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Tumor behavior, including its response to treatments, is influenced by interactions between mesenchymal and malignant cells, as well as their spatial arrangement. To study tumor biology and evaluate anticancer drugs, accurate 3D tumor models are essential. Here, we developed an in vitro biomimetic hepatoma microenvironment model by combining an extracellular matrix (3DM-7721). Initially, the internal grid structure, composed of 10/6 % GelMA/gelatin loaded with SMMC-7721 cells, was printed using 3D bioprinting. The external component consisted of fibroblasts and human umbilical vein endothelial cells loaded with 10/3 % GelMA/gelatin. A control model (3DP-7721) lacked external cell loading. GelMA/gelatin hydrogels provided robust structural support and biocompatibility. The SMMC-7721 cells in the 3DM-7721 model exhibit superior tumor-associated gene expression and proliferation characteristics when compared to the 3DP-7721 model. Furthermore, the 3DM-7721 type exhibited increased resistance to anticancer agents. SMMC-7721 cells in the 3DM-7721 model exhibit significant tumorigenicity in nude mice. The 3DM-7721 model group showed pathological characteristics of malignant tumors, with a high degree of deterioration, and a significant positive correlation between malignant tumor-related gene pathways. This high-fidelity 3DM-7721 tumor microenvironment model is invaluable for studying tumor progression, devising effective treatment strategies, and discovering drugs. STATEMENT OF SIGNIFICANCE., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background: Hepatocellular carcinoma (HCC), a globally common cancer, often presents late and shows high resistance to chemotherapy, resulting in suboptimal treatment efficacy. Components from traditional Chinese medicines have been recognized for their anti-cancer properties., Objective: Exploring the mechanism of Schisandra chinensis lignans and acteoside in suppressing Epithelial-Mesenchymal Transition (EMT) in hepatoma cells through the Extracellular signal-Regulated Kinases (ERK)1/2 pathway and identifying biomarkers, molecular subtypes, and targets via multi-omics for precision oncology., Methods: Proliferation was assessed using cell counting kit-8 (CCK-8) assays, with scratch and transwell assays for evaluating invasion and migration. Flow cytometry quantified apoptosis rates. Expression levels of CCL20, p-ERK1/2, c-Myc, Vimentin, and E-cadherin/N-cadherin were analyzed by real-time PCR and Western blot. Tumor volume was calculated with a specific formula, and growth., Results: The Schisandra chinensis lignans and acteoside combination decreased CCL20 expression, inhibited hepatoma proliferation and migration, and enhanced apoptosis in a dose- and time-dependent manner. Molecular analysis revealed increased E-cadherin and decreased N-cadherin, p-ERK1/2, c-Myc, and Vimentin expression, indicating ERK1/2 pathway modulation. In vivo, treated nude mice showed significantly reduced tumor growth and volume., Conclusion: Schisandra chinensis lignans and acteoside potentially counteract CCL20-induced EMT, invasion, and migration in hepatocellular carcinoma cells via the ERK1/2 pathway, enhancing apoptosis. Multi-omics analysis further aids in pinpointing novel biomarkers for precision cancer therapy., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Ring finger protein 31 (RNF31) has been found to play an important role in tumor immunity. However, the role of RNF31 in liver hepatocellular carcinoma (LIHC) has not been reported. Therefore, we investigated the expression and prognostic value of RNF31 in patients with LIHC and explored its relationship with immune cell infiltration. The Cancer Genome Atlas liver hepatocellular carcinoma (TCGA-LIHC) dataset was downloaded to analyse the impact of RNF31 on the prognosis and immune cell infiltration of LIHC. The Tumor Immune Estimation Resource (TIMER) database was used to analyse the correlation between RNF31 and tumor immune cell infiltration in LIHC. Additionally, we analysed the relationship between RNF31 and tumor necrosis factor (TNF) as well as the interferon-gamma (IFN-γ) signaling pathway. The expression of RNF31 in LIHC was significantly higher than that in normal tissues. Increased RNF31 expression was associated with decreased overall survival (OS) and relapse-free survival (RFS). An increase in RNF31 expression was closely related to the infiltration levels of immune cells (e.g., natural killer (NK) cells, CD8 + T cells, and B cells). RNF31 was also positively correlated with the expression of immune checkpoint genes in LIHC. Moreover, RNF31 may participate in TNF and IFN-γ signaling pathways. In conclusion, RNF31 is a potentially valuable prognostic biomarker in LIHC. RNF31 is also associated with immune cell infiltration in LIHC. RNF31 may be a potential target for immunotherapy of LIHC., (© 2023. The Author(s).)

Background: MicroRNA-421 (miR-421) has been implicated in hepatocellular carcinoma (HCC), but its potential mechanism in HCC remains unclear., Objectives: The study aimed to study the potential mechanism of miR-421 in HCC which is necessary., Methods: The downstream target genes of miR-421 were screened in HCC tissues and cells using miDIP, Targetscan, and starBase databases. Differential analysis, survival analysis, and Pearson correlation analysis were performed between miR-421 and its downstream target genes. Quantitative reverse transcription polymerase chain reaction and western blot were used to assay RNA and protein levels of 4-aminobutyrate aminotransferase (ABAT) and epithelial-mesenchymal transition (EMT)-related proteins. Cell-based assays, including CCK-8, wound healing, transwell, flow cytometry, and metabolic measurements, were implemented to assess proliferation, migration, invasion, cell cycle, and apoptosis of HCC cells with different treatments. Dual-luciferase assay was utilized to detect the targeting relationship between miR-421 and ABAT., Results: miR-421 level was elevated in HCC tissues and cells, and low miR-421 expression hindered phenotype progression of HCC cells. ABAT was identified as a direct target of miR-421 in HCC cells, and miR-421 could inhibit ABAT expression. Rescue assay revealed that miR-421 promoted HCC cell tumorigenesis progress and affected cell metabolic remodeling through down-regulating ABAT., Conclusion: The miR-421/ABAT regulatory axis promoted HCC cell tumorigenesis progress, highlighting its potential as a therapeutic target for HCC., (Copyright: © 2023 Permanyer.)

Background and Aim: The exact place for selective internal radiation therapy (SIRT) in the therapeutic algorithm for hepatocellular carcinoma (HCC) is debated. There are limited data on its indications, efficacy, and safety in Australia., Methods: We performed a multicenter retrospective cohort study of patients undergoing SIRT for HCC in all Sydney hospitals between 2005 and 2019. The primary outcome was overall survival. Secondary outcomes were progression-free survival and adverse events., Results: During the study period, 156 patients underwent SIRT across 10 institutions (mean age 67 years, 81% male). SIRT use progressively increased from 2005 (n = 2), peaking in 2017 (n = 42) before declining (2019: n = 21). Barcelona Clinic Liver Cancer stages at treatment were A (13%), B (33%), C (52%), and D (2%). Forty-four (28%) patients had tumor thrombus. After a median follow-up of 13.9 months, there were 117 deaths. Median overall survival was 15 months (95% confidence interval 11-19). Independent predictors of mortality on multivariable analysis were extent of liver involvement, Barcelona Clinic Liver Cancer stage, baseline ascites, alpha fetoprotein, and model for end-stage liver disease score. Median progression-free survival was 6.0 months (95% confidence interval 5.1-6.9 months). Following SIRT, 11% of patients were downstaged to curative therapy. SIRT-related complications occurred in 17%: radioembolization-induced liver disease (11%), pneumonitis (3%), gastrointestinal ulceration, and cholecystitis (1% each). Baseline ascites predicted for radioembolization-induced liver disease., Conclusion: We present the largest Australian SIRT cohort for HCC. We have identified several factors associated with a poor outcome following SIRT. Patients with early-stage disease had the best survival with some being downstaged to curative therapy., (© 2022 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Background: Currently, the molecular mechanism of the interaction between lncRNAs and microRNAs (miRNAs) and the target of miRNAs in tumor vasculogenic mimicry (VM) formation have not been clarified. Our aim is to study the interaction between lncRNA n339260 and miRNA30e-5p in the formation of VM., Methods: Animal xenografts were established, 104 hepatocellular carcinoma (HCC) patients' frozen tissues were obtained and HCC cells in vitro were used to observe the role of n339260 in HCC progression., Results: In vivo experiment showed lncRNA n339260 promoted tumor growth and VM formation. LncRNA n339260 and miRNA30e-5p were found to be associated with VM formation, metastasis and survival time in HCC patients. In vitro experiment showed that LncRNA n339260 could inhibit miRNA30e-5p expression and TP53INP1 was found to be the downstream targets of miRNA30e-5p. Snail, MMP2, MMP9, VE-cadherin, vimentin and N-cadherin overexpression and the downregulation of TP53INP1 and E-cadherin were observed in HCCLM3 and HepG2 cells overexpressing lncRNA n339260 or in cells with decreased expression of miRNA30e-5p., Conclusion: LncRNA n339260 promotes the development of VM, and lncRNA n339260 may enhance Snail expression by decreasing the expression of miRNA30e-5p, thereby reducing TP53INP1 expression. Therefore, a potential lncRNA n339260- miRNA30e-5p- TP53INP1 regulatory axis was associated with HCC progression., (© 2022. Japanese Society of Gastroenterology.)

CTNNB1 , encoding β-catenin protein, is the most frequently altered proto-oncogene in hepatic neoplasms. In this study, we studied the significance and pathological mechanism of CTNNB1 gain-of-function mutations in hepatocarcinogenesis. Activated β-catenin not only triggered hepatic tumorigenesis but also exacerbated Tp53 deletion or hepatitis B virus infection-mediated liver cancer development in mouse models. Using untargeted metabolomic profiling, we identified boosted de novo pyrimidine synthesis as the major metabolic aberration in β-catenin mutant cell lines and livers. Oncogenic β-catenin transcriptionally stimulated AKT2, which then phosphorylated the rate-limiting de novo pyrimidine synthesis enzyme CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydroorotase) on S1406 and S1859 to potentiate nucleotide synthesis. Moreover, inhibition of β-catenin/AKT2-stimulated pyrimidine synthesis axis preferentially repressed β-catenin mutant cell proliferation and tumor formation. Therefore, β-catenin active mutations are oncogenic in various preclinical liver cancer models. Stimulation of β-catenin/AKT2/CAD signaling cascade on pyrimidine synthesis is an essential and druggable vulnerability for β-catenin mutant liver cancer.

Objectives: This study aimed to characterize current treatment patterns and healthcare resource utilization (HRU) observed among patients with hepatocellular carcinoma (HCC) after the failure of sorafenib in real-world setting in Taiwan., Methods: A chart review was conducted in 130 patients; the inclusion criteria were patients with HCC who were aged 20 years or older and had received systemic therapy or best supportive care after failure of first-line systemic treatment with sorafenib between 2016 and 2018. Anonymized data on patient characteristics, treatment pathways, and survival were abstracted., Results: The mean age of patients was 61.7 years (range 27-84); of these 130 patients, 103 (79%) were male, 81 (62%) had high alpha-fetoprotein (AFP) levels (≥400 ng/mL), and 96 (78.0%) were deceased at the time of data abstraction. After sorafenib therapy, 60 patients (46%) received systemic therapy, including nivolumab monotherapy (42%) and chemotherapy (25%). Oncologist visits at a semiannual per-patient rate of 3.7 (95% confidence interval [CI] 3.4-4.0) and hospitalizations at rate of 1.1 (95% CI 1.0-1.3) were the key contributors to HRU. Semiannual per-patient hospitalization rate was 1.3 (95% CI 1.1-1.5) in the high-AFP group. Median survival from discontinuation of sorafenib was 6.9 months (95% CI 5.9-9.0)., Conclusions: This real-world evidence research on treatment patterns reflected substantial HRU consistent with the severity of HCC, particularly in the high-AFP group. Findings highlighted continuing high mortality in HCC, underlying a need for new treatments that can lengthen survival. Results can inform future evaluations of new HCC treatments that estimate the health economic impact of their adoption in Taiwan., (Copyright © 2022 International Society for Health Economics and Outcomes Research. Published by Elsevier Inc. All rights reserved.)

Hepatocellular carcinoma (HCC) is a prevalent liver malignancy, which ranks third in the cancer-related cause of deaths in worldwide and ninth in the United States. Currently, HCC is typically diagnosed by ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI) scan at its late stage and the survival of HCC patients after diagnosis is usually very poor. Therefore, the development of novel and effective tool for early diagnosis, characterization and staging of HCC patients is of critical importance. Recent studies have demonstrated correlation of HCC with MAGL. In HCC cells, upregulation of MAGL activity enhanced cell invasiveness ability, while pharmacological blockade of MAGL led to significant inhibition of this trend. In this study, we aim to visualize the expression and activity of hepatic MAGL in different HCC cells and HCC patients' samples by taking advantage of positron emission tomography (PET) imaging with our previously developed MAGL radioligand [ 11 C]MAGL-0519. As a result, [ 11 C]MAGL-0519 exhibited higher radioactivity accumulation in HepaG2 and Hepa 1-6 cell lines compared with that of normal liver cells (AML-12 and LX-2), indicating higher MAGL expression levels in these HCC cells. This rationale was then validated by Western blot and immunofluorescent staining analysis. Furthermore, HCC patients' liver sections exhibited significantly increased uptake of [ 11 C]MAGL-0519, which was consistent with the results in cell uptake assays. Taking together, these results provided a biological rationale and built a foundation to use [ 11 C]MAGL-0519 as a potential and effective PET ligand for the diagnosis of HCC., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Aim: Hepatocellular carcinoma (HCC) is considered to be the third leading cause of cancer death. The homologous gene of TP53 is significant in the occurrence and development of cancer. This study explored the relationship between TP53 rs28934571 polymorphism and HCC risk in Guangxi, China. Materials & methods: We first screened the association through bioinformatics. Additionally, a case-control study was performed to further verify the relationship between gene polymorphism and HCC risk after collecting clinical characteristics. Results: Results showed that allele A on TP53 rs28934571 was a risk factor for HCC and mutation from C to A on TP53 rs28934571 would increase the risk of poor prognosis of HCC. Conclusion: Therefore, the study concluded that TP53 rs28934571 may become a diagnostic indicator in judging the prognosis of HCC.

The Na+/K+-ATPase (NKA), has been proposed as a signal transducer involving various pathobiological processes, including tumorigenesis. However, the clinical relevance of NKA in hepatocellular carcinoma (HCC) has not been well studied. This study revealed the upregulation of mRNA of ATP1A1, ATP1B1, and ATP1B3 in HCC using TCGA, ICGC, and GEO database. Subsequently, ATP1B3 was demonstrated as an independent prognostic factor of overall survival (OS) of HCC. To investigate the potential mechanisms of ATP1B3 in HCC, we analyzed the co-expression network using LinkedOmics and found that ATP1B3 co-expressed genes were associated with immune-related biological processes. Furthermore, we found that ATP1B3 was correlated immune cell infiltration and immune-related cytokines expression in HCC. The protein level of ATP1B3 was also validated as a prognostic significance and was correlated with immune infiltration in HCC using two proteomics datasets. Finally, functional analysis revealed that ATP1B3 was increased in HCC cells and tissues, silenced ATP1B3 repressed HCC cell proliferation, migration, and promoted HCC cell apoptosis and epithelial to mesenchymal transition (EMT). In conclusion, these findings proved that ATP1B3 could be an oncogene and it was demonstrated as an independent prognostic factor and correlated with immune infiltration in HCC, revealing new insights into the prognostic role and potential immune regulation of ATP1B3 in HCC progression and provide a novel possible therapeutic strategy for HCC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lu, Cai, Peng, Cheng and Zhang.)

Synergistic photochemical therapy with high performance and weak side effects is of great importance in hepatocellular carcinoma (HCC) treatment, therefore ingenious construct of nano-based therapy agents with accurate drug delivery and high photothermal conversion efficiency is of critical to the cancer therapy. Herein, an organic-inorganic hybrid nanomaterial (MGO@CD-CA-HA) has been constructed successfully by coating the β-cyclodextrin-cholic acid-hyaluronic acid polymer (CD-CA-HA) onto the Fe 3 O 4 -graphene oxide (MGO). The MGO@CD-CA-HA revealed satisfactory multiple-targeted features including the cholic acid supplied hepatic-target, CD44-receptor target of hyaluronic acid and magnetic target of Fe 3 O 4 . Meanwhile, the hydrophobic antitumor drug camptothecin (CPT) was easily loaded by MGO@CD-CA-HA to form the MGO@CD-CA-HA/CPT nanocomposite, and the maximum theoretical adsorption capacity can reach 847.4 mg/g. Based on the facile photothermal response of MGO, the near-infrared radiation (808 nm) induced local hyperthermia was directly generated the apoptosis of tumor cells while triggered the release of CPT. Comparing with other kinds of cancer cells and normal hepatocyte cells, this PCT system provides a significant inhibitory effect for the liver cancer cells in vitro. Furthermore, the synergistic photochemical therapy presented the strong antitumor effect (the tumor inhibition rate > 90 %) in vivo. Thus, this study provided a promising multiple-targeted nanocarrier for chemo-photothermal combination therapy of liver cancer., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Hepatocellular carcinoma (HCC) is a type of common cancer, often accompanied by tumor recurrence and metastasis after surgery with poor prognosis. Therefore, searching into potential biomarkers that can effectively predict the prognosis and progression of HCC is crucial. In this study, we identified 1,981 differentially expressed genes (DEGs) using mRNA expression profiles from the TCGA-LIHC dataset. Subsequently, weighted gene co-expression network analysis found that the turquoise module closely associated with the pathological grade and clinical stage of HCC was identified. Then, from the key genes in the turquoise module and protein-protein interaction network analysis, 13 hub genes significantly related to the prognosis of HCC were screened. Through co-expression and functional enrichment analyses, these 13 hub genes were found to play an important role in mitosis. Finally, we evaluated the relationship between these hub genes and overall survival and disease-free survival through survival analysis. The result indicated that HCC patients with high hub gene expression had a poorer prognosis than HCC patients with low expression. Receiver operating characteristic curves showed that each hub gene could predict the prognosis of HCC patients. In summary, a total of 13 hub genes were identified that play an important role in the progression of HCC, which can be used as potential biomarkers for HCC patients.

Lessons Learned: The combination of ramucirumab (8 mg/kg intravenous, day 1 every 2 weeks) and FOLFOX4 as first-line treatment in patients with advanced hepatocellular carcinoma (HCC) was not sufficiently tolerated. Preliminary efficacy data suggest that the combination may provide clinical benefit to patients with HCC. Dose modification and patient selection should be considered for the future development of ramucirumab plus FOLFOX chemotherapy for advanced HCC., Background: The objective of this study was to investigate the safety, preliminary efficacy, pharmacokinetics, and immunogenicity of ramucirumab plus FOLFOX4 as first-line treatment in patients with advanced hepatocellular carcinoma (HCC)., Methods: Patients received ramucirumab (8 mg/kg) intravenously (IV) on day 1, followed by FOLFOX4 (oxaliplatin 85 mg/m 2 IV on day 1, folinic acid 200 mg/m 2 IV, bolus fluorouracil [5-FU] 400 mg/m 2 , and a continuous infusion of 5-FU 600 mg/m 2 over 22 hours, on days 1 and 2) every 2 weeks. The primary endpoint was to assess the safety and tolerability of the combination therapy., Results: Eight patients (6 men, 2 women) were treated; all eight patients experienced at least one treatment-emergent adverse event (TEAE) of grade ≥3. Dose-limiting toxicities occurred in three patients (37.5%): hepatic hemorrhage (grade 4), blood bilirubin increased (grade 3), and febrile neutropenia (grade 3). Two patients discontinued study because of hepatic hemorrhage (grade 4) and blood bilirubin increase (grade 3). Six deaths occurred due to progressive disease, and no deaths due to TEAEs., Conclusion: There were no unexpected safety findings with ramucirumab plus FOLFOX4 based on the known safety and toxicity of this regimen. The combination was not sufficiently tolerated in patients with advanced HCC at the specified dose and schedule., (© AlphaMed Press; the data published online to support this summary are the property of the authors.)

Objective: This study aims to explore the mechanism of the miR-424-5p/E2F7 axis in hepatocellular carcinoma (HCC) and provide new ideas for targeted therapy of HCC., Methods: Bioinformatics analysis was used to identify the target differentially expressed miRNA in HCC and predict its target gene. qRT-PCR was employed to verify the expression of miR-424-5p and E2F7 mRNA in HCC cells. Western blot was performed to detect the effect of miR-424-5p ectopic expression on the protein expression of E2F7. CCK-8 was used to detect proliferative activity of HCC cells and flow cytometry was carried out for analyzing cell cycle distribution. Dual luciferase reporter assay was conducted to verify the direct targeting relationship between miR-424-5p and E2F7., Results: We observed that miR-424-5p was down-regulated in HCC cells. CCK-8 showed that overexpression of miR-424-5p inhibited cell proliferation, and flow cytometry showed that miR-424-5p could block cells in G0/G1 phase. E2F7 was up-regulated in HCC cells, and E2F7 overexpression could facilitate the proliferative ability of HCC cells and promote the cell cycle progressing from G0/G1 to S phase. Furthermore, dual-luciferase reporter assay indicated that miR-424-5p could directly down-regulate E2F7 expression. Analysis on cell function demonstrated that miR-424-5p inhibited the proliferation of HCC cells and blocked cell cycle at G0/G1 phase by targeting E2F7., Conclusion: Our results proved that E2F7 was a direct target of miR-424-5p, and miR-424-5p could regulate cell cycle and further inhibit the proliferation of HCC cells by targeting E2F7., Competing Interests: The authors have declared that no competing interests exist.

Background: The risk factors for liver metastasis (LM) in patients with non-small-cell lung cancer (NSCLC) remain unknown. Whether LM predicts for the effect of first-line epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) in patients with EGFR-mutant NSCLC needs to be explored., Patients and Methods: A total of 598 NSCLC patients from 3 centers underwent EGFR testing, and 293 had EGFR-mutant NSCLC. Of the 598 NSCLC patients, 99 had LM; 56 patients with EGFR-mutant NSCLC received EGFR-TKIs as first-line therapy., Results: EGFR mutation was not associated with LM in NSCLC patients (relative ratio, 1.305, P = .261). In the EGFR-mutant group that received first-line EGFR-TKIs, patients with LM had shorter progression-free survival (PFS; 7.5 vs. 11.8 months; P = .0003) and overall survival (OS; 20.8 vs. 30.6 months; P = .0190) than patients without LM. The significant difference in PFS was observed in both patients with EGFR exon 19 deletion (19del) and Leu858Arg mutation (L858R). However, patients with EGFR 19del and LM showed marginally significantly shorter OS (P = .0531) and patients with EGFR L858R and LM had OS similar to that of patients without LM (P = .1883). Regardless of EGFR status, patients with LM who received first-line chemotherapy had PFS and OS similar to those of patients without LM. Univariate analyses identified only never smoking (hazard ratio, 0.536; P = .012) was significantly associated with better OS for patients with NSCLC and LM., Conclusion: EGFR mutation is not an independent risk factor for LM in NSCLC patients. However, the presence of LM is a negative predictive factor for first-line EGFR-TKI therapy for patients with EGFR-mutant NSCLC., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Vasculogenic mimicry (VM) is a process by which aggressive tumor cells generate non-endothelial cell-lined channels in malignant tumors including hepatocellular carcinoma (HCC). It has provided new insights into tumor behavior and has surfaced as a potential target for drug therapy. The molecular events underlying the process of VM formation are still poorly understood. In this study, we attempted to elucidate the relationship between Notch4 and VM formation in HCC. An effective siRNA lentiviral vector targeting Notch4 was constructed and transfected into Bel7402, a HCC cell line. VM networks were observed with a microscope in a 3 dimensional cell culture system. Cell migration and invasion were evaluated using wound healing and transwell assays. Matrix metalloproteinases (MMPs) activity was detected by gelatin zymography. Furthermore, the role of Notch4 inhibition in Bel7402 cells in vivo was examined in subcutaneous xenograft tumor model of mice. The results showed that downregulation of Notch4 destroyed VM network formation and inhibited migration and invasion of tumor cells in vitro (P<0.05). In vivo, tumor growth was also inhibited in subcutaneous xenograft model (P<0.05). The potential mechanisms might be related with down-regulation of MT1-MMP, MMP-2, MMP-9 expression and inhibition of the activation of MMP2 and MMP9. These results indicated that Notch4 may play an important role in VM formation and tumor invasion in HCC. Related molecular pathways may be used as novel therapeutic targets for HCC antiangiogenesis therapy.

Purpose: REACH investigated second-line ramucirumab therapy for advanced hepatocellular carcinoma., Results: Median overall survival was 8.2 months for ramucirumab and 6.9 months for placebo (HR, 0.835; 95% CI, 0.634-1.100; p = 0.2046) for East Asians, and 10.1 months for ramucirumab and 8.0 months for placebo (HR, 0.895; 95% CI, 0.690-1.161; p = 0.4023) for non-East Asians. Median overall survival in patients with baseline alpha-fetoprotein ≥ 400 ng/mL was 7.8 months for ramucirumab and 4.2 months for placebo (HR, 0.749; 95% CI, 0.519-1.082; p = 0.1213) for East Asians (n = 139), and 8.2 months for ramucirumab and 4.5 months for placebo (HR, 0.579; 95% CI, 0.371-0.904; p = 0.0149) for non-East Asians (n = 111). The most common grade ≥ 3 treatment-emergent adverse events in East Asians and non-East Asians included hypertension and malignant neoplasm progression., Materials and Methods: A post-hoc analysis of East Asians (N = 252) and non-East Asians (N = 313) in the intent-to-treat population was performed., Conclusions: In East Asians and non-East Asians, ramucirumab did not significantly prolong overall survival. In patients with baseline alpha-fetoprotein ≥ 400 ng/mL, a potentially larger survival benefit was observed in both subgroups. Safety for East Asians was similar to non-East Asians.

Aims: Hepatocellular carcinoma (HCC) is the most common liver cancer worldwide. IFN-α has been used in clinics as a potential therapeutic strategy to treat HCC. In spite of the therapeutic effects, IFN-α caused many side effects due to its short half-life and high dose. Here, we aim to detect the anti-tumor effect of a novel gene delivery system - IFN-α2b gene-modified human bone marrow mesenchymal stem cells (BMSCs) in HCC., Main Methods: Two HCC cell lines, HepG2 and Huh7 were used in the current study. The secretion of IFN-α2b in the BMSC cultured conditioned media (CM) was measured by ELISA. The cell cycle was determined by flow cytometry. The Xenografted NOD/SCID mouse tumor model was generated by subcutaneous inoculation with HepG2 cells., Key Findings: We found that the IFN-α2b-modified BMSC (BMSC/IFN-α2b) could express IFN-α2b stably. The CM from BMSC/IFN-α2b inhibited the proliferation of HCC cells with a much lower growth rate compared with BMSC/vector-CM or DMEM culture group. We further demonstrated that the population of G2/M phase was higher in BMSC/IFN-α2b-CM treated cells than the other two groups. In addition, BMSC/IFN-α2b could significantly inhibit tumor growth in NOD/SCID mice. Moreover, we found that BMSC/IFN-α2b-CM could significantly decrease the mRNA and protein levels of Notch signaling molecules of HCC in vitro and in vivo., Significance: Our data demonstrated that BMSC/IFN-α2b could significantly inhibit HCC cell growth through negatively regulating the Notch signaling, which suggested that IFN-α2b-modified BMSC may be used as an effective therapeutic strategy for hepatomas., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background and Aims: To clarify the role of miR-23a in the onset and development of hepatocarcinoma on the cellular, genetic and molecular levels., Patients and Methods: Seventy-eight patients were included after hepatectomy. Relationships between the clinical pathological factors of tumor and paracancerous tissues were analyzed. Risk factors of overall and recurrence-free survival rates were subject to multi-variable analysis. Tissues were sequenced by digital miRNA expression profiling, and new miRNA was subject to target gene prediction., Results: miR-23a expression was correlated with the stage of the TNM Classification of Malignant Tumours most significantly, followed by tumor size (P=0.041 and 0.047). High miR-23a, vascular invasion, tumor size≥7cm, tumor capsule and late pathological stage were the risk factors of overall survival rate, and those of recurrence-free survival rate also included alpha-fetoprotein level≥200μg/L and multiple tumors. Compared with normal hepatic cell line L-02, the miR-23a expression levels in tumor cell lines SMMC-7721 and HepG2 were up-regulated and down-regulated respectively. Transfecting miR-23a inhibitor suppressed cell growth. Apoptotic rates of the control and those transfected with inhibitor-NC and miR-23a inhibitor for 48h were similar., Conclusion: High miR-23a expression is the independent prognostic factor of overall and recurrence-free survival rates, and miR-23a may be involved in the onset of hepatocarcinoma as an oncogene., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Hepatocellular carcinoma (HCC) is a prevalent primary liver cancer that is derived from hepatocytes and is characterised by high mortality rate and poor prognosis. While HCC is driven by cumulative changes in the hepatocyte genome, it is increasingly recognised that the liver microenvironment plays a pivotal role in HCC propensity, progression and treatment response. The microenvironmental stimuli that have been recognised as being involved in HCC pathogenesis are diverse and include intrahepatic cell subpopulations, such as immune and stellate cells, pathogens, such as hepatitis viruses, and non-cellular factors, such as abnormal extracellular matrix (ECM) and tissue hypoxia. Recently, a number of novel environmental influences have been shown to have an equally dramatic, but previously unrecognized, role in HCC progression. Novel aspects, including diet, gastrointestinal tract (GIT) microflora and circulating microvesicles, are now being recognized as increasingly important in HCC pathogenesis. This review will outline aspects of the HCC microenvironment, including the potential role of GIT microflora and microvesicles, in providing new insights into tumourigenesis and identifying potential novel targets in the treatment of HCC.

Reduction-sensitive shell-sheddable glyco-nanoparticles were designed and developed based on poly(ε-caprolactone)-graft-SS-lactobionic acid (PCL-g-SS-LBA) copolymer for efficient hepatoma-targeting delivery of doxorubicin (DOX). PCL-g-SS-LBA was prepared by ring-opening copolymerization of ε-caprolactone and pyridyl disulfide carbonate followed by postpolymerization modification with thiolated lactobionic acid (LBA-SH) via thiol-disulfide exchange reaction. The dynamic light scattering (DLS) and transmission electron microscopy (TEM) showed that PCL-g-SS-LBA was self-assembled into monodisperse nanoparticles (SS-GNs) with a mean diameter of about 80 nm. SS-GNs while remaining stable under physiological conditions (37 °C, pH 7.4) were prone to rapid shell-shedding and aggregation in the presence of 10 mM dithiothreitol (DTT). DOX was loaded into SS-GNs with a decent loading content of 12.0 wt %. Notably, in vitro release studies revealed that about 80.3% DOX was released from DOX-loaded SS-GNs in 24 h under a reductive condition while low drug release (<21%) was observed for DOX-loaded PCL-g-LBA nanoparticles (reduction-insensitive control) under otherwise the same condition and for DOX-loaded SS-GNs under a nonreductive condition. The flow cytometry and confocal microscopy observations indicated that SS-GNs were efficiently taken up by asialoglycoprotein receptor (ASGP-R)-overexpressing HepG2 cells likely via a receptor-mediated endocytosis mechanism and DOX was released into the nuclei of cells following 4 h incubation. MTT assays showed that DOX-loaded SS-GNs exhibited a high antitumor activity toward HepG2 cells, which was comparable to free DOX and about 18-fold higher than their reduction-insensitive counterparts, while blank SS-GNs were nontoxic up to a tested concentration of 1.0 mg/mL. These shell-sheddable glyco-nanoparticles are promising for hepatoma-targeting chemotherapy.

The therapeutic performance of biodegradable micellar drugs is far from optimal due to existing challenges like poor tumor cell uptake and intracellular drug release. Here, we report on ligand-directed reduction-sensitive shell-sheddable biodegradable micelles based on poly(ethylene glycol)-poly(ε-caprolactone) (PEG-PCL) copolymer actively delivering doxorubicin (DOX) into the nuclei of target cancer cells, inducing superb in vitro antitumor effects. The micelles were constructed from PEG-SS-PCL and galactose-PEG-PCL (Gal-PEG-PCL) block copolymers, in which Gal-PEG-PCL was designed with a longer PEG than that in PEG-SS-PCL (6.0 vs 5.0 kDa) to fully expose Gal ligands onto the surface of micelles for effective targeting to hepatocellular carcinoma cells. PEG-SS-PCL combining with 10 or 20 wt % of Gal-PEG-PCL formed uniform micelles with average sizes of 56.1 and 58.2 nm (denoted as PEG-SS-PCL/Gal10 and PEG-SS-PCL/Gal20, respectively). The in vitro release studies showed that about 81.1 and 75.0% DOX was released in 12 h from PEG-SS-PCL/Gal10 and PEG-SS-PCL/Gal20 micelles under a reducing condition containing 10 mM dithiothreitol (DTT). In contrast, minimal DOX release (<12%) was observed for PEG-SS-PCL/Gal10 and PEG-SS-PCL/Gal20 micelles under nonreducing conditions as well as for reduction-insensitive Gal-PEG-PCL and PEG-PCL/Gal20 micelles in the presence of 10 mM DTT. MTT assays in HeLa and HepG2 cells showed that DOX-loaded PEG-SS-PCL/Gal20 micelles exhibited apparent targetability and significantly enhanced antitumor efficacy toward asialoglycoprotein receptor (ASGP-R)-overexpressing HepG2 cells with a particularly low half maximal inhibitory concentration (IC50) of 1.58 μg DOX equiv/mL, which was comparable to free DOX and approximately six times lower than that for nontargeting PEG-SS-PCL counterparts under otherwise the same conditions. Interestingly, confocal microscopy observations using FITC-labeled PEG-SS-PCL/Gal20 micelles showed that DOX was efficiently delivered and released into the nuclei of HepG2 cells in 8 h. Flow cytometry revealed that cellular DOX level in HepG2 cells treated with DOX-loaded PEG-SS-PCL/Gal20 micelles was much greater than that with reduction-insensitive PEG-PCL/Gal20 and nontargeting PEG-SS-PCL controls, signifying the importance of combining shell-shedding and active targeting. Ligand-directed, reduction-sensitive, shell-sheddable, and biodegradable micelles have emerged as a versatile and potent platform for targeted cancer chemotherapy.

Aims: To study the expression of adhesion molecules in human liver and their possible roles during hepatocarcinogenesis., Methods and Results: The expression of adhesion molecules in normal liver tissues, benign including probable premalignant lesions and malignant lesions was systematically investigated by immunohistochemistry and Western blotting. In normal liver, both hepatocytes and bile duct cells expressed symplekin, desmoglein 1/2, desmocollin 2, desmoplakin and plakophilin 2, but did not express desmocollin 1/3 or plakophilin 1. In benign hepatocyte lesions, expression of the adherens junctions and desmosomes was uniform and slightly increased, but symplekin appeared to show reduced expression in dysplastic lesions. In hepatocellular carcinoma (HCC), the expression of adhesion molecules was often heterogeneous and of abnormal location. Tumour cells with an abnormal distribution or loss of adhesion molecules showed an apolar arrangement of tissue architecture. The expression levels of the adhesion molecules correlated with the differentiation grades of HCC cells., Conclusions: The decreased expression of symplekin may be an early step in the transformation of hepatocytes, whereas alteration of the expression of adherens junctions and desmosomes may indicate more serious changes.

Plasminogen kringle 5 (K5), a proteolytic fragment of plasminogen, is an endogenous angiogenic inhibitor. We have previously shown that K5 inhibits ischemia-induced retinal neovascularization in a rat model. However, its anti-angiogenic potential and application in the treatment of neoplastic diseases have not been well investigated. Our present study was designed to test its effect on the neovascularization and growth of hepatocellular carcinoma, a typical hypervascular tumor. Recombinant human K5 was expressed in E. coli and purified by affinity chromatography. K5 inhibited proliferation and induced apoptosis of primary endothelial cells in dose-dependent manner, but no effect on pericytes from the same origin of endothelial cells, which suggested an endothelial cell-specific inhibition. Moreover, K5 had no effect on the proliferation and apoptosis of mouse HepA and human Bel7402 hepatoma cell lines even in the enhanced concentration range, which suggested K5 having no direct effect on tumor cells. Ventral injection of K5 significantly suppressed the tumor growth in graphed hepatocarcinoma mice model, which was established by injection of mouse HepA hepatoma cells. In xenografted hepatocarcinoma athymic mice model, which mimicked human tumors by injection of human Bel7402 hepatoma cells, K5 significantly suppressed the tumor growth. An average of 68% suppression of primary tumor growth was observed in the K5-treated mice compared with control group. K5 also inhibited intratumoral neovascularization in the two cancer models determined by micro vessel density (MVD) analysis. Injection of K5 significantly induced the cleavage of pro-caspase-3 in tumor tissues of grafted mouse model, which suggested K5 also induced apoptosis of tumor tissues and the decreased intratumoral microvascular density in K5 treated group may correlate with K5-induced endothelial cell apoptosis. These results suggest that tumor growth suppression of K5 depends on its anti-angiogenic activity and K5 could have therapeutic potential in hepatocellular carcinoma.

Objective: To elucidate the relationship between expression of p16 and cyclinD1 proteins and evaluate the role of p16 gene exon 2 mutation in hepatocellular carcinogenesis., Methods: Streptavidin-peroxidase conjugated method(SP) was used to detect expressions of p16 and cyclinD1 proteins in 44 cases of hepatocellular carcinoma(HCC), and polymerase chain reaction single-strand conformation polymorphism analysis(PCR-SSCP) to detect p16 gene mutation in exon 2 in 12 cases of HCC and liver tissues adjacent to HCC., Results: Expression rate and positive signal intensity of p16 protein in HCC were significantly lower(P < 0.01) and those of cyclinD1 protein in HCC were significantly higher (P < 0.05) than those in pericarcinomatous tissues. Of 12 fresh HCC tissues, p16 gene mutation in exon 2 was found in 2 cases, whereas that was not found in pericarcinomatous tissues., Conclusions: 1. Inactivation or/ and deletion of p16 protein may be one of important reasons which result in proliferation unbalance of cells. 2. p16 gene mutation in exon 2 presents in HCC, but it does not frequently occur in Chinese hepatocarcinogenesis. 3. p16 gene abnormality and cyclinD1 over expression may coact in HCC.

Objective: To investigate hepatocarcinogenesis by detecting the effect of HCV NS3 protein on expression of inducible nitric oxide synthase (iNOS) mRNA in hepatocellular carcinoma (HCC) and pericarcinomatous liver tissues(PCLT)., Methods: The expression of HCV NS3 protein and iNOSmRNA was detected by immunohistochemical technique (SP method) and in situ hybridization in specimens of HCC and PCLT from 52 patients with HBV(-). The relationship between the positive rate and the signal strength was tested statistically., Results: The positive rate of HCV NS3 protein in HCC was lower (46.2% vs. 67.3%), and signal strength was weaker than those in PCLT. The signal strength of HCV NS3 protein was correlated with the degree of carcinomatous cells differentiation in HCC (P < 0.01). The positive rates (80.8% and 90.4%) of iNOSmRNA in HCC and PCLT had no statistical difference. Signal intensity of iNOSmRNA in HCC was weaker than that in PCLT. The higher HCVNS3 protein expression level was, the stronger iNOSmRNA signals in PCLT (P < 0.01). In particular, the localization and patterns of HCV NS3 protein were much similar to those of iNOSmRNA in the PCLT of some patients., Conclusions: HCV NS3 protein may exert its hepatocarcinogenic effect in the early stage on host cells by increasing product of nitric oxide, which may bring about transformation of hepatocytes.

Objective: This paper was to evaluate the role of p53 mutation, and p53 and MDM2 proteins expression in hepatocarcinogenesis., Methods: Using streptavidin-peroxidase conjugation method (SP), the expression of p53 and MDM2 proteins was observed in 61 cases of primary hepatocarcinomas (HCC) and 59 cases of corresponding paracancerous tissue, among which p53 mutations in exons 5-8 were detected in 21 cases by polymerase chain reaction single-strand confirmation polymorphism analysis (PCR-SSCP)., Results: Positive nuclear p53 and MDM2 immunostainings were demonstrated in 57.38% (35/61) and 26.23% (16/61) of HCC, and 1.69% (1/59) and 3.39% (2/59) of corresponding paracancerous tissue, respectively. The expressions of p53 and MDM2 proteins in HCCs were significantly higher than those in paracancerous tissues (P < 0.01). The expressions of p53 and MDM2 were not significantly correlated (P > 0.05). There were 42.86% (9/21) mutations in exon 7 of p53 gene and no mutation was observed in exons 5, 6, 8 in HCCs and in paracancerous tissues. In cases of p53 mutations, there were 66.67% (6/9) of p53 overexpression and 11.11% (1/9) of overexpression of both p53 and MDM2. MDM2 overexpression also appeared in 25% (3/12) of cases without mutation., Conclusions: Mutation of p53 gene and functional inactivation of p53 resulting from MDM2 overexpression play an important role in carcinogenesis of HCC. It is possible that p53 mutations and MDM2 overexpression induced by other mechanisms are involved in carcinogenesis of HCC.

Aim: To study the effect of phosphorylation of MAPK and Stat3 and the expression of c-fos and c-jun proteins on hepatocellular carcinogenesis and their clinical significance., Methods: SP immunohistochemistry was used to detect the expression of p42/44(MAPK), p-Stat3, c-fos and c-jun proteins in 55 hepatocellular carcinomas (HCC) and their surrounding liver tissues., Results: The positive rates and expression levels of p42/44(MAPK), p-Stat3, c-fos and c-jun proteins in HCCs were significantly higher than those in pericarcinomatous liver tissues (PCLT). A positive correlation was observed between the expression of p42/44(MAPK) and c-fos proteins,and between p-Stat3 and c-jun but there was no significant correlation between p42/44(MAPK) and p-Stat3 in HCCs and their surrounding liver tissues., Conclusion: The abnormalities of Ras/Raf/MAPK and JAKs/Stat3 cascade reaction may contribute to malignant transformation of hepatocytes. Hepatocytes which are positive for p42/44(MAPK), c-fos or c-jun proteins may be potential malignant pre-cancerous cells. Activation of MAPK and Stat3 proteins may be an early event in hepatocellular carcinogenesis.

The relationship between vascular endothelial growth factor (VEGF) expression and microvessel density was studied with immunohistochemical method in hepatocellular carcinoma (HCC) and pericarcinomatous liver tissue. The positive rate of VEGF in HCCs was significantly lower than in surrounding liver tissues (66.7% vs. 85.4%, P < 0.05). There was no significant difference between HCC and pericarcinomatous liver tissue on expressive intensity of VEGF. The positive signal of VEGF was mainly localized in cytoplasma of cancer cells, pericarcinomatous hepatocytes, and vascular endothelial cells. The microvessel density in HCC was higher than in pericarcinomatous liver tissue and closely correlated to differentiated degree of cancer cells (rs = 0.5870; rs = 0.8235). The poorer cancer cell differentiation, the higher microvessel density. The results suggest that VEGF may not be the sole factor that stimulates angiogenesis in HCC genesis and development. To detect microvessel density in judging prognosis and biological behavior of HCC is more important than that of VEGF.

In situ apoptosis labelling was used for detecting apoptotic cells, and immunohistochemistry for p53, bcl-2 proteins and proliferation cell nuclear antigen(PCNA) in hepatocellular carcinoma(HCC) and liver cirrhosis tissues. The results were that in HCC, the number of apoptotic cells was higher, the density of proliferation cells lower, and expressions of p53 and bcl-2 protein were stronger than that in liver cirrhosis, and they were related to differentiation degree of HCC. The data indicate that overexpression of bcl-2 and mutant p53 proteins, which causes imbalance between cell proliferation and apoptosis, may bring about genesis and development of HCC by selecting proliferation of cells.

Genotypes of hepatitis C virus(HCV) were detected by PCR using type-specific primer in 50 patients' hepatocellular carcinoma(HCC) tissue. The results showed that in the 50 HCC specimens, 30(60%) and 3(6%) were infected with the HCV type II and III, 5(10%) and 8(16%) with type II + III and type II + IV, 2(4%) with type II + I + III in combination, respectively. 2 cases were negative for HCV. These data suggest that HCV type II may be a predominant genotype related to hepatocarcinogenesis in Hunan Province, some cases of HCC may result from coinfection of HCV type II and other genotypes, and only few HCC be separately caused by infection of HCV type III.

Expression of p21 and c-myc protein in hepatocellular carcinomas and their surrounding liver tissue was detected on serial sections by immunohistochemical method. The results showed that the positive rates of p21 expression were 53.3% (16/30) and 96.7% (29/30) in hepatocellular carcinomas and pericarcinomatous liver tissue, and 40% (12/30) and 86.7% (26/30) and 86.7% (26/30) for c-myc protein expression respectively. Their incidences in pericarcinomatous liver tissue were higher than that in cancer tissue (P < 0.01). The patterns of p21 and c-myc protein in cells were cytoplasm, membrane and/or nuclear types. Their expression was more intensive in pericarcinomatous hepatocytes, especially in liver cirrhosis nodes. The results indicate that abnormal activiation and expression of oncogene ras and c-myc may be related to hepatocellular carcinogenesis.