Mitochondrial electron-transport-chain inhibitors of complexes I and II induce autophagic cell death mediated by reactive oxygen species

Abstract

Autophagy is a self-digestion process important for cell survival during starvation. It has also been described as a form of programmed cell death. Mitochondria are important regulators of autophagy-induced cell death and damaged mitochondria are often degraded by autophagosomes. Inhibition of the mitochondrial electron transport chain (mETC) induces cell death through generating reactive oxygen species (ROS). The role of mETC inhibitors in autophagy-induced cell death is unknown. Herein, we determined that inhibitors of complex I (rotenone) and complex II (TTFA) induce cell death and autophagy in the transformed cell line HEK 293, and in cancer cell lines U87 and HeLa. Blocking the expression of autophagic genes (beclin 1 and ATG5) by siRNAs or using the autophagy inhibitor 3-methyladenine (3-MA) decreased cell death that was induced by rotenone or TTFA. Rotenone and TTFA induce ROS production, and the ROS scavenger tiron decreased autophagy and cell death induced by rotenone and TTFA. Overexpression of manganese-superoxide dismutase (SOD2) in HeLa cells decreased autophagy and cell death induced by rotenone and TTFA. Furthermore, blocking SOD2 expression by siRNA in HeLa cells increased ROS generation, autophagy and cell death induced by rotenone and TTFA. Rotenone- and TTFA-induced ROS generation was not affected by 3-MA, or by beclin 1 and ATG5 siRNAs. By contrast, treatment of non-transformed primary mouse astrocytes with rotenone or TTFA failed to significantly increase levels of ROS or autophagy. These results indicate that targeting mETC complex I and II selectively induces autophagic cell death through a ROS-mediated mechanism.

Superoxide is the major reactive oxygen species regulating autophagy

“…Our previous studies indicate that mETC inhibitors (including complex I inhibitor rotenone, complex II inhibitor TTFA, and complex I/SOD inhibitor 2-ME 23,28 ) can induce ROSmediated autophagy and autophagic cell death in cancer cells. 17,18 In these studies, SOD2 overexpression reduced autophagy, supporting the involvement of O 2 KÀ in autophagy induction. However, by measuring ROS only with dihydroethidium (DHE), which is reported to detect O 2 KÀ specifically, 27 we failed to directly address the function of H 2 O 2 .…”

“…Several reports have suggested that starvation-induced autophagy is mediated by ROS. [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] In this study, we investigated the generation of O 2 KÀ and H 2 O 2 under two starvation conditions: starvation of glucose, L-glutamine, pyruvate, and serum (GP), and starvation of amino acids and serum (AA). Cellular levels of O 2 KÀ and H 2 O 2 were measured by flow cytometry using specific fluorescent dyes (see Materials and Methods).…”

“…1 In this study, we detected starvation-induced autophagy using three methods as in our previous studies. 17,18,24 We transiently transfected cells with GFP-LC3, which is recruited to autophagosome membranes during autophagy resulting in a punctate distribution that can be visualized using a fluorescent microscope. We measured the formation of acidic vesicular organelles (AVOs, which include autolysosomes) by flow cytometry using the pHsensitive fluorescent dye acridine orange (AO).…”

Cockayne syndrome group B protein prevents the accumulation of damaged mitochondria by promoting mitochondrial autophagy

“…Upon formation of autophagic vacuoles, cytosolic LC3B (isoform I, LC3B I) is cleaved and inserted into the autopha gic membrane (isoform II, LC3B II), allowing autophagy to be monitored by measuring the LC3B II/LC3B I ratio (Levine and Kroemer, 2008). Previous literature has shown that the mitochondrial toxicant rotenone induces autophagy (Chen et al, 2007). Remarkably, we saw a significant defect in rotenone induced autophagy in CSB deficient CS1AN cells and HSMM after 24 h of treatment (Fig.…”

Molecular Mechanisms of Pesticide Toxicity

“…Recent studies provide strong evidences for the involvement of mitochondrially-generated ROS production in the induction of autophagy (Chen & Gibson, 2008;Chen et al, 2009). Recently, it has been demonstrated that targeting mETC complex I by rotenone, induces autophagic cell death through a ROS-mediated mechanism (Chen et al, 2007).…”