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

Background Idiopathic 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. Methods Primary 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. Results Treatment 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. Conclusions We 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.

Analysis of acetylation status of histone H3-K27 and α-tubulin in vehicle-, LBH589and pirfenidone-treated IPF-fibroblasts Due to highly increased acetylation of this core histone in LBH589-treated IPFfibroblasts in comparison to vehicle and pirfenidone, we had to develop the respective western blot in a very short exposure time (20 sec.) to show an adequate staining for H3K27Ac in LBH589-treatments, despite use of the anti-H3K27Ac antibody (Abcam, ab4729) in a dilution of 1:15000, and thus detection of H3K27Ac in the other treatments could not be visualized within a 20 sec. exposure time (Fig 1A, main manuscript). We therefore repeated the immunoblot by using only fibroblastlysates of vehicle-and pirfendone-treatment, and omitting the LBH589-lysates.
Because we expected only low amounts of H3K27Ac in both conditions, we used the respective antibody in a dilution of 1:2000 and developed the blot after a longer exposure of the luminescence signals (2 min). As shown in Fig 1B (main manuscript), pirfenidone led also to a significant increase of H3K27-acetylation in IPF-fibroblasts, when compared to vehicle.
In addition, LBH589 also resulted in a very strong increase of tubulin-acetylation in IPF-fibroblasts, as compared to vehicle and pirfenidone, and which was (similar to H3K27Ac) already detected after a short exposure time (20 sec.) by using paradoxically a very low concentration of the anti-acetylated α-tubulin antibody (1:60000) (Fig 1C, main manuscript). Again, a clear evaluation of tubulin-acetylation status in vehicle-versus pirfenidone-treated IPF-fibroblasts could not be made, and an additional immunoblot of only vehicle-and pirfenidone-treated IPF-fibroblasts was performed. As shown in Fig 1D (main manuscript), the use of the anti-acetylated αtubulin antibody in the dilution 1:2000 and a longer exposure time of 2 min revealed Korfei et al_Supporting Information 5 basal levels of acetylated α-tubulin in vehicle-, but significantly diminished tubulinacetylation in pirfenidone-treated IPF-fibroblasts.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total cellular RNA was prepared from treated IPF-fibroblasts using the RNeasy  Plus Mini-Kit (Qiagen) and 600 µL RLT Plus-lysis buffer according to the protocol of the manufacturer. The purity and quantity of the isolated RNA was determined by spectrophotometry at 260/280 nm using NanoDrop 2000c photometer (PeqLab).
Reverse transcription (RT) and PCR were performed sequentially in two separate steps. Complementary DNA (cDNA, 2 µg) was first synthesized by reverse transcription (RT) using 2 µg total RNA, and with use of oligo-dT-primers and the Omniscript-RT-Kit (Qiagen). An aliquot of the finished RT reaction/cDNA (100 ng) was then used for PCR amplification employing gene-specific primers for transcripts.
The complete list of primers used is given in S1 Table. Each 40-μL RT reaction contained (final concentration): 2 μg total RNA, 1 μM oligo- extension). The thermal cycler used was from Bio-Rad (model: PTC-1148). In addition, the size of the amplified PCR product for each gene/cDNA, and the number of cycles for amplification of each cDNA, are given in S1 Table. As control experiment, PCR reactions of RNA samples without reverse transcriptase were performed (to exclude amplification of genomic DNA contaminations).
Equal aliquots of the PCR products were electrophoresed through a 2% (w/v) agarose gel containing ethidium bromide in 1× tris-acetate-EDTA (TAE) buffer, and documented by scanning using an UV imager (Gel-Doc XR + , Bio-Rad). Thereafter, band intensities of PCR products were quantified using Image Lab-Software (version 5.2.1, Bio-Rad), and mRNA expression of genes of interest were normalized to the expression of GAPDH.