Hepatocellular carcinoma (HCC) is the most frequent liver cancer, which account for more than 90 % of all liver cancer cases. It is the fifth leading cause of cancer globally and the second leading cause of cancer-related mortality in men. The availability of competent HCC preclinical models is fundamental to the success of mechanistic studies, molecular target identification, and drug testing. However, there are challenges associated with the use of these models. In this review, we provided updates on various cell lines, animals, and human HCC models, their specific preclinic use and associated potential challenges. Overall, the understanding of the merits and demerits of a particular HCC model will improve model selection for various preclinical studies.

BACKGROUND: Liuwei dihuang pills is a famous Traditional Chinese Medicine with various anti-cancer properties. Over 50 pharmaceutical manufacturers produce Liuwei dihuang pills in China and an estimated millions of people around the world orally take it every day. D-glucaro-1,4-lactone (1,4-GL) was quantified to be about 12.0 mg/g in Liuwei dihuang pills and a primary bioactive component of it inhibiting the activity of β-glucuronidase in vivo. 1,4-GL can prevent and effectively inhibit various types of cancer. However, its exact mechanism of action remains unknown. The study would justify the traditional usage of Liuwei dihuang pills against cancers.
OBJECTIVE: 1,4-GL, a bioactive ingredient derived from Liuwei dihuang pills, a famous Traditional Chinese Medicine, could delay the progression of diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in rats. The mechanism underpinning the effect, however, remains poorly understood.
METHODS: Healthy and HCC rats were treated with or without 1,4-GL (40.0 mg/kg) and 1HNMR-based metabonomic analysis was employed. 10 metabolites in uric acid pathway were quantitatively determined by UPLC-MS/MS. The expression of xanthine dehydrogenase (XDH), SLC2A9 mRNA, and SLC2A9 protein was determined using RT-qPCR and Western Blot. The effect of 1,4-GL on HCC-LM3 cells was verified in vitro. The alterations of ROS activity, SLC2A9 and XDH gene levels were observed in NCTC-1469 cells induced by lipopolysaccharide (LPS) after 1,4-GL treatment.
RESULTS: After the intervention of 1,4-GL, improved pathological morphology, liver lesions in HCC rats was observed with restored serum levels of AFP, AST, ALP, γ-GGT and Fisher\'s ratio. Hepatic metabonomics revealed that puring metabolism were significantly regulated by 1,4-GL in HCC rats. Uric acid, xanthine and hypoxanthine levels were quantified by UPLC-MS/MS and found to be nearly restored to control levels after 1,4-GL treatment in HCC rats. Changes in xanthine oxidase activity, XDH mRNA expression, and SLC2A9 mRNA and protein expression were also reversed. 1,4-GL treatment in LM3 HCC cells were consistent with the results in vivo. Furthermore, oxidative stress indicators such as T-SOD, GSH, CAT and MDA in serum and liver were improved after HCC rats treated with 1,4-GL. In vitro, 1,4-GL was observed to reduce lipopolysaccharide-induced ROS levels in NCTC-1469 cells with enhanced mRNA and protein expression of SLC2A9 and decreased mRNA level of XDH.
CONCLUSIONS: The protective effects of 1,4-GL against DEN-induced HCC by reducing uric acid and ROS levels due to down-regulation of uric acid production and up-regulation of SLC2A9 expressions. 1,4-GL may represent a novel treatment that improves recovery from HCC by targeting uric acid-ROS pathway.

BACKGROUND: Calotropis gigantea (L.) Dryand. (C. gigantea) is a traditional medicinal plant, recognized for its effectiveness in managing diabetes, along with its notable antioxidant, anti-inflammatory, and anticancer properties. Type II diabetes mellitus (T2DM) is characterized by chronic metabolic disorders associated with an elevated risk of hepatocellular carcinoma (HCC) due to hyperglycemia and impaired insulin response. The scientific validation of C. gigantea\'s ethnopharmacological efficacy offers advantages in alleviating cancer progression in T2DM complications, enriching existing knowledge and potentially aiding future clinical cancer treatments.
OBJECTIVE: This study aimed to investigate the preventive potential of the dichloromethane fraction of C. gigantea stem bark extract (CGDCM) against diethylnitrosamine (DEN)-induced HCC in T2DM rats, aiming to reduce cancer incidence associated with diabetes while validating C. gigantea\'s ethnopharmacological efficacy.
METHODS: Spontaneously Diabetic Torii (SDT) rats were administered DEN to induce HCC (SDT-DEN-VEH), followed by treatment with CGDCM. Metformin was used as a positive control (SDT-DEN-MET). All the treatments were administered for 10 weeks after the initial DEN injection. Diabetes-related parameters, including serum levels of glucose, insulin, and glycosylated hemoglobin (HbA1c), as well as liver function enzymes (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and gamma-glutamyl transferase), were quantified. Serum inflammation biomarkers interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were evaluated. Liver tissue samples were analyzed for inflammation protein expression (IL-6, TNF-α, transforming growth factor-β1 (TGF-β1), and α-smooth muscle actin (α-SMA)). Histopathological evaluation was performed to assess hepatic necrosis, inflammation, and fibrosis. Liver cell proliferation was determined using immunohistochemistry for Ki-67 expression.
RESULTS: Rats with SDT-DEN-induced HCC treated with CGDCM exhibited reduced serum glucose levels, elevated insulin levels, and decreased HbA1c levels. CGDCM treatment also reduced elevated hepatic IL-6, TNF-α, TGF-β1, and α-SMA levels in SDT-DEN-VEH rats. Additionally, CGDCM treatment prevented hepatocyte damage, fibrosis, and cell proliferation. No adverse effects on normal organs were observed with CGDCM treatment, suggesting its safety for the treatment of HCC complications associated with diabetes. Additionally, the absence of adverse effects in SD rats treated with CGDCM at 2.5 mg/kg further supports the notion of its safe usage.
CONCLUSIONS: These findings suggest that C. gigantea stem bark extract exerts preventive effects against the development of HCC complications in patients with T2DM, expanding the potential benefits of its ethnopharmacological advantages.

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, β-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.

UNASSIGNED: Traditional Chinese medicine (TCM) therapy is an important means to treat hepatocellular carcinoma (HCC), Astragalus (Latin name: Hedysarum Multijugum Maxim; Chinese name: Huangqi, HQ) and Atractylodes (Latin name: Atractylodes Macrocephala Koidz; Chinese name: Baizhu, BZ) (HQBZ), a classic herb pair, is often used in combination to HCC. However, the main components and potential mechanisms of HQBZ therapy in HCC remain unclear. This study aimed to identify the potential active ingredients and molecular mechanisms of action of HQBZ in HCC treatment.
UNASSIGNED: The HQBZ-Compound-Target-HCC network and HQBZ-HCC transcriptional regulatory network were constructed to screen the core active compound components and targets of HQBZ therapy for HCC. Molecular docking techniques are used to verify the stability of binding core active compound components to targets. GO and KEGG enrichment analysis were used to explore the signaling pathway of HQBZ in HCC treatment, the mechanism of HQBZ treatment of HCC was verified based on in vivo H22 tumor bearing mice and in vitro cell experiments.
UNASSIGNED: Network pharmacology and molecular docking studies showed that HQBZ treatment of HCC was related to the targeted regulation of IL-6 and STAT3 by the active compound biatractylolide, KEGG pathway enrichment analysis suggest that HQBZ may play a role in the treatment of HCC through IL-6/STAT3 signaling pathway. In vitro experiment results proved that HQBZ could regulate IL-6/STAT3 signaling pathway transduction on CD8+T cells, inhibit CD8+T cell exhaustion and restore the function of exhausted CD8+T cells. In vivo experiment results proved that HQBZ can regulate IL-6/STAT3 signaling pathway transduction in H22 liver cancer model mouse tumor tissue, increased the proportion of tumor infiltrating CD8+T cells.
UNASSIGNED: This study found that HQBZ may play a therapeutic role in HCC by targeting IL-6 and STAT3 through biatractylolide, its mechanism of action is related to regulating IL-6/STAT3 signaling pathway, reversing T cell failure and increasing tumor infiltration CD8+T cells.

Hepatocellular carcinoma (HCC) is a malignant tumor that occurs in the liver, with a high degree of malignancy and relatively poor prognosis. Gypenoside L has inhibitory effects on liver cancer cells. However, its mechanism of action is still unclear. This study aims to investigate the inhibitory effects of gypenoside L on HCC in vitro and in vivo, and explore its potential mechanisms. The results showed that gypenoside L reduced the cholesterol and triglyceride content in HepG2 and Huh-7 cells, inhibited cell proliferation, invasion and metastasis, arrested cell cycle at G0/G1 phase, promoted cell apoptosis. Mechanistically, it targeted the transcription factor SREPB2 to inhibit the expression of HMGCS1 protein and inhibited the downstream proteins HMGCR and MVK, thereby regulating the mevalonate (MVA) pathway. Overexpression HMGCS1 led to significant alterations in the cholesterol metabolism pathway of HCC, which mediated HCC cell proliferation and conferred resistance to the therapeutic effect of gypenoside L. In vivo, gypenoside L effectively suppressed HCC growth in tumor-bearing mice by reducing cholesterol production, exhibiting favorable safety profiles and minimal toxic side effects. Gypenoside L modulated cholesterol homeostasis, enhanced expression of inflammatory factors by regulating MHC I pathway-related proteins to augment anticancer immune responses. Clinical samples from HCC patients also exhibited high expression levels of MVA pathway-related genes in tumor tissues. These findings highlight gypenoside L as a promising agent for targeting cholesterol metabolism in HCC while emphasizing the effectiveness of regulating the SREBP2-HMGCS1 axis as a therapeutic strategy.

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.

The fungicide fluxapyroxad (BAS 700 F) has been shown to significantly increase the incidence of liver tumours in male Wistar rats at dietary levels of 1500 and 3000 ppm and in female rats at a dietary level of 3000 ppm via a non-genotoxic mechanism. In order to elucidate the mode of action (MOA) for fluxapyroxad-induced rat liver tumour formation a series of in vivo and in vitro investigative studies were undertaken. The treatment of male and female Wistar rats with diets containing 0 (control), 50, 250, 1500 and 3000 ppm fluxapyroxad for 1, 3, 7 and 14 days resulted in a dose-dependent increases in relative weight at 1500 and 3000 ppm from day 3 onwards in both sexes, with an increase in relative liver weight being also observed in male rats given 250 ppm fluxapyroxad for 14 days. Examination of liver sections revealed a centrilobular hepatocyte hypertrophy in some fluxapyroxad treated male and female rats. Hepatocyte replicative DNA synthesis (RDS) was significantly increased in male rats given 1500 and 3000 ppm fluxapyroxad for 3 and 7 days and in female rats given 50-3000 ppm fluxapyroxad for 7 days and 250-3000 ppm fluxapyroxad for 3 and 14 days; the maximal increases in RDS in both sexes being observed after 7 days treatment. The treatment of male and female Wistar rats with 250-3000 ppm fluxapyroxad for 14 days resulted in significant increases in hepatic microsomal total cytochrome P450 (CYP) content and CYP2B subfamily-dependent enzyme activities. Male Wistar rat hepatocytes were treated with control medium and medium containing 1-100 μM fluxapyroxad or 500 μM sodium phenobarbital (NaPB) for 4 days. Treatment with fluxapyroxad and NaPB increased CYP2B and CYP3A enzyme activities and mRNA levels but had little effect on markers of CYP1A and CYP4A subfamily enzymes and of the peroxisomal fatty acid β-oxidation cycle. Hepatocyte RDS was significantly increased by treatment with fluxapyroxad, NaPB and 25 ng/ml epidermal growth factor (EGF). The treatment of hepatocytes from two male human donors with 1-100 μM fluxapyroxad or 500 μM NaPB for 4 days resulted in some increases in CYP2B and CYP3A enzyme activities and CYP mRNA levels but had no effect on hepatocyte RDS, whereas treatment with EGF resulted in significant increase in RDS in both human hepatocyte preparations. Hepatocytes from male Sprague-Dawley wild type (WT) and constitutive androstane receptor (CAR) knockout (CAR KO) rats were treated with control medium and medium containing 1-16 μM fluxapyroxad or 500 μM NaPB for 4 days. While both fluxapyroxad and NaPB increased CYP2B enzyme activities and mRNA levels in WT hepatocytes, only minor effects were observed in CAR KO rat hepatocytes. Treatment with both fluxapyroxad and NaPB only increased RDS in WT and not in CAR KO rat hepatocytes, whereas treatment with EGF increased RDS in both WT and CAR KO rat hepatocytes. In conclusion, a series of in vivo and in vitro investigative studies have demonstrated that fluxapyroxad is a CAR activator in rat liver, with similar properties to the prototypical CAR activator phenobarbital. A robust MOA for fluxapyroxad-induced rat liver tumour formation has been established. Based on the lack of effect of fluxapyroxad on RDS in human hepatocytes, it is considered that the MOA for fluxapyroxad-induced liver tumour formation is qualitatively not plausible for humans.

OBJECTIVE: 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.
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.
RESULTS: 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.
CONCLUSIONS: PS-ASO genetic therapy showed potential to inhibit HCC far more effectively than DXT in rats.

UNASSIGNED: Cinobufotalin injection has obvious curative effects on liver cancer patients with less toxicity and fewer side effects than other therapeutic approaches. However, the core ingredients and mechanism underlying these anti-liver cancer effects have not been fully clarified due to its complex composition.
UNASSIGNED: Multidimensional network analysis was used to screen the core ingredients, key targets and pathways underlying the therapeutic effects of cinobufotalin injection on liver cancer, and in vitro and in vivo experiments were performed to confirm the findings.
UNASSIGNED: By construction of ingredient networks and integrated analysis, eight core ingredients and ten key targets were finally identified in cinobufotalin injection, and all of the core ingredients are tightly linked with the key targets, and these key targets are highly associated with the cell cycle-related pathways, supporting that both cinobufotalin injection and its core ingredients exert anti-liver cancer roles by blocking cell cycle-related pathways. Moreover, in vitro and in vivo experiments confirmed that either cinobufotalin injection or one of its core ingredients, cinobufagin, significantly inhibited cell proliferation, colony formation, cell cycle progression and xenograft tumor growth, and the key target molecules involved in the cell cycle pathway such as CDK1, CDK4, CCNB1, CHEK1 and CCNE1, exhibit consistent changes in expression after treatment with cinobufotalin injection or cinobufagin. Interestingly, some key targets CDK1, CDK4, PLK1, CHEK1, TTK were predicted to bind with multiple of core ingredients of cinobufotalin injection, and the affinity between one of the critical ingredients cinobufagin and key target CDK1 was further confirmed by SPR assay.
UNASSIGNED: Cinobufotalin injection was confirmed to includes eight core ingredients, and they play therapeutic effects in liver cancer by blocking cell cycle-related pathways, which provides important insights for the mechanism of cinobufotalin injection antagonizing liver cancer and the development of novel small molecule anti-cancer drugs.