Cudratricusxanthone O Inhibits H2O2-Induced Cell Damage by Activating Nrf2/HO-1 Pathway in Human Chondrocytes

Osteoarthritis (OA) is a common joint degenerative disease induced by oxidative stress in chondrocytes. Although induced-heme oxygenase-1 (HO-1) has been found to protect cells against oxygen radical damage, little information is available regarding the use of bioactive compounds from natural sources for regulating the HO-1 pathway to treat OA. In this study, we explored the inhibitory effects of cudratricusxanthone O (CTO) isolated from the Maclura tricuspidata Bureau (Moraceae) on H2O2-induced damage of SW1353 chondrocytes via regulation of the HO-1 pathway. CTO promoted HO-1 expression by enhancing the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) into the nucleus without inducing toxicity. Pretreatment with CTO-regulated reactive oxygen species (ROS) production by inducing expression of antioxidant enzymes in H2O2-treated cells and maintained the functions of H2O2-damaged chondrocytes. Furthermore, CTO prevented H2O2-induced apoptosis by regulating the expression of anti-apoptotic proteins. Treatment with the HO-1 inhibitor tin-protoporphyrin IX revealed that these protective effects were exerted due to an increase in HO-1 expression induced by CTO. In conclusion, CTO protects chondrocytes from H2O2-induced damages—including ROS accumulation, dysfunction, and apoptosis through activation of the Nrf2/HO-1 signaling pathway in chondrocytes and, therefore, is a potential therapeutic agent for OA treatment.

CSIG-20. EFFECT OF TUMOR-TREATING FIELDS (TTFIELDS) ON EGFR PHOSPHORYLATION IN GBM CELL LINES

Glioblastoma (GBM) is the most common high grade and most devastating brain tumor among adults. About 50% of GBM express EGFR (epidermal growth factor receptor) and from these another 50% the mutated form EGFRvIII which is associated with a more aggressive disease and poorer prognosis. We have previously shown that expression of different status of EGFR in GBM cell lines drives the 5-ALA induced fluorescence downstream by influencing the rate-limiting enzyme heme oxygenase-1 (HO-1) probably via PI3K/Akt signalling. We demonstrated that 5-ALA-induced protoporphyrin IX (PpIX) fluorescence is pharmacologically influenced by adding different drugs such as deferoxamine (DFO) and tin-protoporphyrin (SnPP), which are an iron chelator of Fe2+ and an inhibitor of HO-1 respectively, or genistein that promotes PpIX accumulation via functional repression of ABCG2 (ABC transporter G2). Our aim is to increase these pharmacological effects on PpIX fluorescence using tumor-treating fields (TTFields). TTFields, a new therapeutic technology for treating newly diagnosed or recurrent GBM, is able to suppress the growth of cancer cells destabilising microtubule elongation and increasing membrane permeability. Interestingly, TTFields, like as other destabilising drugs and compounds, is able to inhibit the phosphorylation of EGFR and subsequent downregulation of EGFR-induced signalling acting for example on the mechanism that regulate the HO-1 activity. Here, we investigate the effects of TTFields on glioma cells, with different EGFR status and consequently different PpIX fluorescence. Exposure to TTFields during or after pharmacological treatments may represent a novel strategy to block or diminish the phosphorylation of EGFR to ameliorate the visualization of PpIX fluorescence in patients where it is not enough to ensure a safe and precise removal of the tumor bulk. In fact, if a combination of TTFields and drug treatment should give the desired results, this strategy could be applied on patients before being subjected to surgical resection.

Carbon Monoxide Partially Mediates Protective Effect of Resveratrol Against UVB-Induced Oxidative Stress in Human Keratinocytes

Based on the antioxidative effect of resveratrol (RES) in mitigating reactive oxygen species (ROS) production through the induction of nuclear factor-erythroid 2-related factor-2 (Nrf2)/heme oxigenase-1 (HO-1) signaling pathway, we investigated whether the protective activity of RES against ROS-mediated cytotoxicity is mediated by intracellular carbon monoxide (CO), a product of HO-1 activity, in ultraviolet B (UVB)-irradiated human keratinocyte HaCaT cells. The cells were exposed to UVB radiation following treatment with RES and/or CO-releasing molecule-2 (CORM-2). RES and/or CORM-2 upregulated HO-1 protein expression, accompanied by a gradual reduction of UVB-induced intracellular ROS levels. CORM-2 reduced intracellular ROS in the presence of tin protoporphyrin IX, an HO-1 inhibitor, indicating that the cytoprotection observed was mediated by intracellular CO and not by HO-1 itself. Moreover, CORM-2 decreased RES-stimulated mitochondrial quantity and respiration and increased the cytosolic protein expressions of radical-scavenging superoxide dismutases, SOD1 and SOD2. Taken together, our observations suggest that RES and intracellular CO act independently, at least partly, in attenuating cellular oxidative stress by promoting antioxidant enzyme expressions and inhibiting mitochondrial respiration in UVB-exposed keratinocytes.

Astrocyte-produced carbon monoxide and the carbon monoxide donor CORM-A1 protect against cerebrovascular dysfunction caused by prolonged neonatal asphyxia

Neonatal asphyxia leads to cerebrovascular disease and neurological complications via a mechanism that may involve oxidative stress. Carbon monoxide (CO) is an antioxidant messenger produced via a heme oxygenase (HO)-catalyzed reaction. Cortical astrocytes are the major cells in the brain that express constitutive HO-2 isoform. We tested the hypothesis that CO, produced by astrocytes, has cerebroprotective properties during neonatal asphyxia. We developed a survival model of prolonged asphyxia in newborn pigs that combines insults of severe hypoxia, hypercapnia, and acidosis while avoiding extreme hypotension and cerebral blood flow reduction. During the 60-min asphyxia, CO production by brain and astrocytes was continuously elevated. Excessive formation of reactive oxygen species during asphyxia/reventilation was potentiated by the HO inhibitor tin protoporphyrin, suggesting that endogenous CO has antioxidant effects. Cerebral vascular outcomes tested 24 and 48 h after asphyxia demonstrated the sustained impairment of cerebral vascular responses to astrocyte- and endothelium-specific vasodilators. Postasphyxia cerebral vascular dysfunction was aggravated in newborn pigs pretreated with tin protoporphyrin to inhibit brain HO/CO. The CO donor CO-releasing molecule-A1 (CORM-A1) reduced brain oxidative stress during asphyxia/reventilation and prevented postasphyxia cerebrovascular dysfunction. The antioxidant and antiapoptotic effects of HO/CO and CORM-A1 were confirmed in primary cultures of astrocytes from the neonatal pig brain exposed to glutamate excitotoxicity. Overall, prolonged neonatal asphyxia leads to neurovascular injury via an oxidative stress-mediated mechanism that is counteracted by an astrocyte-based constitutive antioxidant HO/CO system. We propose that gaseous CO or CO donors can be used as novel approaches for prevention of neonatal brain injury caused by prolonged asphyxia. NEW & NOTEWORTHY Asphyxia in newborn infants may lead to lifelong neurological disabilities. Using the model of prolonged asphyxia in newborn piglets, we propose novel antioxidant therapy based on systemic administration of low doses of a carbon monoxide donor that prevent loss of cerebral blood flow regulation and may improve the neurological outcome of asphyxia.