Myc-nick promotes efferocytosis through M2 macrophage polarization during resolution of inflammation.

A key event required for effective resolution of inflammation is efferocytosis, which is defined as phagocytic removal of apoptotic cells mostly by macrophages acquiring an alternatively activated phenotype (M2). c-Myc has been reported to play a role in alternative activation of human macrophages and is proposed as one of the M2 macrophage markers. We found that M2-like peritoneal macrophages from zymosan A-treated mice exhibited a marked accumulation of Myc-nick, a truncated protein generated by a Calpain-mediated proteolytic cleavage of full-length c-Myc. Further, ectopic expression of Myc-nick in murine bone marrow-derived macrophages promoted the M2 polarization and, consequently, enhanced their efferocytic capability. Notably, Myc-nick-induced efferocytosis was found to be tightly associated with α-tubulin acetylation by K acetyltransferase 2a (Kat2a/Gcn5) activity. These findings suggest Myc-nick as a novel proresolving mediator that has a fundamental function in maintaining homeostasis under inflammatory conditions.-Zhong, X., Lee, H.-N., Kim, S. H., Park, S.-A., Kim, W., Cha, Y.-N., Surh, Y.-J. Myc-nick promotes efferocytosis through M2 macrophage polarization during resolution of inflammation.

Taurine chloramine potentiates phagocytic activity of peritoneal macrophages through up-regulation of dectin-1 mediated by heme oxygenase-1-derived carbon monoxide.

Resolution of inflammation that occurs after microbial infection or tissue damage is an important physiologic process in maintaining or restoring host homeostasis. Taurine chloramine (TauCl) is formed by a reaction between taurine and hypochlorite in leukocytes, and it is especially abundant in activated neutrophils that encounter an oxidative burst. As neutrophils undergo apoptosis, TauCl is released to the extracellular matrix at the inflamed sites, thereby affecting coexisting macrophages in the inflammatory microenvironment. In this study, we investigated the role of TauCl in phagocytosis by macrophages during resolution of fungal infection-induced inflammation. We found that exogenous TauCl substantially increased the phagocytic efficiency of macrophages through up-regulation of dectin-1, a receptor for fungal ß-1,3-glucans, which is present on the membrane of macrophages. Our previous studies demonstrated the induction of heme oxygenase-1 (HO-1) expression in murine peritoneal macrophages treated with TauCl. In the present study, knocking out HO-1 or pharmacologic inhibition of HO-1 with zinc protoporphyrin IX attenuated the TauCl-induced expression of dectin-1 and subsequent phagocytosis. Furthermore, carbon monoxide (CO), a by-product of the HO-1-catalyzed reaction, induced expression of dectin-1 and potentiated phagocytic capability of the macrophages, which appeared to be mediated through up-regulation of peroxisome proliferator-activated receptor γ. Taken together, induction of HO-1 expression and subsequent CO production by TauCl are essential for phagocytosis of fungi by macrophages. Our results suggest that TauCl has important roles in host defense against fungal infection and has therapeutic potential in the management of inflammatory diseases.-Kim, S. H., Zhong, X., Kim, W., Kim, K., Suh, Y.-G., Kim, C., Joe, Y., Chung, H. T., Cha, Y.-N., Surh, Y.-J. Taurine chloramine potentiates phagocytic activity of peritoneal macrophages through up-regulation of dectin-1 mediated by heme oxygenase-1-derived carbon monoxide.

Role of heme oxygenase-1 in potentiation of phagocytic activity of macrophages by taurine chloramine: Implications for the resolution of zymosan A-induced murine peritonitis.

Phagocytosis of pathogens by macrophages is crucial for the successful resolution of inflammation induced by microbial infection. Taurine chloramine (TauCl), an endogenous anti-inflammatory and antioxidative substance, is produced by reaction between taurine and hypochlorous acid by myeloperoxidase activity in neutrophils under inflammatory conditions. In the present study, we investigated the effect of TauCl on resolution of acute inflammation caused by fungal infection using a zymosan A-induced murine peritonitis model. TauCl administration reduced the number of the total peritoneal leukocytes, while it increased the number of peritoneal monocytes. Furthermore, TauCl promoted clearance of pathogens remaining in the inflammatory environment by macrophages. When the macrophages isolated from thioglycollate-treated mice were treated with TauCl, their phagocytic capability was enhanced. In the murine macrophage-like RAW264.7 cells treated with TauCl, the proportion of macrophages clearing the zymosan A particles was also increased. TauCl administration resulted in elevated expression of heme oxygenase-1 (HO-1) in the peritoneal macrophages. Pharmacologic inhibition of HO-1 activity or knockdown of HO-1 in the murine macrophage RAW264.7 cells abolished the TauCl-induced phagocytosis, whereas the overexpression of HO-1 augmented the phagocytic ability of macrophages. Moreover, peritoneal macrophages isolated from HO-1 null mice failed to mediate TauCl-induced phagocytosis. Our results suggest that TauCl potentiates phagocytic activity of macrophages through upregulation of HO-1 expression.

Helicobacter pylori induces Snail expression through ROS-mediated activation of Erk and inactivation of GSK-3ß in human gastric cancer cells.

Helicobacter pylori (H. pylori) infection has been known to be implicated in human gastric carcinogenesis. Snail, the zinc-finger transcription factor known as a key inducer of changes in the cell shape and morphogenetic movement, is aberrantly overexpressed and correlates with lymph node metastasis in gastric cancer. In the present study, we investigated whether H. pylori could induce Snail activation to provoke these changes. Using a cell scatter assay, we noticed that human gastric cancer AGS cells infected with H. pylori underwent morphological changes as well as disruption of cell-cell interaction, which was then reversed by silencing of Snail by use of small interfering RNA (siRNA). In addition, infection with H. pylori resulted in an increased intracellular level of Snail in gastric cancer cells, which was abrogated in the presence of U0126 and LY294002, inhibitors of MEK/Erk and PI3K/Akt pathways, respectively. Cycloheximide pulse-chase experiments coupled with immunocytochemical analysis revealed that the induction of Snail by H. pylori was regulated at multiple levels, including increased transcription of Snail mRNA, inhibition of protein degradation, and enhancement of nuclear translocation of Snail. Pre-treatment of AGS cells with N-acetylcysteine, a well-known reactive oxygen species (ROS) scavenger, attenuated the H. pylori-induced activation of Erk, its binding to Snail promoter, inactivation of GSK-3ß, and accumulation of Snail. Collectively, these findings suggest that the upregulation of Snail expression induced by H. pylori and transformation to a spindle-like shape as a consequence in gastric cancer cells are attributable to ROS-mediated activation of Erk and the inhibition of GSK-3ß signaling. © 2016 Wiley Periodicals, Inc.

Helicobacter pylori Activates IL-6-STAT3 Signaling in Human Gastric Cancer Cells: Potential Roles for Reactive Oxygen Species.

BACKGROUND: Recent studies have shown that Helicobacter pylori (H. pylori) activates signal transducer and activator of transcription 3 (STAT3) that plays an important role in gastric carcinogenesis. However, the molecular mechanism underlying H. pylori-mediated STAT3 activation is still not fully understood. In this study, we investigated H. pylori-induced activation of STAT3 signaling in AGS human gastric cancer cells and the underlying mechanism. MATERIALS AND METHODS: AGS cells were cocultured with H. pylori, and STAT3 activation was assessed by Western blot analysis, electrophoretic mobility shift assay and immunocytochemistry. To demonstrate the involvement of reactive oxygen species (ROS) in H. pylori-activated STAT3 signaling, the antioxidant N-acetylcysteine was utilized. The expression and production of interleukin-6 (IL-6) were measured by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA), respectively. The interaction between IL-6 and IL-6 receptor (IL-6R) was determined by the immunoprecipitation assay. RESULTS: H. pylori activates STAT3 as evidenced by increases in phosphorylation on Tyr(705) , nuclear localization, DNA binding and transcriptional activity of this transcription factor. The nuclear translocation of STAT3 was also observed in H. pylori-inoculated mouse stomach. In the subsequent study, we found that H. pylori-induced STAT3 phosphorylation was dependent on IL-6. Notably, the increased IL-6 expression and the IL-6 and IL-6R binding were mediated by ROS produced as a consequence of H. pylori infection. CONCLUSIONS: H. pylori-induced STAT3 activation is mediated, at least in part, through ROS-induced upregulation of IL-6 expression. These findings provide a novel molecular mechanism responsible for H. pylori-induced gastritis and gastric carcinogenesis.

Resolvin D1 stimulates efferocytosis through p50/p50-mediated suppression of tumor necrosis factor-α expression.

Phagocytosis of apoptotic neutrophils, termed efferocytosis, is essential for the resolution of inflammation as it prevents the tissues surrounding the inflamed site from being exposed to the toxic contents of lytic cells. Resolvin D1 (RvD1), endogenously generated from docosahexaenoic acid during resolution of inflammation, is known to stimulate efferocytosis. However, the molecular mechanism underlying RvD1-mediated enhancement of efferocytosis remains largely unresolved. In the present study, murine macrophage-like RAW264.7 cells treated with lipopolysaccharide (LPS) exhibited markedly reduced efferocytic activity, but this was restored by co-incubation with RvD1. RvD1-induced restoration of the efferocytic activity appears to be mediated by downregulation of LPS-induced TNF-α expression. The inhibitory effect of RvD1 on LPS-induced TNF-α expression was associated with enhanced nuclear localization of p50/p50 homodimer and concomitant reduction of p65/p50 heterodimer accumulation in the nucleus. RvD1 triggered phosphorylation and proteasomal degradation of nuclear factor κB1 (NF-κB1) p105 to generate p50, which was subsequently translocated to the nucleus as a p50/p50 homodimer. Knockdown of NF-κB p50 abolished the ability of RvD1 to suppress TNF-α expression and also to restore efferocytosis, suggesting that the replacement of p65/p50 with p50/p50 homodimer in the nucleus is crucial for RvD1-mediated stimulation of efferocytosis. In a murine peritonitis model, intraperitoneal administration of RvD1 abolished the zymosan-A-induced TNF-α production, thereby stimulating efferocytosis. Taken together, these findings indicate that RvD1 expedites resolution of inflammation through induction of efferocytosis by p50/p50-homodimer-mediated repression of TNF-α production.

Taurine chloramine produced from taurine under inflammation provides anti-inflammatory and cytoprotective effects.

Taurine is one of the most abundant non-essential amino acid in mammals and has many physiological functions in the nervous, cardiovascular, renal, endocrine, and immune systems. Upon inflammation, taurine undergoes halogenation in phagocytes and is converted to taurine chloramine (TauCl) and taurine bromamine. In the activated neutrophils, TauCl is produced by reaction with hypochlorite (HOCl) generated by the halide-dependent myeloperoxidase system. TauCl is released from activated neutrophils following their apoptosis and inhibits the production of inflammatory mediators such as, superoxide anion, nitric oxide, tumor necrosis factor-α, interleukins, and prostaglandins in inflammatory cells at inflammatory tissues. Furthermore, TauCl increases the expressions of antioxidant proteins, such as heme oxygenase 1, peroxiredoxin, thioredoxin, glutathione peroxidase, and catalase in macrophages. Thus, a central role of TauCl produced by activated neutrophils is to trigger the resolution of inflammation and protect macrophages and surrounding tissues from being damaged by cytotoxic reactive oxygen metabolites overproduced during inflammation. This is achieved by attenuating further production of proinflammatory cytokines and reactive oxygen metabolites and also by increasing the levels of antioxidant proteins that are able to scavenge and diminish the production of cytotoxic oxygen metabolites. These findings suggest that TauCl released from activated neutrophils may be involved in the recovery processes of cells affected by inflammatory oxidative stresses and thus TauCl could be used as a potential physiological agent to control pathogenic symptoms of chronic inflammatory diseases.

Guggulsterone induces heme oxygenase-1 expression through activation of Nrf2 in human mammary epithelial cells: PTEN as a putative target.

Guggulsterone (GS) [4,17(20)-pregnadiene-3,16-dione] is a phytosterol found in the gum resin of the Commiphora mukul. GS exists naturally in two stereoisomers: E-GS (cis-GS) and Z-GS (trans-GS). In this study, the effects of both isomers on expression of the cytoprotective enzyme heme oxygenase-1 (HO-1) were evaluated in human mammary epithelial (MCF10A) cells. NF-E2-related factor 2 (Nrf2) is considered a master regulator in activating antioxidant response element (ARE)-driven expression of HO-1 and many other antioxidant/cytoprotective proteins. cis-GS upregulated the transcription and protein expression of HO-1 to a greater extent than did trans-GS. cis-GS treatment enhanced nuclear translocation and ARE-binding activity of Nrf2. MCF10A cells transfected with an ARE luciferase construct exhibited significantly elevated Nrf2 transcriptional activity upon cis-GS treatment compared with cells transfected with the control vector. In addition, silencing of the Nrf2 gene abrogated cis-GS-induced expression of HO-1. Incubation of MCF10A cells with cis-GS increased phosphorylation of Akt. The pharmacological inhibition of phosphoinositide 3-kinase (PI3K), an upstream kinase responsible for Akt phosphorylation, abrogated cis-GS-induced Nrf2 nuclear translocation. Pretreatment with the thiol-reducing agents attenuated Akt phosphorylation, Nrf2 activation and HO-1 expression, suggesting that cis-GS may cause thiol modification of an upstream signaling modulator. Phosphatase and Tensin Homologue Deleted on Chromosome 10 (PTEN) is a negative regulator of the PI3K-Akt axis. The mutation in cysteine 124 present in the catalytic domain of PTEN abolished cis-GS-induced HO-1 expression as well as Akt phosphorylation. Whether this cysteine is a 'bona fide' target of cis-GS in its activation of Nrf2 needs additional investigation.

A protective role of nuclear factor-erythroid 2-related factor-2 (Nrf2) in inflammatory disorders.

Nuclear factor-erythroid 2-related factor-2 (Nrf2) is a key transcription factor that plays a central role in cellular defense against oxidative and electrophilic insults by timely induction of antioxidative and phase-2 detoxifying enzymes and related stress-response proteins. The 5'-flanking regions of genes encoding these cytoprotective proteins contain a specific consensus sequence termed antioxidant response element (ARE) to which Nrf2 binds. Recent studies have demonstrated that Nrf2-ARE signaling is also involved in attenuating inflammation-associated pathogenesis, such as autoimmune diseases, rheumatoid arthritis, asthma, emphysema, gastritis, colitis and atherosclerosis. Thus, disruption or loss of Nrf2 signaling causes enhanced susceptibility not only to oxidative and electrophilic stresses but also to inflammatory tissue injuries. During the early-phase of inflammation-mediated tissue damage, activation of Nrf2-ARE might inhibit the production or expression of pro-inflammatory mediators including cytokines, chemokines, cell adhesion molecules, matrix metalloproteinases, cyclooxygenase-2 and inducible nitric oxide synthase. It is likely that the cytoprotective function of genes targeted by Nrf2 may cooperatively regulate the innate immune response and also repress the induction of pro-inflammatory genes. This review highlights the protective role of Nrf2 in inflammation-mediated disorders with special focus on the inflammatory signaling modulated by this redox-regulated transcription factor.

The positive feedback loop between Nrf2 and phosphogluconate dehydrogenase stimulates proliferation and clonogenicity of human hepatoma cells.

Recent studies report that nuclear factor-erythroid-2-related factor 2 (Nrf2) facilitates tumor progression through metabolic reprogramming in cancer cells. However, the molecular mechanism underlying the oncogenic functions of Nrf2 is not yet well understood. Some of the pentose phosphate pathway (PPP) enzymes are considered to play a role in the cancer progression. The present study was intended to explore the potential role of phosphogluconate dehydrogenase (PGD), one of the PPP enzymes, in the proliferation and migration of human hepatoma HepG2 cells. Genetic ablation of Nrf2 attenuated the expression of PGD at both transcriptional and translational levels. Notably, Nrf2 regulates the transcription of PGD through direct binding to the antioxidant response element in its promoter region. Nrf2 overexpression in HepG2 cells led to increased proliferation, survival, and migration, and these events were suppressed by silencing PGD. Interestingly, knockdown of the gene encoding this enzyme not only attenuated the proliferation and clonogenicity of HepG2 cells but also downregulated the expression of Nrf2. Thus, there seems to exist a positive feedback loop between Nrf2 and PGD which is exploited by hepatoma cells for their proliferation and survival. Treatment of HepG2 cells with ribulose-5-phosphate, a catalytic product of PGD, gave rise to a concentration-dependent upregulation of Nrf2. Collectively, the current study shows that Nrf2 promotes hepatoma cell growth and progression, partly through induction of PGD transcription.

15-Deoxy-Delta12,14-prostaglandin J2 upregulates the expression of heme oxygenase-1 and subsequently matrix metalloproteinase-1 in human breast cancer cells: possible roles of iron and ROS.

Heme oxygenase-1 (HO-1) has recently been found to be involved in angiogenesis and metastasis. In this study, we investigated whether HO-1 could potentiate the metastatic potential of human breast cancer cells. Treatment of MCF-7 and MDA-MB-231 cells with 30 microM of 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) increased the expression of HO-1, which preceded the induction of matrix metalloproteinases (MMPs). The 15d-PGJ2-induced upregulation of MMP-1 was abrogated by the HO-1 inhibitor zinc protoporphyrin IX (ZnPP) as well as introduction of HO-1 short interfering RNA. In addition, HO-1 inducers, such as cobalt protoporphyrin IX and hemin, upregulated the expression of MMP-1. Overexpression of HO-1 in the MCF-7 cells caused the induction of MMP-1 expression. Treatment with the HO-1 inhibitor ZnPP abolished the migrative phenotype of 15d-PGJ2-treated MCF-7 cells. MCF-7 cells treated with 15d-PGJ2 exhibited intracellular accumulation of reactive oxygen species (ROS) which was abolished by ZnPP. We hypothesize that excess iron, released as a consequence HO-1 activity induced by 15d-PGJ2, is transiently available for the stimulation of intracellular ROS generation and subsequently MMP-1 expression. 15d-PGJ2-mediated upregulation of MMP-1 expression was blocked by the iron chelator desferrioxamine and the Fe2+-specific chelator 1,10-phenanthroline. The iron chelators as well as the antioxidant N-acetyl-L-cysteine abrogated ROS formation by 15d-PGJ2. In conclusion, 15d-PGJ2 upregulates MMP-1 expression via induction of HO-1 and subsequent production of iron capable of generating ROS, which may contribute to increased metastasis and invasiveness of the human breast cancer cells.

Production of reactive oxygen and nitrogen species in phagocytes is regulated by taurine chloramine.

Taurine is abundantly present in phagocytic cells and provides protection against cytotoxicity caused by reactive oxygen species (ROS). The reaction between taurine and HOCl, a toxic product of the myeloperoxidase (MPO) system, generates a more stable and less toxic product, taurine chloramine (TauCl). TauCl has also been shown to inhibit the production of superoxide anion (O2-) and nitric oxide (NO). In this review, we compare the effect of taurine and TauCl on the production of these reactive species in phagocytes. First, TauCl inhibit PMA-derived O2- production and this is associated with inhibition of p47phox phosphorylation and of p47phox and p67phox translocation. Second, TauCl inhibits LPS-induced iNOS expression and NO production. This occurs by direct inhibition of Ras activation, ERK1/2 phosphorylation and NF-kappaB activation. Third, TauCl by itself increases the expression of heme oxygenase-1 (HO-1) and enhances HO activity. Carbon monoxide (CO), a product of HO activity, is able to inhibit both O2- and NO production. Combined, these effects of TauCl appear to provide cytoprotection against the inadvertent cytotoxicity caused by overproduction of O2- and NO.

Role of Nrf2-mediated heme oxygenase-1 upregulation in adaptive survival response to nitrosative stress.

Nitrosative stress caused by reactive nitrogen species such as nitric oxide and peroxynitrite overproduced during inflammation leads to cell death and has been implicated in the pathogenesis of many human ailments. However, relatively mild nitrosative stress may fortify cellular defense capacities, rendering cells tolerant or adaptive to ongoing and subsequent cytotoxic challenges, a phenomenon known as 'preconditioning' or 'hormesis'. One of the key components of cellular stress response is heme oxygenase-1 (HO-1), the rate limiting enzyme in the process of degrading potentially toxic free heme into biliverdin, free iron and carbon monoxide. HO-1 is upregulated by a wide array of stimuli and has antioxidant, anti-inflammatory and other cytoprotective functions. This review is intended to provide readers with a welldocumented account of the research done in the area of cellular adaptive survival response against nitrosative stress with special focus on the role of HO-1 upregulation, especially through activation of the transcription factor, Nrf2.

Taurine Chloramine Activates Nrf2, Increases HO-1 Expression and Protects Cells from Death Caused by Hydrogen Peroxide.

Hypochlorous acid (HOCl) is toxic and causes cell death. However, this effect is inhibited by reaction with taurine, which generates taurine chloramine (TauCl), thereby protecting the cells from HOCl-generated toxicity. TauCl has been shown to inhibit the production of inflammatory mediators like O(2) (*-), H(2)O(2) and NO. In this study, RAW 264.7 macrophages treated with TauCl were protected from death caused by H(2)O(2). TauCl increased the expression of peroxiredoxin-1, thioredoxin-1 and heme oxygenase (HO)-1, the anti-oxidant enzymes normally induced by activation of NF-E2-related factor-2 (Nrf2). TauCl increased nuclear translocation of Nrf2 and binding to the anti-oxidant response element. These data suggest that TauCl produced abundantly in the activated neutrophils and released to surrounding cells in the inflamed tissues may induce the expression of cytoprotective anti-oxidant enzymes. Elevation of HO activity via induction of HO-1 expression within neighboring cells may provide protection from cytotoxicity caused by inflammatory oxidants like H(2)O(2).

Vav1 inhibits RANKL-induced osteoclast differentiation and bone resorption.

Vav1 is a Rho/Rac guanine nucleotide exchange factor primarily expressed in hematopoietic cells. In this study, we investigated the potential role of Vav1 in osteoclast (OC) differentiation by comparing the ability of bone marrow mononuclear cells (BMMCs) obtained from Vav1-deficient (Vav1-/-) and wild-type (WT) mice to differentiate into mature OCs upon stimulation with macrophage colony stimulating factor and receptor activator of nuclear kappa B ligand in vitro. Our results suggested that Vav1 deficiency promoted the differentiation of BMMCs into OCs, as indicated by the increased expression of tartrate-resistant acid phosphatase, cathepsin K, and calcitonin receptor. Therefore, Vav1 may play a negative role in OC differentiation. This hypothesis was supported by the observation of more OCs in the femurs of Vav1-/- mice than in WT mice. Furthermore, the bone status of Vav1-/- mice was analyzed in situ and the femurs of Vav1-/- mice appeared abnormal, with poor bone density and fewer number of trabeculae. In addition, Vav1-deficient OCs showed stronger adhesion to vitronectin, an αvß3 integrin ligand important in bone resorption. Thus, Vav1 may inhibit OC differentiation and protect against bone resorption. [BMB Reports 2019; 52(11): 659-664].

Syntheses of tetrahydroisoquinoline derivatives that inhibit NO production in activated BV-2 microglial cells.

Seventeen tetrahydroisoquinoline derivatives were designed, synthesized and evaluated for inhibition of NO production in lipopolysaccharide-stimulated BV-2 microglial cells. Compounds 5a, 9c and 11a potently attenuated NO production by >60%, and 5a and 11a inhibited BH4 production by >48% at 100 microM. In particular, N-ethylcarbonyl-7-hydroxy-6-methoxy-1,2,3,4-tetrahydroisoquinoline (11a) reduced NO production by 64% and tetrahydrobiopterin (BH4) production by 49%. Introducing longer alkyl component at C1 or N2 position led to attenuation of the inhibitory effect. It is possible that 11a inhibits NO production by blocking BH4-dependent dimerization of newly synthesized iNOS monomers.

Role of heme oxygenase-1 and its reaction product, carbon monoxide, in manifestation of breast cancer stem cell-like properties: Notch-1 as a putative target.

Cancer stem cells (CSCs) constitute a subpopulation of transformed cells that possess intrinsic ability to undergo selfrenewal and differentiation, which drive tumour resistance and cancer recurrence. It has been reported that CSCs possess enhanced protection against oxidative stress induced by reactive oxygen species compared with nonstem-like cancer cells. In the present work, we investigated the role of heme oxygenase-1 (HO-1), a representative antioxidant enzyme, on the stemness and selfrenewal of human breast CSCs. We found that pharmacologic or genetic inhibition of HO-1 attenuated the sphere formation, whereas HO-1 inducers enhanced the number and the size of tumourspheres in breast CSCs. Carbon monoxide (CO) is endogenously generated as a consequence of degradation of heme by HO-1. The proportion of populations of CD44+/CD24- cells retaining CSC properties was increased in MDA-MB-231 cells treated with a CO-releasing molecule (CORM-2). Following CORM-2 treatment, the expression of Notch-1 and related genes Jagged-1 and Hes1 was increased, which was accompanied by the mammosphere formation. Taken together, these findings suggest that HO-1-derived CO production stimulates the formation of mammospheres in breast cancer cells through activation of Notch-1 signalling.

Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H-Ras transformed human mammary epithelial cells.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is latent but constitutively activated in many types of cancers. It is well known that STAT3 plays a key role in inflammation-associated tumorigenesis. Curcumin is an anti-inflammatory natural compound isolated from the turmeric (Curcuma longa L., Zingiberaceae) that has been extensively used in a traditional medicine over the centuries. In the present study, we have found that curcumin inhibits STAT3 signaling that is persistently overactivated in H-Ras transformed breast epithelial cells (H-Ras MCF10A). Specific cysteine residues present in STAT3 appear to be critical for the activity as well as conformation of this transcription factor. We identified the cysteine residue 259 of STAT3 as a putative site for curcumin binding. Site-directed mutation of this cysteine residue abolished curcumin-induced inactivation of STAT3 and apoptosis in H-Ras MCF10A cells. The α,ß-unsaturated carbonyl moiety of curcumin appears to be essential in its binding to STAT3 in H-Ras MCF10A cells. Tetrahydrocurcumin that lacks such electrophilic moiety failed to interact with STAT3 and to induce apoptosis in the same cell line. Taken together, our findings suggest that curcumin can abrogate aberrant activation of STAT3 through direct interaction, thereby inhibiting STAT3-mediated mammary carcinogenesis.

Nrf2-mediated heme oxygenase-1 induction confers adaptive survival response to tetrahydropapaveroline-induced oxidative PC12 cell death.

Tetrahydropapaveroline (THP), a dopaminergic isoquinoline neurotoxin, has been reported to contribute to neurodegeneration in parkinsonism. As THP bears two catechol moieties, it undergoes autooxidation or enzymatic oxidation to produce reactive oxygen species (ROS), which may contribute to the THP-induced cell death. Although ROS are cytotoxic, the initial accumulation of ROS may provoke a survival response. In this study, treatment of PC12 cells with THP increased expression of heme oxygenase-1 (HO-1) as an adaptive survival response. Furthermore, THP-induced cytotoxicity was attenuated by the HO-1 inducer (SnCl2) and exacerbated by the HO-1 inhibitor (ZnPP). To elucidate the molecular mechanisms underlying THP-mediated HO-1 expression, we examined the possible involvement of NF-E2-related factor 2 (Nrf2), which plays an important role in the transcriptional regulation of detoxifying/antioxidant genes. THP treatment elevated nuclear translocation of Nrf2 and subsequent binding to antioxidant response element (ARE). PC12 cells transfected with dominant-negative Nrf2 exhibited increased cytotoxicity and decreased HO-1 expression after THP treatment. Moreover, U0126 and LY294002, which are pharmacologic inhibitors of extracellular signal-regulated kinase1/2 and phosphoinositide 3-kinase, respectively, attenuated HO-1 expression as well as Nrf2-ARE binding activity. Taken together, these findings suggest that HO-1 induction via Nrf2 activation may confer a cellular adaptive response against THP-mediated cell death.

Capsaicin induces heme oxygenase-1 expression in HepG2 cells via activation of PI3K-Nrf2 signaling: NAD(P)H:quinone oxidoreductase as a potential target.

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), a major pungent ingredient of red pepper, is reported to have antimutagenic and anticarcinogenic properties. However, the mechanisms underlying its chemoprotective effects remain largely unresolved. In the present study, we found that capsaicin induced expression of heme oxygenase-1 (HO-1) in HepG2 cells. Capsaicin treatment resulted in a transient increase in the phosphorylation of Akt and subsequently nuclear translocation of NF-E2-related factor 2 (Nrf2), enhancing its binding to antioxidant response element (ARE). HepG2 cells treated with capsaicin exhibited increased production of reactive oxygen species (ROS). Prior exposure of cells to N-acetyl-L -cysteine blocked not only the ROS production but also the nuclear translocation of Nrf2 and its ARE binding, as well as HO-1 induction by capsaicin. Immunoblot analysis showed that whereas the level of HO-1 protein was elevated, that of NAD(P)H:quinone oxidoreductase (NQO1) was decreased after the treatment with capsaicin or the inhibitor of NQO1, dicumarol. We hypothesize that quinone metabolites or other reactive forms of capsaicin may bind covalently to NQO1 and thereby inhibit its activity, leading to production of ROS. This, in turn, would trigger the activation of Akt via phosphorylation, increase the nuclear translocation and ARE binding of Nrf2, and upregulate the expression of HO-1.