Abstract
Autophagy is a cellular mechanism by which cells degrade intracellular components in lysosomes, maintaining cellular homeostasis. It has been hypothesized that autophagy could have a role in cancer prevention through the elimination of damaged proteins and organelles; this could explain epidemiological evidence showing the chemopreventive properties of the autophagy-inducer metformin. In this study, we analyzed the autophagy-related effect of metformin in both cancer initiation and progression in non-tumorigenic cells. We also analyzed the induction of tumorigenesis in autophagy-deficient cells, and its correlation with the ER stress. Our results showed that metformin induced massive cell death in preneoplastic JB6 Cl 41-5a cells treated with tumor promoter (phorbol) and in NIH/3T3 treated with H2O2. Inhibiting autophagy with wortmannin or ATG7 silencing, the effect of metformin decreased, indicating an autophagy-related cytotoxic activity under stress conditions. We also found an induction of tumorigenesis in ATG7-silenced NIH/3T3 cell clone (3T3-619C3 cells), but not in wild-type and in scrambled transfected cells, and an upregulation of unfolded protein response (UPR) markers in 3T3-619C3 cells treated with H2O2. These findings suggest that autophagic cell death could be considered as a new mechanism by which eliminate damaged cells, representing an attractive strategy to eliminate potential tumorigenic cells.
Highlights
Tumorigenesis is a complex and multistage process characterized by an accumulation of cellular damage promoted by chronic inflammation and exposure to carcinogens
To evaluate if apoptosis is involved in metformin-induced cell death, cells were treated with the pancaspase inhibitor zVAD-fmk; as shown in Fig. 1A, cytotoxic activity of metformin in Tetradecanoylphorbol 13-acetate (TPA)-stimulated cells did not change after zVAD-fmk treatment, suggesting that apoptosis is not involved in JB6 P+ cell death
The results showed that metformin induced an increase in p62 expression after 24 hours, and a decrease after 48 hours in 3T3-SCRD3 cells; this effect was not revealed in shATG7 3T3-619C3 cells (Fig. 5B), indicating that autophagy was efficiently inhibited in the ATG7-silenced cells
Summary
Tumorigenesis is a complex and multistage process characterized by an accumulation of cellular damage promoted by chronic inflammation and exposure to carcinogens. Despite autophagy is considered a survival mechanism for cancerous cells in the hostile tumor microenvironment, it could prevent chronic tissue stress that can induce cellular damage to proteins, organelles and DNA, inhibiting cancer initiation and progression[2,3,4,5,6]. It has been hypothesized that metformin may have anticancer properties through different mechanisms, independent of its hypoglycemic effect; its main proposed anticancer molecular action is associated with the inhibition of mTORC1 - which is involved in metabolism, growth and differentiation of cancer cells13 - mediated by AMPK activation or in a AMPK-independent manner. Other proposed mechanisms through which metformin could exert its anticancer effects include the induction of cell cycle arrest and/or apoptosis and the inhibition of the unfolded protein response (UPR)[14]. The concurrence between ER stress and autophagy is common in several human pathologies, including neurodegenerative disorders, diabetes and cancer[18]
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