Localized Treatment with a Novel FDA-Approved Proteasome Inhibitor ...

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Localized Treatment with a Novel FDA-Approved Proteasome Inhibitor Blocks the Degradation of Dystrophin and Dystrophin-Associated Proteins in mdx Mice Gloria Bonuccelli1 Federica Sotgia1,2 Franco Capozza1 Elisabetta Gazzerro2 Carlo Minetti2 Michael P. Lisanti1,2*

Abstract

*Correspondence to: Michael P. Lisanti; Kimmel Cancer Center; Departments of Cancer Biology, and Biochemistry & Molecular Biology; Thomas Jefferson University; 233 S. 10th Street; Philadelphia, Pennsylvania 19107 USA; Tel.: 215.503.9295; Fax: 215.923.1098; Email: [email protected]

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Original manuscript submitted: 03/20/07 Manuscript accepted: 03/27/07

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2The Muscular and Neurodegenerative Disease Unit; University of Genova; and G. Gaslini Pediatric Institute; Genova, Italy

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1Kimmel Cancer Center; Departments of Cancer Biology, and Biochemistry and Molecular Biology; Thomas Jefferson University; Philadelphia, Pennsylvania USA

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Duchenne Muscular Dystrophy (DMD) is an incurable inherited disease of childhood, characterized by progressive muscle degeneration and weakness. Our previous findings supported the idea that dystrophin and associated proteins, absent or greatly reduced in DMD, are degraded in dystrophin‑deficient muscle by the proteasomal‑dependent pathway. Indeed, treatment with the proteasome inhibitor MG‑132 of skeletal muscles from mdx mice ‑a spontaneous mouse model of DMD‑ as well as from DMD patients, effectively rescued the expression and correct cellular localization of dystrophin and associated proteins. These promising results led us to further explore the use of protea‑ some inhibitors as a therapy for DMD. Therefore, we directed our attention towards two new dipeptide boronic acid inhibitors blocking the proteasomal‑dependent degrada‑ tion pathway: Velcade (bortezomib or PS‑341) and MLN273 (PS‑273). The exciting aspect of this development is that these drugs have already progressed to preclinical and clinical trials, in different fields than muscular dystrophy. Indeed, Velcade has been already FDA‑approved for treatment of multiple myeloma and its side effects had been already explored and managed. Promisingly, MLN273 is currently in the preclinical trial phase. Here, we test the effectiveness of Velcade and MLN273 by local injection into the gastrocnemius muscle of mdx mice. We show the rescue of expression and membrane localization of a‑dystroglycan, b‑dystroglycan, a‑sarcoglycan, and dystrophin after Velcade and MLN273 localized treatment, versus untreated (PBS only) mdx mice. Intriguingly, we also show that localized treatment with Velcade and MLN273 reduces the activation of Nuclear Factor‑kappaB (NFkB). Because the NFkB pathway has been shown to be involved in inflammation responses in myopathies and DMD, our current results may have important clinical implications. Clearly, more investigations are needed, but our results emphasize the effectiveness of the pharmacological approach as a poten‑ tial treatment for Duchenne muscular dystrophy.

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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=4182

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Key words

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muscular dystrophy, proteasome inhibitor, Velcade, dystrophin, dystrophin complex

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Duchenne muscular dystrophy dystrophin-glycoprotein complex caveolin-3 nuclear factor-kappaB Food and Drug Administration multiple myeloma

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DMD DGC Cav-3 NFkB FDA MM

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Abbreviations

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Acknowledgements

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This work was supported by a grant from the Muscular Dystrophy Association (MDA; to M.P.L.). E.G. and C.M. were supported by Telethon-Italia Grant GGP04166, M.U.R.S.T., and the Italian Ministry of Health (G. Gaslini Institute, Ricerca Finalizzata).

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Introduction

Even though several different approaches1,2 have been explored for the therapeutic treatment of Duchenne Muscular Dystrophy (DMD), DMD remains an incurable inherited disease of childhood, characterized by progressive muscle degeneration and wasting. Mutations in the dystrophin gene are the cause of the Duchenne phenotype.3 Dystrophin4 is a peripheral membrane protein of ~426 kDa important for maintenance of muscle integrity, such that its deficiency results in the structural perturbation of the plasma membrane of skeletal muscle fibers.5 Indeed, dystrophin associates with a large multimeric complex, termed the dystrophin‑glycoprotein complex (DGC), including the dystroglycan complex (a and b‑dystroglycan) and the sarcoglycan complex (a, b, g, d‑sarcoglycan).6 The consequence of loss of dystrophin is the absence or the great reduction of components of the DGC, as described for skeletal muscle fibers from DMD patients and from a mouse model of DMD, named the mdx mouse.7 An exception is caveolin‑3 (Cav‑3), a member of the caveolin protein family,8 which is up‑regulated in the fibers of dystrophin‑deficient muscle.9,10 Our recent studies support the fact that degradation of dystrophin and DGC through the proteasomal‑dependent pathway is involved in the pathogenesis of DMD. Indeed, using mdx mice, we first reported evidence that restoration of functional dystrophin and other proteins of DGC could be achieved by localized and systemic administration of the well‑characterized proteasomal inhibitor MG‑132.11 Interestingly, MG‑132 systemic treatment over an eight‑day period improved muscle integrity and morphology, as judged by H&E staining of skeletal muscle biopsies. Secondly, using the same pharmacological Cell Cycle

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of skeletal muscle were incubated in 1% Triton X‑100 diluted in PBS for 30 minutes at room temperature. Without washing, sections were blocked with 10% normal goat serum in PBS for 1 h at room temperature. In order to reduce the intrinsic background staining, sections were then incubated with the Fab Fragment of unconjugated anti‑mouse IgG in combination with a purified antibody directed against mouse FcgII/III for 90 minutes at room temperature. After washing, sections were incubated with primary antibodies (diluted in PBS containing 5% normal goat serum) overnight at 4°C. The day after, three washes with PBS (5 min each) were done prior to secondary antibody incubation (30 minutes at room temperature): either a lissamine rhodamine B sulfonyl chloride‑conjugated goat anti‑rabbit antibody (5 mg/ml) or goat anti‑mouse antibody (5 mg/ml). Finally, the sections were washed three times with PBS (10 min each). Slides were mounted with Slow‑Fade anti‑fade reagent (Molecular Probes, Inc., Eugene, OR) and observed under an Olympus IX 70 inverted microscope. Western blot analysis. Skeletal muscle tissues were harvested, minced with a scissors, homogenized in a Polytron tissue grinder for 30 s at a medium range speed, and solubilized in a buffer containing 10 mM Tris‑HCl (pH 8.0), 150 mM NaCl, 5mM EDTA, 1% Triton X‑100, and 60 mM octyl glucoside for 45 min at 4°C. Samples were centrifuged at 13,000x g for 10 min at 4°C to remove insoluble debris. Protein concentration was assessed using a BCA kit (Pierce). Soluble proteins were resolved by SDS‑PAGE (10% acrylamide) and transferred to nitrocellulose membranes. Blots were blocked for 1 hour in TBST (10 mM Tris‑HCl, pH 8.0, 150 mM NaCl, 0.2% Tween 20) containing 4% powdered skim milk and 1% BSA. Then, the membranes were incubated for 1 hour with a given primary antibody (or an overnight incubation with the anti‑dystrophin antibody), diluted in TBST/1%BSA. After three washes with TBST, the blots were incubated for 30 minutes with horseradish peroxidase (HRP)‑conjugated secondary antibodies, diluted in TBST/1%BSA. Antibody‑bound proteins were detected using an ECL detection kit (Pierce).

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approach, we successfully rescued the expression of dystrophin, b‑dystroglycan, and a‑sarcoglycan in skeletal muscle explants from DMD patients.12 We strongly believe that these findings might have important clinical implications for an effective treatment of DMD. Here, we investigated the effect of two drugs belonging to a new class of therapeutics that targets the ubiquitin‑proteasome pathway. The exciting aspect of this development is that these drugs, Velcade and MLN273, have already progressed to clinical trials, with applications in different areas than muscular dystrophy. Velcade (bortezomib; formerly PS‑341) is a dipeptide boronic acid proteasome inhibitor that exhibits a potent antitumor activity both in vitro and in vivo, in a variety of tumor types.13‑15 Importantly, Velcade is the first proteasomal inhibitor to be approved by the U.S. Food and Drug Administration (FDA) for the treatment of multiple myeloma (MM).16 MLN273 (PS‑273) is another emerging boronic acid proteasome inhibitor14 with potential application outside of cancer. This drug is currently in the late stages of preclinical testing. Here, we investigate the effectiveness of these new drugs after their local injection into the gastrocnemius muscles of mdx mice. We show that localized administration of Velcade and MLN273 can inhibit dystrophin and DGC degradation in dystrophin‑deficient skeletal muscle fibers. Our findings emphasize that pharmacological treatment can represent an important therapeutic intervention for Duchenne Muscular Dystrophy.

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Experimental Procedures

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Materials. Monoclonal antibodies directed against b‑dystroglycan (NCL‑b‑DG), a‑sarcoglycan (NCL‑a‑sarco) and dystrophin (NCL‑DYS3) were purchased from Novocastra (Newcastle, United Kingdom). A monoclonal antibody against a‑dystroglycan was from Upstate Biotechnology. Anti‑caveolin‑3 IgG (mAb 268) was the generous gift of Dr. Roberto Campos‑Gonzales (BD Pharmingen Laboratories). The antibody directed against mouse FcgII/III (mouse BD FcBlock, CD16/CD32) was from BD Bioscience. An affinitypurified Fab fragment goat anti‑mouse IgG (H+L) was purchased from Jackson Immunoresearch Laboratories. The phospho‑NFkB p65 antibody was from Cell Signaling and the total NFkB p65 antibody from Delta Bioloabs. Velcade (bortezomib or PS‑341) was purchased commercially from Janssen‑Cilag Italia SpA (Cologno Monzese (Milan), Italy). MLN‑273 (or PS‑273) was from Millennium Pharmaceuticals, Inc., Cambridge, MA. Both Velcade and MLN‑273 were dissolved in DMSO as a stock solution of 10 mM and diluted to the various concentrations with PBS. Laboratory animals. Eight‑month‑old male mdx mice (C57BL/ 10ScSn Dmd mdx), purchased from The Jackson Laboratory (JAX mice), were used throughout this study. Skeletal muscle tissues were quickly dissected, flash‑frozen in 2‑methyl butane (isopentane) cooled in liquid nitrogen, and stored at ‑80°C until use. Administration of the proteasomal inhibitors Velcade and PS‑273. Localized administration was performed by injection of the drugs into the gastrocnemius muscles of mdx mice. To visualize the injected area, proteasomal inhibitors were premixed with 1% India Ink in phosphate‑buffered saline (PBS) in a total volume of 100 ml. Mice were sacrificed 24 hours after injection, and skeletal muscles were quickly isolated for further analysis. Immunofluorescence analysis. Tissue samples were isolated from the gastrocnemius muscle, rapidly frozen in liquid nitrogen‑cooled isopentane, and stored at ‑80°C. Unfixed frozen sections (6 mm thick) www.landesbioscience.com

Results Localized treatment with the proteasomal inhibitor Velcade. Our previous studies have shown that in vivo administration of the proteasomal inhibitor MG‑132 effectively restored the expression levels and the localization pattern of dystrophin and of dystrophin‑associated proteins in mdx skeletal muscles.11 Most excitingly, we recently obtained similar results by treating skeletal muscle biopsies from Duchenne and Becker muscular dystrophy patients with MG-132.17 These promising results led us to explore the effects of two novel proteasome inhibitors, Velcade and MLN273, in mdx mice. Importantly, Velcade is the only proteasomal inhibitor that is FDA‑approved for the treatment of leukemia, whereas MLN273 is currently in preclinical trials. We first performed localized treatment, by injecting Velcade into the gastrocnemius of mdx mice. Velcade is a modified boronic acid, and reversible inhibitor of the chymotrypsin‑like activity of the proteasome in mammalian cells.18‑20 To test Velcade efficacy, we administrated two different final concentrations of Velcade (5 and 10 mM). To visualize the injected area, Velcade was premixed with a blue dye (1% India Ink in PBS). After 24 hours, we collected skeletal muscle tissues from treated and untreated hindlimbs, these last injected with PBS only, as an important negative control. Frozen skeletal muscle sections were prepared and examined by immunofluorescence analysis

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Figure 1. Localized treatment of mdx mice with Velcade: Immunohistochemistry. Velcade (final concentrations of 5 and 10 mM) was locally injected onto the right hindlimbs of mdx mice. After 24 hours, mice were sacrificed, and the gastrocnemius muscle was isolated. The left hindlimb of each mouse was injected with PBS only, as an important internal negative control. Frozen sections were prepared from untreated (PBS only) and Velcade‑treated gastrocnemius muscles and were immunostained with antibodies directed against a‑dystroglycan (A), b‑dystroglycan (B), a-sarcoglycan (C), and dystrophin (D). Note that a‑dystroglycan, b‑dystroglycan, a-sarcoglycan, and dystrophin were absent or greatly reduced at the sarcolemma of skeletal muscle fibers from untreated mdx mice (upper panels). On the contrary, a‑dystroglycan, b‑dystroglycan, a-sarcoglycan, and dystrophin appear highly expressed at the plasma membranes of the myofibers after Velcade treatment (lower panels).

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with antibodies directed against a‑dystroglycan (Fig. 1A), b‑dystroglycan (Fig. 1B), a‑sarcoglycan (Fig. 1C), and dystrophin (Fig. 1D). As expected, we observed a lack or great reduction of expression of all those proteins in skeletal muscle fibers from untreated mdx mice (Fig. 1A–D, upper panels). Remarkably, Velcade treatment rescued the expression level and subcellular localization of a‑dystroglycan, b‑dystroglycan, a‑sarcoglycan, and dystrophin. The lower panels of Figure 1A–D show that these proteins are restored at the plasma membrane after treatment with two different concentration of the proteasome inhibitor Velcade (5 and 10 mM). We next verified the reliability of these results by Western blot analysis. For this purpose, we prepared lysates from untreated (PBS only) and Velcade‑treated (5 and 10 mM) gastrocnemius muscles. Figure 2 shows that the expression levels of a‑sarcoglycan, a‑dystroglycan, b‑dystroglycan, and dystrophin are greatly increased in Velcade‑treated skeletal muscles, as compared to untreated (PBS only) controls. Importantly, we observed restoration of expression by treating mdx mice either with 5 mM or 10 mM Velcade, with a more robust effect achieved with the higher concentration of Velcade. Interestingly, a‑dystroglycan migrates as a band of approximately 66 kDa, instead of the expected 97 kDa of the heavily‑glycosylated mature form. We believe that this 66 kDa protein is the precursor form of a‑dystroglycan. In addition, note that, by using a monoclonal antibody against the amino‑terminal domain of dystrophin (NCL‑DYS3), we detect a band of about 115 kDa, instead of the expected full length of 426 kDa. This is not surprising, as the mdx mouse produces a truncated dystrophin protein as a consequence of a premature stop codon in the dystrophin gene.

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Figure 2. Localized treatment of mdx mice with Velcade: Western blot analysis. The right hindlimbs of mdx mice were locally injected mice with Velcade (at final concentrations of 5 and 10 mM). After 24 hours, mice were sacrificed, and gastrocnemius muscles were isolated. The left hindlimb of each mouse was injected with PBS only, and served as an important internal negative control. Protein lysates were prepared from untreated and Velcade‑treated skeletal muscles, and separated by SDS‑PAGE. Blots were incubated with specific antibodies directed against a‑dystroglycan, b‑dystroglycan, a-sarcoglycan, and dystrophin. Interestingly, the expression levels of a‑dystroglycan, b‑dystroglycan, a-sarcoglycan, and dystrophin are increased after treatment with both concentrations of Velcade, as compared to the untreated (PBS only) mice. Note that Velcade treatment rescues the precursor form of a‑dystroglycan.

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Figure 3. Localized treatment of mdx mice with MLN273: Immunohistochemistry. The right hindlimbs of mdx mice were locally injected with MLN273 (at final concentrations of 20, 100 and 160 mM). After 24 hours, mice were sacrificed, and the skeletal muscles were isolated. The left hindlimb of each mouse (injected with PBS only) served as internal control. Frozen sections were prepared from untreated and MLN273‑treated gastrocnemius muscles and were immunostained with antibodies directed against a‑dystroglycan (A), b‑dystroglycan (B), a-sarcoglycan (C), and dystrophin (D). As expected, a‑dystroglycan, b‑dystroglycan, a-sarcoglycan, and dystrophin were absent or greatly reduced in untreated mdx mice. Note that the expression levels of a‑dystroglycan, b‑dystroglycan, a-sarcoglycan, and dystrophin are rescued after treatment with the proteasomal inhibitor.

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Localized treatment with the proteasomal inhibitor MLN273. We next used a similar approach to test another boronic acid proteasomal inhibitor, namely MLN273. Thus, we locally injected MLN273 into the gastrocnemius muscles of mdx mice for a 24‑hour treatment. We administrated different final concentrations of the inhibitor (20 mM, 100 mM and 160 mM) premixed with 1% India ink blue dye. After 24 hours, we collected skeletal muscles from treated and untreated (PBS only) mdx mice. We prepared frozen skeletal muscle sections and examined them by immunofluorescence analysis with antibodies directed against a‑dystroglycan, b‑dystroglycan, a‑sarcoglycan, and dystrophin (Fig. 3). Remarkably, the expression levels and the plasma membrane localization of a‑dystroglycan (Fig. 3A), b‑dystroglycan (Fig. 3B), a‑sarcoglycan (Fig. 3C), and dystrophin (Fig. 3D) are rescued in skeletal muscle fibers after MLN273 treatment, versus untreated (PBS only) controls (Fig. 3A–D, upper panels). Interestingly, the efficacy of treatment is dose‑dependent for a‑sarcoglycan and dystrophin expression levels. We observed some degree of expression restoration at the lower concentration (20 mM), with increasing effects at 100 mM and even stronger signal in myofibers from MLN273‑treated mice at the concentration of 160 mM. www.landesbioscience.com

Next, to validate these results, we evaluated the expression of a‑dystroglycan, b‑dystroglycan, a‑sarcoglycan, and dystrophin by Western blot analysis (Fig. 4). Consistent with the pattern of the immunofluorescence analysis, the levels of a‑dystroglycan, b‑dystroglycan, a‑sarcoglycan were increased after treatment with 20 mM, 100 mM, and 160 mM MLN273. As expected, these same proteins were absent or greatly reduced in untreated (PBS only) mdx muscles. Immunoblotting with dystrophin antibody indicated that only treatment with 100 mM and 160 mM MLN273 effectively rescued the expression levels. However, dystrophin was found absent in untreated muscle samples (as expected) and in muscle lysates treated with 20 mM MLN273. It has been previously demonstrated that maintenance of proper Cav‑3 expression levels is critical for skeletal muscle functioning. For example, Cav‑3 is increased in skeletal muscle fibers from mdx mice and DMD patients,9,10 whereas transgenic over‑expression of Cav‑3 in skeletal muscles induces a Duchenne‑like phenotype.21 As such, we assessed whether treatment with MLN273 would normalize Cav‑3 expression levels. Figure 4 shows that treatment with all three concentrations (20 mM, 100 mM and 160 mM) of MLN273 induces

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Figure 4. Localized treatment of mdx mice with MLN273: Western blot analy‑ sis. The right hindlimbs of mdx mice were locally injected mice with MLN273 (final concentrations of 20, 100 and 160 mM). After 24 hours, the mice were sacrificed, and the gastrocnemius muscles were isolated. The left hindlimb of each mouse (injected with PBS only) served as internal control. Protein lysates were prepared from untreated and MLN273‑treated skeletal muscles and subjected to SDS‑PAGE. Blots were incubated with specific antibodies directed against a‑dystroglycan, b‑dystroglycan, a-sarcoglycan, and dystro‑ phin. Interestingly, the expression levels of a‑dystroglycan, b‑dystroglycan, a-sarcoglycan, and dystrophin are increased after MLN273 treatment as compared to the untreated (PBS only) mice. Except for dystrophin, whose expression is rescued only with 100 and 160 mM MLN273, a‑dystroglycan, b‑dystroglycan, a-sarcoglycan expressions were already augmented after treatment with the lower concentration of MLN273 (20 mM). Note that this treatment rescues the precursor form of the a-dystroglycan. Interestingly, treat‑ ment of mdx mice with MLN273 reduces Cav‑3 expression levels.

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Figure 5. Velcade treatment significantly affects NFkB activity in mdx skel‑ etal muscles. Note that phospho‑NFkB levels are decreased in Velcade treated dystrophin‑deficient skeletal muscles versus untreated controls. Importantly, there is an increased level of the inactive (total) form of NFkB in Velcade‑treated mdx mice (5 and 10 mM), as compared with untreated mdx mice.

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Figure 6. MLN73 treatment significantly affects NFkB activity in mdx skeletal muscles. Note that phospho‑NFkB levels are decreased in MLN273 treated mdx mice (100 and 160 mM) versus untreated. No effects were observed with treatment of skeletal muscle with 20 mM MLN273. Importantly, there is an increased level of the inactive (total) form of NFkB in MLN273‑treated mdx mice (100 and 160 mM), as compared with untreated controls and with mice treated with 20 mM MLN273.

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a downregulation of Cav‑3 expression as compared to untreated (PBS only) controls. In contrast, Velcade treatment did not significantly alter Cav‑3 expression levels in skeletal muscles from mdx mice (data not shown). The possible explanation for the slight differences in expression levels between the results obtained by immunofluorescence (Fig. 3) and corresponding results obtained by Western blot analysis (Fig. 4) might be attributed to the different sensitivity of these two techniques. In conclusion, we show here that the local administration of the novel proteasomal inhibitors Velcade and MLN273, in the skeletal muscles from mdx mice, is effective to rescue the expression and plasma membrane localization of dystrophin and its associated proteins, i.e., a‑dystroglycan, b‑dystroglycan, a‑sarcoglycan. Localized treatment with Velcade and MLN273 reduce the activation of nuclear factor‑kappaB in skeletal muscle fibers. Many authors have reported that sustained activation of the nuclear factor‑kappaB (NFkB) pathway is involved in inflammatory myopathies and DMD. Thus, we next asked whether administration of the novel proteasomal inhibitors could reduce NFkB levels, thus indicating amelioration of the inflammation process. To address this issue, we subjected total skeletal muscle extracts to Western blot analysis with antibodies against the inactive (unphosphorylated) and the active (phosphorylated) form of NFkB. Figure 5 shows that the expression level of the inactive NFkB is increased after treatment 1246

with 5 mM and 10 mM Velcade, as compared with the untreated (PBS only) controls. On the contrary, Western blot with an antibody against phospho NFkB revealed a decrease of the activated form of NFkB in the Velcade‑treated mice compared with untreated controls. Similar results were obtained after localized treatment with MLN273. Indeed, (Fig. 6) demonstrates that the expression level of total NFkB is greatly increased in mice treated with 100 and 160 mM MLN273. Importantly, after treatment with 100 and 160 mM MLN273, the levels of phospho NFkB were decreased in treated skeletal muscle versus untreated controls. The present findings suggest that treatment with Velcade and MLN273 can efficiently block the activation of the transcription factor NFkB, which is importantly involved in the inflammatory reactions in the pathogenesis of Duchenne muscular disease.

Discussion DMD is an incurable disease affecting one of every 3,500 males born, causing progressive muscle degeneration and weakness.4,22 Thus, finding a proper treatment is essential for these patients. Although many treatment strategies have been explored,1,2 currently there is no effective treatment available. Our previous findings supported the fact that dystrophin and other proteins of the DGC are degraded in dystrophin‑deficient muscle by the

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for the binding site to b‑dystroglycan, and that Cav‑3 expression levels may regulate the recruitment of dystrophin to the plasma membrane.30 Thus, we tested whether MLN273 treatment would decrease abnormally elevated Cav‑3 levels in mdx skeletal muscles, which maybe important for improvement of pathological signs. Here, we show a clear decrease in Cav‑3 expression levels in skeletal muscle fibers from MLN273‑treated mdx mice at a higher concentration, as compared with the untreated (PBS only) and MLN273‑treated mdx mice with lower concentrations. Previous reports have shown that proteasomal inhibitors are able to block the activation of nuclear factor‑kB (NFkB). NFkB is a transcription factor, which is involved in the inflammatory and acute stress responses. In addition, NFkB pathway is activated in Duchenne muscular dystrophy, and is involved in muscle degeneration and regeneration in dystrophin‑deficient fibers.31‑33 NFkB forms a dimer with the inhibitory protein IkB, which maintains it in the cytoplasm in an inactive form.34,35 Under a variety of signals, IkB becomes phosphorylated and is targeted to degradation by the proteasome. This allows releasing the inhibition on NFkB, thus leading to NFkB phosphorylation and nuclear translocation. By blocking proteasomal activity, Velcade prevents the activation of NFkB, thus reducing anti‑apoptotic factors and inflammatory molecules.36 With regard to that, we tested lysates of skeletal muscle fibers from mdx mice for the inactive (unphosphorylated) and the active (phosphorylated) form of NFkB, after treatment with Velcade and MLN273. Interestingly, localized treatment with Velcade (5 and 10 mM) and MLN273 (100 and 160 mM) caused an evident decrease in the expression level of the activated form of NFkB. Concomitantly, Velcade and MLN‑273 treatment increased the inactive form of NFkB. This result may be due to the proteasomal inhibitory effect of Velcade and MLN‑273 that, blocking the degradation of inhibitory protein IkB, increase the stability of the inactive form of NFkB. Consequently, the expression level of total inactive NFkB is greatly increased in mice treated with Velcade and MLN‑273, and the expression level of the activated phosphorylated form of NFkB, dependent on the degradation of IkB, is decreased. In conclusion, by localized treatment of mdx mice with two novel inhibitors of the proteasomal pathway (Velcade and MLN273), we blocked the degradation of dystrophin and of dystrophin associated proteins, and rescued their expression and subcellular localization. Moreover, our findings suggest that delivery of Velcade and MLN273 can efficiently block the activation of the transcription factor nuclear factor‑kB (NFkB), which may be important in the pathogenesis of Duchenne muscular dystrophy. Unfortunately, Velcade and MLN273 were not sufficiently available to perform a systemic treatment in mdx mice for an extended period, as we did in our previous study with the proteasome inhibitor MG‑132.11 Clearly, more investigations are necessary, but in any case, our exciting developments emphasize the important potential of these new drugs as therapeutics for DMD. The effective treatment of Duchenne muscular dystrophy might be closer considering that the side effects of Velcade had been already explored and managed25,26 and clinical investigations about MLN273 are in progress.

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proteasomal‑dependent pathway.11,17 Indeed, treatment with the proteasome inhibitor MG‑132 rescued the expression and cellular localization of dystrophin and associated proteins, in mdx mice and skeletal muscle explants from DMD patients. In addition, MG‑132 systemic administration to mdx mice significantly improved muscle integrity. These promising results led us to continue exploring the use of proteasome inhibitors as a therapy for DMD. Therefore, we focused onto two novel drugs (Velcade and MLN273) blocking the proteasomal‑dependent degradation pathway, drugs that have already progressed to preclinical and clinical trials in different fields than muscular dystrophy. Velcade is a potent and a cell permeable dipeptide boronate, and acts as an inhibitor of the proteasome‑ubiquitin pathway. Velcade is employed as anti‑neoplastic agent to arrest tumor growth, tumor spread and angiogenesis in preclinical models of breast, lung, pancreatic, and ovarian tumor types.23‑25 Importantly, on May 2003, the U.S. Food and Drug Administration (FDA) approved Velcade (Millennium Pharmaceuticals, Inc.; Cambridge, MA) for the treatment of refractory or relapsed multiple myeloma (MM).16,26 In the current therapy for MM, Velcade is administered using the i.v. route, but this drug has been shown to be active also orally.13,27 MLN273 is another compound that inhibits the proteasomal activity, and similarly to Velcade, it is a selective, potent, and reversible boronic acid analogue. Millennium Pharmaceuticals is currently testing MLN273 in preclinical studies, in a different area than cancer. The goal of our current study was to further investigate the use of a pharmacological therapy as potential treatment for DMD. To address this issue, we test the effectiveness of Velcade and MLN273 to block protein degradation after local injection into the skeletal muscles of mdx mice. As expected, a lack of expression or a severe reduction of dystrophin and its associated proteins is observed in skeletal muscle fibers from untreated mdx mice. After 24 hours of treatment with Velcade, the expression level and subcellular localization of a‑dystroglycan, b‑dystroglycan, a‑sarcoglycan and dystrophin are restored, as judged by immunofluorescence and western blot analysis. Importantly, these proteins are restored at the plasma membrane and their expression levels are greatly increased in Velcade‑treated skeletal muscle, as compared to untreated controls. We tested two different concentrations of Velcade with similar results. Consistent with our results, recent studies have shown the ability of Velcade to attenuate muscle atrophy after its in vivo administration in gastrocnemius28 and in soleus muscle29 in rats. These findings support the idea that Velcade is effective in the inhibition of muscle wasting due to the accelerated proteasomal‑dependent degradation in skeletal muscle fibers. Regarding MLN273 treatment, we also detected rescue of expression of dystrophin and associated proteins by immunostaining and Western blot analysis. The results show restoration of membrane localization and expression of a‑dystroglycan, b‑dystroglycan, a‑sarcoglycan, and dystrophin after MLN273 localized treatment versus untreated mdx mice. Interestingly, the effects are dose dependent, and the efficacy increases with increasing concentrations of MLN273. It has been previously reported that Cav‑3 expression levels are increased in skeletal muscle fibers from mdx mice and from DMD patients,9,10 whereas transgenic over‑expression of Cav‑3 causes a down‑regulation of dystrophin and of dystrophin‑associated proteins, with origination of a Duchenne‑like phenotype.21 In addition, Cav‑3 was shown to bind b‑dystroglycan at a similar or overlapping site as dystrophin, thus indicating that Cav‑3 and dystrophin may compete www.landesbioscience.com

References 1. Kapsa R, Kornberg AJ, Byrne E. Novel therapies for Duchenne muscular dystrophy. Lancet Neurol 2003; 2:299‑310. 2. Nowak KJ, Davies KE. Duchenne muscular dystrophy and dystrophin: Pathogenesis and opportunities for treatment. EMBO Rep 2004; 5:872‑6. 3. Muntoni F, Torelli S, Ferlini A. Dystrophin and mutations: One gene, several proteins, multiple phenotypes. Lancet Neurol 2003; 2:731‑40. 4. Hoffman EP, Brown RH, Kunkel LM. Dystrophin: The protein product of the Duchenne muscular dystrophy locus. Cell 1987; 51:919‑28.

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32. Carlson CG, Samadi A, Siegel A. Chronic treatment with agents that stabilize cytosolic IkappaB‑alpha enhances survival and improves resting membrane potential in MDX muscle fibers subjected to chronic passive stretch. Neurobiol Dis 2005; 20:719‑30. 33. Messina S, Bitto A, Aguennouz M, Minutoli L, Monici MC, Altavilla D, Squadrito F, Vita G. Nuclear factor kappa‑B blockade reduces skeletal muscle degeneration and enhances muscle function in Mdx mice. Exp Neurol 2006; 198:234‑41. 34. Magnani M, Crinelli R, Bianchi M, Antonelli A. The ubiquitin‑dependent proteolytic system and other potential targets for the modulation of nuclear factor‑kB (NF‑kB). Curr Drug Targets 2000; 1:387‑99. 35. Moynagh PN. The NF‑kappaB pathway. J Cell Sci 2005; 118:4589‑92. 36. Paramore A, Frantz S. Bortezomib. Nat Rev Drug Discov 2003; 2:611‑2.

©

20

07

LA

ND

ES

BIO

SC

IEN

CE

.D

ON

5. Kumar A, Khandelwal N, Malya R, Reid MB, Boriek AM. Loss of dystrophin causes aberrant mechanotransduction in skeletal muscle fibers. Faseb J 2004; 18:102‑13. 6. Ervasti JM, Campbell KP. Membrane organization of the dystrophin‑glycoprotein complex. Cell 1991; 66:1121‑31. 7. Campbell KP. Three muscular dystrophies: Loss of cytoskeleton‑extracellular matrix linkage. Cell 1995; 80:675‑9. 8. Song KS, Scherer PE, Tang ZL, Okamoto T, Li S, Chafel M, Chu C, Kohtz DS, Lisanti MP. Expression of caveolin‑3 in skeletal, cardiac, and smooth muscle cells. Caveolin‑3 is a component of the sarcolemma and cofractionates with dystrophin and dystrophin‑associated glycoproteins. J Biol Chem 1996; 271:15160‑5. 9. Vaghy PL, Fang J, Wu W, Vaghy LP. Increased caveolin‑3 levels in mdx mouse muscles. FEBS Lett 1998; 431:125‑7. 10. Repetto S, Bado M, Broda P, Lucania G, Masetti E, Sotgia F, Carbone I, Pavan A, Bonilla E, Cordone G, Lisanti MP, Minetti C. Increased number of caveolae and caveolin‑3 overexpression in duchenne muscular dystrophy. Biochem Biophys Res Commun 1999; 547‑50. 11. Bonuccelli G, Sotgia F, Schubert W, Park DS, Frank PG, Woodman SE, Insabato L, Cammer M, Minetti C, Lisanti MP. Proteasome inhibitor (MG‑132) treatment of mdx mice rescues the expression and membrane localization of dystrophin and dystrophin‑associated proteins. Am J Pathol 2003; 163:1663‑75. 12. Assereto S, Stringara S, Sotgia F, Bonuccelli G, Broccolini A, Pedemonte M, Traverso M, Biancheri R, Zara F, Bruno C, Lisanti MP, Minetti C. Pharmacological rescue of the dystrophin‑glycoprotein complex in Duchenne and Becker skeletal muscle explants by proteasome inhibitor treatment. Am J Physiol Cell Physiol 2006; 290:C577‑82. 13. Teicher BA, Ara G, Herbst R, Palombella VJ, Adams J. The proteasome inhibitor PS‑341 in cancer therapy. Clin Cancer Res 1999; 5:2638‑45. 14. Adams J, Palombella VJ, Sausville EA, Johnson J, Destree A, Lazarus DD, Maas J, Pien CS, Prakash S, Elliott PJ. Proteasome inhibitors: A novel class of potent and effective antitumor agents. Cancer Res 1999; 59:2615‑22. 15. Adams J. Development of the proteasome inhibitor PS‑341. Oncologist 2002; 7:9‑16. 16. Kane RC, Bross PF, Farrell AT, Pazdur R. Velcade: U.S. FDA approval for the treatment of multiple myeloma progressing on prior therapy. Oncologist 2003; 8:508‑13. 17. Assereto S, Stringara S, Sotgia F, Bonuccelli G, Broccolini A, Pedemonte M, Traverso M, Biancheri R, Zara F, Bruno C, Lisanti MP, Minetti C. Pharmacological rescue of the dystrophin complex in duchenne and becker skeletal muscle explants by proteasomal inhibitor treatment. Am J Physiol Cell Physiol 2005. 18. Groll M, Ditzel L, Lowe J, Stock D, Bochtler M, Bartunik HD, Huber R. Structure of 20S proteasome from yeast at 2.4 A resolution. Nature 1997; 386:463‑71. 19. Heinemeyer W, Fischer M, Krimmer T, Stachon U, Wolf DH. The active sites of the eukaryotic 20 S proteasome and their involvement in subunit precursor processing. J Biol Chem 1997; 272:25200‑9. 20. Arendt CS, Hochstrasser M. Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active‑site formation. Proc Natl Acad Sci USA 1997; 94:7156‑61. 21. Galbiati F, Volonte D, Chu JB, Li M, Fine SW, Fu M, Bermudez J, Pedemonte M, Weidenheim KM, Pestell RG, Minetti C, Lisanti MP. Transgenic overexpression of caveolin‑3 in skeletal muscle fibers induces a Duchenne‑like muscular dystrophy phenotype. Proc Natl Acad Sci USA 2000; 97:9689‑94. 22. Koenig M, Hoffman EP, Bertelson CJ, Monaco AP, Feener C, Kunkel LM. Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 1987; 50:509‑17. 23. Ling YH, Liebes L, Jiang JD, Holland JF, Elliott PJ, Adams J, Muggia FM, Perez‑Soler R. Mechanisms of proteasome inhibitor PS‑341‑induced G2‑M‑phase arrest and apoptosis in human nonsmall cell lung cancer cell lines. Clin Cancer Res 2003; 9:1145‑54. 24. Lenz HJ. Clinical update: Proteasome inhibitors in solid tumors. Cancer Treat Rev 2003; 29(Suppl 1):41‑8. 25. Mack PC, Davies AM, Lara PN, Gumerlock PH, Gandara DR. Integration of the proteasome inhibitor PS‑341 (Velcade) into the therapeutic approach to lung cancer. Lung Cancer 2003; 41(Suppl 1):S89‑96. 26. San Miguel J, Blade J, Boccadoro M, Cavenagh J, Glasmacher A, Jagannath S, Lonial S, Orlowski RZ, Sonneveld P, Ludwig H. A practical update on the use of bortezomib in the management of multiple myeloma. Oncologist 2006; 11:51‑61. 27. Palombella VJ, Conner EM, Fuseler JW, Destree A, Davis JM, Laroux FS, Wolf RE, Huang J, Brand S, Elliott PJ, Lazarus D, McCormack T, Parent L, Stein R, Adams J, Grisham MB. Role of the proteasome and NF‑kappaB in streptococcal cell wall‑induced polyarthritis. Proc Natl Acad Sci USA 1998; 95:15671‑6. 28. Krawiec BJ, Frost RA, Vary TC, Jefferson LS, Lang CH. Hindlimb casting decreases muscle mass in part by proteasome‑dependent proteolysis but independent of protein synthesis. Am J Physiol Endocrinol Metab 2005; 289:E969‑80. 29. Beehler BC, Sleph PG, Benmassaoud L, Grover GJ. Reduction of skeletal muscle atrophy by a proteasome inhibitor in a rat model of denervation. Exp Biol Med (Maywood) 2006; 231:335‑41. 30. Sotgia F, Lee JK, Das K, Bedford M, Petrucci TC, Macioce P, Sargiacomo M, Bricarelli FD, Minetti C, Sudol M, Lisanti MP. Caveolin‑3 directly interacts with the C‑terminal tail of beta‑dystroglycan: Identification of a central WW‑like domain within caveolin family members. J Biol Chem 2000; 275:38048‑58. 31. Monici MC, Aguennouz M, Mazzeo A, Messina C, Vita G. Activation of nuclear factor‑kappaB in inflammatory myopathies and Duchenne muscular dystrophy. Neurology 2003; 60:993‑7.

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