Tumor suppressor Par-4 activates autophagy-dependent ferroptosis.

Ferroptosis is a unique iron-dependent form of non-apoptotic cell death characterized by devastating lipid peroxidation. Whilst growing evidence suggests that ferroptosis is a type of autophagy-dependent cell death, the underlying molecular mechanisms regulating ferroptosis are largely unknown. In this study, through an unbiased RNA-sequencing screening, we demonstrate the activation of a multi-faceted tumor-suppressor protein Par-4/PAWR during ferroptosis. Functional studies reveal that genetic depletion of Par-4 effectively blocks ferroptosis, whereas Par-4 overexpression sensitizes cells to undergo ferroptosis. More importantly, we have determined that Par-4-triggered ferroptosis is mechanistically driven by the autophagic machinery. Upregulation of Par-4 promotes activation of ferritinophagy (autophagic degradation of ferritin) via the nuclear receptor co-activator 4 (NCOA4), resulting in excessive release of free labile iron and, hence, enhanced lipid peroxidation and ferroptosis. Inhibition of Par-4 dramatically suppresses the NCOA4-mediated ferritinophagy signaling axis. Our results also establish that Par-4 activation positively correlates with reactive oxygen species (ROS) production, which is critical for ferritinophagy-mediated ferroptosis. Furthermore, Par-4 knockdown effectively blocked ferroptosis-mediated tumor suppression in the mouse xenograft models. Collectively, these findings reveal that Par-4 has a crucial role in ferroptosis, which could be further exploited for cancer therapy.

Targeting oxeiptosis-mediated tumor suppression: a novel approach to treat colorectal cancers by sanguinarine.

Oxeiptosis is a recently identified reactive oxygen species (ROS)-sensitive, caspase independent, non-inflammatory regulated cell death pathway. The activation of Kelch-like ECH-associated protein 1-Phosphoglycerate mutase 5-Apoptosis inducing factor mitochondria associated 1 (KEAP1-PGAM5-AIFM1) pathway is the key signaling event in the execution of oxeiptosis. In the present study, we demonstrate that sanguinarine (SNG), a quaternary benzophenanthridine alkaloid, induces oxeiptosis in human colorectal cancer (CRC) cells via ROS, specifically hydrogen peroxide (H2O2)-dependent activation of KEAP1-PGAM5-AIFM1 signaling axis. Whilst, knockdown of KEAP1, PGAM5, and AIFM1 largely abolishes SNG-induced oxeiptosis, hence reinforcing the importance of the role of this pathway in the SNG-mediated cytotoxicity. Moreover, extracellular addition of H2O2 sensitizes SNG-induced oxeiptosis in CRC cells, while removal of intracellular ROS by ROS scavengers, not only alleviated the overproduction of ROS caused by SNG, but also reversed the biochemical events associated with oxeiptosis. Finally, in vivo study demonstrates that SNG effectively reduces the tumor growth in HT-29 xenograft mouse model through features associated with oxeiptosis. This study highlights oxeiptosis as a novel tumor suppressive mechanism and further investigation of the role of oxeiptosis in cancer treatment is warranted.

Sanguinarine Induces H2O2-Dependent Apoptosis and Ferroptosis in Human Cervical Cancer.

Sanguinarine (SNG) is a benzophenanthridine alkaloid isolated mainly from Sanguinaria canadensis, Chelidonium majus, and Macleaya cordata. SNG is considered an antineoplastic agent based on its cytotoxic activity against various tumors. However, the exact molecular mechanism through which SNG mediates this activity has not been elucidated. Here, we report that SNG induces death in human cervical cancer (HeLa) cells through activation of two interdependent cell death pathways-apoptosis and ferroptosis. SNG-induced apoptosis was characterized by caspase activation and PARP cleavage, while ferroptosis involved solute carrier family 7 member 11 (SLC7A11) down-regulation, glutathione (GSH) depletion, iron accumulation, and lipid peroxidation (LPO). Interestingly, incubation with caspase inhibitor z-VAD-fmk not only inhibited the features of apoptosis, but also negated markers of SNG-induced ferroptosis. Similarly, pretreatment with ferroptosis inhibitor ferrostatin-1 (Fer-1), apart from rescuing cells from SNG-induced ferroptosis, also curbed the features of SNG-induced apoptosis. Our study implies that, together, apoptosis and ferroptosis act as partners in the context of SNG mediated tumor suppression in HeLa cells. Importantly, SNG increased the generation of reactive oxygen species (ROS), and ROS inhibition blocks the induction of both apoptosis and ferroptosis. These findings highlight the value of continued investigation into the potential use of SNG as an antineoplastic agent.

Caspase cleavage and nuclear retention of the energy sensor AMPK-α1 during apoptosis.

AMP-activated protein kinase (AMPK) coordinates energy homeostasis during metabolic and energy stress. We report that the catalytic subunit isoform AMPK-α1 (but not α2) is cleaved by caspase-3 at an early stage during induction of apoptosis. AMPK-α1 cleavage occurs following Asp529, generating an ∼58-kDa N-terminal fragment (cl-AMPK-α1) and leading to the precise excision of the nuclear export sequence (NES) from the C-terminal end. This cleavage does not affect (1) the stability of pre-formed heterotrimeric complexes, (2) the ability of cl-AMPK-α1 to become phosphorylated and activated by the upstream kinases LKB1 or CaMKK2, or (3) allosteric activation by AMP or A-769662. Importantly, cl-AMPK-α1 is only detectable in the nucleus, consistent with removal of the NES, and ectopic expression of cleavage-resistant D529A-mutant AMPK-α1 promotes cell death induced by cytotoxic agents. Thus, we have elucidated a non-canonical mechanism of AMPK activation within the nucleus, which protects cells against death induced by DNA damage.

Prostate apoptosis response-4 and tumor suppression: it's not just about apoptosis anymore.

The tumor suppressor prostate apoptosis response-4 (Par-4) has recently turned 'twenty-five'. Beyond its indisputable role as an apoptosis inducer, an increasing and sometimes bewildering, new roles for Par-4 are being reported. These roles include its ability to regulate autophagy, senescence, and metastasis. This growing range of responses to Par-4 is reflected by our increasing understanding of the various mechanisms through which Par-4 can function. In this review, we summarize the existing knowledge on Par-4 tumor suppressive mechanisms, and discuss how the interaction of Par-4 with different regulators influence cell fate. This review also highlights the new secretory pathway that has emerged and the likely discussion on its clinical implications.

Acid sphingomyelinase-dependent autophagic degradation of GPX4 is critical for the execution of ferroptosis.

Ferroptosis is a type of regulated cell death characterized by ROS accumulation and devastating lipid peroxidation (LPO). The role of acid sphingomyelinase (ASM), a key enzyme in sphingolipid metabolism, in the induction of apoptosis has been studied; however, to date its role in ferroptosis is unclear. In this study, we report that ASM plays a hitherto unanticipated role in promoting ferroptosis. Mechanistically, Erastin (Era) treatment results in the activation of ASM and generation of ceramide, which are required for the Era-induced reactive oxygen species (ROS) generation and LPO. Inhibition of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) or removal of intracellular ROS, significantly reduced Era-induced ASM activation, suggesting that NADPH oxidase-derived ROS regulated ASM-initiated redox signaling in a positive feedback manner. Moreover, ASM-mediated activation of autophagy plays a critical role in ferroptosis inducers (FINs)-induced glutathione peroxidase 4 (GPX4) degradation and ferroptosis activation. Genetic or pharmacological inhibition of ASM diminishes Era-induced features of autophagy, GPX4 degradation, LPO, and subsequent ferroptosis. Importantly, genetic activation of ASM increases ferroptosis in cancer cells induced by various FINs. Collectively, these findings reveal that ASM plays a novel role in ferroptosis that could be exploited to improve pathological conditions that link to ferroptosis.

Superoxide-mediated ferroptosis in human cancer cells induced by sodium selenite.

Ferroptosis is a novel form of programmed cell death characterized by an iron-dependent increase in reactive oxygen species (ROS). However, the role of ROS in the regulation of ferroptosis remains elusive. In this study, for the first time, we demonstrate that sodium selenite (SS), a well-established redox-active selenium compound, is a novel inducer of ferroptosis in a variety of human cancer cells. Potent ferroptosis inhibitors, such as ferrostatin-1 (Fer-1) and deferoxamine (DFO), rescue cells from SS-induced ferroptosis. Furthermore, SS down-regulates ferroptosis regulators; solute carrier family 7 member 11 (SLC7A11), glutathione (GSH), and glutathione peroxidase 4 (GPx4), while it up-regulates iron accumulation and lipid peroxidation (LPO). These SS-induced ferroptotic responses are achieved via ROS, in particular superoxide (O2-) generation. Antioxidants such as superoxide dismutase (SOD) and Tiron not only scavenged O2- production, but also markedly rescued SLC7A11 down-regulation, GSH depletion, GPx4 inactivation, iron accumulation, LPO, and ferroptosis. Moreover, iron chelator DFO significantly reduces the O2- production, indicating a positive feedback regulation between O2- production and iron accumulation. Taken together, we have identified SS as a novel ferroptosis inducing agent in various human cancer models.

Par-4 regulates autophagic cell death in human cancer cells via upregulating p53 and BNIP3.

Prostate apoptosis response-4 (Par-4) is a tumor suppressor protein that selectively induces apoptosis in cancer cells. Although the mechanism of Par-4-mediated induction of apoptosis has been well studied, the involvement of Par-4 in other mechanisms of cell death such as autophagy is unclear. We investigated the mechanism involved in Par-4-mediated autophagic cell death in human malignant glioma. We demonstrate for the first time that the tumor suppressor lipid, ceramide (Cer), causes Par-4 induction, leading to autophagic cell death in human malignant glioma. Furthermore, we identified the tumor suppressor protein p53 and BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) as downstream targets of Par-4 during Cer-mediated autophagic cell death. RNAi-mediated down-regulation of Par-4 blocks Cer-induced p53-BNIP3 activation and autophagic cell death, while upregulation of Par-4 augmented p53-BNIP3 activation and autophagic cell death. Remarkably, in many instances, Par-4 overexpression alone was sufficient to induce cell death which is associated with features of autophagy. Interestingly, similar results were seen when glioma cells were exposed to classical autophagy inducers such as serum starvation, arsenic trioxide, and curcumin. Collectively, the novel Par-4-p53-BNIP3 axis plays a crucial role in autophagy-mediated cell death in human malignant glioma.

Critical role of H2O2 in mediating sanguinarine-induced apoptosis in prostate cancer cells via facilitating ceramide generation, ERK1/2 phosphorylation, and Par-4 cleavage.

Natural products are a major source of potential anticancer agents, and in order to develop improved and more effective cancer treatments, there is an immense need in exploring and elucidating their mechanism of action. Sanguinarine (SNG), a quaternary benzophenanthridine alkaloid, has been shown to induce cytotoxicity in various human cancers and suppresses various pro-tumorigenic processes such as invasion, angiogenesis, and metastasis in different cancers. Lack of understanding the anticancer mechanism(s) of SNG has impeded the development of this molecule as a potential anticancer agent. Earlier, we have reported that SNG induces reactive oxygen species (ROS)-dependent ceramide (Cer) generation and Akt dephosphorylation, leading to the induction of apoptosis in human leukemic cells. In the present study, we demonstrate that SNG has potent anti-proliferative activity against prostate cancer cells. Our data suggest that SNG induces Cer generation via inhibiting acid ceramidase and glucosylceramide synthase, two important enzymes involved in Cer metabolism. Furthermore, we demonstrate that SNG induces ROS-depended extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation, and prostate apoptosis response-4 (Par-4) cleavage, leading to the induction of apoptosis in human prostate cancer cells. Overall, our findings provide molecular insight into the role of ROS signaling in the anticancer mechanism(s) of SNG. This may provide the basis for its use as a nontoxic and an effective therapeutic agent in the treatment of prostate cancer.

Par-4-dependent p53 up-regulation plays a critical role in thymoquinone-induced cellular senescence in human malignant glioma cells.

Thymoquinone (TQ), the predominant bioactive constituent present in black cumin (Nigella sativa), exerts tumor suppressive activity against a wide variety of cancer cells. Cellular senescence, characterized by stable and long term loss of proliferative capacity, acts as a potent tumor suppressive mechanism. Here, we provide evidence for the first time that TQ suppresses growth of glioma cells by potentially inducing the expression of prostate apoptosis response-4 (Par-4) tumor suppressor protein. In turn, TQ-induced Par-4 expression triggers cellular senescence, as evidenced by increasing cellular size, ß-galactosidase staining, G1 phase arrest, and increased expression of senescence markers such as p53, p21, Rb, and decreased expression of lamin B1, cyclin E and cyclin depended kinase-2 (CDK-2). Further, overexpression of Par-4 significantly increases the expression of p53 and its downstream target p21, and increases ß-galactosidase positive cells, while siRNA/shRNA mediated-knockdown of Par-4 reverses the TQ-induced effects. Altogether, we describe a novel mechanism of cross talk between Par-4 and p53, that plays a critical role in TQ-induced senescence in human malignant glioma cells.

Molecular targets and anticancer potential of sanguinarine-a benzophenanthridine alkaloid.

BACKGROUND: Cancer is an enormous global health burden, and should be effectively addressed with better therapeutic strategies. Currently, over 60% of the clinically approved anticancer agents are either directly isolated from natural sources or are modified from natural lead molecules. Sanguinarine (SNG), a quaternary benzophenanthridine alkaloid has gained increasing attention in recent years as a potential anticancer agent. PURPOSE: There is a large untapped source of phytochemical-based anticancer agents remaining to be explored. This review article aims to recapitulate different anticancer properties of SNG, and describes some of the molecular targets involved in exerting its effect. It also depicts the pharmacokinetic and toxicological properties of SNG, two parameters important in determining the druggability of a molecule. METHODS: Numerous in vivo and in vitro published studies have signified the anticancer properties of SNG. In order to collate and decipher these properties, an extensive literature search was conducted in PubMed, ScienceDirect, and Scopus using keywords followed by the evaluation of the relevant articles where the relevant reports are integrated and analyzed. RESULTS: Apart from inducing cell death, SNG inhibits pro-tumorigenic processes such as invasion, angiogenesis, and metastasis in different cancers. Moreover, SNG has been shown to synergistically enhance the sensitivity of several chemotherapeutic agents and is effective against a variety of multi-drug resistant cancers.

Reactive oxygen species and cancer paradox: To promote or to suppress?

Reactive oxygen species (ROS), a group of highly reactive ions and molecules, are increasingly being appreciated as powerful signaling molecules involved in the regulation of a variety of biological processes. Indeed, their role is continuously being delineated in a variety of pathophysiological conditions. For instance, cancer cells are shown to have increased ROS levels in comparison to their normal counterparts. This is partly due to an enhanced metabolism and mitochondrial dysfunction in cancer cells. The escalated ROS generation in cancer cells contributes to the biochemical and molecular changes necessary for the tumor initiation, promotion and progression, as well as, tumor resistance to chemotherapy. Therefore, increased ROS in cancer cells may provide a unique opportunity to eliminate cancer cells via elevating ROS to highly toxic levels intracellularly, thereby, activating various ROS-induced cell death pathways, or inhibiting cancer cell resistance to chemotherapy. Such results can be achieved by using agents that either increase ROS generation, or inhibit antioxidant defense, or even a combination of both. In fact, a large variety of anticancer drugs, and some of those currently under clinical trials, effectively kill cancer cells and overcome drug resistance via enhancing ROS generation and/or impeding the antioxidant defense mechanism. This review focuses on our current understanding of the tumor promoting (tumorigenesis, angiogenesis, invasion and metastasis, and chemoresistance) and the tumor suppressive (apoptosis, autophagy, and necroptosis) functions of ROS, and highlights the potential mechanism(s) involved. It also sheds light on a very novel and an actively growing field of ROS-dependent cell death mechanism referred to as ferroptosis.

Hydrogen peroxide/ceramide/Akt signaling axis play a critical role in the antileukemic potential of sanguinarine.

Dysregulation of apoptosis is a prime hallmark of leukemia. Therefore, drugs which restore the sensitivity of leukemic cells to apoptotic stimuli are promising candidates in the treatment of leukemia. Recently, we have demonstrated that sanguinarine (SNG), a benzophenanthridine alkaloid, isolated from Sanguinaria canadensis induces ROS-dependent ERK1/2 activation and autophagic cell death in human malignant glioma cells (Pallichankandy et al., 2015; [43]). In this study, we investigated the antileukemic potential of SNG in vitro, and further examined the molecular mechanisms of SNG-induced cell death. In human leukemic cells, SNG activated apoptotic cell death pathway characterized by activation of caspase cascade, DNA fragmentation and down-regulation of anti-apoptotic proteins. Importantly, we have identified a crucial role for hydrogen peroxide (H2O2)-dependent ceramide (Cer) generation in the facilitation of SNG-induced apoptosis. Additionally, we have found that SNG inhibits Akt, a key anti-apoptotic protein kinase by dephosphorylating it at Ser(473), leading to the dephosphorylation of its downstream targets, GSK3ß and mTOR. Interestingly, inhibition of Cer generation, using acid sphingomyelinase inhibitor, significantly reduced the SNG-induced Akt dephosphorylation and apoptosis, whereas, activation of Cer generation using inhibitors of acid ceramidase and glucosylceramide synthase enhanced it. Furthermore, using a group of ceramide activated protein phosphatases (CAPPs) inhibitor (calyculin A, Okadaic acid, and phosphatidic acid), the involvement of protein phosphatase 1 form of CAPP in SNG-induced Akt dephosphorylation and apoptosis was demonstrated. Altogether, these results underscore a critical role for H2O2-Cer-Akt signaling axis in the antileukemic action of SNG.

ROS-dependent activation of autophagy is a critical mechanism for the induction of anti-glioma effect of sanguinarine.

Malignant gliomas are notoriously resistant to therapies that induce apoptosis, but are less resistant to therapies that induce autophagy. Therefore, drugs targeting autophagy are promising candidates in the treatment of malignant gliomas. In this study, we investigated the anti-glioma potential of sanguinarine (SNG) in vitro, and further examined the molecular mechanisms of SNG-induced cell death. In human malignant glioma cells SNG activated autophagic cell death pathway characterized by increased acidic vesicular organelles formation, GFP-LC3 punctate formation, LC3-II conversion, and expression of autophagy related proteins, such as Atg5 and Beclin-1. The autophagy inhibitor bafilomycin A1 or knockdown of Atg5 markedly inhibited the SNG-induced autophagic cell death. Apart from inducing autophagic cell death, SNG has also been shown to induce apoptotic cell death in these cell lines. Importantly, the study also identified the crucial role of reactive oxygen species (ROS)-dependent activation of the extracellular signal-regulated kinase1/2 (ERK1/2) in the facilitation of SNG-induced autophagic cell death. Antioxidants, such as glutathione and N-acetyl cysteine, significantly abrogated ROS production, ERK1/2 activation, and in turn, prevented SNG-induced autophagic cell death. Moreover, scavengers of H2O2 (sodium pyruvate and catalase) significantly attenuated the activity of SNG. Down-regulation of ERK1/2 activity, by using selective ERK1/2 inhibitor (U0126) or siERK1/2, led to an inhibition of SNG-induced autophagic cell death. Furthermore, tumor cells transfected with constitutively active ERK2-MEK1-LA fusion protein accentuated SNG-induced autophagic cell death. Overall, our findings unveil a novel anti-tumor mechanism of action of SNG in human malignant glioma cells, opening up the possibility of using SNG based pro-autophagic drugs for the treatment of malignant glioma.

Tumor suppressive functions of ceramide: evidence and mechanisms.

Studies over the past two decades have identified ceramide as a multifunctional central molecule in the sphingolipid biosynthetic pathway. Given its diverse tumor suppressive activities, molecular understanding of ceramide action will produce fundamental insights into processes that limit tumorigenesis and may identify key molecular targets for therapeutic intervention. Ceramide can be activated by a diverse array of stresses such as heat shock, genotoxic damage, oxidative stress and anticancer drugs. Ceramide triggers a variety of tumor suppressive and anti-proliferative cellular programs such as apoptosis, autophagy, senescence, and necroptosis by activating or repressing key effector molecules. Defects in ceramide generation and metabolism in cancer contribute to tumor cell survival and resistance to chemotherapy. The potent and versatile anticancer activity profile of ceramide has motivated drug development efforts to (re-)activate ceramide in established tumors. This review focuses on our current understanding of the tumor suppressive functions of ceramide and highlights the potential downstream targets of ceramide which are involved in its tumor suppressive action.

ROS-dependent prostate apoptosis response-4 (Par-4) up-regulation and ceramide generation are the prime signaling events associated with curcumin-induced autophagic cell death in human malignant glioma.

Malignant gliomas are extremely resistant to therapies that induce apoptosis, but are less resistant to therapies that induce autophagy. Therefore, drugs targeting autophagy are promising in the management of malignant gliomas. In this study, we investigated the anti-glioma potential of curcumin in vitro, and further examined the molecular mechanisms of curcumin-induced cell death in human malignant glioma. Here, we provide evidence that curcumin is cytotoxic against human malignant glioma cell lines, and the mechanism of cell death caused by curcumin is associated with features of autophagy. Curcumin suppresses the growth of human malignant glioma cells via ROS-dependent prostate apoptosis response-4 (Par-4) induction and ceramide generation. Extracellular supplementation of antioxidants such as glutathione and N-acetylcysteine to glioma cells abrogated the Par-4 induction, ceramide generation, and in turn, prevented curcumin-induced autophagic cell death. Moreover, tumor cells transfected with Par-4 gene sensitized the curcumin-induced autophagic cell death. Overall, this study describes a novel signaling pathway by which curcumin induces ROS-dependent Par-4 activation and ceramide generation, leading to autophagic cell death in human malignant glioma cells.

Role of ceramide in diabetes mellitus: evidence and mechanisms.

Diabetes mellitus is a metabolic disease with multiple complications that causes serious diseases over the years. The condition leads to severe economic consequences and is reaching pandemic level globally. Much research is being carried out to address this disease and its underlying molecular mechanism. This review focuses on the diverse role and mechanism of ceramide, a prime sphingolipid signaling molecule, in the pathogenesis of type 1 and type 2 diabetes and its complications. Studies using cultured cells, animal models, and human subjects demonstrate that ceramide is a key player in the induction of ß-cell apoptosis, insulin resistance, and reduction of insulin gene expression. Ceramide induces ß-cell apoptosis by multiple mechanisms namely; activation of extrinsic apoptotic pathway, increasing cytochrome c release, free radical generation, induction of endoplasmic reticulum stress and inhibition of Akt. Ceramide also modulates many of the insulin signaling intermediates such as insulin receptor substrate, Akt, Glut-4, and it causes insulin resistance. Ceramide reduces the synthesis of insulin hormone by attenuation of insulin gene expression. Better understanding of this area will increase our understanding of the contribution of ceramide to the pathogenesis of diabetes, and further help in identifying potential therapeutic targets for the management of diabetes mellitus and its complications.

Caspase-3 mediated release of SAC domain containing fragment from Par-4 is necessary for the sphingosine-induced apoptosis in Jurkat cells.

BACKGROUND: Prostate apoptosis response-4 (Par-4) is a tumor-suppressor protein that selectively activates and induces apoptosis in cancer cells, but not in normal cells. The cancer specific pro-apoptotic function of Par-4 is encoded in its centrally located SAC (Selective for Apoptosis induction in Cancer cells) domain (amino acids 137-195). The SAC domain itself is capable of nuclear entry, caspase activation, inhibition of NF-κB activity, and induction of apoptosis in cancer cells. However, the precise mechanism(s) of how the SAC domain is released from Par-4, in response to apoptotic stimulation, is not well explored. RESULTS: In this study, we demonstrate for the first time that sphingosine (SPH), a member of the sphingolipid family, induces caspase-dependant cleavage of Par-4, leading to the release of SAC domain containing fragment from it. Par-4 is cleaved at the EEPD131G site on incubation with caspase-3 in vitro, and by treating cells with several anti-cancer agents. The caspase-3 mediated cleavage of Par-4 is blocked by addition of the pan-caspase inhibitor z-VAD-fmk, caspase-3 specific inhibitor Ac-DEVD-CHO, and by introduction of alanine substitution for D131 residue. Moreover, suppression of SPH-induced Akt dephosphorylation also abrogated the caspase dependant cleavage of Par-4. CONCLUSION: Evidence provided here shows that Par-4 is cleaved by caspase-3 during SPH-induced apoptosis. Cleavage of Par-4 leads to the generation of SAC domain containing fragment which may possibly be essential and sufficient to induce or augment apoptosis in cancer cells.