epidermal-growth-factor and Carcinoma--Hepatocellular

The study aims to provide a comprehensive account of the association between the epidermal growth factor (EGF) + 61A/G polymorphism (rs4444903) and susceptibility to virus-related hepatocellular carcinoma (HCC).. Electronic searching of the Chinese National Knowledge Infrastructure, Wanfang, Chinese Scientific Journal Database (VIP), PubMed, Web of Science, and Embase was conducted to select eligible studies. Odds ratios (OR) and 95% confidence intervals (95%CI) were calculated to assess the strength of the association.. In this study, a total of 18 articles were included with 2692 cases and 5835 controls for assessing the association between rs4444903 and HCC risk. The pooled results showed that the EGF + 61A/G polymorphism was significantly associated with the risk of virus-related HCC in all genetic models. Stratified analyses were conducted based on ethnicity, study quality, source of controls, type of controls, number of cases and genotyping method. The results showed that EGF + 61A/G polymorphisms significantly affect HCC susceptibility in different stratified populations. High heterogeneity was observed across included studies, and meta-regression analysis demonstrated that race, type of controls, and study quality contribute to the observed heterogeneity.. This pooled analysis found that EGF + 61A/G polymorphism was significantly associated with the risk of HCC.

Topics: Carcinoma, Hepatocellular; Epidermal Growth Factor; Genetic Predisposition to Disease; Hepatitis; Hepatitis Viruses; Humans; Liver Neoplasms; Polymorphism, Single Nucleotide; Risk Factors

The epidermal growth factor (EGF) rs4444903 polymorphism is associated with aberrant expression of EGF, which was a characteristic of cirrhotic liver diseases, induces highly malignant hepatocellular carcinoma (HCC). Numerous studies have uncovered the association of this polymorphism with the risk of liver disease, but with inconsistent findings.. Therefore, this meta-analysis was performed to evaluate whether EGF rs4444903 polymorphism conferred susceptibility to liver disease. Totally 18 eligible articles were identified by searching PubMed, Google, CNKI and EMBASE up to December 1, 2020.. Our results indicated that there was no significant difference in the minor G allele frequency of rs4444903 polymorphism between HBV/HCV carriers and healthy controls. In other words, EGF rs4444903 polymorphism was not associated with the risk of HBV/HCV. Interestingly, this polymorphism increased the risk of liver cirrhosis in the controls with HCV infection. Additionally, EGF rs4444903 polymorphism is associated with the increased risk of HCC under the five models. Subgroup analysis by ethnicity shows that rs4444903 polymorphism intensifies the risk of HCC among Asians and Caucasians. Strong correlation is also reported in controls with cirrhosis or HCV infection and studies using PCR-RFLP genotyping.. The study supports that EGF rs4444903 polymorphism is a genetic contributor to liver cirrhosis and HCC in the overall population. Nevertheless, this conclusion must be confirmed by larger studies with more diverse ethnic populations.

Topics: Carcinoma, Hepatocellular; Epidermal Growth Factor; Genetic Predisposition to Disease; Hepatitis B virus; Hepatitis C; Humans; Liver Cirrhosis; Liver Neoplasms

The epidermal growth factor (EGF) may play a pathological role in hepatocellular carcinoma (HCC). However, the conclusions of published reports on the relationship between the EGF 61*A/G polymorphism and HCC risk remain controversial. To derive a more precise estimation we performed a meta-analysis based on 14 studies that together included 2,506 cases and 4,386 controls. PubMed, EMBASE, Web of Knowledge and the Chinese National Knowledge Infrastructure (CNKI) databases were used to retrieve articles up to August 1, 2014. The crude odds ratios (ORs) with 95% confidence intervals (95%CIs) were calculated to evaluate the association. Meta-analysis results showed a significant association between the EGF 61*A/G polymorphism and HCC risk in all four genetic models (allele model: OR=1.25, 95%CI=1.12-1.40; dominant model: OR=1.32, 95%CI=1.14-1.54; recessive model: OR=1.33, 95%CI=1.12-1.58; homozygous model: OR=1.59, 95%CI=1.33- 1.90). Moreover, significant associations were observed when stratified by ethnicity, source of controls, etiology and genotype methods. Thus, this meta-analysis suggests that the G-allele of the EGF 61*A/G polymorphism is associated with an increased risk of HCC, especially in Asians and Caucasians, without influence from the source of controls or etiological diversity. Further studies with larger population sizes are needed to confirm these results.

Topics: Alleles; Asian People; Carcinoma, Hepatocellular; Case-Control Studies; Epidermal Growth Factor; Genetic Predisposition to Disease; Genotype; Humans; Liver Neoplasms; Polymorphism, Genetic; Risk; Risk Factors; White People

The association between epidermal growth factor (EGF) gene +61A/G polymorphism (rs4444903) and hepatocellular carcinoma (HCC) susceptibility has been widely reported, but the results were inconsistent. To clarify the effect of this polymorphism on HCC risk, a meta-analysis was performed.. The PubMed, Embase, Cochrane Library, Web of Science, Chinese BioMedical Literature (CBM), Wanfang and Chinese National Knowledge Infrastructure (CNKI) databases were systematically searched to identify relevant studies published up to December 2013. Data were extracted independently by two authors. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated to assess the strength of association.. A total of 16 studies including 2475 HCC cases and 5381 controls were included in this meta-analysis. Overall, a significantly increased HCC risk was observed under all genetic models (G vs. A: OR = 1.383, P < 0.001, 95% CI: 1.174-1.629; GG vs. GA + AA: OR = 1.484, P < 0.001, 95% CI: 1.198-1.838; GG + GA vs. AA: OR = 1.530, P < 0.001, 95% CI: 1.217-1.924; GG vs. AA: OR = 1.958, P < 0.001, 95% CI: 1.433-2.675; GA vs. AA: OR = 1.215, P = 0.013, 95% CI: 1.041-1.418). In the subgroup analyses by ethnicity, a significant association with HCC risk was found in Asian populations (G vs. A: OR = 1.151, P = 0.001, 95% CI: 1.056-1.255), European populations (G vs. A: OR = 1.594, P = 0.027, 95% CI: 1.053-2.413, and African populations (G vs. A: OR = 3.599, P < 0.001, 95% CI: 2.550-5.080), respectively.. Our study shows that EGF +61A/G polymorphism is significantly associated with the increased HCC risk, especially in Asian populations. Further large-scale and well-designed studies are required to confirm this conclusion.

Topics: Asian People; Carcinoma, Hepatocellular; Epidermal Growth Factor; Genetic Association Studies; Genetic Predisposition to Disease; Humans; Liver Neoplasms; Polymorphism, Single Nucleotide; Risk Factors; White People

Hepatocarcinogenesis is a complex process that may be influenced by many factors, including polymorphism in the epidermal growth factor (EGF) gene. Previous work suggests an association between the EGF 61*A/G polymorphism (rs4444903) and susceptibility to hepatocellular carcinoma (HCC), but the results have been inconsistent. Therefore, we performed a meta-analysis of several studies covering a large population to address this controversy.. PubMed, EMBASE, Google Scholar and the Chinese National Knowledge Infrastructure databases were systematically searched to identify relevant studies. Data were abstracted independently by two reviewers. A meta-analysis was performed to examine the association between EGF 61*A/G polymorphism and susceptibility to HCC. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated.. Eight studies were chosen in this meta-analysis, involving 1,304 HCC cases (1135 Chinese, 44 Caucasian and 125 mixed) and 2,613 controls (1638 Chinese, 77 Caucasian and 898 mixed). The EGF 61*G allele was significantly associated with increased risk of HCC based on allelic contrast (OR = 1.29, 95% CI = 1.16-1.44, p<0.001), homozygote comparison (OR = 1.79, 95% CI = 1.39-2.29, p<0.001) and a recessive genetic model (OR = 1.34, 95% CI = 1.16-1.54, p<0.001), while patients carrying the EGF 61*A/A genotype had significantly lower risk of HCC than those with the G/A or G/G genotype (A/A vs. G/A+G/G, OR = 0.66, 95% CI = 0.53-0.83, p<0.001).. The 61*G polymorphism in EGF is a risk factor for hepatocarcinogenesis while the EGF 61*A allele is a protective factor. Further large and well-designed studies are needed to confirm this conclusion.

Topics: Carcinoma, Hepatocellular; Case-Control Studies; Epidermal Growth Factor; Genetic Predisposition to Disease; Hospitals; Humans; Liver Neoplasms; Polymorphism, Genetic; Publication Bias

The association between hepatocellular carcinoma (HCC) and the 61A>G polymorphism in the epidermal growth factor (EGF) gene has been analyzed in several studies, but results have been inconsistent. The aim of this study was to integrate previous findings and explore whether this polymorphism is associated with susceptibility to HCC. A meta-analysis was performed by searching PubMed, Web of Science, and Cochrane Library databases. Data were extracted using predefined form and pooled odds ratios (OR) with 95% confidence intervals (CI) and were calculated to evaluate the strength of this association. Five studies involving 690 cases, 514 healthy controls, and 1419 controls with cancer-free liver diseases were identified. On the basis of healthy controls, the significant main effects on HCC risk were observed in a heterozygote comparison (OR=1.76, 95% CI 1.07-2.90, p=0.02) and a dominant genetic model (OR=1.65, 95% CI 1.03-2.66, p=0.04). On the basis of the controls with cancer-free liver diseases, a significantly increased risk of HCC was found in all the genetic models. Subgroup analyses stratified by ethnicity and etiology of HCC also showed positive associations. The EGF 61G allele is a risk factor for developing HCC without the influence of ethnic and etiological diversity.

Topics: Carcinoma, Hepatocellular; Epidermal Growth Factor; Genetic Predisposition to Disease; Genotype; Humans; Liver Neoplasms; Polymorphism, Genetic; Risk Factors

Hepatocellular cancer (HCC) is the fifth most common cause of cancer worldwide and its incidence is increasing as a result of the dissemination of hepatitis B and C virus infection. Surgical resection and liver transplantation are considered the only cures for HCC, but benefit approximately 10-15% of patients. In addition, radiofrequency ablation may is potentially curative for patients' with small HCC. Some patients with unresectable disease confined to the liver may benefit from embolisation or chemoembolisation. In the presence of disease not amenable to loco-regional therapy, median survival is only a few months. Current systemic therapy with cytotoxic chemotherapy induces relatively few responses and has no clear survival benefit. Current interest is focussed on the potential role of targeted therapies based on the key aspects of molecular pathogenesis of HCC, most notably sorafenib, an oral multikinase inhibitor. Recent developments discussed in this article demonstrate the potential benefits of this drug which seems destined to become first-line therapy for advanced HCC.

Topics: Antineoplastic Agents; Benzenesulfonates; Carcinoma, Hepatocellular; Clinical Trials as Topic; Epidermal Growth Factor; Humans; Liver Neoplasms; Molecular Targeted Therapy; Niacinamide; Phenylurea Compounds; Protein Kinase Inhibitors; Pyridines; Sorafenib; Vascular Endothelial Growth Factor A

Systemic chemotherapy has had a disappointing track record in the management of advanced hepatocellular carcinoma (HCC). Single-agent doxorubicin produces a response rate of 10-15%, but without any survival benefit, and combination chemotherapy has also yielded unimpressive results. With recent advances in the knowledge of hepato-carcinogenesis, there has been encouraging development in the systemic therapy of advanced HCC patients, and particularly in the targeted therapy of advanced HCC. Among the newly identified targets, exciting results have been shown in targeting the anti-angiogenic pathway and the Raf/mitogen-activated protein kinase pathways. Bevacizumab, both as a single agent and in combination with other agents, has shown initial encouraging activity in treating advanced HCC. More recently, single-agent sorafenib, a putative multitargeted kinase inhibitor, has shown to prolong the overall survival of patients with advanced HCC in the pivotal phase III Sorafenib HCC Assessment Randomized Protocol (SHARP) and Oriental study. Currently, sorafenib is the only approved targeted therapy for patients with advanced HCC. In addition, however, promising early results have been reported for other molecular-targeted drugs including erlotinib and sunitinib. Future progress seems likely to depend on using controlled clinical trials to optimize synergistic combination treatments.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Benzenesulfonates; Bevacizumab; Carcinoma, Hepatocellular; Drug Delivery Systems; Epidermal Growth Factor; Humans; Mitogen-Activated Protein Kinases; Neovascularization, Pathologic; Niacinamide; Phenylurea Compounds; Phosphatidylinositol 3-Kinases; Pyridines; Signal Transduction; Sorafenib

Hepatocellular carcinoma (HCC) is one of the 5th most common cancers around the world with a limited number of systemic therapeutic options. Cytotoxic agents, hormonotherapy and immunotherapy have failed to demonstrate benefit compared to best supportive care in patients with advanced HCC. The recent development of targeted therapies provided hope for the treatment of advanced HCC. We reviewed phases II-III trials presented in 2007 and 2008. Results are promising with a clinical benefit reported with molecular therapies targeting EGF/EGFR and VEGF/VEGFR pathways.

Topics: Angiogenesis Inhibitors; Antibodies, Monoclonal; Carcinoma, Hepatocellular; Clinical Trials, Phase II as Topic; Clinical Trials, Phase III as Topic; Epidermal Growth Factor; ErbB Receptors; Forecasting; Humans; Liver Neoplasms; Protein Kinase Inhibitors; Receptors, Vascular Endothelial Growth Factor; Vascular Endothelial Growth Factor A

Topics: Carcinoma, Hepatocellular; Drug Delivery Systems; Epidermal Growth Factor; ErbB Receptors; Humans; Neovascularization, Pathologic

Hepatocellular carcinoma (HCC) is one of the most vascular solid tumors, in which angiogenesis plays an important role. The status of angiogenesis in HCC correlates with the disease progression and prognosis, and thus provides a potential therapeutic target. This review summarizes the vascular changes and molecular and cellular basis of angiogenesis in HCC. Development of HCC is characterized by arterialization of its blood supply and sinusoidal capillarization. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that plays a critical role in mediating angiogenesis in HCC. The VEGF can function on various types of cells, such as endothelial cells, hepatic stellate cells, endothelial progenitor cells and hemangiocytes, to induce vascular changes in HCC. Therefore, blockade of VEGF-mediated pathways, either by anti-VEGF neutralizing antibody or tyrosine kinase inhibitors that target VEGF receptors, suppresses carcinogenesis and angiogenesis in HCC. In addition to VEGF, several other angiogenic factors in HCC have recently been identified. These factors can also regulate angiogenic processes through interaction with VEGF or VEGF-independent pathways. Despite the fact that treatment of HCC remains a tough task due to lack of effective systemic therapy, antiangiogenic therapy has already entered clinical trials in HCC patients and sheds light on a promising novel treatment for this disease.

Topics: Angiogenesis Inhibitors; Angiopoietins; Animals; Carcinoma, Hepatocellular; Endothelial Cells; Epidermal Growth Factor; Humans; Liver Neoplasms; Microcirculation; Neoplastic Stem Cells; Neovascularization, Pathologic; Thymidine Phosphorylase; Vascular Endothelial Growth Factor A

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cetuximab; Clinical Trials, Phase II as Topic; Disease Models, Animal; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Gefitinib; Humans; Ligands; Liver Neoplasms; Multicenter Studies as Topic; Neoplasm Recurrence, Local; Protein Kinase Inhibitors; Quinazolines; Rats; Signal Transduction; Transforming Growth Factor alpha

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Epidermal Growth Factor; fas Receptor; Heparin-binding EGF-like Growth Factor; Hepatitis C; Humans; Intercellular Signaling Peptides and Proteins; Liver Diseases; Mice; Signal Transduction

Hepatocellular carcinoma (HCC) is a common solid malignant tumor occurring worldwide that leads to the third largest cause of death compared to other cancers. Genetic and environmental factors are involved in the pathogenesis of HCC. Epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) can stimulate the proliferation of epidermal and epithelial cells. The EGF signal pathway has a relationship with the growth of the embryo, tissue repairing, and tumorigenesis.. In this study, 416 patients with hepatitis B virus infection (HBV)-related HCC and 645 individuals who had never been infected with HBV of the Chinese Han population were enrolled. Eight single-nucleotide polymorphisms (SNPs), whose minor allele frequency >20% in the EGF and EGFR genes, were genotyped to examine their associations with hepatocarcinogenesis. Genotyping experiments were carried out using TaqMan.. There were significant differences in genotype distributions (p=0.005) and allele frequencies (p=0.001, odds ratio [OR]=1.43, 95% confidence interval [CI]=1.15-1.79) of rs11569017 in the EGF gene between the HCC and control groups. After binary logistic regression to determine independent factors for susceptibility to HCC under an additive model, rs11569017 was still independently associated with the susceptibility to HCC (p=0.021, OR=1.48, 95% CI=1.06-2.07), but no significant differences in other SNPs were found. Additionally, the haplotype T-G constructed by rs11569017 and rs4444903 of the EGF gene might increase the risk of HBV-related HCC (p=0.002, OR=1.44, 95% CI=1.15-1.82).. The rs11569017 T allele was associated with susceptibility to HBV-related HCC.

Topics: Adult; Aged; Alleles; Asian People; Carcinoma, Hepatocellular; China; Epidermal Growth Factor; ErbB Receptors; Female; Hepatitis B; Hepatitis B virus; Humans; Liver Neoplasms; Male; Middle Aged; Polymorphism, Single Nucleotide

Previous studies demonstrated that ASP-3 was a novel calcium-binding protein from Arca subcrenata that effectively inhibited the proliferation of HepG2 cells. To further study the antitumor activity and mechanism of ASP-3, the cytotoxic effects of recombinant ASP-3 were evaluated in HepG2 cells. The results demonstrated that ASP-3 inhibited the proliferation of HepG2 cells by competitively binding to the EGF binding pocket of EGFR and inhibiting the JAK-STAT, RAS-RAF-MEK-ERK, and PI3K-Akt-mTOR signaling pathways mediated by EGFR. ASP-3 significantly inhibited tumor growth in a HepG2 cell subcutaneous xenograft nude mouse model, and its (25 mg/kg and 75 mg/kg) tumor inhibition rates were 46.92 % and 60.28 %, respectively. Furthermore, the crystal structure of ASP-3 was resolved at 1.4 Å. ASP-3 formed as a stable dimer and folded as an EF-Hand structure. ASP-3 stably bound to domain I and domain III of the EGFR extracellular region by using molecular docking and molecular dynamics simulation analysis. Compared with the endogenous ligand EGF, ASP-3 displayed a stronger interaction with EGFR. These experimental results indicated that recombinant ASP-3 possessed an effective anti-hepatoma effect. So, it might be a potential molecule for liver cancer therapy.

Topics: Animals; Calcium-Binding Proteins; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Humans; Liver Neoplasms; Mice; Molecular Docking Simulation; Phosphatidylinositol 3-Kinases; Xenograft Model Antitumor Assays

The effect of targeted therapy for metastatic hepatocellular carcinoma (HCC) is still unsatisfactory. Exploring the underlying mechanism of HCC metastasis is favorable to provide new therapeutic strategies. T-box (TBX) transcription factor family genes, which are crucial regulators in embryo and organ development, are vital for regulating tumor initiation, growth and metastasis. Here we explored the role of TBX19 in HCC metastasis, which is one of the most upregulated TBX family genes in human HCC tissues. TBX19 expression was markedly upregulated in HCC tissues and elevated TBX19 expression predicted poor prognosis. Overexpression of TBX19 enhanced HCC metastasis through upregulating epidermal growth factor receptor (EGFR) and Rac family small GTPase 1 (RAC1) expression. Downregulation of EGFR and RAC1 inhibited TBX19-mediated HCC metastasis, while upregulation of EGFR and RAC1 restored inhibition of HCC metastasis mediated by TBX19 knockdown. Furthermore, epidermal growth factor (EGF)/EGFR signaling upregulated TBX19 expression via the extracellular signal-regulated kinase (ERK)/nuclear factor (NF)-kB axis. Besides, the combined application of EGFR inhibitor Erlotinib and RAC1 inhibitor NSC23766 markedly inhibited TBX19-mediated HCC metastasis. In HCC cohorts, TBX19 expression was positively associated with EGFR and RAC1 expression. Patients with positive coexpression of TBX19/EGFR or TBX19/RAC1 displayed the poorest prognosis. In conclusion, EGF/EGFR signaling upregulated TBX19 expression via ERK/NF-kB pathway and TBX19 fostered HCC metastasis by enhancing EGFR and RAC1 expression, which formed an EGF-TBX19-EGFR positive feedback loop. Targeting this signaling pathway may offer a potential therapeutic strategy to efficiently restrain TBX19-mediated HCC metastasis.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Liver Neoplasms; Neoplasm Metastasis; rac1 GTP-Binding Protein; T-Box Domain Proteins; Transcription Factors

The long non-coding RNA LIMT (lncRNA inhibiting metastasis) acts as a tumor suppressor factor in some cancers. However, the biological role of LIMT in hepatocellular carcinoma (HCC) has not been explored.. Quantitative real-time PCR was performed to evaluate the expression of LIMT in HCC tissue. The effects of LIMT on tumor growth and metastasis were assessed by in vitro experiments, including colony formation and transwell assays, and in vivo in nude mouse models. Western blot analysis was used to evaluate the expression levels of proteins associated with epithelial-mesenchymal transition (EMT). LIMT expression was significantly lower in HCC than in normal liver tissue. Functionally, overexpression of LIMT repressed the proliferation, invasion, and EMT of HCC cells, while LIMT knockdown increased proliferation, invasion, and EMT of HCC cells in vitro. Furthermore, LIMT overexpression suppressed HCC growth and metastasis while silencing of LIMT had an opposite effect in vivo. Finally, LIMT overexpression reversed EGF-induced EMT.. Our results suggest that LIMT could play an anti-cancer effect in HCC and might be a potential novel therapeutic target in HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Liver Neoplasms; Mice; RNA, Long Noncoding

Circular dorsal ruffles (CDRs) are rounded membrane ruffles induced on the dorsal surfaces of cells stimulated by growth factors (GF). They can serve as signal platforms to activate AKT protein kinase. After GF stimulation, phosphatidylinositol 3-kinase (PI3K) generates phosphatidylinositol (3,4,5)-triphosphate (PIP3) in the plasma membrane. PIP3 accumulates inside CDRs, recruits AKT into the structures, and phosphorylates them (pAKT). Given the importance of the PI3K-AKT pathway in GF signaling, CDRs are likely involved in cell growth. Interestingly, some cancer cell lines express CDRs. We hypothesized that CDRs contribute to carcinogenesis by modulating the AKT pathway. In the present study, we identified CDR-expressing cancer cell lines and investigated their cellular functions.. CDR formation was examined in six cancer cell lines in response to epidermal growth factor (EGF) and insulin. The morphology of the CDRs was characterized, and the related signaling molecules were observed using confocal and scanning electron microscopy. The role of CDRs in the AKT pathway was studied using biochemical analysis. The actin inhibitor cytochalasin D (Cyto D) and the PI3K inhibitor TGX221 were used to block CDRs.. GF treatment induced CDRs in the hepatocellular carcinoma (HCC) Hep3B cell line, but not in others, including HCC cell lines HepG2 and Huh7, and the LO2 hepatocyte cell line. Confocal microscopy and western blot analysis showed that the PI3K-PIP3-AKT pathway was activated at the CDRs and that receptor proteins were recruited to the structures. Cyto D and TGX221 completely blocked CDRs and partially attenuated GF-induced pAKT. These results indicate that CDRs regulate the receptor-mediated PI3K-AKT pathway in Hep3B cells and the existence of CDR-independent pAKT mechanisms.. Our results showed that CDRs modulate the AKT pathway in Hep3B cells. Since CDRs were not observed in other HCC and hepatocyte cell lines, we propose that CDRs in Hep3B would determine the carcinoma characteristic of the cell by aberrantly triggering the AKT pathway. Signaling molecules involved in CDR formation are promising therapeutic targets for some types of HCC. Video abstract.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Epidermal Growth Factor; Humans; Liver Neoplasms; Phosphatidylinositol 3-Kinase; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt

m. Univariate Cox regression analysis of 24 mARGs yielded 13 prognostic genes, which were then analyzed for their enriched functions and pathways. After LASSO regression analysis, a prognostic signature was constructed and its reliability validated. Patients were grouped by risk using the signature score, and then the clinical prognosis, the immune landscape, and the oxidative stress landscape between the two groups were analyzed. Drug sensitivity analysis was performed to identify potentially efficient therapeutic agents.. Thirteen prognosis-related mARGs consistently clustered patients with HCC into four groups with significantly different prognosis. Four mARGs (. A prognostic model consisting of mARGs can be used to predict the prognosis of HCC patients. The risk grouping of our model can be used to reveal differences in the tumor immune microenvironment of patients with HCC. Further in-depth study may provide new targets for future treatment.

Topics: Biomarkers, Tumor; Carcinoma, Hepatocellular; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Liver Neoplasms; Oxaliplatin; Oxidative Stress; Prognosis; Reproducibility of Results; Tumor Microenvironment

LIM kinase 1 (LIMK1) is a serine/threonine and tyrosine kinase that is predominantly located in the cytoplasm. In our study, nuclear translocation of LIMK1 in clinical hepatocellular carcinoma (HCC) samples was demonstrated for the first time, especially in samples from those with intravascular tumour thrombus. LIMK1 was overexpressed in HCC tissues, and nuclear LIMK1 expression was associated with poor prognosis in HCC patients. Although the effects of cytoplasmic LIMK1 on cofilin phosphorylation and actin filament dynamics have been well studied, the function of nuclear LIMK1 is still unclear. Gain- and loss-of-function experiments were performed both in vitro and in vivo and demonstrated a correlation between nuclear LIMK1 and the enhanced aggressive phenotype of HCC. EGF could drive the nuclear translocation of LIMK1 by activating the interaction of p-ERK and LIMK1 and facilitating their roles in nuclear shuttling. Moreover, nuclear LIMK1 could directly bind to the promoter region of c-Myc and stimulate c-Myc transcription. Although the EGFR monoclonal antibody cetuximab has a poor therapeutic effect on advanced HCC patients, in vivo animal study showed that cetuximab achieved a significant inhibitory effect on the progression of nuclear LIMK1-overexpressing HCC cells. In addition, recent data have demonstrated the potential of cetuximab in combination therapy for HCC patients with LIMK1 nuclear translocation.

Topics: Carcinoma, Hepatocellular; Disease Progression; Epidermal Growth Factor; Genes, myc; Humans; Lim Kinases; Liver Neoplasms

Hepatocellular carcinoma (HCC), the sixth most common malignancy worldwide, is characterized by a dismal prognosis due to high recurrence and metastasis rates. Thus, the need for the development of novel chemotherapeutic drugs is urgent. Cyclovirobuxine D (CVB-D), a steroidal alkaloid extracted from

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Drugs, Chinese Herbal; Epidermal Growth Factor; Focal Adhesion Protein-Tyrosine Kinases; Hep G2 Cells; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Snail Family Transcription Factors

The therapeutic effects of C16, which is an inhibitor of RNA-dependent protein kinase (PKR), on growth of hepatocellular carcinoma (HCC) cells and tumor progression in vitro and in vivo were evaluated. Huh7 cells, a human HCC cell line, were used. The effects of C16 on cell viability were evaluated with the MTT assay, and real-time RT-PCR was performed. Huh7 cells were grafted into immunodeficient mice, and the in vivo effects of C16 on tumorigenesis were examined. C16 suppressed proliferation of HCC cells in a dose-dependent manner in vitro. Mouse models with xenograft transplantation showed that the inhibitor suppressed the growth of HCC cells in vivo. Moreover, C16 decreased angiogenesis in HCC tissue in the xenograft model. Consistent with these results in mice, transcript levels of vascular endothelial growth factor-A and factor-B, platelet-derived growth factor-A and factor-B, fibroblast growth factor-2, epidermal growth factor, and hepatocyte growth factor, which are angiogenesis-related growth factors, were significantly decreased by C16 in vitro. In conclusion, the PKR inhibitor C16 blocked tumor cell growth and angiogenesis via a decrease in mRNA levels of several growth factors. C16 may be useful in the treatment of HCC.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; eIF-2 Kinase; Epidermal Growth Factor; Female; Fibroblast Growth Factors; Hep G2 Cells; Hepatocyte Growth Factor; Humans; Indoles; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neovascularization, Pathologic; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; RNA, Messenger; Thiazoles; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor B; Xenograft Model Antitumor Assays

DaHuangWan (DHW) is a traditional herbal medicine used by Mongolian to treat liver cancer for many years. Clinical application of the drug has been shown to help control tumor progression, prolong survival and improve quality of life. However, the underlying mechanisms and side effects of this drug remain unclear, which greatly limits the clinical application and further optimization of DHW. In this study, we found that DHW inhibits the proliferation of hepatoma cells by modulating the epithelial growth factor (EGF) signaling pathway. Berberine and Costunolide are the main active ingredients in DHW. Interestingly, the combination of Berberine and Costunolide has a dramatic synergistic effect on inhibiting the proliferation of hepatoma cells. Neither Berberine nor Costunolide directly block EGFR phosphorylation. Berberine promotes endocytosis of activated EGFR, while as Costunolide increases ubiquitination of EGFR and reduces EGFR recycling to cell membrane distribution, thereby inhibiting EGF signaling. Berberine and Costunolide target two different steps in regulating the EGF signaling, which explains the synergistic anti-cancer effect of DHW. Since Berberine and Costunolide do not directly target EGFR phosphorylation, DHW could be a supplementary medicine to tyrosine kinase inhibitors in cancer therapy.

Topics: Apoptosis; Berberine; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; Flow Cytometry; Herbal Medicine; Humans; Liver Neoplasms; Medicine, East Asian Traditional; Mongolia; Plants, Medicinal; Sesquiterpenes

Epithelial-mesenchymal transition (EMT) is a major cellular process in which epithelial cells lose cell polarity and cell-cell adhesion and become motility and invasiveness by transforming into mesenchymal cells. Catechol is one of the natural compounds present in fruits and vegetables and has various pharmacological and physiological activities including anti-carcinogenic effects. However, the effects of catechol on EMT has not been reported. Epidermal growth factor (EGF) is one of the growth factors and is known to play a role in inducing EMT. The present study showed that catechol suppressed not only the morphological changes to the mesenchymal phenotype of epithelial HCC cells, but also the reduction of E-cadherin and the increment of Vimentin, which are typical hallmark of EMT. In addition, catechol suppressed EMT-related steps such as migration, invasion, anoikis resistance acquisition, and stem cell-like characterization through the EGFR-AKT-ERK signaling pathway during liver cancer metastasis. Therefore, these results suggest that catechol may be able to regulate the early metastasis of liver cancer in vitro.

Topics: Carcinoma, Hepatocellular; Catechols; Cell Line, Tumor; Cell Movement; Cell Proliferation; Enzyme Activation; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Neoplastic Stem Cells; Proto-Oncogene Proteins c-akt; Signal Transduction; Snail Family Transcription Factors

The protein Dickkopf-1 (DKK1) is frequently overexpressed at the transcript level in hepatocellular carcinoma (HCC) and promotes metastatic progression through the induction of β-catenin, a Wnt signaling effector. We investigated how

Topics: Acetylation; Animals; Carcinoma, Hepatocellular; Cell Line; Epidermal Growth Factor; Histones; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Male; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Proteins; Phosphorylation; Rats; Rats, Sprague-Dawley; Transcription, Genetic

Epithelial-to-mesenchymal transition (EMT), important cellular process in metastasis of primary tumors, is characterized by loss of their cell polarity, disruption of cell-cell adhesion, and gain certain properties of mesenchymal phenotype that enable migration and invasion. Delphinidin is a member of anthocyanidin belong to flavonoid groups, known as having pharmacological and physiological effects including anti-tumorigenic, antioxidative, anti-inflammatory, and antiangiogenic effects. However, the effects of delphinidin on EMT is rarely investigated. Epidermal growth factor (EGF) is known as a crucial inducer of EMT in various cancer including hepatocellular carcinoma (HCC). To determine whether delphinidin inhibits EGF-induced EMT in HCC cells, antiproliferative effect of delphinidin on Huh7 and PLC/PRF/5 cells were measured by Cell Counting Kit-8 assay. As a result, delphinidin inhibited cell proliferation in a dose-dependent manner. Based on the result of proliferation, to measure the effects of delphinidin on EGF-induced EMT, we designated a proper concentration of delphinidin, which is not affected to cell proliferation. We found that delphinidin inhibits morphological changes from epithelial to mesenchymal phenotype by EGF. Moreover, delphinidin increased the messenger RNA and protein expression of E-cadherin and decreased those of Vimentin and Snail in EGF-induced HCC cells. Also, delphinidin prevented motility and invasiveness of EGF-induced HCC cells through suppressing activation of matrix metalloproteinase 2, EGF receptor (EGFR), AKT, and extracellular signal-regulated kinase (ERK). Taken together, our findings demonstrate that delphinidin inhibits EGF-induced EMT by inhibiting EGFR/AKT/ERK signaling pathway in HCC cells.

Topics: Anthocyanins; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; MAP Kinase Signaling System; Neoplasm Proteins

Patients with advanced hepatocellular carcinoma (HCC) will almost always develop acquired tolerance after sorafenib therapy, and the molecular mechanism of sorafenib tolerance remains poorly characterized. Here, using our established sorafenib-resistant HCC cell and xenograft models, we identified a novel gene, KIAA1199, which was markedly elevated among the differentially expressed genes involved in sorafenib tolerance. Moreover, elevated expression of KIAA1199 was positively correlated with a high risk of recurrence and metastasis and advanced TNM stage in HCC patients. Functionally, loss- and gain-of-function studies showed that KIAA1199 promoted the migration, invasion, and metastasis of sorafenib-resistant HCC cells. Mechanistically, KIAA1199 is required for EGF-induced epithelial-mesenchymal transition (EMT) in sorafenib-resistant HCC cells by aiding in EGFR phosphorylation. In summary, our data uncover KIAA1199 as a novel sorafenib-tolerant promoting gene that plays an indispensable role in maintaining sorafenib-resistant HCC cell metastasis.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Drug Resistance, Neoplasm; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Hep G2 Cells; Heterografts; Humans; Hyaluronoglucosaminidase; Liver Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Metastasis; Phosphorylation; Sorafenib

GP78 is an autocrine motility factor (AMF) receptor (AMFR) with E3 ubiquitin ligase activity that plays a significant role in tumor cell proliferation, motility, and metastasis. Aberrant extracellular signal-regulated kinase (ERK) activation via receptor tyrosine kinases promotes tumor proliferation and invasion. The activation of GP78 leads to ERK activation, but its underlying mechanism is not fully understood. Here, we show that GP78 is required for epidermal growth factor receptor (EGFR)-mediated ERK activation. On one hand, GP78 interacts with and promotes the ubiquitination and subsequent degradation of dual-specificity phosphatase 1 (DUSP1), an endogenous negative regulator of mitogen-activated protein kinases (MAPKs), resulting in ERK activation. On the other hand, GP78 maintains the activation status of EGFR, as evidenced by the fact that EGF fails to induce EGFR phosphorylation in GP78-deficient cells. By the regulation of both EGFR and ERK activation, GP78 promotes cell proliferation, motility, and invasion. Therefore, this study identifies a previously unknown signaling pathway by which GP78 stimulates ERK activation via DUSP1 degradation to mediate EGFR-dependent cancer cell proliferation and invasion.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dual Specificity Phosphatase 1; Epidermal Growth Factor; ErbB Receptors; HEK293 Cells; Humans; Liver Neoplasms; MAP Kinase Signaling System; Neoplasm Invasiveness; Phosphorylation; Proteolysis; Receptors, Autocrine Motility Factor; Ubiquitination

Better understanding of metastasis process would allow for the development of effective approaches to treat hepatocellular carcinoma (HCC). Recent literature has highlighted the fundamental role of interaction between tumor cells and their microenvironment components in tumor metastasis. Aberrant expression of epidermal growth factor (EGF) induces highly malignant HCC, and activated EGF/EGFR signaling is correlated with an aggressive phenotype and intrahepatic metastasis. Thus, EGF in the tumor microenvironment may influence the behavior of HCC cells. In this study, for the first time, we studied the expression of EGF in HCCs, and the potential role of EGF in the motility of HCC cells and the underlying mechanisms. It was demonstrated that EGF was highly expressed in HCCs and positively associated with higher tumor grade. In addition, EGF promoted the migration and invasion of HCC cells mainly via induction of fibronectin (FN) in vitro. Mechanistically, EGF simultaneously increased the nuclear translocation and PKC mediated phosphorylation of p65 which could bind to the -356 bp to -259 bp fragment of FN promoter, leading to a markedly increased activity of FN promoter in HCC cells. These results highlight the potential role of EGF in promoting HCC metastasis, demonstrate a novel pathway for regulation of FN expression and provide potential targets for HCC prevention and treatment.

Topics: Carcinoma, Hepatocellular; Cell Movement; Epidermal Growth Factor; Fibronectins; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Neoplasm Proteins

To determine the expression and function of Delta/Notch-like EGF-related receptor (DNER) in hepatocellular carcinoma (HCC).. The expression of DNER in 84 HCC tissue samples and matched adjacent noncancerous specimens, as well as HCC cells, were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Survival analysis was evaluated using Kaplan-Meier method. For experiments in vitro, cell viability was measured by Cell Counting Kit-8 Assay and Colony Formation Assay. Furthermore, cell invasion and migration assays were performed with Transwell Assay.. The results showed that DNER was overexpressed in the tissues and cell lines of HCC (all, p < 0.05), and the upregulated expression of DNER was significantly correlated with advanced pathologic stage (p = 0.013) and pathologic-M1 (p = 0.012) in HCC patients. Survival analysis revealed that patients with high DNER levels had worse overall survival (OS) than those with low DNER levels (p = 0.004). More importantly, DNER could be an independent predictor of prognosis for OS (HR = 2.582, 95% CI 1.239-5.380, p = 0.011). In vitro, knockdown of DNER significantly suppressed cell proliferation, colony formation, cell invasion, and cell migration in HepG2 cells. Moreover, inhibition of DNER inactivated PI3K/AKT signaling pathway by downregulating the expression of p-PI3K, p-AKT, and p-70s6k.. Taken together, DNER could promote proliferation, migration, and invasion of HCC cells by regulating the activation of PI3K/AKT pathway, and it might act as a potential prognostic biomarker for HCC.

Topics: Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Epidermal Growth Factor; Female; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Invasiveness; Nerve Tissue Proteins; Phosphatidylinositol 3-Kinases; Prognosis; Proto-Oncogene Proteins c-akt; Receptors, Cell Surface; Signal Transduction; Survival Rate; Transfection

The interaction between hepatocellular carcinoma (HCC) cells and their microenvironment plays a fundamental role in tumor metastasis. The HCC microenvironment is rich in epidermal growth factor (EGF) and tumor necrosis factor α (TNFα), which may cooperatively, rather than individually, interact with tumor cells to influence their biological behavior.. Immunohistochemistry was performed to study the expression of EGF and TNFα in HCCs. Western blotting, immunofluorescence, qRT-PCR, wound healing scratch and invasion assay, and chromatin immunoprecipitation assays were used to study the combined roles of EGF and TNFα in the motility of HCC cells in vitro.. We demonstrated that both EGF and TNFα were highly expressed in HCCs, and HCCs with higher expression of both EGF and TNFα were more frequently rated as high-grade tumors. In vitro, EGF and TNFα cooperatively promoted the motility of HCC cells mainly via synergistic induction of an extracellular matrix glycoprotein fibronectin (FN). Mechanistically, EGF and TNFα jointly increased the nuclear translocation and PKC mediated phosphorylation of NF-κB/p65 which could bind to the -356bp to -259bp fragment of the FN promoter, leading to a markedly increased activity of the FN promoter in HCC cells.. HCCs with higher expression of both EGF and TNFα were more frequently rated as high-grade tumors. EGF and TNFα cooperatively promoted the motility of HCC cells mainly through NF-κB/p65 mediated synergistic induction of FN in vitro.. These findings highlight the crosstalk between EGF and TNFα in promoting HCC, and provide potential targets for HCC prevention and treatment.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Fibronectins; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; NF-kappa B; Phosphorylation; Transcription Factor RelA; Tumor Necrosis Factor-alpha

Hepatocellular carcinoma related protein 1 (HCRP1), which is essential for internalization and degradation of ubiquitinated membrane receptors, is downregulated in several tumors and strongly affects the outcomes of cancer patients. It is reported the expression of HCRP1 is inversely related to epidermal growth factor receptor (EGFR) in breast cancer and lead to resistance to cetuximab in ovarian cancer. However, its exact mechanism in the progression of Hepatocellular carcinoma (HCC) remains unknown. Herein, HCRP1 expression and its clinical significance were examined in 101 HCC patients using immunohistochemistry. Cell proliferation, migration and invasion assays were conducted in HCC cell lines. EGFR activation and degradation were then observed after EGF inducing in HCRP1 knockdown HepG2 cells. In addition, we also detected whether epithelial-to-mesenchymal transition (EMT) was involved in the malignancy promoted by HCRP1. The results showed that 59 of the 101 HCC cases exhibited downregulation of HCRP1 expression (P<0.01) as compared to 30 benign liver lesions and 20 normal liver tissues, all of which showed a high level of HCRP1. HCRP1 expression was significantly related to age (P=0.017), pathological grade (P=0.003), tumor encapsulation (P=0.037), recurrence (P=0.039) and death (P=0.015), but unrelated to cirrhosis (P=0.216), tumor size (P=0.273), and distant metastasis (P=0.554). Lower HCRP1 expression was correlated with shorter RFS and OS (P<0.001), and decreased HCRP1 level is an independent prognostic marker in HCC patients (P<0.05). Overexpression of HCRP1 decreased and knockdown increased HCC cell proliferation, migration and invasion. HCRP1 depletion increased EGFR activation and inhibited EGFR degradation. EMT phenotype was promoted after HCRP1 downregulation via increase of Snail and Twist1 and activation of Akt phosphorylation in HepG2 cells. Conversely, upregulation of HCRP1 in SMMC-7721 cells led to the opposite effect. In conclusion, our study indicated that downregulation of HCRP1 is a valuable prognostic factor involved in EGFR regulation and acquisition of the mesenchymal phenotype of HCC cells.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease-Free Survival; Down-Regulation; Endosomal Sorting Complexes Required for Transport; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Liver Neoplasms; Middle Aged; Multivariate Analysis; Neoplasm Invasiveness; Phosphorylation; Proteolysis; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Transfection

Hepatitis C virus (HCV) infection is accelerated following liver transplantation (LT). Single nucleotide polymorphisms (SNPs) near the epidermal growth factor (EGF) (rs4444903), IL28B (rs12979860), and PNPLA3 (rs738409) loci are associated with treatment response, fibrosis, and hepatocellular carcinoma in non-transplant hepatitis C, but allograft population data are limited. We sought to determine the role of these SNPs in 264 patients with HCV who underwent LT between 1990 and 2008. Genotypes were determined from donor wedge/allograft biopsies and recipient explants. Cox proportional hazards model was used to assess time to cirrhosis, liver-related death, and retransplantation, adjusting for donor age and sustained virological response (SVR). Over a median follow-up of 6.3 yr, a trend toward increased progression to graft cirrhosis was observed among recipients of an EGF non-AA vs. AA donor liver (adjusted HR 2.01; 95% CI 0.93-4.34; p = 0.08). No other genotypes predicted cirrhosis development or graft survival. The CC IL28B variant in both recipients and donors was associated with increased rate of SVR (R-CC/D-CC 8/12[67%], R-non-CC/D-CC or R-CC/D-non-CC 23/52[44%], R-non-CC/D-non-CC 12/45[27%], p linear trend = 0.009). Recipient EGF, IL28B, and PNPLA3, and donor IL28B and PNPLA3 genotypes do not predict adverse outcomes in HCV LT recipients. A potential association exists between donor EGF genotype and cirrhosis.

Topics: Adult; Allografts; Antiviral Agents; Carcinoma, Hepatocellular; Cohort Studies; Disease Progression; Epidermal Growth Factor; Female; Follow-Up Studies; Genotype; Graft Rejection; Graft Survival; Hepacivirus; Hepatitis C, Chronic; Humans; Interferons; Interleukins; Lipase; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Male; Membrane Proteins; Middle Aged; Polymorphism, Single Nucleotide; Postoperative Complications; Prognosis; Risk Factors; Tissue Donors; Transplantation, Homologous; Young Adult

The ganglioside GM3 exerts its different effects via various growth factor receptors. The present study investigated and comparatively analyzed the opposing effects exerted by GM3 on the migration of mouse hepatocellular carcinoma Hepa1‑6 cells via epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (HGFR/cMet). The results demonstrated that GM3 inhibited EGF‑stimulated motility, but promoted HGF‑stimulated motility of the Hepa1‑6 cells via phosphatidylinositol 3‑kinase/Akt‑mediated migration signaling. It is well established that the main cytokines modulating cell proliferation, invasion and metastasis are different in different types of tumor. This difference may, at least in part, explain why GM3 exerted its actions in a tumor‑type specific manner.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; ErbB Receptors; G(M3) Ganglioside; Hepatocyte Growth Factor; Mice; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-met; RNA Interference; Sialyltransferases; Signal Transduction

Deregulated activation of oncogenic transcription factors such as signal transducer and activator of transcription 3 (STAT3) plays a pivotal role in proliferation and survival of hepatocellular carcinoma (HCC). Thus, agents which can inhibit STAT3 activation may have an enormous potential for treatment of HCC patients. Hence, in the present report, we investigated the effect of ascochlorin (ASC), an isoprenoid antibiotic on STAT3 activation cascade in various HCC cell lines and orthotopic mouse model. We observed that ASC could substantially inhibit both constitutive and IL-6/EGF inducible STAT3 activation as well as reduce its DNA binding ability. ASC increased the expression of protein inhibitor of activated STAT3 (PIAS3) which could bind to STAT3 DNA binding domain and thereby down-regulate STAT3 activation. Deletion of PIAS3 gene by siRNA abolished the ability of ASC to inhibit STAT3 activation and induce apoptosis in HCC cells. ASC also modulated the expression of diverse STAT3-regulated oncogenic gene products. Finally, when administered intraperitoneally, ASC also inhibited tumor growth in an orthotopic HCC mouse model and reduced STAT3 activation in tumor tissues. Overall our results indicate that ASC mediates its anti-tumor effects predominantly through the suppression of STAT3 signaling cascade, and can form the basis of novel therapy for HCC patients.

Topics: Alkenes; Animals; Anti-Bacterial Agents; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Caspases; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Proliferation; Cell Survival; DNA; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Ki-67 Antigen; Liver Neoplasms; Mice; Molecular Chaperones; Neoplasm Invasiveness; Phenols; Phosphorylation; Platelet Endothelial Cell Adhesion Molecule-1; Protein Binding; Protein Inhibitors of Activated STAT; Protein Transport; RNA, Messenger; Signal Transduction; STAT3 Transcription Factor

Acquired radioresistance of cancer cells interferes with radiotherapy and increases the probability of cancer recurrence. HepG2-8960-R, which is one of several clinically relevant radioresistant (CRR) cell lines, has a high tolerance to the repeated clinically relevant doses of X-ray radiation. In this study, HepG2-8960-R had slightly lower cell proliferation ability than HepG2 in the presence of FBS. In particular, epidermal growth factor (EGF) hardly enhanced cell proliferation and DNA synthesis in HepG2-8960-R. Additionally, EGF could not induce the activation of Erk1/2, because the expression of EGF receptor (EGFR) protein decreased in HepG2-8960-R in accordance with the methylation of the EGFR promoter region. Therefore, cetuximab did not inhibit HepG2-8960-R cell proliferation. Our study showed that HepG2-8960-R had radioresistant and cetuximab-resistant abilities.

Topics: Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Proliferation; Cetuximab; Epidermal Growth Factor; Hep G2 Cells; Humans; Liver Neoplasms; Methylation; Promoter Regions, Genetic; Radiation Tolerance

Hepatocellular carcinoma (HCC) is on the rise and the sixth most common cancer worldwide. To combat HCC effectively research is directed towards its early detection and the development of targeted therapies. Given the fact that epidermal growth factor (EGF) is an important mitogen for hepatocytes we searched for disease regulated proteins to improve an understanding of the molecular pathogenesis of EGF induced HCC. Disease regulated proteins were studied by 2DE MALDI-TOF/TOF and a transcriptomic approach, by immunohistochemistry and advanced bioinformatics.. Mapping of EGF induced liver cancer in a transgenic mouse model identified n = 96 (p < 0.05) significantly regulated proteins of which n = 54 were tumour-specific. To unravel molecular circuits linked to aberrant EGFR signalling diverse computational approaches were employed and this defined n = 7 key nodes using n = 82 disease regulated proteins for network construction. STRING analysis revealed protein-protein interactions of > 70% disease regulated proteins with individual proteins being validated by immunohistochemistry. The disease regulated network proteins were mapped to distinct pathways and bioinformatics provided novel insight into molecular circuits associated with significant changes in either glycolysis and gluconeogenesis, argine and proline metabolism, protein processing in endoplasmic reticulum, Hif- and MAPK signalling, lipoprotein metabolism, platelet activation and hemostatic control as a result of aberrant EGF signalling. The biological significance of the findings was corroborated with gene expression data derived from tumour tissues to evntually define a rationale by which tumours embark on intriguing changes in metabolism that is of utility for an understanding of tumour growth. Moreover, among the EGF tumour specific proteins n = 11 were likewise uniquely expressed in human HCC and for n = 49 proteins regulation in human HCC was confirmed using the publically available Human Protein Atlas depository, therefore demonstrating clinical significance.. Novel insight into the molecular pathogenesis of EGF induced liver cancer was obtained and among the 37 newly identified proteins several are likely candidates for the development of molecularly targeted therapies and include the nucleoside diphosphate kinase A, bifunctional ATP-dependent dihydroyacetone kinase and phosphatidylethanolamine-binding protein1, the latter being an inhibitor of the Raf-1 kinase.

Topics: Animals; Carcinogenesis; Carcinoma, Hepatocellular; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Mice; Neoplasm Proteins; p38 Mitogen-Activated Protein Kinases; Protein Interaction Maps; Proteomics; Signal Transduction

Up-regulation of core fucosylation catalyzed by α1,6-fucosyltransferase (Fut8) has been observed in hepatocellular carcinoma (HCC). Here, to explore the role of Fut8 expression in hepatocarcinogensis, we established the chemical-induced HCC models in the male wild-type (WT; Fut8(+/+)), hetero (Fut8(+/-)), and knockout (KO; Fut8(-/-)) mice by use of diethylnitrosamine (DEN) and pentobarbital (PB). In the Fut8(+/+) and Fut8(+/-) mice, multiple large and vascularized nodules were induced with an increased expression of Fut8 after DEN and PB treatment. However, the formation of HCC in Fut8(-/-) mice was suppressed almost completely. This potent inhibitory effect of Fut8 deficiency on tumorigenesis was also confirmed by the abolished tumor formation of Fut8 KO human hepatoma cell line cells by use of a xenograft tumor model. Furthermore, loss of the Fut8 gene resulted in attenuated responses to epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in the HepG2 cell line, which provides the possible mechanisms for the contribution of Fut8 to hepatocarcinogensis. Taken together, our study clearly demonstrated that core fucosylation acts as a critical functional modulator in the liver and implicated Fut8 as a prognostic marker, as well as a novel, therapeutic target for HCC.

Topics: Animals; Carcinogenesis; Carcinoma, Hepatocellular; Cell Line, Tumor; Down-Regulation; Epidermal Growth Factor; Fucosyltransferases; Hep G2 Cells; Hepatocyte Growth Factor; Humans; Liver Neoplasms; Male; Mice; Signal Transduction

Blood platelet numbers are correlated to growth and aggressiveness of several tumor types, including hepatocellular carcinoma (HCC). We previously found that platelet lysates (hPLs) also stimulated growth and migration, and antagonized the growth-inhibitory and apoptotic effects of both Sorafenib and Regorafenib, two multikinase inhibitors, on three HCC cell lines. In this study, in vitro function of human epidermal growth factor (EGF) with and without Sorafenib or Regorafenib was investigated.. An ELISA kit was used to evaluate the EGF concentrations in hPLs. In vitro function of EGF was assessed with proliferation MTT test. Apoptosis assay, scratch assays, and Transwell assays were performed for apoptosis, invasion, and migration, respectively. MAPK Activation Kit was used to explore MAPK phosphorylation.. EGF antagonized the growth inhibition of Regorafenib on three HCC cell lines. Regorafenib-mediated growth inhibition was blocked by 70 % when the cells were pre-treated with EGF. EGF also blocked Regorafenib-induced apoptosis, as well as Regorafenib-induced decreases in cell migration and invasion. The EGF effects were in turn antagonized by concomitant addition to the cultures of EGF receptor antagonist Erlotinib, showing that the EGF receptor was involved in the mechanisms of EGF-mediated blocking of Regorafenib effects. Erlotinib also partially blocked the effects of hPLs in antagonizing Regorafenib-mediated growth inhibition, showing that EGF was an important component of hPL actions.. All these results show that EGF antagonized Regorafenib-mediated growth and migration inhibition and apoptosis induction in HCC cells and reinforce the idea that microenvironment can influence cancer drug actions.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; Erlotinib Hydrochloride; Hep G2 Cells; Humans; Liver Neoplasms; MAP Kinase Signaling System; Niacinamide; Phenylurea Compounds; Phosphorylation; Pyridines; Quinazolines; Sorafenib

The focal adhesion protein Tensin4, also known as cten (c-terminal tensin like), is structurally distinct from the three other members in the Tensin family. Its expression and potential functions in cancers including hepatocellular carcinoma (HCC) are not well understood. With immunohistochemistry, 43% (13/30) of our human HCC cases showed up-regulation of Tensin4 as compared with their corresponding non-tumorous livers. In HCC cells, treatment with epidermal growth factor (EGF) significantly induced Tensin4 transcript and protein expression, while treatment with pharmacological inhibitors against the MEK1/2 kinases abolished such induction, suggesting that Tensin4 expression was dependent on Ras/MAPK signaling. With immunofluorescence microscopy, the focal adhesion localization of Tensin4 was confirmed in HCC cells. Significantly, detailed examination using a panel of Tensin4 deletion constructs revealed that this specific focal adhesion localization required the N-terminal region together with the C-terminal SH2 domain. Up-regulation of ERK signaling by EGF in the HCC cells resulted in a change to a mesenchymal cell-like morphology through modulation of the actin cytoskeleton. Functionally, stable Tensin4 knockdown in SMMC-7721 HCC cells resulted in reduced cell proliferation and migration in vitro. Taken together, our data suggest that Tensin4 may play a pro-oncogenic role in HCC, possibly functioning as a downstream effector of Ras/MAPK signaling.

Topics: Actin Cytoskeleton; Adult; Aged; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; Female; Focal Adhesions; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Liver Neoplasms; Male; MAP Kinase Signaling System; Mesenchymal Stem Cells; Microfilament Proteins; Microscopy, Fluorescence; Middle Aged; Signal Transduction; src Homology Domains; Tensins; Up-Regulation

Differentially regulated microRNA (miRNA) are associated with hepatic fibrosis; however, their potential usefulness for blocking hepatic fibrosis has not been exploited fully. We examined the expression of miRNA in the liver of a transgenic mouse model in which platelet-derived growth factor C (PDGF-C) is overexpressed (Pdgf-c Tg), resulting in hepatic fibrosis and steatosis and the eventual development of hepatocellular carcinoma (HCC). Robust induction of miR-214 correlated with fibrogenesis in the liver of Pdgf-c Tg mice, atherogenic high-fat diet-induced NASH mice, and patients with chronic hepatitis B or C. Pdgf-c Tg mice were injected with locked nucleic acid (LNA)-antimiR-214 via the tail vein using Invivofectamine 2.0 and the degree of hepatic fibrosis and tumor incidence were evaluated. Pdgf-c Tg mice treated with LNA-antimiR-214 showed a marked reduction in fibrosis and tumor incidence compared with saline or LNA-miR-control-injected control mice. In vitro, LNA-antimiR-214 significantly ameliorated TGF-β1-induced pro-fibrotic gene expression in Lx-2 cells. MiR-214 targets a negative regulator of EGFR signaling, Mig-6. Mimic-miR-214 decreased the expression of Mig-6 and increased the levels of EGF-mediated p-EGFR (Y1173 and Y845) and p-Met (Tyr1234/1235) in Huh-7 cells. Conversely, LNA-antimiR-214 repressed the expression of these genes. In conclusion, miR-214 appears to participate in the development of hepatic fibrosis by modulating the EGFR and TGF-β signaling pathways. LNA-antimiR-214 is a potential therapy for the prevention of hepatic fibrosis.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Gene Expression; HEK293 Cells; Hep G2 Cells; Humans; Incidence; Liver; Liver Cirrhosis; Liver Neoplasms; Lymphokines; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; MicroRNAs; Oligonucleotides; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-met; Signal Transduction; Transforming Growth Factor beta1

To investigate biological mechanisms underlying pyruvate kinase M2 isoform (PKM2) regulation of cell migration and invasion in hepatocellular carcinoma cells.. HepG2 and Huh-7 hepatocellular carcinoma cell lines were stably transfected and cultured in DMEM (HyClone, Logan, UT, United States). To investigate the effects of PKM2 on cellular proliferation, hepatocellular carcinoma cells were subjected to the Cell Counting Kit-8 (Dojindo, Kamimashiki-gun, Kumamoto, Japan). And investigate the effects of PKM2 on cell signal pathway related with migration and invasion, Western immunoblotting were used to find out the differential proteins. All the antibody used was purchaseed from Cell Signal Technology. In order to explore cell motility used Transwell invasion and wound healing assays. The transwell plate with 0.5 mg/mL collagen type I (BD Bioscience, San Jose, CA)-coated filters. The wound-healing assay was performed in 6-well plates. Total RNA was extracted using TRIzol reagent (Invitrogen, CA, United States) and then reverse transcription was conducted. Quantitative reverse transcription-polymerase chain reaction (PCR) analysis was performed with the ABI 7500 real-time PCR system (Applied Biosystems). We further use digital gene expression tag profiling and identification of differentially expressed genes.. The cells seeded in four 96-well plates were measured OD450 by conducted Cell Counting Kit-8. From this conduction we observed that both HepG2 and Huh-7 hepatocellular carcinoma cells with silenced PKM2 turn on a proliferate inhibition; however, cell migration and invasion were enhanced compared with the control upon stimulation with epidermal growth factor (EGF). Our results indicate that the knockdown of PKM2 decreased the expression of E-cadherin and enhanced the activity of the EGF/EGFR signaling pathway, furthermore up-regulate the subsequent signal molecular the PLCγ1 and extracellular signal-regulated kinase 1/2 expression in the hepatocellular carcinoma cell lines HepG2 and Huh-7, which regulates cell motility. These variations we observed were due to the activation of the transforming growth factor beta (TGFβ) signaling pathway after PKM2 knockdown. We also found that the expression of TGFBRI was increased and the phosphorylation of Smad2 was enhanced. Taken together, our findings demonstrate that PKM2 can regulate cell motility through the EGF/EGFR and TGFβ/TGFR signaling pathways in hepatocellular carcinoma cells.. PKM2 play different roles in modulating the proliferation and metastasis of hepatocellular carcinoma cells, and this finding could help to guide the future targeted therapies.

Topics: Carcinoma, Hepatocellular; Carrier Proteins; Cell Movement; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Membrane Proteins; Neoplasm Invasiveness; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; RNA Interference; Signal Transduction; Thyroid Hormone-Binding Proteins; Thyroid Hormones; Time Factors; Transfection; Transforming Growth Factor beta

RFA (radiofrequency ablation) is an established therapy for HCC (hepatocellular carcinoma). The multikinase inhibitor sorafenib prolongs survival in advanced HCC. We examined the effects of RFA alone and in combination with sorafenib on a bystanding tumour in a two-tumour rat model of HCC. A total of 80 rats were implanted with two liver tumours and randomized to four treatment groups: vehicle and sham operation (control), sorafenib and sham operation (Sora/Sham), vehicle and RFA (Vh/RFA), and sorafenib and RFA (Sora/RFA) (n=10/group per time point). RFA or sham-operation was performed on the left lobe tumour on day 15. Animals were killed at day 18 and day 30. Non-RFA-targeted right lobe tumours were analysed for angiogenesis, growth factors [HGF (hepatocyte growth factor), EGF (epidermal growth factor) and VEGF (vascular endothelial growth factor)] and infiltrating immune cells (CD3 and CD68). At day 30, the non-RFA-targeted tumours were significantly smaller in all three treatment groups compared with control (Sora/Sham P≤0.0001, Vh/RFA P=0.005 and Sora/RFA P≤0.0001). The smallest tumours were observed in animals treated with a combination of sorafenib and RFA, whereas the size reduction seen in the RFA-only group indicated an RFA-mediated distant suppression of tumour growth. Growth factor measurement revealed transiently decreased EGF levels after RFA (P=0.008), whereas sorafenib treatment decreased HGF levels (P=0.001). MVD (microvessel density) was reduced by sorafenib (P=0.002) despite increased VEGF levels (P≤0.0001). The immune parameters revealed augmented T-cells and IL-10 (interleukin 10) levels in all three treatment groups; sorafenib additionally increased macrophage numbers (P≤0.0001). RFA and sorafenib alone resulted in significant volume reduction of the non-RFA-targeted tumour; this effect was enhanced when both modalities were combined.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Catheter Ablation; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; Hepatocyte Growth Factor; Interleukin-10; Liver Neoplasms, Experimental; Macrophages; Niacinamide; Phenylurea Compounds; Rats; Sorafenib; T-Lymphocytes; Transforming Growth Factor beta; Vascular Endothelial Growth Factors

The purpose of this study was to evaluate the gene expression of growth factors and growth factor receptors of primary hepatic masses, including hepatocellular carcinoma (HCC) and nodular hyperplasia (NH), in dogs. Quantitative real-time reverse transcriptase-polymerase chain reaction was performed to measure the expression of 18 genes in 18 HCCs, 10 NHs, 11 surrounding non-cancerous liver tissues and 4 healthy control liver tissues. Platelet-derived growth factor-B (PDGF-B), transforming growth factor-α, epidermal growth factor receptor, epidermal growth factor and hepatocyte growth factor were found to be differentially expressed in HCC compared with NH and the surrounding non-cancerous and healthy control liver tissues. PDGF-B is suggested to have the potential to become a valuable ancillary target for the treatment of canine HCC.

Topics: Animals; Carcinoma, Hepatocellular; DNA Primers; Dog Diseases; Dogs; Electrophoresis, Agar Gel; Epidermal Growth Factor; ErbB Receptors; Focal Nodular Hyperplasia; Gene Expression Regulation, Neoplastic; Hepatocyte Growth Factor; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Proto-Oncogene Proteins c-sis; Real-Time Polymerase Chain Reaction; Receptors, Growth Factor; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor alpha

Epidermal growth factor (EGF) and their receptor (EGFR) play an important role in the development of cancer proliferation, and metastasis, although the mechanism remains unclear. The present study aimed at investigating the role of EGF-EGFR signalling pathway in the development of human hepatocellular carcinoma (HCC) inflammatory environment. Gene profiles of inflammatory cytokines from HCC were measured. Cell bio-behaviours of HCC with low or high metastasis were detected by the live cell monitoring system. Cell proliferation was measured by CCK8. The protein level of CXCL5 and CXCL8 was measured by ELISA. The phosphorylation of PI3K, ERK, MAPK was measured by western blot. EGF significantly induced cell proliferation in HepG2 cells, but not in HCCLM3 cells. EGF prompted the cell movement in both HepG2 and HCCLM3 and regulated the production of CXCL5 and CXCL8 from HCC, which were inhibited by EGFR inhibitor, Erk inhibitor (U0126), or PI3K inhibitors (BEZ-235 and SHBM1009). HCC proliferation, metastasis and production of inflammatory cytokines were regulated via EGF-EGFR signal pathways. CXCL5 could interact with CXCL8, possibly by CXCR2 or the cross-talk between CXCR2 and EGFR. EGF-EGFR signaling pathway can be the potential target of therapies for HCC.

Topics: Butadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chemokine CXCL5; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Inflammation; Interleukin-8; Liver Neoplasms; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Quinolines; Receptor Cross-Talk; Receptors, Interleukin-8B; Signal Transduction; Tumor Microenvironment

In this study, we demonstrate that EGF inhibits the TGF-β1-induced apoptosis of Huh7 cells. TGF-β1 up-regulates the expression of PDCD4 causing apoptosis, by stimulating the synthesis of PDCD4 mRNA via the Smad signaling pathway. TGF-β1 also inhibits the activation of S6 kinase 1 which phosphorylates the serine 67 residue of PDCD4 and leads to the phosphorylation of serine 71 and serine 76 in the β-TRCP binding sequence. This phosphorylation sequence causes the protein to be degraded in the ubiquitin-proteasome system. EGF activates S6 kinase 1 via the PI3K-Akt-mTOR signaling pathway and stimulates the degradation of PDCD4. EGF also suppresses PDCD4 mRNA levels. As the mTOR inhibitor rapamycin up-regulated PDCD4 mRNA levels, the PI3K-Akt-mTOR signaling pathway may control the transcription of the PDCD4 gene as well as the degradation of the protein. TPA also inhibited the TGF-β1-induced apoptosis of Huh7 cells, stimulating the degradation of the PDCD4-protein. Analyses using PDCD4 mutants with changes of serines 67, 71 and 76 to alanine revealed that the phosphorylation of serine 67 is not essential for the TPA-induced suppression of the protein. The mitogens could not suppress the PDCD4-mutant proteins with changes of serine 71 and/or serine 76 to alanine, however, indicating that phosphorylations at these residues are necessary for the proteasome-mediated degradation of PDCD4. The phosphor-mimic S71/D and S76/D mutants were able to be degraded in the ubiquitin-proteasome system unlike the mutants with changes of serine to alanine. The expression of S71/D mutant was suppressed with EGF but that of S76/D mutant was not indicating that at least partly the phosphorylation of both sites was mediated by different enzymes.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Hepatocellular; Cell Line, Tumor; Epidermal Growth Factor; Humans; Liver Neoplasms; Mutation; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; RNA-Binding Proteins; RNA, Messenger; Signal Transduction; Sirolimus; Smad Proteins; TOR Serine-Threonine Kinases; Transcription, Genetic; Transcriptional Activation; Transforming Growth Factor beta1

Aberrant regulation of histone deacetylase 2 (HDAC2) contributes to malignant progression in various cancers, but the underlying mechanism leading to the activation of oncogenic HDAC2 remains unknown. In this study, we show that HDAC2 expression is upregulated in a large cohort of patients with human hepatocellular carcinoma, and that high expression of HDAC2 was significantly associated with poor prognosis of patients with hepatocellular carcinoma. We found that mTORC1/NF-κBp50 signaling is necessary for the growth factor-induced HDAC2 and is sustained in hepatocellular carcinoma, but not in normal hepatic cells. Growth factor-induced mTORC1 activates the nuclear translocation of NF-κBp50, where it binds to the intragenic sequences of the HDAC2 gene and promotes its transcription. Hepatocellular carcinoma tissues derived from chemical-induced mouse and rat liver cancer models validated that mTORC1 activation and NF-κBp50 nuclear translocation are essential for the transcriptional activation of oncogenic HDAC2 in hepatocellular carcinoma. In addition, we demonstrate that HDAC2 is required to maintain mTORC1 activity by stabilizing the mTOR/RAPTOR complex. Elevated expression of HDAC2 triggers a positive feedback loop that activates AKT phosphorylation via the transcriptional modulation of phosphoinositide signaling molecules. Bioinformatics analysis of HDAC2 signature and immunoblot analysis of mesenchymal genes also evidenced that HDAC2 plays a role in the malignant behavior of tumor cells by Snail induction and simultaneously E-cadherin suppression in hepatocellular carcinoma cells. These findings establish a molecular mechanism responsible for the activation of oncogenic HDAC2, which explains how growth factor-induced HDAC2 maintains mitogenic signaling and function during hepatocellular malignant progression and provide a novel strategy for therapeutic intervention in liver cancer. Cancer Res; 74(6); 1728-38. ©2014 AACR.

Topics: Animals; Base Sequence; Carcinoma, Hepatocellular; Disease Progression; Epidermal Growth Factor; ErbB Receptors; Feedback, Physiological; Hep G2 Cells; Histone Deacetylase 2; Humans; Kaplan-Meier Estimate; Liver Neoplasms; Mechanistic Target of Rapamycin Complex 1; Mice; Molecular Sequence Data; Multiprotein Complexes; Neoplasm Invasiveness; Neoplasm Transplantation; NF-kappa B p50 Subunit; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; TOR Serine-Threonine Kinases; Transcriptome

Histone deacetylase 2 (HDAC2) is aberrantly regulated and plays a pivotal role in the development of hepatocellular carcinoma (HCC) through regulation of cell-cycle components at the transcriptional level, but the underlying mechanism leading to oncogenic HDAC2 remains unknown. In this study, we show that expression of CK2α (casein kinase II α subunit) was up-regulated in a large cohort of human HCC patients, and that high expression of CK2α was significantly associated with poor prognosis of HCC patients in terms of five-year overall survival. It was also found that CK2α over-expression positively correlated with HDAC2 over-expression in a subset of HCCs. We observed that treatment with epidermal growth factor (EGF) elicited an increase in CK2α expression and Akt phosphorylation, causing induction of HDAC2 expression in liver cancer cells. It was also observed that ectopic expression of dominant-negative CK2α blocked EGF-induced HDAC2 expression, and that ectopic CK2α expression attenuated the suppressive effect of Akt knockdown on HDAC2 expression in liver cancer cells. Targeted disruption of CK2α influenced the cell cycle, causing a significant increase in the number of liver cancer cells remaining in G₂/M phase, and suppressed growth via repression of Cdc25c and cyclin B in liver cancer cells. Taken together, our findings suggest the oncogenic potential of CK2α in liver tumorigenesis. Furthermore, a regulatory mechanism for HDAC2 expression is proposed whereby EGF induces transcriptional activation of HDAC2 by CK2α/Akt activation in liver cancer cells. Therefore, this makes CK2α a promising target in cancer therapy.

Topics: Carcinogenesis; Carcinoma, Hepatocellular; Casein Kinase II; Cell Line, Tumor; Cell Proliferation; Cohort Studies; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Histone Deacetylase 2; Humans; Liver; Liver Neoplasms; Mutant Proteins; Neoplasm Proteins; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Recombinant Proteins; RNA Interference; Signal Transduction; Survival Analysis

Epidermal growth factor (EGF) gene single-nucleotide polymorphism (SNP) is associated with an increased risk of hepatic tumors. The study aimed to elucidate the impact of EGF SNP and EGF receptor (EGFR) expression on the recurrence of hepatocellular carcinoma (HCC) after hepatectomy. To examine the impact of EGF SNP and EGFR on recurrent HCC, we retrospectively analyzed 141 HCC patients with chronic hepatitis C virus infection who underwent curative hepatectomy. The EGF *61 GG allele was present in 69 patients (48.9%), AG in 56 (39.7%) and AA in 16 (11.4%). The AA group had a significantly lower rate of intrahepatic metastasis (0% vs 16.5%, P = 0.02), lower serum EGF concentration (26.3 ± 15.9 pg/mL vs 43.4 ± 30.5 pg/mL, P = 0.02) and lower proportion of early recurrence (≤2 years; 28.6% vs 71.2%, P = 0.03) than the AG/GG group. The AA group had significantly higher recurrence-free survival than the AG/GG group (P = 0.04), but there was no significant difference in overall survival between these two groups (P = 0.97). High versus low EGFR expression analyzed by immunohistochemical staining in cancer cells was not significantly associated with overall survival (P = 0.37) or recurrence-free survival (P = 0.39). Therefore, EGF *61 AA was associated with a lower risk of recurrence after curative hepatectomy for HCC in patients with hepatitis C virus infection than other genotypes, but EGFR expression in cancer cells was not significantly associated with prognosis.

Topics: Aged; Carcinoma, Hepatocellular; Epidermal Growth Factor; ErbB Receptors; Female; Gene Frequency; Genotype; Hepacivirus; Hepatectomy; Hepatitis C, Chronic; Humans; Liver Neoplasms; Male; Neoplasm Recurrence, Local; Polymorphism, Single Nucleotide; Retrospective Studies

Pyruvate kinase isozyme type M2(PKM2) was first found in hepatocellular carcinoma(HCC), and its expression has been thought to correlate with prognosis. A large number of studies have demonstrated that epithelial-mesenchymal transition (EMT) is a crucial event in hepatocellular carcinoma (HCC) and associated metastasis, resulting in enhanced malignancy of HCC. However, the roles of PKM2 in HCC EMT and metastasis remain largely unknown. The present study aimed to determine the effects of PKM2 in EGF-induced HCC EMT and elucidate the molecular mechanisms in vitro. Our results showed that EGF promoted EMT in HCC cell lines as evidenced by altered morphology, expression of EMT-associated markers, and enhanced invasion capacity. Furthermore, the present study also revealed that nuclear translocation of PKM2, which is regulated by ERK pathway, regulated β-catenin-TCF/LEF-1 transcriptional activity and associated EMT in HCC cell lines. These discoveries provide evidence of novel roles of PKM2 in the progression of HCC and potential therapeutic target for advanced cases.

Topics: beta Catenin; Carcinoma, Hepatocellular; Carrier Proteins; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Hep G2 Cells; Humans; Liver Neoplasms; Lymphoid Enhancer-Binding Factor 1; Membrane Proteins; Prognosis; TCF Transcription Factors; Thyroid Hormone-Binding Proteins; Thyroid Hormones; Transcription, Genetic

To investigate signaling pathways for reversal of EGF-mediated multi-drug resistance (MDR) in hepatocellular carcinoma (HCC) models.. HCC MDR cell strain HepG2/adriamycin (ADM) and SMMC7721/ADM models were established using a method of exposure to medium with ADM between low and high concentration with gradually increasing concentration. Drug sensitivity and reversal of multi-drug resistance by EGF were determined and the cell cycle distribution and apoptosis were analyzed by flow cytometry. Phosphorylation of ERK1, ERK2, ERK5 and expression of Bim were detected by Western blotting.. The results showed that HepG2/ADM and SMMC7721/ADM cells were resistant not only to ADM, but also to multiple anticancer drugs. When used alone, EGF had no anti-tumor activity in HepG2/ADM and SMMC7721/ADM cells in vitro, while it increased the cytotoxicity of ADM. EGF induced cell apoptosis and G0/G1 phase cell cycle arrest in HepG2/ADM And SMMC7721/ADM cells, while enhancing activity of p-ERKs and up-regulated expression of BimEL.. EGF might enhance the chemosensitivity of HepG2/ADM and SMMC7721/ADM cells via up-regulating p-ERKs and BimEL protein.

Topics: Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Cycle; Cell Proliferation; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Epidermal Growth Factor; Flow Cytometry; Humans; Liver Neoplasms; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; Tumor Cells, Cultured

Platelets are frequently altered in hepatocellular carcinoma (HCC) patients. Platelet lysates (hPL) can enhance HCC cell growth and decrease apoptosis. The aims were to evaluate whether hPL can modulate the actions of sorafenib or regorafenib, two clinical HCC multikinase antagonists.. Several human HCC cell lines were grown in the presence and absence of sorafenib or regorafenib, with or without hPL. Growth was measured by MTT assay, apoptosis was assessed by Annexin V and by western blot, and autophagy and MAPK growth signaling were also measured by western blot, and migration and invasion were measured by standard in vitro assays.. Both sorafenib and regorafenib-mediated inhibition of cell growth, migration and invasion were all antagonized by hPL. Drug-mediated apoptosis and decrease in phospho-ERK levels were both blocked by hPL, which also increased anti-apoptotic phospho-STAT, Bax and Bcl-xL levels. Preliminary data, obtained with epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I), included in hPL, revealed that these factors were able to antagonized sorafenib in a proliferation assay, in particular when used in combination.. Platelet factors can antagonize sorafenib or regorafenib-mediated growth inhibition and apoptosis in HCC cells. The modulation of platelet activity or numbers has the potential to enhance multikinase drug actions.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Blood Platelets; Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Enzyme Activation; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Hep G2 Cells; Humans; Insulin-Like Growth Factor I; Liver Neoplasms; Neoplasm Invasiveness; Niacinamide; Phenylurea Compounds; Phosphorylation; Protein Kinase Inhibitors; Pyridines; Signal Transduction; Sorafenib; Time Factors

Constitutive activation of signal transducer and activator of transcription signalling 3 (STAT3) has been linked with survival, proliferation and angiogenesis in a wide variety of malignancies including hepatocellular carcinoma (HCC).. We evaluated the effect of lupeol on STAT3 signalling cascade and its regulated functional responses in HCC cells.. Lupeol suppressed constitutive activation of STAT3 phosphorylation at tyrosine 705 residue effectively in a dose- and time-dependent manner. The phosphorylation of Janus-activated kinases (JAKs) 1 and 2 and Src was also suppressed by lupeol. Pervanadate treatment reversed the downregulation of phospho-STAT3 induced by lupeol, thereby indicating the involvement of a phosphatase. Indeed, we observed that treatment with lupeol increased the protein and mRNA levels of SHP-2, and silencing of SHP-2 abolished the inhibitory effects of lupeol on STAT3 activation. Treatment with lupeol also downregulated the expression of diverse STAT3-regulated genes and decreased the binding of STAT3 to VEGF promoter. Moreover, the proliferation of various HCC cells was significantly suppressed by lupeol, being associated with substantial induction of apoptosis. Depletion of SHP-2 reversed the observed antiproliferative and pro-apoptotic effects of lupeol.. Lupeol exhibited its potential anticancer effects in HCC through the downregulation of STAT3-induced pro-survival signalling cascade.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Chemokine CXCL12; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Janus Kinase 1; Janus Kinase 2; Liver Neoplasms; Pentacyclic Triterpenes; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Protein Processing, Post-Translational; Signal Transduction; STAT3 Transcription Factor; Transcriptional Activation; Vascular Endothelial Growth Factor A

The epidermal growth factor receptor (EGFR) is an important surface receptor with N-glycans in its extracellular domain, whose glycosylation is essential for its function, especially in tumor cells. Here, we demonstrated that polylactosamine is markedly increased in H7721 hepatocellular carcinoma cells after treatment with EGF, while it apparently declined after exposure to all-trans retinoic acid (ATRA). In the study of the enzymatic mechanism of this phenomenon, we explored changes in the expression of poly-N-acetyllactosamine (PLN) branching glycosyltransferases using RT-PCR. Among the four glycosyltransferases with altered expression, GnT-V was most elevated by EGF, while GnT-V and B3GNT2 were most declined by ATRA. Next, we conducted co-immunoprecipitation experiments to test whether B3GNT2 and EGFR associate with each other. We observed that EGFR is a B3GNT2-targeting protein in H7721 cells. Taken together, these findings indicated that the altered expression of B3GNT2 will remodel the PLN stucture of EGFR in H7721 cells, which may modify downstream signal transduction.

Topics: Antineoplastic Agents; Blotting, Western; Carcinoma, Hepatocellular; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Glycosylation; Humans; Immunoprecipitation; Liver Neoplasms; N-Acetylglucosaminyltransferases; Phosphorylation; Polysaccharides; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

The aims of this study are to explore the effects of epidermal growth factor (EGF) on hepatocyte proliferation in presence of plasma from patients with acute liver failure (ALF).. HepG2 cells were cultured with 50% plasma from patients with ALF for 6, 12, 24, 48 and 72h with or without different concentrations of EGF. Cell proliferation was determined by the MTT assay and intracellular cyclin D1, cyclin-dependent kinase 4 (CDK4) expressions were analyzed by western blotting.. The proliferation of HepG2 cells was significantly inhibited by treatment with plasma from patients with ALF from 12 to 72 h. Intracellular expression of cyclin D1 and CDK4 was also markedly down-regulated. 5ng/ml, 10ng/ml and 20ng/ml EGF dose dependently induced HepG2 proliferation in presence of plasma from normal control, but only 20ng/ml EGF showed a transient promoting effect on proliferation of HepG2 cells in presence of plasma from patients with ALF.. Plasma from patients with ALF inhibits HepG2 cell proliferation via downregulation of cyclin D1 and CDK4 expression. Plasma from patient with ALF dampens HepG2 cells to EGF induced proliferation response.

Topics: Adolescent; Adult; Apoptosis; Carcinoma, Hepatocellular; Case-Control Studies; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Hep G2 Cells; Humans; Liver Failure, Acute; Liver Neoplasms; Male; Middle Aged; Time Factors

Epithelial-to-mesenchymal transition (EMT) is critical for the development of the invasion and metastasis in human cancers. Recently, signal transducer and activator of transcription 3 (STAT3) activation has been linked to EMT program in breast cancer. However, the actual association of STAT3 activation with EMT, and its mediated tumor invasion and metastasis remains elusive in hepatocellular carcinoma (HCC). The aim of this study was to investigate the correlation between STAT3 activation and EMT, as well as the underlying mechanism involved in HCC progression.. We treated SMMC-7721 cells with a known STAT3 activator, epithelial growth factor (EGF); in the absence or presence of JSI-124, a selective STAT3 inhibitor. The EMT-associated morphologic and molecular changes of cells were analyzed. The EMT-mediated HCC cell invasion, migration and adhesion were evaluated.. In this study, we found that STAT3 activation induced by EGF was associated significantly with morphologic changes, cytoskeleton rearrangement and molecular changes consistent with EMT in SMMC-7721 cells; STAT3 activation-mediated EMT may be transcriptionally induced by Twist. STAT3 activation-mediated EMT also promoted HCC cell invasion, migration and adhesion significantly.. In summary, our study show for the first time that STAT3 activation may induce invasion and metastasis through the mediation of EMT in HCC cells. Activated STAT3 and EMT markers can serve as molecular targets for HCC treatment.

Topics: Carcinoma, Hepatocellular; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Shape; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Humans; Liver Neoplasms; Neoplasm Invasiveness; Signal Transduction; STAT3 Transcription Factor; Triterpenes

Earlier, we reported a highly statistically significant association between T-helper 1 (Th1) and Th2 cytokine genotypes and hepatocellular carcinoma (HCC) risk among natives of southern Guangxi, China, a hyperendemic region for HCC. Epidermal growth factor (EGF) plays a critical role in malignant transformation of hepatocytes and tumor progression. A polymorphism in the EGF gene (61A > G) results in elevation of EGF in liver tissues and blood. Epidemiological data are sparse on the possible association between EGF genetic polymorphism and HCC risk.. The EGF 61A > G polymorphism, multiple Th1 and Th2 genotypes, and environmental risk factors for HCC were determined on 117 HCC cases and 225 healthy control subjects among non-Asians of Los Angeles County, California, a low-risk population for HCC, and 250 HCC cases and 245 controls of southern Guangxi, China.. Following adjustment for all known or suspected HCC risk factors, non-Asians in Los Angeles who possessed at least one copy of the high activity 61*G allele of the EGF gene showed a statistically non-significant, 78% increased risk of HCC compared with those possessing the EGF A/A genotype. This EGF-HCC risk association significantly strengthened among heavy users of alcohol [odds ratio (OR) = 3.44, 95% confidence interval (CI) = 0.93-12.76, P = 0.065)], and among individuals carrying the high-risk Th1/Th2 genotypes for HCC (OR = 3.34, 95% CI = 1.24-9.03, P = 0.017). No association between EGF genotype and HCC risk was observed among Chinese in southern Guangxi, China.. Genetic polymorphism in the EGF gene resulting in elevated level of EGF, may contribute to HCC risk among low-risk non-Asians in Los Angeles.

Topics: Adult; Aged; Alcohol-Related Disorders; Carcinoma, Hepatocellular; Case-Control Studies; China; Epidermal Growth Factor; Female; Genotype; Hepatitis B; Hepatitis C; Humans; Incidence; Liver Neoplasms; Los Angeles; Male; Middle Aged; Polymorphism, Single Nucleotide; Prevalence; Risk Factors; Smoking; Th1 Cells; Th2 Cells

In this article, we present a study on the levels of epidermal growth factor (EGF), its phosphorylated receptor (p-EGFR) and transforming growth factor-β1 (TGF-β1) in the sera of patients with hepatocellular carcinoma (HCC) and chronic hepatitis C (CHC) infection. The results reveal significant higher serum levels of EGF and TGF-β1 in patients with HCC compared to their level in patients with CHC infection and control subjects. The levels of p-EGFR in HCC and CHC patients show a highly significant difference between patients. Based on the best cutoff value of 914 pg/ml, EGF shows 63.3 % sensitivity and 87.5 % specificity for HCC patients where the area under the curve is 0.81. The p-EGFR shows sensitivity of 63.3 % and specificity of 100 % where the area under the curve is 0.87 for HCC patients based on the best cutoff value of 39 U/mg protein. The best cutoff value (370 pg/ml) for serum TGF-β1 displays sensitivity of 86.7 % and specificity of 100 %, where the area under the curve is 0.97 for HCC patients.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Epidermal Growth Factor; ErbB Receptors; Hepacivirus; Humans; Liver Neoplasms; Middle Aged; Transforming Growth Factor beta1; Young Adult

Cyclooxygenase-2 (COX-2), a rate limiting step in arachidonic acid cascade, plays a key role in the biosynthesis of prostaglandin E2 (PGE2) upon inflammatory stimuli, growth factors, hormones and other cellular stresses. Overproduction of PGE2 stimulates proliferation of various cancer cells, confers resistance to apoptosis and favors metastasis and angiogenesis. The steady-state level of PGE2 is maintained by interplay between the biosynthetic pathway including COX and PGE2 synthases and the catabolic pathways involving nicotinamide adenine dinucleotide (NAD(+))-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). 15-PGDH is a crucial enzyme responsible for the biological inactivation of PGE2. Adult hepatocytes fail to induce COX-2 expression regardless of the pro-inflammatory factors used. COX-2 is induced in hepatocytes after partial hepatectomy (PH), in animal models of cirrhosis, in human hepatoma cell lines, in human HCC and after HBV and HCV infection. However, no data are available regarding 15-PGDH expression in HCC. Our results show that 15-PGDH is downregulated in human hepatoma cells with a high COX-2 expression, in chemical and genetic murine models of HCC and in human HCC biopsies. Moreover, 15-PGDH expression is suppressed by EGF (epidermal growth factor) and HGF (hepatocyte growth factor) mainly involving PI3K (phosphatidylinositol-3-kinase), ERK (extracellular signal-regulated kinase) and p38MAPK (mitogen-activated protein kinase) activation. Conversely, ectopic expression of 15-PGDH induces apoptosis in hepatoma cells and decreases the growth of hepatoma cells in nude mice whereas the silencing of 15-PGDH increases the tumor formation. These data suggest a potential therapeutic application of 15-PGDH in HCC.

Topics: Adult; Animals; Apoptosis; Biopsy; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclooxygenase 2; Disease Models, Animal; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Hepatocyte Growth Factor; Humans; Hydroxyprostaglandin Dehydrogenases; Intramolecular Oxidoreductases; Liver Neoplasms; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Phosphatidylinositol 3-Kinases; Prostaglandin-E Synthases; RNA, Messenger

Up to present, EGF 61*A/G, TGF-β1 -509*T/C and TNF-α -308*A/G gene polymorphisms have been analysed in other cancer entities than hepatocellular carcinoma (HCC). We here investigated the frequency of these gene polymorphisms among HCC patients.. A total of 73 HCC patients and 117 cancer-free healthy people were recruited at the Surgical Department of Zhongshan Hospital. Genomic DNA was isolated from peripheral blood and gene polymorphisms were analyzed by PCR-RFLP.. The distribution of EGF 61*G/G homozygotes among HCC patients was more frequent than that in the control group (24.7% vs 11.1%, OR=2.618, 95%CI=1.195-5.738). In parallel, the frequency of the "G" allele in the HCC patient group was also higher than that in the control group (45.9% vs 33.3%, OR= 1.696, 95%CI=1.110-2.592). No difference could be found for the TGF-β1-509 and TNF-α -308 genotypes.. EGF 61*G/G genotype and G allele are significantly increased among patients with HCC. TGF-β1-509*T/C and TNF-α -308*A/G gene polymorphisms are not related to this cancer entity.

Topics: Carcinoma, Hepatocellular; Epidermal Growth Factor; Humans; Liver Neoplasms; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Cancer invasion and metastasis are characterized by epithelial-mesenchymal transition (EMT). Hepatocellular carcinoma (HCC) causes metastasis and significant mortality. Elucidating factors promoting EMT in HCC are necessary to develop effective therapeutic strategies.. The LH86 cell line was developed in our laboratory from well-differentiated HCC without associated hepatitis or cirrhosis and used as a model to study EMT in HCC. Effects of transforming growth factor β-1, epidermal growth factor, hepatocyte growth factor and basic fibroblast growth factor (bFGF) were examined using morphology, molecular markers, effects on migration and tumorigenicity. The involvement of cyclooxygenase-2 (COX-2) and Akt were examined.. LH86 cells display epithelial morphology. Transforming-growth-factor-β-1-, epidermal-growth-factor-, hepatocyte-growth-factor- and basic-fibroblast-growth-factor-induced mesenchymal changes in them were associated with loss of E-cadherin, albumin, α-1 anti-trypsin expression and increased expression of vimentin, collagen I and fibronectin. There was associated increased migration, tumorigenicity and increased expression of COX-2, prostaglandin E2 (PGE2), Akt and phosphorylated Akt. Inhibition of COX-2 and Akt pathways led to inhibition of characteristics of EMT.. Multiple growth factors induce EMT in HCC. COX-2 and Akt may mediate EMT-associated development and progression of HCC and molecular targeting of COX-2 and Akt may be an effective therapeutic or chemopreventive strategy in advanced and metastatic HCC.

Topics: Albumins; alpha 1-Antitrypsin; Animals; Cadherins; Carcinoma, Hepatocellular; Cell Movement; Cell Transplantation; Collagen Type I; Cyclooxygenase 2; Dinoprostone; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Fibroblast Growth Factor 2; Fibronectins; Gene Expression; Hepatocyte Growth Factor; Humans; Mice; Oncogene Protein v-akt; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta1; Tumor Cells, Cultured; Vimentin

Overexpression of epidermal growth factor (EGF) in the liver induces transformation into hepatocellular carcinoma (HCC) in animal models. Polymorphisms in the EGF gene modulate EGF levels.. To evaluate the effect of EGF gene single nucleotide polymorphism and to assess its correlation with the risk of HCC in patients with chronic liver diseases.. The present study included 80 participants divided into four groups: group 1 included 20 asymptomatic healthy control volunteers, group 2 included 20 patients with chronic hepatitis C viral (HCV) infection, group 3 included 20 patients with liver cirrhosis, and group 4 included 20 patients with HCC. For all participants, the following investigations were performed: routine laboratory investigations including complete blood count, liver function tests, sero markers of hepatitis viruses HBsAg, HCV-RNA by quantitative polymerase chain reaction, and α-fetoprotein. DNA was extracted from whole blood for detection of single nucleotide polymorphism of the EGF by polymerase chain reaction, followed by restriction fragment length polymorphism.. We found a significant difference between both patients with HCC and HCV versus controls in terms of the G carrier (GG and GA; 80 vs. 40%, P<0.05). In addition, the cirrhotic and chronic hepatitis C patients with GG had three-fold and 2.3-fold odds ratio for developing HCC, respectively.. The EGF 61GG genotype might be associated with a high risk for the development of HCC in Egyptian patients with chronic liver disease.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Early Diagnosis; Electrophoresis, Agar Gel; Epidermal Growth Factor; Female; Genetic Predisposition to Disease; Genotype; Hepatitis C, Chronic; Heterozygote; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Pilot Projects; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Young Adult

AKR1B10 (aldo-keto reductase 1B10) is overexpressed in liver and lung cancer, and plays a critical role in tumour development and progression through promoting lipogenesis and eliminating cytotoxic carbonyls. AKR1B10 is a secretory protein and potential tumour marker; however, little is known about the regulatory mechanism of AKR1B10 expression. The present study showed that AKR1B10 is induced by mitogen EGF (epidermal growth factor) and insulin through the AP-1 (activator protein-1) signalling pathway. In human HCC (hepatocellular carcinoma) cells (HepG2 and Hep3B), EGF (50 ng/ml) and insulin (10 nM) stimulated endogenous AKR1B10 expression and promoter activity. In the AKR1B10 promoter, a putative AP-1 element was found at bp -222 to -212. Deletion or mutation of this AP-1 element abrogated the basal promoter activity and response to EGF and AP-1 proteins. This AP-1 element bound to nuclear proteins extracted from HepG2 cells, and this binding was stimulated by EGF and insulin in a dose-dependent manner. Chromatin immunoprecipitation showed that the AP-1 proteins c-Fos and c-Jun were the predominant factors bound to the AP-1 consensus sequence, followed by JunD and then JunB. The same order was followed in the stimulation of endogenous AKR1B10 expression by AP-1 proteins. Furthermore, c-Fos shRNA (short hairpin RNA) and AP-1 inhibitors/antagonists (U0126 and Tanshinone IIA) inhibited endogenous AKR1B10 expression and promoter activity in HepG2 cells cultured in vitro or inoculated subcutaneously in nude mice. U0126 also inhibited AKR1B10 expression induced by EGF. Taken together, these results suggest that AKR1B10 is up-regulated by EGF and insulin through AP-1 mitogenic signalling and may be implicated in hepatocarcinogenesis.

Topics: Aldehyde Reductase; Aldo-Keto Reductases; Animals; Base Sequence; Biomarkers, Tumor; Carcinoma, Hepatocellular; DNA Primers; Epidermal Growth Factor; Female; Genes, fos; Hep G2 Cells; Humans; Insulin; Liver Neoplasms; Mice; Mice, Nude; Promoter Regions, Genetic; RNA, Small Interfering; Signal Transduction; Transcription Factor AP-1; Up-Regulation

To determine whether factors secreted by irradiated liver nonparenchymal cells (NPCs) may influence invasiveness and/or metastatic potential of hepatocellular carcinoma (HCC) cells and to elucidate a possible mechanism for such effect.. Primary rat NPCs were cultured and divided into irradiated (10-Gy X-ray) and nonirradiated groups. Forty-eight hours after irradiation, conditioned medium from irradiated (SR) or nonirradiated (SnonR) cultures were collected and added to sublethally irradiated cultures of the hepatoma McA-RH7777 cell line. Then, hepatoma cells were continuously passaged for eight generations (RH10Gy-SR and RH10Gy-SnonR). The invasiveness and metastatic potential of McA-RH7777, RH10Gy-SnonR, and RH10Gy-SR cells were evaluated using an in vitro gelatinous protein (Matrigel) invasion and an in vivo metastasis assay. In addition, SR and SnonR were tested using rat cytokine antibody arrays and enzyme-linked immunosorbent assay (ELISA).. In vitro gelatinous protein invasion assay indicated that the numbers of invading cells was significantly higher in RH10Gy-SR (40 ± 4.74) than in RH10Gy-SnonR (30.6 ± 3.85) cells, and lowest in McA-RH7777 (11.4 ± 3.56) cells. The same pattern was observed in vivo in a lung metastasis assay, as evaluated by number of metastatic lung nodules seen with RH10Gy-SR (28.83 ± 5.38), RH10Gy-SnonR (22.17 ± 4.26), and McA-RH7777 (8.3 ± 3.8) cells. Rat cytokine antibody arrays and ELISA demonstrated that metastasis-promoting cytokines (tumor necrosis factor-α and interleukin-6), circulating growth factors (vascular endothelial growth factor and epidermal growth factor), and metalloproteinases (MMP-2 and MMP-9) were upregulated in SR compared with SnonR.. Radiation can increase invasiveness and metastatic potential of sublethally irradiated hepatoma cells, and soluble mediators released from irradiated NPCs promote this potential. Increased secretion of metastasis-related cytokines and factors from NPCs after irradiation may be a possible mechanism for the radiation-induced invasiveness and metastatic potential of HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Culture Techniques; Collagen; Cytokines; Drug Combinations; Epidermal Growth Factor; Hepatocytes; Interleukin-6; Laminin; Liver Neoplasms; Lung Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Proteoglycans; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

Epidermal growth factor (EGF) signaling is implicated in the invasion and metastasis of hepatoma cells. However, the signaling pathways for EGF-induced motility of hepatoma cells remain undefined.. We found that EGF dose-dependently stimulated the migration of human hepatoma cells HepG2, with the maximal effect at 10 ng/mL. Additionally, EGF increased Arf6 activity, and ectopic expression of Arf6 T27N, a dominant negative Arf6 mutant, largely abolish EGF-induced cell migration. Blocking GEP100 with GEP100 siRNA or GEP100-△PH, a pleckstrin homology (PH) domain deletion mutant of GEP100, blocked EGF-induced Arf6 activity and cell migration. EGF also increased ERK and Rac1 activity. Ectopic expression GEP100 siRNA, GEP100-△PH, or Arf6-T27N suppressed EGF-induced ERK and Rac1 activity. Furthermore, blocking ERK signaling with its inhibitor U0126 remarkably inhibited both EGF-induced Rac1 activation as well as cell migration, and ectopic expression of inactive mutant form of Rac1 (Rac1-T17N) also largely abolished EGF-induced cell migration.. Taken together, this study highlights the function of the PH domain of GEP100 and its regulated Arf6/ERK/Rac1 signaling cascade in EGF-induced hepatoma cell migration. These findings could provide a rationale for designing new therapy based on inhibition of hepatoma metastasis.

Topics: ADP-Ribosylation Factor 6; ADP-Ribosylation Factors; Analysis of Variance; Butadienes; Carcinoma, Hepatocellular; Cell Movement; Epidermal Growth Factor; Guanine Nucleotide Exchange Factors; Hep G2 Cells; Humans; Immunoblotting; In Vitro Techniques; Microscopy, Fluorescence; Mutation, Missense; Neoplasm Metastasis; Nitriles; rac1 GTP-Binding Protein; RNA, Small Interfering; Signal Transduction

EMT plays an essential role in tumor progression and metastasis. Hyperthermia is a potent approach for cancers with low side effects. However, the effect of hyperthermia on EMT of cancer cells is unknown.. Cells were treated with TGF-β1 and epidermal growth factor for 96 h and then exposed to hyperthermia at 43°C for 0.5 h. Cell morphology was observed. Expressions of E-cadherin and vimentin were determined by Western blot. The protein and mRNA expressions of Snail were detected with Western blot and RT-PCR. Cell migratory capacity was evaluated.. TGF-β1 induced EMT in HepG2 cells, which was evidenced by morphological, molecular and functional changes, including the formation of spindle shape and the loss of cell contact. The expression of E-cadherin was decreased but the expression of vimentin increased; also, the migratory capability was increased by 2.1±0.19-fold as compared with untreated cells. However, those effects were inhibited by the treatment of hyperthermia. Furthermore, the protein and mRNA expressions of Snail induced by TGF-β1 were also significantly inhibited by hyperthermia treatment. Hyperthermia can inhibit TGF-β1-induced EMT in HepG2 cells, suggesting that hyperthermia may alter the properties of metastatic potential in cancer cells and inhibit tumor metastasis.

Topics: Antigens, CD; Blotting, Western; Cadherins; Carcinoma, Hepatocellular; Cell Movement; Cell Shape; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Hyperthermia, Induced; Liver Neoplasms; Neoplasm Invasiveness; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Snail Family Transcription Factors; Time Factors; Transcription Factors; Transforming Growth Factor beta1; Vimentin

Taspine, isolated from Radix et Rhizoma Leonticis has demosntrated potential proctiective effects against cancer. Tas13D, a novel taspine derivative synthetized by structure-based drug design, have been shown to possess interesting biological and pharmacological activities. The current study was designed to evaluate its antiproliferative activity and underlying mechanisms.. Antiproliferative activity of tas13D was evaluated by xenograft in athymic mice in vivo, and by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and cell migration assays with human liver cancer (SMMC-7721) cell lines in vitro. Docking between tas13D and VEGFR and EGFR was studied by with a Sybyl/Surflex module. VEGF and EGF and their receptor expression was determined by ELISA and real-time PCR methods, respectively.. Our present study showed that tas13D inhibited SMMC-7721 xenograft tumor growth, bound tightly with the active site of kinase domains of EGFR and VEGFR, and reduced SMMC-7721 cell proliferation (IC=34.7 μmol/L) and migration compared to negative controls. VEGF and EGF mRNAs were significantly reduced by tas13D treatment in a dose-dependent manner, along with VEGF and EGF production.. The obtained results suggest that tas13D inhibits tumor growth and cell proliferation by inhibiting cell migration, downregulating mRNA expression of VEGF and EGF, and decreasing angiogenic factor production. Tas13D deserves further consideration as a chemotherapeutic agent.

Topics: Alkaloids; Animals; Blotting, Western; Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; ErbB Receptors; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A

It is important to understand the mechanisms by which the cells integrate signals from different receptors. Several lines of evidence implicate epidermal growth factor (EGF) receptor (EGFR) in the pathophysiology of hepatocarcinomas. Data also suggest a role of prostaglandins in some of these tumours, through their receptors of the G protein-coupled receptor (GPCR) family. In this study we have investigated mechanisms of interaction between signalling from prostaglandin receptors and EGFR in hepatocarcinoma cells.. The rat hepatocarcinoma cell line MH1C1 and normal rat hepatocytes in primary culture were stimulated with EGF or prostaglandin E2 (PGE2) and in some experiments also PGF2α. DNA synthesis was determined by incorporation of radiolabelled thymidine into DNA, phosphorylation of proteins in signalling pathways was assessed by Western blotting, mRNA expression of prostaglandin receptors was determined using qRT-PCR, accumulation of inositol phosphates was measured by incorporation of radiolabelled inositol, and cAMP was determined by radioimmunoassay.. In the MH1C1 hepatocarcinoma cells, stimulation with PGE2 or PGF2α caused phosphorylation of the EGFR, Akt, and ERK, which could be blocked by the EGFR tyrosine kinase inhibitor gefitinib. This did not occur in primary hepatocytes. qRT-PCR revealed expression of EP1, EP4, and FP receptor mRNA in MH1C1 cells. PGE2 stimulated accumulation of inositol phosphates but not cAMP in these cells, suggesting signalling via PLCβ. While pretreatment with EP1 and EP4 receptor antagonists did not inhibit the effect of PGE2, pretreatment with an FP receptor antagonist blocked the phosphorylation of EGFR, Akt and ERK. Further studies suggested that the PGE2-induced signal was mediated via Ca2+ release and not PKC activation, and that it proceeded through Src and shedding of membrane-bound EGFR ligand precursors by proteinases of the ADAM family.. The results indicate that in MH1C1 cells, unlike normal hepatocytes, PGE2 activates the MEK/ERK and PI3K/Akt pathways by transactivation of the EGFR, thus diversifying the GPCR-mediated signal. The data also suggest that the underlying mechanisms in these cells involve FP receptors, PLCβ, Ca2+, Src, and proteinase-mediated release of membrane-associated EGFR ligand(s).

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Dinoprostone; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Gene Expression Regulation, Neoplastic; Hepatocytes; Humans; Liver Neoplasms; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinazolines; Rats; Signal Transduction

Cytokeratin (CK) 19-positive hepatocellular carcinoma (HCC) has been reported to have a poor prognosis. The mechanism of the development of CK19-positive HCC remains to be studied. To clarify this, in vitro experiments were performed using human HCC cell lines (PLC-5, HepG2), and the phenotypic changes after stimulation with several growth factors were examined using quantitative reverse transcriptase PCR, western blotting, and immunofluorescence staining. In vivo experiments using human HCC specimens obtained from a total of 78 patients and clinicopathological analysis were also performed. Among the growth factors tested, epidermal growth factor (EGF) had prominent effects on inducing CK19 expression in PLC-5 and HepG2, which was accompanied by the reduced expression of α-fetoprotein in PLC-5. The induction of CK19 expression after EGF stimulation was accompanied by the phosphorylation of c-Jun-N-terminal kinase (JNK)/stress-activated protein kinase, which was blocked by the addition of JNK inhibitors. EGF also increased proliferative abilities and invasive properties of the HCC cell lines. In vivo, 9 (12%) of 78 HCC cases showed positive immunohistochemical staining of CK19. The extent of positive immunohistochemical signals of EGF, EGF receptor (EGFR), and JNK expression was significantly intense in CK-19-positive HCC than those of CK19-negative HCC. Clinicopathological analysis showed that CK19-positive HCC had a high incidence of portal vein invasion, extrahepatic metastasis and an early relapse, which was associated with the worsened 2-year disease free survival. These results indicate that the activation of the EGF-EGFR signaling pathway is associated with the development of CK19-positive HCC, and the EGF-induced increase in growth abilities of HCC may account for the poor prognosis of the patients.

Topics: alpha-Fetoproteins; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; In Vitro Techniques; Keratin-19; Liver Neoplasms; Mitogen-Activated Protein Kinase 8; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction

Chronic hepatitis B virus (HBV) infection is a risk factor of hepatocellular carcinoma (HCC) in China. Epidermal growth factor (EGF) plays an important role in tumorigenesis. The association between EGF +61 G/A polymorphism and the risk of HCC is still controversial and ambiguous.. The objective of this study was to investigate the association between EGF +61 G/A polymorphism and the risk of HCC in a Chinese population.. A hospital-based case-control study was designed in a Chinese population. EGF +61 G/A polymorphisms were determined in 120 chronic HBV-infected HCC patients, 120 chronic HBV-infected cirrhotic patients, and 120 healthy controls. The genotype frequency of this polymorphism was determined by using a polymerase chain reaction-restriction fragment length polymorphism assay.. EGF +61 GG (odds ratio=2.76, 95% confidence interval=1.03, 7.38; p=0.04) and G allele frequencies (odds ratio=1.59, 95% confidence interval=1.08, 2.34; p=0.02) in the HCC group were higher than those in the cirrhosis group. EGF +61 A and G allele frequencies in healthy subjects were 28.8% and 71.2%. No relationship between EGF +61 G/A gene polymorphism and HCC risk was found among our recruited HCC patients and healthy controls.. This study suggests that EGF +61 GG genotype is associated with a higher risk of chronic HBV-infected HCC in the Chinese population.

Topics: Aged; Asian People; Carcinoma, Hepatocellular; Case-Control Studies; China; Epidermal Growth Factor; Female; Gene Frequency; Genetic Predisposition to Disease; Genotype; Hepatitis B virus; Hepatitis B, Chronic; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Polymorphism, Genetic; Risk Factors

Aim of this study was the site-specific conjugation of an epidermal growth factor (EGF)-polyethylene glycol (PEG) chain by click chemistry onto a poly(amido amine) (PAMAM) dendron, as a key step toward defined multifunctional carriers for targeted gene delivery. For this purpose, at first propargyl amine cored PAMAM dendrons with ester ends were synthesized. The chain terminal ester groups were then modified by oligoamines with different secondary amino densities. The oligoamine-modified PAMAM dendrons were well biocompatible, as demonstrated in cytotoxicity assays. Among the different oligoamine-modified dendrons, PAMAM-pentaethylenehexamine (PEHA) dendron polyplexes displayed the best gene transfer ability. Conjugation of PAMAM-PEHA dendron with PEG spacer was conducted via click reaction, which was performed before amidation with PEHA. The resultant PEG-PAMAM-PEHA copolymer was then coupled with EGF ligand. pDNA transfections in HuH-7 hepatocellular carcinoma cells showed a 10-fold higher efficiency with the polyplexes containing conjugated EGF as compared to the ligand-free ones, demonstrating the concept of ligand targeting. Overall gene transfer efficiencies, however, were moderate, suggesting that additional measures for overcoming subsequent intracellular bottlenecks in delivery have to be taken.

Topics: Amines; Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Click Chemistry; Dendrimers; DNA; Drug Delivery Systems; Epidermal Growth Factor; Gene Transfer Techniques; Genetic Therapy; Humans; Liver Neoplasms; Magnetic Resonance Spectroscopy; Mice; Plasmids; Polyamines; Polyethylene Glycols; Transfection

Extracellular glycosylation is a critical determinant of malignant character. Here, we report that N-acetylgalactosaminyltransferase 2 (GALNT2), the enzyme that mediates the initial step of mucin type-O glycosylation, is a critical mediator of malignant character in hepatocellular carcinoma (HCC) that acts by modifying the activity of the epidermal growth factor receptor (EGFR). GALNT2 mRNA and protein were downregulated frequently in HCC tumors where these events were associated with vascular invasion and recurrence. Restoring GALNT2 expression in HCC cells suppressed EGF-induced cell growth, migration, and invasion in vitro and in vivo. Mechanistic investigations revealed that the status of the O-glycans attached to the EGFR was altered by GALNT2, changing EGFR responses after EGF binding. Inhibiting EGFR activity with erlotinib decreased the malignant characters caused by siRNA-mediated knockdown of GALNT2 in HCC cells, establishing the critical role of EGFR in mediating the effects of GALNT2 expression. Taken together, our results suggest that GALNT2 dysregulation contributes to the malignant behavior of HCC cells, and they provide novel insights into the significance of O-glycosylation in EGFR activity and HCC pathogenesis.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Female; Gene Knockdown Techniques; Glycosylation; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Mucins; N-Acetylgalactosaminyltransferases; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Polypeptide N-acetylgalactosaminyltransferase; Quinazolines; RNA, Messenger

To analyse the inhibition effect of taspine derivatives on human Liver cancer SMMC7721 cell and its mechanism.. The effects of five taspine derivatives on SMMC7721 cell growth were determined by MTT. The flow cytometry was used to determine the cell cycle. The effects of Tas-D1 on the EGF and VEGF in SMMC7721 cell were determined by ELISA. The mRNA level of EGF and VEGF in SMMC7721 cell was determined by RT-PCR.. The MTT assay demonstrated that the taspine derivative Tas-D1 significantly inhibited the growth of SMMC7721 cell in a dose-dependent manner. Cell was stopped at S phase by Tas-D1. Tas-D1 inhibited the expression of EGF and VEGF and their mRNA in a dose-dependent manner (P<0.05).. The taspine derivative Tas-D1 can inhibit the growth of human Liver cancer SMMC7721 cell and change cell cycle, which may be related to the inhibition of EGF and VEGF expression.

Topics: Alkaloids; Antineoplastic Agents; Carcinoma, Hepatocellular; Caulophyllum; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Epidermal Growth Factor; Flow Cytometry; Humans; Liver Neoplasms; Polymerase Chain Reaction; RNA, Messenger; Vascular Endothelial Growth Factor A

Chronic hepatitis B virus (HBV) infection is an important risk factor for hepatocellular carcinoma (HCC) development in China, while little is known of the genetic susceptibility to hepatocarcinogenesis. The epidermal growth factor (EGF) pathway plays an important role in tumorigenesis, including HCC. EGF polymorphisms are associated with susceptibility to several types of cancers. Therefore, this study aimed to assess whether EGF genetic polymorphisms can influence HCC development.. A total of 338 chronic HBV-infected patients (186 HCC patients and 152 cirrhotic patients) and 186 healthy individuals were enrolled in this study. EGF 61A/G polymorphisms of all subjects and 12 cell lines were assayed with polymerase chain reaction-restriction fragment length polymorphism and the sequencing method. Furthermore, EGF protein levels were measured in the serum and the results were compared with the different genotypes. EGF expression in the liver tissue of the HCC patients was detected by immunohistochemical analysis.. EGF 61A and 61G allele frequencies in healthy subjects were 28.76 and 71.24%. EGF 61GG and G allele frequencies in the HCC group were higher than those in the cirrhosis group. EGF protein levels with the GG genotype were significantly higher than those with either the GA or the AA genotype. About 59.09% of HCC liver tumour tissues assayed showed EGF protein expression.. The EGF 61 GG genotype might be associated with a high risk for the development of chronic HBV infection-related HCC in Chinese patients.

Topics: Adult; Aged; Aged, 80 and over; Asian People; Carcinoma, Hepatocellular; Cell Line, Tumor; DNA Mutational Analysis; Epidermal Growth Factor; Female; Gene Frequency; Genetic Predisposition to Disease; Hepatitis B virus; Hepatitis B, Chronic; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Risk Factors; Young Adult

Gr-1(+)CD11b(+)F4/80(+) cells play important roles in tumor development and have a negative effect on tumor immunotherapy. So far, the mechanisms underlying the regulation of their immunosuppressive phenotype by classical and alternative macrophage activation stimuli are not well elucidated. In this study, we found that molecules from necrotic tumor cells (NTC-Ms) stimulated Gr-1(+)CD11b(+)F4/80(+) cells to induce apoptosis of activated T cells but not nonstimulated T cells. The apoptosis-inducing capacity was determined by higher expression levels of arginase I and IL-10 relative to those of NO synthase 2 and IL-12 in Gr-1(+)CD11b(+)F4/80(+) cells, which were induced by NTC-Ms through TLR4 signaling. The apoptosis-inducing capacity of NTC-Ms-stimulated Gr-1(+)CD11b(+)F4/80(+) cells could be enhanced by IL-10. IFN-gamma may reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells only if their response to IFN-gamma was not attenuated. However, the potential of Gr-1(+)CD11b(+)F4/80(+) cells to express IL-12 in response to IFN-gamma could be attenuated by tumor, partially due to the existence of active STAT3 in Gr-1(+)CD11b(+)F4/80(+) cells and NTC-Ms from tumor. In this situation, IFN-gamma could not effectively reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells. Tumor immunotherapy with 4-1BBL/soluble programmed death-1 may significantly reduce, but not abolish the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells in local microenvironment. Blockade of TLR4 signaling could further reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells and enhance the suppressive effect of 4-1BBL/soluble form of programmed death-1 on tumor growth. These findings indicate the relationship of distinct signaling pathways with apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells and emphasize the importance of blocking TLR4 signaling to prevent the induction of T cell apoptosis by Gr-1(+)CD11b(+)F4/80(+) cells.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Hepatocellular; CD11b Antigen; Cell Line, Tumor; Cells, Cultured; Epidermal Growth Factor; Ligands; Liver Neoplasms; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Monocytes; Necrosis; Receptors, Chemokine; Signal Transduction; T-Lymphocyte Subsets; Toll-Like Receptor 4

The epidermal growth factor receptor (EGFR) is over-expressed in a variety of human cancers. Downstream signalling of this receptor is tightly regulated both spatially and temporally by controlling its internalization and subsequent degradation. Internalization of the EGFR requires dynamin 2 (Dyn2), a large GTPase that deforms lipid bilayers, leading to vesicle scission. The adaptor protein CIN85 (cbl-interacting protein of 85 kDa), which has been proposed to indirectly link the EGFR to the endocytic machinery at the plasma membrane, is also thought to be involved in receptor internalization. Here, we report a novel and direct interaction between Dyn2 and CIN85 that is induced by EGFR stimulation and, most surprisingly, occurs late in the endocytic process. Importantly, disruption of the CIN85-Dyn2 interaction results in accumulation of internalized EGFR in late endosomes that become aberrantly elongated into distended tubules. Consistent with the accumulation of this receptor is a sustention of downstream signalling cascades. These findings provide novel insights into a previously unknown protein complex that can regulate EGFR traffic at very late stages of the endocytic pathway.

Topics: Adaptor Proteins, Signal Transducing; Carcinoma, Hepatocellular; Cell Membrane; Dynamin II; Endocytosis; Endosomes; Epidermal Growth Factor; ErbB Receptors; HeLa Cells; Humans; Immunoprecipitation; Liver Neoplasms; Protein Binding; rab GTP-Binding Proteins; rab7 GTP-Binding Proteins; RNA, Small Interfering; Signal Transduction

Dysregulation of epidermal growth factor and insulin-like growth factor signaling play important roles in human hepatocellular carcinoma (HCC), leading to frequent activation of their downstream targets, the ras/raf/extracellular signal-regulated kinase (ERK) and the phosphoinositide 3-kinase (PI3K)/Akt/mammalian Target of Rapamycin (mTOR) pathways. Salirasib is an S-prenyl-cysteine analog that has been shown to block ras and/or mTOR activation in several non hepatic tumor cell lines. We investigated in vitro the effect of salirasib on cell growth as well as its mechanism of action in human hepatoma cell lines (HepG2, Huh7, and Hep3B) and its in vivo effect in a subcutaneous xenograft model with HepG2 cells.. Salirasib induced a time and dose dependent growth inhibition in hepatocarcinoma cells through inhibition of proliferation and partially through induction of apoptosis. A 50 percent reduction in cell growth was obtained in all three cell lines at a dose of 150 μM when they were cultured with serum. By contrast, salirasib was more potent at reducing cell growth after stimulation with EGF or IGF2 under serum-free conditions, with an IC50 ranging from 60 μM to 85 μM. The drug-induced anti-proliferative effect was associated with downregulation of cyclin A and to a lesser extent of cyclin D1, and upregulation of p21 and p27. Apoptosis induction was related to a global pro-apoptotic balance with caspase 3 activation, cytochrome c release, death receptor upregulation, and a reduced mRNA expression of the apoptosis inhibitors cFLIP and survivin. These effects were associated with ras downregulation and mTOR inhibition, without reduction of ERK and Akt activation. In vivo, salirasib reduced tumour growth from day 5 onwards. After 12 days of treatment, mean tumor weight was diminished by 56 percent in the treated animals.. Our results show for the first time that salirasib inhibits the growth of human hepatoma cell lines through inhibition of proliferation and induction of apoptosis, which is associated with ras and mTOR inhibition. The therapeutic potential of salirasib in human HCC was further confirmed in a subcutaneous xenograft model.

Topics: Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin A; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Farnesol; Female; Hep G2 Cells; Humans; Inhibitor of Apoptosis Proteins; Insulin-Like Growth Factor II; Liver Neoplasms; Mice; Mice, Nude; Microtubule-Associated Proteins; ras Proteins; Reverse Transcriptase Polymerase Chain Reaction; Salicylates; Survivin; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Aberrant Ras/Raf/MAPK and PI3K/AKT/mTOR signaling pathways are found in hepatocellular carcinoma (HCC). This study reports how sorafenib (a multi-kinase inhibitor) and PI-103 (a dual PI3K/mTOR inhibitor) alone and in combination inhibit the proliferation of the HCC cell line, Huh7.. Huh7 proliferation was assayed by 3H-thymidine incorporation and by MTT assay. Western blot was used to detect phosphorylation of the key enzymes in the Ras/Raf and PI3K pathways.. Sorafenib and PI-103, as single agents inhibited Huh7 proliferation and epidermal growth factor (EGF)-stimulated Huh7 proliferation in a dose-dependent fashion; the combination of sorafenib and PI-103 produced synergistic effects. EGF increased phosphorylation of MEK and ERK, key Ras/Raf downstream signaling proteins; this activation was inhibited by sorafenib. However, sorafenib as a single agent increased AKT(Ser473) and mTOR phosphorylation. EGF-stimulated activation of PI3K/AKT/mTOR pathway components was inhibited by PI-103. PI-103 is a potent inhibitor of AKT(Ser473) phosphorylation; in contrast, rapamycin stimulated AKT(Ser473) phosphorylation. It was found that PI-103, as a single agent, stimulated MEK and ERK phosphorylation. However, the combination of sorafenib and PI-103 caused inhibition of all the tested kinases in the Ras/Raf and PI3K pathways.. The combination of sorafenib and PI-103 can significantly inhibit EGF-stimulated Huh7 proliferation by blocking both Ras/Raf/MAPK and PI3K/AKT/mTOR pathways.

Topics: Antineoplastic Combined Chemotherapy Protocols; Benzenesulfonates; Carcinoma, Hepatocellular; Cell Growth Processes; Cell Line, Tumor; Dose-Response Relationship, Drug; Epidermal Growth Factor; Furans; Humans; Liver Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Niacinamide; Phenylurea Compounds; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyridines; Pyrimidines; raf Kinases; ras Proteins; Signal Transduction; Sorafenib; TOR Serine-Threonine Kinases

Transforming growth factor-beta (TGF-beta) induces apoptosis in hepatocytes, through a mechanism mediated by reactive oxygen species (ROS) production. Numerous tumoral cells develop mechanisms to escape from the TGF-beta-induced tumor suppressor effects. In this work we show that in FaO rat hepatoma cells inhibition of the epidermal growth factor receptor (EGFR) with the tyrphostin AG1478 enhances TGF-beta-induced cell death, coincident with an elevated increase in ROS production and GSH depletion. These events correlate with down-regulation of genes involved in the maintenance of redox homeostasis, such as gamma-GCS and MnSOD, and elevated mitochondrial ROS. Nonetheless, not all the ROS proceed from the mitochondria. Emerging evidences indicate that ROS production by TGF-beta is also mediated by the NADPH oxidase (NOX) system. TGF-beta-treated FaO cells induce nox1 expression. However, the treatment with TGF-beta and AG1478 greatly enhanced the expression of another family member: nox4. NOX1 and NOX4 targeted knock-down by siRNA experiments suggest that they play opposite roles, because NOX1 knockdown increases caspase-3 activity and cell death, whilst NOX4 knock-down attenuates the apoptotic process. This attenuation correlates with maintenance of GSH and antioxidant enzymes levels. In summary, EGFR inhibition enhances apoptosis induced by TGF-beta in FaO rat hepatoma cells through an increased oxidative stress coincident with a change in the expression pattern of NOX enzymes.

Topics: Animals; Antioxidants; Apoptosis; Carcinoma, Hepatocellular; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Isoenzymes; Liver Neoplasms; Mitochondria; NADH, NADPH Oxidoreductases; NADPH Oxidase 1; NADPH Oxidase 4; NADPH Oxidases; Oxidative Stress; Protein Kinase Inhibitors; Quinazolines; Rats; Reactive Oxygen Species; RNA, Small Interfering; Transforming Growth Factor beta; Tyrphostins

To observe the effect of Tanshinone II A on the expression of epidermal growth facter (EGF) and epidermal growth facter recepter (EGFR) in human hepatocellular carcinoma cell line SMMC-7721.. The human hepatocellular carcinoma SMMC-7721 cells cultured in vitro was treated with different concentrations of Tanshinone II A. The proliferation of the cells was measured by MTT assay, and the apoptosis of the cells was investigated by flow cytometry and cytochemical staining with Hoechst 33342. The expression of EGF and EGFR was detected by immunocytochemistry method. The levels of EGF in medium were measured by radioimmunoassay.. Tanshinone II A inhibited the growth of SMMC-7721 cells remarkably in a dose-dependent manner. The inhibitory rate reached the peak (72.5%) after 0.5 microg/ml Tanshinone II A was used for 48 h, which was significantly higher than that in the controls (P<0.05). FCM analysis showed that when SMMC-7721 cells were treated with 0.5 microg/ml Tanshinone II A, the apoptosis rates for 24 h, 48 h and 72 h were (4.06+/-0.27)%, (7.58+/-0.56)% and (5.23+/-0.13)%, respectively which were markedly higher than those in the controls (all P<0.01). SMMC-7721 cell apoptosis with cell shrinkage, nuclear chromatin concentration and fragmentation as well as the formation of apoptotic bodies were observed by cytochemical staining when treated with Tanshinone II A. The immunocytochemistry showed that the expressions of EGF and EGFR were down regulated while the concentration of Tanshinone II A was increasing. The high expression rates for EGF and EGFR were 10%, 20%, respectively, and the gray scale was 181.52+/-1.63, 179.37+/-1.59, which were markedly higher than those in the controls (all P<0.05). The levels of EGF in medium measured by radioimmunoassay were decreased significantly after Tanshinone II A treatment.. Tanshinone II A can inhibit cell proliferation and induce apoptosis in hepatocellular carcinoma cell line SMMC-7721, which may be related to the down-regulation of EGF and EGFR protein expression.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Humans; Liver Neoplasms; Phenanthrenes

Hepatocellular carcinoma (HCC) is a major challenge because of its resistance to conventional cytotoxic chemotherapy and radiotherapy. Multi-targeted therapy might be a new option for HCC treatment. Our previous study showed that N-ras gene was activated in HCC and was inhibited by RNA interference. In the present study, we investigated the alternation of gene expression by microarray in N-Ras-siRNA-treated HepG2 cells. The results revealed that the EREG gene, encoding epiregulin, was dramatically up-regulated in response to silence of N-ras. We speculated that the up-regulation of epiregulin was involved in the compensatory mechanism of N-ras knockdown for cell growth. Therefore, we evaluated whether dual silence of N-ras and epiregulin display a greater suppression of cell growth. The results confirmed that dual knockdown of N-ras and epiregulin synergistically inhibited cell growth. Our results also showed that dual knockdown of N-ras and epiregulin significantly induced cell arrest at G0/G1 phase. Furthermore, Western blot assay showed that dual knockdown of N-ras and epiregulin markedly reduced the phosphorylations of ERK1/2, Akt and Rb, and inhibited the expression of cyclin D1. Our findings imply that multi-targeted silence of oncogenes might be an effective treatment for HCC.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Epidermal Growth Factor; Epiregulin; G1 Phase; Gene Knockdown Techniques; Gene Silencing; Humans; Liver Neoplasms; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); Resting Phase, Cell Cycle; Retinoblastoma Protein; RNA, Small Interfering

Epidermal growth factor (EGF) has many biological functions, including mitogenesis, tumorigenesis, and proliferation of epidermal tissues. Previous studies have reported that the EGF +61 (A/G) single nucleotide polymorphism in the 5'-untranslated region of the EGF gene is functional, and is associated with development of hepatocellular carcinoma (HCC) in liver cirrhosis and various malignancy. Our aim was to investigate whether EGF gene A61G polymorphism could be implicated in susceptibility to and/or clinicopathological characteristics of HCC in Chinese patients with chronic hepatitis B virus (HBV) infection.. This polymorphism was studied in 387 patients with chronic HBV infection and in 208 healthy volunteers using restriction fragment-length polymorphism. The patients were divided into two groups: those without (n = 172) and those with HCC (n = 215). These 215 HCC patients with chronic HBV infection were designated as cases, and the remaining 172 patients without HCC served as controls.. There were no significant differences in EGF genotype or allelic frequencies between cases and controls nor was EGF genotype or allelic frequencies associated with tumour number, size, growth phase, stage, and invasiveness. We also found ethnic heterogeneity in the functional EGF polymorphism.. The present results show that although EGF gene A61G polymorphism is associated with development of HCC in liver cirrhosis, it is not sufficient for HCC in Chinese patients with chronic HBV infection.

Topics: 5' Untranslated Regions; Carcinoma, Hepatocellular; Epidermal Growth Factor; Female; Genetic Predisposition to Disease; Genotype; Hepatitis B virus; Hepatitis B, Chronic; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Staging; Polymorphism, Single Nucleotide

In order to quantify the amount of ligands or poly(ethylene glycol) (PEG) on each vector, here we developed a system in which poly-L-glutamic acid (PLG) was used as surface modification loading backbone, to which one PEG (MW 5000, 10000, 20000) or epidermal growth factor (EGF) was linked. The PLG conjugates can electro-statically adsorb upon DNA/ polycation complex with positive charge, and, the amount of EGF or PEG on the surface of complexes could be varied. We have made a series of complexes containing the various PLG conjugates and examined their physicochemical properties, and made a comparison of properties and transfection efficiency between these complexes. EGF- and PEG-modified complexes showed 10-25-folds higher cell transfection efficiency than unmodified complexes in medium with or without serum.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; DNA; Drug Carriers; Drug Delivery Systems; Epidermal Growth Factor; Gene Targeting; Genetic Vectors; Humans; Nanoparticles; Particle Size; Polyglutamic Acid; Transfection

Prostaglandins (PGs) such as PGE2 enhance proliferation in many cells, apparently through several distinct mechanisms, including transactivation of the epidermal growth factor (EGF) receptor (EGFR) as well as EGFR-independent pathways. In this study we found that in primary cultures of rat hepatocytes PGE2 did not induce phosphorylation of the EGFR, and the EGFR tyrosine kinase blockers gefitinib and AG1478 did not affect PGE2-stimulated phosphorylation of ERK1/2. In contrast, PGE2 elicited EGFR phosphorylation and EGFR tyrosine kinase inhibitor-sensitive ERK phosphorylation in MH1C1 hepatoma cells. These findings suggest that PGE2 elicits EGFR transactivation in MH1C1 cells but not in hepatocytes. Treatment of the hepatocytes with PGE2 at 3 h after plating amplified the stimulatory effect on DNA synthesis of EGF administered at 24 h and advanced and augmented the cyclin D1 expression in response to EGF in hepatocytes. The pretreatment of the hepatocytes with PGE2 resulted in an increase in the magnitude of EGF-stimulated Akt phosphorylation and ERK1/2 phosphorylation and kinase activity, including an extended duration of the responses, particularly of ERK, to EGF in PGE2-treated cells. Pertussis toxin abolished the ability of PGE2 to enhance the Akt and ERK responses to EGF. The results suggest that in hepatocytes, unlike MH1C1 hepatoma cells, PGE2 does not transactivate the EGFR, but instead acts in synergism with EGF by modulating mitogenic mechanisms downstream of the EGFR. These effects seem to be at least in part G(i) protein-mediated and include upregulation of signaling in the PI3K/Akt and the Ras/ERK pathways.

Topics: Animals; Carcinoma, Hepatocellular; Cell Culture Techniques; Cells, Cultured; Culture Media, Serum-Free; Cyclin D; Cyclins; Dinoprostone; DNA; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gefitinib; Gene Expression Regulation; Hepatocytes; Liver Neoplasms; Male; Pertussis Toxin; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinazolines; Rats; Rats, Wistar; Signal Transduction; Tyrphostins

Overexpression of epidermal growth factor (EGF) in the liver induces transformation to hepatocellular carcinoma in animal models. Polymorphisms in the EGF gene modulate EGF levels.. To assess the relationship among human EGF gene single-nucleotide polymorphism, EGF expression, and risk of hepatocellular carcinoma.. Molecular mechanisms linking the 61*G allele polymorphism to EGF expression were examined in human hepatocellular carcinoma cell lines and human liver tissue. A case-control study involving 207 patients with cirrhosis was conducted at the Massachusetts General Hospital (1999-2006) and a validation case-control study involving 121 patients with cirrhosis was conducted at Hôpital Paul Brousse (1993-2006). Restriction fragment-length polymorphism was used to determine the EGF gene polymorphism genotype. Logistic regression analysis was used to assess the association between the EGF polymorphism and hepatocellular carcinoma risk.. Mechanisms by which the EGF gene polymorphism modulates EGF levels and associations among EGF gene polymorphism, EGF levels, and hepatocellular carcinoma.. Transcripts from the EGF 61*G allele exhibited more than a 2-fold longer half-life than those from the 61*A allele, and EGF secretion was 2.3-fold higher in G/G hepatocellular carcinoma cell lines than A/A cell lines. Serum EGF levels were 1.8-fold higher in G/G patients than A/A patients, and liver EGF levels were 2.4-fold higher in G/G patients than A/A patients. Among the 207 patients with cirrhosis in the Massachusetts study population, 59 also had hepatocellular carcinoma. Analysis of the distribution of allelic frequencies revealed that there was a 4-fold odds of hepatocellular carcinoma in G/G patients compared with A/A patients in the Massachusetts study population (odds ratio, 4.0; 95% confidence interval [CI], 1.6-9.6; P = .002). Logistic regression analysis demonstrated that the number of copies of G was significantly associated with hepatocellular carcinoma after adjusting for age, sex, race, etiology, and severity of cirrhosis (G/G or A/G vs A/A; hazard ratio, 3.49; 95% CI, 1.29-9.44; P = .01). The significant association was validated in the French patients with alcoholic cirrhosis and hepatocellular carcinoma.. The EGF gene polymorphism genotype is associated with risk for development of hepatocellular carcinoma in liver cirrhosis through modulation of EGF levels.

Topics: Carcinoma, Hepatocellular; Case-Control Studies; Cell Line, Tumor; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Genotype; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Risk Factors

Epidermal growth factor receptor (EGFR) is highly expressed in many human tumors including hepatocellular carcinoma (HCC). Therefore, inhibition of EGF receptors could be a potential target for anticancer therapy. In this study, we investigated the effects of two EGFR tyrosine kinase inhibitors, PD153035 and its analogue 4-[[3-chloro-4-fluorophenyl]amino]-6,7-dimethoxyquinazoline hydrochloride (ANAPD) on human HCC cell lines by cell proliferation assay, flow cytometry and Western blot. Our results demonstrated that both EGFR inhibitors inhibited tumor cell growth in a dose-dependent manner, but ANAPD was more potent than PD153035. These specific inhibitors not only blocked EGF-stimulated EGFR autophosphorylation but also targeted EGFR signaling including MAPK and Akt pathways. Furthermore, EGFR inhibitors induced a delay in cell cycle progression and a G(1) arrest together with a partial G(2)/M block. EGFR inhibitors also induced tumor cells to undergo apoptosis. In conclusion, this study demonstrated that both PD153035 and ANAPD inhibit tumor cell growth in HCC through inhibition of EGFR signaling pathway, and ANAPD is a more potent inhibitor than PD153035. This suggested that blockage of EGF receptors may provide an effective therapeutic approach for human HCC and ANAPD could be a potential drug candidate for the treatment of HCC.

Topics: Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Humans; Liver Neoplasms; Mitogen-Activated Protein Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Quinazolines

The human gene Teratocarcinoma-derived growth factor 1 (TDGF1)/Cripto-1/CR-1 which is expressed in a wide variety of human carcinomas is a member of the EGF-cripto FRL1 cryptic (EGF-CFC) gene family. A majority of human colorectal tumors and hepatomas are known to possess a constitutively active canonical Wnt/beta-catenin/TCF signaling pathway, also express CR-1. Expression of a short form of CR-1 mRNA in colon carcinoma and hepatoma cell lines suggests that there may be differential regulation of CR-1 expression by the canonical Wnt signaling pathway in colon cancer as well as hepatoma cell lines. The present study demonstrates a direct transcriptional regulation of the short form CR-1 expression by the canonical Wnt signaling pathway through an intronic-exonic enhancer element, containing three tandem TCF/LEF binding sites within the CR-1 gene.

Topics: beta Catenin; Binding Sites; Carcinoma; Carcinoma, Hepatocellular; Cell Line, Tumor; Colonic Neoplasms; Enhancer Elements, Genetic; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Genetic Vectors; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Lymphoid Enhancer-Binding Factor 1; Membrane Glycoproteins; Neoplasm Proteins; Protein Isoforms; Recombinant Proteins; RNA, Messenger; TCF Transcription Factors; Transcription, Genetic

Cimetidine, a histamine-2 (H2) receptor antagonist, has been demonstrated to have anticancer effects on colorectal cancer, melanoma and renal cell carcinoma. In the current study, we clarified that cimetidine inhibits both epidermal growth factor (EGF)-induced cell proliferation and migration in hepatocellular carcinoma (HCC) cell lines.. HCC cell lines (Hep3B, HLF, SK-Hep-1, JHH-2, PLC/PRF/5 and HLE) were used and cell proliferation was assessed by [3H]-thymidine incorporation assay. Cell migration was measured by in vitro cell migration assay. Biological effects of cimetidine were assessed with human EGF receptor (EGFR)-expressing mouse fibroblast cells (NR6-WT). The autophosphorylation of EGFR and the activation of other downstream effectors were analyzed by immunoprecipitation and immunoblotting. The concentration of intracellular cyclic AMP (cAMP) was measured by competitive enzyme immunoassay.. Cimetidine inhibited both EGF-induced cell proliferation and migration in Hep3B, HLF, SK-Hep-1 and JHH-2, while cimetidine did not affect EGF-induced cell proliferation and migration in PLC/PRF/5 and HLE. Cimetidine was revealed to disrupt the EGF-induced autophosphorylation of EGFR and its downstream effectors, mitogen activated protein kinases and phospholipase C-gamma. To define the molecular basis of this negative regulation, we identified that cimetidine significantly decreased intracellular cAMP levels and that decrement of cAMP inhibited autophosphorylation of EGFR. The cell permeable cAMP analog, CPT-cAMPS reversed the cimetidine-induced inhibition of EGF-induced cell proliferation and cell migration by restoring autophosphorylation of EGFR.. Cimetidine inhibited EGF-induced cell proliferation and migration in HCC cell lines by decreasing the concentration of intracellular cAMP levels. Cimetidine may be a candidate chemopreventive agent for HCC.

Topics: Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Cimetidine; Epidermal Growth Factor; Histamine H2 Antagonists; Humans; Liver Neoplasms; Tumor Cells, Cultured

The nucleotide excision repair (NER) pathway and its leading gene excision-repair cross-complementary 1 (ERCC1) have been shown to be up-regulated in hepatocellular carcinomas even in the absence of treatment with chemotherapeutics. The aim of this study was to determine the mechanism involved in NER regulation during the liver cell growth observed in hepatocellular carcinoma. Both NER activity and ERCC1 expression were increased after exposure to the epidermal growth factor (EGF) in cultured normal and tumoral human hepatocytes. These increases correlated with the activation of the kinase signaling pathway mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK that is known to be a key regulator in the G(1) phase of the hepatocyte cell cycle. Moreover, EGF-mediated activation of ERCC1 was specifically inhibited by either the addition of U0126, a MEK/ERK inhibitor or small interfering RNA-mediated knockdown of ERK2. Basal expression of ERCC1 was decreased in the presence of the phosphoinositide-3-kinase (PI3K) inhibitor and small hairpin RNA (shRNA) against the PI3K pathway kinase FKBP12-rapamycin-associated protein or mammalian target of rapamycin. Transient transfection of human hepatocytes with constructs containing different sizes of the 5'-flanking region of the ERCC1 gene upstream of the luciferase reporter gene showed an increase in luciferase activity in EGF-treated cells, which correlated with the presence of the nuclear transcription factor GATA-1 recognition sequence. The recruitment of GATA-1 was confirmed by chromatin immunoprecipitation assay. In conclusion, these results represent the first demonstration of an up-regulation of NER and ERCC1 in EGF-stimulated proliferating hepatocytes. The transcription factor GATA-1 plays an essential role in the induction of ERCC1 through the mitogen-activated protein kinase (MAPK) pathway, whereas the PI3K signaling pathway contributes to ERCC1 basal expression.

Topics: Carcinoma, Hepatocellular; DNA Repair; DNA-Binding Proteins; DNA, Neoplasm; Endonucleases; Epidermal Growth Factor; GATA1 Transcription Factor; Humans; Liver Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Tumor Cells, Cultured

Most hepatomas have a defect in prothrombin carboxylation, and can secrete under-carboxylated prothrombin or des-gamma-carboxy-prothrombin (DCP), the function of which is unknown. We considered that the prothrombin-DCP axis might also be involved in growth control. Hepatocytes and hepatoma cells were treated with prothrombin and DNA synthesis and cytoskeletal changes were studied. Prothrombin inhibited DNA synthesis in hepatocytes on fibronectin, but not collagen matrix. Hepatoma cell lines were not inhibited. We found that hepatoma cell matrix conferred resistance to hepatocytes. Prothrombin decreased fibronectin but not collagen amounts, but only in the presence of hepatocytes and not hepatoma cells, indicating that it has a differential action on matrix proteins. It also caused changes in cell shape and actin depolymerization. In vivo, there was a decrease in plasma prothrombin activity after a partial hepatectomy (PH), concomitant with the peak of DNA synthesis in the hepatocytes at 24h after PH. Injection of warfarin at the time of PH, further inhibited PT activity and enhanced this 24h peak of DNA synthesis. Furthermore, repeated injection of prothrombin lowered the peak DNA synthesis after PH. The data support the hypothesis that prothrombin can act as a hepatocyte growth inhibitor, likely at the level of fibronectin loss and result in cytoskeletal changes. Hepatomas resist this action, possibly due to their different matrix proteins. This represents a novel mechanism for growth regulation and provides a possible biological significance for the tumor marker DCP.

Topics: Animals; Carcinoma, Hepatocellular; Cattle; Cell Line, Tumor; Cell Proliferation; Cytoskeleton; DNA; Epidermal Growth Factor; Extracellular Matrix; Hepatectomy; Hepatocytes; Humans; Liver Neoplasms; Liver Regeneration; Male; Prothrombin; Rats; Rats, Inbred F344; Vitamin K

To evaluate the anti-tumor effect of recombinant toxin EGF-TCS against transplanted human hepatocellular carcinoma in nude mice.. Human hepatocellular carcinoma BEL-7,402 cells were inoculated subcutaneously in the right axillary region of nude mice, and 6 days later, EGF-TCS was injected intravenously at 100, 50, and 25 microg/kg. The mice were executed on the next day of drug withdrawal and the tumors were weighed and the tumor inhibition rate calculated. Immunohistochemistry was also performed on the tumor tissues to provide clue for the possible pathways of tumor inhibition.. EGF-TCS markedly inhibited the tumor growth in nude mice, with a tumor inhibition rate of 71.3%, 60.87% and 45.22% corresponding to EGF-TCS dosage of 100, 50, and 25 microg/kg, respectively. Variance analysis suggested that EGF-Linker-TCS could significantly inhibit the tumor growth in the mice (F=8.712, P=0.006), and immunohistochemistry showed significantly inhibited angiogenesis in the tumors by EGF-TCS. No blood vessels were found in the tumor tissues in high dosage group, and there were also reduced blood vessels in the other two smaller dose groups in comparison with the untreated model group, indicating that EGF-TCS inhibited tumor growth and migration by inhibiting tumor angiogenesis.. EGF-TCS can inhibit the growth of solid tumors in nude mice, suggesting the potential value of this preparation in cancer therapy.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Disease Models, Animal; Epidermal Growth Factor; Female; Humans; Immunotoxins; Liver Neoplasms; Male; Mice; Mice, Nude; Neoplasm Transplantation; Random Allocation; Recombinant Fusion Proteins; Trichosanthin

We measured aspartyl (asparaginyl)-beta-hydroxylase (AAH) gene expression in human hepatocelluar carcinoma and surrounding uninvolved liver at both the mRNA and protein level and examined the regulation and function of this enzyme.. Since growth of HCC is mediated by signaling through the insulin-receptor substrate, type 1 (IRS-1), we examined-if AAH is a downstream gene regulated by insulin and IGF-1 in HCC cells. In addition, IRS-1 regulation of AAH was examined in a transgenic (Tg) mouse model in which the human (h) IRS-1 gene was over-expressed in the liver, and an in vitro model in which a C-terminus truncated dominant-negative hIRS-1 cDNA (hIRS-DeltaC) was over-expressed in FOCUS HCC cells. The direct effects of AAH on motility and invasiveness were examined in AAH-transfected HepG2 cells.. Insulin and IGF-1 stimulation increased AAH mRNA and protein expression and motility in FOCUS and Hep-G2 cells. These effects were mediated by signaling through the Erk MAPK and PI3 kinase-Akt pathways. Over-expression of hIRS-1 resulted in high levels of AAH in Tg mouse livers, while over-expression of hIRS-DeltaC reduced AAH expression, motility, and invasiveness in FOCUS cells. Finally, over-expression of AAH significantly increased motility and invasiveness in HepG2 cells, whereas siRNA inhibition of AAH expression significantly reduced directional motility in FOCUS cells.. The results suggest that enhanced AAH gene activity is a common feature of human HCC and growth factor signaling through IRS-1 regulates AAH expression and increases motility and invasion of HCC cells. Therefore, AAH may represent an important target for regulating tumor growth in vivo.

Topics: Animals; Biopsy; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Hypoglycemic Agents; Insulin; Insulin Receptor Substrate Proteins; Insulin-Like Growth Factor I; Liver Neoplasms; Mice; Mice, Transgenic; Mixed Function Oxygenases; Neoplasm Invasiveness; Phosphoproteins; Signal Transduction

Combination of the degradable polymeric gene carriers OEI-HD-1 and LT- OEI-HD-1 with an EGF targeting conjugate resulted in strongly (up to 900-fold) enhanced polyplex activity in EGF-receptor rich HUH7 hepatocellular carcinoma cells. The targeting ligand effect was DNA dose dependent, could be blocked by competitive receptor binding with unbound EGF ligand, and was not observed in receptor-negative control cells. Measures which enhance intracellular endosomal escape, either photochemically enhanced intracellular release (PCI) or the incorporation of a novel membrane-active melittin analog NMA-3, further enhanced gene transfer activity of EGF/OEI-HD-1 polyplexes.

Topics: Animals; Binding, Competitive; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Membrane; DNA; Endosomes; Epidermal Growth Factor; ErbB Receptors; Genes, Reporter; Genetic Therapy; Humans; Liver Neoplasms; Luciferases; Melitten; Mice; Photosensitizing Agents; Plasmids; Polyethyleneimine; Porphyrins; Transfection

Epidermal growth factor is an important mitogen for hepatocytes. Its overexpression promotes hepatocellular carcinogenesis. To identify the network of genes regulated through EGF, we investigated the liver transcriptome during various stages of hepatocarcinogenesis in EGF2B transgenic mice. Targeted overexpression of IgEGF induced distinct hepatocellular lesions and eventually solid tumours at the age of 6-8 months, as evidenced by histopathology. We used the murine MG U74Av2 oligonucleotide microarrays to identify transcript signatures in 12 tumours of small (n=5, pooled), medium (n=4) and large sizes (n=3), and compared the findings with three nontumorous transgenic livers and four control livers. Global gene expression analysis at successive stages of carcinogenesis revealed hallmarks linked to tumour size. A comparison of gene expression profiles of nontumorous transgenic liver versus control liver provided insight into the initial events predisposing liver cells to malignant transformation, and we found overexpression of c-fos, eps-15, TGIF, IGFBP1, Alcam, ets-2 and repression of Gas-1 as distinct events. Further, when gene expression profiles of small manifested tumours were compared with nontumorous transgenic liver, additional changes were obvious and included overexpression of junB, Id-1, minopontin, villin, claudin-7, RR M2, p34cdc2, cyclinD1 and cyclinB1 among others. These genes are therefore strongly associated with tumour formation. Our study provided new information on the tumour stage-dependent network of EGF-regulated genes, and we identified candidate genes linked to tumorigenes and progression of disease.

Topics: Animals; Carcinoma, Hepatocellular; Epidermal Growth Factor; Expressed Sequence Tags; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, fos; Homeodomain Proteins; Liver Neoplasms; Mice; Mice, Transgenic; Precancerous Conditions; Proto-Oncogene Protein c-ets-2; Proto-Oncogene Proteins; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; Trans-Activators; Transcription, Genetic

We have reported that the selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, gefitinib ('Iressa', ZD1839), suppressed intrahepatic metastasis of hepatocellular carcinoma CBO140C12 cells. In this study, we focused on the tumour necrosis factor-alpha (TNF-alpha) signalling pathways. Real-time reverse transcription-polymerase chain reaction showed that TNF-alpha mRNA was expressed in large quantities in the implanted tumour. Gefitinib inhibited EGF- but not hepatocyte growth factor (HGF)-induced activation of mitogen-activated protein kinase (MAPK) cascades, suggesting selectivity of the inhibitor. However, gefitinib inhibited the TNF-alpha-induced activation of MAPKs and Akt. In addition, TNF-alpha-induced metastatic properties including adhesion to fibronectin, mRNA expression of integrin alpha v, production of matrix metalloproteinase-9 and invasion were inhibited by gefitinib without affecting cell proliferation. Furthermore, the TNF-alpha-induced responses except for NF-kappaB activation were blocked by metalloprotease inhibitors, suggesting that gefitinib inhibited the transactivation of EGFR induced by TNF-alpha. These results suggest that the TNF-alpha signalling pathway is a possible target of gefitinib in suppressing the intrahepatic metastasis of hepatocellular carcinoma.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Epidermal Growth Factor; Female; Gefitinib; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinases; Neoplasm Metastasis; Protein Kinase Inhibitors; Quinazolines; Tumor Necrosis Factor-alpha

Hepatitis B virus (HBV) preferentially replicates in quiescent cells. It was analyzed whether HBV affects cell cycle control.. The amount of EGF-receptor (EGFR) and the binding capacity for 125I-EGF was determined. Expression of mdm2 and p21 and relevance of p53 for it were analyzed by reporter gene assays and western blotting. Cyclin A/E associated cdk2 activities were determined by immunocomplex assays. Cell proliferation was quantified by measurement of BrdU incorporation.. In HBV producing cells a significant reduction of EGFR expression, diminished 125I-EGF-binding capacity and insensitivity to EGF-stimulation were observed as compared to the control. Moreover, c-Raf-1-dependent induction of mdm2-P2 and p21cip1/waf1-promoter and elevated amounts of the respective proteins were observed in HBV producing cells. Whereas activation of mdm2-P2-promoter requires p53, activation of p21cip1/waf1-promoter is mediated partially by a p53-independent process. Induction of p21cip1/waf1 is reflected by a reduction of cyclin A associated cdk2 activity and an increase of cyclin E associated cdk2 activity. In accordance with this proliferation rate of HBV-producing hepatocytes is reduced as compared to control cells.. These results describe novel cell-cycle inhibitory functions of HBV that correlate well with the general concept of enhanced HBV replication in quiescent cells.

Topics: Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cells, Cultured; Cyclin A; Cyclin E; Epidermal Growth Factor; ErbB Receptors; Genes, Reporter; Hepatitis B virus; Humans; Liver Neoplasms; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Virus Replication

Caspase-8 is the apical caspase essential for triggering Fas-induced apoptosis. In this study, we investigated caspase-8 expression in hepatocellular carcinomas (HCCs) using recently described HCC mouse models (c-myc and IgEGF transgenes).. HCCs were isolated from c-myc and IgEGF transgenic animals. Expression of caspase-8 was monitored by reverse-transcription polymerase chain reaction. The murine caspase-8 promoter was characterized by luciferase-reporter analysis and the analysis of promoter methylation was performed by bisulfite genomic sequencing.. In HCCs investigated, we frequently found a lack of caspase-8 messenger RNA expression. Genomic deletions at the caspase-8 locus did not contribute to caspase-8 silencing. We examined tumor-derived promoter sequences and found significant hypermethylation at distinct CpG sites. In parallel, we characterized the murine caspase-8 promoter and identified a 30-bp promoter element that is indispensable for basal promoter activity. This minimal promoter element contained SP1 binding motifs that are colocalized with CpG sites and were methylated in tumor-derived promoter sequences. Electrophoretic mobility shift assay analysis showed that methylation of these SP1 sites is sufficient to prevent SP1 complex formation. To support our data, we mimicked the methylation pattern of a tumor-derived caspase-8 promoter in vitro using CpG methylase and found a strong reduction of promoter activity.. We show that HCCs are correlated frequently with silencing of caspase-8 expression and provide data suggesting that caspase-8 silencing is a direct consequence of inhibiting SP1-dependent transactivation caused by CpG methylation at its essential binding sites in the promoter region. Our data support the hypothesis that inhibition of apoptosis triggers hepatocarcinogenesis.

Topics: Animals; Base Sequence; Carcinoma, Hepatocellular; Caspase 8; Caspases; Cell Line, Tumor; Consensus Sequence; CpG Islands; DNA Methylation; Epidermal Growth Factor; Gene Silencing; Genes, myc; Genes, Reporter; Humans; Liver Neoplasms; Luciferases; Mice; Mice, Transgenic; Molecular Sequence Data; Promoter Regions, Genetic; Sp1 Transcription Factor

Previously, we reported that, in hepatocyte growth factor (HGF)-induced HepG2 cells, protein kinase C (PKC) decreased the duration of intensive Erk1/Erk2 MAP kinase activation. This study shows that the inhibition of PKC enhanced significantly the HGF-induced integrin expression. Beside the prolonged activation of Erk1/Erk2, the activity of phosphatidylinositol 3-kinase (PI 3K) was required for growth factor-induced integrin expression. PI 3-kinase was activated to a higher extent in response to HGF than to epidermal growth factor (EGF), though the activation was transient in both cases. In EGF-induced cells, PI 3K activation was terminated by the loss of phosphotyrosine docking sites for PI 3K. To the contrary, the decrease of PI 3K activation, which followed the HGF-induced increase was not accompanied by the loss of phosphotyrosine docking sites and was prevented by the inhibition of PKC. The negative modulator effects of PKC on integrin expression and PI 3-kinase activation correlated with its ability to limit the HGF-induced motogen response.

Topics: Carcinoma, Hepatocellular; Cell Movement; Enzyme Inhibitors; Epidermal Growth Factor; Hepatocyte Growth Factor; Humans; Integrins; MAP Kinase Signaling System; Phorbol Esters; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinase C; Tumor Cells, Cultured

Osteopontin (OPN) is a phosphorylated glycoprotein with diverse functions including cancer development, progression and metastasis. It is unclear how osteopontin is regulated in HepG2 cells. The aim of this study was to investigate the effect of epidermal growth factor on the expression of osteopontin in HepG2 cells, and to explore the signal transduction pathway mediated this expression.. Osteopontin expression was detected by RNAase protection assay and Western blot. Wortmannin, a specific inhibitor of PI3K, was used to see if PI3K signal transduction was involved in the induction of osteopontin gene expression.. HepG2 cells constitutively expressed low levels of osteopontin. Treatment with epidermal growth factor increased osteopontin mRNA and protein level in a dose- and time-dependent manner. Application of wortmannin caused a dramatic reduction of epidermal growth factor-induced osteopontin expression.. Osteopontin gene expression can be induced by treatment of HepG2 cells with epidermal growth factor. Epidermal growth factor may regulate osteopontin gene expression through PI3K signaling pathway. Several potential targets in the pathway can be manipulated to block the synthesis of osteopontin and inhibit liver cancer metastasis.

Topics: Androstadienes; Carcinoma, Hepatocellular; Cell Line, Tumor; Enzyme Inhibitors; Epidermal Growth Factor; Gene Expression; Humans; Liver Neoplasms; Osteopontin; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Sialoglycoproteins; Signal Transduction; Wortmannin

The mitogenic signaling pathway of epidermal growth factor receptor (EGFR) is activated in a variety of cancers, including hepatocellular carcinoma (HCC). The recently cloned EGFR-related protein (ERRP) has been proposed to be a negative regulator of EGFR. The expression of ERRP in non-malignant liver cells and in HCC, and its relation to cell proliferation, have not been examined.. Paraffin sections of formalin-fixed HCC specimens of 51 HCCs and 10 normal liver specimens were immunostained with anti-ERRP and anti-p53 antibodies. To determine the relationship between ERRP expression and cell proliferation, we performed double staining for ERRP and proliferating cell nuclear antigen (PCNA) in the same HCC specimens.. In normal liver, ERRP was expressed in 100% of specimens, while in non-malignant liver tissue from HCC, ERRP expression was present in 50% of the specimens (p < 0.001). In contrast, ERRP expression was present only in 14% of the HCC specimens. The expression of ERRP in cancer cells was inversely correlated with proliferative activity and tumor size (p < 0.001, p < 0.05, respectively). No significant correlation was found between p53 expression and the expression of ERRP.. Human HCC, which has been shown to be associated with increased activation of EGFR, exhibits a substantial reduction in ERRP expression. The inverse relationship between ERRP expression, proliferative activity of tumor cells and tumor size suggests that in some HCC, ERRP is a negative regulator of HCC growth.

Topics: Carcinoma, Hepatocellular; Cell Division; Epidermal Growth Factor; ErbB Receptors; Genes, p53; Glycoproteins; Humans; Immunohistochemistry; Liver Neoplasms; Molecular Sequence Data; Oncogene Proteins; Proliferating Cell Nuclear Antigen; Signal Transduction; Tumor Cells, Cultured

Many evidences demonstrated that the epidermal growth factor receptor (EGFR) subfamily played an important role in they initiation and progression of various cancers. But it is not clear whether there is a relationship between EGFR and hepatocellular carcinoma (HCC). The aims of the present study were to explore the expression and significance of the epidermal growth factor (EGF) mRNA and EGFR mRNA in human HCC tissues.. Reverse transcription polymerase chain reaction (RT-PCR) was employed to determine the expression of EGF mRNA and EGFR mRNA in 60 HCC tissues and their adjacent liver tissues.. The positive rate of EGF mRNA was significantly lower in the HCC tissue (60%, 36/60) than in the adjacent liver tissue (80%, 48/60) (P< 0.05). The positive rate of EGFR mRNA in the HCC tissue (60%, 36/60) was markedly higher than in the adjacent liver tissue (41.67%, 25/60) (P< 0.05). The expression of EGFR mRNA in the HCC tissue was significantly correlated with the clinical stage, the portal vein tumor thrombus, the presence of extrahepatic metastasis, and the recurrence of tumor, the number of tumor, but not correlated with the diameter of tumor, the level of serum alpha-fetoprotein (AFP), the differentiation of tumor and the liver cirrhosis in the adjacent tissue. The detectable rate of EGF mRNA was correlated with the diameter of tumor but not correlated with the clinical stage, the portal vein tumor thrombus, the presence of extrahepatic metastasis, the recurrence of tumor, the number of tumor, the level of serum AFP, the differentiation of tumor and the liver cirrhosis in the adjacent tissue.. EGF may not be involved in the initiation and progression of HCC, whereas, EGFR relates to the initiation and progression and the recurrence of HCC. EGFR can be considered as a marker for predicting the metastasis and recurrence of HCC.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Epidermal Growth Factor; ErbB Receptors; Female; Follow-Up Studies; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; RNA, Messenger

To investigate the effect of catenin p120 (p120ctn) translocation on the malignant features of hepatocellular carcinoma and its interrelation with beta-catenin in E-cadherin-mediated cell signaling.. Expression and translocation of p120ctn, tyrosine phosphorylation, and its binding capacity to E-cadherin were detected by DNA transfection, immunoblotting and immunoprecipitation. Cellular localization of p120ctn and beta-catenin was detected by immunofluorescent microscopy. Cell adhesion, cell migration and cell proliferation were also studied.. Expression of p120ctn increased after cells transfected with p120ctn isoform 3A, and it was located mainly at cell-cell contact region. Its binding to E-cadherin was enhanced. After EGF stimulation, tyrosine phosphorylation of p120ctn was increased, membrane expression of p120ctn and beta-catenin was decreased while cytosol expression was increased. It was translocated into the nucleus, cell adhesiveness was increased but mobility decreased. With over-expression of p120ctn, beta-catenin was recruited by nucleus export. Cell proliferation was reduced but it was increased after EGF treatment.. p120tn plays an important role in cell adhesion, migration and proliferation of hepatocellular carcinoma, and its tyrosine phosphorylation might contribute to this mechanism. There might be a competitive relationship between p120ctn and beta-catenin.

Topics: beta Catenin; Cadherins; Carcinoma, Hepatocellular; Catenins; Cell Adhesion; Cell Adhesion Molecules; Cell Line, Tumor; Cell Membrane; Cell Movement; Cell Nucleus; Cell Proliferation; Cytoskeletal Proteins; Cytosol; Delta Catenin; Epidermal Growth Factor; Humans; Liver Neoplasms; Phosphoproteins; Phosphorylation; Protein Transport; Trans-Activators; Tyrosine

Tumor growth is accelerated by induction of angiogenesis regulated by many cytokines and growth factors. In a tumor mass, various angiogenic factors and their receptors are simultaneously expressed and the overlapped expressions of various factors may contribute to the aggressive growth of tumor. However, the possible combined effect and mechanism of growth factors involved in angiogenesis are still under investigated. Insulin-like growth factor-II (IGF-II) has been identified as an angiogenic factor and highly expressed in solid tumors. Here we demonstrated that another angiogenic factor, epidermal growth factor (EGF), synergistically induced the angiogenic activity when co-treated with IGF-II in vivo. We performed mouse Matrigel plug assay. Cotreatment of IGF-II and EGF resulted in a significant induction of functional new vessels in mouse Matrigel plug more than additive amount of vessels induced by each growth factor. However, synergism of these two factors was not found in in vitro angiogenic assays, i.e., in migration and proliferation assays. The metalloproteinase-2 (MMP-2) protein level was enhanced by co-treatment of IGF-II and EGF, similar to that of individual treatment of them or PMA or bFGF. EGF down-regulated hypoxia-induced IGF-II binding protein-3 (IGFBP-3), which may contribute to the enhancement of free IGF-II accessibility to its receptors in vivo. Moreover, the combination of IGF-II with EGF significantly induced bFGF mRNA level, a potent angiogenic molecule, which may also contribute to synergistic effect in vivo. These results suggest that IGF-II and EGF may synergistically cooperate to induce angiogenesis in vivo by indirect mechanisms, i.e., synergistic induction of another angiogenic factor and modulation of IGF-II's bioavailability.

Topics: Angiogenesis Inducing Agents; Animals; Blood Vessels; Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Chick Embryo; Collagen; Drug Combinations; Drug Synergism; Drug Therapy, Combination; Endothelium, Vascular; Epidermal Growth Factor; Fibroblast Growth Factor 2; Humans; Hypoxia; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor II; Laminin; Liver Neoplasms; Male; Matrix Metalloproteinase 2; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Proteoglycans; RNA, Messenger; Tumor Cells, Cultured; Umbilical Veins

Rab proteins comprise a family of monomeric GTPases that control cellular membrane traffic. Rab21 is a poorly characterised member with no known function. Human Rab21 cDNA from K562 cells was subcloned into GFP expression vectors to generate Rab21 and Rab21 mutants defective in either GTP hydrolysis (Rab21 Q78L) or binding (Rab21 T33N) for transfection studies in HeLa cells. Confocal fluorescence microscopy and ultrastructural studies revealed Rab21 to be predominantly localised to the early endocytic pathway, on vesicles containing earlyendosomal antigen 1 EEA1, transferrin receptor and internalised ligands. EEA1 was localised to enlarged endosomes in Rab21 wild-type expressing cells but the GTP hydrolysis and GDP binding mutants had unique phenotypes labelling tubular reticular structures and the trans-Golgi network, respectively. Early endosome localisation for Rab21 was confirmed in a hepatoma cell line that allowed analysis of the subcellular distribution of the endogenous protein. Comparison of the localisation of Rab21 with other Rabs revealed extensive colocalisation with early endocytic variants Rab4, Rab5, Rab17 and Rab22 but much less overlap with those associated with late endosomes, recycling endosomes and the early secretory pathway. Cells expressing Rab21 T33N had defects in endocytosis of transferrin and epidermal growth factor and failed to effectively deliver the latter ligand to late endosomes and lysosomes for degradation. Collectively, our data provide the first characterisation of Rab21 function in early endosome dynamics.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cloning, Molecular; Endocytosis; Endosomes; Epidermal Growth Factor; Golgi Apparatus; Green Fluorescent Proteins; HeLa Cells; Humans; Immunohistochemistry; Membrane Proteins; Microscopy, Confocal; Mutation; Precipitin Tests; Protein Binding; rab GTP-Binding Proteins; rab4 GTP-Binding Proteins; rab5 GTP-Binding Proteins; Transfection; Transferrin; Vesicular Transport Proteins

Many growth factors, such as epidermal growth factor (EGF), are associated with the carcinogenesis. EGF plays its role in the proliferation of hepatoma cells through binding with EGF receptor (EGFR) and a series of signal transduction. But the postreceptor pathway is still not clear. In the present experiment, we studied the effect of tyrosine kinase, protein kinase C, Na(+)/H(+) exchange, calmodulin and voltage-dependent Ca(2+) channel on EGF-induced hepatoma cell proliferation.. Hepatoma cell line SMMC7721 was cultured in RPMI1640 serum-free medium. In order to study the effect of thyrosine kinase, protein kinase C, Na(+)/H(+) exchange, calmodulin and voltage-dependent Ca(2+) channel on human heptoma cell proliferation induced by epidermal growth factor (EGF), DNA synthesis rate of hepatoma cells was measured by the method of (3)H-TdR incorporation.. EGF (10(-9) M) stimulated the proliferation of heptoma cells significantly ((3)H-TdR incorporation was 1 880+/-281 cpm/well, P<0.05), and this effect was significantly inhibited by tyrosine kinase inhibitor genistein ((3)H-TdR incorporation was 808+/-209 cpm/well, P<0.001). Calmodulin inhibitor W-7, protein kinase C inhibitor H-7 and Na(+)/H(+) exchange inhibitor amiloride individually had significant inhibiting effect on EGF-induced proliferation of hepatoma cells ((3)H-TdR incorporation was 978+/-87.3 cpm/well, 1 241+/-147 cpm/well, 1 380+/-189 cpm/well, respectively, P<0.001, P<0.01, P<0.05), but they all had no effect on the basal level proliferation of cultured hepatoma cells ((3)H-TdR incorporation was 1 284+/-260 cpm/well, 1 179+/-150 cpm/well, 1 392+/-152 cpm/well, respectively, (3)H-TdR incorporation of the control was 1 353+/-175 cpm/well, P>0.05). Voltage-dependent Ca(2+) channel inhibitor verapamil had no inhibition on EGF-induced proliferation of hepatoma cells ((3)H-TdR incorporation was 1 637+/-133 cpm/well, P>0.05), it also had no effect on the basal level proliferation of cultured hepatoma cells ((3)H-TdR incorporation was 1 196+/-112 cpm/well, P>0.05).. Our data suggest that tyrosine kinase, Ca(2+)-calmodulin-dependent pathway, protein kinase C and Na(+)/H(+) exchange play a critical role in EGF-induced proliferation of hepatoma cells and that the effect of EGF is independent of voltage-dependent Ca(2+) channel.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Carcinoma, Hepatocellular; Cell Division; Enzyme Inhibitors; Epidermal Growth Factor; Genistein; Liver Neoplasms; Sodium-Hydrogen Exchangers; Sulfonamides; Tumor Cells, Cultured

Emerging data suggest that signaling by heparin-binding growth factors is influenced by the sulfation state of N-acetylglucosamine residues of heparan sulfate proteoglycans (HSPGs). Here we report that the recently identified protein HSulf-1, a heparin-degrading endosulfatase, encodes a cell surface-associated enzyme that diminishes sulfation of cell surface HSPGs. The message encoding this enzyme is readily detectable in a variety of normal tissues, including normal ovarian surface epithelial cells, but is undetectable in 5 of 7 ovarian carcinoma cell lines and markedly diminished or undetectable in approximately 75% of ovarian cancers. Similar down-regulation is also observed in breast, pancreatic, renal cells, and hepatocellular carcinoma lines. Re-expression of HSulf-1 in ovarian cancer cell lines resulted in diminished HSPG sulfation, diminished phosphorylation of receptor tyrosine kinases that require sulfated HSPGs as co-receptors for their cognate ligands, and diminished downstream signaling through the extracellular signal-regulated kinase pathway after treatment with fibroblast growth factor-2 or heparin-binding epidermal growth factor. Consistent with these changes, HSulf-1 re-expression resulted in reduced proliferation as well as sensitivity to induction of apoptosis by the broad spectrum kinase inhibitor staurosporine and the chemotherapeutic agent cisplatin. Collectively, these observations provide evidence that HSulf-1 modulates signaling by heparin-binding growth factors, and HSulf-1 down-regulation represents a novel mechanism by which cancer cells can enhance growth factor signaling.

Topics: 5' Untranslated Regions; Breast Neoplasms; Carcinoma, Hepatocellular; Cell Division; Cloning, Molecular; Epidermal Growth Factor; Epithelial Cells; Female; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Ovarian Neoplasms; Ovary; Recombinant Proteins; Signal Transduction; Sulfotransferases; Transfection; Tumor Cells, Cultured

Stat3 is activated by cytokines and growth factors via specific tyrosine phosphorylation, dimerization, and nuclear translocation. However, the mechanism involved in its nuclear translocation is unclear. In this study, by systematic deletion and site-directed mutagenesis we identified Arg-214/215 in the alpha-helix 2 region of the coiled-coil domain of Stat3 as a novel sequence element essential for its nuclear translocation, stimulated by epidermal growth factor as well as by interleukin-6. Furthermore, we identified Arg-414/417 in the DNA binding domain as also required for the nuclear localization of Stat3. This sequence element corresponds to Lys-410/413 of Stat1, a reported sequence for Stat1 nuclear translocation. On the other hand, Leu-411 of Stat3, corresponding to Leu-407 of Stat1, a necessary residue for Stat1 nuclear transport, is not essential for Stat3 nuclear import. The mutant of Arg-214/215 or Arg-414/417 was shown to be tyrosyl-phosphorylated normally but failed to enter the nucleus in response to epidermal growth factor or interleukin-6. The defect, however, can be rescued by the wild-type Stat3 but cannot be compensated by these two mutants. Mutations on Arg-414/417, but not Arg-214/215, destroy the DNA binding activity of Stat3. Our data for the first time identified a sequence element located in the coiled-coil domain that is involved in the ligand-induced nuclear translocation of Stat3. This novel sequence together with a conserved sequence element in the DNA binding domain coordinates to mediate the nuclear translocation of Stat3.

Topics: Amino Acid Sequence; Animals; Arginine; Binding Sites; Biological Transport; Carcinoma, Hepatocellular; Cell Fractionation; Cell Nucleus; Conserved Sequence; COS Cells; DNA; DNA-Binding Proteins; Epidermal Growth Factor; Fluorescent Antibody Technique; Gene Deletion; Gene Expression; Humans; Immunosorbent Techniques; Leucine; Liver Neoplasms; Mice; Models, Molecular; Molecular Structure; Mutagenesis, Site-Directed; Phosphorylation; Polymerase Chain Reaction; Protein Structure, Secondary; Signal Transduction; STAT3 Transcription Factor; Structure-Activity Relationship; Trans-Activators; Transfection; Tumor Cells, Cultured; Tyrosine

To investigate the expression of TNF-related apoptosis -inducing Ligand (TRAIL) receptors and antitumor effects of TRAIL in hepatocellular carcinoma (HCC).. Expression of TRAIL receptors was determined in 60 HCC tissues, 20 normal liver samples and two HCC cell lines (HepG2 and SMMC-7721). The effects of TRAIL on promoting apoptosis in HCC cell lines were analyzed after the cells were exposed to the recombinant TRAIL protein, as well as transfected with TRAIL-expression construct. In vivo effects of TRAIL on tumor growth were investigated by using nude mice HCC model of hepG2.. Both death receptors were expressed in all HCC tissues and normal hepatic samples. In contrast, 54 HCC tissues did not express DcR1 and 25 did not express DcR2. But both DcR were detectable in all of the normal liver tissues. The expression patterns of DR and DcR in HCC samples (higher DR expression level and lower DcR expression level) were quite different from those in normal tissue. DR5, DR4, and DcR2 expressed in both cell lines, while no DcR1 expression was detected. Recombinant TRAIL alone was found to have a slight activity as it killed a maximum of 15 % of HCC cells within 24 h. Transfection of the TRAIL cDNA failed to induce extensive apoptosis in HCC lines. In vivo administration of TRAIL gene could not inhibit tumor growth in nude mice HCC model. However, chemotherapeutic agents or anticancer cytokines dramatically augmented TRAIL-induced apoptosis in HCC cell lines.. Loss of DcR (especially DcR1) in HCC may contribute to antitumor effects of TRAIL to HCC.HCC is insensitive towards TRAIL-mediated apoptosis, suggesting that the presence of mediators can inhibit the TRAIL cell-death-inducing pathway in HCC. TRAIL and chemotherapeutic agents or anticancer cytokines combination may be a novel strategy for the treatment of HCC.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Drug Synergism; Epidermal Growth Factor; Flow Cytometry; Gene Expression Regulation, Neoplastic; Genetic Therapy; GPI-Linked Proteins; Humans; Interleukin-2; Jurkat Cells; Liver Neoplasms; Mice; Mice, Nude; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Member 10c; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor Decoy Receptors

In hepatocellular carcinoma cells, the tyrosine phosphorylation of p120(ctn) was stimulated by epidermal growth factor (EGF) to investigate the relationship between the tyrosine phosphorylation of p120(ctn) and the translocation of p120(ctn), also the relationship between the tyrosine phosphorylation of p120(ctn) and the biological behaviour of hepatocellular carcinoma cells. The role of p120(ctn) in the cell adhesion and signaling of hepatocellular carcinoma is to be investigated.. In BEL-7404 human hepatocellular carcinoma cells, the tyrosine phosphotyrosine of p120(ctn) stimulated by EGF were detected by immunoprecipitation (IP) and Immunoblotting (IB). The cellular distribution and translocation of p120(ctn) and beta-catenin were detected and examined by indirect intracellular immunofluorescence. Cell morphology and cell adhesion potential were also detected using correspondent methods. Antisense nucleotide of p120(ctn) was transfected into BEL-7404 cells.. The tyrosine phosphorylation of p120(ctn) was enhanced after EGF treatment than control, especially at 20min after EGF treatment; When BEL-7404 cells were transfected with antiseuse nucleotide of p120(ctn) before EGF treatment, the tyrosine phosphorylation of p120(ctn) stimulated by EGF was obviously lowered. We also observed that the tyrosine phosphorylation of p120(ctn) stimulated by EGF was accompanied by the nuclear translocation of p120(ctn); the similar translocation was also observed in beta-catenin after EGF stimulation. At the meantime, cell adhesion potential was reduced after EGF treatment and cell morphology became thin, elongated and irregular, speudopods increased.. In BEL-7404 cells,the tyrosine phosphorylation of p120(ctn) could be stimulated by EGF, which was accompanied by the nuclear accumulation of p120(ctn). The tyrosine phosphorylation of p120(ctn) stimulated by EGF was also in correlation with the changes of cell adhesion and cell morphology. The results indicated that the tyrosine phosphorylation of p120(ctn) cell correlated with the translocation of p120(ctn) and the biological behavior of cells.

Topics: Active Transport, Cell Nucleus; beta Catenin; Carcinoma, Hepatocellular; Catenins; Cell Adhesion; Cell Adhesion Molecules; Cell Line, Tumor; Cytoskeletal Proteins; Delta Catenin; Epidermal Growth Factor; Humans; Liver Neoplasms; Phosphoproteins; Phosphorylation; Trans-Activators; Transfection; Tyrosine

The hepatocyte growth factor-regulated tyrosine kinase substrate Hrs is an early endosomal protein that is thought to play a regulatory role in the trafficking of growth factor/receptor complexes through early endosomes. Stimulation of cells with epidermal growth factor (EGF) rapidly leads to phosphorylation of Hrs, raising the question whether the receptor tyrosine kinase phosphorylates Hrs directly. Here, we present evidence that a downstream kinase, rather than the active receptor kinase is responsible. We show that the nonreceptor tyrosine kinase Src is able to phosphorylate Hrs in vitro and in vivo, but that Hrs is nevertheless phosphorylated in Src-, Yes- and Fyn-negative cells. Moreover, we show that only 10-20% of Hrs is phosphorylated following EGF stimulation, and that phosphorylation occurs at multiple tyrosines located in different parts of Hrs. These results suggest that Hrs is a substrate for several kinases downstream of the EGF receptor.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line; CSK Tyrosine-Protein Kinase; Endosomal Sorting Complexes Required for Transport; Endosomes; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Genes, src; HeLa Cells; Humans; Liver Neoplasms; Mice; Neoplasm Proteins; Phosphoproteins; Phosphorylation; Phosphotyrosine; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fyn; Proto-Oncogene Proteins c-yes; Recombinant Fusion Proteins; Signal Transduction; src-Family Kinases; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

We have developed a time-resolved fluorescent assay using Wallac's DELFIA system (DELFIA assay) to monitor changes in the phosphorylation level of insulin receptor from rat hepatoma (KRC-7) cells in response to ligand and the nonspecific, protein-tyrosine phosphatase inhibitor pervanadate. In this system, a biotinylated antiinsulin receptor antibody was used to capture the insulin receptor and an europium-labeled antiphosphotyrosine antibody was used to assess tyrosine phosphorylation. This assay provides a highly sensitive, nonradioactive readout of receptor phosphorylation. We have validated the DELFIA assay by directly comparing receptor phosphorylation using the well-established technique of immunoblotting. The utility of the DELFIA assay in measuring the phosphorylation status of other receptors has also been demonstrated using epidermal growth factor receptor from A431 cells.

Topics: Animals; Antibodies, Monoclonal; Biotinylation; Blotting, Western; Carcinoma, Hepatocellular; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Europium; Fluoroimmunoassay; Insulin; Phosphorylation; Phosphotyrosine; Precipitin Tests; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Rats; Receptor, Insulin; Tumor Cells, Cultured

Systemic tumor-targeted gene delivery is attracting increasing attention as a promising alternative to conventional therapeutical strategies. To be considered as a viable option, however, the respective transgene has to be administered with high tumor specificity. Here, we describe novel polyethylenimine (PEI)-based DNA complexes, shielded by covalent attachment of polyethylene glycol (PEG), that make use of epidermal growth factor (EGF) as a ligand for targeting gene delivery to EGF receptor-expressing human hepatocellular carcinoma (HCC) cells. In vitro transfection of luciferase reporter DNA resulted in high levels of gene expression in the human HCC cell lines Huh-7 and HepG2. An excess of free EGF during transfection clearly reduced expression levels, indicating a specific EGF receptor-mediated uptake of the DNA particles. Following intravenous injection into human HCC xenograft-bearing SCID mice, luciferase expression was predominantly found in the tumor, with levels up to 2 logs higher than in the liver, which was the highest expressing major organ. Histologic investigation showed reporter gene expression (beta-galactosidase) localized to tumor cells. Assessing DNA distribution within the tumor by immunofluorescence microscopy, rhodamine-labelled transgene DNA was found to be mainly associated with HCC cells. In the liver, DNA was taken up almost exclusively by Kupffer cells and, as indicated by the low expression, subsequently degraded. In conclusion, we have shown that intravenous injection of PEGylated EGF-containing DNA/PEI complexes allows for highly specific expression of a transgene in human HCC tumors.

Topics: Animals; Carcinoma, Hepatocellular; Electrochemistry; Epidermal Growth Factor; Gene Expression; Genes, Reporter; Genetic Therapy; Humans; In Vitro Techniques; Injections, Intravenous; Liver Neoplasms; Luciferases; Mice; Mice, SCID; Neoplasm Transplantation; Particle Size; Polyethylene Glycols; Polyethyleneimine; Transfection; Transgenes; Transplantation, Heterologous; Tumor Cells, Cultured

To investigate the correlation between epidermal growth factor (EGF) and overexpression of vascular endothelial growth factor (VEGF) in hepatocellular carcinoma (HCC).. Expressions of EGF, VEGF and microvessel density were studied through immunohistochemical SABC method in 36 HCC specimens, their paraneoplastic liver tissues and 6 normal liver tissues with the correlation between these parameters analyzed. Recombinant human EGF was used to stimulate HepG(2) cells and semi-quantitative reverse transcription PCR was adopted to detect the expression of VEGF in HepG(2) cells.. The positive rates of EGF and VEGF expression in HCC tissue were 75.0% and 88.9%. There was positive correlation between EGF and VEGF expression (P < 0.01, r = 0.462). Recombinant human EGF could induce the expression of VEGF in HepG(2) cells in a dose and time dependent manner.. The expression of EGF in HCC underlies the overexpression of VEGF in HCC.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Endothelial Growth Factors; Epidermal Growth Factor; Female; Humans; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Lymphokines; Male; Middle Aged; Neovascularization, Pathologic; Statistics as Topic; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Thrombospondin-1 (TSP-1), a multifunctional matrix protein, affects tumor growth through modulation of angiogenesis and other stromal biological functions. In several of nine human cancer cell lines derived from liver, brain, pancreas, and bone, expression of TSP-1 was up-regulated in response to the two most representative growth factors, epidermal growth factor (EGF) and transforming growth factor beta1 (TGFbeta1). Expression of TSP-1 was markedly enhanced in hepatic HuH-7 cells by EGF but not by TGFbeta1. In contrast, expression of TSP-1 was markedly enhanced by TGFbeta1, but not by EGF, in osteosarcoma MG63 cells. EGF induced activation of TSP-1 promoter-driven luciferase activity in HuH-7 cells, and the elements between -267 and -71 on the 5' region of TSP-1 gene containing two GC boxes to which Sp1 bound, were found to be responsible for the promoter activation by EGF. However, EGF did not alter TSP-1 mRNA stability in hepatic cells. On the other hand, no such enhancement of the TSP-1 promoter activity by TGFbeta1 appeared in MG63 cells. Enhanced expression of TSP-1 by TGFbeta1 in MG63 cells was partially blocked by exogenous addition of SB203580, an inhibitor of p38 mitogen-activated protein kinase. TGFbeta was found to induce marked elongation of TSP-1 mRNA longevity in osteosarcoma cells when mRNA degradation was assayed in the presence of alpha-amanitin. The up-regulation of TSP-1 by EGF and TGFbeta might play a critical role in modulation of angiogenesis and formation of matrices in tumor stroma.

Topics: Bone Neoplasms; Carcinoma, Hepatocellular; Epidermal Growth Factor; Gene Expression Regulation; Humans; Liver Neoplasms; Osteosarcoma; RNA Stability; Thrombospondin 1; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

Intrinsic expression of the multidrug resistance (MDR) transporter P-glycoprotein (Pgp) may be regulated by reactive oxygen species (ROS). A transient expression of Pgp was observed during the growth of multicellular tumor spheroids. Maximum Pgp expression occurred in tumor spheroids with a high percentage of quiescent, Ki-67-negative cells, elevated glutathione levels, increased expression of the cyclin-dependent kinase inhibitors p27Kip1 and p21WAF-1 as well as reduced ROS levels and minor activity of the mitogen-activated kinase (MAPK) members c-Jun amino-terminal kinase (JNK), extracellular signal-regulated kinase ERK1,2, and p38 MAPK. Raising intracellular ROS by depletion of glutathione with buthionine sulfoximine (BSO) or glutamine starvation resulted in down-regulation of Pgp and p27Kip1, whereas ERK1,2 and JNK were activated. Down-regulation of Pgp was furthermore observed with low concentrations of hydrogen peroxide and epidermal growth factor, indicating that ROS may regulate Pgp expression. The down-regulation of Pgp following BSO treatment was abolished by agents interfering with receptor tyrosine kinase signaling pathways, i.e. the protein kinase C inhibitors bisindolylmaleimide I (BIM-1) and Ro-31-8220, the p21ras farnesyl protein transferase inhibitor III, the c-Raf inhibitor ZM 336372 and PD98059, which inhibits ERK1,2 activation. ROS involved as second messengers in receptor tyrosine kinase signaling pathways may act as negative regulators of Pgp expression.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Buthionine Sulfoximine; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Drug Resistance, Multiple; Enzyme Inhibitors; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Glutathione; Humans; Hydrogen Peroxide; JNK Mitogen-Activated Protein Kinases; Kinetics; Liver Neoplasms; Male; MAP Kinase Signaling System; Melanoma; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Prostatic Neoplasms; Protein-Tyrosine Kinases; Reactive Oxygen Species; Tumor Cells, Cultured; Tumor Suppressor Proteins

The expressions of Lewis (Le) antigens, alpha-1,3/1,4 fucosyltransferases (alpha-1,3/1,4 FuTs), and metastatic potential after the treatment of 2 differentiation inducers, all- trans retinoic acid (ATRA), 8-bromo-cyclic 3',5'adenosine monophosphate (8-Br-cAMP); and 2 proliferation inducers, epidermal growth factor (EGF) and phobol-12-myristate-13-acetate (PMA), on 7721 human hepatocarcinoma cell line were studied. Cell adhesion to human umbilical vein endothelial cells (HUVEC), cell migration through transwell and invasion through matrigel were selected as the indexes of metastatic potential-related phenotypes. Using fluorescence-labelled antibodies and flow-cytometric analysis, it was found that 7721 cells mainly expressed sialyl Lewis X (SLe(x)) and a less amount of sialyl dimeric Lewis X (SDLe(x)) antigens on the cell surface. Their expressions were down-regulated by ATRA, and up-regulated by EGF. SLe(x)antigen was also decreased and increased by the treatment of 8-Br-cAMP and PMA respectively. With Northern blot to detect the mRNAs of alpha-1,3/1,4 FuTs, the main enzymatic basis for the change in SLe(x)expression was found to be the alteration of the expression of alpha-1,3 FuT-VII. It was evidenced by the observations that alpha-1,3 FuT-VII was the main alpha-1,3/1,4 FuT in 7721 cells, while alpha-1,3/1,4 FuT-III and alpha-1,3 FuT-VI were expressed rather low. The changes in the expressions of SLe(x)antigen and alpha-1,3 FuT-VII resulted in the altered cell adhesion to tumour necrosis factor-alpha stimulated HUVEC, since only the monoclonal antibody of the SLe(x), but not other monoclonal antibodies blocked the adhesion of 7721 cells to HUVEC. The migration and invasion of 7721 cells were also reduced by the treatment of ATRA or 8-Br-cAMP, and elevated by EGF or PMA. The above findings indicate that the metastatic potential of 7721 cells is suppressed by differentiation-inducers and promoted by proliferation-inducers.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Adhesion; Cell Differentiation; Cell Division; Cell Movement; Epidermal Growth Factor; Fucosyltransferases; Humans; Lewis Blood Group Antigens; Lewis X Antigen; Liver Neoplasms; Neoplasm Metastasis; Phenotype; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

After internalization from the plasma membrane, activated EGF receptors (EGFRs) are delivered to multivesicular bodies (MVBs). Within MVBs, EGFRs are removed from the perimeter membrane to internal vesicles, thereby being sorted from transferrin receptors, which recycle back to the plasma membrane. The phosphatidylinositol (PI) 3'-kinase inhibitor, wortmannin, inhibits internal vesicle formation within MVBs and causes EGFRs to remain in clusters on the perimeter membrane. Microinjection of isotype-specific inhibitory antibodies demonstrates that the PI 3'-kinase required for internal vesicle formation is hVPS34. In the presence of wortmannin, EGFRs continue to be delivered to lysosomes, showing that their removal from the recycling pathway and their delivery to lysosomes does not depend on inward vesiculation. We showed previously that tyrosine kinase-negative EGFRs fail to accumulate on internal vesicles of MVBs but are recycled rather than delivered to lysosomes. Therefore, we conclude that selection of EGFRs for inclusion on internal vesicles requires tyrosine kinase but not PI 3'-kinase activity, whereas vesicle formation requires PI 3'-kinase activity. Finally, in wortmannin-treated cells there is increased EGF-stimulated tyrosine phosphorylation when EGFRs are retained on the perimeter membrane of MVBs. Therefore, we suggest that inward vesiculation is involved directly with attenuating signal transduction.

Topics: Androstadienes; Antibodies, Monoclonal; Autocrine Communication; Carcinoma, Hepatocellular; Coated Vesicles; Endosomes; Epidermal Growth Factor; ErbB Receptors; Humans; Lysosomes; Microinjections; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Phosphotyrosine; Protein Transport; Tumor Cells, Cultured; Wortmannin

We have used a transgenic animal model, which constitutively develops hepatocarcinoma (Antithrombin III SV40 T large Antigen: ASV), to study the involvement of Annexin 1 (ANX1) in liver regeneration and malignant transformation. Primary hepatocytes isolated from normal mice did not express ANX1. In contrast, ANX1 was strongly expressed in hepatocytes of transgenic mice during constitutive development of hepatocarcinoma. In ASV transgenic mice, an elevated ANX1 level preceded the appearance of the tumor, indicating that it could be a good marker in the diagnosis of cancer. One-third hepatectomy in normal mice resulted in stimulation of ANX1 synthesis and phosphorylation. This upregulation correlated with increased synthesis of EGF and consequently with increased phosphorylation of the EGF receptor (EGF-R). Stable transfection of a hepatocyte cell line derived from ASV transgenic mice (mhAT2) with antisense complementary DNA for ANX1 reduced the proliferation rate as well as cytosolic phospholipase A(2) (cPLA(2)) activity. Thus, ANX1 expression and phosphorylation could be a factor implicated in liver regeneration and tumorigenesis, either through modulation of cPLA(2) activity or EGF-R function.

Topics: Animals; Annexin A1; Antigens, Polyomavirus Transforming; Antithrombin III; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Cells, Cultured; Epidermal Growth Factor; Hepatectomy; Liver Neoplasms; Liver Regeneration; Mice; Mice, Inbred Strains; Mice, Transgenic; Phosphorylation; Postoperative Period; Promoter Regions, Genetic; Up-Regulation

The effects of all-trans retinoic acid (ATRA) and epidermal growth factor (EGF) on the expression of nm23-H1, a metastasis suppressor gene, were studied in a human 7721 hepatocarcinoma cell line. It was discovered that the expression of nm23-H1 mRNA was up-regulated by ATRA. This was compatible with the observation that the metastasis-associated phenotypes, such as chemotaxic cell migration and invasion, were both reduced in the ATRA-treated and nm23-H1-cDNA-transferred 7721 cells. However, ability of cells to adhere to fibronectin and laminin was not altered identically in the ATRA-treated and nm23-H1-cDNA-transfected 7721 cells. In contrast, the expression of nm23-H1 mRNA in 7721 cells was down-regulated both by the treatment with EGF and by the transfection of c-erbB-2/neu cDNA, which codes a protein homologous to the EGF receptor. EGF is a compound with biological effects opposite to those of ATRA, and c-erbB-2/neu is known to be a metastasis-promoting gene. These results reveal that the metastasis-preventing effect of ATRA may partly result from the up-regulation of nm23-H1, and the metastasis-promoting effects of EGF and c-erbB-2/neu were probably mediated in part by the down-regulation of nm23-H1.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Liver Neoplasms; Monomeric GTP-Binding Proteins; Neoplasm Metastasis; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Promoter Regions, Genetic; RNA, Messenger; Transcription Factors; Tretinoin; Tumor Cells, Cultured

In rat liver epithelial cells constitutively expressing transforming growth factor alpha (TGFalpha), c-Met is constitutively phosphorylated in the absence of its ligand, hepatocyte growth factor. We proposed that TGFalpha and the autocrine activation of its receptor, epidermal growth factor receptor (EGFR), leads to phosphorylation and activation of c-Met. We found that there is constitutive c-Met phosphorylation in human hepatoma cell lines and the human epidermoid carcinoma cell line, A431 which express TGFalpha, but not in normal human hepatocytes. Constitutive c-Met phosphorylation in A431, HepG2, AKN-1, and HuH6 cells was inhibited by neutralizing antibodies against TGFalpha and/or EGFR. Exposure to exogenous TGFalpha or EGF increased the phosphorylation of c-Met in the human epidermoid carcinoma cell line, A431. The increase of c-Met phosphorylation by TGFalpha in A431 cells was inhibited by neutralizing antibodies against TGFalpha and/or EGFR and by the EGFR-specific inhibitor tyrphostin AG1478. These results indicate that constitutive c-Met phosphorylation, and the increase of c-Met phosphorylation by TGFalpha or EGF, in tumor cell lines is the result of the activation via EGFR. We found that c-Met in tumor cells co-immunoprecipitates with EGFR regardless of the existence of their ligands in tumor cells, but not in normal human hepatocytes. We conclude that c-Met associates with EGFR in tumor cells, and this association facilitates the phosphorylation of c-Met in the absence of hepatocyte growth factor. This cross-talk between c-Met and EGFR may have significant implications for altered growth control in tumorigenesis.

Topics: Animals; Carcinoma, Hepatocellular; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Hepatocyte Growth Factor; Humans; Liver Neoplasms; Phosphorylation; Protein Binding; Proto-Oncogene Proteins c-met; Quinazolines; Rats; Receptor Cross-Talk; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tyrphostins

In order to study the dynamics of gap junctions in living cells, a cDNA was expressed in hepatocellular carcinoma-derived PLC cells coding for chimerical polypeptide Cx.EGFP-1, which consists of rat connexin32 and enhanced green fluorescent protein (EGFP). Cx.EGFP-1 was integrated into gap junctions, and the emitted epifluorescence reliably reported the distribution of the chimera. Therefore, stably transfected PLC clone PCx-9 was used to examine the dynamic behavior of gap junctions by time-lapse fluorescence microscopy. The pleomorphic fluorescent junctional plaques were highly motile within the plasma membrane. They often fused with each other or segregated into smaller patches, and fluctuation of fluorescence was detected within individual gap junctions. Furthermore, the uptake of junctional fragments into the cytoplasm of live cells was documented as originating from dynamic invaginations that form long tubulovesicular structures that pinch off. Endocytosis and subsequent lysosomal degradation, however, appeared to contribute only a little to the rapid gap junction turnover (determined half-life of 3.3 h for Cx.EGFP-1), since most cytoplasmic Cx.EGFP-1 fluorescence did not colocalize with the endocytosed fluid phase marker horseradish peroxidase or the receptor-specific endocytotic ligand transferrin and since it was distinct from lysosomes. Disassembly of gap junctions was monitored in the presence of the translation-inhibitor cycloheximide and showed increased endocytosis and continuous reduction of junctional plaques. Highly motile cytoplasmic microvesicles, which were detectable as multiple, weakly fluorescent puncta in all movies, are proposed to contribute significantly to gap junction morphogenesis by the transport of small subunits between biosynthetic, degradative, and recycling compartments.

Topics: Animals; Carcinoma, Hepatocellular; Connexins; Cycloheximide; DNA, Complementary; Endocytosis; Epidermal Growth Factor; Epithelial Cells; Gap Junction beta-1 Protein; Gap Junctions; Gene Expression; Genes, Reporter; Green Fluorescent Proteins; Indicators and Reagents; Luminescent Proteins; Microscopy, Fluorescence; Microscopy, Video; Protein Synthesis Inhibitors; Rats; Recombinant Fusion Proteins; Tumor Cells, Cultured

STAT proteins are a family of latent transcription factors that mediate the response to various cytokines and growth factors. Upon stimulation by cytokines, STAT proteins are recruited to the receptors via their SH2 domains, phosphorylated on a specific tyrosine, dimerized, and translocated into the nucleus, where they bind specific DNA sequences and activate the target gene transcription. STATs share highly conserved structures, including an N-domain, a coiled-coil domain, a DNA-binding domain, a linker domain, and an SH2 domain. To investigate the role of the coiled-coil domain, we performed a systematic deletion analysis of the N-domain and each of the alpha-helices and mutagenesis of conserved residues in the coiled-coil region of Stat3. Our results indicate that the coiled-coil domain is essential for Stat3 recruitment to the receptor and the subsequent tyrosine phosphorylation and tyrosine phosphorylation-dependent activities, such as dimer formation, nuclear translocation, and DNA binding, stimulated by epidermal growth factor (EGF) or interleukin-6 (IL-6). Single mutation of Asp170 or, to a lesser extent, Lys177 in alpha-helix 1 diminishes both receptor binding and tyrosine phosphorylation. Furthermore, the Asp170 mutant retains its ability to bind to DNA when phosphorylated on Tyr705 by Src kinase in vitro, implying a functional SH2 domain. Finally, we demonstrate a direct binding of Stat3 to the receptor. Taken together, our data reveal a novel role for the coiled-coil domain that regulates the early events in Stat3 activation and function.

Topics: Animals; Antigens, CD; Carcinoma, Hepatocellular; COS Cells; Cytokine Receptor gp130; Dimerization; DNA; DNA-Binding Proteins; Epidermal Growth Factor; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Liver Neoplasms; Membrane Glycoproteins; Mice; Neoplasm Proteins; Phosphorylation; Point Mutation; Protein Binding; Protein Processing, Post-Translational; Protein Structure, Secondary; Receptors, Interleukin-6; Recombinant Fusion Proteins; src Homology Domains; STAT3 Transcription Factor; Structure-Activity Relationship; Trans-Activators; Transcription, Genetic; Transfection; Tumor Cells, Cultured

In 6 HCC cell lines, clear expressions of EGFR and TGF-alpha were found in flow cytometry, while expressions of EGF, HB-EGF and AR were quite low. TGF-alpha secretion into culture supernatants became measurable when TPA 0.5 microM was added. TPA accelerated the proliferation of KYN-3 cells, and anti-TGF-alpha neutralizing antibody suppressed this proliferation in a dose-dependent manner. Addition of exogenous TGF-alpha, EGF, AR, or HB-EGF with heparin accelerated cell proliferation. In non-stimulated cultures, cell proliferation was suppressed by anti-EGFR neutralizing antibody, but not by the antibodies for EGF, TGF-alpha, AR and HB-EGF. HCC may possess a paracrine system regulated by these 4 ligands, and an autocrine system, under a certain condition, via TGF-alpha and EGFR.

Topics: Amphiregulin; Antibodies; Carcinoma, Hepatocellular; Cell Division; Cell Line; Culture Media; EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; Glycoproteins; Growth Substances; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Ligands; Liver Neoplasms; Transforming Growth Factor alpha

The peroxisome proliferators are rodent non-genotoxic hepatocarcinogens that suppress apoptosis and induce DNA replication, cell proliferation and liver tumours. In order to investigate the effect of peroxisome proliferators on cell cycle progression, we arrested the well-differentiated rat hepatoma cell line FaO in the G1 phase of the cell cycle. Under these conditions, CDK2 and CDK4 protein expression remained unchanged compared with proliferating cells, but expression of cyclin D1 and p27(KIP1) was down-regulated and cyclin E accumulated in the inactive form. G1-arrested cells were able to enter the cell cycle on addition of exogenous growth factors such as epidermal growth factor (EGF) or hepatocyte growth factor (HGF) and replicate their DNA within 12 to 24 h of re-stimulation. Upon release from G1 arrest, CDK2 protein expression was down-regulated and, surprisingly, p27(KIP1) expression was restored. Cyclin D1 and phosphorylated cyclin E accumulated at 12 h but were degraded by 24 h after addition of EGF. Importantly, the peroxisome proliferator nafenopin and tumour necrosis factor alpha were able to induce DNA replication. Thus, the profile of expression of cell cycle regulatory proteins upon stimulation with nafenopin is comparable with that induced by growth factors such as EGF.

Topics: Animals; Butyrates; Carcinogens; Carcinoma, Hepatocellular; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Down-Regulation; Enzyme Inhibitors; Epidermal Growth Factor; G1 Phase; Microtubule-Associated Proteins; Nafenopin; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Rats; Time Factors; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Tumor Suppressor Proteins

Protein kinases play key roles in the control of cell proliferation, differentiation and metabolism. In this work, we studied the effect of coumarin and its derivatives, including daphnetin, esculin, 2-OH-coumarin, 4-OH-coumarin and 7-OH-coumarin, on the activity of protein kinases. It was found that, in these compounds, only daphnetin was a protein kinase inhibitor. This compound inhibited tyrosine-specific protein kinase, EGF receptor (IC(50) = 7.67 microM), and serine/threonine-specific protein kinases, including cAMP-dependent protein kinase (PKA) (IC(50) = 9.33 microM) and protein kinase C (PKC) (IC(50) = 25.01 microM) in vitro. The inhibition of EGF receptor tyrosine kinase by daphnetin was competitive to ATP and non-competitive to the peptide substrate. The inhibition of EGF-induced tyrosine phosphorylation of EGF receptor by daphnetin was not observed in human hepatocellular carcinoma HepG2 cells. The structural comparison of daphnetin with coumarin and other coumarin derivatives suggests that the hydroxylation at C8 may be required for daphnetin acting as a protein kinase inhibitor.

Topics: Adenosine Triphosphate; Binding, Competitive; Carcinoma, Hepatocellular; Coumarins; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Esculin; Genistein; Humans; Hydroxylation; Inhibitory Concentration 50; Kinetics; Phosphorylation; Protein Kinase C; Protein Kinase Inhibitors; Protein Kinases; Tumor Cells, Cultured; Umbelliferones

A new human hepatocellular (HCC) cell line, HAK-3, was established from a resected HCC of a Japanese, female patient. HAK-3 retains morphologic features of the original HCC, and proliferates in a monolayered sheet (doubling time: 26 h). HAK-3 is a single aneuploid cell population with a DNA index of 2.42, the karyotype is human, chromosomes are 80-85 (mode: 83), and secretes fibronectin and tissue polypeptide antigen. Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) dose-dependently accelerated the cell proliferation, while deletion-type hepatocyte growth factor (dHGF) tended to suppress the proliferation, and transforming growth factor (TGF)-alpha showed almost no influence. dHGF induced the decrease of cell adhesiveness, changed the cell morphology to spindle-shaped cells, increased cell movement, and showed chemotactic effects with the increase of its concentration gradient in cultures. HAK-3 would be useful in studies on the acceleration mechanisms of cancer cell proliferation by growth factors and of chemotaxis by dHGF.

Topics: Carcinoma, Hepatocellular; Cell Division; Cell Movement; Cell Size; Chemotaxis; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Growth Substances; Hepatocyte Growth Factor; Humans; Karyotyping; Liver Neoplasms; Middle Aged; Ploidies; Transforming Growth Factor alpha; Tumor Cells, Cultured

Transforming growth factor-beta1 (TGF-beta1) has been shown to induce apoptosis in normal or transformed hepatocytes. To elucidate the biochemical pathways leading to apoptosis induced by TGF-beta1 in human hepatoma cells (HuH-7), we examined the expression of Bcl-2-related proteins and X-chromosome-linked inhibitor of apoptosis (XIAP), and activation of the caspase cascade following TGF-beta1 treatment. Bcl-xL expression began to decline at 12 hours after TGF-beta1 treatment and progressively decreased to very low levels in a time-dependent manner. Bax expression showed a little change throughout the experiment. On the other hand, activation of caspase-8 was clearly observed at 36 hours after TGF-beta1 treatment, followed by activation of caspase-9, and caspase-3 was activated at 48 hours after treatment at which time apoptosis of HuH-7 cells was observed. TGF-beta1 significantly decreased XIAP expression in HuH-7 cells. Addition of an inhibitor of caspase-8 or caspase-3 (IETD-FMK or DEVD-CHO) markedly inhibited TGF-beta1-induced apoptosis of HuH-7 cells. Fas/Fas ligand (FasL) interactions in HuH-7 cells were not involved in the apoptotic process. Furthermore, epidermal growth factor (EGF) also completely inhibited TGF-beta1-induced apoptosis of HuH-7 cells by inhibiting activation of the caspase cascade. Our results suggested that activation of caspase-3 initiated through caspase-8 activation is involved in the apoptotic process induced by TGF-beta1 in HuH-7 cells. Our results also showed that down-regulation of the expression of Bcl-xL and XIAP by TGF-beta1 may facilitate activation of caspase-3 in these cells.

Topics: Apoptosis; bcl-X Protein; Carcinoma, Hepatocellular; Caspase 3; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cell Division; Cysteine Proteinase Inhibitors; Enzyme Activation; Epidermal Growth Factor; fas Receptor; Gene Expression Regulation, Neoplastic; Humans; In Situ Nick-End Labeling; Kinetics; Liver Neoplasms; Oligopeptides; Proto-Oncogene Proteins c-bcl-2; Time Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

Heparin-binding epidermal growth factor-like growth factor is an hepatotrophic factor expressed in non-parenchymal liver cells but not in hepatocytes in regenerating rat liver after partial hepatectomy. Human hepatocellular carcinoma cells also produce this growth factor. In this study, the expression of the growth factor in the hepatocytes of fibrotic liver during hepatocarcinogenesis was investigated.. Hepatic fibrosis was induced in rats by oral administration of 0.05% thioacetamide. Hepatocytes were isolated by in situ perfusion methods. Growth factor gene and protein expression were investigated by northern hybridization and immunohistochemistry, respectively. Expression of glutathione s-transferase P, which is expressed when hepatocytes undergo neoplastic transformation, was also investigated.. Some hepatocytes in fibrotic liver, but not in normal liver, stained positively by immunohistochemistry for heparin-binding epidermal growth factor-like growth factor. The growth factor and glutathione s-transferase P gene transcript were present in hepatocytes isolated from fibrotic liver, but not in those isolated from normal liver. Immunohistochemical localization of both proteins in fibrotic liver revealed similar patterns.. In essence, hepatocytes in fibrotic rat liver produce heparin-binding epidermal growth factor-like growth factor. Expression of this growth factor may occur as hepatocytes are transformed to a neoplastic phenotype.

Topics: Animals; Carcinoma, Hepatocellular; Electrophoresis, Agar Gel; Epidermal Growth Factor; Glutathione Transferase; Heparin-binding EGF-like Growth Factor; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Liver; Liver Cirrhosis, Experimental; Liver Neoplasms; Male; Rats; Rats, Sprague-Dawley; Thioacetamide

We have shown that a synthetic vitamin K analog, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or compound 5 (Cpd 5), potently inhibits cell growth and suggested that the analog exerts its effects mainly via sulfhydryl arylation rather than redox cycling. Since protein-tyrosine phosphatases (PTPases), which have pivotal roles in many cellular functions, have a critical cysteine in their active site, we have proposed PTPases as likely targets for Cpd 5. To test this hypothesis, we examined the effects of Cpd 5 on protein tyrosine phosphorylation of cellular proteins and on the activity of PTPases. We found that Cpd 5 rapidly induced protein tyrosine phosphorylation in a human hepatocellular carcinoma cell line (Hep3B) at growth inhibitory doses, and the effect was blocked by thiols but not by non-thiol antioxidants or tyrosine kinase inhibitors. Cpd 5 inhibited PTPase activity, which was also significantly antagonized by reduced glutathione. Furthermore, the well studied PTPase inhibitor orthovanadate also induced protein tyrosine phosphorylation and growth inhibition in Hep3B cells. These results suggest that inhibition of cellular PTPases by sulfhydryl arylation and subsequent perturbation of protein tyrosine phosphorylation may be involved in the mechanisms of Cpd 5-induced cell growth inhibition.

Topics: Carcinoma, Hepatocellular; Catechols; Cell Division; Cell Line; Enzyme Inhibitors; Epidermal Growth Factor; Genistein; Humans; Kinetics; Liver Neoplasms; Mercaptoethanol; Naphthoquinones; Nitriles; Phosphorylation; Phosphotyrosine; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Sulfhydryl Compounds; Tumor Cells, Cultured; Tyrphostins; Vanadates; Vitamin K

Butein, a plant polyphenol, was shown to be a specific protein tyrosine kinase inhibitor. This compound inhibited not only the epidermal growth factor (EGF)-stimulated auto-phosphotyrosine level of EGF receptor in HepG2 cells but also tyrosine-specific protein kinase activities of EGF receptor (IC50 = 65 microM) and p60c-src (IC50 = 65 microM) in vitro. The inhibition was competitive to ATP and non-competitive to the phosphate acceptor, poly (Glu, Ala, Tyr) 6:3:1 for EGF receptor tyrosine kinase. In contrast, butein non-significantly inhibited the activities of serine- and threonine-specific protein kinases, such as protein kinase C (PKC) and cAMP-dependent protein kinase (PKA).

Topics: Carcinoma, Hepatocellular; Chalcone; Chalcones; CSK Tyrosine-Protein Kinase; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Flavonoids; Humans; Kinetics; Molecular Structure; Phenols; Phosphorylation; Polymers; Polyphenols; Protein Kinase C; Protein-Tyrosine Kinases; Receptor Protein-Tyrosine Kinases; src-Family Kinases; Tumor Cells, Cultured

We recently showed that heparin-binding EGF-like growth factor (HB-EGF) has hepatotrophic effects. In this study, we developed an ELISA system with high specificity and sensitivity for human plasma HB-EGF. In 14 patients who underwent partial hepatectomy, plasma HB-EGF levels were measured serially after surgery. In patients who underwent gross hepatectomy (lobectomy and segmentectomy), plasma HB-EGF levels increased, reaching maximal levels approximately 5 to 7 days after surgery. In patients who underwent minor hepatectomy (subsegmentectomy), plasma HB-EGF levels did not increase. Maximal plasma HB-EGF levels were significantly higher in patients who had a percent increased volume of the remaining liver (%ILV) above 20% than those who had a %ILV below 20% (32.4 +/- 19.6 pg/ml vs 7.4 +/- 2.7, P < 0.05). The plasma HB-EGF values did not correlate with WBC counts, C-reactive protein, or alanine aminotransferase. Plasma HB-EGF may be a marker for liver regeneration after hepatectomy in humans.

Topics: Aged; Biomarkers; Carcinoma, Hepatocellular; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Female; Heparin-binding EGF-like Growth Factor; Hepatectomy; Humans; Intercellular Signaling Peptides and Proteins; Liver Regeneration; Male; Middle Aged; Tomography

The influence of p53 on cytokine-triggered Janus kinase-STAT signaling was investigated in human hepatoma Hep3B cell lines engineered to constitutively express the temperature-sensitive Val135 mutant of p53. In comparison to the parental p53-free Hep3B cells, these p53-Val135-containing Hep3B cell lines displayed a reduced response to IL-6 at the wild-type-like p53 temperature (32.5 degrees C). In these cells, IL-6 induced a marked reduction in the immunologic accessibility of cytoplasmic and nuclear STAT3 and STAT5 within 20 to 30 min that lasted 2 to 4 h (STAT-masking) provided that the cells had been previously cultured at 32.5 degrees C for at least 18 to 20 h. The onset of IL-6-induced STAT-masking required protein tyrosine kinase, protein tyrosine phosphatase, proteasomal, phospholipase C, and mitogen-activated protein kinase kinase 1 activities. The maintenance of IL-6-induced STAT-masking was dependent on continued signaling through the phosphatidylinositol-dependent phospholipase C pathway. Despite a reduction in IL-6-induced STAT3 DNA binding activity in the nuclear compartment during STAT-masking, there was increased and prolonged accumulation of tyrosine-phosphorylated STAT3 in both the cytoplasmic and nuclear compartments, indicating that the capacity of tyrosine-phosphorylated STAT3 to bind DNA was reduced during STAT-masking. Thus, IL-6-induced STAT-masking, as dramatically evident on immunomicroscopy, is a visible consequence of a novel cellular process by which a p53-Val135-induced gene product(s) regulates the association of masking protein(s) with and the DNA-binding capacity of STAT3.

Topics: Amino Acid Substitution; Carcinoma, Hepatocellular; Cytokines; DNA-Binding Proteins; Dose-Response Relationship, Immunologic; Epidermal Growth Factor; Humans; Interferon-gamma; Interleukin-6; Milk Proteins; Mutation; Phenotype; Phosphatidylinositol Diacylglycerol-Lyase; Phosphorylation; Signal Transduction; STAT3 Transcription Factor; STAT5 Transcription Factor; Temperature; Time Factors; Trans-Activators; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Type C Phospholipases; Tyrosine; Valine

The ability of TGF-beta 1 (transforming growth factor-beta 1) to suppress growth factor induced proliferation of many cell types in vitro is well documented; however, TGF-beta 1 increases within a similar time frame as the hepatocyte mitogens HGF (hepatocyte growth factor), EGF (epidermal growth factor), and TGF-alpha (transforming growth factor-alpha) prior to hepatocyte proliferation during liver regeneration. This has raised the issue that TGF-beta 1 may have effects on hepatocytes additional to mito-inhibition and that these effects may be relevant to the regenerative process. To this end, we examined the effect of TGF-beta 1 on both the mitogenesis and the motility of growth factor stimulated primary rat hepatocytes and the hepatoblastoma cell line HepG2 in vitro. TGF-beta 1 significantly enhanced the chemotactic motility of EGF or TGF-alpha, and not HGF, stimulated hepatocytes on a collagen I substratum. TGF-beta 1 was not chemotactic when added alone and decreased the DNA synthesis of all hepatocyte cultures to near control levels. HepG2 cells were chemotactic toward HGF, EGF, and TGF-beta 1 alone and displayed an additive chemotactic response when TGF-beta 1 was added to either HGF or EGF. Additionally, HepG2 cells were refractory to the growth stimulatory effects of HGF or EGF and the growth inhibitory effects of TGF-beta 1. Hepatocytes plated onto other collagen-containing substrates (collagen IV, Matrigel, or ECL, an entactin-collagen IV-laminin matrix), but not on fibronectin or laminin alone, also displayed enhanced EGF stimulated motility by TGF-beta 1. The data indicate that an additional, novel role for TGF-beta 1 during liver tissue remodeling following PHx may include the synergistic enhancement EGF stimulated hepatocyte motility responses, and this enhancement is observed only on collagen-containing extracellular matrices.

Topics: Animals; Carcinoma, Hepatocellular; Cell Division; Cell Movement; Collagen; DNA; Drug Synergism; Epidermal Growth Factor; Extracellular Matrix; Hepatocyte Growth Factor; Liver Regeneration; Neoplasm Metastasis; Rats; Transforming Growth Factor beta; Tumor Cells, Cultured

Met and ron proto-oncogenes encode the cell surface receptors for hepatocyte growth factor (HGF) and hepatocyte growth factor-like (HLP) protein, respectively, and induce mitogenesis, motogenesis, morphogenesis, and metastatic activity in various cell types. Overexpression of met in human carcinoma has been reported by several groups including ours; however, the mechanisms that control met gene expression are thus far unclear. The present study focuses on the expression and regulation of the Met and Ron receptors in human hepatocellular carcinoma (HCC). We report here that abnormal expression of met and ron proteins occurs in some cases of human HCC. Using several HCC cell lines as a model system, we show that HGF, as well as other cytokines, such as epidermal growth factor (EGF), interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha), induce met and ron expression. Using several chimeric constructs consisting of various lengths of the met promoter region fused to the reporter gene of chloramphenicol acetyl transferase (CAT), and by performing transient transfection of these constructs into HepG2 cells, we show that induction of met gene expression by HGF and other cytokines is, at least in part, through up-regulation of met gene promoter activity. The DNA region conferring responsiveness to cytokine induction was located within 0.2 kb of the met core promoter. Interestingly, EGF did not stimulate met promoter activity in any of the met-CAT chimeric constructs. These results provide evidence that met and ron are modulated in the liver by a similar cytokine network. In the case of met expression, the 0.2-kb region in the met gene promoter may play an important role in mediating its gene induction in response to HGF and other cytokines. Our results also suggest that unregulated expression of met and ron may be associated with pathological conditions, such as HCC, in the liver.

Topics: Animals; Carcinoma, Hepatocellular; Epidermal Growth Factor; Gene Expression Regulation; Hepatocyte Growth Factor; Humans; Interleukin-1; Interleukin-6; Liver; Liver Neoplasms; Mice; Proto-Oncogene Proteins c-met; Receptor Protein-Tyrosine Kinases; Receptors, Cell Surface; Transcriptional Activation; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Topics: Carcinoma, Hepatocellular; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Humans; Liver Neoplasms

Synthetic 4,5-didehydro GGA (geranylgeranoic acid), a potent ligand both for cellular retinoic acid-binding protein and for nuclear retinoid receptors, induced apoptosis in human hepatoma-derived cell line HuH-7 but not in primary hepatocytes, although all-trans or 9-cis retinoic acid did not induce any growth inhibition. Either exogenous transforming growth factor-alpha (TGF alpha) or epidermal growth factor(EGF) prevented the cells from apoptosis in the presence of 4,5-didehydro GGA, but hepatocyte growth factor, insulin-like growth factor-II, insulin or triiodothyronine was essentially inactive. 4,5-Didehydro GGA down-regulated the cellular levels of TGF alpha mRNA as early as 30 min after the treatment. Either anti-TGF alpha or anti-EGF receptor monoclonal antibody induced apoptosis in HuH-7 cells without using the acid. Taken together, the present study strongly suggests that 4,5-didehydro GGA induced apoptosis in HuH-7 cells through the destruction of autocrine loop consisting of TGF alpha and EGF receptor, due to the down regulation of TGF alpha gene expression.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Line; Cell Survival; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Humans; Insulin-Like Growth Factor II; Kinetics; Liver; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transforming Growth Factor alpha; Tretinoin; Triiodothyronine; Tumor Cells, Cultured

In this work, using the ECL Western blotting assay system, it was found that genistein, a specific tyrosine kinase inhibitor, was able to inhibit EGF-induced EGF receptor degradation and tyrosine phosphorylation in human hepatoma HepG2 cells. This inhibition was increased with increasing genistein concentration. With treatment of HepG2 cells with genistein at 37 degrees C for 30 min, the amount of internalized EGF, which was measured by the detection of the sorting of 125I-EGF in the cells, was remarkably decreased. Under the same conditions, in cells untreated with genistein, the degradation of EGF was significantly increased. After preincubation of HepG2 cells with and without genistein for 120 min at 37 degrees C, the ratio between degraded and released EGF was 16 and 24, respectively. These results suggest that EGF-induced internalization and degradation of EGF-EGF receptor complexes in HepG2 cells depend on EGF receptor tyrosine kinase activity.

Topics: Carcinoma, Hepatocellular; Cell Line; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Genistein; Humans; Isoflavones; Kinetics; Liver Neoplasms; Phosphotyrosine; Protein-Tyrosine Kinases; Tumor Cells, Cultured

Epidermal growth factor (EGF) receptor was detected in the human Chang liver and human hepatoma HepG2 cells. Both cell lines were found to be able to bind EGF. The expression of EGF receptor in Chang liver and HepG2 cells was 1.3 x 10(5) EGF receptors/cell and 1.8 x 10(5) EGF receptors/cell, respectively. In both cells, this receptor was identified by ECL Western blotting using monoclonal anti-EGF receptor antibody and by immunohistochemical assay using polyclonal anti-EGF receptor antibody. Some of internalized EGF was recycled in Chang liver cells, but not in HepG2 cells.

Topics: Carcinoma, Hepatocellular; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Humans; Immunohistochemistry; Liver; Liver Neoplasms; Molecular Weight; Tumor Cells, Cultured

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family and is highly expressed in hepatoma tissues but not in normal liver. However, it is unknown when HB-EGF is induced during hepatocarcinogenesis and what are the mechanisms underlying its high expression in hepatoma. To address this issue, the expression of HB-EGF was investigated during hepatocarcinogenesis in LEC (Long-Evans with a cinnamon-like coat color) rats, which spontaneously develop hepatitis and hepatoma. LEA (Long-Evans with an agouti coat color) rats were used as controls. Furthermore, the induction of HB-EGF mRNA by various agents was investigated in a rat hepatoma cell line and hepatocytes in primary culture. Expression of HB-EGF mRNA in the liver was very low at the stage of acute and chronic hepatitis and markedly increased at the stage of hepatoma in LEC rats. Non-involved tissues adjacent to hepatoma showed low expression of HB-EGF mRNA. Immunochemical studies revealed positive staining in hepatoma tissues. Induction of HB-EGF mRNA by several growth factors was observed in a hepatoma cell line but not in normal hepatocytes. Our results suggest that HB-EGF is associated with the early progression steps of hepatoma.

Topics: Animals; Carcinoma, Hepatocellular; Epidermal Growth Factor; Heparin-binding EGF-like Growth Factor; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Male; Neoplasm Proteins; Rats; Rats, Mutant Strains; Rats, Sprague-Dawley; RNA, Messenger; RNA, Neoplasm

The mechanisms of hepatocarcinogenesis are still poorly understood. The development of hepatocellular carcinoma has recently been shown to be associated with increased DNA synthesis in cirrhosis. The aim of this work was to determine whether the high rate of hepatocyte regeneration observed in cirrhotic liver with hepatocellular carcinoma is associated with the presence of a growth factor that could be detectable in the serum.. Adult human hepatocytes in primary culture, allowing the evaluation of the release of circulating hepatotrophic factors, were used. These cultures were treated for 48 h with serum from patients with cirrhosis with and without hepatocellular carcinoma, from patients with liver metastasis, and from healthy subjects. The rate of DNA synthesis in these cultures was assessed by measuring the amount of [3H]-thymidine incorporation into genomic DNA.. On average, the synthesis of DNA was increased 2.5-, 2.2-, 2.1-, and 2.3-fold, respectively, in response to serum from patients with cirrhosis with hepatocellular carcinoma, from patients with cirrhosis without hepatocellular carcinoma, from patients with liver metastasis, and from healthy subjects.. We conclude that the hepatotrophic activity of the serum is not significantly different in patients with cirrhosis with or without hepatocellular carcinoma. These results suggest that the increased DNA synthesis in hepatocytes of cirrhotic liver with hepatocellular carcinoma might be due to proliferative factor(s) acting by paracrine or autocrine pathways.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Case-Control Studies; Cell Division; Cells, Cultured; DNA; Epidermal Growth Factor; Female; Humans; Liver; Liver Cirrhosis; Liver Neoplasms; Liver Regeneration; Male; Middle Aged; Recombinant Proteins; Reference Values

Heparin-binding EGF-like growth factor (HB-EGF) is a recently identified potent mitogen for smooth muscle cells and fibroblasts. HB-EGF has been shown to be an EGF receptor ligand, and also to stimulate epithelial cell growth. A human hepatoma-derived cell line, Mahlavu, was analyzed for the production of HB-EGF mRNA and active HB-EGF protein. It was found that the cell line synthesized very low or undetectable basal level of HB-EGF mRNA. However, the addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) led to a rapid and transient rise in HB-EGF mRNA level. HB-EGF in Mahlavu cells appears to be regulated by a protein kinase C (PKC) pathway, since PKC inhibitors, H7, staurosporin, and calphostin C, abrogated the induction of HB-EGF mRNA by TPA. Unlike vascular smooth muscle cells, induction of HB-EGF gene transcription by TPA was blocked completely by incubation with cycloheximide, suggesting that protein synthesis may be a prerequisite for HB-EGF gene transcription in Mahlavu cells. Mahlavu cells were also found to release a bioactive HB-EGF-like protein into conditioned medium which stimulates DNA synthesis in EP170.7 cells. This activity was neutralized by an anti-HB-EGF antibody. These results indicate that HB-EGF gene transcription is regulated via a PKC pathway, resulting in secretion of active HB-EGF into the culture medium of hepatoma-derived Mahlavu cells.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Carcinoma, Hepatocellular; Cycloheximide; Epidermal Growth Factor; Gene Expression Regulation; Heparin; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Naphthalenes; Protein Kinase C; RNA, Messenger; Staurosporine; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tumor Cells, Cultured

To target gene expression to malignant hepatic cells, we have constructed recombinant retroviral vectors containing a reporter gene encoding nuclear beta-galactosidase (nls-LacZ) under transcriptional control of regulatory sequences from the rat alpha-fetoprotein (AFP) or human insulinlike growth factor II (IGFII) genes. The AFP and IGFII P3 promoters activate transcription during fetal development and are often reactivated in hepatocellular carcinoma (HCC). Infection of several cultured cell types with the retroviral vector containing the IGFII P3 sequence resulted in expression of the reporter gene in all cell lines tested, including those that do not produce IGFII. In contrast, selective expression was achieved by vectors containing the AFP transcriptional regulatory sequence. Nuclear beta-galactosidase activity was detectable in cells from lines that produce AFP, and not in cells that do not express the AFP gene. In most infected cell lines, retroviral RNA synthesis from the 5' LTR was inhibited, and deletion of the retroviral LTR enhancer did not change expression from either the IGFII P3-nls-LacZ or the AFP-nls-LacZ cassettes. After treatment of cells with 12-O-tetradecanoylphorbol-13-acetate and epidermal growth factor (EGF), the decrease in concentrations of endogenous AFP messenger RNA (mRNA) and nls-LacZ mRNA transcribed from the transferred AFP regulatory sequence were similar. In the context of an integrated provirus, the AFP transcriptional regulatory sequence is therefore subject to similar regulatory control as that of the endogenous gene. These data show that the AFP sequence, and not the IGFII P3 promoter we used, is suitable for targeting gene expression to malignant hepatic cells.

Topics: 3T3 Cells; alpha-Fetoproteins; Animals; beta-Galactosidase; Blotting, Northern; Carcinoma, Hepatocellular; DNA; Epidermal Growth Factor; Gene Expression; Gene Transfer Techniques; HeLa Cells; Humans; Insulin-Like Growth Factor II; Liver Neoplasms; Mice; Promoter Regions, Genetic; Rats; Regulatory Sequences, Nucleic Acid; Retroviridae; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tumor Cells, Cultured

Epidermal growth factor (EGF) stimulation of HepG2 and NIH 3T3 cells expressing high levels of the human EGF receptor (3T3/ER) resulted in the tyrosine phosphorylation of a 115-kDa protein that was co-immunoprecipitated with the Src homology 2 (SH2) domain containing protein tyrosine phosphatase, SHPTP2. In contrast, activation of the EGF receptor resulted in a relatively low level (< 1%) of the total SHPTP2 pool associated with the tyrosine-autophosphorylated EGF receptor itself. Similarly, quantitative immunoprecipitations also demonstrated that only trace amounts of the total EGF receptor pool were associated with SHPTP2. Further, activation of the EGF receptor did not result in any significant tyrosine phosphorylation of SHPTP2 and/or the association of the 115-kDa protein with Grb2. In comparison, activation of Jurkat cells with a T cell receptor agonist monoclonal antibody resulted in the co-immunoprecipitation of a 120-kDa tyrosine-phosphorylated protein with Grb2 and a 105-kDa protein with SHPTP2. Thus, these data have identified the 115- and 105-kDa proteins as the predominant SHPTP2-associated phosphotyrosine proteins in EGF- and T cell receptor-activated cells, respectively.

Topics: 3T3 Cells; Animals; Blotting, Western; Carcinoma, Hepatocellular; Cell Line; Epidermal Growth Factor; ErbB Receptors; Humans; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Mice; Molecular Weight; Phosphoproteins; Phosphorylation; Phosphotyrosine; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases; SH2 Domain-Containing Protein Tyrosine Phosphatases; Tumor Cells, Cultured; Tyrosine

The growth characteristics of a newly established cell line, Hep40, derived from a human hepatoma are described. An absolute requirement was found for serum to mediate cell growth. Neither EGF, TGF-alpha, nor HGF altered cell growth in the presence or absence of serum. A partial suppression of cell growth was achieved by several TGF-beta family proteins. Affinity crosslinking gels using 125I-labeled TGF-beta showed a significant decrease in the TGF-beta cell-surface type II receptor in Hep40 cells, compared to the TGF-beta-sensitive Hep3B cell line. However, growth could be completely suppressed by addition of vitamins K to the culture medium in both Hep40 and several other hepatoma cell lines. Growth suppression by vitamins K was accompanied by an increased level of transcripts for c-myc, c-jun, and prothrombin genes, in contrast to the actions of TGF-beta 1 protein, which caused a decrease in the level of c-myc transcripts. These data show that this new human hepatoma cell line has partial resistance to growth inhibition by TGF-beta with a unique TGF-beta receptor defect. However, growth was completely suppressed by vitamins K. The differing gene expression patterns in response to TGF-beta as compared to vitamin K suggest that these two growth inhibitors act through differing pathways.

Topics: Animals; Carcinoma, Hepatocellular; Cell Division; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Prothrombin; Proto-Oncogene Proteins c-jun; Proto-Oncogene Proteins c-myc; Rats; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Vitamin K

We have shown previously that approximately 1 in 10,000 primary hepatocytes isolated from untreated rats undergo clonal growth in soft agar in vitro in response to the synergistic action of nafenopin, a peroxisome proliferator (PP) and epidermal growth factor (EGF), a naturally occurring liver growth regulator. Here, we demonstrate that prior treatment of the animals with the genotoxic hepatocarcinogen diethylnitrosamine (DEN) caused a dose-dependent increase in soft agar colony numbers formed in vitro. These data suggest that the colony assay may offer a method of detecting in vitro hepatocytes transformed in vivo by DEN. It is known that rats treated with DEN develop enzyme altered foci prior to the development of tumours. The majority of these foci express high levels of gamma-glutamyl transpeptidase (GGT). However, foci promoted by PPs do not show this increased enzyme activity. In the present study, the colonies we have generated in vitro mimicked this pattern since the majority (approximately 80%) of the spontaneous colonies expressed GGT whereas colonies promoted by the synergistic action of nafenopin and EGF were mainly (75%) GGT negative. The proportion of colonies positive for GGT were similar using either hepatocytes isolated from control or from DEN-initiated rats. Further studies are required to assess if the hepatocytes selected for clonal expansion by this EGF/nafenopin regime reflect the presumed pre-neoplastic cells induced by genotoxin in vivo and associated with an increased propensity to cancer.

Topics: Agar; Animals; Carcinogenicity Tests; Carcinoma, Hepatocellular; Cells, Cultured; Contact Inhibition; Diethylnitrosamine; Dose-Response Relationship, Drug; Drug Synergism; Epidermal Growth Factor; gamma-Glutamyltransferase; Gene Expression Regulation, Neoplastic; Liver Neoplasms; Male; Nafenopin; Rats; Rats, Wistar; Tumor Stem Cell Assay

Intracellular pH (pHi) plays an important role in the metabolic activation of quiescent cells after a proliferative stimulus, and Na+/H+ exchange activity is required for growth in some extrahepatic tumors. To investigate intracellular acid/base homeostasis in hepatoma cells and the effects of putative liver growth factors on Na+/h+ exchange activity, we have studied intracellular pH (pHi) regulation in Hep G2 cells, a well-differentiated hepatoma cell line, both in resting conditions and after administration of epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), and insulinlike growth factor-II (IGF-II). The effects of fetal calf serum, TGF alpha, and amiloride on 3H-Thymidine incorporation were also studied. Amiloride (1 mmol/L) and external Na+ removal decreased baseline pHi in both HEPES and KRB. In HEPES, cells recovered from an acid load (20 mmol/L NH4Cl) by an amiloride inhibitable Na+/H+ exchange. In KRB, an additional, DIDS-inhibitable, Na(+)- and HCO3- dependent, but Cl(-)-independent acid extruder (Na:HCO3 cotransport) was activated. No evidence was found for a Cl/HCO3 exchange acting as acid loader. Administration of EGF and TGF alpha, but not of IGF-II, induced a dose-dependent, amiloride-inhibitable increase in baseline pHi, together with an increase in Na+/H+ exchange activity, shifting to the right the JH/pHi curve. Finally, 3H-thymidine incorporation in Hep G2 cells, in the presence of FCS or TGF alpha, was strongly inhibited by amiloride. In conclusion, in Hep G2 cells, pHi is mainly regulated by Na+/H+ exchange, which activity can be stimulated by EGF and TGF alpha, but not by IGF-II. Administration of TGF alpha stimulates DNA synthesis, an effect that is blocked by amiloride, an inhibitor of Na+/H+ exchanger. These data suggest that Na+/H+ exchange activation may play a critical role in the growth of some hepatic tumors.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Ammonium Chloride; Animals; Carcinoma, Hepatocellular; Carrier Proteins; Cattle; Cell Division; DNA; Epidermal Growth Factor; Fetal Blood; Fluoresceins; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Insulin-Like Growth Factor II; Liver Neoplasms; Sodium-Bicarbonate Symporters; Sodium-Hydrogen Exchangers; Transforming Growth Factor alpha; Tumor Cells, Cultured

The rat liver ectoATPase has reportedly been cloned. The cDNA, a member of the carcinoembryonic antigen (CEA) gene family, was shown to increase aggregation of transfected cells, but ATPase activity was not evaluated. Using this cDNA as a probe to clone the mercurial-insensitive ectoATPase (MI-ectoATPase) of human hepatoma Li-7A cells, the cDNA obtained was that of CEA which has no ATPase activity. The probe also did not detect increased transcription when MI-ectoATPase activity was induced in Li-7A cells. It is concluded that the "rat liver ectoATPase cDNA" codes for a cell adhesion molecule but does not code for an ectoATPase. It was also discovered that expression of four CEA transcripts in Li-7A cells was markedly stimulated by a single growth modulator, EGF, and was further stimulated by a cAMP elevating agent, cholera toxin.

Topics: Adenosine Triphosphatases; Animals; Base Sequence; Carcinoembryonic Antigen; Carcinoma, Hepatocellular; Cell Adhesion Molecules; Cell Line; DNA, Complementary; Enzyme Induction; Epidermal Growth Factor; Gene Library; Humans; Liver Neoplasms; Mercury Compounds; Molecular Sequence Data; Multigene Family; Rats; Sequence Homology, Nucleic Acid; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Transforming growth factor alpha (TGF-alpha) is a polypeptide closely associated with hepatocyte proliferation in vivo and in vitro. In order to investigate the mechanisms by which TGF-alpha contributes to hepatocyte replication and transformation, we isolated hepatocytes from mice bearing a human TGF-alpha transgene and examined their growth properties and gene expression in defined, serum-free culture. The transgenic hepatocytes continued to overexpress human TGF-alpha mRNA and peptide, and were able to proliferate without exogenous growth factors in primary culture, in contrast to nontransgenic mouse hepatocytes. In short-term culture the transgenic hepatocytes underwent 1 wave of DNA replication at 72-96 h in culture before senescing, similar to nontransgenic hepatocytes supplemented with epidermal growth factor. Constitutive expression of TGF-alpha rendered the transgenic hepatocytes unresponsive to further growth stimulation by exogenous TGF-alpha, as well as other mitogens such as epidermal growth factor and hepatocyte growth factor. However, it did not alter their sensitivity to growth inhibition by TGF beta 1, 2 and 3. The addition of nicotinamide to the culture medium enabled both transgenic and epidermal growth factor-supplemented normal hepatocytes to replicate repeatedly and survive for > or = 2 months in primary culture while maintaining differentiated traits. From these long-term primary cultures of transgenic and nontransgenic hepatocytes, we established immortalized cell lines (designated TAMH and NMH lines, respectively). Both lines continued to express differentiated adult hepatocytic markers such as albumin, alpha-1-antitrypsin, transferrin, and connexin 26 and 32 mRNAs, but also expressed mRNAs for the oncofetal markers alpha-fetoprotein and insulin-like growth factor II. Unlike the near-diploid NMH hepatocyte line, the transgenic TAMH hepatocyte line was quasi-tetraploid, strongly expressed human TGF-alpha mRNA, and was highly tumorigenic in nude mice. Well-differentiated hepatocellular carcinomas developed in nude mice given injections of the TAMH line, and these appeared similar to the primary liver tumors seen in TGF-alpha transgenic mice with regard to histology and strong expression of mouse and human TGF-alpha, insulin-like growth factor II, and alpha-fetoprotein mRNAs. Our data show that TGF-alpha overexpression causes autonomous hepatocyte proliferation and contributes to neoplasia but that additional cellular alterations mus

Topics: alpha-Fetoproteins; Animals; Carcinoma, Hepatocellular; Cell Division; Cell Line; Cell Transformation, Neoplastic; Culture Media, Serum-Free; DNA Replication; Epidermal Growth Factor; Insulin-Like Growth Factor II; Liver; Male; Mice; Mice, Nude; Mice, Transgenic; Niacinamide; Ploidies; RNA, Messenger; Time Factors; Transforming Growth Factor alpha

Previous studies have suggested that both cAMP-dependent signal transduction pathway and Ca2+/protein kinase C-dependent pathway are involved in GSH efflux from hepatocytes. In the present study, GSH efflux from Hep G2 cells, a human-derived hepatoma cell line, was further characterized. Both epidermal growth factor (0.1-10 ng/ml) and insulin (1 microgram/ml) significantly increased GSH efflux from Hep G2 cells. A fall in the membrane potential produced by the replacement of Na+ with equivalent K+ did not affect GSH efflux significantly. Neither ouabain, a Na+/K+ ATPase inhibitor, vanadate, a Ca2+ ATPase inhibitor, nor BaCl2, a K+ channel blocker, significantly affected the GSH efflux. Methionine (1mM) decreased GSH efflux from the cells, although total GSH content in the cells was not affected during the incubation time of 60 min. Signal transductions through tyrosine kinase-coupled receptors may also be involved in GSH efflux from hepatocytes.

Topics: Adenosine Triphosphatases; Carcinoma, Hepatocellular; Cell Line; Epidermal Growth Factor; Glutathione; Humans; Insulin; Liver Neoplasms; Methionine; Potassium Channels; Receptor Protein-Tyrosine Kinases; Signal Transduction; Tumor Cells, Cultured

I examined the effects of intracellular pH (pHi) and the growth factors, epidermal growth factor (EGF) and human hepatocyte growth factor (HGF), on DNA synthesis in non-regenerating hepatocytes, regenerating hepatocytes and hepatoma cells. Non-regenerating and regenerating hepatocytes were isolated from the livers of intact adult rat and of the adult rat 24 hours after 70% hepatectomy, respectively. Hep G2 cells, a human hepatoma cell line, was employed as hepatoma cells. Regenerating hepatocytes and Hep G2 cells, but not non-regenerating hepatocytes displayed increased DNA synthesis with increasing pHi in the absence of EGF and HGF. However, non-regenerating hepatocytes displayed little increase, regenerating hepatocytes displayed substantial increase in DNA synthesis with increasing pHi in the presence of EGF or HGF. In contrast, Hep G2 cells displayed decreased DNA synthesis in the presence of HGF but not EGF. These findings indicate that pHi influences the fashion of proliferation in hepatocytes and cancer cells. EGF and HGF stimulate DNA synthesis in hepatocyte and inhibit that in cancer cell, suggesting that increasing pHi and administration of these growth factors may be one of the effective treatment for hepatoma.

Topics: Animals; Carcinoma, Hepatocellular; Cells, Cultured; DNA; DNA, Neoplasm; Epidermal Growth Factor; Hepatocyte Growth Factor; Humans; Hydrogen-Ion Concentration; Liver; Liver Neoplasms; Liver Regeneration; Male; Rats; Rats, Inbred F344; Tumor Cells, Cultured

Growth factors are involved in the development and progression of cancer. The purpose of this study was to evaluate the possible role of heparin-binding epidermal growth factor-like growth factor (HB-EGF), which is a member of the EGF family, in the neoplastic transformation of hepatocytes.. Gene expression and protein production of HB-EGF were investigated in samples of human hepatocellular carcinoma (HCC) from 17 patients using Northern hybridization and immunohistochemical methods.. The amount of HB-EGF messenger RNA was increased in the patients' HCC specimens compared with the surrounding liver tissues. In noncancerous hepatic tissues, HB-EGF was faintly positive in hepatocytes. Immunoreactive HB-EGF-producing cells were identified in HCC cells of all 17 patients with HCC, indicating that HB-EGF was produced in HCC cells themselves. However, none of the specimens from 10 patients with metastatic adenocarcinoma in the liver was positive for HB-EGF. The EGF receptor, which binds to HB-EGF, was also expressed on HCC cells.. It is hypothesized that the enhanced expression of immunoreactive HB-EGF on the cell suggests a possible role of HB-EGF in the development or progression of human HCC in an autocrine and/or a juxtacrine manners.

Topics: Adult; Blotting, Northern; Carcinoma, Hepatocellular; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression; Heparin; Heparin-binding EGF-like Growth Factor; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Liver; Liver Neoplasms; Male; Middle Aged; RNA, Messenger

We found that thapsigargin (Tg), a non-phorbol ester type tumor promoter that specifically inhibits endoplasmic reticulum Ca(2+)-ATPase, transiently increased the level of cytosolic free calcium ([Ca2+]i) and subsequently induced chromatin condensation, nuclear fragmentation, and internucleosomal DNA cleavage in cultured PLC/PRF/5 human hepatoma cells. These alterations were followed by the loss of plasma membrane integrity and by cell death. Epidermal growth factor (EGF) and vasopressin similarly elevated [Ca2+]i without causing DNA fragmentation which is characteristic of apoptosis. Consequently, the elevation of [Ca2+]i itself was not sufficient for causing Tg-induced cell death. On the other hand, preculturing the cells with Tg completely suppressed Ca2+ mobilization induced by EGF and vasopressin; a result that strongly suggests that Tg depleted the endoplasmic reticulum Ca2+ pool. Such depletion is hypothesized to induce apoptotic cell death in this hepatoma cell line by changing the nuclear Ca2+ levels which probably produce a structural change in chromatin.

Topics: Apoptosis; Calcium; Carcinoma, Hepatocellular; Cell Division; DNA Topoisomerases, Type II; DNA, Neoplasm; Epidermal Growth Factor; Humans; Liver Neoplasms; Terpenes; Thapsigargin; Tumor Cells, Cultured

We describe the establishment and characterization of a novel hepatoma cell line. This cell line, designated RBHF-1, was established from a hepatocellular carcinoma of a 67-yr-old man with a history of genetic hemochromatosis. At this writing, the cells have been maintained in RPMI-1640 tissue-culture medium and fetal calf serum without any additional supplements for 30 mo. The cells form colonies on soft agar and are not tumorigenic in nude mice. The cell line is polymorphic and displays characteristics of mature hepatocytes by synthesizing albumin, alpha 2-macroglobulin, fibronectin and alpha-fetoprotein. Cytogenetic analysis shows multiple chromosomal aberrations, with a consistent deletion in the long arm and deletions or rearrangements in the short arm of chromosome 1. There is no evidence for hepatitis B or hepatitis C virus infection of the cell line. The cells contain no detectable intracellular iron after staining with Perls' stain. Unlike other hepatoma cell lines, there is no detectable binding of epidermal growth factor to RBHF-1 cells. This is the first cell line to be established from a patient with hemochromatosis, and it provides a potentially important model for the study of hepatocyte transformation in association with iron overload.

Topics: Aged; Animals; Carcinoma, Hepatocellular; Chromosome Aberrations; Epidermal Growth Factor; Hemochromatosis; Humans; Iron; Liver Neoplasms; Male; Mice; Mice, Nude; Neoplasm Transplantation; Tumor Cells, Cultured

Epidermal growth factor and transforming growth factor alpha stimulated DNA synthesis in primary cultures of adult rat hepatocytes. Neurotensin amplified epidermal growth factor-stimulated or transforming growth factor alpha-stimulated DNA synthesis by three- to eightfold. Neurotensin by itself did not stimulate DNA synthesis. Amplification of DNA synthesis by neurotensin was observed as low as 10(-10) M, and it was increased in a dose-dependent manner with maximal effects at 10(-8) M. These results were obtained when hepatocytes were cultured in Williams' medium E, but not in Leibovitz L-15 medium, suggesting that a minor component(s) in the medium is required for hepatocytes to fully respond to neurotensin. Neurotensin effect on DNA synthesis was observed not only in normal rat hepatocytes but also in partially hepatectomized rat hepatocytes, although its effect was stronger in normal hepatocytes. Amplified DNA synthesis was inhibited by transforming growth factor beta. Secondary mitogens (co-mitogens) such as insulin, vasopressin, or angiotensin II interacted additively with low concentrations of epidermal growth factor as well as with neurotensin. Neurotensin-related peptides such as kinetensin or neuromedin-N, which was released from blood plasma by pepsin digestion, did not have this amplifying effect on DNA synthesis at any concentrations tested. Neurotensin mRNA was found in several organs including brain and intestine, but not liver. These results suggest that neurotensin can be regarded as a new secondary mitogen and that it may be involved in cell proliferation, including regenerating liver as a gastrointestinal hormone and/or a neurotransmitter.

Topics: Animals; Carcinoma, Hepatocellular; Cells, Cultured; Culture Media; DNA; Dose-Response Relationship, Drug; Drug Synergism; Epidermal Growth Factor; Liver; Liver Neoplasms; Liver Regeneration; Male; Neuropeptides; Neurotensin; Rats; Rats, Inbred F344; RNA, Messenger; Tissue Distribution; Transforming Growth Factor alpha

To investigate whether atrial natriuretic factor regulates the growth of hepatocytes and to determine the receptor subtype involved in such modulation, we studied the effect of atrial natriuretic factor 103-126 and clearance receptor binding analogs of atrial natriuretic factor, (des-(Q116, S117, G118, L119, G120) atrial natriuretic factor 102-121 and des-(C105,121) atrial natriuretic factor 104-126) on growth of human hepatoblastoma cells. Atrial natriuretic factor 103-126 and des-(Q116, S117, G118, L119, G120) atrial natriuretic factor 102-121 inhibited thymidine incorporation into human hepatoblastoma cells cultured in the presence of bovine serum albumin and epidermal growth factor but not in cells cultured in bovine serum albumin alone. Moreover, atrial natriuretic factor 103-126, des-(Q116, S117, G118, L119, G120) atrial natriuretic factor 102-121 and des-(C105,121) atrial natriuretic factor 104-126, in a concentration-dependent manner, inhibited thymidine incorporation and cell proliferation. As monitored by the ability of des-(Q116, S117, G118, L119, G120) atrial natriuretic factor 102-121 to displace 125I-labeled atrial natriuretic factor, epidermal growth factor increased the expression of cell surface clearance receptors. Epidermal growth factor also transiently increased the cellular content of atrial natriuretic factor clearance receptor messenger RNA without altering the levels of guanylyl cyclase-linked atrial natriuretic factor receptor messenger RNA levels. Maximal increase in atrial natriuretic factor clearance receptor messenger RNA coincided with the maximal increase in des-(Q116, S117, G118, L119, G120) atrial natriuretic factor 102-121-displaceable 125I-atrial natriuretic factor binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-Methyl-3-isobutylxanthine; Atrial Natriuretic Factor; Carcinoma, Hepatocellular; Cell Division; Cyclic AMP; Cyclic GMP; DNA Replication; Epidermal Growth Factor; Kinetics; Liver Neoplasms; Peptide Fragments; Receptors, Atrial Natriuretic Factor; RNA, Messenger; RNA, Neoplasm; Thymidine; Tritium; Tumor Cells, Cultured

Topics: Apoptosis; Calcium; Calcium-Transporting ATPases; Carcinoma, Hepatocellular; Epidermal Growth Factor; Humans; Liver Neoplasms; Terpenes; Thapsigargin; Tumor Cells, Cultured

Human hepatoma Li-7A cells exhibit two cell surface ATPase (ectoATPase) activities distinguishable by their different biochemical properties. The activity of the minor ectoATPase, ectoCa(2+)-ATPase, is enhanced severalfold when Li-7A cells are treated simultaneously by epidermal growth factor (EGF) and cAMP elevating agents (Knowles, A. F., 1990, Arch. Biochem. Biophys. 283, 114-119). Here we report that the human ectoCa(2+)-ATPase is biochemically similar to the major rat hepatocyte ectoATPase/cell adhesion molecule (cell-CAM 105) with respect to response to divalent ions and sulfhydryl reagents. Furthermore, the binding of rat liver ectoATPase antibody increased markedly in EGF/cholera toxin/hydrocortisone-treated Li-7A cells compared to untreated cells. Western blot analysis revealed cross-reactivity of the antibody with a 125-kDa protein. Partial purification of ectoCa(2+)-ATPase from EGF/cholera toxin/hydrocortisone-treated Li-7A cells confirmed that enrichment of the 125-kDa protein correlated with an increase in ATPase activity. We conclude that EGF and increased levels of cAMP lead to increased synthesis of the ectoCa(2+)-ATPase in Li-7A cells. The present demonstration of similarity between the ectoCa(2+)-ATPase and a rat liver cell adhesion molecule, cell-CAM 105, contributes significantly to an understanding of the implication of down-regulation of ectoCa(2+)-ATPase during hepatocyte-hepatoma transformation.

Topics: Adenosine Triphosphatases; Animals; Blotting, Western; Calcium; Calcium-Transporting ATPases; Carcinoma, Hepatocellular; Cell Membrane; Cholera Toxin; Cross Reactions; Cyclic AMP; Enzyme Induction; Epidermal Growth Factor; Extracellular Space; Humans; Hydrocortisone; In Vitro Techniques; Liver; Liver Neoplasms; Magnesium; Molecular Weight; Rats; Tumor Cells, Cultured

The roles of epidermal growth factor (EGF) on cell growth control and phosphatidylinositol signal transduction pathway in human hepatoma cell lines with different differentiated states were evaluated. Ligand binding study showed that only one receptor type with similar affinity was found in all three cell lines. Under serum-free conditions, EGF (10(-8)-10(-11) M) enhanced DNA synthesis and cell proliferation of the three cell lines in a dose-dependent manner. The response of the poorly differentiated HA22T/VGH hepatoma cells was most obvious whereas much smaller effects were found in Hep3B and Chang liver cells. The metabolism of phosphoinositides also increased in HA22T/VGH cells as compared with both Hep3B and Chang liver cells under basal and EGF-treated conditions. Our data indicated that EGF had different effects on different human hepatoma cell lines and its role might be more important in poorly differentiated hepatoma cells than in well differentiated ones.

Topics: Carcinoma, Hepatocellular; Cell Differentiation; Cell Division; DNA, Neoplasm; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Humans; Inositol Phosphates; Kinetics; Liver; Liver Neoplasms; Signal Transduction; Thymidine; Tritium; Tumor Cells, Cultured

Teleocidin, a phorbol ester-type tumor promoter, inhibits cell proliferation and calcium mobilization induced by epidermal growth factor and vasopressin in PLC/PRF/5 hepatoma cells. These inhibitory effects of teleocidin were observed even after a prolonged exposure of the hepatoma cells to this promoter, suggesting the presence of down-regulation-resistant protein kinase C in this hepatoma cell line. Column chromatography of cytosolic fractions showed three separate peaks of protein kinase C activity, two being down-regulation-sensitive while one was down-regulation-resistant. This down-regulation-resistant PKC is suggested to be responsible for the inhibitory effect of teleocidin on cell proliferation and calcium mobilization induced by epidermal growth factor and vasopressin.

Topics: Calcium; Carcinoma, Hepatocellular; Cell Count; Cell Division; Cell Line; Down-Regulation; Epidermal Growth Factor; Humans; Lyngbya Toxins; Protein Kinase C; Tumor Cells, Cultured; Vasopressins

The expression of the cellular gene coding for the epidermal growth factor (EGF) receptor (EGF-R) was assayed in the presence of hepatitis B virus (HBV) gene expression under different experimental conditions in human hepatoma-derived cells. First, transfection experiments of the well-differentiated HepG2 human hepatoma cell line using different expression vectors of the HBV X-region demonstrated that the X-gene product is capable of inducing EGF-R gene overexpression; in addition, by using a stable in vitro expression system for HBV, it was shown that EGF-R gene expression in these cells is greater than in the uninfected parent cells, and that this results in a three-fold increase in 125I-EGF binding. Finally, a CAT-expression assay was performed, indicating that regulatory regions of the EGF-R-gene are target sequences for X-protein trans-activation.

Topics: Carcinoma, Hepatocellular; Chloramphenicol O-Acetyltransferase; Epidermal Growth Factor; ErbB Receptors; Genetic Vectors; Hepatitis B virus; Humans; Liver Neoplasms; Promoter Regions, Genetic; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Trans-Activators; Transcriptional Activation; Transfection; Tumor Cells, Cultured; Viral Regulatory and Accessory Proteins; Virus Replication

We carried out the following three experiments to clarify the mechanism of hepatocarcinogenesis in Long-Evans Cinnamon (LEC) rats. (1) Sensitivity to diethylnitrosamine (DEN): LEC rats (8 and 25 weeks old) without and with hepatitis and age-matched F344 rats were administered an intraperitoneal injection of a low dose of DEN. Eight weeks after the injection, the numbers of glutathione-S-transferase placental-form (GST-P)-positive foci in the 33-week-old LEC rat liver were significantly higher than those in the livers of the other three groups of rats. (2) Potential for unscheduled DNA synthesis (UDS): Isolated hepatocytes of 25-week-old LEC rats with chronic hepatitis showed about one-third the level of UDS induced by UV irradiation, as compared to that of age-matched F344 rats, while no significant difference was found between the UDS of isolated hepatocytes of 8-week-old LEC rats and age-matched F344 rats. (3) Potential for proliferation: Isolated hepatocytes from 8-week-old LEC rats responded well to epidermal growth factor (EGF) in culture, to almost the same degree as F344 rat hepatocytes, while a remarkable decrease in the responsiveness of hepatocytes isolated from 25-week-old LEC rats to EGF was found. These results suggested that LEC rat hepatocellular carcinoma could be naturally initiated after the onset of hepatitis by carcinogens contaminating food and the environment, probably due to the reduction of DNA repair activity, after which initiated hepatocytes selectively proliferate in response to growth stimuli endogenously produced as a result of continuous loss of hepatocytes (chronic hepatitis), because of a decrease in growth activity of non-initiated hepatocytes.

Topics: Animals; Carcinoma, Hepatocellular; Cell Division; Chronic Disease; Diethylnitrosamine; DNA; Epidermal Growth Factor; Glutathione Transferase; Hepatitis, Animal; Liver; Male; Rats; Rats, Inbred F344; Rats, Mutant Strains; Ultraviolet Rays

Epidermal growth factor (EGF) was found to induce a rapid 2-fold increase in the amount of insulin-like growth factor binding protein-1 (IGFBP-1) mRNA in human hepatoma Hep2G cells, and this was accompanied by a 2-fold increase in IGFBP-1 secretion. A protein synthesis inhibitor cycloheximide (CHX) caused a 2-3-fold increase in the amount of IGFBP-1 mRNA, which could be accounted for the observed stabilization in decay of IGFBP-1 mRNA after CHX treatment. In nuclear run-on transcription experiments neither EGF nor CHX affected the transcription rate of the IGFBP-1 gene. It is concluded that EGF increases IGFBP-1 secretion rapidly by enhancing IGFBP-1 mRNA accumulation, and the addition of a protein synthesis inhibitor results in a specific increment of IGFBP-1 mRNA, suggesting that a labile protein repressor protein is involved in the turnover IGFBP-1 mRNA.

Topics: Blotting, Northern; Carcinoma, Hepatocellular; Carrier Proteins; Cell Nucleus; Cycloheximide; DNA Probes; Epidermal Growth Factor; Humans; Insulin-Like Growth Factor Binding Protein 1; Kinetics; Liver Neoplasms; RNA, Messenger; Somatomedins; Transcription, Genetic; Tumor Cells, Cultured

The effect of epidermal growth factor (EGF) receptor overexpression on ligand-induced EGF receptor downregulation was examined using a hepatoma-derived cell line, PLC/PRF/5, which expresses normal amounts of the EGF receptor, and a subline, NPLC/PRF/5, which expresses 10-fold more receptors at its cell surface. PLC/PRF/5 cells efficiently downregulated surface receptor levels upon exposure to saturating and subsaturating concentrations of EGF; the rate of receptor downregulation corresponded to that of ligand-receptor internalization. Upon internalization, EGF receptors were degraded and receptor biosynthesis remained at basal levels. EGF surface receptor remained downregulated for as long as cells were exposed to EGF. By contrast, surface EGF receptor abundance in NPLC/PRF/5 cells decreased by only 5-15% after 1-4 h incubation with subsaturating doses of EGF and actually increased by 67% within 20 h. Exposure of these cells to saturating concentrations of EGF induced modest decreases in surface receptor abundance during the initial 12 h incubation, followed by a progressive decline to 30% of initial values by 24 h. Relative ligand-receptor internalization rates in NPLC/PRF/5 cells were lower than those in PLC/PRF/5, although their surface receptor population was even higher than that predicted by the decreased internalization rates. EGF receptor degradation in NPLC/PRF/5 cells was also inhibited; exposure to saturating levels of EGF for more than 16 h was necessary before significant degradation occurred. Receptor protein and mRNA biosynthesis in NPLC/PRF/5 were stimulated by 8 h exposure to EGF but when saturating concentrations of EGF were present for 16 h, receptor biosynthesis was inhibited. EGF receptor overexpression circumvents the downregulatory effect of EGF by decreasing the rate of receptor internalization, inhibiting degradation of the internalized receptor pool, and stimulating EGF receptor biosynthesis. Conversely, receptor downregulation becomes pronounced at late times when receptor degradation is high and biosynthesis is inhibited.

Topics: Carcinoma, Hepatocellular; Down-Regulation; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Humans; In Vitro Techniques; Inositol Phosphates; Liver Neoplasms; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured

A high level of expression of the functional product of the epidermal growth factor receptor (EGFR) gene was detected in the human hepatocarcinoma cell line Li7A and it was found to correlate with gene amplification. The karyotype was paratriploid, with 15 rearranged chromosomes, several of which contained abnormally banded regions (ABRs). The search for DNA sequences co-amplified with the EGFR gene, using the in-gel renaturation technique, allowed us to detect an amplified DNA band (La1) of about 30 kb. This DNA was used as a probe for in situ hybridization on chromosomes, to locate the amplified segment. In normal lymphocytes, the DNA of band La1 hybridized to chromosome regions in which repetitive DNAs are located, i.e. on juxtacentromeric regions, the site of alphoid and CCATT satellite DNA, and on the short arms of acrocentrics, the site of ribosomal RNA (RNR) genes. In Li7A cells, it hybridized to the same regions and, in addition, to several chromosome arms corresponding to ABRs. The same ABRs hybridized to EGFR and RNR probes, but neither Alu sequences nor various probes for other repetitive sequences were recognized. They also exhibited nucleolus organizer region staining characterizing functionally active (RNR) genes. It was concluded that transcriptionally active genes were co-amplified in the same ABRs, although they originated from different chromosomes, i.e. chromosome 7 for EGFR and acrocentrics for RNR genes.

Topics: Blotting, Northern; Blotting, Southern; Carcinoma, Hepatocellular; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Humans; Liver Neoplasms; Nucleic Acid Hybridization; Plasmids; Precipitin Tests; Radioligand Assay; RNA, Ribosomal; Transcription, Genetic; Tumor Cells, Cultured

To characterize epidermal growth factor-related transforming growth factors in the urine of patients with hepatocellular carcinoma, gel filtration with Bio-Gel P-30 was performed in seven hepatocellular carcinoma patients and seven sex-matched and age-matched healthy controls. Distinct profiles of soft agar growth assay in the hepatocellular carcinoma patients and the normal controls were seen. Three peaks (A, B and C) in the urine were examined. Peak C in most hepatocellular carcinoma patients was higher than that in healthy controls. Similar profiles were detected with epidermal growth factor radioreceptor assay and cellular DNA synthesis assay. This result might indicate that transforming growth factors with low molecular weight were found in the urine of hepatocellular carcinoma patients. An exceptional HCC patient had an additional peak (A') that corresponded to the high molecular weight protein. We concluded that there were transforming growth factors with functional activity in the urine of patients with hepatocellular carcinoma.

Topics: Adult; Agar; Aged; Carcinoma, Hepatocellular; Cell Division; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Humans; Liver Neoplasms; Middle Aged; Radioligand Assay; Transforming Growth Factors; Tumor Cells, Cultured

Culture of Hep G2 cells in medium containing 2% (v/v) dimethyl sulphoxide (DMSO) resulted in a slowing of growth and a marked change in morphological appearance. By day 6, cultures containing DMSO had one-third the number of cells compared with parallel control cultures. Measurement of 125I-epidermal-growth-factor (EGF) binding to DMSO-treated cells revealed a striking time-dependent elevation in specific EGF binding to their cell surface. Increased binding was detectable within 24 h of the start of DMSO treatment, reaching, by 6 days, levels almost 25 times greater than those for control cells. Addition of EGF to DMSO-treated cells caused a rapid down-regulation of the EGF receptor, but did not alter their proliferation rate. Slowing of growth by other means, such as serum starvation, growth to confluence or culture in the presence of sodium butyrate, did not affect 125I-EGF binding, indicating a specific effect of DMSO on these cells.

Topics: Carcinoma, Hepatocellular; Cell Differentiation; Cell Division; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Humans; In Vitro Techniques; Microscopy, Electron, Scanning; Tumor Cells, Cultured

To clarify sex differences in DNA synthesis in hepatocellular carcinoma (HCC), bromodeoxyuridine labeling indices (BrdU LI) of HCC cells included in tumor biopsy specimens from 12 consecutive male patients and from 5 consecutive female patients all with liver cirrhosis and HCC were examined using an in vitro labeling technique. The mean BrdU LI +/- SE of HCC from male patients (7.7 +/- 0.8%) was significantly (P less than 0.05) higher than that from female patients (4.4 +/- 1.0%). While 7 of the 12 male HCC patients belonged to the high DNA synthesis group (BrdU LI greater than or equal to 7.0%), none of the 5 female HCC patients showed high DNA synthesis (P less than 0.05). We conclude that DNA synthesis in HCC was higher in males than in females.

Topics: Aged; Bromodeoxyuridine; Carcinoma, Hepatocellular; DNA, Neoplasm; Epidermal Growth Factor; Female; Humans; Liver Neoplasms; Male; Middle Aged; Sex Factors

Expression of epidermal growth factor (EGF) and fibroblast growth factor (FGF) was examined in 56 patients with hepatocellular carcinoma (HCC) using an immunohistochemical method. EGF and FGF were expressed on carcinoma cells in 14 (25%) and 23 cases (41%), respectively. In the 23 FGF-positive cases, 11 cases were positive for both acidic and basic FGF, while 18 were positive for acidic FGF, and 16 were positive for basic FGF. In non-cancerous hepatic tissues, FGF was weakly positive in macrophages, hepatocytes and vascular endothelial cells in some cases, while EGF was totally negative. There were no significant correlations between the expression of EGF or FGF on carcinoma cells and the various clinicopathologic factors examined. These data suggest that EGF and FGF are produced by human HCC cells in vivo. The roles of the expression of these growth factors in the development and progression of HCC remain only speculative.

Topics: Adult; Carcinoma, Hepatocellular; Epidermal Growth Factor; Female; Fibroblast Growth Factors; Humans; Immunohistochemistry; Liver Neoplasms; Male

Plasminogen activator inhibitor type-1 (PAI-1) is a physiologic modulator of the fibrinolytic system. We have shown previously that PAI-1 biosynthesis in cultured cells depends on several factors in serum. Because platelets are richly endowed with specific growth factors and because the release reaction is an integral part of thrombosis, the present study was performed to determine whether platelets augment PAI-1 production and if so, to define mediators responsible. Hep G2 cells were used to determine whether platelet lysates increased PAI-1 synthesis in a dose and time-dependent manner. In cells labeled metabolically with 35S-methionine for 6 h, an increase in labeled PAI-1 was elicited indicative of de novo synthesis as well as increased secretion of PAI-1 mediated by platelet lysates. Steady state levels of both the 3.2 and 2.2 kb forms of PAI-1 mRNA increased after 2 h and peaked in 3-5 h in a dose-dependent fashion as well. Incubation of Hep G2 cells with collagen activated platelets resulted in a similar induction of PAI-1 mRNA. The increase in PAI-1 mRNA occurred with exposure of the cells to platelet lysates for intervals as brief as 15 min and was not inhibited by cycloheximide indicating its independence of new protein synthesis. In order to identify the factors in platelets responsible for the induction of PAI-1 synthesis in the Hep G2 cell model system, neutralizing antibodies were used to inhibit specific platelet associated growth factors. Antibodies to transforming growth factor-beta (TGF-beta) and to the epidermal growth factor (EGF)/transforming growth factor alpha (TGF-alpha) receptor inhibited the platelet lysate-mediated increase in PAI-1 protein by 77%.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Blood Platelets; Carcinoma, Hepatocellular; Epidermal Growth Factor; Humans; Liver Neoplasms; Plasminogen Inactivators; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Liver cells are considered the principal source of plasma vitronectin. The human hepatoma cell line HepG2 produces vitronectin into its culture medium. In the current work we have analyzed the regulation of vitronectin by transforming growth factor-beta 1 (TGF beta 1) in this hepatoma cell line by Northern hybridization, polypeptide and immunoprecipitation analyses and compared the response to another TGF beta-regulated gene, plasminogen activator inhibitor (PAI-1). Rabbit antibodies raised against human plasma-derived vitronectin were used in immunodetection. Polypeptide and immunoprecipitation analyses of the medium and cells, as well as immunoblotting analysis of the cells and their extracellular matrices, indicated enhanced TGF beta 1-induced production and extracellular deposition of vitronectin. Accordingly, TGF beta 1 enhanced the expression of vitronectin mRNA at picomolar concentrations (2-20 ng/ml) as shown by Northern hybridization analysis. Comparison of the temporal TGF beta induction profiles of vitronectin and PAI-1 mRNAs showed that vitronectin was induced more slowly but the vitronectin mRNAs persisted longer. In addition, platelet-derived and epidermal growth factors had an effect on vitronectin expression, but it was of lower magnitude. TGF beta 1 enhanced the expression of PAI-1 but, unlike previous reports, epidermal growth factor did not have any notable effect on PAI-1 in these cells. The results indicate that TGF beta 1 is an efficient regulator of the production of vitronectin by HepG2 cells and that PAI-1 and vitronectin are not coordinately regulated. In addition, with affinity purified antibodies to vitronectin receptor, we observed strong enhancement of the alpha subunit of the receptor in response to TGF beta 1. These effects of TGF beta are probably involved in various processes of the liver where matrix induction and controlled pericellular proteolysis is needed, as in tissue repair.

Topics: Blood Proteins; Carcinoma, Hepatocellular; Cell Line; Cycloheximide; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Glycoproteins; Growth Substances; Humans; Kinetics; Liver Neoplasms; Plasminogen Inactivators; Poly A; Receptors, Immunologic; Receptors, Vitronectin; RNA, Messenger; Transforming Growth Factor beta; Vitronectin

The mechanisms by which growth factors are degraded and the role this process plays in the regulation of cell growth are not well understood. Insulin degradation is believed to be mediated by a specific metalloprotease, insulin-degrading enzyme (IDE). We have previously shown that IDE can also degrade transforming growth factor-alpha (TGF alpha), but not epidermal growth factor (EGF), in vitro. This selectivity was surprising, since TGF alpha and EGF are structurally similar and bind to the same receptor with comparable affinities. Using a spectrum of protease inhibitors, we have now analyzed the degradation of TGF alpha, EGF, and insulin by human hepatoma HepG2 cells. The results suggest that bacitracin-sensitive metalloproteases are involved in the degradation of TGF alpha and EGF as well as insulin, and that the degradation of TGF alpha, but not EGF, is mediated in part by IDE. Inhibiting the activity of these metalloproteases decreased growth factor depletion, suggesting that these enzymes play an important role in the control of extracellular growth factor levels. The existence of separate degradative pathways for EGF and TGF alpha may explain how the two factors exert differential effects in some systems, and degradation of TGF alpha by IDE would provide a possible mechanism for interaction between the insulin and TGF alpha/EGF signalling systems.

Topics: Bacitracin; Carcinoma, Hepatocellular; Endopeptidases; Epidermal Growth Factor; Humans; Insulin; Liver Neoplasms; Metalloendopeptidases; Protease Inhibitors; Transforming Growth Factor alpha; Tumor Cells, Cultured

The structure of coagulation factor XII (Hageman factor), inferred from its DNA sequence, includes two epidermal growth factor (EGF)-homologous domains in its amino-terminal region. This suggests that factor XII may exhibit EGF-like activities. Reciprocal antigenic cross-reactivity between factor XII and EGF was shown by exposing purified human factor XII or mouse EGF to anti-mouse EGF or anti-human factor XII. Western blot analysis showed that anti-mouse EGF recognized intact factor XII at 80 kDa. Together, these results suggest that the EGF-homologous domains are accessible for anti-EGF binding in native factor XII. To determine whether factor XII has mitogenic activity, HepG2 or L cells (10(4) cells per well) were grown in serum-free medium in the presence or absence of factor XII or kaolin-activated factor XII (factor XIIa). Both factors XII and XIIa (6.0 micrograms/ml) enhanced cell proliferation by approximately 2-fold (P less than 0.001 and P less than 0.005, respectively). In contrast, L cells, which are not EGF target cells, were not affected by either factor XII or factor XIIa. Various doses of factor XII enhanced cell proliferation, [3H]thymidine incorporation, and [3H]leucine incorporation in HepG2 cells cultured under the same conditions. These data indicate that factor XII, like EGF, is a mitogen for HepG2 cells and suggest a possible autocrine role in the liver.

Topics: Antibodies; Antigens; Carcinoma, Hepatocellular; Cell Division; DNA; Epidermal Growth Factor; Factor XII; Factor XIIa; Humans; L Cells; Liver Neoplasms; Mitogens; Tumor Cells, Cultured

Chang liver cells cultured in the simultaneous presence of novobiocin and butyrate stopped proliferating and changed into fibroblast-like cells with remarkably elongated cytoplasm. In these fibroblast-like cells, the cellular content of both protein and DNA was increased 2- to 3-fold. In addition, the production of specific proteins such as type III procollagen, actin, and tubulin was increased and the expression of proliferation-associated nuclear antigen which was reactive with Ki-67 monoclonal antibody was reduced remarkably. Therefore, Chang liver cells cultured with novobiocin and butyrate were considered to be arrested at the premitotic G2 phase of the cell cycle and then enter into the noncycling resting state. These actions of novobiocin and butyrate were not mediated by the pathway of epidermal growth factor action or modulated by the diacylglycerol agonist 1-oleoyl-2-acetylglycerol and the C kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine. On the other hand, novobiocin was disclosed to be a stimulator of [3H]acetate uptake and acted synergistically with butyrate in enhancing nuclear protein acetylation. From these results it can be speculated that novobiocin and butyrate chemically modulate nuclear proteins and thereby alter the gene expression and the differentiation state of Chang liver cells.

Topics: Acetylation; Blotting, Western; Butyrates; Carcinoma, Hepatocellular; Cell Cycle; Cell Differentiation; Cell Division; Collagen; Epidermal Growth Factor; Fibroblasts; Gene Expression; Humans; In Vitro Techniques; Liver; Liver Neoplasms; Molecular Weight; Novobiocin; Nuclear Proteins; Proliferating Cell Nuclear Antigen; Tumor Cells, Cultured

Epidermal growth factor (EGF)-induced increases in cytosolic Ca2+ and inositol polyphosphate production were compared in a human hepatocellular carcinoma-derived cell line, PLC/PRF/5, and in an EGF receptor-overexpressing subline, NPLC/PRF/5. Formation of these second messengers was correlated to EGF receptor display at the cell surface by monitoring ligand-induced EGF receptor down-regulation. Both cell lines exhibited a strikingly similar cytosolic Ca2+ increase upon exposure to EGF. The initial inositol phosphate responses were also similar in the two cell lines; inositol 1,4,5-trisphosphate increased within 10-15 s and returned to prestimulatory values after 2 min in both cell lines, while inositol tetrakisphosphate and inositol 1,3,4-trisphosphate were elevated after a 2-min exposure to EGF. At later times the responses were markedly different; NPLC/PRF/5 cells exhibited prolonged production of inositol 1,3,4-trisphosphate and inositol tetrakisphosphate (maximum at 1-3 h) but PLC/PRF/5 cells showed decreased levels of these isomers after 10 min and a return to basal values by 1 h. Exposure of PLC/PRF/5 cells to EGF caused a progressive decrease in the amount of EGF receptor at the cell surface whereas such treatment did not change the surface receptor levels in NPLC/PRF/5 cells. Kinetic analysis of EGF receptor down-regulation showed that receptor internalization was rapid enough to account for the transient nature of the inositol phosphate response in PLC/PRF/5 cells. Thus, the divergent patterns of signaling exhibited by the two cell lines may reflect differences in the efficiency of EGF-induced down-regulation of surface receptors.

Topics: Angiotensin II; Calcium; Carcinoma, Hepatocellular; Cell Line; Chromatography, High Pressure Liquid; Cytosol; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Humans; Inositol Phosphates; Ligands; Liver Neoplasms; Molecular Weight; Second Messenger Systems

The human hepatoma cell line (Li-7A) possesses a high concentration of epidermal growth factor (EGF) receptors and exhibits ectoATPase activity in the presence of either MgATP or CaATP (Knowles: J. Cell. Physiol., 134:109-116, 1988). Growth for 96 hours in the presence of both EGF and cholera toxin or another cyclic AMP elevating agent induced an ectoATPase activity which was more active with CaATP and resistant to inhibition by the sulfydryl reagent, p-chloromercuriphenylsulfonate (pCMPS) (Knowles: Arch. Biochem. Biophys., 263: 264-271, 1988). In contrast, treatment of cells with butyrate, a short chain organic acid which can be derived from the analogue, dibutyryl cyclic AMP, resulted in a 4-7-fold increase of an ectoATPase which was more active with MgATP and highly sensitive to pCMPS inhibition. Maximal induction by butyrate required 48 hours and was dependent on butyrate concentration, but was independent of EGF and cyclic AMP elevating agents. Of six organic acids tested, butyrate was most effective in the induction of the ectoMg2(+)-ATPase. The increase in the ectoMg2(+)-ATPase activity could be prevented with actinomycin D and cycloheximide, indicating that both transcription and translation were necessary for induction. In addition to the induction of the ectoMg2(+)-ATPase, butyrate induced alkaline phosphatase activity, but had no effect on a third ectoenzyme 5'-nucleotidase. These data further support our proposal that two distinct ectoATPases exist in the plasma membrane of Li-7A hepatoma cells.

Topics: 4-Chloromercuribenzenesulfonate; Adenosine Triphosphatases; Biological Transport, Active; Bucladesine; Butyrates; Carcinoma, Hepatocellular; Cell Division; Cycloheximide; Dactinomycin; Dose-Response Relationship, Drug; Enzyme Induction; Epidermal Growth Factor; Fatty Acids; Humans; Kinetics; Liver Neoplasms; Neoplasm Proteins; Time Factors; Tumor Cells, Cultured

Sex hormone-binding globulin (SHBG) is a glycoprotein whose production has been demonstrated to be regulated by both sex steroids, as well as by thyroid hormone and peptide hormones such as insulin. However, none of these regulatory factors would explain the marked decrease in serum SHBG seen throughout the prepubertal and pubertal time period in both boys and girls. Furthermore, current in vitro data show that both androgens and estrogens can stimulate SHBG production by the human hepatoblastoma cell line Hep G2; yet, in vivo androgens appear to suppress SHBG levels, while estrogens are associated with elevated levels. This study was undertaken to determine possible mechanisms to explain this phenomenon. Hep G2 cell cultures were incubated with insulin-like growth factor I (IGF-I), epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), or dehydroepiandrosterone (DHEA). Significant decreases in the level of SHBG in the culture medium relative to control cultures occurred for each of the growth factors (P less than .01), whereas an increase in SHBG levels was observed in the medium of DHEA-treated cells. When cells were coincubated with IGF-I and thyroxine (T4), which alone stimulates SHBG production both in vivo and in vitro, the SHBG response to T4 was blunted. These results suggest that growth factors, as well as insulin, may be important determinants in SHBG production.

Topics: Carcinoma, Hepatocellular; Cell Line; Dehydroepiandrosterone; Epidermal Growth Factor; Growth Substances; Humans; Insulin-Like Growth Factor I; Liver Neoplasms; Sex Hormone-Binding Globulin; Thyroxine; Tumor Cells, Cultured

A mercurial-insensitive ectoATPase, which was more active with CaATP than with MgATP, was induced when human hepatoma (Li-7A) cells were cultured in the presence of epidermal growth factor (EGF) and cholera toxin. Cholera toxin could be replaced by forskolin, 8-Br-cAMP, butyryl-cAMP, and dibutyryl-cAMP. Requirement for EGF was specific, but EGF was ineffective if added more than 24 h after the addition of forskolin or cholera toxin. It was concluded that induction of the ectoCa2(+)-ATPase was a consequence of the synergistic actions of EGF and cyclic AMP. The tyrosine kinase activity of the EGF receptor was essential for the induction of ectoCa2(+)-ATPase, since enzyme induction was abolished by a tyrosine kinase inhibitor, genistein. Cycloheximide and actinomycin D were also inhibitory to enzyme induction, indicating that enhancement of enzyme activity by EGF and cAMP was not due to post-translational modification. The results of this and previous investigations established that the two ectoATPases of Li-7A cells are under different regulation.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Bucladesine; Calcium-Transporting ATPases; Carcinoma, Hepatocellular; Cell Line; Cholera Toxin; Colforsin; Cyclic AMP; Dimethyl Sulfoxide; Epidermal Growth Factor; Humans; Hydrocortisone; Kinetics; Liver Neoplasms; Time Factors

In human hepatoma cell line Hep3B/T2, the human heat-shock-inducible gene hsp70 could be induced by insulin. The dose-dependent insulin effect correlates very well with the dissociation constant of the insulin receptor, indicating that the insulin effect is mediated by the insulin receptor. The expression of hsp70 gene was neither significantly induced by growth factors of epidermal and platelet-derived growth factors, nor by tumor promoter, calcium ionophore, cAMP, and glucocorticoids. These results indicate that the induction of expression of hsp70 gene by insulin is very specific and not cell cycle-related. Furthermore, the insulin-induced expression of hsp70 gene is transient, occurring specifically from 4 to 8 h after insulin addition.

Topics: Carcinoma, Hepatocellular; Cycloheximide; DNA; Dose-Response Relationship, Drug; Epidermal Growth Factor; Gene Expression Regulation; Heat-Shock Proteins; Hepatitis B Surface Antigens; Humans; Insulin; Kinetics; Liver Neoplasms; Platelet-Derived Growth Factor; Receptor, Insulin; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured

The culture system of hepatocellular carcinoma (HCC) has been established and the effect of hepatotrophic factors on these cell lines have been studied. During this study the effect of the human epidermal growth factor intensifying anti-tumor action of anti-tumor agents will be evaluated. HCC cell lines (C-HC-4, C-HC-20) show logarithmic growth in the serum-free medium. These growth curves after administration of 1 x 10(-12- -8) M of hEGF were measured, and the existence of the hEGF receptors was evaluated using the ABC method. The growth curve of HC-4 transplanted into nude mice was measured in 4 groups; the control group, the hEGF group, the gamma Interferon (gamma IFN) group, and the hEGF + gamma IFN group. The hEGF enhanced the cell growth of C-HC-4 and C-HC-20, and EGF receptors were confirmed immunohistologically. In the hEGF+ gamma IFN group, growth inhibition seems to be most important. The hEGF may intensify the anti-tumor action of the anti-tumor agents.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Cycle; Cell Line; Epidermal Growth Factor; Humans; Interferon-gamma; Liver Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation; Transplantation, Heterologous; Tumor Cells, Cultured

Data are presented from a comparative research on expression of epidermal growth factor (EGF) receptors and response to EGF of six independently established cell lines derived from human hepatoma. These lines differ in terms of the degree of differentiation, presence of hepatitis B virus (HBV) DNA copies in integrated form and expression of HBV genes. Our results indicate differential expression of membrane EGF receptors and differential response to EGF under serum- and hormone-free culture conditions. Furthermore, a significant difference in affinity could be detected between EGF receptors of the two highly dedifferentiated cell lines (HA22T/VGH and Li7A) whose replication is inhibited by EGF concentrations capable of stimulating more differentiated phenotypes.

Topics: Blotting, Southern; Carcinoma, Hepatocellular; Cell Division; DNA, Viral; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Genes, Viral; Hepatitis B virus; Humans; Liver Neoplasms; Tumor Cells, Cultured

A cDNA encoding transforming growth factor type alpha (TGF alpha) was fused to the 5' end of a gene encoding a modified form of Pseudomonas exotoxin A (PE), which is devoid of the cell recognition domain (domain Ia). The chimeric molecule, termed TGF alpha-PE40, was expressed in Escherichia coli and isolated from the periplasm or inclusion bodies depending on the construction expressed. TGF alpha-PE40 was found to be extremely cytotoxic to cells displaying epidermal growth factor (EGF) receptors. Comparison with a similar molecule in which TGF alpha was placed at the carboxyl end of PE40 demonstrated the importance of the position of the cell recognition element; TGF alpha-PE40 was found to be about 30-fold more cytotoxic to cells bearing EGF receptors than PE40-TGF alpha. In addition, TGF alpha-PE40 was shown to be extremely cytotoxic to a variety of cancer cell lines including liver, ovarian, and colon cancer cell lines, indicating high levels of EGF receptor expression in these cells.

Topics: ADP Ribose Transferases; Bacterial Toxins; Base Sequence; Binding, Competitive; Carcinoma, Hepatocellular; Cell Survival; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Escherichia coli; Exotoxins; Female; Gene Expression; Liver Neoplasms; Molecular Sequence Data; Ovarian Neoplasms; Pseudomonas aeruginosa Exotoxin A; Recombinant Fusion Proteins; Structure-Activity Relationship; Transforming Growth Factor alpha; Transforming Growth Factors; Tumor Cells, Cultured; Virulence Factors

Epidermal growth factor receptor (EGFR) gene expression and growth stimulation of EGF on human hepatoma cells of cell lines BEL-7404 and SMMC-7721 were studied. 125I-EGF binding assay was used to measure the binding characteristics and the amounts of EGFR on these cells. The binding time course and the binding competition assay showed that the binding of 125I-EGF to 7404 cells was saturable and specific. Scatchard analysis of EGF binding curve indicated that 7404 and 7721 cells expressed approximately 1.1 x 10(5) and 0.7 x 10(5) EGFRs per cell with binding affinity (Kd) 2.1 nM and 1.8 nM respectively. Northern hybridization and immunoblotting analysis showed the EGFR gene expression products in 7404 and 7721 cells were 5.6 Kb mRNA and 170 Kilo-dalton glycoprotein. Anchorage-dependent growth of 7404 and 7721 cells was stimulated in the presence of nanogram quantities of EGF in medium containing 10% calf serum or 0.5% calf serum. The factors in serum appeared to act synergitically in stimulating of cell proliferation. EGF also stimulated the anchorage-independent growth of 7404 and 7721 cells in soft agar. The results suggest that EGFR is actively expressed in human hepatoma 7404 and 7721 cells and EGF may be one of the mitogens needed for the growth of hepatoma cells.

Topics: Animals; Carcinoma, Hepatocellular; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Mice; Tumor Cells, Cultured

A cloned human hepatoma cell line (Li-7A), possessing epidermal growth factor (EGF) receptors numbering in the range of 10-20 pmol/10(6) cells, was inhibited in its growth by EGF as well as an antagonist monoclonal antibody (MoAb) to the EGF receptor. The mode of action of the two ligands of EGF receptors appeared to be different as indicated by the following results: 1) EGF induced marked alteration in cell morphology, whereas the antibody did not; 2) cellular protein accumulated in the EGF-treated cells but not in the antibody treated cells; and 3) ectoATPase activities were greatly enhanced in Li-7A cells treated with EGF and cholera toxin but were unaffected in cells treated with antibody and cholera toxin. The last result also suggests that expression of ectoATPase activities is under the regulation of both EGF and cholera toxin. Li-7A cells provide an additional valuable experimental system for the study of EGF action, as well as the interactive effects of EGF and cholera toxin. The enrichment of the ATPase activities in the EGF-cholera toxin-treated cells can be exploited for the detailed study and isolation of these enzymes and elucidation of their physiological functions.

Topics: Adenosine Triphosphatases; Antibodies, Monoclonal; Carcinoma, Hepatocellular; Cell Division; Cholera Toxin; Clone Cells; Epidermal Growth Factor; ErbB Receptors; Humans; Liver Neoplasms

Expression of the epidermal growth factor (EGF) was analyzed in six human hepatocellular carcinoma-derived and one human hepatoblastoma-derived cell line, each of which retained the differentiated phenotype and functions of the parenchymal hepatocyte. The level of receptor expression of each hepatoma cell line was similar to that of the normal human fibroblast, approximately 10(5) molecules per cell. However, NPLC/PRF/5, a subline of the PLC/PRF/5 cell line obtained following reestablishment of a xenograft tumor in vitro, was found to express 4 x 10(6) high-affinity EGF receptor molecules per cell. Proliferation of the NPLC/PRF/5 cell line was inhibited in the presence of nanomolar quantities of ligand. Receptor overexpression was found to result from EGF receptor gene amplification without apparent rearrangement of the EGF receptor coding sequences. Although cell-specific variability in posttranslational processing of EGF receptor N-linked oligosaccharides in the hepatoma cell lines was found, no difference between the receptors in PLC/PRF/5 and NPLC/PRF/5 was observed and no aberrant receptor-related species were detected. EGF receptor gene amplification in the NPLC/PRF/5 cell line is probably a reflection of genome instability and selection of variants with augmented growth potential in limiting concentrations of EGF in vivo. When viewed in this light, EGF receptor overexpression could represent a manifestation of tumor progression in the EGF-responsive hepatocyte.

Topics: Carcinoma, Hepatocellular; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Gene Expression Regulation; Glycoproteins; Glycosylation; Isoelectric Point; Karyotyping; Liver Neoplasms; Molecular Weight; Protein Processing, Post-Translational

A human hepatoma cell line (Li-7A) possesses ectoATPase activity which is activated by either Mg2+ or Ca2+. Both ectoMg2+-ATPase and ectoCa2+-ATPase hydrolyze other nucleoside triphosphates, are inactive with ADP and AMP, and are inhibited by both p-chloromercuriphenyl sulfonate (pCMPS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Different Km values for ATP and pH curves are obtained for ectoMg2+-ATPase and ectoCa2+-ATPase. The specific activities of the two ATPases remain relatively constant through several days of cell growth after an initial decrease. In contrast, the specific activities of the two ATPases, especially the ectoCa2+-ATPase, increases continuously in Li-7A cells cultured in the presence of EGF, cholera toxin, and hydrocortisone. The ATPases of the factor-treated cells are also indiscriminate with respect to nucleoside triphosphate substrates; however, the kinetic constants for substrates are altered when compared to that of the untreated cells. Most strikingly, the sensitivity to inhibitors is greatly reduced. It is concluded that the long-term effect of EGF, cholera toxin, and hydrocortisone on the Li-7A cells is the induction or activation of a new or minor component of the ectoATPases, which is preferentially activated by Ca2+ and insensitive to pCMPS.

Topics: Adenosine Triphosphatases; Calcium; Carcinoma, Hepatocellular; Cholera Toxin; Epidermal Growth Factor; Humans; Hydrocortisone; Liver Neoplasms; Magnesium; Neoplasm Proteins; Substrate Specificity; Tumor Cells, Cultured

A human hepatocellular carcinoma-derived cell line, PLC/PRF/5, was examined for its ability to respond to epidermal growth factor (EGF) exposure with increased phosphatidylinositol 4,5-bisphosphate hydrolysis. Upon addition of EGF (25 ng/ml), a rapid (10-15 s) but transient increase in Ins(1,4,5)P3 levels and large, prolonged (2 min) increases in Ins(1,3,4,5)P4 and Ins(1,3,4)P3 levels were detected. Increases in cytosolic Ca2+ were observed after a 10 to 20 s lag, reaching peak value at 1 min, and remaining elevated for 10 min. The initial burst of cytosolic Ca2+ occurred in the absence of extracellular Ca2+ and probably reflects mobilization of intracellular Ca2+ stores. In cells pretreated with EGTA, the sustained component of the Ca2+ response was not observed. Comparison of the inositol phosphate and Ca2+ responses of PLC/PRF/5 cells to responses reported in other cell types indicates that this cell line is a good model for EGF action in liver.

Topics: Calcium; Carcinoma, Hepatocellular; Chromatography, High Pressure Liquid; Cytosol; Egtazic Acid; Epidermal Growth Factor; Humans; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Liver Neoplasms; Sugar Phosphates; Tumor Cells, Cultured

To identify factors potentially influencing expression of type 1 plasminogen activator inhibitor (PAI-1), we characterized the human tissue-specific distribution of PAI-1 mRNA and the influence of epidermal growth factor (EGF) on expression of steady state levels of PAI-1 mRNA and secretion of PAI-1 by Hep G2 cells. Two species of PAI-1 mRNA (3.2 and 2.2 kilobases) were detected, and the ratio of the two varied among tissues (3 to 5:1) in contrast to the 1:1 ratio detected in Hep G2 cells. Expression of PAI-1 mRNA was inversely related to the distribution of tissue-type plasminogen activator mRNA (2.3 kilobases). Nu-Serum, a growth media supplement, increased steady state levels of PAI-1 mRNA 5-fold within 3 h. Factors responsible were found to be trypsin-sensitive and dialysis-resistant. Antisera to EGF attenuated Nu-Serum-induced increases of PAI-1 mRNA by 57%, suggesting that EGF or EGF homologous peptides contributed to the response. EGF elicited increases of PAI-1 mRNA levels in a dose-dependent manner. Induction was rapid (7-fold at 3 h with 5 ng/ml) and complete within 10 h. The response was not attenuated by cycloheximide (25 micrograms/ml). Factor X and glyceraldehyde-3-phosphate dehydrogenase mRNA did not increase. Increased levels of PAI-1 antigen were detected in conditioned media of Hep G2 cells by 4 h and were maximal at 8 h (6-fold). We conclude that the expression of PAI-1 mRNA is tissue-specific and regulated by epidermal growth factor in Hep G2 cells.

Topics: Carcinoma, Hepatocellular; Cell Line; Epidermal Growth Factor; Factor X; Female; Gene Expression Regulation; Glyceraldehyde-3-Phosphate Dehydrogenases; Glycoproteins; Humans; Kinetics; Liver Neoplasms; Organ Specificity; Plasminogen Activators; Plasminogen Inactivators; RNA, Messenger; Transcription, Genetic

A radioimmunoassay for transforming growth factor alpha (TGF-alpha) using synthetic rat sarcoma transforming growth factor and its rabbit polyclonal antibody has been developed. Using radioimmunoassays, the urinary TGF-alpha and epidermal growth factor (EGF) concentrations in 31 patients with hepatocellular carcinoma (HCC), 15 probable HCC, four metastatic liver cancer, and 33 age, sex-matched healthy controls were determined. For the first time, we have shown that the average TGF-alpha concentration for HCC patients was 21.5 +/- 20.3 micrograms per g creatinine, significantly higher than that of healthy subjects, 4.9 +/- 2.8 micrograms per g creatinine (P less than 0.001). There was no statistical difference in the level of EGF between HCC patients and controls (40.9 +/- 29.3 versus 46.2 +/- 16.6 micrograms per g creatinine; P greater than 0.05). The ratio of EGF/TGF-alpha between HCC patients (3.37 +/- 4.42) and controls (15.5 +/- 13.0) was significantly different (P less than 0.001). Among patients, 65% (20 of 31) of HCC cases and 87% (13 of 15) of probable HCC cases showed a marked elevation of TGF-alpha levels. We found only 16% (five of 31) of HCC cases with increased EGF level. EGF excretion was inversely age related. Serum total protein concentration and alkaline phosphatase activity were positively correlated to EGF concentration (r = 0.522, P less than 0.01 and rt = 0.393, P less than 0.05, respectively). There was no correlation between biochemical functions of liver and TGF-alpha concentration in HCC patients. Our results also suggested that TGF-alpha may be a useful complementary tumor marker for management of patients with clinical manifestation of HCC who have low alpha-fetoprotein levels.

Topics: Adult; alpha-Fetoproteins; Carcinoma, Hepatocellular; Creatinine; Epidermal Growth Factor; Female; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Peptides; Reference Values; Transforming Growth Factors

Topics: Carcinoma, Hepatocellular; Cell Line; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Humans; Liver Neoplasms

Several human cell lines derived from primary cancer of the liver are able to grow under serum-free conditions and produce spreading and growth factors which are released into the culture medium. Since this autocrine growth under hormone-free conditions might play a basic role in malignant transformation, we studied the effect on cell replication and the presence of specific membrane receptors of epidermal growth factor (EGF) and insulin on a dedifferentiated human hepatoma cell line, named HA22T/VGH. Our results point to a similar inhibitory effect on cell replication in the presence of both EGF and insulin, in spite of detecting different affinities of binding.

Topics: Carcinoma, Hepatocellular; Cell Division; Cell Line; DNA, Viral; Epidermal Growth Factor; ErbB Receptors; Growth Hormone; Hepatitis B Surface Antigens; Hepatitis B virus; Humans; Insulin; Liver Neoplasms; Receptor, Insulin

Three preparations known to be angiogenic in vivo and which stimulate production of latent collagenase by cultured bovine capillary endothelial (BCE) cells were tested for their ability to stimulate production of latent collagenase by cultured human umbilical vein endothelial (HUVE) cells. Bovine retinal extract and murine adipocyte-conditioned medium had no effect on production of latent collagenase by HUVE cells at concentrations that were effective in stimulating production of latent collagenase by BCE cells. However, with higher concentrations of bovine retinal extract, production of latent collagenase by HUVE cells was stimulated. Human hepatoma cell sonicate stimulated production of latent collagenase by HUVE cells in a dose-dependent manner. The concentration of human hepatoma cell sonicate which stimulated production of latent collagenase by HUVE cells was lower than the concentration that was effective for the stimulation of production of latent collagenase by BCE cells. Plasminogen activator production by HUVE cells was unaffected by human hepatoma cell sonicate. Varying the concentration of serum in HUVE cultures did not affect the stimulation of latent collagenase production by human hepatoma cell sonicate, suggesting that serum components neither block nor stimulate the action of the collagenase-inducing factor. Although human hepatoma cell sonicate is reported to stimulate endothelial cell multiplication, purified and partially purified endothelial cell mitogens had no effect on production of latent collagenase. Thus, at least two preparations which contain angiogenic activity will stimulate production of latent collagenase by HUVE cells.

Topics: Adipose Tissue; Angiogenesis Inducing Agents; Blood; Carcinoma, Hepatocellular; Cell Survival; Cells, Cultured; Endothelium; Enzyme Induction; Epidermal Growth Factor; Fibroblast Growth Factors; Growth Substances; Humans; Liver Neoplasms; Microbial Collagenase; Mitogens; Plasminogen Activators; Retina; Tetradecanoylphorbol Acetate; Tissue Extracts; Umbilical Veins

We have obtained a cloned cell line (Li-7A) from primary cultures of a human hepatoma xenograft (Li-7). Li-7A was able to grow in the absence of serum. Growth was stimulated 0-3 fold by addition of newborn calf serum, but was inhibited in DME/F12 media containing nine growth factors. The ectoMg2+-ATPase was 1.5-2 fold higher than the ectoCa2+-ATPase activity in cells grown in media with or without serum. In cells grown in media supplemented with the nine factors, the ectoCa2+-ATPase activity exceeded the ectoMg2+-ATPase, and there was also a 5-10 fold increase in its specific activity. Inhibition of growth was due to epidermal growth factor alone. The increased expression of the ectoCa2+-ATPase was absolutely dependent on EGF, but also required hydrocortisone and cholera toxin. The characteristics of Li-7A cells make it a suitable system for studying both the mechanism of action of EGF and plasma membrane ATPases.

Topics: Adenosine Triphosphatases; Carcinoma, Hepatocellular; Cell Division; Cell Line; Cholera Toxin; Clone Cells; Culture Media; Epidermal Growth Factor; Humans; Hydrocortisone; Insulin; Liver Neoplasms; Transferrin

Since methods to disperse and culture hepatocytes were developed 15 years ago, numerous investigations have shown that primary cultures of mature hepatocytes retain most liver functions and respond as well to various hormones as those in vivo. Thus they are the most suitable system in vitro for studies on the liver. Moreover, recently it was found that differentiated hepatocytes in culture can grow under certain conditions and that this growth is regulated not only by several hormones, such as insulin, epidermal growth factor and serum growth factor, but also by a cell membrane factor and proteins in the environmental matrix through cell contact. This article describes the biochemical characterization of regulatory factors for hepatocyte growth and functions and their reciprocal expression. The mechanisms of liver regeneration, differentiation and carcinogenesis and the importance of the tissue architecture for these events are discussed mainly on the basis of our findings.

Topics: Animals; Carcinoma, Hepatocellular; Cell Differentiation; Cell Division; Cell Membrane; Cells, Cultured; Contact Inhibition; DNA Replication; Epidermal Growth Factor; Gene Expression Regulation; Growth Substances; Insulin; Liver; Liver Neoplasms; Liver Regeneration; Proline; Rats; Tryptophan Oxygenase

Topics: Alkaloids; Biological Transport; Carcinogens; Carcinoma, Hepatocellular; Cell Division; Cell Line; Cell Nucleus; Epidermal Growth Factor; Humans; Kinetics; Liver Neoplasms; Lyngbya Toxins

The tumor promoter teleocidin inhibited the proliferation of human HUH hepatoma cells in tissue culture. HUH cells cultured with teleocidin developed elongated cytoplasm. In addition, these biological effects of teleocidin were associated with a remarkable decrease in the amount of cellular fibronectin. The synthesis of structural proteins and the number of cell surface receptors for epidermal growth factor were not altered in cells treated with teleocidin, suggesting that teleocidin acted selectively on some of the cellular constituents such as fibronectin. The present data also suggest a possible relationship between the cellular fibronectin content and the biological characters of HUH cells.

Topics: Alkaloids; Carcinoma, Hepatocellular; Cells, Cultured; Epidermal Growth Factor; Fibronectins; Humans; Liver Neoplasms; Lyngbya Toxins; Receptors, Cell Surface

Epidermal growth factor (EGF) bound specifically to the human hepatoma cell line PLC/PRF/5. Treatment of these cells with nanomolar concentrations of EGF for 4-6 hr resulted in a 2- to 6-fold increase in ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity. 12-O-Tetradecanoylphorbol 13-acetate also produced an increase in enzyme activity in these cells and exhibited an additive effect with EGF. It did not inhibit the binding of 125I-labeled EGF to these cells. The stimulation of enzyme activity by EGF was not inhibited by cycloheximide or actinomycin D, although these agents did cause a significant decrease in enzyme levels when added without EGF. Also, colchicine, chloroquine, ammonium chloride, and methylamine, compounds that inhibit EGF degradation in various cells types, did not interfere with the ability of EGF to elevate enzyme levels in the human hepatoma cells.

Topics: Ammonium Chloride; Carboxy-Lyases; Carcinoma, Hepatocellular; Cell Line; Chloroquine; Colchicine; Cycloheximide; Dactinomycin; Enzyme Induction; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Liver Neoplasms; Ornithine Decarboxylase; Peptides; Receptors, Cell Surface

Protein breakdown in many cell lines is inhibited by growth factors. The response is specific, rapid and additive only at suboptimal concentrations. Transformed cells are typically more sensitive to growth factors than nontransformed cells while the effectiveness of growth factors in human fibroblasts is diminished with senescence. Bovine colostrum has been shown to be an extremely rich source of growth factors as compared to serum.

Topics: Animals; Carcinoma, Hepatocellular; Cattle; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Colostrum; Epidermal Growth Factor; Fibroblast Growth Factors; Fibroblasts; Growth Substances; Humans; Insulin; Liver Neoplasms; Mice; Peptides; Proteins; Somatomedins