Diverse functions of cytochrome c in cell death and disease.

The redox-active protein cytochrome c is a highly positively charged hemoglobin that regulates cell fate decisions of life and death. Under normal physiological conditions, cytochrome c is localized in the mitochondrial intermembrane space, and its distribution can extend to the cytosol, nucleus, and extracellular space under specific pathological or stress-induced conditions. In the mitochondria, cytochrome c acts as an electron carrier in the electron transport chain, facilitating adenosine triphosphate synthesis, regulating cardiolipin peroxidation, and influencing reactive oxygen species dynamics. Upon cellular stress, it can be released into the cytosol, where it interacts with apoptotic peptidase activator 1 (APAF1) to form the apoptosome, initiating caspase-dependent apoptotic cell death. Additionally, following exposure to pro-apoptotic compounds, cytochrome c contributes to the survival of drug-tolerant persister cells. When translocated to the nucleus, it can induce chromatin condensation and disrupt nucleosome assembly. Upon its release into the extracellular space, cytochrome c may act as an immune mediator during cell death processes, highlighting its multifaceted role in cellular biology. In this review, we explore the diverse structural and functional aspects of cytochrome c in physiological and pathological responses. We summarize how posttranslational modifications of cytochrome c (e.g., phosphorylation, acetylation, tyrosine nitration, and oxidation), binding proteins (e.g., HIGD1A, CHCHD2, ITPR1, and nucleophosmin), and mutations (e.g., G41S, Y48H, and A51V) affect its function. Furthermore, we provide an overview of the latest advanced technologies utilized for detecting cytochrome c, along with potential therapeutic approaches related to this protein. These strategies hold tremendous promise in personalized health care, presenting opportunities for targeted interventions in a wide range of conditions, including neurodegenerative disorders, cardiovascular diseases, and cancer.

Involvement of Bid in the crosstalk between ferroptotic agent-induced ER stress and TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces death receptor-mediated extrinsic apoptosis, specifically in cancer cells, and Bid (BH3-interacting domain death agonist) plays an important role in TRAIL-induced apoptosis. Ferroptosis is a newly defined form of regulated cell death known to be distinct from other forms of cell death. However, our previous studies have shown that ferroptosis shares common pathways with other types of programmed cell death such as apoptosis. In this study, we investigated the role of Bid in the crosstalk between the ferroptotic agent-induced endoplasmic reticulum (ER) stress response and TRAIL-induced apoptosis. When human colorectal carcinoma HCT116 cells were treated with the ferroptosis-inducing agents artesunate and erastin in combination with TRAIL, TRAIL-induced activation of caspase-8 was enhanced, and subsequently, the truncation of Bid was increased. Similar results were observed when ovarian adenocarcinoma OVCAR-3 cells were treated with the ferroptotic agents in combination with TRAIL. Results from studies with Bid mutants reveal that the truncation of Bid and the presence of intact BH3 domains are critical for synergistic apoptosis. Nonfunctional Bid mutants were not able to activate the mitochondria-dependent apoptosis pathway, which is required for the conversion of p19 to p17, the active form of caspase-3. These results indicate that Bid plays a critical role in the crosstalk between the ferroptotic agent-induced ER stress response and TRAIL-induced apoptosis.

Ferroptosis Inducer Improves the Efficacy of Oncolytic Virus-Mediated Cancer Immunotherapy.

Ferroptosis is a type of programmed cell death dependent on iron and characterized by the accumulation of lipid peroxides. In this study, we explore the combination of a ferroptosis activator with an oncolytic vaccinia virus in tumor models. Erastin induced cell death in hepatoma, colon, and ovarian cancer cells, but not in melanoma cancer cells. Erastin, not the oncolytic vaccinia virus (OVV), induced the expression of key marker genes for ferroptosis in cancer cells. In hepatocellular carcinoma and colon cancer models, either erastin or OVV inhibited tumor growth, but a combination of the two yielded the best therapeutic effects, as indicated by inhibited tumor growth or regression and longer host survival. Immunological analyses indicate that erastin alone had little or no effect on systemic immunity or local immunity in the tumor. However, when combined with OV, erastin enhanced the number of activated dendritic cells and the activity of tumor-infiltrating T lymphocytes as indicated by an increase in IFN-γ+CD8+ and PD-1+CD8+ T cells. These results demonstrate that erastin can exert cytotoxicity on cancer cells via ferroptosis, but has little effect on immune activity by itself. However, when combined with an OVV, erastin promoted antitumoral immunity and efficacy by increasing the number of activated dendritic cells and promoting the activities of tumor specific CD8+ T cells in the tumor.

The emerging role of selenium metabolic pathways in cancer: New therapeutic targets for cancer.

Selenium (Se) is incorporated into the body via the selenocysteine (Sec) biosynthesis pathway, which is critical in the synthesis of selenoproteins, such as glutathione peroxidases and thioredoxin reductases. Selenoproteins, which play a key role in several biological processes, including ferroptosis, drug resistance, endoplasmic reticulum stress, and epigenetic processes, are guided by Se uptake. In this review, we critically analyze the molecular mechanisms of Se metabolism and its potential as a therapeutic target for cancer. Sec insertion sequence binding protein 2 (SECISBP2), which is a positive regulator for the expression of selenoproteins, would be a novel prognostic predictor and an alternate target for cancer. We highlight strategies that attempt to develop a novel Se metabolism-based approach to uncover a new metabolic drug target for cancer therapy. Moreover, we expect extensive clinical use of SECISBP2 as a specific biomarker in cancer therapy in the near future. Of note, scientists face additional challenges in conducting successful research, including investigations on anticancer peptides to target SECISBP2 intracellular protein.

The anti-fibrotic drug pirfenidone inhibits liver fibrosis by targeting the small oxidoreductase glutaredoxin-1.

Activation of the hepatic stellate cells (HSCs) is a key pathogenic event in liver fibrosis. Protein S-glutathionylation (PSSG) of cysteine residues is a distinct form of oxidative response that modifies protein structures and functions. Glutaredoxin-1 (GLRX) reverses PSSG by liberating glutathione (GSH). In this study, we showed that pirfenidone (PFD), an anti-lung fibrosis drug, inhibited HSC activation and liver fibrosis in a GLRX-dependent manner. Glrx depletion exacerbated liver fibrosis, and decreased GLRX and increased PSSG were observed in fibrotic mouse and human livers. In contrast, overexpression of GLRX inhibited PSSG and liver fibrosis. Mechanistically, the inhibition of HSC activation by GLRX may have been accounted for by deglutathionylation of Smad3, which inhibits Smad3 phosphorylation, leading to the suppression of fibrogenic gene expression. Our results have established GLRX as the therapeutic target of PFD and uncovered an important role of PSSG in liver fibrosis. GLRX/PSSG can be both a biomarker and a therapeutic target for liver fibrosis.

Glucose deprivation-induced endoplasmic reticulum stress response plays a pivotal role in enhancement of TRAIL cytotoxicity.

Abnormalities of the tumor vasculature result in insufficient blood supply and development of a tumor microenvironment that is characterized by low glucose concentrations, low extracellular pH, and low oxygen tensions. We previously reported that glucose-deprived conditions induce metabolic stress and promote tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity. In this study, we examined whether the metabolic stress-associated endoplasmic reticulum (ER) stress response pathway plays a pivotal role in the enhancement of TRAIL cytotoxicity. We observed no significant cytotoxicity when human colorectal cancer SW48 cells were treated with various doses of TRAIL (2-100 ng/ml) for 4 h or glucose (0-25 mM) for 24 h. However, a combination of TRAIL and low glucose-induced dose-dependent apoptosis through activation of caspases (-8, -9, and -3). Studies with activating transcription factor 4 (ATF4), C/EBP-homologous protein (CHOP), p53 upregulated modulator of apoptosis (PUMA), or death receptor 5 (DR5)-deficient mouse embryonic fibroblasts or HCT116 cells suggest that the ATF4-CHOP-PUMA axis and the ATF4-CHOP-DR5 axis are involved in the combined treatment-induced apoptosis. Moreover, the combined treatment-induced apoptosis was completely suppressed in BH3 interacting-domain death agonist (Bid)- or Bcl-2-associated X protein (Bax)-deficient HCT116 cells, but not Bak-deficient HCT116 cells. Interestingly, the combined treatment-induced Bax oligomerization was suppressed in PUMA-deficient HCT116 cells. These results suggest that glucose deprivation enhances TRAIL-induced apoptosis by integrating the ATF4-CHOP-PUMA axis and the ATF4-CHOP-DR5 axis, consequently amplifying the Bid-Bax-associated mitochondria-dependent pathway.

Improved chemosensitivity following mucolytic therapy in patient-derived models of mucinous appendix cancer.

Abundant intraperitoneal (IP) accumulation of extracellular mucus in patients with appendiceal mucinous carcinoma peritonei (MCP) causes compressive organ dysfunction and prevents delivery of chemotherapeutic drugs to cancer cells. We hypothesized that reducing extracellular mucus would decrease tumor-related symptoms and improve chemotherapeutic effect in patient-derived models of MCP. Mucolysis was achieved using a combination of bromelain (BRO) and N-acetylcysteine (NAC). Ex vivo experiments of mucolysis and chemotherapeutic drug delivery/effect were conducted with MCP and non-MCP tissue explants. In vivo experiments were performed in mouse and rat patient-derived xenograft (PDX) models of early and late (advanced) MCP. MCP tumor explants were less chemosensitive than non-MCP explants. Chronic IP administration of BRO + NAC in a mouse PDX model of early MCP and a rat PDX model of late (advanced) MCP converted solid mucinous tumors into mucinous ascites (mucolysis) that could be drained via a percutaneous catheter (rat model only), significantly reduced solid mucinous tumor growth and improved the efficacy of chemotherapeutic drugs. Combination of BRO + NAC efficiently lyses extracellular mucus in clinically relevant models of MCP. Conversion of solid mucinous tumors into mucinous ascites decreases tumor bulk and allows for minimally invasive drainage of liquified tumors. Lysis of extracellular mucus removes the protective mucinous coating surrounding cancer cells and improves chemotherapeutic drug delivery/efficacy in cancer cells. Our data provide a preclinical rationale for the clinical evaluation of BRO + NAC as a therapeutic strategy for MCP.

Synergistic apoptosis following endoplasmic reticulum stress aggravation in mucinous colon cancer.

BACKGROUND: Mucinous colon cancers (MCC) are characterized by abundant production of mucin 2 (MUC2) protein and are less sensitive to standard systemic chemotherapy. We postulated that severe/persistent endoplasmic reticulum stress (ERS) aggravation in MCC would overwhelm compensatory cytoprotective pathways and induce apoptosis. RESULTS: Basal levels of ERS markers were higher in MCC and dnTCF-LS174T cells than non-mucinous tumors and these levels were significantly increased by combinatorial treatment with ERS aggravators celecoxib + orlistat. Combination treatment inhibited cell viability and synergistically induced apoptosis. Treatment-induced cell death was ERS-dependent, apoptotic pathways were not activated following knockdown of ERS protein CHOP. Dual drug treatment significantly reduced mucinous tumor growth in vivo and induced ERS and apoptosis, consistent with in vitro experiments. CONCLUSIONS: Novel therapies are needed since MCC are more resistant to standard systemic chemotherapy. This study suggests ERS aggravation is a viable therapeutic strategy to reduce tumor growth in MCC.

BAX-dependent mitochondrial pathway mediates the crosstalk between ferroptosis and apoptosis.

Ferroptosis is considered a distinctive form of cell death compared to other types of death such as apoptosis. It is known to result from iron-dependent accumulation of lipid peroxides rather than caspase activation. However, we reported recently that ferroptosis interplays with apoptosis. In this study, we investigated a possible mechanism of this interplay between ferroptosis and apoptosis. Results from our studies reveal that combined treatment of the ferroptotic agent erastin and the apoptotic agent TRAIL effectively disrupted mitochondrial membrane potential (ΔΨm) and subsequently promoted caspase activation. The alterations of mitochondrial membrane potential are probably due to an increase in oligomerization of BAX and its accumulation at the mitochondria during treatment with erastin and TRAIL. Interestingly, the combined treatment-promoted apoptosis was effectively inhibited in BAX-deficient HCT116 cells, but not BAK-deficient cells. These results indicate that the BAX-associated mitochondria-dependent pathway plays a pivotal role in erastin-enhanced TRAIL-induced apoptosis.

Ferroptotic agent-induced endoplasmic reticulum stress response plays a pivotal role in the autophagic process outcome.

Ferroptosis has been reported as a unique form of cell death. However, in recent years, researchers have increasingly challenged the uniqueness of ferroptosis compared to other types of cell death. In this study, we examined whether ferroptosis shares cell death pathways with other types of cell death, especially autophagy, via the autophagic process. Here, we observed that ferroptosis inducers (artesunate [ART] and erastin [ERA]) and autophagy inducers (bortezomib [BOR] and XIE62-1004) led to autophagosome formation via the endoplasmic reticulum (ER) stress response. Unlike XIE62-1004, ART, ERA, and BOR, which affect glutathione production or utilization, induced oxidative stress responses-an increase in the levels of heme oxygenase-1 and lipid peroxidation. Oxidative stress responses were attenuated by deletion of autophagy-related gene-5 or treatment with autophagy inhibitors (bafilomycin and chloroquine). Our studies provide an overview of common death pathways-the ER stress response-associated autophagic process in ferroptosis and autophagy. We also highlight the role played by glutathione redox system in the outcome of the autophagic process.

Rational application of targeted therapeutics in mucinous colon/appendix cancers with positive predictive factors.

Molecular-targeted therapies have demonstrated disappointing results against most advanced solid cancers. This may largey be attributed to irrational drug use against unselected cancers. We investigated the efficacy of dual MEK-PI3K drug therapy against KRAS mutated mucin 2 (MUC2)-secreting LS174T cells and patient-derived ex vivo and in vivo models of KRAS mutated mucinous colon/appendix cancers. These tumors demonstrate unique phenotypic and genotypic features that likely predict sensitivity to this targeted co-therapy. Co-treatment with MEK inhibitor (trametinib) and PI3K inhibitor (pictilisib)-induced synergistic cytotoxicity and intrinsic mitochondrial-mediated apoptosis in LS174T cells and tumor explants in vitro. Dual drug therapy also induced endoplasmic reticulum stress (ERS)-associated proteins (GRP78/BiP, ATF4, and CHOP). However, CHOP knock-down assays demonstrated that mitochondrial-mediated apoptosis in LS174T cells was not ERS-dependent. Dual drug therapy also significantly decreased MUC2 expression, MUC2 post-translational modification (palmitoylation) and secretion in LS174T cells, suggesting a simultaneous cytotoxic and mucin suppressive mechanism of action. We also demonstrated effective mucinous tumor growth suppression in ex vivo epithelial organoid (colonoid) cultures and in in vivo intraperitoneal patient-derived xenograft models derived from mucinous colon/appendix cancer. These promising preclinical data support a role for dual MEK-PI3K inhibitor therapy in mucinous colon/appendix cancers. We postulate that mucinous KRAS mutated cancers are especially vulnerable to this co-treatment based on their unique phenotypic and genotypic characteristics.

Genomic profiling of the residual disease of advanced high-grade serous ovarian cancer after neoadjuvant chemotherapy.

The goal of our study was to demonstrate the spectrum of genomic alterations present in the residual disease of patients with advanced high-grade serous ovarian cancer (HGSOC) after neoadjuvant chemotherapy (NAC), including matched pretreatment biopsies. During the study period between 2006 and 2017, we collected pre-NAC and post-NAC tumor tissue samples from patients with advanced HGSOC. We performed combined next-generation sequencing and immunohistochemistry to identify actionable targets and pathway activation in post-NAC residual tumors. We also examined whether post-NAC profiling of residual HGSOC identified targetable molecular lesions in the chemotherapy-resistant component of tumors. Among 102 post-NAC samples, 41 (40%) of patients had mutations in homologous recombination repair (HRR) genes (HRR deficiency). Patients with HRR mutations had higher tumor mutation burdens (p < 0.001) and higher alterations in the PI3K-AKT-mTOR pathway (p = 0.004) than patients without these HRR mutations. Nevertheless, we found no significant differences in progression-free survival (p = 0.662) and overall survival (OS; p = 0.828) between the two groups. Most patients (91%) had alterations in at least one of the targetable pathways, and those patients with cell cycle (p = 0.004) and PI3K-AKT-mTOR signaling (p = 0.005) pathway alterations had poorer OS (Bonferroni-corrected threshold = 0.0083, 0.05/6). We showed the genomic landscape of tumor cells remaining in advanced HGSOC after NAC. Once validated, these data can help inform biomarker-driven adjuvant studies in targeting residual tumors to improve the outcomes of patients with advanced HGSOC after NAC.

Ferroptosis-inducing agents enhance TRAIL-induced apoptosis through upregulation of death receptor 5.

Ferroptosis is considered genetically and biochemically distinct from other forms of cell death. In this study, we examined whether ferroptosis shares cell death pathways with other types of cell death. When human colon cancer HCT116, CX-1, and LS174T cells were treated with ferroptotic agents such as sorafenib (SRF), erastin, and artesunate, data from immunoblot assay showed that ferroptotic agents induced endoplasmic reticulum (ER) stress and the ER stress response-mediated expression of death receptor 5 (DR5), but not death receptor 4. An increase in the level of DR5, which is activated by binding to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and initiates apoptosis, was probably responsible for synergistic apoptosis when cells were treated with ferroptotic agent in combination with TRAIL. This collateral effect was suppressed in C/EBP (CCAAT-enhancer-binding protein)-homologous protein (CHOP)-deficient mouse embryonic fibroblasts or DR5 knockdown HCT116 cells, but not in p53-deficient HCT116 cells. The results from in vitro studies suggest the involvement of the p53-independent CHOP/DR5 axis in the synergistic apoptosis during the combinatorial treatment of ferroptotic agent and TRAIL. The synergistic apoptosis and regression of tumor growth were also observed in xenograft tumors when SRF and TRAIL were administered to tumor-bearing mice.

PARK7 modulates autophagic proteolysis through binding to the N-terminally arginylated form of the molecular chaperone HSPA5.

Macroautophagy is induced under various stresses to remove cytotoxic materials, including misfolded proteins and their aggregates. These protein cargoes are collected by specific autophagic receptors such as SQSTM1/p62 (sequestosome 1) and delivered to phagophores for lysosomal degradation. To date, little is known about how cells sense and react to diverse stresses by inducing the activity of SQSTM1. Here, we show that the peroxiredoxin-like redox sensor PARK7/DJ-1 modulates the activity of SQSTM1 and the targeting of ubiquitin (Ub)-conjugated proteins to macroautophagy under oxidative stress caused by TNFSF10/TRAIL (tumor necrosis factor [ligand] superfamily, member 10). In this mechanism, TNFSF10 induces the N-terminal arginylation (Nt-arginylation) of the endoplasmic reticulum (ER)-residing molecular chaperone HSPA5/BiP/GRP78, leading to cytosolic accumulation of Nt-arginylated HSPA5 (R-HSPA5). In parallel, TNFSF10 induces the oxidation of PARK7. Oxidized PARK7 acts as a co-chaperone-like protein that binds the ER-derived chaperone R-HSPA5, a member of the HSPA/HSP70 family. By forming a complex with PARK7 (and possibly misfolded protein cargoes), R-HSPA5 binds SQSTM1 through its Nt-Arg, facilitating self-polymerization of SQSTM1 and the targeting of SQSTM1-cargo complexes to phagophores. The 3-way interaction among PARK7, R-HSPA5, and SQSTM1 is stabilized by the Nt-Arg residue of R-HSPA5. PARK7-deficient cells are impaired in the targeting of R-HSPA5 and SQSTM1 to phagophores and the removal of Ub-conjugated cargoes. Our results suggest that PARK7 functions as a co-chaperone for R-HSPA5 to modulate autophagic removal of misfolded protein cargoes generated by oxidative stress.

Correction: Molecular crosstalk between ferroptosis and apoptosis: Emerging role of ER stress-induced p53-independent PUMA expression.

[This corrects the article DOI: 10.18632/oncotarget.23046.].

Peripheral cannabinoid 1 receptor blockade mitigates adipose tissue inflammation via NLRP3 inflammasome in mouse models of obesity.

AIM: To analyze the metabolic parameters and adipose tissue inflammation via NLRP3 inflammasome following chronic treatment of mouse models of obesity with AJ5018 as the peripherally restricted cannabinoid 1 receptor (CB1R) antagonist. MATERIALS AND METHODS: The selectivity for CB1R over CB2R, brain/plasma concentration ratio, and centrally mediated neurobehavioural effects of AJ5018, were assessed. The long-term effects of AJ5018 and rimonabant on the metabolic parameters and adipose tissue inflammation were analyzed in diet-induced obese (DIO) mice and diabetic db/db mice. RESULTS: AJ5018 had a higher degree of selectivity for CB1R over CB2R and markedly reduced brain penetrance, as reflected by the lower brain/plasma concentration ratio and the attenuated centrally mediated neurobehavioural effects, compared with its brain-penetrant parent compound rimonabant. In DIO and db/db mice, AJ5018 exhibited comparable effects to rimonabant in improving metabolic abnormalities and suppressing macrophage infiltration into white adipose tissue, activation of the NLRP3 inflammasome, and production of proinflammatory cytokines. CONCLUSIONS: These results suggest that peripheral CB1R blockade improves obesity-induced insulin resistance by suppressing adipose tissue inflammation via the NLRP3 inflammasome.

Crosstalk Between Apoptosis and Autophagy Is Regulated by the Arginylated BiP/Beclin-1/p62 Complex.

Emerging evidence demonstrates that autophagy and apoptosis are interconnected and their interplay greatly affects cell death. However, the key regulators in this crosstalk remain elusive. Therefore, the role of N-terminal arginylated BiP (R-BiP)/Beclin-1/p62 complex was examined in the crosstalk between apoptosis and autophagy during combination chemotherapy with mitomycin C and bortezomib using immunoblot, immunoprecipitation, and cellular imaging assays in wild-type (WT) and genetically engineered colorectal cancer cells. In addition, the tumoricidal efficacy of the combinatorial treatment in a nude mouse tumor xenograft model of colorectal cancer was assessed. Bortezomib combined with mitomycin C synergistically induced cytotoxicity and apoptosis rather than autophagy. Mechanistically, this combination inactivated Akt and subsequently induced Beclin-1 (BECN1) dephosphorylation at Ser 234/295. Dephosphorylation of Beclin-1 resulted in increased cleavage of Beclin-1 and disruption of the R-BiP/Beclin-1/p62 complex, which led to switching autophagy to the synergistic induction of apoptosis. Importantly, the combination significantly suppressed LS174T intraperitoneal xenograft tumor growth, induced Akt inactivation and Beclin-1 cleavage, and decreased autophagy in vivo Moreover, the tumoricidal efficacy of the combinatorial treatment was less effective, in vitro and in vivo, in HCT116 tumors harboring a Beclin-1 caspase 8 cleavage site mutant knock-in.Implications: This study uncovers that the R-BiP/Beclin-1/p62 complex has an important role in the crosstalk between apoptosis and autophagy. The results also propose how mono-drug resistance can be overcome using potent combinations to improve anticancer therapy. Mol Cancer Res; 16(7); 1077-91. ©2018 AACR.

Ferroptosis-Induced Endoplasmic Reticulum Stress: Cross-talk between Ferroptosis and Apoptosis.

Since its discovery in 2012, ferroptosis has been well characterized by the accumulation of lipid peroxides due to the failure of glutathione-dependent antioxidant defenses. It is known as an iron-dependent form of programmed cell death, which is distinct from other forms of cell death such as apoptosis and necrosis. Nonetheless, little is known about the ferroptotic agent-induced endoplasmic reticulum (ER) stress response and its role in cell death. Recent studies reveal that the ferroptotic agent-induced ER stress response plays an important role in the cross-talk between ferroptosis and other types of cell death. Ferroptotic agents induce the unfolded protein response and subsequently ER stress-mediated activation of the PERK-eIF2α-ATF4-CHOP pathway. CHOP (C/EBP homologous protein) signaling pathway-mediated p53-independent PUMA (p53 upregulated modulator of apoptosis) expression is involved in the synergistic interaction between ferroptosis and apoptosis. This review highlights the recent literature on ferroptotic and apoptotic agent interactions through the ER stress-mediated PERK-eIF2α-ATF4-CHOP-PUMA pathway and implicates combined treatment to effectively enhance tumoricidal efficacy as a novel therapeutic strategy for cancer. Mol Cancer Res; 16(7); 1073-6. ©2018 AACR.