In Vivo and In Vitro Models of Hepatocellular Carcinoma: Current Strategies for Translational Modeling.

Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide and the third leading cause of cancer-related death globally. HCC is a complex multistep disease and usually emerges in the setting of chronic liver diseases. The molecular pathogenesis of HCC varies according to the etiology, mainly caused by chronic hepatitis B and C virus infections, chronic alcohol consumption, aflatoxin-contaminated food, and non-alcoholic fatty liver disease associated with metabolic syndrome or diabetes mellitus. The establishment of HCC models has become essential for both basic and translational research to improve our understanding of the pathophysiology and unravel new molecular drivers of this disease. The ideal model should recapitulate key events observed during hepatocarcinogenesis and HCC progression in view of establishing effective diagnostic and therapeutic strategies to be translated into clinical practice. Despite considerable efforts currently devoted to liver cancer research, only a few anti-HCC drugs are available, and patient prognosis and survival are still poor. The present paper provides a state-of-the-art overview of in vivo and in vitro models used for translational modeling of HCC with a specific focus on their key molecular hallmarks.

Beneficial effects of anthocyanin-rich peels of Myrtaceae fruits on chemically-induced liver fibrosis and carcinogenesis in mice.

Hepatocellular carcinoma (HCC) arising from fibrosis/cirrhosis is the most common type of primary liver cancer. Conversely, a higher intake of fruits and vegetables might play a protective role in HCC risk. Recently, Myrtaceae family tropical fruits have raised great interest due to the high levels of anthocyanins especially in their peels, which are usually discarded upon consumption. Anthocyanins are antioxidant pigments known to have beneficial effects in vivo/in vitro cancer bioassays. Thus, we evaluated whether dietary Myrciaria jaboticaba, Syzygium cumini, and Syzygium malaccense fruit peel powders reduce fibrosis and hepatocarcinogenesis in mice. Female C3H/HeJ mice were submitted to the model of diethylnitrosamine/carbon tetrachloride-induced liver fibrosis and carcinogenesis. Concomitantly, mice received a basal diet containing 2% of M. jaboticaba, S. cumini, or S. malaccense fruit peel powders, obtained by convective drying, for 10 weeks. M. jaboticaba peel powder showed the highest levels of total anthocyanins, while S. cumini peel powder displayed the greatest diversity of these pigments. All Myrtaceae family peel powders reduced the serum levels of the liver injury marker alanine aminotransferase. M. jaboticaba peel feeding reduced the incidence of liver preneoplastic foci, hepatocyte proliferation (Ki-67), and the protein levels of hepato-mitogen tumor necrosis factor-alpha (TNF-α). M. jaboticaba peel feeding also diminished liver lipid peroxidation and increased total glutathione levels. S. cumini peel feeding reduced hepatic collagen, lipid peroxidation, and TNF-α levels while increased catalase activity. Although S. malaccense peel powder, which displayed the lowest anthocyanin levels, decreased oxidative stress, and cytokine levels, no effects were observed on liver fibrosis or preneoplastic lesion outcomes. Findings indicate a protective effect of anthocyanin-rich M. jaboticaba and S. cumini peel powder feeding on preneoplastic lesion development and fibrosis, respectively. Results indicate that differential biological responses may be attributed to distinct anthocyanin profiles and levels, assigning a functional/market value to the underutilized peel fraction.

The combination of coffee compounds attenuates early fibrosis-associated hepatocarcinogenesis in mice: involvement of miRNA profile modulation.

Aberrant microRNA expression implicates on hepatocellular carcinoma (HCC) development. Conversely, coffee consumption reduces by ~40% the risk for fibrosis/cirrhosis and HCC, while decaffeinated coffee does not. It is currently unknown whether these protective effects are related to caffeine (CAF), or to its combination with other common and/or highly bioavailable coffee compounds, such as trigonelline (TRI) and chlorogenic acid (CGA). We evaluated whether CAF individually or combined with TRI and/or CGA alleviates fibrosis-associated hepatocarcinogenesis, examining the involvement of miRNA profile modulation. Then, male C3H/HeJ mice were submitted to a diethylnitrosamine/carbon tetrachloride-induced model. Animals received CAF (50 mg/kg), CAF+TRI (50 and 25 mg/kg), CAF+CGA (50 and 25 mg/kg) or CAF+TRI+CGA (50, 25 and 25 mg/kg), intragastrically, 5×/week, for 10 weeks. Only CAF+TRI+CGA combination reduced the incidence, number and proliferation (Ki-67) of hepatocellular preneoplastic foci while enhanced apoptosis (cleaved caspase-3) in adjacent parenchyma. CAF+TRI+CGA treatment also decreased hepatic oxidative stress and enhanced the antioxidant Nrf2 axis. CAF+TRI+CGA had the most pronounced effects on decreasing hepatic pro-inflammatory IL-17 and NFκB, contributing to reduce CD68-positive macrophage number, stellate cell activation, and collagen deposition. In agreement, CAF+TRI+CGA upregulated tumor suppressors miR-144-3p, miR-376a-3p and antifibrotic miR-15b-5p, frequently deregulated in human HCC. CAF+TRI+CGA reduced the hepatic protein levels of pro-proliferative EGFR (miR-144-3p target), antiapoptotic Bcl-2 family members (miR-15b-5p targets), and the number of PCNA (miR-376a-3p target) positive hepatocytes in preneoplastic foci. Our results suggest that the combination of most common and highly bioavailable coffee compounds, rather than CAF individually, attenuates fibrosis-associated hepatocarcinogenesis by modulating miRNA expression profile.

Fibrosis-associated hepatocarcinogenesis revisited: Establishing standard medium-term chemically-induced male and female models.

Hepatocellular carcinoma causes ~10% of all cancer-related deaths worldwide, usually emerging in a background of liver fibrosis/cirrhosis (70%-90% of cases). Chemically-induced mouse models for fibrosis-associated hepatocarcinogenesis are widely-applied, resembling the corresponding human disease. Nonetheless, a long time is necessary for the development of preneoplastic/neoplastic lesions. Thus, we proposed an early fibrosis-associated hepatocarcinogenesis model for male and female mice separately, focusing on reducing the experimental time for preneoplastic/neoplastic lesions development and establishing standard models for both sexes. Then, two-week old susceptible C3H/HeJ male and female mice (n = 8 animals/sex/group) received a single dose of diethylnitrosamine (DEN, 10 or 50 mg/Kg). During 2 months, mice received 3 weekly doses of carbon tetrachloride (CCl4, 10% corn oil solution, 0.25 to 1.50 µL/g b.wt.) and they were euthanized at week 17. DEN/CCl4 protocols for males and females displayed clear liver fibrosis, featuring collagen accumulation and hepatic stellate cell activation (α-SMA). In addition, liver from males displayed increased CD68+ macrophage number, COX-2 protein expression and IL-6 levels. The DEN/CCl4 models in both sexes impaired antioxidant defense as well as enhanced hepatocyte proliferation and apoptosis. Moreover, DEN/CCl4-treated male and female developed multiple preneoplastic altered hepatocyte foci and hepatocellular adenomas. As expected, the models showed clear male bias. Therefore, we established standard and suitable fibrosis-associated hepatocarcinogenesis models for male and female mice, shortening the experimental time for the development of hepatocellular preneoplastic/neoplastic lesions in comparison to other classical models.