A model of alcoholic liver disease based on different hepatotoxics leading to liver cancer.

The worst-case scenario related to alcoholic liver disease (ALD) arises after a long period of exposure to the harmful effect of alcohol consumption along with other hepatotoxics. ALD encompasses a broad spectrum of liver-associated disorders, such as steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Based on the chronic administration of different hepatotoxics, including ethanol, sucrose, lipopolysaccharide, and low doses of diethylnitrosamine over a short period, here we aimed to develop a multiple hepatotoxic (MHT)-ALD model in the mouse that recapitulates the human ALD-associated disorders. We demonstrated that the MHT-ALD model induces ADH1A and NXN, an ethanol metabolizer and a redox-sensor enzyme, respectively; promotes steatosis associated with the induction of the lipid droplet forming FSP27, inflammation identified by the infiltration of hepatic neutrophils-positive to LY-6G marker, and the increase of MYD88 level, a protein involved in inflammatory response; and stimulates the early appearance of cellular senescence identified by the senescence markers SA-ß-gal activity and p-H2A.XSer139. It also induces fibrosis associated with increased desmin, a marker of hepatic stellate cells whose activation leads to the deposition of collagen fibers, accompanied by cell death and compensatory proliferation revealed by increased CASP3-mediated apoptosis, and KI67- and PCNA-proliferation markers, respectively. It also induces histopathological traits of malignancy and the level of the HCC marker, GSTP1. In conclusion, we provide a useful model for exploring the chronological ALD-associated alterations and stages, and addressing therapeutic approaches.

Annexins A2 and A5 are potential early biomarkers of hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is a highly lethal liver cancer with late diagnosis; therefore, the identification of new early biomarkers could help reduce mortality. We determine the tissue and plasma status of five annexins during hepatocarcinogenesis by diethylnitrosamine-induced cirrhosis-HCC. We found that Anxa5 was the earliest upregulated gene at week 12 after HCC initiation, while Anxa1 and Anxa2 were upregulated in advanced HCC stages (weeks 18 and 22). Furthermore, the protein level of Annexin A1, A2, A5 and A10 was increased from the early stages. Immunofluorescence and subcellular fractionation revealed Annexin A1, A2, and A5 in the cytoplasm and nuclei of tumor cells. Notably, increased plasma levels of Annexin A5 significantly (r2 = 0.8203) correlated with Annexin A5 levels in liver tissue from week 12 and gradually increased until week 22. Using the TCGA database, we found that the expression of ANXA2 (HR = 1.7, p = 0.0046) and ANXA5 (HR = 1.8, p = 0.00077) was associated with poor survival in HCC patients. In conclusion, we have identified Annexin A1 and A5 as potentially useful early biomarkers for poor prognosis in HCC patients.

Comparative subcellular localization of NRF2 and KEAP1 during the hepatocellular carcinoma development in vivo.

The activation of Nuclear Factor, Erythroid 2 Like 2 - Kelch Like ECH Associated Protein 1 (NRF2-KEAP1) signaling pathway plays a critical dual role by either protecting or promoting the carcinogenesis process. However, its activation or nuclear translocation during hepatocellular carcinoma (HCC) progression has not been addressed yet. This study characterizes the subcellular localization of both NRF2 and KEAP1 during diethylnitrosamine-induced hepatocarcinogenesis in the rat. NRF2-KEAP1 pathway was continuously activated along with the increased expression of its target genes, namely Nqo1, Hmox1, Gclc, and Ptgr1. Similarly, the nuclear translocation of NRF2, MAF, and KEAP1 increased in HCC cells from weeks 12 to 22 during HCC progression. Likewise, colocalization of NRF2 with KEAP1 was higher in the cell nuclei of HCC neoplastic nodules than in surrounding cells. Moreover, immunofluorescence analyses revealed that the interaction of KEAP1 with filamentous Actin was disrupted in HCC cells. This disruption may be contributing to the release and nuclear translocation of NRF2 since the cortical actin cytoskeleton serves as anchoring of KEAP1. In conclusion, this evidence indicates that NRF2 is progressively activated and promotes the progression of experimental HCC.

Chronic administration of diethylnitrosamine to induce hepatocarcinogenesis and to evaluate its synergistic effect with other hepatotoxins in mice.

Hepatocellular carcinoma (HCC) arises after a long period of exposition to etiological factors that might be either independent or collectively contributing. Several rodent models resemble human HCC; however, the major limitation of these models is the lack of chronic injury that reproducibly mimics the molecular alterations as it occurs in humans. Thus, we hypothesized that chronic administration of different DEN treatments identifies the best-fit dose to induce the HCC and/or to determine whether small DEN doses act synergistically with other known hepatotoxins to induce HCC in mice. C57BL/6 J male mice were intraperitoneally injected twice a week for 6 weeks with different DEN doses ranging from 2.5 to 40 mg/kg body weight; then, selected doses (2.5, 5 and 20 mg/kg) for 6, 10, 14, and 18 weeks. We demonstrated that DEN at 20 mg/kg promoted reactive oxygen species and 4-hydroxynonenal production, cell proliferation inflammatory infiltrate, and fibrosis, which in turn induced liver cancer by week 18. These parameters were established by evaluating histopathological changes, HCC markers such as glutathione S-transferase placental-1 (Gstp1), Cytokeratin-19 (Ck19) and prostaglandin reductase-1 (Ptgr1); that of Cyp2e1, a DEN metabolizing enzyme; and the expression of the proliferation marker Ki67. While DEN at 2.5 and 5 mg/kg increased Gstp1 and Ck19, DEN at 20 mg/kg decreased them and Cyp2e1 expression and activity. In summary, our results demonstrate that DEN chronically administrated at 20 mg/kg induces the HCC, while DEN at 2.5 and 5 mg/kg could be useful in elucidating its synergistic effect with other hepatotoxic agents in mice.

The activation of peroxisome proliferator-activated receptor γ is regulated by Krüppel-like transcription factors 6 & 9 under steatotic conditions.

Liver steatosis is characterised by lipid droplet deposition in hepatocytes that can leads to an inflammatory and fibrotic phenotype. Peroxisome proliferator-activated receptors (PPARs) play key roles in energetic homeostasis by regulating lipid metabolism in hepatic tissue. In adipose tissue PPARγ regulates the adipocyte differentiation by promoting the expression of lipid-associated genes. Within the liver PPARγ is up-regulated under steatotic conditions; however, which transcription factors participate in its expression is not completely understood. Krüppel-like transcription factors (KLFs) regulate various cellular mechanisms, such as cell proliferation and differentiation. KLFs are key components of adipogenesis by regulating the expression of PPARγ and other proteins such as the C-terminal enhancer binding protein (C/EBP). Here, we demonstrate that the transcript levels of Klf6, Klf9 and Pparγ are increased in response to a steatotic insult in vitro. Chromatin immunoprecipitation (ChIp) experiments showed that klf6 and klf9 are actively recruited to the Pparγ promoter region under these conditions. Accordingly, the loss-of-function experiments reduced cytoplasmic triglyceride accumulation. Here, we demonstrated that KLF6 and KLF9 proteins directly regulate PPARγ expression under steatotic conditions.