Carole Nicco

Accumulation of hydrogen peroxide is an early and crucial step for paclitaxel-induced cancer cell death both in vitro and in vivo.

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− and H2O2 levels was detected by spectrofluorometry. Addition of N‐acetylcysteine (NAC) or glutathione, two H2O2 scavenger, induced a 4‐fold increase in paclitaxel IC50. 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 H2O2 accumulation, but did not modify O°2− levels. Co‐incubation with diphenylene iodonium suggested that paclitaxel induced‐O°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 H2O2. 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 H2O2 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.

Reactive Oxygen Species Controls Endometriosis Progression

Endometriosis is associated with chronic inflammation, and reactive oxygen species (ROS) are proinflammatory mediators that modulate cell proliferation. We have investigated whether the dysregulation of ROS production in endometriotic cells correlates with a pro-proliferative phenotype and can explain the spreading of this disease. Stromal and epithelial cells were purified from ovarian endometrioma and eutopic endometrium from 14 patients with endometriosis to produce four primary cell lines from each patient. ROS production, detoxification pathways, cell proliferation, and mitogen-activated protein kinase pathway activation were studied and compared with epithelial and stromal cell lines from 14 patients without endometriosis. Modulation of the proliferation of endometriosis by N-acetyl-cysteine, danazol, and mifepristone was tested in vitro and in 28 nude mice implanted with endometriotic tissue of human origin. Endometriotic cells displayed higher endogenous oxidative stress with an increase in ROS production, alterations in ROS detoxification pathways, and a drop in catalase levels, as observed for tumor cells. This increase in endogenous ROS correlated with increased cellular proliferation and activation of ERK1/2. These phenomena were abrogated by the antioxidant molecule N-acetyl-cysteine both in vitro and in a mouse model of endometriosis. Human endometriotic cells display activated pERK, enhanced ROS production, and proliferative capability. Our murine model shows that antioxidant molecules could be used as safe and efficient treatments for endometriosis.

Platelet CD154 Potentiates Interferon-α Secretion by Plasmacytoid Dendritic Cells in Systemic Lupus Erythematosus

In the autoimmune disease lupus, platelets activated by self-antigens contribute to pathology by triggering the secretion of interferon from immune cells. Taming the Big Bad Wolf Systemic lupus erythematosus (SLE)—a name some attribute to this disorder’s wolf-like ability to “devour” the affected organs—is an autoimmune inflammatory disease. It can affect virtually any part of the body, but often targets skin, kidney, and joints. A variety of immunological factors have been proposed to contribute to SLE, in particular the type I interferon (IFN) system, which is normally activated in response to viruses. Here, Duffau et al. point to platelets as the culprits in causing aberrant activation of IFN-α (a member of the type I IFN group) in lupus patients and suggest that a drug that blocks platelet activation could be a promising new treatment. A protein called CD154 (CD40 ligand) is found on T cells, where it helps to defend the body by activating cytotoxic immune cells during viral infections. It is also found on the surface of platelets that are activated for clotting and may contribute to the pathogenesis of inflammatory states such as atherosclerosis and autoimmune disorders, including SLE. Here, the authors collected platelets from patients experiencing SLE flare-ups of varying severity, as well as healthy controls, and demonstrated that CD154 abundance and shedding from platelets correlated with disease severity. Moreover, exposure of platelets from healthy donors to serum from patients with active SLE or to immune complexes similar to those in SLE patients triggered an increase in activation and CD154 production. These activated platelets, in turn, signaled to antigen-presenting cells to produce IFN-α, thus propagating an inflammatory cycle, both in vitro and in a murine model of lupus. To further test these ideas, Duffau et al. depleted the platelets in lupus-prone mice, which decreased inflammation in the animals’ kidneys, a commonly affected organ in lupus. They achieved a similar outcome by treating the mice with clopidogrel, an inhibitor of platelet activation already commonly used in patients with heart disease and stroke. In addition to experiencing less kidney damage, the clopidogrel-treated mice with lupus lived for an extra 3 months. The current mainstay of treatment for SLE is immunosuppressive therapy, achieved with steroids and chemotherapy-like medications. These drugs have numerous toxic effects, not the least of which is the immunosuppression itself, which predisposes patients to infections. Being able to treat lupus with an antiplatelet medication such as clopidogrel, which has few side effects, would markedly improve these patients’ safety and quality of life. A similar approach may prove useful in other autoimmune diseases such as rheumatoid arthritis, where it would also provide a badly needed alternative to immunosuppression. Systemic lupus erythematosus (SLE) is a systemic inflammatory autoimmune disease characterized by the involvement of multiple organs and an immune response against nuclear components. Although its pathogenesis remains poorly understood, type I interferon (IFN) and CD40 ligand (CD154) are known to contribute. Because platelets are involved in inflammatory processes and represent a major reservoir of CD154, we hypothesized that they participate in SLE pathogenesis. Here, we have shown that in SLE patients, platelets were activated by circulating immune complexes composed of autoantibodies bound to self-antigens through an Fc-γ receptor IIa (CD32)–dependent mechanism. Further, platelet activation correlated with severity of the disease and activated platelets formed aggregates with antigen-presenting cells, including monocytes and plasmacytoid dendritic cells. In vitro, activated platelets enhanced IFN-α secretion by immune complex–stimulated plasmacytoid dendritic cells through a CD154-CD40 interaction. Finally, in lupus-prone mice, depletion of platelets or administration of the P2Y(12) receptor antagonist (clopidogrel) improved all measures of disease and overall survival; transfusion of activated platelets worsened the disease course. Together, these data identify platelet activation as an important contributor to SLE pathogenesis and suggest that this process and its sequelae may provide a new therapeutic target.

Selective Oxidation of DNA Topoisomerase 1 Induces Systemic Sclerosis in the Mouse

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 H2O2 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 H2O2 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.

Pivotal role of superoxide anion and beneficial effect of antioxidant molecules in murine steatohepatitis

Nonalcoholic fatty liver disease, frequently associated with obesity, can lead to nonalcoholic steatohepatitis (NASH) and cirrhosis. The pathophysiology of NASH is poorly understood, and no effective treatment is available. In view of a potential deleterious role for reactive oxygen species (ROS), we investigated the origin of ROS overproduction in NASH. Mitochondrial production of ROS and its alterations in the presence of antioxidant molecules were studied in livers from ob/ob mice that bear a mutation of the leptin gene and develop experimental NASH. N‐acetyl‐cysteine and the superoxide dismutase (SOD) mimics ambroxol, manganese [III] tetrakis (5,10,15,20 benzoic acid) (MnTBAP), and copper [II] diisopropyl salicylate (CuDIPS) were used to target different checkpoints of the oxidative cascade to determine the pathways involved in ROS production. Liver mitochondria from ob/ob mice generated more O2°− than those of lean littermates (P < .01). Ex vivo, all three SOD mimics decreased O2°− generation (P < .001) and totally inhibited lipid peroxidation (P < .001) versus untreated ob/ob mice. Those modifications were associated with in vivo improvements: MnTBAP and CuDIPS reduced weight (P < .02) and limited the extension of histological liver steatosis by 30% and 52%, respectively, versus untreated ob/ob mice. In conclusion, these data demonstrate deleterious effects of superoxide anions in NASH and point at the potential interest of nonpeptidyl mimics of SOD in the treatment of NASH in humans. (HEPATOLOGY 2004;39:1277–1285.)

Production of Superoxide Anions by Keratinocytes Initiates P. acnes-Induced Inflammation of the Skin

Acne vulgaris is a chronic inflammatory disorder of the sebaceous follicles. Propionibacterium acnes (P. acnes), a gram-positive anareobic bacterium, plays a critical role in the development of these inflammatory lesions. This study aimed at determining whether reactive oxygen species (ROS) are produced by keratinocytes upon P. acnes infection, dissecting the mechanism of this production, and investigating how this phenomenon integrates in the general inflammatory response induced by P. acnes. In our hands, ROS, and especially superoxide anions (O2 •−), were rapidly produced by keratinocytes upon stimulation by P. acnes surface proteins. In P. acnes-stimulated keratinocytes, O2 •− was produced by NAD(P)H oxidase through activation of the scavenger receptor CD36. O2 •− was dismuted by superoxide dismutase to form hydrogen peroxide which was further detoxified into water by the GSH/GPx system. In addition, P. acnes-induced O2 •− abrogated P. acnes growth and was involved in keratinocyte lysis through the combination of O2 •− with nitric oxide to form peroxynitrites. Finally, retinoic acid derivates, the most efficient anti-acneic drugs, prevent O2 •− production, IL-8 release and keratinocyte apoptosis, suggesting the relevance of this pathway in humans.

Radical oxygen species production induced by advanced oxidation protein products predicts clinical evolution and response to treatment in systemic sclerosis

Objectives: To investigate the role of reactive oxygen species (ROS) in the development of the various patterns of systemic sclerosis (SSc) and the mechanisms of ROS production by endothelial cells and fibroblasts. Methods: Production of hydrogen peroxide (H2O2), nitric oxide (NO) and cellular proliferation were determined following incubation of endothelial cells and fibroblasts with 56 SSc and 30 healthy sera. Correlations were established between those markers, the type and the severity of the clinical involvements, and the response to treatment. The factors leading to ROS production were determined. Results: H2O2 production by endothelial cells and fibroblasts was higher after incubation with SSc sera than with normal sera (p<0.001) and with sera from SSc patients with severe complications than sera from other patients (p<0.05). Sera from patients with lung fibrosis triggered the proliferation of fibroblasts more than other SSc sera (p<0.001), whereas sera from patients with vascular complications exerted no proliferative effect on fibroblasts, but inhibited endothelial cell growth (p<0.05) and induced NO overproduction (p<0.05). Bosentan reduced NO release by 32%, whereas N-acetylcystein potentiated 5-fluorouracil (5FU) to inhibit fibroblast proliferation by 78%. Those serum-mediated effects did not involve antibodies but advanced oxidation protein products that selectively triggered cells to produce H2O2 or NO. Conclusions: SSc sera induce the production of different types of ROS that selectively activate endothelial cells or fibroblasts, leading to vascular or fibrotic complications. Assaying serum-induced ROS production allows clinical activity of the disease to be followed and appropriate treatments to be selected.

Targeting ADAM-17/notch signaling abrogates the development of systemic sclerosis in a murine model

OBJECTIVE Systemic sclerosis (SSc) is characterized by the fibrosis of various organs, vascular hyperreactivity, and immunologic dysregulation. Since Notch signaling is known to affect fibroblast homeostasis, angiogenesis, and lymphocyte development, we undertook this study to investigate the role of the Notch pathway in human and murine SSc. METHODS SSc was induced in BALB/c mice by subcutaneous injections of HOCl every day for 6 weeks. Notch activation was analyzed in tissues from mice with SSc and from patients with scleroderma. Mice with SSc were either treated or not treated with the γ-secretase inhibitor DAPT, a specific inhibitor of the Notch pathway, and the severity of the disease was evaluated. RESULTS As previously described, mice exposed to HOCl developed a diffuse cutaneous SSc with pulmonary fibrosis and anti-DNA topoisomerase I antibodies. The Notch pathway was hyperactivated in the skin, lung, fibroblasts, and splenocytes of diseased mice and in skin biopsy samples from patients with scleroderma. ADAM-17, a proteinase involved in Notch activation, was overexpressed in the skin of mice and patients in response to the local production of reactive oxygen species. In HOCl-injected mice, DAPT significantly reduced the development of skin and lung fibrosis, decreased skin fibroblast proliferation and ex vivo serum-induced endothelial H(2)O(2) production, and abrogated the production of anti-DNA topoisomerase I antibodies. CONCLUSION Our results show the pivotal role of the ADAM-17/Notch pathway in SSc following activation by reactive oxygen species. The inhibition of this pathway may represent a new treatment of this life-threatening disease.

Synthesis and Selective Anticancer Activity of Organochalcogen Based Redox Catalysts

Many tumor cells exhibit a disturbed intracellular redox state resulting in higher levels of reactive oxygen species (ROS). As these contribute to tumor initiation and sustenance, catalytic redox agents combining significant activity with substrate specificity promise high activity and selectivity against oxidatively stressed malignant cells. We describe here the design and synthesis of novel organochalcogen based redox sensor/effector catalysts. Their selective anticancer activity at submicromolar and low micromolar concentrations was established here in a range of tumor entities in various biological systems including cell lines, primary tumor cell cultures, and animal models. In the B-cell derived chronic lymphocytic leukemia (CLL), for instance, such compounds preferentially induce apoptosis in the cancer cells while peripheral blood mononuclear cells (PBMC) from healthy donors and the subset of normal B-cells remain largely unaffected. In support of the concept of sensor/effector based ROS amplification, we are able to demonstrate that underlying this selective activity against CLL cells are pre-existing elevated ROS levels in the leukemic cells compared to their nonmalignant counterparts. Furthermore, the catalysts act in concert with certain chemotherapeutic drugs in several carcinoma cell lines to decrease cell proliferation while showing no such interactions in normal cells. Overall, the high efficacy and selectivity of (redox) catalytic sensor/effector compounds warrant further, extensive testing toward transfer into the clinical arena.

The mTOR/AKT Inhibitor Temsirolimus Prevents Deep Infiltrating Endometriosis in Mice

Deep infiltrating endometriosis (DIE) is a particular clinical and histological entity of endometriosis responsible for chronic pelvic pain and infertility. Here we characterize the proliferative phenotype of DIE cells, to explore the cellular and molecular mechanisms that could explain their aggressive potential. In addition, the inhibition of mTOR/AKT pathway was tested, as a potential treatment of DIE. Included were 22 patients with DIE and 12 control patients without endometriosis. Epithelial and stromal cells were extracted from biopsies of eutopic endometrium and deep infiltrating endometriotic nodules from patients with DIE. Cell proliferation was determined by thymidine incorporation. Oxidative stress was assayed by spectrofluorometry. The ERK and mTOR/AKT pathways were analyzed in vitro by Western blot and for AKT in vivo in a mouse model of DIE. The proliferation rate of eutopic endometrial cells and of deep infiltrating endometriotic cells from DIE patients was higher than that of endometrial cells from controls. The hyperproliferative phenotype of endometriotic cells was associated with an increase in endogenous oxidative stress, and with activation of the ERK and mTOR/AKT pathways. mTOR/AKT inhibition by temsirolimus decreased endometriotic cell proliferation both in vitro and in vivo in a mouse model of DIE. Blocking the mTOR/AKT pathway offers new prospects for the treatment of DIE.