Intracellular events following paclitaxel binding to microtubules that lead to cell death remain poorly understood. Because reactive oxygen species (ROS) are involved in the cytotoxicity of anticancer agents acting through independent molecular targets, we explored the role of ROS in paclitaxel cytotoxicity. Within 15 min after in vitro exposure of A549 human lung cancer cells to paclitaxel, a concentration-dependent intracellular increase in O 2°2 and H 2 O 2 levels was detected by spectrofluorometry. Addition of N-acetylcysteine (NAC) or glutathione, two H 2 O 2 scavenger, induced a 4-fold increase in paclitaxel IC 50 . Delaying NAC co-incubation by 4 hr, resulted in a 3-fold reduction in cell protection. The glutathione synthesis inhibitor, buthionine sulfoximine significantly increased paclitaxel cytotoxicity and H 2 O 2 accumulation, but did not modify O 2°2 levels. Co-incubation with diphenylene iodonium suggested that paclitaxel induced-O 2°2 production was in part associated with increased activity of cytoplasmic NADPH oxidase. Concomitant treatment with inhibitors of caspases 3 and 8 increased cell survival but did not prevent the early accumulation of H 2 O 2. To evaluate the role of ROS in paclitaxel antitumoral activity, mice were injected with LLC1 lung cancer cells and treated with paclitaxel i.p. and/or NAC. The antitumoral activity of paclitaxel in mice was abolished by NAC. In conclusion, the accumulation of H 2 O 2 is an early and crucial step for paclitaxel-induced cancer cell death before the commitment of the cells into apoptosis. These results suggest that ROS participate in vitro and in vivo to paclitaxel cytotoxicity. ' 2006 Wiley-Liss, Inc.
Systemic sclerosis (SSc) is a connective tissue disorder of great clinical heterogeneity. Its pathophysiology remains unclear. Our aim was to evaluate the relative roles of reactive oxygen species (ROS) and of the immune system using an original model of SSc. BALB/c and immunodeficient BALB/c SCID mice were injected s.c. with prooxidative agents (hydroxyl radicals, hypochlorous acid, peroxynitrites, superoxide anions), bleomycin, or PBS everyday for 6 wk. Skin and lung fibrosis were assessed by histological and biochemical methods. Autoantibodies were detected by ELISA. The effects of mouse sera on H 2 O 2 production by endothelial cells and on fibroblast proliferation, and serum concentrations in advanced oxidation protein products (AOPP) were compared with sera from patients with limited or diffuse SSc. We observed that s.c. peroxynitrites induced skin fibrosis and serum anti-CENP-B Abs that characterize limited SSc, whereas hypochlorite or hydroxyl radicals induced cutaneous and lung fibrosis and anti-DNA topoisomerase 1 autoantibodies that characterize human diffuse SSc. Sera from hypochlorite-or hydroxyl radical-treated mice and of patients with diffuse SSc contained high levels of AOPP that triggered endothelial production of H 2 O 2 and fibroblast hyperproliferation. Oxidized topoisomerase 1 recapitulated the effects of whole serum AOPP. SCID mice developed an attenuated form of SSc, demonstrating the synergistic role of the immune system with AOPP in disease propagation. We demonstrate a direct role for ROS in SSc and show that the nature of the ROS dictates the form of SSc. Moreover, this demonstration is the first that shows the specific oxidation of an autoantigen directly participates in the pathogenesis of an autoimmune disease. The Journal of Immunology, 2009, 182: 5855-5864. S ystemic sclerosis (SSc)4 is a connective tissue disorder of unknown etiology characterized by vascular hyperreactivity, fibrosis of skin and visceral organs, and immunological alterations, including a distinct pattern of autoantibodies in the sera (1). The mechanisms that determine the clinical manifestations of the disease remain unclear (2). Several reports have suggested that reactive oxygen species (ROS) are involved in the pathogenesis of SSc (3-9). Indeed, skin fibroblasts from SSc patients spontaneously produce large amounts of ROS that trigger collagen synthesis (7, 10). In addition, autoantibodies found in SSc patients against the platelet-derived growth factor receptor expressed on fibroblasts also induce the production of ROS (11). Recently, we have demonstrated that sera from patients with SSc could induce not only the production of ROS by endothelial cells but also the hyperproliferation of fibroblasts (6).However, no direct proof for the involvement of oxidative stress in SSc pathogenesis has been brought forth to date. Moreover, the origin and nature of the oxidative stress remain to be elucidated. Environmental factors, and in particular silica dust, may be involved, which generate hydroxyl radicals (OH⅐) (12...
Sorafenib is presently the only effective therapy in advanced hepatocellular carcinoma (HCC). Because most anticancer drugs act, at least in part, through the generation of reactive oxygen species, we investigated whether sorafenib can induce an oxidative stress. The effects of sorafenib on intracellular ROS production and cell death were assessed in vitro in human (HepG2) and murine (Hepa 1.6) HCC cell lines and human endothelial cells (HUVEC) as controls. In addition, 26 sera from HCC patients treated by sorafenib were analyzed for serum levels of advanced oxidation protein products (AOPP). Sorafenib significantly and dosedependently enhanced in vitro ROS production by HCC cells. The SOD mimic MnTBAP decreased sorafenibinduced lysis of HepG2 cells by 20% and of Hepa 1.6 cells by 75% compared with HCC cells treated with 5 mg/L sorafenib alone. MnTBAP significantly enhanced by 25% tumor growth in mice treated by sorafenib. On the other hand, serum levels of AOPP were higher in HCC patients treated by sorafenib than in sera collected before treatment (P < 0.001). An increase in serum AOPP concentration !0.2 mmol/L chloramine T equivalent after 15 days of treatment is a predictive factor for sorafenib response with higher progression free survival (P < 0.05) and overall survival rates (P < 0.05). As a conclusion, sorafenib dose-dependently induces the generation of ROS in tumor cells in vitro and in vivo. The sera of Sorafenib-treated HCC patients contain increased AOPP levels that are correlated with the clinical effectiveness of sorafenib and can be used as a marker of effectiveness of the drug.
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