pyrimidinones and Carcinoma--Hepatocellular

Studies in the past decades have shown that active hepatitis B virus (HBV) replication is the key driver of liver injury and disease progression, and thus sustained viral suppression is of paramount importance in the management of chronic HBV infection. The nucleos(t)ide analogues lamivudine, adefovir, entecavir, telbivudine and tenofovir are potent inhibitors of HBV polymerase/reverse transcriptase activity and are highly effective in the suppression of HBV replication, but rarely eliminate the virus. Long-term therapy is usually required to achieve sustained hepatitis B e antigen seroconversion, HBV DNA suppression, ALT normalization and fibrosis reversal. Maintained long-term therapy has been demonstrated to significantly lower the rate of hepatic decompensation and development of cirrhosis or hepatocellular carcinoma. However, drug resistance is a serious risk on prolonged nucleos(t)ide analogue therapy, and this poses a critical challenge. Prevention and proper management of drug resistance are crucial to ensure long-term success.

Topics: Adenine; Adenine Nucleotides; Alanine Transaminase; Carcinoma, Hepatocellular; DNA, Viral; Drug Administration Schedule; Drug Resistance, Viral; Drug Therapy, Combination; Guanine; Hepatitis B e Antigens; Hepatitis B virus; Hepatitis B, Chronic; Humans; Lamivudine; Liver Cirrhosis; Liver Neoplasms; Nucleosides; Organophosphonates; Pyrimidinones; Randomized Controlled Trials as Topic; Reverse Transcriptase Inhibitors; Telbivudine; Thymidine; Treatment Outcome; Virus Replication

The ultimate goal of treatment for chronic hepatitis B (CHB) is to prevent hepatocellular carcinoma (HCC). During the last decade, great strides have been made in the treatment of hepatitis B virus (HBV) infections. Six highly effective anti-HBV agents are currently available and more agents are on the horizon. Prospective and retrospective studies of large numbers of CHB patients with advanced liver disease, including cirrhosis, have demonstrated that the treatment with lamivudine not only delays the disease progression but also reduces the incidence of HCC. In a large prospective study of 3,653 HBV carriers in Taiwan, 164 persons developed HCC in a 12-year follow-up period; an extensive analysis of their condition led to the conclusion that the most important risk factor for HCC is an increased serum level of HBV DNA >10,000 copies/mL regardless of the HBeAg status, alanine aminotransferase levels or presence of cirrhosis. The incidence of HCC correlated with serum HBV DNA level at entry in a dose-response relationship. These pivotal studies re-emphasize the need for an active anti-HBV therapy for CHB patients with viral replication as the ultimate prevention and/or delay for the development of HCC.

Topics: Antiviral Agents; Carcinoma, Hepatocellular; Hepatitis B, Chronic; Humans; Interferons; Lamivudine; Liver Neoplasms; Nucleosides; Pyrimidinones; Telbivudine; Thymidine

The long-term goals of therapy for chronic hepatitis B are to reduce serum HBV DNA to low or undetectable levels and ultimately reduce or prevent the development of cirrhosis and hepatocellular carcinoma.. To review the current treatment of chronic hepatitis B, with a focus on diagnosis and management of resistance and active management of suboptimal responses.. A systematic review of the literature, with a focus on recent guidelines, was undertaken.. Among the six drugs licensed for the treatment of chronic hepatitis B in the US, the preferred agents in 2008 will include entecavir, peginterferon alfa-2a, possibly telbivudine, and tenofovir following licensure. When using an oral agent, a major focus of management is on the selection of a drug with high potency and low rate of resistance, and active on-treatment management to optimize therapy. Preventing the sequelae of antiviral drug resistance and appropriate management when resistance is initially detected are also the major focus of current management. The addition of an antiviral agent that is not cross-resistant is critical to restore suppression of viral replication.. Newer agents and modified treatment strategies, especially using combination therapy, hold promise to optimize the management of patients with chronic hepatitis B by achieving the high potency and the lowest rate of resistance.

Topics: Adenine; Antiviral Agents; Carcinoma, Hepatocellular; Deoxycytidine; Drug Resistance, Viral; Emtricitabine; Guanine; Hepatitis B virus; Hepatitis B, Chronic; Humans; Interferon alpha-2; Interferon-alpha; Lamivudine; Liver Cirrhosis; Nucleosides; Organophosphonates; Polyethylene Glycols; Pyrimidinones; Recombinant Proteins; Reverse Transcriptase Inhibitors; Telbivudine; Tenofovir; Thymidine; Virus Replication

The combination of trametinib and sorafenib has an acceptable safety profile, albeit at doses lower than approved for monotherapy. Maximum tolerated dose is trametinib 1.5 mg daily and sorafenib 200 mg twice daily. The limited anticancer activity observed in this unselected patient population does not support further exploration of trametinib plus sorafenib in patients with hepatocellular carcinoma.. The RAS/RAF/MEK/ERK signaling pathway is associated with proliferation and progression of hepatocellular carcinoma (HCC). Preclinical data suggest that paradoxical activation of the MAPK pathway may be one of the resistance mechanisms of sorafenib; therefore, we evaluated trametinib plus sorafenib in HCC.. This was a phase I study with a 3+3 design in patients with treatment-naïve advanced HCC. The primary objective was safety and tolerability. The secondary objective was clinical efficacy.. A total of 17 patients were treated with three different doses of trametinib and sorafenib. Two patients experienced dose-limiting toxicity, including grade 4 hypertension and grade 3 elevation of aspartate aminotransferase (AST)/alanine aminotransferase (ALT)/bilirubin over 7 days. Maximum tolerated dose was trametinib 1.5 mg daily and sorafenib 200 mg twice a day. The most common grade 3/4 treatment-related adverse events were elevated AST (37%) and hypertension (24%). Among 11 evaluable patients, 7 (63.6%) had stable disease with no objective response. The median progression-free survival (PFS) and overall survival (OS) were 3.7 and 7.8 months, respectively. Phosphorylated-ERK was evaluated as a pharmacodynamic marker, and sorafenib plus trametinib inhibited phosphorylated-ERK up to 98.1% (median: 81.2%) in peripheral blood mononuclear cells.. Trametinib and sorafenib can be safely administered up to trametinib 1.5 mg daily and sorafenib 200 mg twice a day with limited anticancer activity in advanced HCC.

Topics: Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Hepatocellular; Humans; Leukocytes, Mononuclear; Liver Neoplasms; Niacinamide; Phenylurea Compounds; Pyridones; Pyrimidinones; Sorafenib

1. This study assessed the mass balance, metabolism and disposition of [(14)C]trametinib, a first-in-class mitogen-activated extracellular signal-related kinase (MEK) inhibitor, as an open-label, single solution dose (2 mg, 2.9 MBq [79 µCi]) in two male subjects with advanced cancer. 2. Trametinib absorption was rapid. Excretion was primarily via feces (∼81% of excreted dose); minor route was urinary (∼19% of excreted dose). The primary metabolic elimination route was deacetylation alone or in combination with hydroxylation. Circulating drug-related component profiles (composed of parent with metabolites) were similar to those found in elimination together with N-glucuronide of deacetylation product. Metabolite analysis was only possible from <50% of administered dose; therefore, percent of excreted dose (defined as fraction of percent of administered dose recovery over total dose recovered in excreta) was used to assess the relative importance of excretion and metabolite routes. The long elimination half-life (∼10 days) favoring sustained targeted activity was important in permitting trametinib to be the first MEK inhibitor with clinical activity in late stage clinical studies. 3. This study exemplifies the challenges and adaptability needed to understand the metabolism and disposition of an anticancer agent, like trametinib, with both low exposure and a long elimination half-life.

Topics: Absorption; Administration, Oral; Aged; Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Dose-Response Relationship, Drug; Humans; Liver Neoplasms; Male; Melanoma; Middle Aged; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; Radiometry; Radiopharmaceuticals; Rats; Skin Neoplasms

Topics: Adenylyl Cyclases; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Line, Tumor; Cytoskeletal Proteins; Humans; Liver Neoplasms; Neoplastic Stem Cells; Oligopeptides; Pyrimidinones; Sorafenib

Multidrug resistance (MDR) counteracts the efficiency of sorafenib, an important first-line therapy for hepatocellular carcinoma (HCC). Sirtuins (SIRTs) 1 and 2 are associated with tumor progression and MDR. We treated 2D and 3D cultures (which mimic the features of in vivo tumors) from HCC cells with sorafenib alone or in the presence of SIRTs 1 and 2 inhibitors (cambinol or EX-527; combined treatments). Cultures subjected to combined treatments showed a greater fall in cellular viability, proliferation (PCNA, cyclin D1 and Ki-67 expression and cell cycle analysis), migration and invasion when compared with cultures treated only with sorafenib. Similarly, combined treatments produced more apoptosis (annexin V/PI, caspase-3/7 activity) than sorafenib alone. Since cell cycle dysregulation and apoptotic blockage are reported mechanisms of MDR, the modulation found in PCNA, cyclin D1, Ki-67 and caspase-3/7 proteins by cambinol and EX-527 are probably playing a role in enhancing the sensitivity of HCC cell lines to sorafenib. EX-527 reduced MRP3 and BCRP expression in sorafenib-treated HCC cells. Since ABC transporters contribute to MDR, MRP3 and BCRP could be also influencing in the response of HCC cells to sorafenib. Overall, 2D and 3D cultures behave similarly except that 3D cultures were less sensitive to treatments, reinforcing the clinical relevance of the current study. Findings presented in this manuscript support a potential application for SIRTs 1 and 2 inhibitors since we demonstrated that these compounds enhance the inhibitory effect of sorafenib upon treatment of hepatocellular carcinoma cells lines.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; ATP Binding Cassette Transporter, Subfamily G, Member 2; Carbazoles; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Movement; Cell Proliferation; Drug Resistance, Neoplasm; Hep G2 Cells; Histone Deacetylase Inhibitors; Humans; Liver Neoplasms; Multidrug Resistance-Associated Proteins; Naphthalenes; Neoplasm Invasiveness; Neoplasm Proteins; Pyrimidinones; Signal Transduction; Sirtuin 1; Sirtuin 2; Sorafenib; Spheroids, Cellular

Targeting the cell cycle checkpoints and DNA damage response are promising therapeutic strategies for cancer. Adavosertib is a potent inhibitor of WEE1 kinase, which plays a critical role in regulating cell cycle checkpoints. However, the effect of adavosertib on hepatocellular carcinoma (HCC) treatment, including sorafenib-resistant HCC, has not been thoroughly studied. In this study, we comprehensively investigated the efficacy and pharmacology of adavosertib in HCC therapy. Adavosertib effectively inhibited the proliferation of HCC cells in vitro and suppressed tumor growth in HCC xenografts and patient-derived xenograft (PDX) models in vivo. Additionally, adavosertib treatment effectively inhibited the motility of HCC cells by impairing pseudopodia formation. Further, we revealed that adavosertib induced DNA damage and premature mitosis entrance by disturbing the cell cycle. Thus, HCC cells accumulating DNA damage underwent mitosis without G2/M checkpoint arrest, thereby leading to mitotic catastrophe and apoptosis under adavosertib administration. Given that sorafenib resistance is common in HCC in clinical practice, we also explored the efficacy of adavosertib in sorafenib-resistant HCC. Notably, adavosertib still showed a desirable inhibitory effect on the growth of sorafenib-resistant HCC cells. Adavosertib markedly induced G2/M checkpoint arrest and cell apoptosis in a dose-dependent manner, confirming the similar efficacy of adavosertib in sorafenib-resistant HCC. Collectively, our results highlight the treatment efficacy of adavosertib in HCC regardless of sorafenib resistance, providing insights into exploring novel strategies for HCC therapy.

Topics: Animals; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Survival; Dose-Response Relationship, Drug; Drug Delivery Systems; Drug Resistance, Neoplasm; Enzyme Inhibitors; Hep G2 Cells; Humans; Liver Neoplasms; Male; Mice; Mice, Nude; Middle Aged; Phenotype; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidinones; Xenograft Model Antitumor Assays

Accurate discrimination of inflammations and cancers as well as differential inhibition of cancers are significant for early diagnoses and timely treatments. Nanoparticles have become new modalities for diagnosis and therapy. However, they are still challenged by the efficient delivery of multiple reagents into living cells, discriminating multisignals without any interference, and differential treatments of different diseases. Here, multifunctional spiky topological nanocapsules (STNs) are prepared for the discrimination and differential inhibition of inflammation and cancer. With unique spiky hollow architectures, STNs' advantages including excellent loading capacity, enhanced cellular uptake, DNAs' protection against degradation, target-controlled drug release, and efficient endo-/lysosome escape are demonstrated. Therefore, sequential detection of inflammation-related miR-155 (by external modified hairpin DNAs) and the cancer target of monocarboxylate transporter 1 (MCT1) (by internal loaded pH-sensitive carbon dots and MCT1 inhibitor-AZD3965) are achieved. Furthermore, the release of AZD3965 from the cavities of STNs is controlled by the miR-155 amount (first target). Therefore, the released drug of AZD3965 realizes the stage-dependent differential treatment of diseases via cellular acidosis induced by MCT1 inhibition. Via in vivo evaluations of normal, inflammatory, and liver cancer cells/mice, as well as the efficient inhibition of tumor growth, the possibility of STN-based discrimination and differential treatment is confirmed. This would encourage new strategies for multidiagnosis and differential treatment of early-stage cancer.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Drug Liberation; Female; Humans; Inflammation; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Monocarboxylic Acid Transporters; Nanocapsules; Pyrimidinones; Symporters; Thiophenes; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Radiotherapy (RT) has a promising anti-tumor effect depending on its effects on both cancer cells and tumor immune microenvironment (TIME). As one of the most common alterations in hepatocellular carcinoma (HCC), wnt/β-catenin pathway activation, has been reported to induce radioresistance and suppressive TIME. In this study, we aim to explore the effect of wnt/β-catenin inhibitor ICG-001 on radiosensitivity and RT-related TIME of HCC and the underlying mechanism.. C57BL/6 and nude mouse tumor models were used to evaluate the efficacy of different treatments on tumor growth, recurrence and mice survival. Flow cytometry was performed to assess tumor infiltrating lymphocytes (TILs). DNA damage response (DDR) and radioresistance was investigated by colony formation assays, γ-H2AX and micronuclei measurements.. Our findings showed that ICG-001 displayed both local and systematic effects by increasing radiosensitivity and improving immunity in HCC, which indicated that ICG-001 might be a potential synergetic treatment for radiotherapy and radioimmunotherapy in HCC patients.

Topics: Animals; beta Catenin; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Hepatocellular; CD8-Positive T-Lymphocytes; Cell Line, Tumor; DNA Damage; Humans; Liver Neoplasms; Mice; Mice, Inbred C57BL; Mice, Nude; Neoplasm Recurrence, Local; Pyrimidinones; Tumor Microenvironment

The tumor suppressor gene

Topics: Carcinoma, Hepatocellular; Cell Cycle Proteins; CRISPR-Cas Systems; Humans; Liver Neoplasms; Nuclear Proteins; Protein-Tyrosine Kinases; Pyrimidinones; X-linked Nuclear Protein

Phosphatidylinositol-3-phosphate (PI3P) is a lipid that accumulates in the early endosomal membrane, and acts as a scaffold to recruit proteins that contain a PI3P-binding domain, such as the FYVE domain. In this study, we examined the effect of PI3P depletion on the insulin response in rat hepatoma-derived H4IIEC3 cells. We found that insulin treatment induced the transient formation of an actin domain structure, a mesh-like tangled network of actin filaments where phosphorylated Akt, endosomal proteins, and PI3P accumulated. Actin domain formation was repressed by the depletion of PI3P by SAR405, an inhibitor of the class III PI3 kinase, Vps34, by the inhibition of PI3P function by the competitive binding of an excess amount of GST-fused 2xFYVE protein to intracellular PI3P, and by the use of diabetic model cells, in which PI3P was depleted. SAR405 did not affect the phosphorylation level of Akt, and the transcriptional regulation of gluconeogenic and cholesterol synthetic genes after insulin treatment. Interestingly, insulin-induced DNA synthesis was specifically inhibited by SAR405, cytochalasin B, and also in diabetic model cells. These results suggest that PI3P is required for the formation of actin domains, which affected a signaling pathway downstream of Akt associated with DNA synthesis in H4IIEC3 cells.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Class III Phosphatidylinositol 3-Kinases; Cytochalasin B; DNA, Neoplasm; Insulin; Liver Neoplasms; Phosphatidylinositol Phosphates; Protein Domains; Proto-Oncogene Proteins c-akt; Pyridines; Pyrimidinones; Rats

Hepatocellular carcinoma (HCC) is an aggressive malignancy. In HCC, mitogen-activated protein kinase (MAPK) signaling is overactivated. The MAPK kinase (MEK) inhibitor trametinib has been approved to treat several types of advanced cancers with a BRAF mutation. Herein, we examined whether trametinib has efficacy against HCC.. The effects of trametinib on cell viability, proliferation and tumor growth were assessed in HCC-derived cell lines and mouse xenograft models. Western blot analysis and immunohistochemistry were used to identify key regulators critical for HHC cell proliferation and tumor growth.. We found that trametinib dose-dependently inhibited the viability and proliferation of HCC cells. We also found that a strong suppression of MEK by trametinib downregulated the pro-survival protein MYC, but upregulated the pro-apoptotic protein BIM. This dual differential regulation of MYC and BIM was found to be accompanied by upregulation of a MYC-targeted cyclin dependent kinase inhibitor, p27. Collectively, our data indicate that trametinib exhibits efficacy in treating HCC cells via distinct regulation of the MYC and BIM pathways. As such, targeting MEK to block MAPK signaling with trametinib may provide novel treatment opportunities for HCC.

Topics: Animals; Bcl-2-Like Protein 11; Carcinoma, Hepatocellular; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Mice, Nude; Mitogen-Activated Protein Kinase Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-myc; Pyridones; Pyrimidinones; Tumor Burden; Xenograft Model Antitumor Assays

The epidermal growth factor receptor (EGFR) pathway and Hippo signaling play an important role in the carcinogenesis of hepatocellular carcinoma (HCC). However, the crosstalk between these two pathways and its implications in targeted therapy remains unclear. We found that the activated EGFR signaling could bypass RhoA to promote the expression of YAP(Yes-associated protein), the core effector of the Hippo signaling, and its downstream target Cyr61. Further studies indicated that EGFR signaling mainly acted through the PI3K-PDK1 (Phosphoinositide 3-kinase-Phosphoinositide-dependent kinase-1) pathway to activate YAP, but not the AKT and MAPK pathways. While YAP knockdown hardly affected the EGFR signaling. In addition, EGF could promote the proliferation of HCC cells in a YAP-independent manner. Combined targeting of YAP and EGFR signaling by simvastatin and the EGFR signaling inhibitors, including the EGFR tyrosine kinase inhibitor (TKI) gefitinib, the RAF inhibitor sorafenib and the MEK inhibitor trametinib, presented strong synergistic cytotoxicities in HCC cells. Therefore, the EGFR-PI3K-PDK1 pathway could activate the YAP signaling, and the activated EGFR signaling could promote the HCC cell growth in a YAP-independent manner. Combined use of FDA-approved inhibitors to simultaneously target YAP and EGFR signaling presented several promising therapeutic approaches for HCC treatment.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; Adaptor Proteins, Signal Transducing; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Hepatocellular; Cell Proliferation; ErbB Receptors; Gefitinib; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Molecular Targeted Therapy; Phosphatidylinositol 3-Kinase; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; Signal Transduction; Simvastatin; Sorafenib; Transcription Factors; YAP-Signaling Proteins

Sirtuins (SIRTs) 1 and 2 deacetylases are overexpressed in hepatocellular carcinoma (HCC) and are associated with tumoral progression and multidrug resistance (MDR). In this study we analyzed whether SIRTs 1 and 2 activities blockage was able to affect cellular survival and migration and to modulate p53 and FoxO1 acetylation in HepG2 and Huh7 cells. Moreover, we analyzed ABC transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein 3 (MRP3) expression. We used cambinol and EX-527 as SIRTs inhibitors. Both drugs reduced cellular viability, number of colonies and cellular migration and augmented apoptosis. In 3D cultures, SIRTs inhibitors diminished spheroid growth and viability. 3D culture was less sensitive to drugs than 2D culture. The levels of acetylated p53 and FoxO1 increased after treatments. Drugs induced a decrease in ABC transporters mRNA and protein levels in HepG2 cells; however, only EX-527 was able to reduce MRP3 mRNA and protein levels in Huh7 cells. This is the first work demonstrating the regulation of MRP3 by SIRTs. In conclusion, both drugs decreased HCC cells survival and migration, suggesting SIRTs 1 and 2 activities blockage could be beneficial during HCC therapy. Downregulation of the expression of P-gp and MRP3 supports the potential application of SIRTs 1 and 2 inhibitions in combination with conventional chemotherapy.

Topics: Acetylation; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B; Carbazoles; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Survival; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Liver Neoplasms; Multidrug Resistance-Associated Proteins; Naphthalenes; Neoplasm Proteins; Protein Processing, Post-Translational; Pyrimidinones; Sirtuin 1; Sirtuin 2

Monastrol and its analog oxomonastrol differ by replacement of the sulfur atom present in monastrol to an oxygen atom in oxomonastrol. Monastrol inhibits the mitotic kinesin family member 11 (EG5), which has been studied for its potential use in cancer therapy. The aim of this study was to investigate the effect of monastrol and oxomonastrol on HepG2/C3A cells. Our results showed that monastrol induced DNA damage, reduced cell proliferation, and up-regulated the cytochrome P450 family 1 subfamily A member 1 (CYP1A1) mRNA levels. However, oxomonastrol was cytotoxic only at the highest concentrations used, without reducing cell proliferation and viability. Moreover, no genotoxic damage or alteration of levels of mRNA were found. Our results suggest that monastrol has greater antiproliferative activity compared to oxomonastrol, and this effect is probably related to the DNA damage induced by monastrol and its possible bioactivation demonstrated by the increase in CYP1A1 mRNA expression. Moreover, these effects appear to be related to the presence of the sulfur atom in its structure.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Proliferation; Cell Survival; Comet Assay; Cytochrome P-450 CYP1A1; DNA Damage; Drug Screening Assays, Antitumor; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Kinetics; Liver Neoplasms; Pyrimidines; Pyrimidinones; RNA, Messenger; Spindle Apparatus; Thiones

It has been a challenge to identify liver tumor suppressors or oncogenes due to the genetic heterogeneity of these tumors. We performed a genome-wide screen to identify suppressors of liver tumor formation in mice, using CRISPR-mediated genome editing.. We identified 4 candidate liver tumor suppressor genes not previously associated with liver cancer (Nf1, Plxnb1, Flrt2, and B9d1). CRISPR-mediated knockout of Nf1, a negative regulator of RAS, accelerated liver tumor formation in mice. Loss of Nf1 or activation of RAS up-regulated the liver progenitor cell markers HMGA2 and SOX9. RAS pathway inhibitors suppressed the activation of the Hmga2 and Sox9 genes that resulted from loss of Nf1 or oncogenic activation of RAS. Knockdown of HMGA2 delayed formation of xenograft tumors from cells that expressed oncogenic RAS. In human HCCs, low levels of NF1 messenger RNA or high levels of HMGA2 messenger RNA were associated with shorter patient survival time. Liver cancer cells with inactivation of Plxnb1, Flrt2, and B9d1 formed more tumors in mice and had increased levels of mitogen-activated protein kinase phosphorylation.. Using a CRISPR-based strategy, we identified Nf1, Plxnb1, Flrt2, and B9d1 as suppressors of liver tumor formation. We validated the observation that RAS signaling, via mitogen-activated protein kinase, contributes to formation of liver tumors in mice. We associated decreased levels of NF1 and increased levels of its downstream protein HMGA2 with survival times of patients with HCC. Strategies to inhibit or reduce HMGA2 might be developed to treat patients with liver cancer.

Topics: Animals; Benzimidazoles; Blotting, Western; Butadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; CRISPR-Cas Systems; Cytoskeletal Proteins; DNA, Neoplasm; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genes, Neurofibromatosis 1; Genome-Wide Association Study; Hepatocytes; High-Throughput Nucleotide Sequencing; HMGA Proteins; HMGA2 Protein; Humans; Immunohistochemistry; Liver Neoplasms; Liver Neoplasms, Experimental; Membrane Glycoproteins; Mice; Mice, Knockout; Mice, Nude; Mitogen-Activated Protein Kinases; Nerve Tissue Proteins; Niacinamide; Nitriles; Phenylurea Compounds; Prognosis; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-myc; Pyridones; Pyrimidinones; ras Proteins; Real-Time Polymerase Chain Reaction; Receptors, Cell Surface; Sequence Analysis, DNA; Sorafenib; Survival Analysis; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Wee1 kinase inhibitors are effective radiosensitizers in cells lacking a G1 checkpoint. In this study we examined the potential effect of Wee1 kinase inhibition on inducing replication stress in hepatocellular carcinoma (HCC).. Five independent datasets from the Oncomine database comparing gene expression in HCC compared to normal tissue were combined and specific markers associated with Wee1 sensitivity were analyzed. We then performed a series of in vitro experiments to study the effect of Wee1 inhibition on irradiated HCC cell lines with varying p53 mutational status. Clonogenic survival assays and flow cytometry using anti-γH2AX and phospho-histone H3 antibodies with propidium iodide were performed to study the effect of AZD1775 on survival, cell cycle, and DNA repair. Additionally, nucleoside enriched medium was used to examine the effect of altering nucleotide pools on Wee1 targeted radiation sensitization.. Our analysis of the Oncomine database found high levels of CDK1 and other cell cycle regulators indicative of Wee1 sensitivity in HCC. In our in vitro experiments, treatment with AZD1775 radiosensitized and chemosensitized Hep3B, Huh7, and HepG2 cell lines and was associated with delayed resolution of γH2AX foci and the induction of pan-nuclear γH2AX staining. Wee1 inhibition attenuated radiation-induced G2 arrest in the Hep3B (TP53 null) and Huh7 (TP53 mutant) cell lines but not in the TP53 wild-type cell line HepG2. Supplementation with nucleosides reversed the radiation-sensitizing effect of AZD1775 and reduced the amount of cells with pan-nuclear γH2AX staining after radiation.. Radiation sensitization with Wee1 inhibition occurs in cells regardless of their p53 mutational status. In this study we show for the first time that replication stress via the overconsumption of nucleotides plays an important role in AZD1775-induced radiation sensitization.

Topics: Carcinoma, Hepatocellular; CDC2 Protein Kinase; Cell Cycle Proteins; DNA Damage; DNA Replication; G2 Phase Cell Cycle Checkpoints; Genes, p53; Hep G2 Cells; Histones; Humans; Liver Neoplasms; Mutation; Nuclear Proteins; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones; Radiation-Sensitizing Agents; Stress, Physiological

Sorafenib, a multikinase inhibitor, is currently the only approved drug for advanced hepatocellular carcinoma (HCC). The current study tested the hypothesis whether inhibition of the Wnt/β-catenin signaling pathway could improve the anti-tumor effects of sorafenib in HCC. ICG-001, a small molecule which blocks the interaction of β-catenin with its transcriptional coactivator CBP, dose-dependently enhanced the growth-suppressive and apoptosis-induction effects of sorafenib in multiple HCC cell lines. Downregulation of β-catenin by RNA interference increased sorafenib sensitivity, whereas overexpression of β-catenin reduced sorafenib sensitivity in Huh7 cells. The sorafenib-sensitization effect of short hairpin RNA (shRNA)-mediated β-catenin downregulation in Huh7 cells was attenuated by β-catenin overexpression. Mechanistically, sorafenib combined with ICG-001 or shRNA-mediated β-catenin downregulation augmented the induction of apoptosis, and resulted in a significant downregulation of Mcl-1 in HCC cells. In Huh7 cell mouse xenograft model, the combination of ICG-001 and sorafenib showed a more significant growth-retarding effect than single agent treatment of sorafenib or ICG-001. Our data indicate that inhibition of the Wnt/β-catenin signaling pathway improves the antitumor effects of sorafenib against HCC in vitro and in vivo.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; beta Catenin; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Hepatocellular; Cell Proliferation; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; Myeloid Cell Leukemia Sequence 1 Protein; Niacinamide; Phenylurea Compounds; Protein Kinase Inhibitors; Pyrimidinones; RNA Interference; RNAi Therapeutics; Sorafenib; Transfection; Wnt Signaling Pathway; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC), the third most common cause of cancer-related deaths worldwide, lacks effective medical therapy. Large subsets of HCC demonstrate Wnt/β-catenin activation, making this an attractive therapeutic target. We report strategy and characterization of a novel small-molecule inhibitor, ICG-001, known to affect Wnt signaling by disrupting β-catenin-CREB binding protein interactions. We queried the ZINC online database for structural similarity to ICG-001 and identified PMED-1 as the lead compound, with ≥70% similarity to ICG-001. PMED-1 significantly reduced β-catenin activity in hepatoblastoma and several HCC cells, as determined by TOPflash reporter assay, with an IC50 ranging from 4.87 to 32 μmol/L. Although no toxicity was observed in primary human hepatocytes, PMED-1 inhibited Wnt target expression in HCC cells, including those with CTNNB1 mutations, and impaired cell proliferation and viability. PMED-1 treatment decreased β-catenin-CREB binding protein interactions without affecting total β-catenin levels or activity of other common kinases. PMED-1 treatment of Tg(OTM:d2EGFP) zebrafish expressing GFP under the β-catenin/Tcf reporter led to a notable decrease in β-catenin activity. The PMED effect on β-catenin signaling lasted from 12 to 24 hours in vitro and 6 to 15 hours in vivo. Thus, using a rapid and cost-effective computational methodology, we have identified a novel and specific small-molecule inhibitor of Wnt signaling that may have implications for HCC treatment.

Topics: Animals; beta Catenin; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Hepatocellular; Cell Line, Tumor; CREB-Binding Protein; Drug Discovery; Humans; Inhibitory Concentration 50; Liver Neoplasms; Pyrimidinones; Structure-Activity Relationship; Wnt Signaling Pathway; Zebrafish

Sirtuins (SIRT1-7) are a highly conserved family of NAD(+)-dependent enzymes that control the activity of histone and nonhistone regulatory proteins. SIRT1 is purposed to promote longevity and to suppress the initiation of some cancers. Nevertheless, SIRT1 is reported to function as a tumor suppressor as well as an oncogenic protein. Our data show that compared with normal liver or surrounding tumor tissue, SIRT1 is strongly overexpressed in human hepatocellular carcinoma (HCC). In addition, human HCC cell lines (Hep3B, HepG2, HuH7, HLE, HLF, HepKK1, skHep1) were screened for the expression of the sirtuin family members and only SIRT1 was consistently overexpressed compared with normal hepatocytes. To determine its effect on HCC growth, SIRT1 activity was inhibited either with lentiviruses expressing short hairpin RNAs or with the small molecule inhibitor, cambinol. Knockdown or inhibition of SIRT1 activity had a cytostatic effect, characterized by an altered morphology, impaired proliferation, an increased expression of differentiation markers, and cellular senescence. In an orthotopic xenograft model, knockdown of SIRT1 resulted in 50% fewer animals developing tumors and cambinol treatment resulted in an overall lower tumor burden. Taken together, our data show that inhibition of SIRT1 in HCC cells impairs their proliferation in vitro and tumor formation in vivo. These data suggest that SIRT1 expression positively influences the growth of HCC and support further studies aimed to block its activity alone or in combination as a novel treatment strategy.

Topics: Animals; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Disease Models, Animal; Female; Gene Expression; Gene Knockdown Techniques; Humans; Liver Neoplasms; Mice; Naphthalenes; Pyrimidinones; Sirtuin 1; Transplantation, Heterologous

Sirtuins and hypoxia-inducible transcription factors (HIF) have well-established roles in regulating cellular responses to metabolic and oxidative stress. Recent reports have linked these two protein families by demonstrating that sirtuins can regulate the activity of HIF-1 and HIF-2. Here we investigated the role of SIRT1, a NAD+-dependent deacetylase, in the regulation of HIF-1 activity in hypoxic conditions. Our results show that in hepatocellular carcinoma (HCC) cell lines, hypoxia did not alter SIRT1 mRNA or protein expression, whereas it predictably led to the accumulation of HIF-1α and the up-regulation of its target genes. In hypoxic models in vitro and in in vivo models of systemic hypoxia and xenograft tumor growth, knockdown of SIRT1 protein with shRNA or inhibition of its activity with small molecule inhibitors impaired the accumulation of HIF-1α protein and the transcriptional increase of its target genes. In addition, endogenous SIRT1 and HIF-1α proteins co-immunoprecipitated and loss of SIRT1 activity led to a hyperacetylation of HIF-1α. Taken together, our data suggest that HIF-1α and SIRT1 proteins interact in HCC cells and that HIF-1α is a target of SIRT1 deacetylase activity. Moreover, SIRT1 is necessary for HIF-1α protein accumulation and activation of HIF-1 target genes under hypoxic conditions.

Topics: Animals; Benzamides; Blotting, Western; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Liver Neoplasms, Experimental; Mice; Mice, Knockout; Mice, Nude; Naphthalenes; Naphthols; Protein Binding; Pyrimidinones; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Sirtuin 1; Transcriptional Activation; Transplantation, Heterologous; Tumor Burden

Over the past decade, advances in the antiviral therapy in patients with chronic hepatitis B have enabled the sustained suppression of hepatitis B viral replication and the prevention of progressive liver disease. Hepatitis B surface antigen (HBsAg) has been used to diagnose patients with hepatitis B virus infection. Recently, test for quantitative HBsAg titers are available and on-treatment HBsAg quantitations are used to predict treatment outcome. Serum HBV DNA levels have been shown to predict natural course of chronic hepatitis B infection. The HBV DNA levels have been reported to be positively correlated with the development of cirrhosis, hepatocellular carcinoma and related death. The baseline and on-treatment levels of HBV DNA are important factors for predicting treatment outcomes. In this article, we will discuss the role of HBV DNA and HBsAg quantitation during antiviral therapy.

Topics: Antiviral Agents; Carcinoma, Hepatocellular; DNA, Viral; Hepatitis B Surface Antigens; Hepatitis B virus; Hepatitis B, Chronic; Humans; Lamivudine; Liver Cirrhosis; Liver Neoplasms; Nucleosides; Pyrimidinones; Telbivudine; Thymidine

Hepatocellular carcinoma (HCC) is the 5th most common cancer worldwide. It is intrinsically resistant toward standard chemotherapy, making it imperative to develop novel selective chemotherapeutic agents. The Wnt/beta-catenin pathway plays critical roles in development and oncogenesis, and is dysregulated in HCC. Our study aims to evaluate the activity of 3 small molecule antagonists of the Tcf4/beta-catenin complex (PKF118-310, PKF115-584 and CGP049090) on HCC cell lines in vitro and in vivo. All 3 chemicals displayed dose-dependent cytotoxicity in vitro against all 3 HCC cell lines (HepG2, Hep40 and Huh7), but were at least 10 times less cytotoxic to normal hepatocytes (from 3 donors) by using ATP assay. In HepG2 and Huh7 cells, treatment with the antagonists decreased Tcf4/beta-catenin binding capability and transcriptional activity, associated with downregulation of the endogenous Tcf4/ beta-catenin target genes c-Myc, cyclin D1 and survivin. In HepG2 and Huh7 cells, treatment with the antagonists induced apoptosis and cell cycle arrest at the G1/S phase. All antagonists suppressed in vivo tumor growth in a HepG2 xenograft model, associated with apoptosis and reduced c-Myc, cyclin D1 and survivin expressions. Our results suggest that these 3 antagonists of the Tcf4/beta-catenin complex are potential chemotherapeutic agents which may offer a pathway specific option for the clinical management of HCC.

Topics: Animals; Antineoplastic Agents; Apoptosis; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; beta Catenin; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclin D1; Dose-Response Relationship, Drug; Down-Regulation; Drug Screening Assays, Antitumor; Gene Expression Regulation, Neoplastic; Genes, myc; Hepatocytes; Humans; Immunohistochemistry; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Mice; Mice, Nude; Microtubule-Associated Proteins; Perylene; Pyrimidinones; Random Allocation; Signal Transduction; Survivin; Transcription Factor 4; Transcription Factors; Triazines; Wnt Proteins; Xenograft Model Antitumor Assays

Topics: Animals; Carbon Isotopes; Carcinoma, Hepatocellular; Cell Nucleolus; Centrifugation, Density Gradient; Humans; Liver; Liver Neoplasms; Liver Regeneration; Male; Microscopy, Electron; Neoplasms, Experimental; Nucleotides; Orotic Acid; Phosphorus Isotopes; Purines; Pyrimidinones; Rats; RNA, Neoplasm