Induction Of Heme Oxygenase-1 Research Articles

Synthesis of Bifunctional Lipoxin‐Derived Enzyme‐Triggered CO‐Releasing Molecules (LipET‐CORMs)

AbstractIn an attempt to develop new anti‐inflammatory agents which act by co‐release of carbon monoxide (CO) and a specialized pro‐resolving mediator, we designed conjugates of a lipoxin A4 analogue and an acyloxycyclohexadiene‐Fe(CO)3 complex as an esterase‐triggered CO‐releasing molecule (ET‐CORM). After adjustment of the protecting group strategy, two of such compounds were successfully prepared by total synthesis (12 steps; 4–5 % overall yield) starting from deoxy‐d‐ribose and exploiting a Wittig olefination and an intermolecular Heck reaction as key C−C bond‐forming steps. A crucial late reduction of an aryl‐ketone moiety in the presence of a highly sensitive dienol ester functionality was achieved with BH3‐SMe2 in the presence of catalytic amounts of NaBH4. Both target compounds were dose‐dependently toxic towards cultured human umbilical vein endothelial cells (HUVEC), with LipET‐CORM 1‐A being slightly more toxic. While induction of heme oxygenase 1 (HO‐1) in HUVEC was observed for both compounds, they did not inhibit TNF‐α‐mediated VCAM‐1 expression in these cells. In M2 polarized macrophages HO‐1 expression was more pronounced as compared to M1 polarized macrophages. In both types of macrophages HO‐1 expression was downregulated by lipopolysaccharide, but only in M2 macrophages HO‐1 expression was rescued by LipET‐CORM. 15‐Lipoxygenase (15‐LO) was only expressed in M2 macrophages and was not influenced by LipET‐CORM. Collectively our data demonstrate that LipET‐CORMs induce HO‐1 expression in endothelial cells and M2 polarized macrophages. The role of the intra‐cellular released lipoxin A4 in resolution of inflammation, however, remains to be assessed.

Anti-Inflammatory Property of 5-Demethylnobiletin (5-Hydroxy-6, 7, 8, 3', 4'-pentamethoxyflavone) and Its Metabolites in Lipopolysaccharide (LPS)-Induced RAW 264.7 Cells.

Hydroxylated polymethoxyflavones (PMFs) are a unique class of flavonoid compounds mainly found in citrus plants. We investigated the anti-inflammatory effects of one major 5-hydroxy PMF, namely 5-demethylnobiletin (5DN) and its metabolites 5, 3'-didemethylnobiletin (M1), 5, 4'-didemethylnobiletin (M2), and 5, 3', 4'-tridemethylnobiletin (M3) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. The results showed that M2 and M3 produced stronger inhibitory effects on the production of nitric oxide (NO) than their parent compound at non-cytotoxic concentrations. Western blotting and real-time PCR analyses demonstrated that M2 and M3 significantly decreased iNOS and COX-2 gene expression. The results also showed that M1 and M3 induced heme oxygenase-1(HO-1) gene expression. Overall, our results demonstrated that metabolites of 5DN significantly inhibited LPS-induced inflammation in RAW 264.7 macrophage cells and generally possessed more potent anti-inflammatory activity than the parent compound, 5DN.

Identification of herbal teas and their compounds eliciting antiviral activity against SARS-CoV-2 in vitro

BackgroundThe SARS-CoV-2/COVID-19 pandemic has inflicted medical and socioeconomic havoc, and despite the current availability of vaccines and broad implementation of vaccination programs, more easily accessible and cost-effective acute treatment options preventing morbidity and mortality are urgently needed. Herbal teas have historically and recurrently been applied as self-medication for prophylaxis, therapy, and symptom alleviation in diverse diseases, including those caused by respiratory viruses, and have provided sources of natural products as basis for the development of therapeutic agents. To identify affordable, ubiquitously available, and effective treatments, we tested herbs consumed worldwide as herbal teas regarding their antiviral activity against SARS-CoV-2.ResultsAqueous infusions prepared by boiling leaves of the Lamiaceae perilla and sage elicit potent and sustained antiviral activity against SARS-CoV-2 when applied after infection as well as prior to infection of cells. The herbal infusions exerted in vitro antiviral effects comparable to interferon-β and remdesivir but outperformed convalescent sera and interferon-α2 upon short-term treatment early after infection. Based on protein fractionation analyses, we identified caffeic acid, perilla aldehyde, and perillyl alcohol as antiviral compounds. Global mass spectrometry (MS) analyses performed comparatively in two different cell culture infection models revealed changes of the proteome upon treatment with herbal infusions and provided insights into the mode of action. As inferred by the MS data, induction of heme oxygenase 1 (HMOX-1) was confirmed as effector mechanism by the antiviral activity of the HMOX-1-inducing compounds sulforaphane and fraxetin.ConclusionsIn conclusion, herbal teas based on perilla and sage exhibit antiviral activity against SARS-CoV-2 including variants of concern such as Alpha, Beta, Delta, and Omicron, and we identified HMOX-1 as potential therapeutic target. Given that perilla and sage have been suggested as treatment options for various diseases, our dataset may constitute a valuable resource also for future research beyond virology.

Chloroquine is a safe autophagy inhibitor for sustaining the expression of antioxidant enzymes in trophoblasts

Inhibition of autophagy contributes to the pathophysiology of preeclampsia. Although chloroquine (CHQ) is an autophagy inhibitor, it can reduce the occurrence of preeclampsia in women with systemic lupus erythematosus. To clarify this important clinical question, this study aimed to address the safety of CHQ in trophoblast cells from the viewpoint of homeostasis, in which the anti-oxidative stress (OS) response and autophagy are involved. We used Western blotting to evaluate the protein levels in the trophoblast cells. The expression levels of heme oxygenase-1 (HO-1), an anti-OS enzyme, mediate resistance to OS induced by hydrogen peroxide (H2O2) in trophoblast cell lines. Among the autophagy modulators, bafilomycin A1 (BAF), an autophagy inhibitor, but not autophagy activators, suppressed HO-1 expression in BeWo cells; CHQ did not suppress HO-1 expression in BeWo cells. To clarify the role of autophagy in HO-1 induction, we observed no difference in HO-1 induction by H2O2 between autophagy-normal and autophagy-deficient cells. As for the mechanism of HO-1 induction by OS, BAF suppressed HO-1 induction by downregulating the expression of neighbor of BRCA1 gene 1 (NBR1) in the selective p62-NBR1-nuclear factor erythroid 2-related factor 2 (Nrf2) autophagy pathway. CHQ did not inhibit HO-1 expression by sustaining NBR1 expression in human villous tissues compared to BAF treatment. In conclusion, CHQ is a safer medicine than BAF for sustaining NBR1, which resist against OS in trophoblasts by connecting selective autophagy and the anti-OS response.

Hemin versus erythropoietin: Possible role in Nrf2/HO-1 signaling pathway in rats with nephrotoxicity

Background and aimGentamycin-induced nephrotoxicity is related to stimulation of oxidative stress and inflammatory cascades leading to apoptotic renal damage. Heme oxygenase-1 (HO-1) induction considered to be an adaptive response against oxidative tissue damage. Our study aimed to investigate the possible nephroprotective role of HO-1 inducers (hemin and erythropoietin (EPO)) and elucidate their potential underlying molecular mechanisms by assessing their antioxidant, anti-apoptotic, and anti-inflammatory properties. MethodsKidney function markers (urea and creatinine), lipid peroxidation and antioxidant markers (MDA and GPx), inflammation and apoptotic markers (IL-6 and Bcl-2), and the relative gene expression levels of Nrf2 and HO-1 were assessed. Histopathological changes of the kidney were examined. ResultsNephrotoxic rats pretreated with hemin showed significant decrease in serum level of urea, creatinine, and MDA, compared to non-treated group. The kidney tissues also showed significant elevation of Bcl2 level, but significant decrease of IL-6, compared to non-treated group. Moreover, hemin pre-treatment significantly upregulated gene expression of Nrf2 and HO-1 in kidney tissue to near the normal control group. On the other hand, pretreatment with EPO showed significant upregulation of HO-1 gene expression but didn’t show significant difference in Nrf2 gene expression compared to control group. The histopathological examination of kidney supported the biochemical results. ConclusionThe current results proved that hemin rather than EPO, showed reno-protective effects in rats, which was mediated by activation of Nrf2 signaling pathway. This could be also attributed to the observed anti-inflammatory, antioxidant, and anti-apoptotic properties of hemin. In this regard, EPO showed lower potency.

The Inhibitory Effect of Phycocyanin Peptide on Pulmonary Fibrosis In Vitro.

Phycocyanin is an excellent antioxidant with anti-inflammatory effects on which recent studies are growing; however, its specific target remains unclear. Linear tetrapyrrole compounds such as bilirubin have been shown to lead to the induction of heme oxygenase 1 expression in vivo, thus achieving antioxidant and anti-inflammatory effects. Phycocyanin is bound internally with linear tetrapyrrole phycocyanobilin in a similar structure to bilirubin. We speculate that there is probably a way of inducing the expression of heme oxygenase 1, with which tissue oxidative stress and inflammation can be inhibited, thus inhibiting pulmonary fibrosis caused by oxidative damage and inflammation of lung. By optimizing the enzymatic hydrolysis process, phycocyanobilin-bound phycocyanin peptide were obtained, and its in vitro antioxidant, anti-inflammatory, and anti-pulmonary fibrosis activities were investigated. The results show that the phycocyanobilin peptide was able to alleviate oxidative and inflammatory damage in cells through the Keap1-Nrf2-HO-1 pathway, which in turn relieved pulmonary fibrosis symptoms.

Microglial Hemoxygenase-1 Deletion Reduces Inflammation in the Retina of Old Mice with Tauopathy.

Tauopathies such as Alzheimer's disease are characterized by the accumulation of neurotoxic aggregates of tau protein. With aging and, especially, in Alzheimer's patients, the inducible enzyme heme oxygenase 1 (HO-1) progressively increases in microglia, causing iron accumulation, neuroinflammation, and neurodegeneration. The retina is an organ that can be readily accessed and can reflect changes that occur in the brain. In this context, we evaluated how the lack of microglial HO-1, using mice that do not express HO-1 in microglia (HMO-KO), impacts retinal macro and microgliosis of aged subjects (18 months old mice) subjected to tauopathy by intrahippocampal delivery of AAV-hTauP301L (TAU). Our results show that although tauopathy, measured as anti-TAUY9 and anti-AT8 positive immunostaining, was not observed in the retina of WT-TAU or HMO-KO+TAU mice, a morphometric study of retinal microglia and macroglia showed significant retinal changes in the TAU group compared to the WT group, such as: (i) increased number of activated microglia, (ii) retraction of microglial processes, (iii) increased number of CD68+ microglia, and (iv) increased retinal area occupied by GFAP (AROA) and C3 (AROC3). This retinal inflammatory profile was reduced in HMO-KO+TAU mice. Conclusion: Reduction of microglial HO-1 could be beneficial to prevent tauopathy-induced neuroinflammation.

Expression Level and Biomarker Potential of Hemoxygenase-1 in Pancreatic Cancer

<h3>Purpose/Objective(s)</h3> In pancreatic ductal adenocarcinoma (PDAC), hypoxia is key mediator of resistance to chemo-radiotherapy and a prominent feature of PDAC tumor microenvironment (TME). Heme oxygenase-1 (HMOX-1) is an Nrf2-regulated gene that plays a critical role in modulation of oxidative stress and ROS production. Previous research suggested that HMOX-1 is expressed in the hypoxic PDAC TME and is induced upon exposure to chemotherapy. Utilizing a tumor microarray (TMA) of PDAC patients treated at our institution, we sought to determine baseline expression of HMOX-1 in the tumor and the TME and whether it could be a biomarker of resistance to neoadjuvant treatment (NAT). <h3>Materials/Methods</h3> Four TMAs containing 80 cores (of normal and cancerous pancreatic tissue) on each slide were stained with HMOX-1 antibody. Slides were manually evaluated and computer-assisted morphometric analysis of digital images was performed using the Aperio Image Analysis software™. Immunohistochemistry (IHC) staining was manually scored as follows: Tumor cells staining, Normal and hyperplasia cell staining, TME staining, Intensity of IHC stain (negative = 0, borderline = +/-, minimal = 1, moderate = 2, or severe = 3); Localization of stain and its distribution (homogenous, heterogeneous, focal, multifocal, and/or variable). The Positive Pixel Count algorithm was used to quantify the amount of stain present in a scanned slide image and derive the H-score which represents a combination of number weak positives, moderate positives, and strong positives/Number of Total positive/ pixels on each core. <h3>Results</h3> Through the Oncology Research Information Exchange Network (ORIEN) program, we developed 6 TMAs of PDAC patients that were treated at our institution between 2013-2020. Four out of 6 available PDAC TMAs were evaluated for expression of HMOX-1 equaling to 320 cores representing 101 patients. 35% of those patients received NAT. Seven out of 14 patients who received RT as part of their NAT were treated with SBRT. A total of 175 eligible cores were included in the final analysis. Overall, there was minimal cytoplasmic cell staining in pancreatic cancer cells. Only 5 out of 175 eligible cores were considered true positives. The 5 positive cores represented 5 different patients. None of these patients received NAT. One true positive underwent whole genome sequencing and was found to have a BRIP1 (BRCA-1 interacting protein) germline mutation. There was no staining in the TME and mild inflammatory cell staining (macrophages and subset of lymphocytes). <h3>Conclusion</h3> We showed that HMOX-1 is not overexpressed at baseline in pancreatic cancer tissue, limiting its use as a predictive marker for resistance to NAT. However, given its inducible nature, it could be primarily expressed upon exposure to these therapies. Future research is needed to investigate mechanism of HMOX-1 induction after treatment. Our future goal is to utilize this clinically rich resource to investigate other relevant proteins that may predict response to therapy.

Heat shock-induced heme oxygenase-1 expression in a mouse hepatoma cell line is dependent on HSF1 and modified by NRF2 and BACH1.

The induction mechanism of heme oxygenase-1 (HO-1) by heat shock (HS) is still unknown. Here, we discovered that HS activates the HO-1 expression in a mouse hepatoma cell line (Hepa 1-6). Knockdown experiments showed that the HS-induced HO-1 expression was dependent on HS factor 1 (HSF1). A chromatin immunoprecipitation (ChIP) assay demonstrated that the HS-activated HSF1 bound to the HS elements (HSEs) in the upstream enhancer 1 region (E1). Unexpectedly, HS also facilitates the BTB and CNC homology 1 (BACH1) binding to the Maf recognition elements (MAREs) in E1. We examined the effects of a catalytically inactive CRISPR-associated 9 nucleases (dCas9) with short guide RNAs (sgRNAs), and demonstrated that the HSF1 binding to HSEs in E1 was indispensable for the HS-induced HO-1 expression. Heme treatment (HA) dissociates BACH1 from MAREs and facilitated the binding of nuclear factor-erythroid-2-related factor 2 (NRF2) to MAREs. Following treatment with both HS and HA, the HO-1 induction and the HSF1 binding to HSEs in E1 were most notably observed. These results indicate that the HS-induced HO-1 expression is dependent on the HSF1 binding to HSEs in E1, although modulated by the BACH1 and NRF2 binding to MAREs within the same E1.

Fatty Liver/Adipose Tissue Dual-Targeting Nanoparticles with Heme Oxygenase-1 Inducer for Amelioration of Obesity, Obesity-Induced Type 2 Diabetes, and Steatohepatitis.

Persistent uptake of high-calorie diets induces the storage of excessive lipid in visceral adipose tissue. Lipids secreted from obese adipose tissue are accumulated in peripheral tissues such as the liver, pancreas, and muscle, and impair insulin sensitivity causing type 2 diabetes mellitus (T2DM). Furthermore, the accumulation of inflammatory cytokines and lipids in the liver induces apoptosis and fibrogenesis, and ultimately causes nonalcoholic steatohepatitis (NASH). To modulate obese tissue environments, it is challenged to selectively deliver inducers of heme oxygenase-1 (HO-1) to adipose tissue with the aid of a prohibitin targeting drug delivery system. Prohibitin binding peptide (PBP), an oligopeptide targeting prohibitin rich in adipose tissue, is conjugated on the surface of Hemin- or CoPP-loaded poly(lactide-co-glycolide) nanoparticles (PBP-NPs). PBP-NPs efficiently differentiate lipid storing white adipocytes into energy-generating brown adipocytes in T2DM and NASH models. In addition, PBP-NPs are found to target prohibitin overexpressed fatty liver in the NASH model and inhibit hepatic uptake of circulating lipids. Furthermore, PBP-NPs switch phenotypes of inflammatory macrophages in damaged organs and lower inflammation. Taken together, dual-targeted induction of HO-1 in fatty adipose and liver tissues is proven to be a promising therapeutic strategy to ameliorate obesity, insulin resistance, and steatohepatitis by lowering lipids and cytokines.

Heme Oxygenase-1 Overexpression Promotes Uveal Melanoma Progression and Is Associated with Poor Clinical Outcomes.

Uveal melanoma (UM) is the most common primary intraocular tumor in adults. To date, the main strategies to counteract its progression consist of focal radiation on the tumor site and ocular enucleation. Furthermore, many UM patients develop liver metastasis within 10 years following diagnosis, eventually resulting in a poorer prognosis for those patients. Dissecting the molecular mechanism involved in UM progression may lead to identify novel prognostic markers with significative clinical applications. The aim of the present study was to evaluate the role of Heme Oxygenase 1 (HO-1) in regulating UM progression. UM cell lines (92.1) were treated with Hemin (CONC e time), a strong inducer of HO-1, and VP13/47, a selective inhibitor of its enzymatic activity. Interestingly, our results showed an enhanced 92.1 cellular proliferation and wound healing ability following an HO-1 increase, overall unveiling the role played by this protein in tumor progression. Similar results were obtained following treatment with two different CO releasing molecules (CORM-3 and CORM-A1). These results were further confirmed in a clinical setting using our UM cohort. Our results demonstrated an increased median HO-1 expression in metastasizing UM when compared to nonmetastasizing patients. Overall, our results showed that HO-1 derived CO plays a major role in UM progression and HO-1 protein expression may serve as a potential prognostic and therapeutical factor in UM patients.

Target induction of heme oxygenase-1 attenuates anti-HLA I antibody induced transplant vasculopathy in an allogeneic aortic transplantation model in mice

Abstract Introduction After solid organ transplantation every second patient develops transplant vasculopathy (TV), which still remains the major obstacle for long-term graft survival. The main risk factor for developing TV are de novo synthesized donor-specific antibodies binding molecules of the MHC complex (HLA complex in humans, respectively) on the surface of endothelial cells (EC). EC become activated, vascular smooth muscle cells proliferate, and monocytes are recruited, contributing the development of the neointima a hallmark of TV. We have demonstrated that the anti-HLA I antibody (w6/32) significantly increases transmigration of monocytes across an endothelial monolayer in vitro. These interactions were dependent on modulating the anti-inflammatory enzyme heme oxygenase (HO)-1. Purpose The aim of the project is to investigate which adhesion molecule might mediate the antibody-induced transmigration of monocytes sensitive to HO-1 modulation and whether this modulation has beneficial effects in an allogenic aortic transplantation model in mice. Methods Before w6/32 stimulation, HO-1 was modulated by pretreating Human Coronary Artery Endothelial Cells (HCAEC) either with CoPPIX as a HO-1 activator or with statins to test for a therapeutic approach. Expression of a variety of established adhesion receptors in HCAEC was screened after HO-1 modulation and w6/32 stimulation. Antibody-induced transmigration and adhesion of THP-1 were quantified in the presence of CD62E-blocking antibodies. Thoracic aorta was transplanted into the abdominal aorta between fully MHC mismatched mice and applying the corresponding anti-MHC I antibody (SF1-1.1) induced TV. Mice were treated with CoPPIX, statins or CORM to evaluate the effects of HO-1 modulation or its metabolite CO on TV development. Statistical evaluation was performed with t-test or ANOVA. Results HO-1 modulation in HCAEC diminished w6/32-induced THP-1 transmigration. CD62E-expression in HCAECs was enhanced after w6/32 stimulation on mRNA and protein level, but preincubation with CoPPIX or statins reduced these effects. Incubation of HCAEC with a CD62E-blocking antibody after w6/32 stimulation significantly diminished the rate of transmigration normalized to isotype-stimulated HCAEC (isotype 100%, w6/32 + IgG 226% vs. w6/32 + CD62E-block 138%, p&amp;lt;0.05.). Treatment of mice with CoPPIX resulted in a significant reduction of the SF1-1.1-induced number of transmigrated monocytes (SF1-1.1: 10 vs. CoPPIX: 5.4; p&amp;lt;0.05). Application of statins (SF1-1.1: 10 vs. statin 6.6; p&amp;lt;0.05) or CORM (CORM: 6.1 vs. iCORM: 11.3, p&amp;lt;0.05) significantly reduces monocyte number as well. In all treatment groups, the SF1-1.1-induced neointima formation was ameliorated. Conclusion w6/32-induced monocyte transmigration is CD62E-dependent. SF1-1.1-induced monocytic infiltrate and intimal hyperplasia in a murine aortic transplantation model is also CD62E dependent and amenable to HO-1 modulation. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This sudy was funded by the German Research Foundation to Jan Larmann

491 Pel overexpression by Pseudomonas aeruginosa reduces induction of heme-oxygenase 1 in macrophages

491 Pel overexpression by Pseudomonas aeruginosa reduces induction of heme-oxygenase 1 in macrophages

Morin: a promising nutraceutical therapy for modulation of the NF-kB/NOX-2/IL-6/HO-1 signaling pathways in paracetamol-induced liver toxicity

Introduction: Paracetamol overdose potentially causes liver injury. This study aimed to evaluate the impact of morin against paracetamol overdose-induced hepatotoxicity. Methods: Thirty albino rats weighing 185 ± 5 g were randomly selected from five groups: group I: orally administered with 1% Tween 80; group II: administered with 1 g paracetamol; group III: administered with 1 g paracetamol and 50 mg morin; group IV: administered with 100 mg paracetamol and morin; and group V: administered with 100 mg paracetamol and silymarin, with all treatments administered for 14 days. Results: Morin and silymarin significantly protected the rats against induced alterations in the plasma total cholesterol, triacylglycerol, and HDL-C levels as well as the liver ALT, AST, ALP, LDH, protein thiol, GSH, SOD, CAT, MDA, and tumor necrosis factor-alpha levels. Furthermore, morin significantly inhibited the expression of NF-κB, NADPH oxidase-2, and interleukin-6 and induction of heme oxygenase-1 compared with paracetamol. The histological results indicated that morin protected the liver tissues against the toxic effect of paracetamol. Conclusion: Morin significantly depletes the side effects of paracetamol and protects the liver tissue from the resulting free radicals.

Transcriptomic Analysis of Genes Associated with Oxidative Stress in Chronic Rhinosinusitis Patients with Nasal Polyps: Identifying Novel Genes Involved in Nasal Polyposis.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a complicated inflammatory disease, and the underlying mechanism remains unclear. While some reactive oxygen/nitrogen species-related gene products are reported to participate in CRSwNP, a systemic and full analysis of oxidative-stress-associated genes in CRSwNP has not been extensively studied. Therefore, this study sought to catalog the gene-expression patterns related to oxidative stress and antioxidant defense in control and CRSwNP patients. In total, 25 control and 25 CRSwNP patients were recruited. The distribution and expression of 4-hydroxynonenal and 3-nitrotyrosine as markers of oxidative stress—which is represented by lipid peroxidation and the protein nitration of tyrosine residues in CRSwNP nasal polyps (NPs)—were more apparently increased than those found in the control nasal mucosae, as determined by immunohistochemistry (IHC). The expression of 84 oxidative-stress-related genes in nasal mucosae and NP tissues was analyzed via real-time PCR, which showed that 19 genes and 4 genes were significantly up- and downregulated, respectively; among them, inducible nitric oxide synthase (iNOS) and heme oxygenase 1 (HO-1) were notably upregulated, whereas lactoperoxidase (LPO), myeloperoxidase (MPO), and superoxide dismutase 3 (SOD3) were highly downregulated. Changes in the mRNA and protein levels of these redox proteins were confirmed with a customized, real-time PCR array and RT-PCR analysis, as well as Western blotting and IHC assays. A receiver operating characteristic curve analysis further suggested that LPO, MPO, SOD3, HO-1, and iNOS are possible endotype predictors of CRSwNP development. Collectively, we present an oxidative-stress-related gene profile of CRSwNP NP tissues, providing evidence that the systemic changes in oxidative stress and the antioxidative defense system, including novel iNOS, heme peroxidases, and other genes, are closely linked to CRSwNP pathology, development, and progression.

Identification of Cytoprotective Small-Molecule Inducers of Heme-Oxygenase-1.

Acute kidney injury (AKI) is a major public health concern with significant morbidity and mortality and no current treatments beyond supportive care and dialysis. Preclinical studies have suggested that heme-oxygenase-1 (HO-1), an enzyme that catalyzes the breakdown of heme, has promise as a potential therapeutic target for AKI. Clinical trials involving HO-1 products (biliverdin, carbon monoxide, and iron), however, have not progressed beyond the Phase ½ level. We identified small-molecule inducers of HO-1 that enable us to exploit the full therapeutic potential of HO-1, the combination of its products, and yet-undefined effects of the enzyme system. Through cell-based, high-throughput screens for induction of HO-1 driven by the human HO-1 promoter/enhancer, we identified two novel small molecules and broxaldine (an FDA-approved drug) for further consideration as candidate compounds exhibiting an Emax ≥70% of 5 µM hemin and EC50 <10 µM. RNA sequencing identified shared binding motifs to NRF2, a transcription factor known to regulate antioxidant genes, including HMOX1. In vitro, the cytoprotective function of the candidates was assessed against cisplatin-induced cytotoxicity and apoptosis. In vivo, delivery of a candidate compound induced HO-1 expression in the kidneys of mice. This study serves as the basis for further development of small-molecule HO-1 inducers as preventative or therapeutic interventions for a variety of pathologies, including AKI.

Differential Effects of Arsenic in Drinking Water on Mouse Hepatic and Intestinal Heme Oxygenase-1 Expression.

Arsenic exposure has been associated with the risks of various diseases, including cancers and metabolic diseases. The aim of this study was to examine the effects of arsenic exposure via drinking water on the expression of heme oxygenase-1 (HO-1), a major responsive gene to arsenic-induced oxidative stress, in mouse intestinal epithelial cells which is the first site of exposure for ingested arsenic, and the liver, a known target of arsenic toxicity. The expression of HO-1 was determined at mRNA, protein, or enzymic activity levels in mice exposed to sodium arsenite through drinking water, at various doses (0, 2.5, 10, 25, 100 ppm), and for various time periods (1, 3, 7, or 28 days). HO-1 was significantly induced in the intestine, but not liver, at arsenic doses of 25 ppm or lower. The intestinal HO-1 induction was seen in both males and females, plateaued within 1–3 days of exposure, and was accompanied by increases in microsomal HO activity. In mice exposed to 25-ppm of arsenite for 7 days, total arsenic and As(III) levels in intestinal epithelial cells were significantly higher than in the liver. These findings identify intestinal epithelial cells as likely preferential targets for arsenic toxicity and support further studies on the functional consequences of intestinal HO-1 induction.

Antiproliferative Effect of Plant-Derived Bioactive Compounds Endowed with Antioxidant Activity on Breast Cancer Cells

Several lines of evidence indicate that plant-derived antioxidant compounds can be used as anticancer agents to support conventional pharmacological therapy. In this context, heme-oxygenase-1 (HO-1) modulation has been proven to represent a valid approach for reducing cancer cells’ proliferation through the activation of apoptosis and ferroptosis. This study focused on three little studied HO-1 inducers (paeonol, rosolic acid and dimethoxy resveratrol) in order to evaluate their efficacy as antiproliferative compounds on breast cancer cells (MCF-7 and MDA-MB 231). Cell viability data showed an interesting selectivity of dimethoxy resveratrol (DMR) for MDA-MB 231 cells. The ineffectiveness of Ferrostatin-1 and Trolox treatment led to the exclusion of ferroptosis involvement; meanwhile, cell viability reduction was associated with caspase 3/7 activation and apoptosis. Taken together, our results suggest a potential role of DMR as an adjuvant in conventional chemotherapy for breast cancer treatment.

Mitigation of Cadmium Toxicity through Modulation of the Frontline Cellular Stress Response

Cadmium (Cd) is an environmental toxicant of public health significance worldwide. Diet is the main Cd exposure source in the non-occupationally exposed and non-smoking populations. Metal transporters for iron (Fe), zinc (Zn), calcium (Ca), and manganese (Mn) are involved in the assimilation and distribution of Cd to cells throughout the body. Due to an extremely slow elimination rate, most Cd is retained by cells, where it exerts toxicity through its interaction with sulfur-containing ligands, notably the thiol (-SH) functional group of cysteine, glutathione, and many Zn-dependent enzymes and transcription factors. The simultaneous induction of heme oxygenase-1 and the metal-binding protein metallothionein by Cd adversely affected the cellular redox state and caused the dysregulation of Fe, Zn, and copper. Experimental data indicate that Cd causes mitochondrial dysfunction via disrupting the metal homeostasis of this organelle. The present review focuses on the adverse metabolic outcomes of chronic exposure to low-dose Cd. Current epidemiologic data indicate that chronic exposure to Cd raises the risk of type 2 diabetes by several mechanisms, such as increased oxidative stress, inflammation, adipose tissue dysfunction, increased insulin resistance, and dysregulated cellular intermediary metabolism. The cellular stress response mechanisms involving the catabolism of heme, mediated by heme oxygenase-1 and -2 (HO-1 and HO-2), may mitigate the cytotoxicity of Cd. The products of their physiologic heme degradation, bilirubin and carbon monoxide, have antioxidative, anti-inflammatory, and anti-apoptotic properties.

A Randomized Phase 2 Study of 5-Aminolevulinic Acid Hydrochloride and Sodium Ferrous Citrate for the Prevention of Nephrotoxicity Induced by Cisplatin-Based Chemotherapy of Lung Cancer

Introduction: Cisplatin-based chemotherapy was established in the 1980s, and it has been improved by the development of a short hydration protocol in lung cancer therapy. However, cisplatin-based chemotherapy is still associated with renal toxicity. Because 5-aminolevulinic acid (5-ALA) with sodium ferrous citrate (SFC) is known to be a mitochondrial activator and a heme oxygenase-1 (HO-1) inducer, 5-ALA with SFC is speculated to mitigate cisplatin-induced renal inflammation. Methods: We investigated the effects of oral administration of 5-ALA with SFC for preventing cisplatin-based nephrotoxicity in patients with lung cancer and evaluated its benefits for patients who received cisplatin-based chemotherapy. The primary endpoint was the significance of the difference between the serum creatinine (sCr) levels of the patients administered 5-ALA with SFC and those given placebo after course 1 of chemotherapy. The difference in the estimated glomerular filtration rate (eGFR) between the two groups was also evaluated as the secondary endpoint. Results: The double-blind, randomized two-arm studies were conducted at 15 medical facilities in Japan; 54 male and 20 female patients with lung cancer who received cisplatin-based chemotherapy between the ages of 42 and 75 years were included in the study. The compliance rate was greater than 94% in the primary assessment and subsequent drug administration periods. All enrolled patients completed the four cycles of cisplatin-based chemotherapy with short hydration. The average level of sCr on day 22 of course 1 was 0.707 mg/dL in the group treated with 5-ALA and SFC and 0.735 mg/dL in the placebo group, respectively, and the sCr in the test group was significantly lower than that in the placebo group (p = 0.038). In addition, the eGFR was significantly higher in the SPP-003 group than in the placebo group up to day 1 of course 3 (84.66 and 75.68 mL/min/1.73 m², respectively, p = 0.02) and kept better even after the last administration of the study drug (82.37 and 73.49 mL/min/1.73 m², respectively, p = 0.013). Conclusions: The oral administration of 5-ALA with SFC is beneficial to patients undergoing cisplatin-based chemotherapy for lung cancer with short hydration.