Proteomic analysis of DEN and CCl4-induced hepatocellular carcinoma mouse model.

Hepatocellular carcinoma (HCC) seriously threatens human health, mostly developed from liver fibrosis or cirrhosis. Since diethylnitrosamine (DEN) and carbon tetrachloride (CCl4)-induced HCC mouse model almost recapitulates the characteristic of HCC with fibrosis and inflammation, it is taken as an essential tool to investigate the pathogenesis of HCC. However, a comprehensive understanding of the protein expression profile of this model is little. In this study, we performed proteomic analysis of this model to elucidate its proteomic characteristics. Compared with normal liver tissues, 432 differentially expressed proteins (DEPs) were identified in tumor tissues, among which 365 were up-regulated and 67 were down-regulated. Through Gene Ontology (GO) analysis, Ingenuity Pathway Analysis (IPA), protein-protein interaction networks (PPI) analysis and Gene-set enrichment analysis (GSEA) analysis of DEPs, we identified two distinguishing features of DEN and CCl4-induced HCC mouse model in protein expression, the upregulation of actin cytoskeleton and branched-chain amino acids metabolic reprogramming. In addition, matching DEPs from the mouse model to homologous proteins in the human HCC cohort revealed that the DEN and CCl4-induced HCC mouse model was relatively similar to the subtype of HCC with poor prognosis. Finally, combining clinical information from the HCC cohort, we screened seven proteins with prognostic significance, SMAD2, PTPN1, PCNA, MTHFD1L, MBOAT7, FABP5, and AGRN. Overall, we provided proteomic data of the DEN and CCl4-induced HCC mouse model and highlighted the important proteins and pathways in it, contributing to the rational application of this model in HCC research.

Pregnane X receptor (PXR) deficiency promotes hepatocarcinogenesis via induction of Akr1c18 expression and prostaglandin F2α (PGF2α) levels.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Pregnane X receptor (PXR), a xenobiotic-sensing nuclear receptor, plays a critical role in the metabolism of endogenous and exogenous substances in the liver. Here, we investigate whether PXR plays a role in pathogenesis of HCC. We show that liver tumors were developed in diethylnitrosamine (DEN)-treated in PXR knockout (KO) mice. Hepatic levels of prostaglandin F2α (PGF2α) and aldo-keto reductase family 1 member C18 (Akr1c18), a prostaglandin synthase of catalyzing reduction of PGH2 to PGF2α, were significantly elevated in DEN-treated PXR KO mice. Hepatic mRNA levels of alpha fetoprotein (AFP), cyclin D1 (Ccnd1), fibroblast growth factor 21 (FGF21), and inflammatory cytokine interleukin 6 (IL-6) were significantly increased in DEN-treated PXR KO mice. Other members of Akr1c family, liver metabolizing enzymes including Cyp1a2, Cyp2b10 and Cyp3a11, and bile acid synthesis enzyme Cyp7a1 mRNA levels were significantly decreased in DEN-treated PXR KO mice. Our findings revealed that PXR deficiency promoted DEN-induced HCC in mice via induction of Akr1c18 expression and PGF2α levels and the increased PGF2α levels synthetized by Akr1c18 enhanced hepatocytes proliferation and induced inflammatory cytokine production, which accelerated liver tumor development after DEN treatment, suggesting that PXR deficiency may create a microenvironment that is more prone to DEN-induced liver tumors and targeting PXR and Akr1c18 to reduce PGF2α biosynthesis may be a potential and novel therapeutic strategy for HCC.

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

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, ß-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.

H-ras-targeted genetic therapy remarkably surpassed docetaxel treatment in inhibiting chemically induced hepatic tumors in rats.

AIMS: Hepatocellular carcinoma (HCC) is still a leading cause of cancer-related death worldwide. But its chemotherapeutic options are far from expectation. We here compared H-ras targeted genetic therapy to a commercial docetaxel formulation (DXT) in inhibiting HCC in rats. MAIN METHODS: After the physicochemical characterization of phosphorothioate-antisense oligomer (PS-ASO) against H-ras mutated gene, the PS-ASO-mediated in vitro hemolysis, in vivo hepatic uptake, its pharmacokinetic profile, tissue distribution in some highly perfused organs, its effect in normal rats, antineoplastic efficacy in carcinogen-induced HCC in rats were evaluated and compared against DXT treatment. Mutated H-ras expression by in situ hybridization, hep-par-I, CK-7, CD-15, p53 expression patterns by immunohistochemical methods, scanning electron microscopic evaluation of hepatic architecture, various hepatic marker enzyme levels and caspase-3/9 apoptotic enzyme activities were also carried out in the experimental rats. KEY FINDINGS: PS-ASO showed low in vitro hemolysis (<3 %), and had a sustained PS-ASO blood residence time in vivo compared to DTX, with a time-dependent hepatic uptake. It showed no toxic manifestations in normal rats. PS-ASO distribution was although initially less in the lung than liver and kidney, but at 8 h it accumulated more in lung than kidney. Antineoplastic potential of PS-ASO (treated for 6 weeks) excelled in inhibiting chemically induced tumorigenesis compared to DTX in rats, by inhibiting H-ras gene expression, some immonohistochemical modulations, and inducing caspase-3/9-mediated apoptosis. It prevented HCC-mediated lung metastatic tumor in the experimental rats. SIGNIFICANCE: PS-ASO genetic therapy showed potential to inhibit HCC far more effectively than DXT in rats.

No mutations found in exons of TP53, H-RAS and K-RAS genes in liver of male Wistar rats submitted to a medium-term chemical carcinogenesis assay

The standart protocol to evaluate the carcionogenic potencial of chemicals is the long-term bioassay in rodents, not performed in developing countries due to its high cost and complex operational procedures. Our laboratory has established an alternative an alternative medium-term bioassay in Wistar rats, also DMBDD assay, based on the paradigm iniation/promotion of chemical carcinogenesis. This method was accepted by the Brazilian Environment Agency (IBAMA) as an official source of evidence of carcinogenity. The aim of this study was to evaluate alterations in exons 5 to 8 of the tumor suppressor gene TP53 and exons 1 and 2 of oncogenes K-RAS and H-RAS in preneoplastic hepatic lesions observed in DMBDD assay. The characterization of these alterations may contribute to the recognition of patterns of damage in critical genes, as well as to suggest mechanisms of action of the compounds tested in the protocol. Sixty male wistar rats were separeted into3 groups? the first was treated with no chemicals; the second received five initiaing agents and the third received initiation followed by phenobarbital. Liver DNA samples (obtained from formalin-fixed and parafin-fixed and paraffin-embedded tissues after histological analysis) were evaluated by the non-isotopic PCR-SSCP technique. No changes in any analyzed exons were detected by the PCR-SSCP banding pattern in all experimental groups. This suggests that liver in exons 5 to 8 of TP53 and exons 1 and 2 of H-RAS are not among the early molecular alterations occuring in the hepatic carcinogenesis process by the DMBDD protocol in male Wistar rats

Mecanismo teórico de la hepatocarcinogénesis / Theoretical mechanism of ethionine hepatocarcinogenesis

En resumen, parece ser que el proceso necesario, al menos conceptualmente que le permita a uno construir una noción adecuada del mecanismo involucrado durante la carcinogénesis inducida por etionina, puede ser relevante a un proceso más general y fundamental aplicable a todos los carcinógenos. Se ha descrito un intento que trata de aportar un mecanismo considerado en este artículo, también enfatiza la importancia de la activación de proteínas embriónicas como un ejemplo de un proceso más general que se requiere para la carcinogénesis

Diferencias entre los ácidos ribonucleicos mensajeros de hígado normal y de hepatoma de Novikoff / Differences between the messenger ribonucleic acids of the normal liver and Novikoff's hepatoma

En la activación de varios genes en algunas células cancerosas aparentemente se requiere que se desencadene una cascada de eventos, que trae como consecuencia la transcripción de diversos genes; al mismo tiempo, otros genes son reprimidos. El propósito de este trabajo fue investigar si existen diferencias en la transcripción del ácido ribonucleico mensajero (mRNA) del hígado normal y del hepatoma de Novikoff. En todos los experimentos se utilizaron ratas macho adultas Sprague Dawley. La extracción del RNA total de bazo, hígado y células del hepatoma de Novikoff se llevó a cabo con el método de Schueltz y la separación del mRNA se realizó utilizando una columna de celulosa-ácido oligotimidílico, de acuerdo a lo señalado por Aviv y Leder. La cuantificación del mRNA mostró que las células del hepatoma de Novikoff tienen el doble de esta macromolécula comparadas con el hígado normal de rata