miR-379 links glucocorticoid treatment with mitochondrial response in Duchenne muscular dystrophy.

Duchenne Muscular Dystrophy (DMD) is a lethal muscle disorder, caused by mutations in the DMD gene and affects approximately 1:5000-6000 male births. In this report, we identified dysregulation of members of the Dlk1-Dio3 miRNA cluster in muscle biopsies of the GRMD dog model. Of these, we selected miR-379 for a detailed investigation because its expression is high in the muscle, and is known to be responsive to glucocorticoid, a class of anti-inflammatory drugs commonly used in DMD patients. Bioinformatics analysis predicts that miR-379 targets EIF4G2, a translational factor, which is involved in the control of mitochondrial metabolic maturation. We confirmed in myoblasts that EIF4G2 is a direct target of miR-379, and identified the DAPIT mitochondrial protein as a translational target of EIF4G2. Knocking down DAPIT in skeletal myotubes resulted in reduced ATP synthesis and myogenic differentiation. We also demonstrated that this pathway is GC-responsive since treating mice with dexamethasone resulted in reduced muscle expression of miR-379 and increased expression of EIF4G2 and DAPIT. Furthermore, miR-379 seric level, which is also elevated in the plasma of DMD patients in comparison with age-matched controls, is reduced by GC treatment. Thus, this newly identified pathway may link GC treatment to a mitochondrial response in DMD.

BCAT1 controls metabolic reprogramming in activated human macrophages and is associated with inflammatory diseases.

Branched-chain aminotransferases (BCAT) are enzymes that initiate the catabolism of branched-chain amino acids (BCAA), such as leucine, thereby providing macromolecule precursors; however, the function of BCATs in macrophages is unknown. Here we show that BCAT1 is the predominant BCAT isoform in human primary macrophages. We identify ERG240 as a leucine analogue that blocks BCAT1 activity. Selective inhibition of BCAT1 activity results in decreased oxygen consumption and glycolysis. This decrease is associated with reduced IRG1 levels and itaconate synthesis, suggesting involvement of BCAA catabolism through the IRG1/itaconate axis within the tricarboxylic acid cycle in activated macrophages. ERG240 suppresses production of IRG1 and itaconate in mice and contributes to a less proinflammatory transcriptome signature. Oral administration of ERG240 reduces the severity of collagen-induced arthritis in mice and crescentic glomerulonephritis in rats, in part by decreasing macrophage infiltration. These results establish a regulatory role for BCAT1 in macrophage function with therapeutic implications for inflammatory conditions.

Codon-optimized filovirus DNA vaccines delivered by intramuscular electroporation protect cynomolgus macaques from lethal Ebola and Marburg virus challenges.

Cynomolgus macaques were vaccinated by intramuscular electroporation with DNA plasmids expressing codon-optimized glycoprotein (GP) genes of Ebola virus (EBOV) or Marburg virus (MARV) or a combination of codon-optimized GP DNA vaccines for EBOV, MARV, Sudan virus and Ravn virus. When measured by ELISA, the individual vaccines elicited slightly higher IgG responses to EBOV or MARV than did the combination vaccines. No significant differences in immune responses of macaques given the individual or combination vaccines were measured by pseudovirion neutralization or IFN-γ ELISpot assays. Both the MARV and mixed vaccines were able to protect macaques from lethal MARV challenge (5/6 vs. 6/6). In contrast, a greater proportion of macaques vaccinated with the EBOV vaccine survived lethal EBOV challenge in comparison to those that received the mixed vaccine (5/6 vs. 1/6). EBOV challenge survivors had significantly higher pre-challenge neutralizing antibody titers than those that succumbed.

Antitumor activity of efrapeptins, alone or in combination with 2-deoxyglucose, in breast cancer in vitro and in vivo.

Efrapeptins (EF), a family of fungal peptides, inhibit proteasomal enzymatic activities and the in vitro and in vivo growth of HT-29 cells. They are also known inhibitors of F(1)F(0)-ATPase, a mitochondrial enzyme that functions as an Hsp90 co-chaperone. We have previously shown that treatment of cancer cells with EF results in disruption of the Hsp90:F(1)F(0)-ATPase complex and inhibition of Hsp90 chaperone activity. The present study examines the effect of EF on breast cancer growth in vitro and in vivo. As a monotherapy, EF inhibited cell proliferation in vitro with an IC(50) value ranging from 6 nM to 3.4 µM. Inhibition of Hsp90 chaperone function appeared to be the dominant mechanism of action and the factor determining cellular sensitivity to EF. In vitro inhibition of proteasome became prominent in the absence of adequate levels of Hsp90 and F(1)F(0)-ATPase as in the case of the relatively EF-resistant MDA-MB-231 cell line. In vivo, EF inhibited MCF-7 and MDA-MB-231 xenograft growth with a maximal inhibition of 60% after administration of 0.15 and 0.3 mg/kg EF, respectively. 2-Deoxyglucose (2DG), a known inhibitor of glycolysis, acted synergistically with EF in vitro and antagonistically in vivo. In vitro, the synergistic effect was attributed to a prolonged endoplasmic reticulum (ER) stress. In vivo, the antagonistic effect was ascribed to the downregulation of tumoral and/or stromal F(1)F(0)-ATPase by 2DG.

F1F0-ATP synthase functions as a co-chaperone of Hsp90-substrate protein complexes.

Inhibition of heat shock protein 90 (Hsp90) has emerged as a novel intervention for the treatment of solid tumors and leukemias. Here, we report that F(1)F(0)-ATP synthase, the enzyme responsible for the mitochondrial production of ATP, is a co-chaperone of Hsp90. F(1)F(0)-ATP synthase co-immunoprecipitates with Hsp90 and Hsp90-client proteins in cell lysates of MCF-7, T47D, MDA-MB-453, and HT-29 cancer cells. Inhibition of F(1)F(0)-ATP synthase by efrapeptins results in the disruption of the Hsp90 complexing with its substrate proteins and, in most cases, in the degradation of the latter. Hsp90-client proteins affected by the inhibition of F(1)F(0)-ATP synthase included ERalpha, mutated p53 (m.p53), Hsp70, Hsp27, and caspase-3 but not Raf-1. This is the first report identifying caspase-3 as a substrate protein of Hsp90. Unlike typical Hsp90 inhibitors, efrapeptin treatment triggers Hsp70 downregulation in parallel with depletion of Hsp90. This suggests that suppression of Hsp90 chaperone function through inhibition of F(1)F(0)-ATP synthase does not result in activation of transcription factor HSF-1, a generally unfavorable consequence of anti-cancer treatments based on Hsp90 inhibition.

Generation of a specific immunological response to FGF-2 does not affect wound healing or reproduction.

Angiogenesis, the process of new capillary formation from pre-existing vessels, has been established as an important mechanism involved in pathologic processes, such as cancer, as well as in normal physiology (Ribatti, D.; Vacca, A.; Roncali, L.; Dammacco, F. Angiogenesis under normal and pathological conditions. Haematologica 1991, 76 (4), 311-320). Basic fibroblast growth factor (FGF-2) is a critical mediator of angiogenesis that is important for normal reproduction and wound healing. FGF-2 mediates its pro-angiogenic effects by binding to heparin sulfate proteoglycan in addition to a tyrosine kinase receptor (Baird, A.; Schubert, D.; Ling, N.; Guillemin, R. Receptor and heparin-binding domain of basic fibroblast growth factor. Proc. Natl. Acad. Sci. U. S. A. 1998, 5 (7), 2324-2328; Richard, C.; Roghani, M.; Moscatelli, D. Fibroblast growth factor (FGF)-2 mediates cell attachment through interactions with two FGF receptor-1 isoforms and extracellular matrix or cell-associated heparin sulfate proteoglycans. Biochem. Biophys. Res. Commun. 2000, 276 (2), 399-405; Casu, B.; Guerrini, M.; Naggi, A.; Perez, M.; Torri, G.; Ribatti, D.; Carminati, P.; Giannini, G.; Penco, S.; Pisano, C.; Belleri, M.; Rusnati, M.; Presta, M. Short heparin sequences spaced by glycol-split urinate residues are antagonists of fibroblast growth factor 2 and angiogenesis inhibitors. Biochemistry 2002, 41 (33), 10519-10528; Murphy, P.V.; Pitt, N.; O'Brien, A.; Enright, P.M.; Dunne, A.; Wilson, S.J.; Duane, R.M.; O'Boyle, K.M. Identification of novel inhibitors of fibroblast growth factor (FGF-2) binding to heparin and endothelial cell survival from a structurally diverse carbohybrid library. Bioorg. Med. Chem. Lett. 2002, 12 (22), 3287-3290). We developed a liposomal-based peptide vaccine, L(HBD) that targets the heparin binding domain of the FGF-2 molecule. This vaccine, when inoculated into mice, inhibits angiogenesis in response to FGF-2 in a hepatic sponge model as well as tumor progression in two models of pulmonary metastatic disease. In the present studies, we further characterize the immunological and physiological responses to this vaccine. Vaccinated animals generated a specific anti-FGF-2 antibody (titer of 1:5000) that was able to inhibit FGF-2 binding to heparin sulfate in a dose dependent fashion. Cell mediated immunity was evidenced by a delayed type hypersensitivity response following challenge with the heparin binding domain peptide. Despite an immune response toward FGF-2, vaccination with L(HBD) did not result in alterations in mean time to wound healing when compared to unvaccinated animals or those treated with a liposome control. In reproductive studies, vaccinated females were not impaired in their ability to: 1) become pregnant, 2) support the growth and development of their embryos, and 3) deliver viable offspring. Furthermore, when assessed histologically, these offspring did not demonstrate any alterations in organogenesis when compared to pups born to untreated or liposome control treated females. Thus, while vaccination against FGF-2 induces a specific FGF-2 antibody response, and inhibits angiogenesis and tumor development in a pathological setting, it does not adversely alter normal physiological events dependent on FGF-2.

Synergistic activity of recombinant human endostatin in combination with adriamycin: analysis of in vitro activity on endothelial cells and in vivo tumor progression in an orthotopic murine mammary carcinoma model.

PURPOSE: Current combination treatment strategies in malignancy are designed to evaluate the use of cytotoxic drugs and antiangiogenic agents. Endostatin, a fragment of collagen XVIII, specifically inhibits proliferation, migration, and differentiation of endothelial cells in vitro as well as angiogenesis and tumor progression in in vivo models. In this study, we determine the antitumor effect of rhEndostatin administered alone or in combination with Adriamycin against established orthotopic murine mammary carcinoma. EXPERIMENTAL DESIGN: Mice bearing orthotopically established DA-3 mammary adenocarcinoma tumors received varying doses of rhEndostatin alone and in combination with Adriamycin to assess tumor growth inhibition. Additional studies of this in vivo combination included a determination of Adriamycin-induced cardiotoxicity and in vitro effects on human umbilical vein endothelial cell proliferation and cord formation. RESULTS: For single-agent activity, optimal tumor growth inhibition was observed after s.c. administration of 50 mg/kg/day rhEndostatin or 5 mg/kg Adriamycin injected i.v. every 4 days. Combination of Adriamycin with optimal or suboptimal doses of rhEndostatin resulted in synergistic inhibition of DA-3 tumor growth. Importantly, unlike other antiangiogenic agents, rhEndostatin did not exacerbate the cardiotoxicity of Adriamycin. The synergistic interaction between rhEndostatin and Adriamycin was also observed in vitro for inhibition of human umbilical vein endothelial cell proliferation and inhibition of cord formation. CONCLUSIONS: These data suggest that the synergy observed with rhEndostatin in combination with Adriamycin is exerted at the level of the endothelial cell and can result in enhanced tumor growth inhibition. The potential benefit of Adriamycin used in combination with rhEndostatin is being considered for clinical evaluation.