Inhibits Hepatocellular Carcinoma Development Research Articles

Retracted Article: MiR-206 reduced the malignancy of hepatocellular carcinoma cells in vitro by inhibiting MET and CTNNB1 gene expressions.

The anti-cancer role of miR-206 in hepatocellular carcinoma (HCC) cells has been reported, but its mechanism of action remains poorly understood. This research aimed to investigate the anti-HCC mechanism of miR-206. We analyzed 25 pairs of HCC and adjacent tissue specimens from HCC patients. Two patient-derived HCC cell lines were established. MiR-206 levels in tissue specimens and cell lines were detected by qRT-PCR. MiR-206 overexpression was mimicked by miR-206 mimic transfection. MET or CTNNB1 gene was overexpressed by transient transfection. Protein and protein phosphorylation levels of interests were assessed by western blotting. HCC cell malignancy in vitro was evaluated by cell proliferation, apoptosis, colony formation, trans-well invasion assays as well as western blotting assessing the marker proteins of epithelial or mesenchymal phenotype. We found that miR-206 level was significantly lower in HCC tissue specimens in comparison to adjacent counterparts. Two patient-derived HCC cell lines showed lower miR-206 level than L02 human hepatocytes. MiR-206 mimic transfection significantly reduced phosphorylation levels of pan-Akt Ser9, Erk1 Thr202/Tyr204 and Gsk-3beta Ser308 as well as protein levels of beta-catenin and c-Met in primary HCC cells in vitro. Luciferase reported assay and AGO2-RNA co-immunoprecipitation assays results demonstrated that miR-206 reduced MET and CTNNB1 gene expressions in HCC cells by interacting with the 3′ UTR of their mRNAs. Restoring c-Met or beta-catenin protein level by MET or CTNNB1 transient overexpression partially restored the malignancy of HCC cells in vitro. We concluded that miR-206 might inhibit HCC development by targeting MET and CTNNB1 gene expression.

Abstract 4004: The H2 receptor antagonist nizatidine inhibits carcinogenesis in two rodent models of hepatocellular carcinoma

Abstract Introduction: The purpose of this study was to examine whether nizatidine could reduce liver fibrosis and subsequent tumor burden in two rodent models of hepatocellular carcinoma (HCC). Methods: An in silico screen identified nizatidine as a compound that can reverse a previously identified gene signature associated with disease progression and HCC development in human cirrhosis patients. We tested the ability of nizatidine to inhibit HCC development in two rodent models. In the first study, male Wistar rats received weekly intraperitoneal injections of 50 mg/kg diethylnitrosamine (DEN). This model has previously been shown to reliably recapitulate the histologic and molecular features of human HCC development including fibrosis after 8 weeks, cirrhosis after 12 weeks, and HCC by 18 weeks. DEN-injured rats were randomized to receive oral gavage of nizatidine (n=7) or vehicle control (n=9) after 8 weeks. In the second study, male C57BL/6J mice received a single intraperitoneal injection of 25 mg/kg DEN at day 15 followed by initiation of a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) at 6 weeks of age. DEN+CDAHFD-injured mice were randomized to receive oral gavage of nizatidine (n=7) or vehicle control (n=9) at 12 weeks of age and were sacrificed at 30 weeks of age after development of HCC in the setting of nonalcoholic steatohepatitis (NASH). Results: As expected, repeated injections of DEN in rats resulted in progressive fibrosis followed by HCC formation. Treatment with nizatidine resulted in a 35% reduction of tumor nodules relative to vehicle controls (p<0.18). Liver sections were stained by picrosirius red to assess fibrosis and nizatidine reduced collagen deposition in DEN-injured rats (collagen proportional area = 5±0.03 vs. 9.2±0.04; p<0.05). This histologic observation was further confirmed by gene expression analysis with reduction in several profibrotic markers, such as alpha-smooth muscle actin and collagen type I, after treatment with nizatidine. All mice receiving DEN+CDAHFD developed HCC. Treatment with nizatidine resulted in a 60% reduction in tumor nodules relative to vehicle controls (p<0.0001). Nizatidine treatment also resulted in a significant reduction in liver to body weight (p<0.01). Nizatidine treatment reduced collagen proportional area (11±0.05 vs. 15±.01; p<0.05) and expression of profibrotic markers. Nizatidine treatment also reduced the expression of several proinflammatory markers including CD68, interferon gamma, and interleukin-6. Conclusion: Our data suggest that the H2 receptor antagonist nizatidine reduces fibrosis and subsequent HCC development. This could be beneficial in patient management given the low cost and ready availability of this agent. Citation Format: Shen Li, Sarani Ghoshal, Gunisha Arora, Derek J. Erstad, Mozhdeh Sojoodi, Michael Lanuti, Yujin Hoshida, Thomas Baumert, Kenneth K. Tanabe, Bryan C. Fuchs. The H2 receptor antagonist nizatidine inhibits carcinogenesis in two rodent models of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4004.

IL-37 mediates the anti-tumor activity in hepatocellular carcinoma via T cell activation and angiogenesis suppression

Abstract IL-37 has been shown to play an immunosuppressive role against inflammatory and autoimmune diseases. However, the role of IL-37 in tumor development is still controversial. Recent studies demonstrated that IL-37 could recruit NK cells, stimulate CD4+ T cell activation, as well as inhibit tumor angiogenesis to suppress tumor progression in different tumor models. Other studies suggested that IL-37 could affect DC function to suppress adaptive immune response. In the current study, we found that IL-37 could directly inhibit hepatocellular carcinoma (HCC) cell expansion and promote its apoptosis. The in vivo results showed that IL-37 could inhibit HCC development through enhancing the T cell cytokine production and killing capacity in murine orthotopic HCC model and DEN-induced HCC model. This anti-tumor effect was fully abrogated in NOD-SCID mice. Furthermore, we found that IL-37 could also inhibit tumor angiogenesis in peritumoral areas. The in vitro study showed that recombinant IL-37 protein could directly promote the migration, tube formation and IL-1R8, VEGF, MMP2 expression in HUVEC cells. However, the supernatant from IL-37 expressing HCC cells showed an inhibition on the HUVEC cell migration and tube formation, suggesting its secondary role on angiogenesis through tumor cells could be dominant in the tumor microenvironment. Overall, these results showed that IL-37 could suppress HCC development through T cell activation and angiogenesis suppression.

IL-17C inhibits the development of hepatocellular carcinoma through the modulation of gut microbiota

Abstract IL-17C has been recently reported to play an important role in infections and inflammatory diseases. However, its function in anti-tumor immune response is not clear. Here, we investigated the role of IL-17C in murine hepatocellular carcinoma (HCC) models and found that genetic depletion of IL-17C significantly promoted HCC growth, whilst overexpression of IL-17C inhibited HCC development. Flow cytometry analysis further demonstrated that the percentage of CD8+ T cells was reduced in IL-17C−/− mice. Moreover, the activation of T cells was also significantly reduced when IL-17C was deficient. Interestingly, depletion of gut microbiota with antibiotics treatment diminished the difference in tumor burdens between WT and IL-17C−/− groups, indicating microbiota might be involved in the process of IL-17C regulating HCC development. In addition, 16sRNA sequencing results revealed that IL-17C deficiency significantly reduced the Lactobacillus acidophilus colonization. Collectively, we found that IL-17C suppressed HCC growth in a gut microbiota-dependent manner. Further studies are needed to confirm the contribution of L.acidophilus to HCC development and investigate the detailed mechanisms of IL-17C regulating anti-tumor immune response.

Role of exosomes and exosomal microRNAs in hepatocellular carcinoma: Potential in diagnosis and antitumour treatments (Review).

Communication between hepatocellular carcinoma (HCC) cells and their environment is essential for the development and progression of HCC. Exosomes, which are microvesicles secreted by a number of cell types, are carriers of intercellular information and regulate the tumour microenvironment. Studies have demonstrated that exosomes are involved in the communication between HCC cells, endothelial cells and stem cells, and that they serve important roles in the metastasis and invasion, immune evasion and immunotherapy of HCC. In addition, the mechanism of HCC-derived exosome-mediated microRNA (miRNA) transfer is important in the environmental modulation of HCC growth and progression. As exosomes can be used for detecting and monitoring HCC, they can potentially serve as specific biomarkers for early-stage tumours and the tumour metastasis of HCC. Moreover, mesenchymal stem cell-derived exosomes can be transfected with miRNAs to inhibit HCC development. Therefore, as nucleic acid delivery vehicles, exosomes show a tremendous potential for effective treatment against HCC. In the present review, recent advances in our understanding of the source, composition and function of exosomes in HCC, and their potential value in the early diagnosis and treatment of HCC, are summarized.

An endogenous DNA adduct as a prognostic biomarker for hepatocarcinogenesis and its prevention by Theaphenon E in mice.

These results support γ-OHPdG as a mechanism-based, biologically relevant biomarker for predicting the risk of HCC and its recurrence. (Hepatology 2018;67:159-170).

Abstract IA08: p62/SQSTM1 accumulation links chronic tissue damage to malignant progression in liver and pancreas

Abstract Despite extensive genomic exploration, the mechanisms that initiate malignancy remain obscure. We addressed this issue by studying how chronic liver diseases progress to hepatocellular carcinoma (HCC). We focused our studies on the role of p62/SQSTM1, whose accumulation is a hallmark of premalignant liver disease. Using mice, we found that hepatocyte-specific p62 ablation inhibits HCC development in four distinct models, with strongest effect exerted in etiologies involving mTORC1 activation. Forced p62 expression in autophagy-competent livers suffices for HCC induction. Amongst p62 activities including aggregate formation, binding ubiquitinated proteins, mTORC1 stimulation and transcription factor activation, most critical is the NRF2 pathway, which is mutationally activated in 14% of human HCCs. We propose that environmentally-induced p62-dependent NRF2 signaling is a key early event inHCC pathogenesis that together with mTORC1 promotes survival and expansion of superoxide-accumulating HCC-initiating cells. Indeed, high p62 expression in preneoplastic human liver predicts rapid HCC recurrence after curative ablation. Citation Format: Michael Karin. p62/SQSTM1 accumulation links chronic tissue damage to malignant progression in liver and pancreas. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr IA08.

Differential regulation of the expression of aquaporins 3 and 9 by Auphen and dbcAMP in the SMMC-7721 hepatocellular carcinoma cell line

Aquaglycero-aquaporins (agAQPs) are the structural foundation of rapid water transport and they appear to participate in cancer proliferation and malignancy. AQP3 expression is increased and AQP9 expression is decreased in hepatocellular carcinoma (HCC) compared to normal liver, which suggests their possible use as targets for cancer treatment. AQP-based modifiers, such as Auphen and dibutyryladenosine 3′, 5′-cyclic monophosphate (dbcAMP), might be used to treat several diseases and as chemical tools for assessing the functions of AQPs in biological systems. We investigated the effects of both Auphen on AQP3 and dbcAMP on AQP9 in SMMC-7721 cells. We used western blotting, real-time quantitative polymerase chain reaction (qPCR) and immunohistochemistry to evaluate changes in AQP3 and AQP9 expression in SMMC-7721 cells after culturing with Auphen and dbcAMP, respectively. We also determined the proliferation of SMMC-7721 cells. We found that compared to HL-7702 (L02) liver cells, Auphen increased AQP3 expression in tumor cells, whereas dbcAMP decreased expression of AQP9 in these cells. Also, high concentrations of Auphen and dbcAMP inhibited proliferation of SMMC-7721 cells in vitro. Auphen and dbcAMP may inhibit HCC development and could be considered targets for HCC diagnosis and therapy.

Estradiol and Estrogen Receptor Agonists Oppose Oncogenic Actions of Leptin in HepG2 Cells.

Obesity is a significant risk factor for certain cancers, including hepatocellular carcinoma (HCC). Leptin, a hormone secreted by white adipose tissue, precipitates HCC development. Epidemiology data show that men have a much higher incidence of HCC than women, suggesting that estrogens and its receptors may inhibit HCC development and progression. Whether estrogens antagonize oncogenic action of leptin is uncertain. To investigate potential inhibitory effects of estrogens on leptin-induced HCC development, HCC cell line HepG2 cells were treated with leptin in combination with 17 β-estradiol (E2), estrogen receptor-α (ER-α) selective agonist PPT, ER-β selective agonist DPN, or G protein-coupled ER (GPER) selective agonist G-1. Cell number, proliferation, and apoptosis were determined, and leptin- and estrogen-related intracellular signaling pathways were analyzed. HepG2 cells expressed a low level of ER-β mRNA, and leptin treatment increased ER-β expression. E2 suppressed leptin-induced HepG2 cell proliferation and promoted cell apoptosis in a dose-dependent manner. Additionally E2 reversed leptin-induced STAT3 and leptin-suppressed SOCS3, which was mainly achieved by activation of ER-β. E2 also enhanced ERK via activating ER-α and GPER and activated p38/MAPK via activating ER-β. To conclude, E2 and its receptors antagonize the oncogenic actions of leptin in HepG2 cells by inhibiting cell proliferation and stimulating cell apoptosis, which was associated with reversing leptin-induced changes in SOCS3/STAT3 and increasing p38/MAPK by activating ER-β, and increasing ERK by activating ER-α and GPER. Identifying roles of different estrogen receptors would provide comprehensive understanding of estrogenic mechanisms in HCC development and shed light on potential treatment for HCC patients.

Abstract 1427: Liver selective Acetyl-CoA Carboxylase inhibition by ND-654 decreases hepatocellular carcinoma development in cirrhotic rats

Abstract Background: Hepatocellular carcinoma (HCC) is increasing in incidence worldwide. This increase is in part due to the obesity epidemic which is an independent risk factor for both nonalcoholic fatty liver disease (NAFLD) and HCC. In fact, recent evidence suggests that enhanced de novo lipogenesis is a critical mediator for HCC development. Current treatment options for HCC are limited, and as such, prognosis is extremely poor with a 5-year survival less than 12%. Therefore, there is an urgent medical need for new therapeutic strategies. Simultaneous inhibition of the acetyl-CoA carboxylase isozymes, ACC1 and ACC2, results in concomitant inhibition of fatty acid synthesis (FASyn) and stimulation of fatty acid oxidation (FAOxn) in cultured cells and in animals, reduces hepatic steatosis, and impedes tumor cell growth in vitro. Here, we test the hypothesis that simultaneous reduction of hepatic FASyn through inhibition of ACC1 and stimulation of hepatic FAOxn through inhibition ACC2 is a potential therapeutic target for HCC. Results: Using state-of-the-art structure-based drug design and crystal structures of the human ACC2 biotin carboxylase domain, we identified ND-654, a hepatoselective (2700:5:1 liver to plasma to muscle exposure), allosteric inhibitor that binds to the ACC subunit dimerization site, inhibits the enzymatic activity of both ACC1 (IC50 = 3 nM) and ACC2 (IC50 = 8 nM), and inhibits FASyn in HepG2 cells (IC50 = 14 nM) and in rats (ED50 = 0.3 mg/kg). Male Wistar rats were treated weekly with 50 mg/kg diethylnitrosamine (DEN) to induce sequential development of fibrosis, cirrhosis and HCC. After establishment of cirrhosis (13 wks), rats were treated daily by oral gavage with either vehicle or 10 mg/kg ND-654. At the end of the study (18 wks), rats were sacrificed and tumor nodules were counted. Liver tissue and plasma were also analyzed to assess the effects of ND-654 on disease progression. When measured four hours after the final dose, ND-654 showed preferential liver exposure, with steady state levels reaching 3.7±1.8 µM in cirrhotic tissue and 2.2±1.8 µM in tumor tissue. ND-654 did not alter body weight or food consumption, but reduced serum triglyceride levels by 35%. Importantly, ND-654 significantly (p < 0.01) inhibited HCC development in DEN-injured livers from an average of 15.8 tumor nodules in controls to an average of 7.1 tumor nodules in ND-654-treated rats. Consistently, ND-654 reduced the DEN-induced increase in liver weight by 48%, and decreased the number of tumor related deaths by 67% (6 of 8 controls vs 2 of 8 ND-654-treated rats). Conclusions: These results provide further evidence that de novo lipogenesis is an important mediator of hepatic carcinogenesis and that selective inhibition of hepatic ACC is a potential therapeutic strategy for HCC. Citation Format: Danielle K. DePeralta, Lan Wei, Geraldine Harriman, Jeremy Greenwood, Sathesh Bhat, William Westlin, H. James Harwood, Rosana Kapeller, Kenneth K. Tanabe, Bryan C. Fuchs. Liver selective Acetyl-CoA Carboxylase inhibition by ND-654 decreases hepatocellular carcinoma development in cirrhotic rats. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1427. doi:10.1158/1538-7445.AM2014-1427

1, 25(OH)<sub>2</sub>D<sub>3</sub> Inhibits Hepatocellular Carcinoma Development Through Reducing Secretion of Inflammatory Cytokines from Immunocytes

Epidemiological and clinical studies have indicated that low vitamin D activity is not only associated with an increased cancer risk and a more aggressive tumor growth, but also connected with an aggravated liver damage caused by chronic inflammation. Meanwhile, increasing evidence has demonstrated that 1,25(OH)₂D₃ (the most biologically active metabolite of vitamin D) can inhibit inflammatory response in some chronic inflammatory associated cancer, which is considered to have the anti-tumor potency. However, the interaction between 1,25(OH)₂D₃ and inflammation during hepatocellular carcinoma (HCC) initiation and progression is not yet clear. Here, we report an anti-tumorigenesis effect of 1,25(OH)₂D₃ via decreasing inflammatory cytokine secretion in HCC and hypothesize the possible underlying mechanism. Firstly, we show that the enhanced tumor growth is associated with elevated inflammatory cytokine IL-6 and TNF-α in 1α(OH)ase gene-knockout mice. Secondly, 1,25(OH)₂D₃ can inhibit vitamin D receptor (VDR) shRNA interfered tumor cell growth through decreasing inflammatory cytokine secretion in vitro and in vivo. Finally, using p27(kip1) gene knock-out mouse model, we demonstrate that the effect of 1,25(OH)₂D₃ in inhibiting immune cell related inflammatory cytokine secretion, exerts in a p27(kip1) gene dependent way. Collectively, 1,25(OH)₂D₃ inhibits HCC development through up-regulating the expression of p27(kip1) in immune cell and reducing inflammatory cytokine production.

Anti-platelet therapy in the prevention of hepatitis B virus-associated hepatocellular carcinoma

Previous studies in mouse models of self-limited viral hepatitis showed that platelets contribute to acute liver damage by promoting the intrahepatic accumulation of virus-specific CD8 T cells and, secondarily, virus-non-specific inflammatory cells. Built on these observations, a recent preclinical study took advantage of a previously established hepatitis B virus (HBV) transgenic mouse model of immune-mediated chronic hepatitis that progresses to hepatocellular carcinoma (HCC), to demonstrate that clinically achievable doses of the anti-platelet drugs aspirin and clopidogrel - administered continuously after the onset of liver disease - can prevent hepatocarcinogenesis and greatly improve overall survival. These outcomes were preceded by and associated with reduced hepatic accumulation of virus-specific CD8 T cells and virus-non-specific inflammatory cells, reduced hepatocellular injury and hepatocellular proliferation, and reduced severity of liver fibrosis. The observation that anti-platelet therapy inhibits HCC development identifies platelets as key players in the pathogenesis of HBV-associated liver cancer and supports the notion that a sustained immune-mediated necroinflammatory liver disease is sufficient to trigger HCC. The results abovementioned and their clinical implications are discussed in this report.

Effects of Saikosaponin-D on syndecan-2, matrix metalloproteinases and tissue inhibitor of metalloproteinases-2 in rats with hepatocellular carcinoma

ObjectiveTo investigate effects of Saikosaponin D (SSd) on syndecan-2, matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases-2 (TIMP-2) in livers of rat with hepatocellular carcinoma (HCC). MethodsMale SD rats were divided into control (n=10), model (n=20) and SSd (n=20) groups, and model and SSd groups given intragastric 0.2% (w/v) N-diethylnitrosamine to induce HCC. SSd group received 0.03% (w/v) SSd in saline. Liver samples were analysed immunohistochemically for syndecan-2, MMP-2, MMP-13 and TIMP-2 at 16 weeks. ResultsThe model group had more malignant nodules than the SSd group; all model-group HCC cells were grade III; SSd-group HCC cells were grades I-II. Controls showed normal hepatic cell phenotypes and no syndecan-2 + staining. Syndecan-2 + staining was greater in the model group (35.2%, P≤0.001) than in controls or the SSd group (16.5%, P ≤ 0.001). The model group had more intense MMP-2 + staining than controls (0.37 vs 0.27, P≤0.01) or the SSd group (0.31 vs 0.37, P≤0.05); and higher MMP-13+ staining (72.55%) than in controls (12.55%, P≤0.001) and SSd group (20.18%, P≤0.01). The model group also had more TIMP-2 + staining (57.2%) than controls (20.9%, P≤0.001) and SSd group (22.7%, P≤0.001). Controls and SSd group showed no difference in TIMP-2+ rates. ConclusionSSd inhibited HCC development, and downregulated expression of syndecan-2, MMP-2, MMP-13 and TIMP-2 in rat HCC liver tissue.

Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, accounting for an estimated 600,000 deaths annually. Aberrant methylation, consisting of DNA hypomethylation and/or promoter gene CpG hypermethylation, is implicated in the development of a variety of solid tumors, including HCC. We analyzed the global levels of DNA methylation as well as the methylation status of 105 putative tumor suppressor genes and found that the extent of genome-wide hypomethylation and CpG hypermethylation correlates with biological features and clinical outcome of HCC patients. We identified activation of Ras and downstream Ras effectors (ERK, AKT, and RAL) due to epigenetic silencing of inhibitors of the Ras pathway in all HCC. Further, selective inactivation of SPRY1 and -2, DAB2, and SOCS4 and -5 genes and inhibitors of angiogenesis (BNIP3, BNIP3L, IGFBP3, and EGLN2) was associated with poor prognosis. Importantly, several epigenetically silenced putative tumor suppressor genes found in HCC were also inactivated in the nontumorous liver. Our results assign both therapeutic and chemopreventive significance to methylation patterns in human HCC and open the possibility of using molecular targets, including those identified in this study, to effectively inhibit HCC development and progression.

Ursodiol Use Is Possibly Associated with Lower Incidence of Hepatocellular Carcinoma in Hepatitis C Virus–Associated Liver Cirrhosis

Abstract In a previous study of patients with hepatitis C virus (HCV)–associated liver cirrhosis (HCV-LC), we showed that increased liver inflammation, as assessed by higher serum alanine aminotransferase (ALT), was associated with increased risk for the development of hepatocellular carcinoma (HCC). This suggested that suppression of inflammation might inhibit HCC development in HCV-LC. Several agents have been suggested to possess chemopreventive potential against the development of HCC in chronic HCV-associated liver disease, including herbal medicines, such as Stronger-Neo-Minophagen C (glycyrrhizin) and Sho-saiko-to (TJ-9). Ursodiol [ursodeoxycholic acid (UDCA)], a bile acid widely used to treat cholestatic liver diseases, also possesses anti-inflammatory properties in liver disease. We hypothesized that suppression of liver inflammation, as assessed by decreases in serum ALT, might inhibit HCC occurrence in patients with HCV-LC. In this study, the preventive effect of UDCA on HCC was examined in patients with early-stage HCV-LC. One hundred two patients with HCV-LC (Child stage A) were treated with anti-inflammatory drugs, Stronger-Neo-Minophagen C,Sho-saiko-to, or UDCA, with the goal of lowering the average serum ALT level to <80 IU. Iftheaverage ALT level did not remain <80 IU after treatment with one agent, multiagent therapy was initiated. The patients were followed up for >5 years and were retrospectively subdivided into two groups: 56 UDCA users (group A) and 46 UDCA nonusers (group B). The mean ± SD dosage of UDCA administered in group A was 473.7 ± 183.0 mg/d. The average duration of UDCA administration in group A was 37.3 ± 15.9 months over the 5-year study period. The cumulative incidence of HCC was recorded. The 5-year incidence of HCC in group A was 17.9% (10 of 56) and was significantly lower than that in group B (39.1%, 18 of 46; P = 0.025). The risk for HCC incidence, calculated by a logistic regression model, showed that the administration of UDCA significantly decreased hepatocarcinogenesis (P = 0.036). The herbal medicines used were comparable in dosage and treatment duration in the UDCA and non-UDCA groups. In conclusion, UDCA might prevent HCC development in HCV-LC. Interestingly, because the serum ALT trends over time were nearly the same in both groups, the chemopreventive effectiveness of UDCA was not accompanied by greater reductions in ALT compared with the UDCA nonusers.

Different effects of the liver mitogens triiodo-thyronine and ciprofibrate on the development of rat hepatocellular carcinoma.

Previous work has shown that treatment with thyroid hormone (T3) decreased the incidence of rat hepatocellular carcinoma (HCC). The present study was designed to determine whether the inhibitory effect of T3 on HCC development was limited to early steps of the carcinogenetic process or, whether a similar effect could also be exerted by starting T3 treatment at later stages. Hepatic nodules were induced in Fischer rats by a single dose of DENA, followed by a 2-week exposure of the animals to 2-AAF and partial hepatectomy. Rats were then divided into 3 groups: group 1 was maintained on basal diet: group 2 was fed a diet containing 4 mg/kg T3 for a week, every month/7 months, starting 9 weeks after DENA administration: group 3 was exposed to cycles of T3 starting 8 months after initiation. Results demonstrate that inhibition of HCC development was essentially similar in rats exposed to T3 starting either 9 weeks or 8 months after initiation (50% inhibition compared to control rats). We have previously shown that T3-induced nodule regression and HCC inhibition occurred in spite of its mitogenic effect. Therefore, we next wished to determine whether a similar antitumoral effect could be exerted by other liver mitogens, such as peroxisome proliferators. Rats exposed to the initiation-promotion protocol described previously, were subjected to 11 cycles of a T3 or a ciprofibrate-supplemented diet, each cycle consisting of 7 days/month: the incidence of HCC and lung metastases was determined 13.5 months after initiation. Results showed that although treatment with T3 strongly inhibited HCC development (only 31% of T3+ rats showed HCC vs 91% of controls), rats given ciprofibrate developed the same number of HCC as T3-untreated rats. In conclusion, the results of this study showed that the anticarcinogenic effect of T3 is maintained also when treatment begins late in the process, and its antitumoral property appears to be specific and may not be shared by other liver mitogens.

KDR/Flk-1 is a major regulator of vascular endothelial growth factor-induced tumor development and angiogenesis in murine hepatocellular carcinoma cells.

Vascular endothelial growth factor (VEGF), which is one of the most potent angiogenic factors, has been shown to play a pivotal role in tumor angiogenesis, including hepatocellular carcinoma (HCC). The effects of VEGF are mediated mainly through two distinct receptors, flt-1 and KDR/Flk-1. It has been suggested that KDR/Flk-1 plays an important role in tumor development. However, the role of KDR/Flk-1 in HCC has not been examined. We previously reported that VEGF tightly regulated murine HCC development, based on the results of a study using a retroviral tetracycline-regulated (Retro-Tet) gene expression system. This system allows VEGF gene expression to be manipulated in vivo by providing tetracycline in the drinking water. In the present study, we combined the KDR/Flk-1-specific neutralizing monoclonal antibody (KDR/Flk-1mAb) and the Retro-Tet system to elucidate the role of KDR/Flk-1 in VEGF-induced tumor development and angiogenesis in a murine HCC experimental model. In a xenograft study, tumor augmentation induced by VEGF overexpression was almost abolished by means of KDR/Flk-1mAb treatment, with accompanying inhibition of angiogenesis, KDR/Flk-1 autophosphorylation, but not interference of flt-1 activation. This inhibitory effect was achieved even on established tumors and regardless of whether the tumor size was small or large. On the contrary, KDR/Flk-1mAb treatment significantly increased the apoptosis in the tumor. With orthotopic transplantation, KDR/Flk-1mAb also inhibited HCC development in the liver. These results suggest that KDR/Flk-1 is a major regulator of VEGF-mediated HCC development and angiogenesis not only at the initial stage, but also after the tumor has fully developed.