Yon Rojanasakul

Mechanisms of nanoparticle-induced oxidative stress and toxicity.

The rapidly emerging field of nanotechnology has offered innovative discoveries in the medical, industrial, and consumer sectors. The unique physicochemical and electrical properties of engineered nanoparticles (NP) make them highly desirable in a variety of applications. However, these novel properties of NP are fraught with concerns for environmental and occupational exposure. Changes in structural and physicochemical properties of NP can lead to changes in biological activities including ROS generation, one of the most frequently reported NP-associated toxicities. Oxidative stress induced by engineered NP is due to acellular factors such as particle surface, size, composition, and presence of metals, while cellular responses such as mitochondrial respiration, NP-cell interaction, and immune cell activation are responsible for ROS-mediated damage. NP-induced oxidative stress responses are torch bearers for further pathophysiological effects including genotoxicity, inflammation, and fibrosis as demonstrated by activation of associated cell signaling pathways. Since oxidative stress is a key determinant of NP-induced injury, it is necessary to characterize the ROS response resulting from NP. Through physicochemical characterization and understanding of the multiple signaling cascades activated by NP-induced ROS, a systemic toxicity screen with oxidative stress as a predictive model for NP-induced injury can be developed.

Inflammation and Lung Cancer: Roles of Reactive Oxygen/Nitrogen Species

The lung is a highly specialized organ that facilitates uptake of oxygen and release of carbon dioxide. Due to its unique structure providing enormous surface area to outside ambient air, it is vulnerable to numerous pathogens, pollutants, oxidants, gases, and toxicants that are inhaled continuously from air, which makes the lung susceptible to varying degrees of oxidative injury. To combat these unrelenting physical, chemical, and biological insults, the respiratory epithelium is covered with a thin layer of lining fluid containing several antioxidants and surfactants. Inhaled toxic agents stimulate the generation of reactive oxygen/nitrogen species (ROS/RNS), which in turn provoke inflammatory responses resulting in the release of proinflammatory cytokines and chemokines. These subsequently stimulate the influx of polymorphonuclear leukocytes (PMNs) and monocytes into the lung so as to combat the invading pathogens or toxic agents. In addition to the beneficial effects, persistent inhalation of the invading pathogens or toxic agents may result in overwhelming production of ROS/RNS, producing chronic inflammation and lung injury. During inflammation, enhanced ROS/RNS production may induce recurring DNA damage, inhibition of apoptosis, and activation of proto-oncogenes by initiating signal transduction pathways. Therefore, it is conceivable that chronic inflammation-induced production of ROS/RNS in the lung may predispose individuals to lung cancer. This review describes the complex relationship between lung inflammation and carcinogenesis, and highlights the role of ROS/RNS in cancer development.

Oxygen radical-mediated pulmonary toxicity induced by some cationic liposomes.

AbstractPurpose. The objectives of this study are to investigate the toxicityassociated with polycationic liposomes and to elucidate the underlyingmechanism. We tested the hypothesis that the positive charge of liposomesis a key determinant of toxicity by testing differently chargedliposomes in mice. Methods. Differently charged liposomal systems including cationicliposomes, LipofectAMINE and DOTAP, and neutral and negativeliposomes were evaluated for their toxicity after pulmonaryadministration in mice. LDH assay and differential cell counts were performedto measure toxicity and pulmonary inflammation, respectively. Reactiveoxygen intermediates (ROI) were assessed by chemiluminescence. Results. Instillation of cationic liposomes eliciteddose-dependent toxicity and pulmonary inflammation. This effect was more pronouncedwith the multivalent cationic liposome LipofectAMINE as comparedto the monovalent cationic DOTAP. Neutral and negative liposomes didnot exhibit lung toxicity. Toxicity associated with cationic liposomescorrelated with the oxidative burst induced by the liposomes.LipofectAMINE induced a dose-dependent increase in ROI generation. Thiseffect was less pronounced with DOTAP and absent with neutral andnegative liposomes. Conclusions. ROI play a key role in cationic lipid-mediated toxicity.Polyvalent cationic liposomes cause a release of ROI which areresponsible for the pulmonary toxicity.

An NLRP7-Containing Inflammasome Mediates Recognition of Microbial Lipopeptides in Human Macrophages

Cytosolic pathogen- and damage-associated molecular patterns are sensed by pattern recognition receptors, including members of the nucleotide-binding domain and leucine-rich repeat-containing gene family (NLR), which cause inflammasome assembly and caspase-1 activation to promote maturation and release of the inflammatory cytokines interleukin-1β (IL-1β) and IL-18 and induction of pyroptosis. However, the contribution of most of the NLRs to innate immunity, host defense, and inflammasome activation and their specific agonists are still unknown. Here we describe identification and characterization of an NLRP7 inflammasome in human macrophages, which is induced in response to microbial acylated lipopeptides. Activation of NLRP7 promoted ASC-dependent caspase-1 activation, IL-1β and IL-18 maturation, and restriction of intracellular bacterial replication, but not caspase-1-independent secretion of the proinflammatory cytokines IL-6 and tumor necrosis factor-α. Our study therefore increases our currently limited understanding of NLR activation, inflammasome assembly, and maturation of IL-1β and IL-18 in human macrophages.

S-Nitrosylation of Bcl-2 Inhibits Its Ubiquitin-Proteasomal Degradation A NOVEL ANTIAPOPTOTIC MECHANISM THAT SUPPRESSES APOPTOSIS

Bcl-2 is a key apoptosis regulatory protein of the mitochondrial death pathway whose function is dependent on its expression levels. Although Bcl-2 expression is controlled by various mechanisms, post-translational modifications, such as ubiquitination and proteasomal degradation, have emerged as important regulators of Bcl-2 function. However, the underlying mechanisms of this regulation are unclear. We report here that Bcl-2 undergoes S-nitrosylation by endogenous nitric oxide (NO) in response to multiple apoptotic mediators and that this modification inhibits ubiquitin-proteasomal degradation of Bcl-2. Inhibition of NO production by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and by NO synthase inhibitor aminoguanidine effectively inhibited S-nitrosylation of Bcl-2, increased its ubiquitination, and promoted apoptotic cell death induced by chromium (VI). In contrast, the NO donors dipropylenetriamine NONOate and sodium nitroprusside showed opposite effects. The effect of NO on Bcl-2 stability was shown to be independent of its dephosphorylation. Mutational analysis of Bcl-2 further showed that the two cysteine residues of Bcl-2 (Cys158 and Cys229) are important in the S-nitrosylation process and that mutations of these cysteines completely inhibited Bcl-2 S-nitrosylation. Treatment of the cells with other stress inducers, including Fas ligand and buthionine sulfoxide, also induced Bcl-2 S-nitrosylation, suggesting that this is a general phenomenon that regulates Bcl-2 stability and function under various stress conditions. These findings indicate a novel function of NO and its regulation of Bcl-2, which provides a key mechanism for the control of apoptotic cell death and cancer development.

Reactive Oxygen Species Mediate Caspase Activation and Apoptosis Induced by Lipoic Acid in Human Lung Epithelial Cancer Cells through Bcl-2 Down-Regulation

The antioxidant α-lipoic acid (LA) is a naturally occurring compound that has been shown to possess promising anticancer activity because of its ability to preferentially induce apoptosis and inhibit proliferation of cancer cells relative to normal cells. However, the molecular mechanisms underlying the apoptotic effect of LA are not well understood. We report here that LA induced reactive oxygen species (ROS) generation and a concomitant increase in apoptosis of human lung epithelial cancer H460 cells. Inhibition of ROS generation by ROS scavengers or by overexpression of antioxidant enzymes glutathione peroxidase and superoxide dismutase effectively inhibited LA-induced apoptosis, indicating the role of ROS, especially hydroperoxide and superoxide anion, in the apoptotic process. Apoptosis induced by LA was found to be mediated through the mitochondrial death pathway, which requires caspase-9 activation. Inhibition of caspase activity by the pan-caspase inhibitor (z-VAD-FMK) or caspase-9-specific inhibitor (z-LEHD-FMK) completely inhibited the apoptotic effect of LA. Likewise, the mitochondrial respiratory chain inhibitor rotenone potently inhibited the apoptotic and ROS-inducing effects of LA, supporting the role of mitochondrial ROS in LA-induced cell death. LA induced down-regulation of mitochondrial Bcl-2 protein through peroxide-dependent proteasomal degradation, and overexpression of the Bcl-2 protein prevented the apoptotic effect of LA. Together, our findings indicate a novel pro-oxidant role of LA in apoptosis induction and its regulation by Bcl-2, which may be exploited for the treatment of cancer and related apoptosis disorders.

Protein Nanoparticles as Drug Delivery Carriers for Cancer Therapy

Nanoparticles have increasingly been used for a variety of applications, most notably for the delivery of therapeutic and diagnostic agents. A large number of nanoparticle drug delivery systems have been developed for cancer treatment and various materials have been explored as drug delivery agents to improve the therapeutic efficacy and safety of anticancer drugs. Natural biomolecules such as proteins are an attractive alternative to synthetic polymers which are commonly used in drug formulations because of their safety. In general, protein nanoparticles offer a number of advantages including biocompatibility and biodegradability. They can be prepared under mild conditions without the use of toxic chemicals or organic solvents. Moreover, due to their defined primary structure, protein-based nanoparticles offer various possibilities for surface modifications including covalent attachment of drugs and targeting ligands. In this paper, we review the most significant advancements in protein nanoparticle technology and their use in drug delivery arena. We then examine the various sources of protein materials that have been used successfully for the construction of protein nanoparticles as well as their methods of preparation. Finally, we discuss the applications of protein nanoparticles in cancer therapy.

Activation of Inflammasomes Requires Intracellular Redistribution of the Apoptotic Speck-Like Protein Containing a Caspase Recruitment Domain

Activation of caspase 1 is essential for the maturation and release of IL-1β and IL-18 and occurs in multiprotein complexes, referred to as inflammasomes. The apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is the essential adaptor protein for recruiting pro-caspase 1 into inflammasomes, and consistently gene ablation of ASC abolishes caspase 1 activation and secretion of IL-1β and IL-18. However, distribution of endogenous ASC has not yet been examined in detail. In the present study, we demonstrated that ASC localized primarily to the nucleus in resting human monocytes/macrophages. Upon pathogen infection, ASC rapidly redistributed to the cytosol, followed by assembly of perinuclear aggregates, containing several inflammasome components, including caspase 1 and Nod-like receptors. Prevention of ASC cytosolic redistribution completely abolished pathogen-induced inflammasome activity, which affirmed that cytosolic localization of ASC is essential for inflammasome function. Thus, our study characterized a novel mechanism of inflammasome regulation in host defense.

Cadmium Increases HIF-1 and VEGF Expression through ROS, ERK, and AKT Signaling Pathways and Induces Malignant Transformation of Human Bronchial Epithelial Cells

Cadmium is categorized as a human carcinogen especially involved in lung cancers. Angiogenesis is considered a fundamental requirement for tumorigenesis, but the mechanisms underlying the tumor angiogenesis induced by cadmium are poorly understood. Using in vitro and in vivo models, we investigated the angiogenic mechanisms of cadmium in human bronchial epithelial cells and tumor formation. Our results demonstrated that cadmium (CdCl(2)) activated extracellular signal-regulated kinases (ERK) and AKT signaling and elevated the expression of a key downstream proangiogenic molecule hypoxia-inducible factor-1 (HIF-1) in immortalized human lung epithelial BEAS-2B cells. Cadmium also induced reactive oxygen species (ROS) production, which could be inhibited by ROS scavengers, catalase and diphenyleneiodonium chloride. Inhibition of ROS generation also attenuated ERK, AKT, p70S6K1 activation, and HIF-1α expression. Similar results were obtained in normal human bronchial epithelial (NHBE) cells, showing that cadmium induced HIF-1 expression via ROS/ERK/AKT signaling pathway. Furthermore, cadmium induced vascular endothelial growth factor expression and transcriptional activation through ROS, ERK, and AKT pathways. Finally, cadmium transformed human bronchial epithelial cells in culture; the transformed cells induced tube formation in vitro, angiogenesis on chicken chorioallantoic membrane, and formed tumors in nude mice. Taken together, the results of this study provide explanation for the role and molecular mechanisms of cadmium in promoting angiogenesis in lung epithelial cells and malignant transformation and will be helpful for improved occupational protection, prevention, as well as chemotherapy of human lung cancers caused by heavy metal cadmium.

Cobalt-mediated generation of reactive oxygen species and its possible mechanism

Electron spin resonance spin trapping was utilized to investigate free radical generation from cobalt (Co) mediated reactions using 5,5-dimethyl-1-pyrroline (DMPO) as a spin trap. A mixture of Co with water in the presence of DMPO generated 5,5-dimethylpyrroline-(2)-oxy(1) DMPOX, indicating the production of strong oxidants. Addition of superoxide dismutase (SOD) to the mixture produced hydroxyl radical (.OH). Catalase eliminated the generation of this radical and metal chelators, such as desferoxamine, diethylenetriaminepentaacetic acid or 1,10-phenanthroline, decreased it. Addition of Fe(II) resulted in a several fold increase in the .OH generation. UV and O2 consumption measurements showed that the reaction of Co with water consumed molecular oxygen and generated Co(II). Since reaction of Co(II) with H2O2 did not generate any significant amount of .OH radicals, a Co(I) mediated Fenton-like reaction [Co(I) + H2O2-->Co(II) + .OH + OH-] seems responsible for .OH generation. H2O2 is produced from O2.- via dismutation, O2.- is produced by one-electron reduction of molecular oxygen catalyzed by Co. Chelation of Co(II) by biological chelators, such as glutathione or beta-ananyl-3-methyl-L-histidine alters, its oxidation-reduction potential and makes Co(II) capable of generating .OH via a Co(II)-mediated Fenton-like reaction [Co(II) + H2O2-->Co(III) + .OH + OH-]. Thus, the reaction of Co with water, especially in the presence of biological chelators, glutathione, glycylglycylhistidine and beta-ananyl-3-methyl-L-histidine, is capable of generating a whole spectrum of reactive oxygen species, which may be responsible for Co-induced cell injury.