Down-regulation of mcl-1 by small interfering RNA sensitizes resistant melanoma cells to fas-mediated apoptosis

Resistance of malignant melanoma cells to Fas-mediated apoptosis is among the mechanisms by which they escape immune surveillance. However, the mechanisms contributing to their resistance are not completely understood, and it is still unclear whether antiapoptotic Bcl-2-related family proteins play a role in this resistance. In this study, we report that treatment of Fas-resistant melanoma cell lines with cycloheximide, a general inhibitor of de novo protein synthesis, sensitizes them to anti-Fas monoclonal antibody (mAb)-induced apoptosis. The cycloheximide-induced sensitization to Fas-induced apoptosis is associated with a rapid down-regulation of Mcl-1 protein levels, but not that of Bcl-2 or Bcl-xL. Targeting Mcl-1 in these melanoma cell lines with specific small interfering RNA was sufficient to sensitize them to both anti-Fas mAb-induced apoptosis and activation of caspase-9. Furthermore, ectopic expression of Mcl-1 in a Fas-sensitive melanoma cell line rescues the cells from Fas-mediated apoptosis. Our results further show that the expression of Mcl-1 in melanoma cells is regulated by the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and not by phosphatidylinositol 3-kinase/AKT signaling pathway. Inhibition of ERK signaling with the mitogen-activated protein/ERK kinase-1 inhibitor or by expressing a dominant negative form of mitogen-activated protein/ERK kinase-1 also sensitizes resistant melanoma cells to anti-Fas mAb-induced apoptosis. Thus, our study identifies mitogen-activated protein kinase/ERK/Mcl-1 as an important survival signaling pathway in the resistance of melanoma cells to Fas-mediated apoptosis and suggests that its targeting may contribute to the elimination of melanoma tumors by the immune system.     

Dimethylsulfoxide (DMSO) induces downregulation of heme oxygenase-1 (HO-1) in HL-60 cells: involvement of HO-1 in HL-60 cell differentiation

Heme oxygenase-1 (HO-1), an inducible enzyme with broad tissue expression, is wel1-regulated in response to hematopoietic stress and preserves vascular homeostasis. We investigated the involvement of HO-1 in HL-60 cell differentiation. Dimethyl sulfoxide (DMSO) completely decreased HO-1 expression in a time-dependent manner, but clearly induced HL-60 cell differentiation, as evidenced by a marked increase in CD11b expression. Interestingly, zinc protoporphyrin (ZnPP), a strong inhibitor of HO-1, induced HL-60 cell differentiation. In contrast, treatment with cobalt protoporphyrin (CoPP), an activator of HO-1, decreased CD11b expression. Additionally, ZnPP downregulated HO-1 protein expression in HL-60 cells, whereas CoPP induced upregulation. These results suggest that HO-1 might have a negative function in DMSO-induced HL-60 cell differentiation. This study provides the first evidence that HO-1 plays an important role in DMSO-induced HL-60 cell differentiation.     

Antiapoptotic role of heme oxygenase (HO) and the potential of HO as a target in anticancer treatment

Heme oxygenase-1 (HO-1) is an inducible enzyme that catalyzes oxidative degradation of heme to form biliverdin, carbon monoxide (CO), and free iron. Biliverdin is subsequently reduced to bilirubin by the enzyme biliverdin reductase. Increasing evidence has indicated the critical role of HO-1 in cytoprotection and more diverse biological functions. Induction of HO-1 by various chemical inducers that are primarily cell stress inducers or by HO-1 gene transfection confers a protective capacity to cultured cells as well as to cells in several in vivo animal models. In addition, HO-1-deficient mice exhibit a significant increase in susceptibility to tissue injury. The cytoprotective action of HO-1 seems to be mainly a function of the antiapoptotic effects of the enzyme. HO-1 is believed to exert this antiapoptotic action by multiple mechanisms: (a) decreased intracellular pro-oxidant levels, (b) increased bilirubin levels, and (c) elevated CO production. CO may produce an antiapoptotic effect by inhibiting both expression of p53 and release of mitochondrial cytochrome c. HO-1 may also be a target in antitumor therapy because the growth of most tumors depends on HO-1. Our preliminary studies with an HO inhibitor showed a promising antitumor effect. This preliminary work warrants continued investigation for possible novel anticancer chemotherapy.     

Overexpression of DRG2 suppresses the growth of Jurkat T cells but does not induce apoptosis

Developmentally regulated GTP-binding protein (DRG) is a new subfamily within the superfamily of GTP-binding proteins. Its expression is regulated during embryonic development. To investigate the effect of the expression of DRG2 on cell growth, we constructed a human Jurkat-T-cell line that overexpresses DRG2. Overexpression of DRG2 suppressed the growth and the aggregation of Jurkat cells but did not induce apoptotic cell death. We used cDNA microarray analysis to examine the global changes in gene expression induced by an overexpression of DRG2. DNA array analyses identified genes that may suppress cell growth at a number of levels in multiple signaling cascades in Jurkat cells and also several prosurvival genes that may protect cells from apoptosis.     

Caveolin-1 is a competitive inhibitor of heme oxygenase-1 (HO-1) with heme: identification of a minimum sequence in caveolin-1 for binding to HO-1

Heme oxygenase (HO) catalyzes the O(2)-dependent degradation of heme to biliverdin IXα, carbon monoxide (CO), and free ferrous iron through a multistep mechanism. Electrons required for HO catalysis in mammals are provided by NADPH-cytochrome P450 reductase. Recently, Kim et al. reported for the first time that HO, especially inducible HO-1, appears in caveolae and showed that caveolin-1, a principal isoform of the caveolin family, physically interacts with HO-1 [ Jung , N. H. et al. ( 2003 ) IUBMB Life 55 , 525 - 532 ; Kim , H. P. et al. ( 2004 ) FASEB J. 18 , 1080 - 1089 ]. In the present study, we confirmed by immunoprecipitation experiments that rat HO-1 and rat caveolin-1 (residues 1-101) directly interact with each other and that the HO-1 activity is inhibited by caveolin-1 (1-101). The 82-101 residues of caveolin-1 (CAV(82-101)), called the caveolin scaffolding domain, play essential roles in caveolin-related protein-protein interactions. The HO-1 activity is also inhibited by CAV(82-101) in a competitive manner with hemin, and a hemin titration experiment showed that CAV(82-101) interferes with hemin binding to HO-1. The enzyme kinetics and surface plasmon resonance experiments gave comparable K(i) and K(D) values of 5.2 and 1.0 μM for CAV(82-101), respectively, with respect to the interaction with HO-1. These observations indicated that CAV(82-101) and hemin share a common binding site within the HO-1 protein. The identified caveolin binding motif (FLLNIELF) of rat HO-1 is incomplete compared to the proposed consensus sequence. The affinity between HO-1 and CAV(82-101), however, was almost completely or remarkably eliminated by replacement of Phe(207) and/or Phe(214) with Ala, indicating that HO-1 binds to caveolin-1 via this motif. Among the peptide fragments derived from CAV(82-101), i.e., CAV(82-91), CAV(87-96), CAV(92-101), and CAV(97-101), CAV(92-101) and CAV(97-101) are able to inhibit the HO-1 activity to a similar extent; thus, the five-amino acid sequence (residues 97-101) is considered to be a minimum sequence for binding to HO-1.     

Resolution of the rat brain heme oxygenase activity: absence of a detectable amount of the inducible form (HO-1)

In the present study we report on the detection of a distinct pattern of heme oxygenase isoform composition in the rat brain. In this organ only the noninducible form of heme oxygenase, HO-2, could be clearly detected. This pattern of composition distinguishes the brain from other organs tested to date, namely the liver, testis, and spleen. The rat brain microsomal fraction displayed a rather impressive rate of heme oxygenase activity. This fraction also exhibited a rate of NADPH-cytochrome P-450 reductase activity that was sufficient to fully support the oxygenase activity. The brain microsomal fraction was solubilized and subjected to ion-exchange chromatography on DEAE-Sephacel. The chromatographic elution pattern of heme oxygenase activity was compared with those of the liver and testis. In the brain only one peak of heme oxygenase activity was detected. The peak exhibited an elution profile similar to that of HO-2 of the liver and the testis. The presence of an activity peak was not detected in the elution profile at the region where the inducible isoform of heme oxygenase, HO-1, was expected. Cross-reactivity was observed between the solubilized brain microsomal fraction and antiserum to the testis HO-2 when subjected to Ouchterlony double diffusion immunoanalysis. A reaction was not observed when antiserum to liver HO-1 was employed. The presence of HO-2 in the brain microsomal preparation was also established by Western immunoblotting analysis. A protein having a mobility that was identical to the purified testicular HO-2 (Mr 36,000) was present in the brain microsomal preparation when probed with antiserum to HO-2. However, our attempts to demonstrate the presence of HO-1 in the brain microsomal preparation by a similar technique, but using antiserum to HO-1, were not successful. It is proposed that HO-2 is responsible for the bulk, if not all, of the brain microsomal heme oxygenase activity. It is further proposed that tissue-specific regulatory mechanisms are responsible for both the refractory response of the brain heme oxygenase to known metallic inducers and the absence of a detectable amount of the HO-1 isoform.     

Crystal structures of ferrous and CO-, CN(-)-, and NO-bound forms of rat heme oxygenase-1 (HO-1) in complex with heme: structural implications for discrimination between CO and O2 in HO-1

Heme oxygenase (HO) catalyzes heme degradation by utilizing O(2) and reducing equivalents to produce biliverdin IX alpha, iron, and CO. To avoid product inhibition, the heme[bond]HO complex (heme[bond]HO) is structured to markedly increase its affinity for O(2) while suppressing its affinity for CO. We determined the crystal structures of rat ferrous heme[bond]HO and heme[bond]HO bound to CO, CN(-), and NO at 2.3, 1.8, 2.0, and 1.7 A resolution, respectively. The heme pocket of ferrous heme-HO has the same conformation as that of the previously determined ferric form, but no ligand is visible on the distal side of the ferrous heme. Fe[bond]CO and Fe[bond]CN(-) are tilted, whereas the Fe[bond]NO is bent. The structure of heme[bond]HO bound to NO is identical to that bound to N(3)(-), which is also bent as in the case of O(2). Notably, in the CO- and CN(-)-bound forms, the heme and its ligands shift toward the alpha-meso carbon, and the distal F-helix shifts in the opposite direction. These shifts allow CO or CN(-) to bind in a tilted fashion without a collision between the distal ligand and Gly139 O and cause disruption of one salt bridge between the heme and basic residue. The structural identity of the ferrous and ferric states of heme[bond]HO indicates that these shifts are not produced on reduction of heme iron. Neither such conformational changes nor a heme shift occurs on NO or N(3)(-) binding. Heme[bond]HO therefore recognizes CO and O(2) by their binding geometries. The marked reduction in the ratio of affinities of CO to O(2) for heme[bond]HO achieved by an increase in O(2) affinity [Migita, C. T., Matera, K. M., Ikeda-Saito, M., Olson, J. S., Fujii, H., Yoshimura, T., Zhou, H., and Yoshida, T. (1998) J. Biol. Chem. 273, 945-949] is explained by hydrogen bonding and polar interactions that are favorable for O(2) binding, as well as by characteristic structural changes in the CO-bound form.     

Anti-inflammatory effects of HO-1 activity in vascular endothelial cells, commentary on "Carbon monoxide donors or heme oxygenase (HO-1) overexpression blocks interleukin-18-mediated NF-kappaB-PTEN-dependent human cardiac endothelial cell death"

In this issue of Free Radical Biology & Medicine, Zabalgoitia et al. show that IL-18-dependent cell death of human microvascular endothelial cells (EC) is due to activation of p38alpha and NF-kappaB and suppression of p38beta activity. Most interestingly, IL-18 and heme oxygenase-1 (HO-1) activities appear to oppose each other in these cells. IL-18 suppresses HO-1, an effect that is mediated by instability of the HO-1 mRNA. Though the contribution of HO-1 metabolites remains somewhat a mystery, treatment with carbon monoxide releasing molecules (CORMs) also induces these same effects, implicating carbon monoxide (CO) as a major player. HO-1 and CO act to suppress IL-18-mediated activation of p38alpha and to restore p38beta activity, which is suppressed by IL-18. Furthermore, HO-1 and CO suppress NF-kappaB activation by IL-18. This suppression of NF-kappaB reduces levels of PTEN which relieves IL-18-mediated suppression of Akt activity. Thus, HO-1 and CO oppose multiple proinflammatory and pro-cell death effects of IL-18 in human microvascular endothelial cells. The results of this study imply that induction of HO-1 or application of CORMs should be protective to the microvascular endothelium. Clinical trials to test the effects of CORMs in pulmonary inflammation are ongoing. The study by Zabalgoitia et al. provides mechanistic information pertaining to the homeostatic balance of IL-18 and HO-1 activities and may be useful for designing new clinical studies and for interpretation of data from ongoing studies.     

Manganese superoxide dismutase inactivation during Fas (CD95)-mediated apoptosis in Jurkat T cells

Manganese superoxide dismutase (MnSOD, SOD2) is an essential primary antioxidant enzyme which converts superoxide radical to hydrogen peroxide within the mitochondrial matrix. MnSOD plays a prominent role in protection against many apoptotic stimuli. Its absence may therefore impair the cellular redox balance and enhance apoptosis. Our data show that in Jurkat T cells, following oligomerization of the Fas receptor, MnSOD is selectively degraded during apoptosis. In the presence of cycloheximide, an inhibitor of protein synthesis, the rates of cell death and MnSOD degradation were accelerated. Fas-induced MnSOD cleavage was partially inhibited in the presence of the pan-caspase inhibitor, z-VAD-fmk. MnSOD in the mitochondrial fractions was cleaved in vitro by treatment with the cytosolic fraction of Fas-activated cells. Moreover, two possible cleavage sites of recombinant hMnSOD by direct interaction with recombinant caspase-3 were noted. Cellular and mitochondrial factors were found to be necessary for the interaction. These factors include intracellular mobilization of calcium. Our data indicate that inactivation of MnSOD in receptor-mediated apoptosis by caspase-specific degradation would render the mitochondria sensitive to the steady-state production of superoxide, decrease the steady-state flux of H₂O₂, expedite the loss of mitochondrial function, and potentiate apoptosis.     

ApoA1: Mimetic peptide reverses adipocyte dysfunction in vivo and in vitro via an increase in heme oxygenase (HO-1) and Wnt10b

Insulin resistance is a risk factor in the development of type 2 diabetes and is a major cause of atherosclerosis. Reduction in heme oxygenase (HO-1) has been shown to exacerbate vascular dysfunction and insulin resistance in obese mice and involves a decrease in adiponectin levels. Adiponectin is released from mesenchymal stem cell (MSC)-derived adipocytes, its levels are decreased in type 2 diabetes. We hypothesized that the apoA1 mimetic peptide, L-4F, will target the expression of the HO-1-adiponectin axis and reverse adipocyte dysfunction both in vivo and in vitro. The administration of L-4F [2 mg/Kg/daily (i.p.) for 4-week to 8-week-old obese (ob) mice restored adipocyte function, increased adiponectin release (p < 0.05) and decreased the levels of IL-1 and IL-6 (p < 0.05)]. These perturbations were associated with an increase in insulin sensitivity (p < 0.01 vs. untreated ob mice) and decreased glucose levels (309 + 42 vs. 201 + 8 mg/d after L-4F treatment). Treatment of both mesenchymal stem cell (MSC)-derived adipocytes with L-4F (50 µg/ml) increased adiponectin (p < 0.05), decreased IL-1 and IL-6 (p < 0.05) levels and increased MSC-derived adipocyte cell numbers by 50% in S phase (p < 0.05). MSC-derived adipocytes treated with L-4F increased WNT10b and decreased Peg 1/Mest. Inhibition of HO activity reversed the decrease in the adipogenic response gene, Peg 1/Mest. An increase of HO-1 expression by L-4F increased insulin-receptor phosphorylation. These findings support the hypothesis that L-4F increases early adipocyte markers, HO-1-adiponectin, WNT10b and decreases Peg1/Mest, negative regulators of adipocyte differentiation.Key words: diabetes, osteoporosis, osteoblasts, BMP2, heme oxygenase     

Specific cleavage of Mcl-1 by caspase-3 in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in Jurkat leukemia T cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces programmed cell death through the caspase activation cascade and translocation of cleaved Bid (tBid) by the apical caspase-8 to mitochondria to induce oligomerization of multidomain Bax and Bak. However, the roles of prosurvival Bcl-2 family proteins in TRAIL apoptosis remain elusive. Here we showed that, besides the specific cleavage and activation of Bid by caspase-8 and caspase-3, TRAIL-induced apoptosis in Jurkat T cells required the specific cleavage of Mcl-1 at Asp-127 and Asp-157 by caspase-3, while other prototypic antiapoptotic factors such as Bcl-2 or Bcl-X(L) seemed not to be affected. Mutation at Asp-127 and Asp-157 of Mcl-1 led to cellular resistance to TRAIL-induced apoptosis. In sharp contrast to cycloheximide-induced Mcl-1 dilapidation, TRAIL did not activate proteasomal degradation of Mcl-1 in Jurkat cells. We further established for the first time that the C-terminal domain of Mcl-1 became proapoptotic as a result of caspase-3 cleavage, and its physical interaction and cooperation with tBid, Bak, and voltage-dependent anion-selective channel 1 promoted mitochondrial apoptosis. These results suggested that removal of N-terminal domains of Bid by caspase-8 and Mcl-1 by caspase-3 enabled the maximal mitochondrial perturbation that potentiated TRAIL-induced apoptosis.     

A Jurkat T cell variant resistant to death receptor-induced apoptosis. Correlation with heat shock protein (Hsp) 27 and 70 levels

Ligation of Fas induces an apoptotic program in Jurkat cells (Jd). We describe a Jurkat T cell variant (Jr) which shows total resistance to Fas-mediated apoptosis but which exhibits sensitivity to non-death-receptor pro-apoptotic stimuli such as staurosporine. Resistance to Fas-induced apoptosis in Jr cells is correlated with high expression of Hsps. A prior heat-shock increases Hsp27 and 70 expression and protects Jd and Jr cells from Fas- and staurosporine-induced apoptosis. Staurosporine, but not the anti-Fas antibody CH11, abrogates constitutive Hsp70 expression at 37 degrees C and staurosporine also inhibit Hsp27 expression in Jd and Jr cells at 42 degrees C. These data suggest that constitutive expression of Hsp27 inhibits Fas-mediated apoptosis, but only induced expression of Hsp70 can protect T cells from staurosporine-induced apoptosis. Thus, Hsp27 could play a role in the regulation of death receptor-mediated apoptosis, while Hsp70 could regulate mitochondrial-dependent cell death.     

Synergistic induction of delta-aminolevulinate synthase by glutethimide and iron: relationship to the synergistic induction of heme oxygenase

Relationships between activities of delta-aminolevulinate synthase and heme oxygenase, respectively the rate-limiting enzymes of heme biosynthesis and degradation, have been studied in chick embryo liver cell cultures following exposure of the cultures to glutethimide and iron, a combination known to produce a synergistic induction of both enzymes. In time-course experiments, synergistic induction of heme oxygenase activity by glutethimide and iron preceded that of delta-aminolevulinate synthase by 4 h. Effects of selective inhibitors of both heme synthesis and degradation have also been studied with respect to effects on delta-aminolevulinate synthase and heme oxygenase activities. The synergistic induction of heme oxygenase by glutethimide and iron appears to be dependent upon cellular heme synthesis because addition of inhibitors of heme biosynthesis, 4,6-dioxoheptanoic acid or N-methyl-mesoporphyrin abolishes this synergistic induction. Exposure of cultures to tin-mesoporphyrin, a potent inhibitor of heme oxygenase, prevented the synergistic induction of delta-aminolevulinate synthase produced by glutethimide and iron, or, when added after induction was already established, promptly halted any further induction. These results suggest that the level of activity of heme oxygenase can reciprocally modulate intracellular heme levels and thus activity of delta-aminolevulinate synthase.     

Human T-lymphotropic virus type 1 mitochondrion-localizing protein p13II sensitizes Jurkat T cells to Ras-mediated apoptosis

Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia. In addition to typical retroviral structural and enzymatic gene products, HTLV-1 encodes unique regulatory and accessory proteins, including a singly spliced pX open reading frame II (ORF II) product, p13(II). We have demonstrated that proviral clones of HTLV-1 which are mutated in pX ORF II fail to obtain typical proviral loads and antibody responses in a rabbit animal model. p13(II) localizes to mitochondria and reduces cell growth and tumorigenicity in mice, but its function in human lymphocytes remains undetermined. For this study, we analyzed the functional properties of Jurkat T cells expressing p13(II), using both transient and stable expression vectors. Our data indicate that p13(II)-expressing Jurkat T cells are sensitive to caspase-dependent, ceramide- and FasL-induced apoptosis. p13(II)-expressing Jurkat T cells also exhibited reduced proliferation when cultured at a high density. Furthermore, preincubation of the p13(II)-expressing cells with a farnesyl transferase inhibitor, which blocks the posttranslational modification of Ras, markedly reduced FasL-induced apoptosis, indicating the participation of the Ras pathway in p13(II)'s influence on lymphocyte survival. Our data are the first to demonstrate that p13(II) alters Ras-mediated apoptosis in T lymphocytes, and they reveal a potential mechanism by which HTLV-1 alters lymphocyte proliferation.     

Delta9-tetrahydrocannabinol-induced apoptosis in Jurkat leukemia T cells is regulated by translocation of Bad to mitochondria

Plant-derived cannabinoids, including Delta9-tetrahydrocannabinol (THC), induce apoptosis in leukemic cells, although the precise mechanism remains unclear. In the current study, we investigated the effect of THC on the upstream and downstream events that modulate the extracellular signal-regulated kinase (ERK) module of mitogen-activated protein kinase pathways primarily in human Jurkat leukemia T cells. The data showed that THC down-regulated Raf-1/mitogen-activated protein kinase/ERK kinase (MEK)/ERK/RSK pathway leading to translocation of Bad to mitochondria. THC also decreased the phosphorylation of Akt. However, no significant association of Bad translocation with phosphatidylinositol 3-kinase/Akt and protein kinase A signaling pathways was noted when treated cells were examined in relation to phosphorylation status of Bad by Western blot and localization of Bad to mitochondria by confocal analysis. Furthermore, THC treatment decreased the Bad phosphorylation at Ser(112) but failed to alter the level of phospho-Bad on site Ser(136) that has been reported to be associated with phosphatidylinositol 3-kinase/Akt signal pathway. Jurkat cells expressing a constitutively active MEK construct were found to be resistant to THC-mediated apoptosis and failed to exhibit decreased phospho-Bad on Ser(112) as well as Bad translocation to mitochondria. Finally, use of Bad small interfering RNA reduced the expression of Bad in Jurkat cells leading to increased resistance to THC-mediated apoptosis. Together, these data suggested that Raf-1/MEK/ERK/RSK-mediated Bad translocation played a critical role in THC-induced apoptosis in Jurkat cells.     

AT 101 induces early mitochondrial dysfunction and HMOX1 (heme oxygenase 1) to trigger mitophagic cell death in glioma cells

In most cases, macroautophagy/autophagy serves to alleviate cellular stress and acts in a pro-survival manner. However, the effects of autophagy are highly contextual, and autophagic cell death (ACD) is emerging as an alternative paradigm of (stress- and drug-induced) cell demise. AT 101 ([-]-gossypol), a natural compound from cotton seeds, induces ACD in glioma cells as confirmed here by CRISPR/Cas9 knockout of ATG5 that partially, but significantly rescued cell survival following AT 101 treatment. Global proteomic analysis of AT 101-treated U87MG and U343 glioma cells revealed a robust decrease in mitochondrial protein clusters, whereas HMOX1 (heme oxygenase 1) was strongly upregulated. AT 101 rapidly triggered mitochondrial membrane depolarization, engulfment of mitochondria within autophagosomes and a significant reduction of mitochondrial mass and proteins that did not depend on the presence of BAX and BAK1. Conversely, AT 101-induced reduction of mitochondrial mass could be reversed by inhibiting autophagy with wortmannin, bafilomycin A₁ and chloroquine. Silencing of HMOX1 and the mitophagy receptors BNIP3 (BCL2 interacting protein 3) and BNIP3L (BCL2 interacting protein 3 like) significantly attenuated AT 101-dependent mitophagy and cell death. Collectively, these data suggest that early mitochondrial dysfunction and HMOX1 overactivation synergize to trigger lethal mitophagy, which contributes to the cell killing effects of AT 101 in glioma cells.

Abbreviations: ACD, autophagic cell death; ACN, acetonitrile; AT 101, (-)-gossypol; BAF, bafilomycin A₁; BAK1, BCL2-antagonist/killer 1; BAX, BCL2-associated X protein; BH3, BCL2 homology region 3; BNIP3, BCL2 interacting protein 3; BNIP3L, BCL2 interacting protein 3 like; BP, Biological Process; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; CC, Cellular Component; Con, control; CQ, chloroquine; CRISPR, clustered regularly interspaced short palindromic repeats; DMEM, Dulbecco’s Modified Eagle Medium; DTT, 1,4-dithiothreitol; EM, electron microscopy; ER, endoplasmatic reticulum; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GO, Gene Ontology; HAcO, acetic acid; HMOX1, heme oxygenase 1; DKO, double knockout; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; LPL, lipoprotein lipase, MEFs, mouse embryonic fibroblasts; mPTP, mitochondrial permeability transition pore; MTG, MitoTracker Green FM; mt-mKeima, mito-mKeima; MT-ND1, mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1; PBS, phosphate-buffered saline; PE, phosphatidylethanolamine; PI, propidium iodide; PRKN, parkin RBR E3 ubiquitin protein ligase; SDS, sodium dodecyl sulfate; SQSTM1/p62, sequestome 1; STS, staurosporine; sgRNA, single guide RNA; SILAC, stable isotope labeling with amino acids in cell culture; TFA, trifluoroacetic acid, TMRM, tetramethylrhodamine methyl ester perchlorate; WM, wortmannin; WT, wild-type