Lack of mitochondrial DNA impairs chemical hypoxia-induced autophagy in liver tumor cells through ROS-AMPK-ULK1 signaling dysregulation independently of HIF-1α.
Alterations in mitochondrial DNA (mtDNA) and autophagy activation are common events in tumors. Here we have investigated the effect of mitochondrial genome depletion on chemical hypoxia-induced autophagy in liver tumor cells. Human SK-Hep-1 wild-type and mtDNA-depleted (Rho) cells were exposed to the hypoxia mimetic agents CoCl2and deferoxamine (DFO). Up-regulation of HIF-1α, but not HIF-2α was observed. The expression of several HIF-1α target genes was also found. In human SK-Hep-1 and mouse Hepa 1-6 liver tumor cells, but not in the counterpart Rho derived lines, chemical hypoxia increased the abundance of autophagosomes and autolysosomes. In wild-type and Rho cells, chemical hypoxia induced down-regulation of HIF-1α-dependent autophagy inhibitors Bcl-2 and mTOR, whereas activation of AMPK/ULK1-mediated pro-autophagy pathway occurred only in wild-type cells. Chemical (compound C) and genetic (shRNA) inhibition of AMPK activation resulted in reduced autophagy. ATP levels were similar in both cell types, whereas constitutive and chemical hypoxia-induced reactive oxygen species (ROS) generation was lower in Rho cells. In wild-type cells, the antioxidant N-acetylcysteine blocked CoCl2- and DFO-induced AMPK and autophagy activation, but not endoplasmic reticulum stress induced by CoCl2. Enhanced Bax-α/Bcl-2 ratio and cell death was induced by hypoxia mimetic agents more markedly in wild-type than in Rho cells. Upon blocking autophagy activation with 3-methyladenine, DFO-induced cell death was partially prevented whereas that induced by CoCl2was increased, but only in wild-type cells. These results suggest that mitochondrial dysfunction associated with the lack of mtDNA impairs the signaling pathways mediated by ROS, controlling autophagy activation in liver tumor cells, which may contributes to cancer development.
Laboratory of Experimental Hepatology and Drug Targeting, IBSAL, CIBERehd. University of Salamanca, 37007 Salamanca, Spain.
Jose J G
JJG
Marin
Laboratory of Experimental Hepatology and Drug Targeting, IBSAL, CIBERehd. University of Salamanca, 37007 Salamanca, Spain.
Elisa
E
Lozano
Laboratory of Experimental Hepatology and Drug Targeting, IBSAL, CIBERehd. University of Salamanca, 37007 Salamanca, Spain; University Hospital of Salamanca, IECSCYL-IBSAL, 37007 Salamanca, Spain. Electronic address: mjperez@usal.es.
Maria J
MJ
Perez
D055372:AMP-Activated Protein Kinases / Q000235:genetics* / Q000378:metabolism*; D000111:Acetylcysteine / Q000494:pharmacology; D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects*; D000071189:Autophagy-Related Protein-1 Homolog / Q000235:genetics / Q000378:metabolism*; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D003035:Cobalt / Q000494:pharmacology*; D004272:DNA, Mitochondrial / Q000378:metabolism*; D003676:Deferoxamine / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D057809:HEK293 Cells; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D010078:Oxazines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction
D000975:Antioxidants; D004272:DNA, Mitochondrial; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D047908:Intracellular Signaling Peptides and Proteins; D010078:Oxazines; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D003035:Cobalt; C025946:3-methyladenine; C085142:oxadin; D000071189:Autophagy-Related Protein-1 Homolog; C526181:PRKAA2 protein, human; C484754:ULK1 protein, human; D055372:AMP-Activated Protein Kinases; C526180:PRKAA1 protein, human; C018021:cobaltous chloride; D003676:Deferoxamine; D000225:Adenine; D000111:Acetylcysteine
AMPK; Autophagy; HIF-1α; Hepatocarcinoma; Hypoxia mimetic agents; Mitochondria; Reactive oxygen species; Retrograde regulation; Rho cells