Ampelopsin-induced autophagy protects breast cancer cells from apoptosis through Akt-mTOR pathway via endoplasmic reticulum stress.
Bottom Line: Blockage of autophagy augmented AMP-induced cell death, suggesting that autophagy has cytoprotective effects.Meanwhile, AMP treatment suppressed Akt-mammalian target of rapamycin (mTOR) pathway as evidenced by dose- and time-dependent decrease of the phosphorylation of Akt, mTOR and ribosomal protein S6 kinase (p70S6K), whereas Akt activator insulin-like growth factor-1 (IGF-1) pretreatment partially restored Akt-mTOR pathway inhibited by AMP and decreased AMP-inuduced autophagy, signifying that AMP activated autophagy via inhibition of the Akt-mTOR pathway.Additionally, blocking ER stress not only reduced autophagy induction, but also alleviated inhibition of the Akt-mTOR pathway induced by AMP, suggesting that activation of ER stress was involved in induction of autophagy and inhibition of the Akt-mTOR pathway.
Affiliation: Research Center for Nutrition and Food Safety, Institute of Military Preventive Medicine, Third Military Medical University, Chongqing Key Laboratory of Nutrition and Food Safety, Research Center for Medical Nutrition, Chongqing, China.Show MeSH
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Mentions: To investigate whether AMP could induce autophagy in MCF-7 and MDA-MB-231 cells, the autophagic activity was measured by the formation of autophagosomes under transmission electron microscopy (TEM), which is one of the most widely accepted methods for monitoring autophagy.26 The formation of double-membraned autophagic vacuoles, which is indicative of autophagy induction, was frequently observed in cells treated with 60 μM AMP, but not in control cells (Fig.1a). To confirm these observations, we further tested the expressions of MAP1LC3B-II (LC3B-II), which is a marker of autophagy,27 and p62/SQSTM1, which acts as a cargo receptor for autophagic degradation of ubiquitinated targets, by western blotting. AMP treatment induced LC3B activation and time-dependently increased the expression of LC3B, enhanced the conversion of LC3B-I to autophagosome-associated LC3B-II in both breast cancer cell lines, along with downregulation of p62/SQSTM1 (Fig.1b,d). The formation of punctate spots (puncta) with green fluorescent protein (GFP)-MAPLC3B reporter is a well-characterized marker for visualizing autophagosomes formation and represents the accumulation of LC3B-II on autophagic vesicles.27 Moreover, we assessed the formation of GFP-MAPLC3B puncta as an autophagic marker under fluorescence microscopy. MCF-7 and MDA-MB-231 cells after treatment with 60 μM AMP for 24 h showed a significant increase in the percentage of cells containing GFP-MAP1LC3B puncta, and autophagy inhibitor 3-methyladenine (3-MA, 5 mM) partially blocked the increase of GFP-MAP1LC3B puncta induced by AMP (P < 0.05) (Fig.1c).
Affiliation: Research Center for Nutrition and Food Safety, Institute of Military Preventive Medicine, Third Military Medical University, Chongqing Key Laboratory of Nutrition and Food Safety, Research Center for Medical Nutrition, Chongqing, China.