FoxO3a is Essential for the Antiproliferative and Apoptogenic Effects of Sunitinib in MDA-MB231 Cell Line.

BACKGROUND: Sunitinib (SUN), a tyrosine kinase inhibitor, is a promising treatment for triple-negative breast cancer (TNBC), the most aggressive and fast-growing type of breast cancer. Yet, the protective effect of SUN against TNBC is poorly investigated and the role of Forkhead box type O (FOXO3a) transcription factor is still unknown. MATERIALS AND METHODS: Cell proliferation was evaluated using the MTT assay. The mRNA and protein expression of apoptotic, oxidative stress and cell cycle genes were determined by real-time polymerase chain reaction (RT-PCR) and western blot analyses, respectively. Percentage of the apoptotic cells were determined by flow cytometry. The role of FOXO3a was knock-downed using siRNA. RESULTS: SUN caused suppression of MDA-MB231 cell growth associated with induction of apoptosis, cell cycle arrest, oxidative stress markers and FOXO3a gene. Importantly, silencing of FOXO3a mRNA using siRNA significantly rescued MDA-MB231 cells from SUN-induced cell-proliferative arrest. CONCLUSION: SUN inhibits TNBC MDA-MB231 cell proliferation through activation of FOXO3a expression.

Differential Effects of Sunitinib on the Expression Profiles of Xenobiotic-Metabolizing Enzymes and Transporters in Rat Liver and Kidneys.

Sunitinib (SUN) is a multi-targeted tyrosine kinase inhibitor that was recently approved for the treatment of gastrointestinal tract and renal cancers. To date, very little is known about the effects of SUN on the expression of hepatic and renal xenobiotic-metabolizing enzymes (XMEs) and transporters. The present study was designed to investigate the capacity of chronic SUN treatment to modulate the mRNA and protein expression levels of phase I cytochrome P450 (CYP), phase II conjugating enzymes, and phase III transporters in rat liver and kidneys. For this purpose, SUN (25, 50 and 100 mg/kg) was injected IP into Wistar albino rats for 4 weeks; thereafter, the mRNA and protein expression levels of several XMEs and transporters were determined by RT-PCR and Western blot analysis, respectively. Real-time PCR analysis showed that SUN significantly induced the hepatic and renal CYP1A1, 1A2, 1B1, 2E1 and 4F4, whereas it inhibited CYP2C11 and 4A2. Furthermore, SUN specifically induced renal, but not hepatic, CYP2J3 and 3A2, while it induced only hepatic CYP4A1. With regard to phase II, SUN induced hepatic GSTA1 and UGT1A and renal NQO1 and UGT1A mRNA levels, whereas it inhibited renal GST1A expression. On the other hand, both renal and hepatic P-gp, MRP2 and BCRP transporters were significantly induced by SUN at the mRNA and protein expression levels. Importantly, these differential effects were associated with changes in oxidative stress genes and lipid peroxidation levels. In conclusion, SUN can serve as XME and transporters modulator, which potentially may counteract the efficacy of the treatment, adverse reactions and drug interactions in SUN treatment.

Prophylactic and Therapeutic Potential of Acetyl-L-carnitine against Acetaminophen-Induced Hepatotoxicity in Mice.

Prophylactic and therapeutic effects of acetylcarnitine against acetaminophen-induced hepatotoxicity were studied in mice. To evaluate the prophylactic effects of acetylcarnitine, mice were supplemented with acetylcarnitine (2 mmol/kg/day per oral (p.o.) for 5 days) before a single dose of acetaminophen (350 mg/kg intraperitoneal (i.p.)). Animals were sacrificed 6 h after acetaminophen injection. Acetaminophen significantly increased the markers of liver injury, hepatic reactive oxygen species, and nitrate/nitrite, and decreased hepatic glutathione (GSH) and the antioxidant enzymes. Acetylcarnitine supplementation resulted in reversal of all biochemical parameters toward the control values. To explore the therapeutic effects of acetylcarnitine, mice were given a single dose of acetylcarnitine (0.5, 1, and 2 mmol/kg p.o.) 1.5 h after acetaminophen. Animals were sacrificed 6 h after acetaminophen. Acetylcarnitine administration resulted in partial reversal of liver injury only at 2 mmol/kg p.o. At equimolar doses, N-acetylcystiene was superior as therapeutic agent to acetylcarnitine. However, acetylcarnitine potentiated the effect of N-acetylcystiene in the treatment of acetaminophen toxicity.

Proteinase activated receptor-2-mediated dual oxidase-2 up-regulation is involved in enhanced airway reactivity and inflammation in a mouse model of allergic asthma.

Airway epithelial cells (AECs) express a variety of receptors, which sense danger signals from various aeroallergens/pathogens being inhaled constantly. Proteinase-activated receptor 2 (PAR-2) is one such receptor and is activated by cockroach allergens, which have intrinsic serine proteinase activity. Recently, dual oxidases (DUOX), especially DUOX-2, have been shown to be involved in airway inflammation in response to Toll-like receptor activation. However, the association between PAR-2 and DUOX-2 has not been explored in airways of allergic mice. Therefore, this study investigated the contribution of DUOX-2/reactive oxygen species (ROS) signalling in airway reactivity and inflammation after PAR-2 activation. Mice were sensitized intraperitoneally with intact cockroach allergen extract (CE) in the presence of aluminium hydroxide followed by intranasal challenge with CE. Mice were then assessed for airway reactivity, inflammation, oxidative stress (DUOX-2, ROS, inducible nitric oxide synthase, nitrite, nitrotyrosine and protein carbonyls) and apoptosis (Bax, Bcl-2, caspase-3). Challenge with CE led to up-regulation of DUOX-2 and ROS in AECs with concomitant increases in airway reactivity/inflammation and parameters of oxidative stress, and apoptosis. All of these changes were significantly inhibited by intranasal administration of ENMD-1068, a small molecule antagonist of PAR-2 in allergic mice. Administration of diphenyliodonium to allergic mice also led to improvement of allergic airway responses via inhibition of the DUOX-2/ROS pathway; however, these effects were less pronounced than PAR-2 antagonism. The current study suggests that PAR-2 activation leads to up-regulation of the DUOX-2/ROS pathway in AECs, which is involved in regulation of airway reactivity and inflammation via oxidative stress and apoptosis.

Acute glutathione depletion leads to enhancement of airway reactivity and inflammation via p38MAPK-iNOS pathway in allergic mice.

Glutathione (GSH) plays a major role in allergic airway responses through a variety of mechanism which include direct scavenging of oxidative species, being a reducing equivalent and regulation of cellular signaling through redox sensitive mechanisms. Therefore, the aim of the present study was to evaluate the role of acute GSH depletion on airway reactivity, inflammation and NO signaling in a mouse model of allergic asthma. Buthionine sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase was used for depletion of GSH levels. Acute depletion of GSH with BSO worsened allergen induced airway reactivity and inflammation through increase in nitrosative stress as reflected by increased inducible NO synthase (iNOS) expression, total nitrates and nitrites (NOx), nitrotyrosine, protein carbonyls, and decreased total antioxidant capacity. Treatment with p38 mitogen-activated protein kinase (MAPK) and iNOS inhibitors attenuated the effects of GSH depletion on airway reactivity and inflammation through attenuation of nitrosative stress as evidenced by a decrease in NOx, nitrotyrosine, protein carbonyls and increase in total antioxidant capacity (TAC). In conclusion, these data suggest that acute depletion of glutathione is associated with alteration of airway responses through an increase in nitrosative stress in allergic airways of mice.

Airway and systemic oxidant-antioxidant dysregulation in asthma: a possible scenario of oxidants spill over from lung into blood.

Asthma is characterized by repeated episodes of airway obstruction and an ongoing cycle of airway inflammation. Reactive oxygen species (ROS) produced by inflammatory cells in the lung play a key role in the pathogenesis as well as amplification of inflammation in asthmatic airways. Several enzymatic and non-enzymatic antioxidants are available in the lung and systemic circulation to counteract ROS-mediated damage on various biomolecules such as lipid membranes, proteins, and DNA; however during asthmatic inflammation these defenses are overwhelmed due to excessive production of ROS thereby leading to inflammatory events in the airways/systemic circulation. ROS-mediated damage may result in increased vascular permeability, mucus hypersecretion, smooth muscle contraction, epithelial shedding and impairment in the responsiveness of ß-adrenergic receptors. Strategies aimed to boost the endogenous antioxidants either through dietary or pharmacological intervention to redress oxidant-antioxidant imbalance in asthma is the current area of research in many laboratories throughout the world. This review aims at providing a comprehensive overview of the available literature on oxidative stress and antioxidants imbalance in asthma with a focus both on lung and blood components and bring forth correlations between lung/blood oxidative stress/antioxidant parameters and lung function.

Glutathione modulation during sensitization as well as challenge phase regulates airway reactivity and inflammation in mouse model of allergic asthma.

Glutathione, being a major intracellular redox regulator has been shown to be implicated in regulation of airway reactivity and inflammation. However, no study so far has investigated the effect of glutathione depletion/repletion during sensitization and challenge phases separately, which could provide an important insight into the pathophysiology of allergic asthma. The aim of the present study was to evaluate the role of glutathione depletion/repletion during sensitization and challenge phases separately in a mouse model of allergic asthma. Buthionine sulphoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase or N-acetyl cysteine (NAC), a thiol donor were used for depletion or repletion of glutathione levels respectively during both sensitization and challenge phases separately followed by assessment of airway reactivity, inflammation and oxidant-antioxidant balance in allergic mice. Depletion of glutathione with BSO during sensitization as well as challenge phase worsened allergen induced airway reactivity/inflammation and caused greater oxidant-antioxidant imbalance as reflected by increased NADPH oxidase expression/reactive oxygen species (ROS) generation/lipid peroxides formation and decreased total antioxidant capacity. On the other hand, repletion of glutathione pool by NAC during sensitization and challenge phases counteracted allergen induced airway reactivity/inflammation and restored oxidant-antioxidant balance through a decrease in NADPH oxidase expression/ROS generation/lipid peroxides formation and increase in total antioxidant capacity. Taken together, these findings suggest that depletion or repletion of glutathione exacerbates or ameliorates allergic asthma respectively by regulation of airway oxidant-antioxidant balance. This might have implications towards increased predisposition to allergy by glutathione depleting environmental pollutants.