Anees Rahman

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

ABSTRACT 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. HIGHLIGHTSROS are a heterogeneous group of highly reactive ions and molecules.Cancer cells are shown to have increased ROS levels.Increased ROS levels have been linked to various tumorigenic processes.Many anticancer drugs kill cancer cells via increasing ROS to toxic level.Elevated ROS in cancer cells is a promising avenue for selectively targeting them.

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 β-cell apoptosis, insulin resistance, and reduction of insulin gene expression. Ceramide induces β-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.

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.

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.

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, GSK3β 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.

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.

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.

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, β-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 β-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.

Sphingosine analogue FTY720 sensitizes arsenic trioxide-induced autophagic cell death in human malignant glioma cells

Malignant gliomas are known to be resistant to therapies that induce apoptosis. Recently, several lines of evidence indicate that glioblastoma cells may be less resistant to therapies that induce autophagy. Therefore, drugs targeting autophagy are considered as promising in the management of malignant gliomas. The purpose of this study was to investigate the antitumour potential of FTY720, a novel sphingosine analogue, and to elucidate the molecular mechanism of its cytotoxic effects on malignant glioma cells. We demonstrate that FTY720 induces autophagy but not apoptosis in malignant glioma cells. We found that FTY720 sensitizes arsenic trioxide-induced autophagic cell death in glioma cells and that FTY720–arsenic trioxide (ATO) combined treatment induces autophagy through the inhibition of the Akt/mTOR signalling pathway. Our findings provide a possible base for combinatorial therapeutic application of FTY720–ATO in the management of malignant gliomas.