Cr(VI)-Induced Autophagy Protects L-02 Hepatocytes from Apoptosis Through the ROS-AKT-mTOR Pathway

Abstract

Background/Aims: Hexavalent chromium [Cr(VI)] pollution has become a global concern for both ecosystems and human health. Our previous study revealed Cr(VI) could induce both apoptosis and autophagy in L-02 hepatocytes. Here, we sought to explore the underlying mechanism of Cr(VI)-induced autophagy and its exact role in cell death. Methods: Autophagy ultrastructure was observed under transmission electron microscope (TEM), autophagy flux was measured with double-tagged mCherry-green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) assay, long-lived protein degradation assay, and LC3II expression assay in the presence of lysosomal inhibitor, bafilomycin A1 (BafA1). Reactive oxygen species (ROS) level was determined using fluorescent probe dichloro-dihydrofluorescein diacetate (DCFH-DA). The expression levels of Beclin-1, LC3, p62/ SQSTM1, and AKT-mammalian target of rapamycin (mTOR) pathway-related molecules including phosphorylation (p)-AKT, AKT, p-mTOR, and mTOR were examined using real-time polymerase chain reaction (RT-PCR) and western blotting. Apoptosis was determined using Annexin V- fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. Results: Our results demonstrated Cr(VI) exposure activated autophagy in L-02 hepatocytes, as evidenced by the accumulation of autophagosomes, the increase of LC3-II and degradation of p62/ SQSTM1, and the enhanced overall degradation of proteins. We also confirmed Cr(VI)-induced LC3-II elevation mainly came from autophagy induction rather than lysosomal degradation impairment. ROS-AKT-mTOR pathway was associated with Cr(VI)-induced autophagy, and ROS scavenger N-acetylcysteine (NAC) pretreatment inhibited Cr(VI)-induced autophagy by alleviating the inhibition of the AKT-mTOR pathway. Autophagy inhibitors 3-methyladenine (3-MA) and chloroquine diphosphate (CDP) promoted Cr(VI)-induced apoptotic death. Conclusion: These findings indicated Cr(VI)-induced autophagy protected L-02 hepatocytes from apoptosis through the ROS-AKT-mTOR pathway.

The crosstalk between mitochondrial dysfunction and endoplasmic reticulum stress promoted ATF4‐mediated mitophagy induced by hexavalent chromium

“…Several articles including our previous study, have demonstrated that Cr (VI) induced autophagy in cancer cells 23,25 . Autophagy acts as a cytoprotective mechanism in cells in order to maintain homeostasis primarily by degrading damaged organelles, malfolded proteins, and alien matter.…”

“…Several articles including our previous study, have demonstrated that Cr (VI) induced autophagy in cancer cells. 23,25 Autophagy acts as a cytoprotective mechanism in cells in order to maintain homeostasis primarily by degrading damaged organelles, malfolded proteins, and alien matter. Mitophagy is a selective form of macro-autophagy in which dysfunctional mitochondria are specifically targeted for autophagic degradation.…”

“…The generation of intracellular ROS was measured using the 2,7‐dichlorofluorescein diacetate (DCFH‐DA) assay 25 . DCFH‐DA can penetrate the cells and be hydrolyzed by intracellular esterases to the nonfluorescent DCFH.…”

“…The generation of intracellular ROS was measured using the 2,7-dichlorofluorescein diacetate (DCFH-DA) assay. 25…”

Aegle marmelos extract rich in marmelosin exacted ameliorative effect against chromium‐induced oxidative stress and apoptosis through regulation of Gadd45 in HepG2 cell line

“…Our previous study has suggested that Cr (VI)-induced oxidative stress can cause upregulated ROS, RNS, mitochondrial membrane damage, and elevated Bax: Bcl-2 ratio, and lead to apoptotic cell death (Navya et al, 2017). Some of the recent studies have confirmed that Cr (VI)-induced autophagy by activation of ROS-AKT-mTOR pathway mechanism (Liang et al, 2018). Apoptotic cell death is another important toxic mechanism of Cr (VI) toxicity, which lead to cell shrinkage, LDH leakage, mitochondrial membrane potential damage via activation of caspase cascades (Wu et al, 2020).…”