Anand Krishnan V. Iyer

Role of oxidative/nitrosative stress-mediated Bcl-2 regulation in apoptosis and malignant transformation

Bcl‐2 is a key apoptosis regulatory protein of the mitochondrial death pathway. The oncogenic potential of Bcl‐2 is well established, with its overexpression reported in various cancers. The antiapoptotic function of Bcl‐2 is closely associated with its expression levels. Reactive oxygen and nitrogen species (ROS/RNS) are important intracellular signaling molecules that play a key role in various physiological processes including apoptosis. We have recently reported that ROS and RNS can regulate Bcl‐2 expression levels, thereby impacting its function. Superoxide anion (·O2–) plays a proapoptotic role by causing downregulation and degradation of Bcl‐2 protein through the ubiquitin‐proteasomal pathway. In contrast, nitric oxide (NO)‐mediated S‐nitrosylation of Bcl‐2 prevents its ubiquitination and subsequent proteasomal degradation, leading to inhibition of apoptosis. Interestingly, NO‐mediated S‐nitrosylation and stabilization of Bcl‐2 protein was the primary mechanism involved in the malignant transformation of nontumorigenic lung epithelial cells in response to long‐term carcinogen exposure. We describe a novel mechanism of Bcl‐2 regulation by ·O2– and NO, providing a new dimension to reactive species‐mediated Bcl‐2 stability, apoptotic cell death, and cancer development.

Cell surface restriction of EGFR by a tenascin cytotactin-encoded EGF-like repeat is preferential for motility-related signaling.

The 14th EGFL‐repeat (Ten14) of human tenascin cytotactin activates the epidermal growth factor receptor (EGFR) with micromolar affinity; however, unlike EGF, Ten14‐mediated activation of EGFR does not lead to receptor internalization. As the divergent signaling pathways downstream of EGFR have been shown to be triggered from plasma membrane and cytosolic locales, we investigated whether Ten14‐mediated surface restriction of EGFR resulted in altered biochemical and cellular responses as compared to EGF. Molecules associated with migratory cascades were activated to a relatively greater extent in response to Ten14, with very weak activation of proliferation‐associated cascades. Activation of phospholipase C γ (PLCγ) and m‐calpain, associated with lamellipod protrusion and tail retraction, respectively, were noted at even at sub‐saturating doses of Ten14. However, activation of ERK/MAPK, p90RSK, and Elk1, factors affecting proliferation, remained low even at high Ten14 concentrations. Similar activation profiles were observed for EGF‐treated cells at 4°C, a maneuver that limits receptor internalization. We demonstrate a concurrent effect of such altered signaling on biophysical responses—sustained migration was observed at levels of Ten14 that activated PLCγ, but did not stimulate proliferation significantly. Here, we present a novel class of EGFR ligands that can potentially signal as a part of the extracellular matrix, triggering specific intracellular signaling cascades leading to a directed cellular response from an otherwise pleiotropic receptor. This work extends the signaling paradigm of EGFL repeat being presented in a restricted fashion as part of the extracellular matrix. J. Cell. Physiol. 214: 504–512, 2008.

Phosphatidylinositol-3-Kinase/Akt Regulates Bleomycin-Induced Fibroblast Proliferation and Collagen Production

Abnormal repair and dysregulated angiogenesis have been implicated in the pathogenesis of pulmonary fibrosis, but the underlying mechanisms of regulation are not well understood. The present study investigated the role of phosphatidylinositol-3-kinase (PI3K)/Akt in fibrogenesis of human lung fibroblasts and its regulation by reactive oxygen species (ROS). Exposure of lung fibroblasts to bleomycin, a known inducer of fibrosis, resulted in rapid activation of PI3K/Akt and a parallel increase in fibroblast proliferation and collagen production, characteristics of lung fibrosis. Bleomycin had no significant effect on total Akt protein expression but induced phosphorylation of the protein at threonine 308 and serine 473 positions. Inhibition of this phosphorylation by PI3K inhibitors or by dominant-negative Akt (T308A/S473A) expression abrogated the effects of bleomycin on fibroblast proliferation and collagen production, suggesting the role of PI3K/Akt in the fibrogenic process. Activation of PI3K/Akt by bleomycin also led to transcriptional activation and protein expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor, which contributed to the fibroproliferative and collagen-inducing effects of bleomycin. The fibrogenic effects of bleomycin were dependent on ROS generation, particularly superoxide anion and hydrogen peroxide, which were induced by bleomycin. Inhibition of ROS generation by antioxidant enzymes, catalase and superoxide dismutase mimetic MnTBAP, abrogated the fibrogenic effects of bleomycin as well as its induction of PI3K/Akt and HIF-1alpha activation. Together, our results indicate a novel role of PI3K/Akt in fibrogenesis of human lung fibroblasts and its regulation by ROS, which could be exploited for the treatment of pulmonary fibrosis and related disorders.

Role of S-Nitrosylation in Apoptosis Resistance and Carcinogenesis

Nitric oxide (NO) has been widely recognized as a positive regulator of tumorigenesis and cancer progression through its ability to regulate important proteins in various signal transduction pathways. S-Nitrosylation, or covalent attachment of NO to protein sulphydryl groups, has gained prominence as an important mechanism by which NO modulates physiologic and pathologic cellular responses. In this article, we discuss S-nitrosylation of two key apoptosis-regulatory proteins of the intrinsic and extrinsic death pathways, namely B-cell lymphoma-2 (Bcl-2) and FLICE-inhibitory protein (FLIP). These proteins have been shown to be upregulated in a variety of tumors and have been implicated with cancer chemoresistance through dysregulation of apoptosis. S-Nitrosylation of these proteins precludes their ubiquitination and subsequent degradation by the proteasome, thus accentuating their anti-apoptotic effect which is critical in the context of tumorigenic potential and cancer progression. We propose that such post-translational modifications of proteins by NO may be a general mechanism that tumor cells exploit to tilt the scales towards survival and proliferation by evading cell death.

Tenascin cytotactin epidermal growth factor-like repeat binds epidermal growth factor receptor with low affinity

Select epidermal growth factor (EGF)‐like (EGFL) repeats of human tenascin cytotactin (tenascin C) can stimulate EGF receptor (EGFR) signaling, but activation requires micromolar concentrations of soluble EGFL repeats in contrast to subnanomolar concentrations of classical growth factors such as EGF. Using in silico homology modeling techniques, we generated a structure for one such repeat, the 14th EGFL repeat (Ten14). Ten14 assumes a tight EGF‐like fold with truncated loops, consistent with circular dichroism studies. We generated bound structures for Ten14 with EGFR using two different approaches, resulting in two distinctly different conformations. Normal mode analysis of both structures indicated that the binding pocket of EGFR exhibits a significantly higher mobility in Ten14–EGFR complex compared to that of the EGF–EGFR complex; we hypothesized this may be attributed to loss of key high‐affinity interactions within the Ten14–EGFR complex. We proved the efficacy of our in silico models by in vitro experiments. Surface plasmon resonance measurements yielded equilibrium constant KD of 74 µM for Ten14, approximately three orders of magnitude weaker than that of EGF. In accordance with our predicted bound models, Ten14 in monomeric form does not bind EGFR with sufficient stability so as to induce degradation of receptor, or undergo EGFR‐mediated internalization over either the short (20 min) or long (48 h) term. This transient interaction with the receptor on the cell surface is in marked contrast to other EGFR ligands which cause EGFR transit through, and signaling from intracellular locales in addition to cell surface signaling. J. Cell. Physiol. 211: 748–758, 2007.

Reactive oxygen species-mediated p38 MAPK regulates carbon nanotube-induced fibrogenic and angiogenic responses

Abstract Single-walled carbon nanotubes (SWCNTs) are fibrous nanoparticles that are being used widely for various applications including drug delivery. SWCNTs are currently under special attention for possible cytotoxicity. Recent reports suggest that exposure to nanoparticles leads to pulmonary fibrosis. We report that SWCNT-mediated interplay of fibrogenic and angiogenic regulators leads to increased angiogenesis, which is a novel finding that furthers the understanding of SWCNT-induced cytotoxicity. SWCNTs induce fibrogenesis through reactive oxygen species-regulated phosphorylation of p38 mitogen-activated protein kinase (MAPK). Activation of p38 MAPK by SWCNTs led to the induction of transforming growth factor (TGF)-β1 as well as vascular endothelial growth factor (VEGF). Both TGF-β1 and VEGF contributed significantly to the fibroproliferative and collagen-inducing effects of SWCNTs. Interestingly, a positive feedback loop was observed between TGF-β1 and VEGF. This interplay of fibrogenic and angiogenic mediators led to increased angiogenesis in response to SWCNTs. Overall this study reveals key signalling molecules involved in SWCNT-induced fibrogenesis and angiogenesis.

Superoxide-mediated proteasomal degradation of Bcl-2 determines cell susceptibility to Cr(VI)-induced apoptosis

Hexavalent chromium [Cr(VI)] compounds are redox cycling environmental carcinogens that induce apoptosis as the primary mode of cell death. Defects in apoptosis regulatory mechanisms contribute to carcinogenesis induced by Cr(VI). Activation of apoptosis signaling pathways is tightly linked with the generation of reactive oxygen species (ROS). Likewise, ROS have been implicated in the regulation of Cr(VI)-induced apoptosis and carcinogenicity; however, its role in Cr(VI)-induced apoptosis and the underlying mechanism are largely unknown. We report that ROS, specifically superoxide anion (.O(-)(2), mediates Cr(VI)-induced apoptosis of human lung epithelial H460 cells. H460 rho(0) cells that lack mitochondrial DNA demonstrated a significant decrease in ROS production and apoptotic response to Cr(VI), indicating the involvement of mitochondrial ROS in Cr(VI)-induced apoptosis. In agreement with this observation, we found that Cr(VI) induces apoptosis mainly through the mitochondrial death pathway via caspase-9 activation, which is negatively regulated by the antiapoptotic protein Bcl-2. Furthermore, .O(-)(2) induced apoptosis in response to Cr(VI) exposure by downregulating and degrading Bcl-2 protein through the ubiquitin-proteasomal pathway. This study reveals a novel mechanism linking .O(-)(2) with Bcl-2 stability and provides a new dimension to ROS-mediated Bcl-2 downregulation and apoptosis induction.

Nitric Oxide–Mediated Bcl-2 Stabilization Potentiates Malignant Transformation of Human Lung Epithelial Cells

Hexavalent chromium (Cr(VI)) compounds are known human carcinogens associated with the incidence of lung cancer. Although a direct correlation between Cr(VI) exposure and lung cancer has been established, several studies aimed at generating animal models for Cr(VI) have yielded inconsistent data that do not affirmatively support findings from epidemiologic studies. Because the lack of a good animal model has hindered the identification of molecular mechanisms involved in Cr(VI) exposure, we developed an in vitro model that facilitates mechanistic studies of Cr(VI)-induced carcinogenesis. We report here that long-term exposure to Cr(VI) leads to the malignant transformation of nontumorigenic human lung epithelial cells. Cr(VI)-transformed cells exhibited loss of contact inhibition, colony formation, and increased rates of cell invasion, migration, and proliferation, as compared with passage-matched control cells. Cr(VI)-transformed cells evaded apoptosis by a mechanism involving S-nitrosylation and stabilization of Bcl-2 protein in a nitric oxide-dependent manner. This study establishes an important in vitro model that facilitates mechanistic studies of Cr(VI)-induced carcinogenesis, and elucidates a novel mechanism that causes apoptosis-resistant malignant transformation of nontumorigenic lung cells in response to a human carcinogen.

Role of apoptosis-related miRNAs in resveratrol-induced breast cancer cell death.

Breast cancer is the most frequently diagnosed cancer in women, and one of the leading causes of cancer-related deaths worldwide. Recent evidences indicate that dietary agents such as resveratrol may inhibit cancer progression through modulation of microRNAs (miRNAs). We demonstrate that resveratrol regulates apoptotic and cell cycle machinery in breast cancer cells by modulating key tumor-suppressive miRNAs including miR-125b-5p, miR-200c-3p, miR-409-3p, miR-122-5p and miR-542-3p. Resveratrol-mediated miRNA modulation regulates key anti-apoptotic and cell cycle proteins including Bcl-2, X-linked inhibitor of apoptosis protein and CDKs, which are critical for its activity. Modulating miRNAs with mimics or inhibitors further validated a key role for miR-542-3p in MCF-7 and miR-122-5p in MDA-MB-231 breast cancer cell death in response to resveratrol. In conclusion, this study reveals novel miRNAs modulated by resveratrol that have a key role in breast cancer cell death.

Dependence of Reactive Oxygen Species and FLICE Inhibitory Protein on Lipofectamine-Induced Apoptosis in Human Lung Epithelial Cells

Cationic liposomes such as Lipofectamine (LF) are widely used as nonviral gene delivery vectors; however, their clinical application is limited by their cytotoxicity. These agents have been shown to induce apoptosis as the primary mode of cell death, but their mechanism of action is not well understood. The present study investigated the mechanism of LF-induced apoptosis and examined the role of reactive oxygen species (ROS) in this process. We found that LF induced apoptosis of human epithelial H460 cells through a mechanism that involves caspase activation and ROS generation. Inhibition of caspase activity by pan-caspase inhibitor (z-VAD-fmk) or by specific caspase-8 inhibitor (z-IETD-fmk) or caspase-9 inhibitor (z-LEHD-fmk) inhibited the apoptotic effect of LF. Overexpression of FLICE-inhibitory protein (FLIP) or B-cell lymphoma-2, which are known inhibitors of the extrinsic and intrinsic death pathways, respectively, similarly inhibited apoptosis by LF. Induction of apoptosis by LF was shown to require ROS generation because its inhibition by ROS scavengers or by ectopic expression of antioxidant enzyme superoxide dismutase and glutathione peroxidase strongly inhibited the apoptotic effect of LF. Electron spin resonance studies showed that LF induced multiple ROS; however, superoxide was found to be the primary ROS responsible for LF-induced apoptosis. The mechanism by which ROS mediate the apoptotic effect of LF involves down-regulation of FLIP through the ubiquitination pathway. In demonstrating the role of FLIP and ROS in LF death signaling, we document a novel mechanism of apoptosis regulation that may be exploited to decrease cytotoxicity and increase gene transfection efficiency of cationic liposomes.