Doxorubicin is a highly effective cancer treatment whose use is severely limited by dose-dependent cardiotoxicity. It is well established that doxorubicin increases reactive oxygen species (ROS) production. In this study, we investigated contributions to doxorubicin cardiotoxicity from Nox2 NADPH oxidase, an important ROS source in cardiac cells, which is known to modulate several key processes underlying the myocardial response to injury. Nox2-deficient mice (Nox2 −/− ) and wild-type (WT) controls were injected with doxorubicin (12 mg/kg) or vehicle and studied 8 weeks later. Echocardiography indicated that doxorubicin-induced contractile dysfunction was attenuated in Nox2 −/− versus WT mice (fractional shortening: 29.5 ± 1.4 versus 25.7 ± 1.0%; P < 0.05). Similarly, in vivo pressure-volume analysis revealed that systolic and diastolic function was preserved in doxorubicin-treated Nox2 −/− versus WT mice (ejection fraction: 52.6 ± 2.5 versus 28.5 ± 2.3%, LVdP/dt min : −8,379 ± 416 versus −5,198 ± 527 mmHg s −1 ; end-diastolic pressure-volume relation: 0.051 ± 0.009 versus 0.114 ± 0.012; P < 0.001). Furthermore, in response to doxorubicin, Nox2 −/− mice exhibited less myocardial atrophy, cardiomyocyte apoptosis, and interstitial fibrosis, together with reduced increases in profibrotic gene expression (procollagen IIIαI, transforming growth factor-β 3 , and connective tissue growth factor) and matrix metalloproteinase-9 activity, versus WT controls. These alterations were associated with beneficial changes in NADPH oxidase activity, oxidative/nitrosative stress, and inflammatory cell infiltration. We found that adverse effects of doxorubicin were attenuated by acute or chronic treatment with the AT1 receptor antagonist losartan, which is commonly used to reduce blood pressure. Our findings suggest that ROS specifically derived from Nox2 NADPH oxidase make a substantial contribution to several key processes underlying development of cardiac contractile dysfunction and remodeling associated with doxorubicin chemotherapy.

Purpose: Antiangiogenic therapies can be an important adjunct to the management of many malignancies. Here we investigated a novel protein, FKBPL, and peptide derivative for their antiangiogenic activity and mechanism of action.Experimental Design: Recombinant FKBPL (rFKBPL) and its peptide derivative were assessed in a range of human microvascular endothelial cell (HMEC-1) assays in vitro. Their ability to inhibit proliferation, migration, and Matrigel-dependent tubule formation was determined. They were further evaluated in an ex vivo rat model of neovascularization and in two in vivo mouse models of angiogenesis, that is, the sponge implantation and the intravital microscopy models. Antitumor efficacy was determined in two human tumor xenograft models grown in severe compromised immunodeficient (SCID) mice. Finally, the dependence of peptide on CD44 was determined using a CD44-targeted siRNA approach or in cell lines of differing CD44 status.Results: rFKBPL inhibited endothelial cell migration, tubule formation, and microvessel formation in vitro and in vivo. The region responsible for FKBPL's antiangiogenic activity was identified, and a 24-amino acid peptide (AD-01) spanning this sequence was synthesized. It was potently antiangiogenic and inhibited growth in two human tumor xenograft models (DU145 and MDA-231) when administered systemically, either on its own or in combination with docetaxel. The antiangiogenic activity of FKBPL and AD-01 was dependent on the cell-surface receptor CD44, and signaling downstream of this receptor promoted an antimigratory phenotype.Conclusion: FKBPL and its peptide derivative AD-01 have potent antiangiogenic activity. Thus, these agents offer the potential of an attractive new approach to antiangiogenic therapy.

The somatic JAK2 valine-to-phenylalanine (V617F) mutation has been detected in up to 90% of patients with polycythemia and in a sizeable proportion of patients with other myeloproliferative disorders such as essential thrombocythemia and idiopathic myelofibrosis. Suppressor of cytokine signaling 3 (SOCS3) is known to be a strong negative regulator of erythropoietin (EPO) signaling through interaction with both the EPO receptor (EPOR) and JAK2. We report here that JAK2 V617F cannot be regulated and that its activation is actually potentiated in the presence of SOCS3. Instead of acting as a suppressor, SOCS3 enhanced the proliferation of cells expressing both JAK2 V617F and EPOR. Additionally, although SOCS1 and SOCS2 are degraded in the presence of JAK2 V617F, turnover of SOCS3 is inhibited by the JAK2 mutant kinase and this correlated with marked tyrosine phosphorylation of SOCS3 protein. We also observed constitutive tyrosine phosphorylation of SOCS3 in peripheral blood mononuclear cells (PBMCs) derived from patients homozygous for the JAK2 V617F mutant. These findings suggest that the JAK2 V617F has overcome normal SOCS regulation by hyperphosphorylating SOCS3, rendering it unable to inhibit the mutant kinase. Thus, JAK2 V617F may even exploit SOCS3 to potentiate its myeloproliferative capacity. IntroductionThe somatic valine-to-phenylalanine (V617F) mutation in JAK2 has been associated with a variety of myeloproliferative disorders (MPD), including polycythemia vera (PV), essential thrombocythemia (ET), and idiopathic myelofibrosis (IMF). [1][2][3][4][5] In wild-type JAKs the JH2 domain inhibits the JH1 kinase domain through interactions at 2 interfaces, with the region containing V617 being predicted to preserve the inactive conformation of the activation loop. 6 The V617F mutation might alter this conformation and perhaps stabilize the activation loop in an active state, or it may prevent access of other proteins to the catalytic domain. The V617 residue of JAK2 is conserved in the JH2 domain of JAK1 and TYK2, whereas in JAK3 it is replaced by methionine. Like JAK2 V617F, analogous mutations in JAK1 or TYK2 also results in their constitutive activation. 7 Janus kinases require the JH2 pseudokinase domain for normal physiologic activation of the JH1 catalytic domain. Therefore, it seems that the V617F mutation may disrupt the putative inhibition of the catalytic domain by the pseudokinase domain and create a constitutively activated kinase. However, the Janus kinases are also potently regulated by the suppressor of cytokine signaling (SOCS) proteins that are thought to bind to the JH1 catalytic loop and target the kinases for degradation. Whether SOCS can regulate the JAK2 V617F mutant has not been explored. 8 SOCS1 and SOCS3 bind to the catalytic groove of JAK2 via their kinase inhibitory region (KIR) to inhibit catalytic activity. 8 Both of these SOCS proteins can also target TEL-JAK2 and wild-type JAK2 for ubiquitination and degradation via their SOCS box ECS ubiquitin E3 ligase interaction motif. 9,10 A...