The carbon monoxide-releasing molecule CORM-2 inhibits the inflammatory response induced by cytokines in Caco-2 cells.
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ABSTRACT: Recent evidence indicates that carbon monoxide-releasing molecules (CO-RMs) exhibit potential anti-inflammatory properties. In the present study, we have investigated whether tricarbonyl dichloro ruthenium(II) dimer (CORM-2) can control the inflammatory response induced by cytokines in a human colonic epithelial cell line, Caco-2.Caco-2 cells were preincubated with CORM-2 for 30 minutes and then stimulated with interleukin (IL)-1beta, tumor necrosis factor-alpha and interferon-gamma for different times. Gene expression was analyzed by real-time PCR. Protein expression was investigated by Western blot and ELISA. Transcription factor activation was determined by the luciferase method.We have shown that CORM-2 significantly decreased the mRNA expression of nitric oxide synthase-2 (NOS-2) and the production of nitrite, in Caco-2 cells stimulated with cytokines. IL-8, IL-6 and metalloproteinase-7 (MMP-7) mRNA and protein were also significantly reduced by CORM-2. Time-course and small interfering RNA studies suggest that inhibition of IL-6 plays a role in the regulation of MMP-7 expression by CORM-2. These effects of CORM-2 can be dependent on the modulation of nuclear factor-kappaB (NF-kappaB), activator protein-1, CCAT/enhancer binding protein and the phosphorylated forms of NF-kappaB inhibitory protein-alpha, c-Jun N-terminal protein kinase 1/2, p38 and extracellular signal-regulated kinase 1/2.CORM-2 can regulate a number of genes relevant in intestinal inflammation and cancer progression. These findings provide new insights into the anti-inflammatory properties and potential applications of this class of compounds.
Project description:Chronic infections resulting from biofilm formation are difficult to eradicate with current antimicrobial agents and consequently new therapies are needed. This work demonstrates that the carbon monoxide-releasing molecule CORM-2, previously shown to kill planktonic bacteria, also attenuates surface-associated growth of the gram-negative pathogen Pseudomonas aeruginosa by both preventing biofilm maturation and killing bacteria within the established biofilm. CORM-2 treatment has an additive effect when combined with tobramycin, a drug commonly used to treat P. aeruginosa lung infections. CORM-2 inhibited biofilm formation and planktonic growth of the majority of clinical P. aeruginosa isolates tested, for both mucoid and non-mucoid strains. While CORM-2 treatment increased the production of reactive oxygen species by P. aeruginosa biofilms, this increase did not correlate with bacterial death. These data demonstrate that CO-RMs possess potential novel therapeutic properties against a subset of P. aeruginosa biofilm related infections.
Project description:Severe forms of malaria infection, such as cerebral malaria (CM) and acute lung injury (ALI), are mainly caused by the apicomplexan parasite Plasmodium falciparum. Primary therapy with quinine or artemisinin derivatives is generally effective in controlling P. falciparum parasitemia, but mortality from CM and other forms of severe malaria remains unacceptably high. Herein, we report the design and synthesis of a novel carbon monoxide-releasing molecule (CO-RM; ALF492) that fully protects mice against experimental CM (ECM) and ALI. ALF492 enables controlled CO delivery in vivo without affecting oxygen transport by hemoglobin, the major limitation in CO inhalation therapy. The protective effect is CO dependent and induces the expression of heme oxygenase-1, which contributes to the observed protection. Importantly, when used in combination with the antimalarial drug artesunate, ALF492 is an effective adjunctive and adjuvant treatment for ECM, conferring protection after the onset of severe disease. This study paves the way for the potential use of CO-RMs, such as ALF492, as adjunctive/adjuvant treatment in severe forms of malaria infection.
Project description:The delivery of controlled amounts of carbon monoxide (CO) to biological targets is of significant current interest. Very few CO-releasing compounds are currently known that can be rigorously controlled in terms of the location and amount of CO released. To address this deficiency, we report herein a new metal-free, visible-light-induced CO-releasing molecule (photoCORM) and its prodrug oxidized form, which offer new approaches to controlled, localized CO delivery. The new photoCORM, based on a 3-hydroxybenzo[ g]quinolone framework, releases 1 equiv of CO upon visible-light illumination under a variety of biologically relevant conditions. This nontoxic compound can be tracked prior to CO release using fluorescence microscopy and produces a nontoxic byproduct following CO release. An oxidized prodrug form of the photoCORM is reduced by cellular thiols, providing an approach toward activation in the reducing environment of cancer cells. Strong noncovalent affinity of the nonmetal photoCORM to albumin enables use of an albumin:photoCORM complex for targeted CO delivery to cancer cells. This approach produced cytotoxicity IC50 values among the lowest reported to date for CO delivery to cancer cells by a photoCORM. This albumin:photoCORM complex is also the first CO delivery system to produce significant anti-inflammatory effects when introduced at nanomolar photoCORM concentration.
Project description:As an endogenous signaling molecule, carbon monoxide (CO) has emerged as an increasingly promising option regarding as gas therapy due to its positive pharmacological effects in various diseases. Owing to the gaseous nature and potential toxicity, it is particularly important to modulate the CO release dosages and targeted locations to elucidate the biological mechanisms of CO and facilitate its clinical applications. Based on these, diverse CO-releasing molecules (CORMs) have been developed for controlled release of CO in biological systems. However, practical applications of these CORMs are limited by several disadvantages including low stability, poor solubility, weak releasing controllability, random diffusion, and potential toxicity. In light of rapid developments and diverse advantages of nanomedicine, abundant nanomaterials releasing CO in controlled ways have been developed for therapeutic purposes across various diseases. Due to their nanoscale sizes, diversified compositions and modified surfaces, vast CO-releasing nanomaterials (CORNMs) have been constructed and exhibited controlled CO release in specific locations under various stimuli with better pharmacokinetics and pharmacodynamics. In this review, we present the recent progress in CORNMs according to their compositions. Following a concise introduction to CO therapy, CORMs and CORNMs, the representative research progress of CORNMs constructed from organic nanostructures, hybrid nanomaterials, inorganic nanomaterials, and nanocomposites is elaborated. The basic properties of these CORNMs, such as active components, CO releasing mechanisms, detection methods, and therapeutic applications, are discussed in detail and listed in a table. Finally, we explore and discuss the prospects and challenges associated with utilizing nanomaterials for biological CO release.
Project description:Flavonoids are naturally occurring compounds found in fruits, vegetables, and other plant-based foods, and they are known for their health benefits, such as UV protection, antioxidant, anti-inflammatory, and antiproliferative properties. This study investigates whether flavonoids, such as quercetin and 2,3-dehydrosilybin, can act as photoactivatable carbon monoxide (CO)-releasing molecules under physiological conditions. CO has been recently recognized as an important signaling molecule. Here, we show that upon direct irradiation, CO was released from both flavonoids in PBS with chemical yields of up to 0.23 equiv, which increased to almost unity by sensitized photooxygenation involving singlet oxygen. Photoreleased CO reduced cellular toxicity caused by high flavonol concentrations, partially restored mitochondrial respiration, reduced superoxide production induced by rotenone and high flavonol levels, and influenced the G0/G1 and G2/M phases of the cell cycle, showing antiproliferative effects. The findings highlight the potential of quercetin and 2,3-dehydrosilybin as CO-photoreleasing molecules with chemopreventive and therapeutic implications in human pathology and suggest their possible roles in plant biology.
Project description:Neurodegenerative disorders and brain damage are initiated by excessive production of reactive oxygen species (ROS), which leads to tissue injury, cellular death and inflammation. In cellular anti-oxidant systems, heme oxygenase-1 (HO-1) is an oxidative-sensor protein induced by ROS generation or carbon monoxide (CO) release. CO releasing molecules (CORMs), including CORM-3, exert anti-oxidant and anti-inflammatory effects. However, the molecular mechanisms of CORM-3-induced HO-1 expression and protection against interleukin (IL)-1?-induced inflammatory responses have not been fully elucidated in rat brain astrocytes (RBA-1). To study the regulation of CORM-3-induced HO-1 expression, signaling pathways, promoter activity, mRNA and protein expression were assessed following treatment with pharmacological inhibitors and gene-specific siRNA knockdown. We found that CORM-3 mediated HO-1 induction via transcritional and translational processes. Furthermore, CORM-3-induced HO-1 expression was mediated by phosphorylation of several protein kinases, such as c-Src, Pyk2, protein kinase C? (PKC?) and p42/p44 mitogen-activated protein kinase (MAPK), which were inhibited by respective pharmacological inhibitors or by gene-specific knockdown with siRNA transfections. Next, we found that CORM-3 sequentially activated the c-Src/Pyk2/PKC?/p42/p44 MAPK pathway, thereby up-regulating mRNA for the activator protein (AP)-1 components c-Jun and c-Fos; these effects were attenuated by an AP-1 inhibitor (Tanshinone IIA; TSIIA) and other relevant inhibitors. Moreover, CORM-3-induced upregulation of HO-1 attenuated the IL-1?-induced cell migration and matrix metallopeptidase-9 mRNA expression in RBA-1 cells. These effects were reversed by an matrix metalloproteinase (MMP)2/9 inhibitor or by transfection with HO-1 siRNA.
Project description:PurposeDespite the role of carbon monoxide in ameliorating ischemia-reperfusion injury (IRI), its use in the clinical setting is restricted owing to its toxicity. Herein, we investigated the in vivo effects of carbon monoxide-releasing molecule-3 (CORM-3) on IRI.Materials and methodsFifteen rats were equally and randomly divided into three groups: sham (right nephrectomy), control (right nephrectomy and left renal ischemia), and CORM-3 (right nephrectomy and CORM-3 injection before left renal ischemia). Kidney tissues and blood samples collected from sacrificed rats were evaluated to determine the renoprotective effect and mechanism of CORM-3.ResultsConcentrations of serum creatinine and kidney injury molecule-1 in the CORM-3 group were significantly lower than in the control group after 75 minutes of IRI (1.2 vs. 2.4 mg/dL, p=0.01, and 292 vs. 550 pg/mL, p<0.001, respectively). Furthermore, the CORM-3 group exhibited a higher portion of normal tubules and glomeruli. TUNEL staining revealed fewer apoptotic renal tubular cells in the CORM-3 group than in the control group. The expression of 960 genes in the CORM-3 group was also altered. Pretreatment with CORM-3 before renal IRI produced a significant renoprotective effect. Fifteen of the altered genes were found to be involved in the peroxisome proliferator-activated receptors signaling pathway, and the difference in the expression of these genes between the CORM-3 and control groups was statistically significant (p<0.001).ConclusionsCORM-3 ameliorates IRI by decreasing apoptosis and may be a novel strategy for protection against renal warm IRI.
Project description:ObjectiveTo investigate the protective effects and mechanisms of carbon monoxide-releasing molecule-2 (CORM-2) on barrier function of intestinal epithelial cells.Materials and methodsAfter pre-incubation with CORM-2 for 1 hour, cultured intestinal epithelial IEC-6 cells were stimulated with 50 µg/ml lipopolysaccharides (LPS). Cytokines levels in culture medium were detected using ELISA kits. Trans-epithelial electrical resistance (TER) of IEC-6 cell monolayers in Transwells were measured with a Millipore electric resistance system (ERS-2; Millipore) and calculated as Ω/cm2 at different time points after LPS treatment. The permeability changes were also measured using FITC-dextran. The levels of tight junction (TJ) proteins (occludin and ZO-1) and myosin light chain (MLC) phosphorylation were detected using Western blotting with specific antibodies. The subsequent structural changes of TJ were visualized using transmission electron microscopy (TEM).ResultsCORM-2 significantly reduced LPS-induced secretion of TNF-α and IL-1β. The LPS-induced decrease of TER and increase of permeability to FITC-dextran were inhibited by CORM-2 in a concentration dependent manner (P<0.05). LPS-induced reduction of tight junction proteins and increase of MLC phosphorylation were also attenuated. In LPS-treated cells, TEM showed diminished electron-dense material and interruption of TJ and desmosomes between the apical lateral margins of adjoining cells, which were prevented by CORM-2 treatment.ConclusionsThe present study demonstrates that CORM-2, as a novel CO-releasing molecule, has ability to protect the barrier function of LPS-stimulated intestinal epithelial cells. Inhibition of inflammatory cytokines release, restoration of TJ proteins and suppression of MLC phosphorylation are among the protective effects of CORM-2.
Project description:The immunoresponsive gene 1 (IRG1) protein has crucial functions in embryonic implantation and neurodegeneration. IRG1 promotes endotoxin tolerance by increasing A20 expression in macrophages through reactive oxygen species (ROS). The cytoprotective protein heme oxygenase-1 (HO-1), which generates endogenous carbon monoxide (CO), is expressed in the lung during Lipopolysaccharide (LPS) tolerance and cross tolerance. However, the detailed molecular mechanisms and functional links between IRG1 and HO-1 in the innate immune system remain unknown. In the present study, we found that the CO releasing molecule-2 (CORM-2) and chemical inducers of HO-1 increased IRG1 expression in a time- and dose-dependent fashion in RAW264.7 cells. Furthermore, inhibition of HO-1 activity by zinc protoporphyrin IX (ZnPP) and HO-1 siRNA significantly reduced expression of IRG1 under these conditions. In addition, treatment with CO and HO-1 induction significantly increased A20 expression, which was reversed by ZnPP and HO-1 siRNA. LPS-stimulated TNF-? was significantly decreased, whereas IRG1 and A20 were increased by CORM-2 application and HO-1 induction, which in turn were abrogated by ZnPP. Interestingly, siRNA against IRG1 and A20 reversed the effects of CO and HO-1 on LPS-stimulated TNF-? production. Additionally, CO and HO-1 inducers significantly increased IRG1 and A20 expression and downregulated TNF-? production in a LPS-stimulated sepsis mice model. Furthermore, the effects of CO and HO-1 on TNF-? production were significantly reversed when ZnPP was administered. In conclusion, CO and HO-1 induction regulates IRG1 and A20 expression, leading to inhibition of inflammation in vitro and in an in vivo mice model.