There is a correlation between oxidative stress generated by diethylnitrosamine (DEN) metabolism and liver cancer development. Quercetin is a flavonoid with anti-carcinogenic and antioxidant properties. This study demonstrates the mechanism of action for the chemopreventive effect of quercetin. A 10 mg/kg dose of quercetin produced drastic effect, when it is administrated 2 h before DEN; at 24 days post-DEN, a 70.3% and 66.2% decrease in total area and number of preneoplastic lesions were observed, respectively. At 12 h post-DEN, quercetin inhibited levels of lipid peroxidation by 40%. Quercetin increased the levels of both GSH and of total glutathione, it increased the GSH/GSSG index and it caused a rapid and simultaneous elevation in the activities of superoxide dismutase, glutathione peroxidase and catalase. In conclusion, the quercetin mechanism of action is due to promote the enzymatic and non-enzymatic antioxidant defense system during the initiation of hepatocarcinogenesis.

We conclude that liver injury up-regulates TGF-β1 that inhibits parenchymal cell proliferation, but stimulates HSC proliferation leading to the production of ECM and type I collagen resulting in fibrosis.

BackgroundRecently, extremely low frequency electromagnetic fields (ELF-EMF) have been studied with great interest due to their possible effects on human health. In this study, we evaluated the effect of 4.5 mT - 120 Hz ELF-EMF on the development of preneoplastic lesions in experimental hepatocarcinogenesis.MethodsMale Fischer-344 rats were subjected to the modified resistant hepatocyte model and were exposed to 4.5 mT - 120 Hz ELF-EMF. The effects of the ELF-EMF on hepatocarcinogenesis, apoptosis, proliferation and cell cycle progression were evaluated by histochemical, TUNEL assay, caspase 3 levels, immunohistochemical and western blot analyses.ResultsThe application of the ELF-EMF resulted in a decrease of more than 50% of the number and the area of γ-glutamyl transpeptidase-positive preneoplastic lesions (P = 0.01 and P = 0.03, respectively) and glutathione S-transferase placental expression (P = 0.01). The number of TUNEL-positive cells and the cleaved caspase 3 levels were unaffected; however, the proliferating cell nuclear antigen, Ki-67, and cyclin D1 expression decreased significantly (P ≤ 0.03), as compared to the sham-exposure group.ConclusionThe application of 4.5 mT - 120 Hz ELF-EMF inhibits preneoplastic lesions chemically induced in the rat liver through the reduction of cell proliferation, without altering the apoptosis process.

Akt kinase isoforms (Akt1, Akt2, and Akt3) have generally been thought to play overlapping roles in phosphoinositide 3-kinase (PI3K)-mediated-signaling. However, recent studies have suggested that they display isoform-specific roles in muscle and fat. To determine whether such isoform-specificity is observed with respect to alcoholic liver disease (ALD) progression, we examined the role of Akt1, Akt2, and Akt3 in hepatic inflammation, and pro-fibrogenic proliferation and migration using Kupffer cells, hepatic stellate cells (HSC), and hepatocytes in an ethanol and lipopolysaccharide (LPS)-induced two-hit model in vitro and in vivo. We determined that siRNA-directed silencing of Akt2, but not Akt1, significantly suppressed cell inflammatory markers in HSC and Kupffer cells. Although both Akt1 and Akt2 inhibited cell proliferation in HSC, only Akt2 inhibited cell migration. Both Akt1 and Akt2, but not Akt3, inhibited fibrogenesis in hepatocytes and HSC. In addition, our in vivo results show that administration of chronic ethanol, binge ethanol and LPS (EBL) in wild-type C57BL/6 mice activated all three Akt isoforms with concomitant increases in activated forms of phosphoinositide dependent kinase-1 (PDK1), mammalian target-of-rapamycin complex 2 (mTORC2), and PI3K, resulting in upregulation in expression of inflammatory, proliferative, and fibrogenic genes. Moreover, pharmacological blocking of Akt2, but not Akt1, inhibited EBL-induced inflammation while blocking of both Akt1 and Akt2 inhibited pro-fibrogenic marker expression and progression of fibrosis. Our findings indicate that Akt isoforms play unique roles in inflammation, cell proliferation, migration, and fibrogenesis during EBL-induced liver injury. Thus, close attention must be paid when targeting all Akt isoforms as a therapeutic intervention.

S-adenosylmethionine (SAM), N-acetylcysteine (NAC) and quercetin exhibit a chemoprotective effect. Likely this effect is mediated by counteracting, oxidative stress and NF-kB activation. To test this hypothesis F344 rats were subjected to hepatocarcinogenesis with or without antioxidants. NAC decreased foci in number and area, SAM and quercetin decreased area. Lipid-peroxidation was decreased by antioxidants, but only SAM increased glutathione. SAM, in its regulation from IKK downwards, abolished the NF-kB activation. NAC decreased IKK and IkB-a phosphorylation, and Rel-A/p65 and NF-kB binding, though the last two were affected with less intensity compared to the NF-kB inhibitor. Quercetin decreased Rel-A/p65, without modifying upstream signalling. Although all antioxidants inhibited oxidative stress as shown by reduction of lipid peroxidation, not all exerted the same effect on NF-kB signalling pathway and only SAM increased GSH. The mechanisms exerted by SAM in the reduction of foci makes this compound a potential liver cancer therapeutic agent.

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Caffeic acid phenethyl ester (CAPE), a natural component of propolis, shows anticarcinogenic properties in the modified resistant hepatocyte model when administered before initiation or promotion of hepatocarcinogenesis process; however, information about the mechanism underlying this chemoprotection is limited. The aim of this work was to characterize the effect of CAPE on cytochrome P450 (CYP), which is involved in diethylnitrosamine (DEN) metabolism during the initiation stage of chemical hepatocarcinogenesis. Male Fischer-344 rats were treated as in the modified resistant hepatocyte model. Liver samples were obtained at four different times: at 12 h after pretreatment with CAPE and at 12 and 24 h and 25 days after DEN administration. Liver damage was determined by histology with hematoxylin and eosin, measurement of total CYP levels and enzyme activity, and gamma-glutamyl transpeptidase-positive (GGT+) staining of hepatocyte foci. CAPE administration prevented DEN-induced necrosis at 24 h. It also decreased O-dealkylation of 7-ethoxy-resorufin (EROD), O-dealkylation of 7-methoxyresorufin (MROD), and 7-pentoxy-resorufin activities at 12 h after its administration and EROD and MROD activities at 12 h after administration of DEN. CAPE treatment decreased GGT+ foci by 59% on day 25. Our results suggest that CAPE modifies the enzymatic activity of CYP isoforms involved in the activation of DEN, such as CYP1A1/2 and CYP2B1/2. These findings describe an alternative mechanism for understanding the ability of CAPE to protect against chemical hepatocarcinogenesis.

Alcohol-induced liver fibrosis and eventually cirrhosis is a leading cause of death. Acetaldehyde, the first metabolite of ethanol, up-regulates expression of the human α2(I) collagen gene (COL1A2). Early acetaldehyde-mediated effects involve phosphorylation and nuclear translocation of SMAD3/4-containing complexes that bind to COL1A2 promoter to induce fibrogenesis. We used human and mouse hepatic stellate cells to elucidate the mechanisms whereby acetaldehyde up-regulates COL1A2 by modulating the role of Ski and the expression of SMADs 3, 4, and 7. Acetaldehyde induced up-regulation of COL1A2 by 3.5-fold, with concomitant increases in the mRNA (threefold) and protein (4.2- and 3.5-fold) levels of SMAD3 and SMAD4, respectively. It also caused a 60% decrease in SMAD7 expression. Ski, a member of the Ski/Sno oncogene family, is colocalized in the nucleus with SMAD4. Acetaldehyde induces translocation of Ski and SMAD4 to the cytoplasm, where Ski undergoes proteasomal degradation, as confirmed by the ability of the proteasomal inhibitor lactacystin to blunt up-regulation of acetaldehyde-dependent COL1A2, but not of the nonspecific fibronectin gene (FN1). We conclude that acetaldehyde up-regulates COL1A2 by enhancing expression of the transactivators SMAD3 and SMAD4 while inhibiting the repressor SMAD7, along with promoting Ski translocation from the nucleus to cytoplasm. We speculate that drugs that prevent proteasomal degradation of repressors targeting COL1A2 may have antifibrogenic properties.