Interaction of Mitochondrial and Epigenetic Regulation in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a pathology preceded mainly by cirrhosis of diverse etiology and is associated with uncontrolled dedifferentiation and cell proliferation processes. Many cellular functions are dependent on mitochondrial function, among which we can mention the enzymatic activity of PARP-1 and sirtuin 1, epigenetic regulation of gene expression, apoptosis, and so on. Mitochondrial dysfunction is related to liver diseases including cirrhosis and HCC; the energetic demand is not properly supplied and mitochondrial morphologic changes have been observed, resulting in an altered metabolism. There is a strong relationship between epigenetics and mitochondrion since the first one is dependent on the correct function of the last one. There is an interest to improve or to maintain mitochondrial integrity in order to prevent or reverse HCC; such is the case of IFC-305 that has a beneficial effect on mitochondrial function in a sequential model of cirrhosis-HCC. In this model, IFC-305 downregulates the expression of PCNA, thymidylate synthase, HGF and its receptor c-Met and upregulates the cell cycle inhibitor p27, thereby decreasing cell proliferation. Both effects, improvement of mitochondria function and reduction of tumor proliferation, suggest its use as HCC chemoprevention or as an adjuvant in chemotherapy

Mitoepigenetics and hepatocellular carcinoma

Mitochondria are the center of energy production in eukaryotic cells and are crucial for several cellular processes. Dysfunctional mitochondria have been associated with cancer progression. Mitochondria contain their own circular DNA (mtDNA), which codes for 13 proteins, 2rRNA, 22tRNA and non-coding RNAs. Recent evidence showed the presence of 5-methylcytosine and 5-hydroximethylcytosine in mtDNA suggesting that the level of gene expression could be modulated like a nuclear DNA by direct epigenetic modifications. Mitoepigenetics is a bidirectional phenomenon in the epigenetic regulation of mitochondrial genes encoded in both the nucleus and the mitochondrion. This process is affected by SAM-mediated methylation and hydroxymethylation of mtDNA and by nuclear chromatin modulators from mitochondria, such as Acetyl-CoA and NAD+. There is some information about physiological and pathological methylated profiles, but information is scarce for hepatocellular carcinoma (HCC). The aim of this review is to summarize the mitoepigenetic knowledge in HCC already reported so far, through a keywords search in Medline. In addition, the deregulation of energy intermediaries needed for the mitoepigenetic regulation is described. As this is a new area of study, a rigorous analysis and careful interpretation and integration of results are needed

IFC-305 partially prevented fatty acid uptake by Huh-7 cells.

Huh-7 cells were treated with the indicated concentrations of FFAs and IFC-305 for 24 h and then stained with oil red reagent, and the absorbance was read at 510 nm. All results are shown as the mean ± SD (n = 3) from a representative experiment. Statistically significant differences were determined by one-way analysis of variance (ANOVA) with multiple comparisons. “a” indicates statistically significant differences from the control group, P < 0.01. “b” indicates statistically significant differences from the 1 mM FFA group, P < 0.01. “c” indicates statistically significant differences from the 2 mM FFA group, P < 0.01.</p

IFC-305 prevents alterations in the hepatic parenchyma and the development of steatosis.

(A) Representative liver sections from each experimental group were stained with H&E and observed at 20X magnification. Macrovesicular and microvesicular steatosis are indicated by ↑ and ▲, respectively. (B) Hepatic triglyceride and (C) cholesterol content. (D) H&E-stained liver sections observed at 20X magnification were analyzed according to semiquantitative Kleiner’s criteria. Statistically significant differences were determined by one-way analysis of variance (ANOVA) with multiple comparisons. “a” indicates a significant difference compared to the control group; “b” indicates a significant difference compared to the HFS group; “d” indicates a significant difference compared to the IFC group. A difference was considered significant when p≤0.05.</p

Morphological changes evaluated by electron microscopy.

Each panel shows representative samples from the control, HFS, HFS+IFC-305 and IFC-305 groups. Each group was observed at different magnifications. MT: mitochondria, RER: rough endoplasmic reticulum.</p

Inflammation induced by a high-fat diet and sucrose was inhibited by IFC-305.

Determination of serum cytokines. The levels of the proinflammatory cytokines IL-1β, TNF-α, IL-6, and IFN-γ; the macrophage-recruiting chemokines MCP-1 and VEGF; and the anti-inflammatory interleukin IL-10 were measured. Statistically significant differences were determined by one-way analysis of variance (ANOVA) with multiple comparisons. “a” indicates a statistically significant difference compared to the control group; “b” indicates a significant difference compared to the HFS group. Differences were considered significant when P≤0.05.</p

S1 Data -

Metabolic syndrome is a multifactorial disease with high prevalence worldwide. It is related to cardiovascular disease, diabetes, and obesity. Approximately 80% of patients with metabolic syndrome have some degree of fatty liver disease. An adenosine derivative (IFC-305) has been shown to exert protective effects in models of liver damage as well as on elements involved in central metabolism; therefore, here, we evaluated the effect of IFC-305 in an experimental model of metabolic syndrome in rats induced by a high-fat diet and 10% sucrose in drinking water for 18 weeks. We also determined changes in fatty acid uptake in the Huh-7 cell line. In the experimental model, increases in body mass, serum triglycerides and proinflammatory cytokines were induced in rats, and the adenosine derivative significantly prevented these changes. Interestingly, IFC-305 prevented alterations in glucose and insulin tolerance, enabling the regulation of glucose levels in the same way as in the control group. Histologically, the alterations, including mitochondrial morphological changes, observed in response to the high-fat diet were prevented by administration of the adenosine derivative. This compound exerted protective effects against metabolic syndrome, likely due to its action in metabolic regulation, such as in the regulation of glucose blood levels and hepatocyte fatty acid uptake.</div

mRNA expression of ACSL1 and ACSL3 in HepG2 cells.

mRNA expression of ACSL1 and ACSL3 in HepG2 cells. HepG2 cells were cultured in serum-free DMEM supplemented with 1% BSA-free FFAs (control) in the presence of 1 mM IFC-305 or FFA with or without 1 mM IFC-305 for 24 h. Real-time quantitative polymerase chain reaction (qPCR) analysis of the mRNA expression of (A) ACSL1 and (B) ACSL3 isoforms normalized to β-actin. The relative mRNA levels were calculated using the comparative ΔΔCt method. mRNA expression is expressed as the mean value ± SEM from three independent experiments. **P (TIFF)</p

Food and water consumption.

Food and water intake (or water+sucrose solution for HFS and HFS+IFC groups) were measured twice a week. Captured data are expressed as mean values ± SEM from 0, 8, 12 and 18 weeks of treatment. Statistical differences were determined with ANOVA followed by a multiple comparison test where “a” indicates a significant difference compared to the control group, P≤0.05. (DOCX)</p

Changes in body weight and elements associated with metabolic syndrome induced by HFS and its regulation by IFC-305.

(A) Weight gain was measured twice a week for 18 weeks. Statistical differences were analyzed with ANOVA followed by Fisher’s test without corrections. Differences between groups were estimated considering each treatment mean value throughout time and were considered significant when p≤0.05. (B), Triglycerides, (C) LDL cholesterol, and (D) insulin were determined at the end of the treatments. (E) Glucose intolerance test. Blood glucose concentrations were measured 0, 15, 30, 60, 90, 120 and 150 minutes after oral glucose administration (3 g/kg). (F) Blood glucose concentrations 0, 15, 30, 60, 90 and 120 minutes after insulin administration (100 IU/ml). Glucose levels from the glucose and insulin tolerance tests were quantified as the mean from the area under the curve (AUC) of each animal (G and H, respectively). Differences were considered significant when p≤0.05. “a” indicates a significant difference compared to the control group; “b” indicates a significant difference compared to the HFS group; “c” indicates a significant difference compared to the HFS+IFC-305 group. The glucose levels from the glucose and insulin tolerance tests were quantified as the area under the curve (AUC) values.</p