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Validation of the 2nd Generation Proteasome Inhibitor Oprozomib for Local Therapy of Pulmonary Fibrosis.

ABSTRACT: Proteasome inhibition has been shown to prevent development of fibrosis in several organs including the lung. However, effects of proteasome inhibitors on lung fibrosis are controversial and cytotoxic side effects of the overall inhibition of proteasomal protein degradation cannot be excluded. Therefore, we hypothesized that local lung-specific application of a novel, selective proteasome inhibitor, oprozomib (OZ), provides antifibrotic effects without systemic toxicity in a mouse model of lung fibrosis. Oprozomib was first tested on the human alveolar epithelial cancer cell line A549 and in primary mouse alveolar epithelial type II cells regarding its cytotoxic effects on alveolar epithelial cells and compared to the FDA approved proteasome inhibitor bortezomib (BZ). OZ was less toxic than BZ and provided high selectivity for the chymotrypsin-like active site of the proteasome. In primary mouse lung fibroblasts, OZ showed significant anti-fibrotic effects, i.e. reduction of collagen I and ? smooth muscle actin expression, in the absence of cytotoxicity. When applied locally into the lungs of healthy mice via instillation, OZ was well tolerated and effectively reduced proteasome activity in the lungs. In bleomycin challenged mice, however, locally applied OZ resulted in accelerated weight loss and increased mortality of treated mice. Further, OZ failed to reduce fibrosis in these mice. While upon systemic application OZ was well tolerated in healthy mice, it rather augmented instead of attenuated fibrotic remodelling of the lung in bleomycin challenged mice. To conclude, low toxicity and antifibrotic effects of OZ in pulmonary fibroblasts could not be confirmed for pulmonary fibrosis of bleomycin-treated mice. In light of these data, the use of proteasome inhibitors as therapeutic agents for the treatment of fibrotic lung diseases should thus be considered with caution.

SUBMITTER: Semren N

PROVIDER: S-EPMC4560391 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Validation of the 2nd Generation Proteasome Inhibitor Oprozomib for Local Therapy of Pulmonary Fibrosis.

Semren Nora N Habel-Ungewitter Nunja C NC Fernandez Isis E IE Königshoff Melanie M Eickelberg Oliver O Stöger Tobias T Meiners Silke S

PloS one 20150904 9

Proteasome inhibition has been shown to prevent development of fibrosis in several organs including the lung. However, effects of proteasome inhibitors on lung fibrosis are controversial and cytotoxic side effects of the overall inhibition of proteasomal protein degradation cannot be excluded. Therefore, we hypothesized that local lung-specific application of a novel, selective proteasome inhibitor, oprozomib (OZ), provides antifibrotic effects without systemic toxicity in a mouse model of lung ...[more]

PMID: 26340365

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Project description:BackgroundMultiple myeloma (MM) remains an incurable malignancy, despite the advent of therapies such as proteosome inhibitors (PIs) that disrupt protein homeostasis and induce ER stress. We have pursued inhibition of geranylgeranyl diphosphate synthase (GGDPS) as a novel mechanism by which to target protein homeostasis in MM cells. GGDPS inhibitors (GGSI) disrupt Rab geranylgeranylation, which in turn results in perturbation of Rab-mediated protein trafficking, leading to accumulation of intracellular monoclonal protein, induction of ER stress and apoptosis. Our lead GGSI, RAM2061, has demonstrated favorable pharmacokinetic properties and in vivo efficacy. Here we sought to evaluate if combination therapy with GGSI and PI would result in enhanced disruption of the unfolded protein response (UPR) and increase anti-MM efficacy.MethodsMTT assays were conducted to evaluate the cytotoxic effects of combining RAM2061 with bortezomib in human MM cells. The effects of RAM2061 and/or PI (bortezomib or carfilzomib) on markers of UPR and apoptosis were evaluated by a combination of immunoblot (ATF4, IRE1, p-eIF2a, cleaved caspases and PARP), RT-PCR (ATF4, ATF6, CHOP, PERK, IRE1) and flow cytometry (Annexin-V). Induction of immunogenic cell death (ICD) was assessed by immunoblot (HMGB1 release) and flow cytometry (calreticulin translocation). Cell assays were performed using both concurrent and sequential incubation with PIs. To evaluate the in vivo activity of GGSI/PI, a flank xenograft using MM.1S cells was performed.ResultsIsobologram analysis of cytotoxicity data revealed that sequential treatment of bortezomib with RAM2061 has a synergistic effect in MM cells, while concurrent treatment was primarily additive or mildly antagonistic. The effect of PIs on augmenting RAM2061-induced upregulation of UPR and apoptotic markers was dependent on timing of the PI exposure. Combination treatment with RAM2061 and bortezomib enhanced activation of ICD pathway markers. Lastly, combination treatment slowed MM tumor growth and lengthened survival in a MM xenograft model without evidence of off-target toxicity.ConclusionWe demonstrate that GGSI/PI treatment can potentiate activation of the UPR and apoptotic pathway, as well as induce upregulation of markers associated with the ICD pathway. Collectively, these findings lay the groundwork for future clinical studies evaluating combination GGSI and PI therapy in patients with MM.

Dataset Information

Validation of the 2nd Generation Proteasome Inhibitor Oprozomib for Local Therapy of Pulmonary Fibrosis.

Publications

Validation of the 2nd Generation Proteasome Inhibitor Oprozomib for Local Therapy of Pulmonary Fibrosis.

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