Shazib Pervaiz

Annexin 1: the new face of an old molecule

The annexin superfamily consists of 13 calcium or calcium and phospholipid binding proteins with a significant degree of biological and structural homology (40–60%). First described in the late 1970s and subsequently referred to as macrocortin, renocor‐tin, lipomodulin, lipocortin‐1, and more recently Annexin I, this 37 kDa calcium and phospholipid binding protein is a strong inhibitor of glucocorticoid‐induced eicosanoid synthesis and PLA2. Recent interest in the biological activity of this intriguing molecule has unraveled important functional attributes of Annexin 1 in a variety of inflammatory pathways, on cell proliferation machinery, in the regulation of cell death signaling, in phagocytic clearance of apoptosing cells, and most importantly in the process of carcinogenesis. Here we attempt to present a short review on these diverse biological activities of an interesting and important molecule, which could be a potential target for novel therapeutic intervention in a host of disease states.—Lim, L. H. K., Pervaiz, S. Annexin 1: the new face of an old molecule. FASEB J. 21, 968–975 (2007)

TNF receptor superfamily-induced cell death: redox-dependent execution

Tumor necrosis factor (TNF) superfamily is a group of cytokines with important functions in immunity, inflammation, differentiation, control of cell proliferation, and apoptosis. TNFα is the founding member of the 19 different proteins that have so far been identified within this family. TNF family members exert their biological effects through the TNF receptor (TNFR) superfamily of cell surface receptors that share a stretch of ∼ 80 amino acids within their cytoplasmic region, the death domain (DD), critical for recruiting the death machinery. Work over the last decade has unraveled critical signaling networks involved in TNFR‐induced cell death, specifically using the constitutively expressed TNFR1 as a prototype. Of particular interest is the intermediary role of intracellular reactive oxygen species (ROS) in signal transduction after ligation of the TNFR1. With the increasing understanding of the of death receptor signaling pathways, the exact role of ROS in TNFα‐induced execution is now believed to be far more complicated than originally thought. Recently, some important discoveries have underscored the critical role of ROS in TNFα signaling, notably in TNFα‐mediated activation of nuclear factor‐κB (NF‐κB) and c‐Jun N‐terminal kinase (c‐Jun NH2‐terminal kinase, JNK), as well as in cell death (apoptotic and necrotic) pathways. Here we attempt to reviewthe existing knowledge on the involvement of ROS in death receptor signaling using TNFα‐TNFR1 as the model system, specifically addressing the involvement of intracellular ROS in TNFα‐induced cell death and in TNFα‐induced activation of NF‐κB and JNK and their crosstalk.—Shen, H‐M., Pervaiz, S. TNF receptor superfamily‐induced cell death: redox‐dependent execution. FASEB J. 20, 1589–1598 (2006)

Apoptosis induced by hydrogen peroxide is mediated by decreased superoxide anion concentration and reduction of intracellular milieu

Hydrogen peroxide (H2O2) is considered to be a mediator of apoptotic cell death but the mechanism by which it induces apoptosis is unclear. Here, we show that cells undergoing apoptosis from exposure to H2O2 display a significant decrease in intracellular concentration of superoxide (O− 2) which is associated with a reduction of the intracellular milieu, as measured by an increase in the GSH/GSSG ratio and a decrease in intracellular pH. The notion that a decrease in intracellular O− 2 concentration triggers apoptosis is supported by the observation that H2O2‐mediated apoptosis could be retarded in cells in which the intracellular O− 2 concentration is maintained at or above the cellular baseline level by inhibition of the major O− 2 scavenger superoxide dismutase (Cu/Zn SOD). Taken together, our observations indicate that a decrease in the intracellular O− 2 concentration, reduction and acidification of the intracellular milieu constitute a signal for H2O2‐mediated apoptosis, thereby inducing a reductive as opposed to an oxidative stress.

Simultaneous Induction of Non-Canonical Autophagy and Apoptosis in Cancer Cells by ROS-Dependent ERK and JNK Activation

Background Chemotherapy-induced reduction in tumor load is a function of apoptotic cell death, orchestrated by intracellular caspases. However, the effectiveness of these therapies is compromised by mutations affecting specific genes, controlling and/or regulating apoptotic signaling. Therefore, it is desirable to identify novel pathways of cell death, which could function in tandem with or in the absence of efficient apoptotic machinery. In this regard, recent evidence supports the existence of a novel cell death pathway termed autophagy, which is activated upon growth factor deprivation or exposure to genotoxic compounds. The functional relevance of this pathway in terms of its ability to serve as a stress response or a truly death effector mechanism is still in question; however, reports indicate that autophagy is a specialized form of cell death under certain conditions. Methodology/Principal Findings We report here the simultaneous induction of non-canonical autophagy and apoptosis in human cancer cells upon exposure to a small molecule compound that triggers intracellular hydrogen peroxide (H2O2) production. Whereas, silencing of beclin1 neither inhibited the hallmarks of autophagy nor the induction of cell death, Atg 7 or Ulk1 knockdown significantly abrogated drug-induced H2O2-mediated autophagy. Furthermore, we provide evidence that activated extracellular regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) are upstream effectors controlling both autophagy and apoptosis in response to elevated intracellular H2O2. Interestingly, inhibition of JNK activity reversed the increase in Atg7 expression in this system, thus indicating that JNK may regulate autophagy by activating Atg7. Of note, the small molecule compound triggered autophagy and apoptosis in primary cells derived from patients with lymphoma, but not in non-transformed cells. Conclusions/Significance Considering that loss of tumor suppressor beclin 1 is associated with neoplasia, the ability of this small molecule compound to engage both autophagic and apoptotic machineries via ROS production and subsequent activation of ERK and JNK could have potential translational implications.

Intracellular acidification triggered by mitochondrial-derived hydrogen peroxide is an effector mechanism for drug-induced apoptosis in tumor cells

We recently showed that two photoproducts of merocyanine 540, C2 and C5, triggered cytochrome C release; however, C5 was inefficient in inducing caspase activity and apoptosis in leukemia cells, unlike C2. Here we show that HL60 cells acidified upon exposure to C2 but not C5. The intracellular drop in pH and caspase activation were dependent upon hydrogen peroxide production, and were inhibited by scavengers of hydrogen peroxide. On the contrary, caspase inhibitors did not block hydrogen peroxide production. In turn, increased intracellular hydrogen peroxide concentration was downstream of superoxide anion produced within 2 h of exposure to C2. Inhibitor of NADPH oxidase diphenyleneiodonium neither inhibited superoxide production nor caspase activation triggered by C2. However, exposure of purified mitochondria to C2 resulted in significantly increased superoxide production. Furthermore, cytochrome C release from isolated mitochondria induced by C2 was completely inhibited in the presence of scavengers of hydrogen peroxide. Contrarily, scavenging hydrogen peroxide had no effect on the cyclosporin A-sensitive mitochondrial permeability transition induced by C5. Our data suggest a scenario where drug-induced hydrogen peroxide production induces intracellular acidification and release of cytochrome C, independent of the inner membrane pore, thereby creating an intracellular environment permissive for caspase activation.

hTERT Overexpression Alleviates Intracellular ROS Production, Improves Mitochondrial Function, and Inhibits ROS-Mediated Apoptosis in Cancer Cells

The human telomerase reverse transcriptase (hTERT) is the catalytic subunit of the telomerase holoenzyme. Evidence is accumulating to link hTERT to activities other than telomere maintenance and immortalization. Here, we show that hTERT overexpression not only reduces the basal cellular reactive oxygen species (ROS) levels but also inhibits endogenous ROS production in response to stimuli that induce intracellular ROS generation. Conversely, siRNA-mediated gene silencing of hTERT potentiated the increase in cellular ROS levels following exposure to oxidative stress. This antioxidant effect of hTERT is mediated via a significant increase in the ratio of reduced to oxidized glutathione (GSH:GSSG) as well as efficient recovery of the oxidized peroxiredoxin to its nonoxidized form. Our data also provide evidence for mitochondrial localization of hTERT, and a significantly higher activity of cytochrome C oxidase, the rate-limiting enzyme in the mitochondrial electron transport chain, in hTERT overexpressing cells. To ascertain whether the improved mitochondrial function and antioxidant effect of hTERT could provide cancer cells with a survival advantage, the effect of oxidative stress on mitochondrial apoptosis was evaluated. Indeed, hTERT overexpressing cells inhibited cytosolic acidification, translocation of Bax, the drop in mitochondrial transmembrane potential, the release of cytochrome C to the cytosol, and cell death. Taken together, these data demonstrate a hitherto undefined role of hTERT in alleviating cellular ROS levels by way of potentiating the cellular antioxidant defense systems, and in doing so endowing cancer cells with the ability to evade death stimuli.

Activation of the RacGTPase inhibits apoptosis in human tumor cells

The small GTP-binding protein Rac is a downstream effector of the oncogene product p21-ras. Rac is involved in actin polymerization, Jun kinase activation, and intracellular superoxide anion production, through distinct pathways in tumor cells. Here we investigated the role of activated Rac in the response of tumor cells to apoptosis triggered by anti-cancer drugs or the cell surface death receptor CD95. Using M14 melanoma cells stably transfected with a constitutively active form of Rac1, we show that activated Rac inhibits tumor cell response to apoptosis. The inhibitory effect of activated Rac on apoptotic signaling is mediated by the interaction of Rac with intracellular oxidase and the subsequent production of superoxide, which is supported by experiments performed with M14 and NIH3T3 cells transiently transfected with the loss-of-function mutants of Rac in an activated RacV12 background. Consistent with these findings, we also demonstrate that inhibition of the Rac pathway in the HaRas-expressing T24 bladder carcinoma cell line induces a decrease in superoxide anion concentration, and results in a significant increase in tumor cell sensitivity to apoptosis. These findings demonstrate the existence of a novel Rac-dependent survival pathway mediated by intracellular superoxide in tumor cells.

Caspase‐1 dependent macrophage death induced by Burkholderia pseudomallei

Burkholderia pseudomallei is the causative agent for melioidosis, an infectious disease endemic in South‐east Asia and northern Australia. Infection can result in a wide spectrum of clinical outcomes, including asymtomatic, acute or chronic conditions. The ability of the bacteria to survive intracellularly within phagocytes and non‐phagocytes is postulated to help this pathogen persist in the body during latent chronic conditions. In some Gram‐negative bacteria, such as Shigella and Salmonella, the ability to evade macrophage killing involves inducing rapid macrophage cell death. In several of these instances, these bacteria activate cellular caspase‐1 to induce cell death, which is increasingly described to exhibit features more characteristic of oncosis than classical apoptosis. We found that B. pseudomallei is also capable of inducing caspase‐1 dependent death in macrophages and this process requires a functional bsa Type III Secretion System (TTSS). Bacterial internalization and pore formation in the cell membrane is necessary for death. Furthermore, cell death is accompanied by the release of IL‐1β and IL‐18. We believe that this novel description of macrophage death induced by B. pseudomallei could shed light on the pathogenesis of the bacteria in disease.

Hydrogen Peroxide-Mediated Cytosolic Acidification Is a Signal for Mitochondrial Translocation of Bax during Drug-Induced Apoptosis of Tumor Cells

Absence of the proapoptotic protein Bax renders tumor cells resistant to drug-induced apoptosis. We have shown that hydrogen peroxide (H2O2)-mediated cytosolic acidification is an effector mechanism during drug-induced apoptosis of tumor cells. Here, we report that Bax is critical in determining the sensitivity of tumor cells to H2O2-induced apoptosis. More importantly, exposure of colorectal carcinoma (HCT116) and leukemia cells (HL60 and CEM) to H2O2 or its intracellular production during drug-induced apoptosis is a signal for mitochondrial translocation of Bax. Furthermore, we provide evidence that drug-induced H2O2-mediated Bax translocation in tumor cells is caspase independent but involves cytosolic acidification. Inhibiting cytosolic acidification prevents Bax translocation, and contrarily enforced acidification of the intracellular milieu results in mitochondrial recruitment of Bax, even in the absence of a trigger. These findings provide a novel mechanism for mitochondrial translocation of Bax and directly implicate H2O2-mediated cytosolic acidification in the recruitment of the mitochondrial pathway during drug-induced apoptosis of tumor cells.

Art and science of photodynamic therapy.

1 Photodynamic therapy is an established modality for the treatment of solid tumours and other accessible lesions. Although the concept and practice of combining light with a photosensitizing agent for the treatment of disease states has been around for almost a century, the understanding of the art and science therein has been tremendously enhanced over the past few years. 2 Photosensitized reactions are dependent on the generation of reactive oxygen species, in particular singlet oxygen, which accounts for the damaging effects on biological macromolecules, such as membrane lipids and proteins. Therefore, compounds that give a good yield of 1O2 are used as photosensitizers. 3 The main photosensitizers used in the clinical setting belong to the photofrin family; however, newer and more effective sensitizers are being evaluated for their potential clinical effectiveness. 4 Light sources have moved from the use of white light with specific filters in the old days to the more recent use of monochromatic light sources, such as lasers, to more sophisticated light‐emitting diodes. However, dosimetry remains a big issue mainly because of difficulties in establishing the optimum treatment conditions for an approach that requires the fine‐tuning of several variables, such as sensitizer and light doses and drug‐to‐light interval, as well as the issues of skin photosensitivity and low selectivity. A newer development to circumvent these and provide a broader application for this concept has been the phenomenon of photo‐activation, whereby photo‐exposure of chromophores to generate novel, small biologically active compounds has been demonstrated successfully. 5 The aim of the present review was to provide a general overview of the art and science of photodynamic therapy and to highlight some of the issues and recent developments in further advancing this modality of treatment.