Comparison of the antifibrotic effects of the pan-histone deacetylase-inhibitor panobinostat versus the IPF-drug pirfenidone in fibroblasts from patients with idiopathic pulmonary fibrosis

Abstract

BackgroundIdiopathic pulmonary fibrosis (IPF) is a devastating lung disease with a poor prognosis. Pirfenidone is the first antifibrotic agent to be approved for IPF-treatment as it is able to slow down disease progression. However, there is no curative treatment other than lung transplantation. Because epigenetic alterations are associated with IPF, histone deacetylase (HDAC)-inhibitors have recently been proven to attenuate fibrotic remodeling in vitro and in vivo. This study compared the effects of pirfenidone with the pan-HDAC-inhibitor panobinostat/LBH589, a FDA-approved drug for the treatment of multiple myeloma, head-to-head on survival, fibrotic activity and proliferation of primary IPF-fibroblasts in vitro.MethodsPrimary fibroblasts from six IPF-patients were incubated for 24h with vehicle (0.25% DMSO), panobinostat (LBH589, 85 nM) or pirfenidone (2.7 mM), followed by assessment of proliferation and expression analyses for profibrotic and anti-apoptosis genes, as well as for ER stress and apoptosis-markers. In addition, the expression status of all HDAC enzymes was examined.ResultsTreatment of IPF-fibroblasts with panobinostat or pirfenidone resulted in a downregulated expression of various extracellular matrix (ECM)-associated genes, as compared to vehicle-treated cells. In agreement, both drugs decreased protein level of phosphorylated (p)-STAT3, a transcription factor mediating profibrotic responses, in treated IPF-fibroblasts. Further, an increase in histone acetylation was observed in response to both treatments, but was much more pronounced and excessive in panobinostat-treated IPF-fibroblasts. Panobinostat, but not pirfenidone, led to a significant suppression of proliferation in IPF-fibroblasts, as indicated by WST1- and BrdU assay and markedly diminished levels of cyclin-D1 and p-histone H3. Furthermore, panobinostat-treatment enhanced α-tubulin-acetylation, decreased the expression of survival-related genes Bcl-XL and BIRC5/survivin, and was associated with induction of ER stress and apoptosis in IPF-fibroblasts. In contrast, pirfenidone-treatment maintained Bcl-XL expression, and was neither associated with ER stress-induction nor any apoptotic signaling. Pirfenidone also led to increased expression of HDAC6 and sirtuin-2, and enhanced α-tubulin-deacetylation. But in line with its ability to increase histone acetylation, pirfenidone reduced the expression of HDAC enzymes HDAC1, -2 and -9.ConclusionsWe conclude that, beside other antifibrotic mechanisms, pirfenidone reduces profibrotic signaling also through STAT3 inactivation and weak epigenetic alterations in IPF-fibroblasts, and permits survival of (altered) fibroblasts. The pan-HDAC-inhibitor panobinostat reduces profibrotic phenotypes while inducing cell cycle arrest and apoptosis in IPF-fibroblasts, thus indicating more efficiency than pirfenidone in inactivating IPF-fibroblasts. We therefore believe that HDAC-inhibitors such as panobinostat can present a novel therapeutic strategy for IPF.

The Multi-Modal Effect of the Anti-fibrotic Drug Pirfenidone on NSCLC

“…Furthermore, core mediators of the signaling pathway showed effects on their expression at the mRNA and the protein level, where Pirfenidone induced a differential gene expression response in three well characterized gene sets comprising SMAD3 downstream genes (19) or TGFβ pathway members. A similar effect on cell proliferation was recently observed in mesothelioma cell lines (33), hepatocellular carcinoma cells (34), and pancreatic cancer cells (16), although there was no effect on SMAD expression or phosphorylation but rather on p-ERK 1/2 (33) in mesothelioma cells or p-Stat3 in fibroblasts of IPF patients (35). Pirfenidone affected also cellular mobility as indicated by reduced wound healing ability and migration that was also recently observed independently and linked to the uPA/PAI-1 system (17) and resulted in our experiments in a reduced vital tumor area in vivo.…”

Pirfenidone regulates LPS mediated activation of neutrophils

“…Apart from antimicrobial degranulation, neutrophils fight infection by phagocytosis and NETosis which utilize cellular cytoskeleton machinery 41 . As kinases and MMPs (particularly MMP8 53 ) are involved in triggering phagocytosis by re-organizing cellular cytoskeleton proteins, we observed that Pf treatment, which targets kinases 18,54 , resulted in a reduced phagocytic uptake of Escherichia coli bioparticle in LPS-stimulated neutrophils 55,56 . Furthermore, for host defense, neutrophils employ a unique suicidal mechanism, also driven by kinases, to trap microbes (NETosis) by releasing intracellular histones and DNA to form NETs 57 .…”

Dual inhibition of HDAC and tyrosine kinase signaling pathways with CUDC-907 attenuates TGFβ1 induced lung and tumor fibrosis

“…The activities of all Class I and II HDACs were reported to be significantly upregulated in IPF lung tissues 33 . Many studies have used HDAC inhibitors to block lung fibrotic process and tumor growth, and SAHA, a nonselective HDAC Class I and II inhibitor, has been reported to have the ability to inhibit the differentiation of TGFβ1-induced myofibroblasts 34 – 36 . Similarly, Trichostatin A, a recently proved HDAC inhibitor, works at a micromolar level and sufficiently inhibits fibroblasts in vitro 17 .…”