Targeting Heme Oxygenase-1 Research Articles

Genetic and Pharmacologic Targeting of Glycogen Synthase Kinase 3β Reinforces the Nrf2 Antioxidant Defense against Podocytopathy.

Evidence suggests that the glycogen synthase kinase 3 (GSK3)-dictated nuclear exclusion and degradation of Nrf2 is pivotal in switching off the self-protective antioxidant stress response after injury. Here, we examined the mechanisms underlying this regulation in glomerular disease. In primary podocytes, doxorubicin elicited cell death and actin cytoskeleton disorganization, concomitant with overactivation of GSK3β (the predominant GSK3 isoform expressed in glomerular podocytes) and minimal Nrf2 activation. SB216763, a highly selective small molecule inhibitor of GSK3, exerted a protective effect that depended on the potentiated Nrf2 antioxidant response, marked by increased Nrf2 expression and nuclear accumulation and augmented production of the Nrf2 target heme oxygenase-1. Ectopic expression of the kinase-dead mutant of GSK3β in cultured podocytes reinforced the doxorubicin-induced Nrf2 activation and prevented podocyte injury. Conversely, a constitutively active GSK3β mutant blunted the doxorubicin-induced Nrf2 response and exacerbated podocyte injury, which could be abolished by treatment with SB216763. In murine models of doxorubicin nephropathy or nephrotoxic serum nephritis, genetic targeting of GSK3β by doxycycline-inducible podocyte-specific knockout or pharmacologic targeting by SB216763 significantly attenuated albuminuria and ameliorated histologic signs of podocyte injury, including podocytopenia, loss of podocyte markers, podocyte de novo expression of desmin, and ultrastructural lesions of podocytopathy (such as foot process effacement). This beneficial outcome was likely attributable to an enhanced Nrf2 antioxidant response in glomerular podocytes because the selective Nrf2 antagonist trigonelline abolished the proteinuria-reducing and podocyte-protective effect. Collectively, our results suggest the GSK3β-regulated Nrf2 antioxidant response as a novel therapeutic target for protecting podocytes and treating proteinuric glomerulopathies.

Targeting Heme Oxygenase-1 Improves Poor Therapeutical Outcomes of BCR-ABL-Positive Adult B-Allwithikaros Isoform 6

Genomic profiling studies have demonstrated that the dominant-negative Ikaros isoform 6 (IK6) is overexpressed in BCR-ABL1-positive B-lineage acute lymphoblastic leukemia (BCR-ABL1-positive B-ALL), and it is strongly associated with a poor outcome. In addition, IK6 expression leads to BCR-ABL-positive B-ALL patients insensitive to tyrosine kinase inhibitors (TKIs). Moreover, a study has revealed that Heme oxygenase-1 (HO-1), an essential survival factor, could be a potential target in treating ALL. Therefore, we were wondering whether targeting HO-1 could improve poor therapeutical outcomes of BCR-ABL-positive adult B-ALL with IK6. Firstly, we detected that IK6 existed in 20 of 42 (47.6%) adult BCR-ABL1-positive B-ALL by using reverse transcribed polymerase chain reaction (PCR) and nucleotide sequencing. We also found that early clinical response was poor in BCR-ABL1-positive B-ALL patients with IK6, and they were usually high WBC counts at diagnosis and a high relapse rate. Compared to healthy donors' CD34+ cells, the levels of HO-1 expression were higher in primary CD34+ cells derived from adult BCR-ABL1-positive B-ALL patients with IK6 by qRT-PCR and western blot studies. And there was a strong correlation between the expression of IK6 and HO-1. In primary CD34+ leukemic cells derived from our IK6-positive patients' pool, silencing HO-1 resulted in the reduce of proliferation and a substantial proapoptotic effect, including a significantly enriched subG1 population, an increase of Annexin V-positive cells and of caspase3 cleavage. Furthermore, silencing HO-1 also increased their sensitivity to TKIs. This phenomenon was also supported by the observation that ZnPP IX substantially augmented the growth-inhibitory effects of imatinib on primary leukemic cells. Finally, we found that IK6 led to STAT5 activiation, and HO-1 was one of the downstream target genes of STAT5. In conclusion, the existence of extra IK6 seems to exacerbate poor prognosis and risks of relapse in adult BCR-ABL1-positive B-ALL. HO-1 plays an important role as a survival factor in BCR-ABL1-positive B-ALL with IK6, and targeting HO-1 can probably improve the clinical outcomes. DisclosuresNo relevant conflicts of interest to declare.

Bach1 deficiency reduces severity of osteoarthritis through upregulation of heme oxygenase-1.

IntroductionBTB and CNC homology 1 (Bach1) is a transcriptional repressor of Heme oxygenase-1 (HO-1), which is cytoprotective through its antioxidant effects. The objective of this study was to define the role of Bach1 in cartilage homeostasis and osteoarthritis (OA) development using in vitro models and Bach1-/- mice.MethodsHO-1 expression in Bach1-/- mice was analyzed by real-time PCR, immunohistochemistry and immunoblotting. Knee joints from Bach1-/- and wild-type mice with age-related OA and surgically-induced OA were evaluated by OA scoring systems. Levels of autophagy proteins and superoxide dismutase 2 (SOD2) were determined by immunohistochemistry. The relationship between HO-1 and the protective effects for OA was determined in chondrocytes treated with small interfering RNA (siRNA) targeting HO-1 gene.ResultsHO-1 expression decreased with aging in articular cartilages and menisci of mouse knees. Bach1-/- mice showed reduced severity of age-related OA and surgically-induced OA compared with wild-type mice. Microtubule-associated protein 1 light chain 3 (LC3), autophagy marker, and SOD2 were increased in articular cartilage of Bach1-/- mice compared with wild-type mice. Interleukin-1β (IL-1β) induced a significant increase in Adamts-5 in wild-type chondrocytes but not in Bach1-/- chondrocytes. The expression of SOD2 and the suppression of apoptosis in Bach1-/- chondrocytes were mediated by HO-1.ConclusionsBach1 deficiency reduces the severity of OA-like changes. This may be due to maintenance of cartilage homeostasis and joint health by antioxidant effects through HO-1 and downregulation of extracellular matrix degrading enzymes. These results suggest that inactivation of Bach1 is a novel target and signaling pathway in OA prevention.

Triptolide Attenuates Myocardial Ischemia/Reperfusion Injuries in Rats by Inducing the Activation of Nrf2/HO-1 Defense Pathway.

Triptolide is a bioactive component of Chinese herbal plant Tripterygium wilfordii Hook F that has recently been noted to attenuate hepatic and cerebral ischemia/reperfusion (I/R) injuries in rodents. To investigate whether triptolide could protect against myocardial I/R injuries, triptolide (25, 50 or 100μg/kg) was administrated in Wistar rats that underwent left anterior descending coronary artery ligation in this study. Our data showed that triptolide pretreatment could attenuate myocardial infarction, increase the fractional shortening and left ventricular systolic pressure and decrease the left ventricular end-diastolic pressure in ischemic rats. Also, triptolide was noted to inhibit the activities of lactate dehydrogenase and creatine kinase in I/R rats. Moreover, triptolide administration suppressed macrophage infiltration, inhibited the overproduction of tumor necrosis factor-α, interleukin (IL)-1β, IL-6 and malondialdehyde (MDA) in reperfused myocardium tissues and upregulated the activities of antioxidative superoxide dismutase (SOD), glutathione (GSH) and glutathione peroxidase (GPx). In addition, nuclear accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) and the activity of its downstream target Heme oxygenase-1 (HO-1) in ischemic myocardium tissues were enhanced by triptolide pretreatment. In addition, the HO-1 inhibitor, zinc protoporphyrin-IX, abrograted the cardiac protection mediated by triptolide. Our study reveals a novel cardioprotective effect of triptolide in rats with I/R injuries, wherein the activation of Nrf2/HO-1 signaling was involved.

Activation of NRF2 pathway in spleen, thymus as well as peripheral blood mononuclear cells by acute arsenic exposure in mice

Arsenic has already been demonstrated to activate the nuclear factor erythroid 2-related factor 2 (NRF2) in many different organs and cell lines. The present study tried to explore the expression of NRF2 pathway by acute arsenic exposure in immune system in vivo. Our results showed that treatment with arsenic (sodium arsenite, 5, 10 and 20mg/kg, intra-gastrically) increased the expression of NRF2 and its downstream targets heme oxygenase-1 (HO-1), glutathione-S-transferase (GST), glutamate–cysteine ligase (GCL) and glutathione reductase (GR) consistently in spleen, thymus, as well as peripheral blood mononuclear cells (PBMCs), as early as treatment from 6h. Arsenic was also detected to up-regulate the mRNA levels of Hmox1, NAD(P)H: quinine oxidoreductase 1 (Nqo1), Gclc and Gclm in spleen and thymus. Besides, we detected the enhancement of Kelch-like ECH-associated protein (KEAP1) expression in these immune organs and immunocytes. What's more, our results also found the imbalanced oxidative redox status under the circumstances that arsenic activated NRF2 pathway, reflected by the generation of lipid peroxidation, as well as the reduction of antioxidative capacities in both spleen and thymus. Taken together, our results here strongly suggested the expression and activation of NRF2 pathway by acute arsenic exposure in immune system in vivo. Further studies are being investigated to explore the possible roles and functions of NRF2 pathway stimulation in the regulation of immune responses of this metalloid.

Abstract 1262: The transcription factor ATF4 regulates resistance to anoikis and promotes metastasis in fibrosarcoma via cooperative upregulation of Heme Oxygenase-1 with Nrf2

Abstract The Integrated Stress Response (ISR) plays a critical role in cancer cell survival, and targeting the ISR results in inhibition of tumor progression. A critical aspect of ISR involves the preferential translation of activating transcription factor 4(ATF4), a transcriptional factor regulating genes involved in metabolism, nutrient uptake, and anti-oxidant responses. We previously demonstrated that ATF4 expression is significantly increased in tumors compared to corresponding normal tissue, and that ablation of ATF4 compromised primary tumor growth in mice. Based on the central roles that ATF4 target genes play in pro-survival processes, we hypothesized that ATF4 might also play a role in tumor metastasis. Upon loss of matrix attachment, a critical step in the metastatic process, we found induction of phosphorylation of the translation factor eIF2α mediated by upstream kinase PERK in human adenocarcinoma HT1080 cells and colorectal adenocarcinoma DLD1 cells. Increased eIF2α phosphorylation resulted in translational upregulation of ATF4 and its transcriptional targets CHOP, ASNS and ATF3. Interestingly, failure to induce ISR and upregulate ATF4 resulted in increased apoptosis following matrix detachment - a process known as anoikis. Furthermore, we demonstrate that ATF4 promotes anoikis resistance by activating a coordinated program of autophagy and anti-oxidant responses. Upon detachment, ATF4 activates cytoprotective autophagy by transcriptionally upregulating several key autophagic genes (Atg5, Atg7 and Ulk1). Simultaneously, ATF4 also induces the expression of the heme oxygenase 1 (HO-1) - a major antioxidant enzyme. Activation of HO-1 following matrix detachment occurs by coordinated upregulation of ATF4 and the antioxidant PERK dependent transcription factor Nrf2, which converge to bind on antioxidant regulatory elements (ARE) in the HO-1 promoter. Failure to either initiate the autophagy response or HO-1 induction sensitizes cells to anoikis. In agreement with our in vitro observations, HT1080 cells harboring ATF4 shRNA (shATF4) injected through tail vein of nude mice fails to establish metastatic lung colonization after 4 weeks compared to the non-targeting (shNT) counterparts. Immunohistochemical analysis on the tumor bearing lungs show high and colocalized expression of ATF4 and HO-1. Reconstituting either ATF4 or HO-1 expression in ATF4-deficient cells, rescues the tumor lung colonization phenotype. Finally, higher expression of HO-1 and ATF4 was found in human primary as well as metastatic tumors compared to normal epithelium or stromal tissue and correlated with reduced overall survival of lung adenocarcinoma and glioblastoma patients. Collectively, these results establish ATF4 as a major player in tumor metastasis, and the combined activity of its downstream targets HO-1 and Nrf2 as a critical mediator in this process. Citation Format: Souvik Dey, Carly M. Sayers, Stacey L. Lehman, Yi Cheng, George J. Cerniglia, Stephen W. Tuttle, Michael D. Feldman, Paul J.L. Zhang, Serge Y. Fuchs, J. Alan Diehl, Constantinos Koumenis. The transcription factor ATF4 regulates resistance to anoikis and promotes metastasis in fibrosarcoma via cooperative upregulation of Heme Oxygenase-1 with Nrf2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1262. doi:10.1158/1538-7445.AM2015-1262

Targeting heme oxygenase-1 and carbon monoxide for therapeutic modulation of inflammation.

The heme oxygenase-1 (HO-1) enzyme system remains an attractive therapeutic target for the treatment of inflammatory conditions. HO-1, a cellular stress protein, serves a vital metabolic function as the rate-limiting step in the degradation of heme to generate carbon monoxide (CO), iron, and biliverdin-IXα (BV), the latter which is converted to bilirubin-IXα (BR). HO-1 may function as a pleiotropic regulator of inflammatory signaling programs through the generation of its biologically active end products, namely CO, BV and BR. CO, when applied exogenously, can affect apoptotic, proliferative, and inflammatory cellular programs. Specifically, CO can modulate the production of proinflammatory or anti-inflammatory cytokines and mediators. HO-1 and CO may also have immunomodulatory effects with respect to regulating the functions of antigen-presenting cells, dendritic cells, and regulatory T cells. Therapeutic strategies to modulate HO-1 in disease include the application of natural-inducing compounds and gene therapy approaches for the targeted genetic overexpression or knockdown of HO-1. Several compounds have been used therapeutically to inhibit HO activity, including competitive inhibitors of the metalloporphyrin series or noncompetitive isoform-selective derivatives of imidazole-dioxolanes. The end products of HO activity, CO, BV and BR may be used therapeutically as pharmacologic treatments. CO may be applied by inhalation or through the use of CO-releasing molecules. This review will discuss HO-1 as a therapeutic target in diseases involving inflammation, including lung and vascular injury, sepsis, ischemia-reperfusion injury, and transplant rejection.

Anti-inflammatory activities of carbon monoxide-releasing molecules (CO-RMs) in the brain.

The transcription factor Nrf2 and its downstream target heme oxygenase-1 (HO-1) are ubiquitous protective systems against oxidative stress, inflammation and tissue injury. The products of HO-1 enzymatic activity, biliverdin and carbon monoxide (CO), possess interesting anti-oxidant and anti-inflammatory properties suggesting that exploitation of this pathway as a target for drug discovery may offer therapeutic avenues in a variety of disorders [1]. In this context we have developed carbon monoxide-releasing molecules (CO-RMs), a class of compounds that deliver CO in a controlled fashion and exert a variety of pharmacological effects [2]. Data will be presented showing that CO-RMs modulate neuroinflammation and neuroprotection in vitro and in vivo. In BV2 microglia cells challenged with thrombin and interferon gamma, CO-RMs reduced the production of inflammatory mediators both in normoxic and hypoxic conditions. Similarly, in a rat model of collagenase-induced intracerebral hemorrhage, CO-RMs modulated microglia activation and the production of TNF-α while partially protecting against brain damage [3]. The mechanism(s) by which CO liberated from CO-RMs exert protection remains elusive. However, our laboratory has recently produced findings in BV2 microglia pointing to mitochondria as a plausible target for the anti-inflammatory action of CO. Studies are also currently ongoing on the synthesis and characterization of novel hybrid molecules that potently activate Nrf2 and simultaneously release CO in order to maximize the anti-inflammatory effects of the HO-1 pathway [4].

ROS/Autophagy/Nrf2 Pathway Mediated Low-Dose Radiation Induced Radio-Resistance in Human Lung Adenocarcinoma A549 Cell

Low-dose ionizing radiation (LDIR) can induce radio-resistance to following high dose radiation in various mammalian cells. The protective role of LDIR has been thought to be associated with the overall outcomes of cancer radiotherapy. NF-E2 related factor 2 (Nrf2) is a transcription factor that plays pivotal roles in maintaining cellular oxidative equilibrium. Since oxidative stress has been indicated to be a mediator of LDIR induced radio-resistance, the role of Nrf2 in this process was investigated in this research. Our results showed that in human lung adenocarcinoma A549 cell, 5cGy alpha particle induced radio-resistance to following 75cGy alpha particle radiation. The expression level of Nrf2 and its target Heme Oxygenase-1(HO-1) increased after 5cGy radiation. Both the shRNA of Nrf2 and the chemical inhibitor of HO-1 suppressed the induced radio-resistance, indicating the involvement of Nrf2 antioxidant pathway in this process. Further, we found 5cGy radiation stimulated autophagy process in A549. Inhibition of the autophagy process resulted in suppression of the radio-resistance and the induced expression of Nrf2 and HO-1. ROS scavenger N-acetyl-L-cysteine (NAC) blocked the autophagy process induced by 5cGy alpha particle, the upregulation of Nrf2 and HO-1, as well as the induced radio-resistance. In conclusion, ROS elevation caused by LDIR promoted Autophagy/Nrf2-HO-1 and conferred radio-resistance in A549.

TI-I-174, a Synthetic Chalcone Derivative, Suppresses Nitric Oxide Production in Murine Macrophages via Heme Oxygenase-1 Induction and Inhibition of AP-1.

Chalcones (1,3-diaryl-2-propen-1-ones), a flavonoid subfamily, are widely known for their anti-inflammatory properties. Propenone moiety in chalcones is known to play an important role in generating biological responses by chalcones. In the present study, we synthesized chalcone derivatives structurally modified in propenone moiety and examined inhibitory effect on nitric oxide (NO) production and its potential mechanisms. Among the chalcone derivatives used for this study, TI-I-174 (3-(2-Hydroxyphenyl)-1-(thiophen-3-yl)prop-2-en-1-one) most potently inhibited lipopolysaccharide (LPS)-stimulated nitrite production in RAW 264.7 macrophages. TI-I-174 treatment also markedly inhibited inducible nitric oxide synthase (iNOS) expression. However, TI-I-174 did not significantly affect production of IL-6, cyclooxygenase-2 (COX-2) and tumor necrosis factor-α (TNF-α), implying that TI-I-174 inhibits production of inflammatory mediators in a selective manner. Treatment of macrophages with TI-I-174 significantly inhibited transcriptional activity of activator protein-1 (AP-1) as determined by luciferase reporter gene assay, whereas nuclear factor-κB (NF-κB) activity was not affected by TI-I-1744. In addition, TI-I-174 significantly inhibited activation of c-Jun-N-Terminal kinase (JNK) without affecting ERK1/2 and p38MAPK, indicating that down-regulation of iNOS gene expression by TI-I-174 is mainly attributed by blockade of JNK/AP-1 activation. We also demonstrated that TI-I-174 treatment led to an increase in heme oxygenase-1 (HO-1) expression both at mRNA and protein level. Transfection of siRNA targeting HO-1 reversed TI-I-174-mediated inhibition of nitrite production. Taken together, these results indicate that TI-I-174 suppresses NO production in LPS-stimulated RAW 264.7 macrophages via induction of HO-1 and blockade of AP-1 activation.

Design and synthesis of new hybrid molecules that activate the transcription factor Nrf2 and simultaneously release carbon monoxide.

The transcription factor Nrf2 and its downstream target heme oxygenase-1 (HO-1) are essential protective systems against oxidative stress and inflammation. The products of HO-1 enzymatic activity, biliverdin and carbon monoxide (CO), actively contribute to this protection, suggesting that exploitation of these cellular systems may offer new therapeutic avenues in a variety of diseases. Starting from a CO-releasing compound and a chemical scaffold exhibiting electrophilic characteristics (esters of fumaric acid), we report the synthesis of hybrid molecules that simultaneously activate Nrf2 and liberate CO. These hybrid compounds, which we termed "HYCOs", release CO to myoglobin and activate the CO-sensitive fluorescent probe COP-1, while also potently inducing nuclear accumulation of Nrf2 and HO-1 expression and activity in different cell types. Thus, we provide here the first example of a new class of pharmacologically active molecules that target the HO-1 pathway by combining an Nrf2 activator coordinated to a CO-releasing group.

Macrophages in diabetic gastroparesis--the missing link?

Diabetic gastroparesis results in significant morbidity for patients and major economic burden for society. Treatment options for diabetic gastroparesis are currently directed at symptom control rather than the underlying disease and are limited. The pathophysiology of diabetic gastroparesis includes damage to intrinsic and extrinsic neurons, smooth muscle, and interstitial cells of Cajal (ICC). Oxidative damage in diabetes appears to be one of the primary insults involved in the pathogenesis of several complications of diabetes, including gastroparesis. Recent studies have highlighted the potential role of macrophages as key cellular elements in the pathogenesis of diabetic gastroparesis. Macrophages are important for both homeostasis and defense against a variety of pathogens. Heme oxygenase 1 (HO1), an enzyme expressed in a subset of macrophages has emerged as a major protective mechanism against oxidative stress. Activation of macrophages with high levels of HO1 expression protects against development of delayed gastric emptying in animal models of diabetes, while activation of macrophages that do not express HO1 are linked to neuromuscular cell injury. Targeting macrophages and HO1 may therefore be a therapeutic option in diabetic gastroparesis. This report briefly reviews the pathophysiology of diabetic gastroparesis with a focus on oxidative damage and how activation and polarization of different subtypes of macrophages in the muscularis propria determines development of delay in gastric emptying or protects against its development.

Free radical scavenging and antioxidant activity of betanin: Electron spin resonance spectroscopy studies and studies in cultured cells

Betanin is a red pigment present in red beetroot. Recently, potential health benefits of betanin-rich beetroot have been suggested. However, little is known regarding the free radical scavenging and antioxidant activity of betanin. Electron spin resonance spectroscopy (ESR) and spin trapping techniques were applied to evaluate the ability of betanin to scavenge hydroxyl, superoxide, 2,2 diphenyl-1-picrylhydrazyl (DPPH), and galvinoxyl free radicals. In addition, we tested in cultured cells the ability of betanin to prevent DNA damage and to induce the transcription factor Nrf2 (nuclear factor (erythroid-derived 2)-like 2) as well as its down-stream target heme oxygenase1 (HO-1), paraoxonase1 (PON1) and glutathione (GSH). Betanin dose-dependently scavenged DPPH-, galvinoxyl-, superoxide-, and hydroxyl-radicals in the ESR and spin trapping studies and prevented hydrogen peroxide induced DNA damage as determined by the Comet assay. Furthermore, betanin treatment induced the transcription factor Nrf2 and resulted in an increase of HO-1 protein levels, PON1-transactivation and cellular GSH. Present data suggest that betanin is both a free radical scavenger and an inducer of antioxidant defense mechanism in cultured cells.

The PTEN/NRF2 Axis Promotes Human Carcinogenesis

A recent study conducted in mice reported that liver-specific knockout of tumor suppressor Pten augments nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcriptional activity. Here, we further investigated how phosphatase and tensin homolog deleted on chromosome 10 (PTEN) controls NRF2 and the relevance of this pathway in human carcin ogenesis. Drug and genetic targeting to PTEN and phosphoproteomics approaches indicated that PTEN leads to glycogen synthase kinase-3 (GSK-3)-mediated phosphorylation of NRF2 at residues Ser(335) and Ser(338) and subsequent beta-transducin repeat containing protein (β-TrCP)-dependent but Kelch-like ECH-associated protein 1 (KEAP1)-independent degradation. Rescue experiments in PTEN-deficient cells and xerographs in athymic mice indicated that loss of PTEN leads to increased NRF2 signature which provides a proliferating and tumorigenic advantage. Tissue microarrays from endometrioid carcinomas showed that 80% of PTEN-negative tumors expressed high levels of NRF2 or its target heme oxygenase-1 (HO-1). These results uncover a new mechanism of oncogenic activation of NRF2 by loss of its negative regulation by PTEN/GSK-3/β-TrCP that may be relevant to a large number of tumors, including endometrioid carcinomas. Increased activity of NRF2 due to loss of PTEN is instrumental in human carcinogenesis and represents a novel therapeutic target.

Repair of radiation damage of U2OS osteosarcoma cells is related to DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity

The present study was to investigate the effects of DNA-PKcs deficiencies on radiation sensitivity of human osteosarcoma U2OS cells to γ-ray and to explore the underlying molecular mechanism. In vitro, U2OS cells were transfected with different DNA-PKcs siRNAs or control siRNAs to establish stably siRNA-transfected cell lines U2OS-Si and U2OS-Sc, respectively. Cell viability and apoptosis after irradiation were analyzed using cell counting kit (CCK-8) and flow cytometric assay, respectively. Expressions of apoptosis-related and oxidative stress-responded proteins were assessed using Western blot. The tumorigenesis activity was examined in nude mice xenograft osteosarcoma mode. Results showed that DNA-PKcs siRNA significantly could inhibit U2OS viability and cell proliferation after exposure to irradiation. Compared with the U2OS and U2OS-Sc cells, the U2OS-Si cells induced higher apoptosis rate and loss of mitochondrial membrane potentials, accompanying with more reactive oxygen species (ROS) and malondialdehyde (MDA) production, increased DNA double-strand breaks (DSBs) induced by irradiation. Protein levels of the anti-apoptotic Bcl-2 were downregulated most obviously in U2OS-Si cells after irradiation, while pro-apoptotic factor Bax and caspase-3 upregulated. Moreover, the antioxidants protein expression levels of Nuclear factor-erythroid 2-related factor 2 (Nrf2) and its target heme oxygenase-1 (HO-1) were also significantly reduced in parallel to DNA-PKcs inhibition in U2OS-Si cells. In nude mice xenograft model, DNA-PKcs siRNA remarkably inhibited tumor growth and dissemination. In conclusion, DNA-PKcs siRNA might have a potential for osteosarcoma treatment by sensitizing osteosarcoma cells to γ-ray through modulation on oxidative stress-mediated DNA DSBs repair and mitochondrial pathway apoptosis.

MiR-200c sensitizes clear-cell renal cell carcinoma cells to sorafenib and imatinib by targeting heme oxygenase-1

Clear-cell renal cell carcinoma is a highly treatment-resistant tumor type. Heme oxygenase-1 plays an anti-apoptotic role in cancer chemotherapeutic inducing tumor-progression. The miR-200 family was involved in the process of mesenchymal-epithelial transition (MET) during renal development. In the present study, we demonstrated the regulatory relationship between miR-200c and HO-1. We provided evidences to elucidate that miR-200c could sensitize ccRCC cells to sorafenib or imatinib to inhibit cell proliferation, at least partly by targeting HO-1. Moreover, the correlation between miR-200c and HO-1 expression level and drug resistance in ccRCC was also determined. Combined application with chemotherapeutic drugs, miR-200c, a HO-1 inhibitor, may enhance the efficiency of therapy by promoting both apoptosis and autophagy.

Induction Of Ferroportin, Erg-1, p21 and IKBα In Sickle Cell Disease May Contribute To HIV-1 Inhibition

▪ BackgroundHypoxia and low iron induce hypoxia-induced factor 1(HIF-1) by stabilizing its alpha subunit and deregulate HIV-1 which transcription and several other steps of life cycle depend on cellular iron [1]. HIV-1 transcription is inhibited at low oxygen levels and reduced cellular iron through deregulation of CDK9/cyclin T1 and CDK2/cyclin E. Sickle cell disease has low odds of ratio for HIV-1 infection [2]. Sickle cell disease (SCD) leads to hemolytic anemia which results in local ischemia and release of heme. Induction of heme oxygenase-1 (HO-1) by hemin was shown to inhibit HIV-1 [1], although the mechanism of the inhibition was not clarified. Iron depletion by iron chelators or through the expression of ferroportin, an iron export protein, inhibits CDK2 and CDK9 activities and blocks HIV-1 transcription [1]. Because neither CDK2 nor CDK9 require iron for the enzymatic activity, we analyzed the expression of hypoxia and iron –dependent factors that may deregulate HIV-1 infection in SCD. ResultsExpression profiling followed by real-time PCR analysis showed induction of HO-1, p21, Erg-1, IKBα, HIF-1 and ferroportin mRNA and decrease of hepcidin mRNA in PBMCs from SCD patients. HIV-1 replication was reduced in SCD PBMCs comparing to normal controls, and also in THP1 cells treated with hemin. Subsequent treatment with hepcidin restored HIV-1 replication in SCD PBMC and in hemin-treated THP-1 cells, suggesting that ferroportin played a key role in the HIV-1 inhibition in these settings. Stable ferroportin knock down in THP-1 cells led to the inability of hemin to inhibit HIV-1, suggesting that ferroportin played a key role in the heme-meidated HIV-1 inhibition. Stable HIF-1a knockdown in promonocytic THP1 cells increased HIV replication suggesting that HIF1α is a restriction factor for HIV-1. Iron chelators induced the expression of IKBα, an inhibitor of NF-kB and also induced the expression of HIF-1 and p21. Iron chelators also inhibited enzymatic activity of CDK2 and shifted CDK9/cyclin T1 from the large to the small complex making it unavailable for HIV-1 Tat recruitment. Hemin treatment induced expression of HO-1, ferroportin, IkBα, HIF1α and p21 thus mimicking the effect of iron chelators. ConclusionsHemolytic conditions of sickle cell disease upregulate hypoxia and iron regulatory pathways leading to refraction of HIV-1. Targeting cellular iron, ferroportin and HO-1 may lead to novel anti-HIV-1 therapeutics.AcknowledgmentsThis project was supported by NIH Research Grants 1SC1GM082325, 2G12RR003048, and P30HL107253.

Sulforaphane induces Nrf2 and protects against CYP2E1-dependent binge alcohol-induced liver steatosis

BackgroundThe mechanism(s) by which alcohol causes cell injury are still not clear but a major mechanism appears to be the role of lipid peroxidation and oxidative stress in alcohol toxicity. CYP2E1-generated ROS contributes to the ethanol-induced oxidant stress and inhibition of CYP2E1 activity decreases ethanol-induced fatty liver. The transcription factor Nrf2 regulates the expression of many cytoprotective enzymes which results in cellular protection against a variety of toxins. MethodThe current study was designed to evaluate the ability of sulforaphane, an activator of Nrf2, to blunt CYP2E1-dependent, ethanol-induced steatosis in vivo and in vitro. ResultsThe sulforaphane treatment activated Nrf2, increased levels of the Nrf2 target heme oxygenase-1 and subsequently lowered oxidant stress as shown by the decline in lipid peroxidation and 3-nitrotyrosine protein adducts and an increase in GSH levels after the acute ethanol treatment. It decreased ethanol-elevated liver levels of triglycerides and cholesterol and Oil Red O staining. Similar results were found in vitro as addition of sulforaphane to HepG2 E47 cells, which express CYP2E1, elevated Nrf2 levels and decreased the accumulation of lipid in cells cultured with ethanol. Sulforaphane treatment had no effect on levels of or activity of CYP2E1. ConclusionsSulforaphane proved to be an effective in vivo inhibitor of acute ethanol-induced fatty liver in mice. General significanceThe possible amelioration of liver injury which occurs under these conditions by chemical activators of Nrf2 is of clinical relevance and worthy of further study.

Sulforaphane preconditioning of the Nrf2/HO-1 defense pathway protects the cerebral vasculature against blood–brain barrier disruption and neurological deficits in stroke

Disruption of the blood–brain barrier (BBB) and cerebral edema are the major pathogenic mechanisms leading to neurological dysfunction and death after ischemic stroke. The brain protects itself against infarction via activation of endogenous antioxidant defense mechanisms, and we here report the first evidence that sulforaphane-mediated preactivation of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream target heme oxygenase-1 (HO-1) in the cerebral vasculature protects the brain against stroke. To induce ischemic stroke, Sprague-Dawley rats were subjected to 70min middle cerebral artery occlusion (MCAo) followed by 4, 24, or 72h reperfusion. Nrf2 and HO-1 protein expression was upregulated in cerebral microvessels of peri-infarct regions after 4–72h, with HO-1 preferentially associated with perivascular astrocytes rather than the cerebrovascular endothelium. In naïve rats, treatment with sulforaphane increased Nrf2 expression in cerebral microvessels after 24h. Upregulation of Nrf2 by sulforaphane treatment prior to transient MCAo (1h) was associated with increased HO-1 expression in perivascular astrocytes in peri-infarct regions and cerebral endothelium in the infarct core. BBB disruption, lesion progression, as analyzed by MRI, and neurological deficits were reduced by sulforaphane pretreatment. As sulforaphane pretreatment led to a moderate increase in peroxynitrite generation, we suggest that hormetic preconditioning underlies sulforaphane-mediated protection against stroke. In conclusion, we propose that pharmacological or dietary interventions aimed to precondition the brain via activation of the Nrf2 defense pathway in the cerebral microvasculature provide a novel therapeutic approach for preventing BBB breakdown and neurological dysfunction in stroke.

Porphyromonas gingivalis lipopolysaccharide increases lipid accumulation by affecting CD36 and ATP-binding cassette transporter A1 in macrophages

Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS) promotes macrophage-derived foam cell formation, however, the mechanisms are not well established. In macrophages, lipid uptake is mediated by scavenger receptors including SR-A and CD36, while the cholesterol efflux is mediated by ATP-binding cassette transporter G1 (ABCG1), ABCA1 and SR-BI. We further investigated the mechanisms underlying the dysregulation by P. gingivalis LPS of these regulators resulting in the promotion of lipid accumulation in THP-1-derived macrophages. Our results showed that P. gingivalis LPS exacerbated lipid accumulation in oxidized low-density lipoprotein (oxLDL)-treated macrophages. However, cholesterol efflux was inhibited by P. gingivalis LPS in THP-1-derived macrophages. In oxLDL-untreated macrophages, P. gingivalis LPS treatment caused an increase in CD36 mRNA and protein levels, and a decrease in ABCA1 mRNA and protein levels, while having no effect on SR-A, SR-BI or ABCG1 expression. Upregulation of CD36 by P. gingivalis LPS resulted from activation of c-Jun/AP-1, and this was confirmed by the inhibition of increased CD36 expression after AP-1 inhibition using SP600125. However, the decreased protein stability of ABCA1 by P. gingivalis LPS was a result of increased calpain activity. Moreover, small hairpin RNA (shRNA) targeting heme oxygenase-1 (HO-1) augmented the P. gingivalis LPS-induced atherogenic effects on the expression of c-Jun/AP-1, CD36, ABCA1 and calpain activity. Accordingly, P. gingivalis LPS-regulated promotion of lipid accumulation in foam cells was also exacerbated by HO-1 shRNA. These results indicate that P. gingivalis LPS confers a exacerbation effect on the formation of foam cells by a novel HO-1-dependent mediation of cholesterol efflux and lipid accumulation in macrophages.