effect on human colon cancer HCT116 cells(P <0.05)and with concentrationand time-dependent.The wound healing experiment results showed that RAA9 inhibited the migration ability of HCT116 cells with concentration-dependent.The effects RAA9 on autophagy in HCT116 cells:①Acridine orange staining results suggest that RAA9 can induce autophagy in HCT116 cells,and intracellular autophagic vesicles increase with increasing of RAA9 concentration.②PCR and Western blot results showed that RAA9 could up-regulate the expression of LC3 and Beclin-1,and promote cell autophagy.(3)Western blot results suggest that RAA9 can significantly down-regulate the expression of PI3 K / Akt / mTOR pathway-related proteins Raptor,P-AKT,S6K1,P-S6K1,4ebp1(P <0.05).Conclusion:(1)The rosmarinic acid derivative RAA9 can significantly inhibit the proliferation and migration of colon cancer HCT116 cells.(2)Rosemary acid derivative RAA9 can induce autophagy in colon cancer HCT116 cells by up-regulating LC3 and Beclin-1.(3)The induction of autophagy in colon cancer HCT116 cells by rosmarinic acid derivative RAA9 may be achieved by inhibiting PI3 K / AKT / mTOR pathway activity.