Pirfenidone Reverts Global DNA Hypomethylation, Promoting DNMT1/UHRF/PCNA Coupling Complex in Experimental Hepatocarcinoma

Abstract

Hepatocellular carcinoma (HCC) development is associated with altered modifications in DNA methylation, changing transcriptional regulation. Emerging evidence indicates that DNA methyltransferase 1 (DNMT1) plays a key role in the carcinogenesis process. This study aimed to investigate how pirfenidone (PFD) modifies this pathway and the effect generated by the association between c-Myc expression and DNMT1 activation. Rats F344 were used for HCC development using 50 mg/kg of diethylnitrosamine (DEN) and 25 mg/kg of 2-Acetylaminofluorene (2-AAF). The HCC/PFD group received simultaneous doses of 300 mg/kg of PFD. All treatments lasted 12 weeks. On the other hand, HepG2 cells were used to evaluate the effects of PFD in restoring DNA methylation in the presence of the inhibitor 5-Aza. Histopathological, biochemical, immunohistochemical, and western blot analysis were carried out and our findings showed that PFD treatment reduced the amount and size of tumors along with decreased Glipican-3, β-catenin, and c-Myc expression in nuclear fractions. Also, this treatment improved lipid metabolism by modulating PPARγ and SREBP1 signaling. Interestingly, PFD augmented DNMT1 and DNMT3a protein expression, which restores global methylation, both in our in vivo and in vitro models. In conclusion, our results suggest that PFD could slow down HCC development by controlling DNA methylation.

Keywords: DNMT1; DNMT3a; c-Myc; hepatocellular carcinoma; β-catenin.

Figure 1

Liver damage caused by chemicals is prevented by PFD. (A) Established experimental design; NT, non-treated group; HCC, hepatocellular carcinoma group injected weekly with DEN (50 mg/kg/i.p.) plus 2AAF (25 mg/kg/p.o.); HCC/PFD group, HCC treatment plus 300 mg/kg PFD from week 0. All experimental groups were euthanized after 12 weeks. (B) Weekly logging of body weight. (C) Representative images of livers after 12 weeks of treatment. Greater size and number of dysplastic nodules were observed in HCC group than in the HCC/PFD group (white asterisks). (D) Graph of the liver weight at the end of treatment. (E) The ratio of liver weight to body weight of animals in each study group. (F) Serum gamma-glutamyl transferase (GTP) assay. (G) Serum alanine transaminase (ALT) assay. Data are presented as mean ± SD using ANOVA followed by Tukey’s multiple comparison test. ns, not significantly different, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

PFD prevented alteration of hepatic architecture, fibrosis, and neoplastic lesions. (A) Photomicrograph representative of H&E staining (H&E) of groups at 12 weeks. Deformed portal tracts and thickened hepatic plaques are evident in HCC (asterisks). (B) Quantification of atypical hepatocytes. Cells with many nuclei and changes in the nuclear–cytoplasmic ratio (asterisk), in addition to numerous steatosis sites (yellow arrows). (C) Masson’s trichrome staining (MT). (D) Quantification of the percentage of collagen fibers deposited in the liver tissues of the different groups. (E) Representative expression of GPC3. Hepatic GPC-3 was analyzed in tissue by immunohistochemistry using primary anti-rabbit GPC-3 antibody. (F) Percentage of areas positive for GPC-3. (G) Representative western blot of cytoplasmic β-catenin. (H) Quantification of β-catenin expression. (I) Western blotting representative of the nuclear fraction of β-catenin and c-Myc. (J) Quantification of β-catenin nuclear expression. (K) Quantification of c-Myc nuclear expression. Significantly different at * p < 0.05, ** p < 0.001, **** p < 0.00001. ns, not significantly different.

Figure 3

PFD modulates the expression and subcellular localization of PPAR isoforms and SREBP1. (A) Western blot representative of the cytoplasmic expression of different PPAR isoforms. (B) Densitometry determination of PPARα expression, (C) PPARγ and (D) PPARγ2. (E) Western blotting representative of the nuclear expression of different isoforms of PPARs. (F) Densitometry determination of the nuclear expression of PPARα (G) PPARγ and (H) PPARγ2. (I) Western blot representative of total and phosphorylated SREBP expression. (J) Graph of the determination of SREBP expression and (K) pSREBP-1c (ser372). The results are shown as the mean ± standard deviation (SD) of triplicate assays. One-way ANOVA and Tukey’s post-hoc tests were performed. Significantly different at * p < 0.05, ** p < 0.001, *** p < 0.0001 and **** p < 0.00001. ns, not significantly different.

Figure 4

Pirfenidone modulates the expression of enzymes that modify DNA and promote global methylation. (A) Representative western blots of DNMT1, DNMT1Ac, DNMT3a, DNMT3b, UHRF1, and PCNA. (B) Graphs showing the relative expression levels of DNMT1, (C) DNMT1Ac, (D) DNMT3a, (E) DNMT3b, (F) UHRF1, and (G) PCNA. (H) Representative images of the nuclear localization of DNMT1 and 5mC in liver tissues. Nuclei were stained with DAPI (blue), DNMT1 (green) and 5-Methylcytosine (5mC) (red). Images were captured using an epifluorescence microscope. Dysplastic nodule (DN). White asterisks indicate positivity to the different markers analyzed. (I) Graph showing the number of positive hepatocytes for DNMT1. (J) Dot blot representative of global DNA methylation through the detection of 5mC. (K) Quantification of densitometry results of the relative levels of 5mC. (L) Determination of overall percentage of methylated DNA. One-way ANOVA and Tukey’s post-hoc tests were performed. Significantly different at * p < 0.05, ** p < 0.001, *** p < 0.0001, **** p < 0.00001. ns, not significantly different.

Figure 5

In vitro, pirfenidone affects the synthesis of enzymes altering DNA global methylation (A) Representative western blots for DNMT1, acDNMT1, DNMT3a, DNMT3b, UHRF1, and PCNA. Relative expression of DNMT1 (B), acDNMT1 (C), DNMT3a (D), DNMT3b (E), UHRF1 (F) and PCNA (G). Lamin-B1 was used as a loading control. (H) Representative western blots for p53, β-Catenin and c-Myc. Relative expression of p53 (I), β-Catenin (J) and c-Myc (K). Lamin-B1 was used as a loading control. (L) HepG2 cell nuclei were stained with DNMT1, DNMT3a and DNMT3B (green) and 5mC (red). White asterisks indicate positivity to the different markers analyzed. (M) Dot blot representative of global DNA methylation through 5mC detection. (N) quantification for 5mC relative levels, methylene blue was used as a DNA loading control. The results are shown as the mean ± standard deviation (SD) of triplicate assays. One-way ANOVA and Tukey’s post-hoc tests were performed. Significantly different at * p < 0.05, ** p < 0.001, *** p < 0.0001 and **** p < 0.00001. ns, not significantly different.

Figure 6

Analysis of the protein–protein interaction between PPARγ and DNMT1. (A) STRING database was used to generate a comprehensive protein–protein interaction (PPI) network between PPARγ and DNMT1. (B) The interaction between PFD and PPARγ is depicted, along with a linear representation of PPARγ and DNMT1 and their corresponding structural domains. (C) The 3D structures of PPARγLBD (3QT0), PPARγDBD-RXR (3DZY) and DNMT1 (3PTA) were obtained from the Protein Data Bank (https://www.rcsb.org/ (accessed on 28 May 2024)). (D) The high-scoring docking poses from the docking simulation of PPARγLBD-DNMT1 proteins are shown, with PPARγ (cyan blue) serving as a possible receptor and DNMT1 (green) as a possible ligand. Interacting amino acids are highlighted in red. (E) The docking poses from the docking simulation of PPARγDBD-RXR-DNMT1 proteins are shown, with PPARγ (cyan blue) serving as a possible receptor and DNMT1 (green) as a possible ligand. Interacting amino acids are highlighted in red.

Figure 7

Modulation of epigenetic markers induced during HCC is the proposed mechanism exerted by PFD. Left panel: Molecular mechanisms activated during HCC growth: β-catenin crosses into the nucleus, facilitating c-Myc oncogene transcription. Right panel: PFD is a PPARγ ligand/agonist that alters DNMT1 and DNMT3a function, promoting DNA hypermethylation, and reducing c-Myc expression. These mechanisms together could suppress aberrant cell division leading to HCC.