P- 43 PIRFENIDONE PREVENTS NEOPLASTIC LESIONS DEVELOPMENT BY OXIDATIVE, FIBROGENIC, ANTIPROLIFERATIVE AND EPIGENETIC MECHANISMS REGULATION IN A MODEL OF CHEMICAL HEPATOCARCINOGENESIS

Introduction and Objectives

Hepatocellular carcinoma (HCC) is the most frequent hepatic neoplasia, where oxidative, fibrogenic, proliferative, and epigenetic processes are altered. Pirfenidone (PFD) has been shown to have important hepatoprotective properties. However, its efficacy in HCC development is unknown. This study aimed to 1) To determine whether PFD has antioxidative, antifibrogenic and antiproliferative effects. 2) To determine PFD effects on epigenetic regulation mechanisms.

Materials and Methods

Male Fischer-344 rats were divided into three groups. Group 1. Control, NT; Group 2. Damage, HCC, generated by diethylnitrosamine weekly administration; (50mg/kg, i.p.) and 2-acetylaminofluorene (25mg/kg, p.o.) for 12 weeks; and Group 3. HCC/PFD: with the same treatment as Group 2, plus PFD (300 mg/kg, p.o./day). Liver enzyme activity was quantified in serum; lipoperoxidation and GSH levels were evaluated in liver tissue samples; histopathological analyzes were performed. In addition, fibrogenic, antioxidant, anti-proliferative and epigenetic regulation markers were determined by Western blot. Finally, global DNA methylation was determined by Dot-blot and ELISA. The data obtained were analyzed using one-way ANOVA and a Tukey post hoc test.

Results

We demonstrate that PFD treatment reduces the number and size of neoplastic lesions, prevents damage to hepatic architecture and collagen deposition, and decreases the presence of the histopathological marker Glypican-3. On the other hand, it positively regulates antioxidant markers such as GSH, MDA, Nrf2, GSTP1 and Catalase. It was also effective to decrease c-Myc expression and β-catenin redistribution from the nucleus to the cytoplasm. Finally, PFD stimulated the nuclear transfer of several isoforms of PPARs, SIRT1 and DNMT1, increasing epigenetic mechanisms of global DNA methylation (figure 1).

Conclusions

PFD prevents neoplastic lesions development by modulating antifibrogenic, antioxidant, and antiproliferative processes and modulating epigenetic marks to reverse global DNA hypomethylation.

Figure 1. Analysis of global DNA methylation. A) Representative dot blot using anti-5mC which recognizes global methylated DNA, anti-IgG as negative control and methylene blue staining as total DNA loading control. B) Graphs shows mean ± standard deviation of 5mC densitometry brand intensity of study groups. C) Graph that represents the percentage of global methylation of the DNA analyzed with ELISA.A one-way ANOVA statistical test and a Tukey post hoc test were performed. Group NT: only received vehicle; Group HCC: damage group induced by weekly administration of DEN and 2-AAF for 12 weeks; and Group HCC/PFD: which received the same treatment as Group HCC, plus PFD (300 mg/kg) (**p<0.005)