Biphasic Effect Of Inducing Proliferation And Autophagic Cell Death In Magnolol Treated H460 Cell

Magnolol,isolated from the stem bark of Magnolia sp.,has been traditionally used to treat for cough,diarrhea,and allergic rhinitis.In this study,we investigate the mechanism of H460 proliferation or autophagy in different concentrations induced by Magnolol.H460 cell viability was measured by MTT.H460 cells morphological changes were detected by Wright-Giemsa staining assay.Cytoplasmic vacuoles were stained with the autophagic marker monodansylcadaverine(MDC).DNA synthesis was assayed using DNA synthesis detection kit.Lactate dehydrogenase(LDH)activity was assessed using a standardized kinetic determination.Western blot analysis was used to evaluate the level of PTEN and Akt expression.The results showed that Magnolol presented a biphasic effect in DNA synthesis and displayed a stimulatory effect at low concentrations(≤20μM)whilst inhibitory effect at high concentrations(≥40μM)in H460 cell,the inhibition of DNA synthesis was accompanied by the appearance of an intense intracytoplasmic vacuoles.The characterization of these vacuoles can be labelled by MDC and further confirmed by 3-MA.The result of the LDH Activity assay showed that Magnolol greatly inhibited H460 cell growth via autophagic pathway at high concentration for 24 h,but apoptotic process was negligible.Furthermore, many proteins,including Akt and PTEN were related to both proliferation and autophagy.In conclusion,Magnolol induces H460 cell proliferation or autophagy in different concentrations, in which ClassⅠPI3K/PTEN/Akt pathway is involved.But the classⅢPI3K stimulates macroautophagy in this process.These results provide new insights into the signaling events that control induction of autophagy by Magnolol in human cancer cells and suggest that magnolol warrant further study as potential cancer chemopreventive agents.Meanwhile,we also investigate the effect of low concentrations Magnolol preserved H460 cells from an oxidative agent tert-butylhydroperoxide(TBHP)-induced cell death. Magnolol(20μM)augmented cell survival ratio after TBHP challenged.The protective action of this drug was more efficacious than that of N-acetylcysteine(NAC)which is a putative antioxidant.DNA damage,detected by the comet assay,was diminished after treatment of Magnolol.The cells viability decreased after treatment with 0.15 mM TBHP for 24 h, accompanied by inducing apoptotic death of the cells.Cytotoxicity and apoptosis induced by TBHP were significantly inhibited or attenuated after pretreatment with 20μM Magnolol. Magnolol contributes to the cells survival through downregulated the p53 phosphorylation and PTEN expression,and upregulated Akt phosphorylation.Taken together,Magnolol was effective against DNA single strand breaks(SSB)formation,cytotoxicity and lipid peroxidation induced by TBHP,and its effects on p53 phosphorylation,PTEN and Akt phosphorylation were due to its antioxidative function,and partially via a p53 dependent mechanism in this protective effects.Taken together,Magnolol is effective against DNA SSB formation,cytotoxicity,and lipid peroxidation induced by TBHP at low concentrations,and can induce cancer cells autophagy leading to S phase block at high concentrations.On growing evidence of the Magnolol-mediated antioxidation,it may be a potential candidate as an anti-oxidative or anti-cancer agent.Further efforts to develop potential therapeutic strategies with Magnolol might be considered.