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Metabolites are important indicators of cancer and mutations in genes involved in amino acid metabolism may influence tumorigenesis. Immunotherapy is an effective cancer treatment option; however, its relationship with amino acid metabolism has not been reported. In this study, RNA-seq data for 371 liver cancer patients were acquired from TCGA and used as the training set. Data for 231 liver cancer patients were obtained from ICGC and used as the validation set to establish a gene signature for predicting liver cancer overall survival outcomes and immunotherapeutic responses. Four reliable groups based on 132 amino acid metabolism-related DEGs were obtained by consistent clustering of 371 HCC patients and a four-gene signature for prediction of liver cancer survival outcomes was developed. Our data show that in different clinical groups, the overall survival outcomes in the high-risk group were markedly low relative to the low-risk group. Univariate and multivariate analyses revealed that the characteristics of the 4-gene signature were independent prognostic factors for liver cancer. The ROC curve revealed that the risk characteristic is an efficient predictor for 1-, 2-, and 3-year HCC survival outcomes. The GSVA and KEGG pathway analyses revealed that high-risk score tumors were associated with all aspects of the degree of malignancy in liver cancer. There were more mutant genes and greater immune infiltrations in the high-risk groups. Assessment of the three immunotherapeutic cohorts established that low-risk score patients significantly benefited from immunotherapy. Then, we established a prognostic nomogram based on the TCGA cohort. In conclusion, the 4-gene signature is a reliable diagnostic marker and predictor for immunotherapeutic efficacy., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Ethnopharmacological Relevance: Lobelia chinensis Lour. (LCL) is a common herb used for clearing heat and detoxifying, and it has antitumor activity. Quercetin is one of its important components, which may play an important role in the treatment of hepatocellular carcinoma (HCC)., Aim of the Study: To study the active ingredients of LCL, their mechanism of action on HCC, and lay the foundations for the development of new drugs for the treatment of HCC., Materials and Methods: Network pharmacology was used to examine the probable active ingredients and mechanisms of action of LCL in HCC treatment. Based on an oral bioavailability of ≥30% and a drug-likeness index of ≥0.18, relevant compounds were selected from the Traditional Chinese Medicine Systems Pharmacology database and TCM Database@Taiwan. HCC-related targets were identified using gene cards and the Online Mendelian Inheritance in Man (OMIM) database. A Venn diagram was created to assess the relationship between the intersection of disease and medication targets by creating a protein-protein interaction network, and the hub targets were selected by topology. Gene Ontology enrichment analyses were performed using the DAVID tool. Finally, in vivo and in vitro experiments (qRT-PCR, western blotting, hematoxylin and eosin staining, transwell assays, scratch tests, and flow cytometry assays) verified that LCL demonstrated notable therapeutic effects on HCC., Results: In total, 16 bioactive LCL compounds met the screening criteria. The 30 most important LCL therapeutic target genes were identified. Of these, AKT1 and MAPK1 were the most important target genes, and the AKT signaling pathway was identified as the key pathway. Transwell and scratch assays showed that LCL prevented cell migration, and flow cytometry tests revealed that the LCL-treated group showed a considerably higher rate of apoptosis than the control group. LCL reduced tumor formation in mice in vivo, and Western blot analysis of tumor tissues treated with LCL indicated variations in PTEN, p-MAPK and p-AKT1 levels. The results show that LCL may inhibit the progression of HCC through the PTEN/AKT signaling pathway to achieve the goal of treating HCC., Conclusion: LCL is a broad-spectrum anticancer agent. These findings reveal potential treatment targets and strategies for preventing the spread of cancer, which could aid in screening potential traditional Chinese medicine for anticancer and clarifying their mechanisms., 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 © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Hyperlipidemia is a common complication after liver transplantation (LT) and develops mostly in the early posttransplant period. Recently, some studies have reported a positive correlation between hyperlipidemia and favorable prognosis in patients with hepatocellular carcinoma (HCC) undergoing hepatectomy. This study aimed to evaluate the possibility of predicting prognosis in HCC patients receiving LT by early posttransplant dyslipidemia., Methods: From January 2015 to December 2017, a total of 806 HCC patients from China Liver Transplant Registry database were retrospectively enrolled. The prognostic relevance of early posttransplant hypertriglyceridemia or hypercholesterolemia was examined using survival analysis, and subgroup analysis was implemented based on LT criteria., Results: Early posttransplant hypercholesterolemia (EPHC) was independently inversely associated with the risk of recurrence [hazard ratio (HR) = 0.630; P = 0.022], but was not significantly correlated with the mortality. However, early posttransplant hypertriglyceridemia was not related to prognosis. Intriguingly, with further classification, we found that borderline EPHC (B-EPHC), instead of significant EPHC, was a predictor of lower risk for both recurrence (HR = 0.504; P = 0.006) and mortality (HR = 0.511; P = 0.023). Compared with non-EPHC patients, B-EPHC patients achieved significantly superior 1-year and 3-year tumor-free survival (89.6% and 83.7% vs. 83.8% and 72.7% respectively; P = 0.023), and 1-year and 3-year overall survival (95.8% and 84.8% vs. 94.6% and 77.6% respectively; P = 0.039). In the subgroup analysis, B-EPHC remained an independent predictor of better prognosis in patients beyond Milan criteria and those within Hangzhou criteria; whereas there was no significant relationship between B-EPHC and prognosis in patients within Milan criteria and those beyond Hangzhou criteria. More interestingly, patients beyond Milan criteria but within Hangzhou criteria were identified as the crucial subpopulation who benefited from B-EPHC (recurrence HR = 0.306, P = 0.011; mortality HR = 0.325, P = 0.031)., Conclusions: B-EPHC could assist transplant teams in dynamically evaluating prognosis after LT for HCC as a postoperative non-oncological biomarker, especially in patients beyond Milan criteria but within Hangzhou criteria., (Copyright © 2022. Published by Elsevier B.V.)

Angiogenesis is vital for tumor growth and metastasis. Emerging evidence suggests that metabolic reprogramming in endothelial cells (EC) may affect angiogenesis. Here, we showed that multiple regulators in the fructose metabolism pathway, especially fructose transporter SLC2A5 and fructose-metabolizing enzyme ketohexokinase (KHK), were upregulated in tumor endothelial cells from hepatocellular carcinoma (HCC). In mouse models with hepatoma xenografts or with Myc/sgp53-induced liver cancer, dietary fructose enhanced tumor angiogenesis, tumor growth, and metastasis, which could be attenuated by treatment with an inhibitor of SLC2A5. Furthermore, vessel growth was substantially increased in fructose-containing Matrigel compared with PBS-Matrigel. Inhibiting fructose metabolism in EC cells in vivo using EC-targeted nanoparticles loaded with siRNA against KHK significantly abolished fructose-induced tumor angiogenesis. Fructose treatment promoted the proliferation, migration, and tube formation of ECs and stimulated mitochondrial respiration and ATP production. Elevated fructose metabolism activated AMPK to fuel mitochondrial respiration, resulting in enhanced EC migration. Fructose metabolism was increased under hypoxic conditions as a result of HIF1α-mediated upregulation of multiple genes in the fructose metabolism pathway. These findings highlight the significance of fructose metabolism in ECs for promoting tumor angiogenesis. Restricting fructose intake or targeting fructose metabolism is a potential strategy to reduce angiogenesis and suppress tumor growth., Significance: Fructose metabolism in endothelial cells fuels mitochondrial respiration to stimulate tumor angiogenesis, revealing fructose metabolism as a therapeutic target and fructose restriction as a dietary intervention for treating cancer., (©2023 American Association for Cancer Research.)

Objective: To construct a predictive model for pain in patients undergoing hepatic arterial chemoembolization (TACE) in interventional operating room., Methods: Through literature review and expert interviews, a questionnaire was prepared for the assessment of pain factors in patients with hepatic arterial chemoembolization. A prospective cohort study was used to select 228 patients with hepatic arterial chemoembolization in a tertiary and first-class hospital. The data of the patients in the pain group and the non-pain group were compared, and a rapid screening prediction model was constructed by univariate analysis and logistic regression analysis, and its prediction effect was tested., Results: Tumor size, liver cancer stage, and chemoembolization with drug-loaded microspheres and pirarubicin hydrochloride (THP) mixed with lipiodol were independent predictors of pain in patients after hepatic arterial chemoembolization. Finally, the pain prediction model after TACE was obtained. The results of Hosmer-Lemeshow test showed that the model fit was good (χ 2  = 13.540, p = 0.095). The area under the receiver operating characteristic curve was 0.798, p < 0.001., Conclusion: The rapid screening and prediction model of pain in patients undergoing hepatic arterial chemoembolization has certain efficacy, which is helpful for clinical screening of patients with high risk of pain, and can provide reference for predictive pain management decision-making., (© 2023. The Author(s).)

Chronic hepatitis B virus (HBV) infection can lead to fibrosis, liver cirrhosis, and primary hepatocellular carcinoma. Investigating host factors that regulate HBV replication helps to identify antiviral targets. In the current study, we identified Nicotinamide N-Methyltransferase gene (NNMT) as a novel factor that regulates HBV transcription. NNMT is up-regulated at both the mRNA and protein levels in HepG2.2.15 cells compared to HepG2 cells. Overexpression of NNMT reduces HBV replication in several cell models, while knockdown of NNMT enhances HBV DNA levels. Mechanistically, NNMT suppresses HBV DNA replication by inhibiting HBV RNA transcription. The region required for the inhibitory effect of NNMT was narrowed to nt 1672-1708 in enhancer II by luciferase assays. On the other hand, ChIP assays and EMSA results showed that NNMT does not bind to this region substantially, either directly or indirectly. Next, a collection of hepatic nuclear receptor transcription factors was screened to determine whether they were affected by NNMT overexpression. NR5A1, a positive regulator of HBV replication, decreased significantly after NNMT overexpression. Collectively, the findings of this study shed light on the regulation of HBV transcription., 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 © 2022 Elsevier Inc. All rights reserved.)

Bicyclol, a synthetic hepatoprotective and anti-inflammatory agent approved in China, was widely used to treat various hepatitis accompanied by elevated serum aminotransferases. However, the pharmacological effects and mechanisms of bicyclol on advanced liver diseases, such as fibrosis/cirrhosis and hepatocellular carcinoma (HCC), remain to be explored. Here, we revealed that bicyclol prevents from formatting severe fibrosis, slows the progression of moderate liver fibrosis, accelerates the regression of moderate liver fibrosis, decreases the malignancy of HCC in rat models induced by diethylnitrosamine (DEN), and also blocks steatohepatitis to HCC in mice induced by western diet plus carbon tetrachloride and DEN. The detailed pharmacological mechanism showed that bicyclol alleviates chronic progressive liver diseases by inhibiting the levels of IL-6 and subsequent phosphorylated STAT3. Conclusion: Bicyclol plays significant protective roles in multiply stages of fibrosis/cirrhosis-HCC and nonalcoholic fatty liver disease-related HCC via inhibiting IL-6/STAT3 signaling pathway. Therefore, bicyclol might be a promising therapeutic strategy for treating advanced liver diseases., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Objective: To evaluate the efficacy of three endoscopic therapies of isolated gastric varices (IGV) with modified tissue adhesive., Methods: A retrospective analysis was conducted with the clinical data of 73 IGV patients who were treated between January 2008 and December 2019 at Beijing Ditan Hospital. Patient clinical data on age, sex, etiology, biochemistry findings, Child-Pugh classification, the type of spontaneous shunt, preoperative bleeding history, and the presence or absence of liver cancer were collected. The three therapies evaluated were endoscopic intravenous injection of tissue glue combined with lauromacrogol, endoscopic clip-assisted intravenous injection of tissue glue combined with lauromacrogol, and endoscopic clip and LOOP-assisted intravenous injection of tissue glue combined with lauromacrogol. Their respective clinical treatment outcomes, including ectopic embolism rate, survival rate, rebleeding rate, amount of lauromacrogol and tissue glue used, the number of endoscopic clips used, and the number of times of the procedure the patient underwent, were evaluated., Results: In the patient baseline data, Child-Pugh grade, preoperative thrombus formation, and the presence or absence of liver cancer, showed significant difference between the three therapies ( P <0.05). There was no significant difference in the rates of ectopic embolism among the three methods ( P >0.05), but no ectopic embolism occurred after endoscopic clip-assisted intravenous injection of tissue glue combined with lauromacrogol, or after endoscopic clip and LOOP-assisted intravenous injection of tissue glue combined with lauromacrogol. There was no significant difference in the survival rate, the rebleeding rate, amount of lauromacrogol and tissue glue used for the three therapies, but there was significant difference in the number of endoscopic clips used and the number of times the procedure was conducted within one year ( P <0.05)., Conclusion: The two endoscopic therapies of intravenous injection of modified tissue glue, one assisted by clip and the other assisted by clip and LOOP, can help reduce the number of procedures IGV patients undergo within one year., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Sciences).)

Introduction and Objectives: Optimal treatment of hepatocellular carcinoma (HCC) involving portal vein tumor thrombus (PVTT) remains controversial., Materials and Methods: A total of 627 HCC patients with PVTT after initial treatment with one of the following at Affiliated Tumor Hospital of Guangxi Medical University: liver resection (LR, n = 225), transarterial chemoembolization (TACE, n = 298) or sorafenib (n = 104) were recruited and randomly divided into the training cohort (n = 314) and internal validation cohort (n = 313). Survival analysis were repeated after stratifying patients by Cheng PVTT type., Results: Resection led to significantly higher OS than the other two treatments among patients with type I or II PVTT. TACE worked significantly better than the other two treatments for patients with type III. All three treatments were associated with similar OS among patients with type IV. These findings were supported by the internal validation cohort., Conclusions: Our results suggest that the optimal treatment for HCC involving PVTT depends on the type of PVTT. LR may be more appropriate for type I or II PVTT; TACE, for type III Sorafenib may be more appropriate than invasive treatments for patients with type IV PVTT., Competing Interests: Conflicts of interest No authors declare any conflicts of interest., (Copyright © 2021 Fundación Clínica Médica Sur, A.C. Published by Elsevier España, S.L.U. All rights reserved.)

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, develops rapidly and has a high mortality rate. Relapsed metastasis is the most important factor affecting prognosis and is also the main cause of death for patients with HCC. Cantharidin is a kind of folk medicine for malignant tumors in China. Because of its cytotoxicity, the application of cantharidin is very limited. Magnesium demethylcantharidate (MDC) is a derivative of cantharidin independently developed by our laboratory. Our results show that MDC has anticancer activity and exhibited lower toxicity than cantharidin. However, whether MDC affects the invasion and metastasis of HCC cells and the underlying molecular mechanisms remain obscure. Transwell and Matrigel assays showed that MDC could effectively inhibit the invasion and metastasis of the HCC cell lines SMMC-7721 and SK-Hep1 in a dose-dependent manner. Moreover, MDC significantly inhibited the expression of invasion and metastasis related proteins MMP-2 and MMP-9. In addition, our study found that MDC inhibited the invasion and metastasis of HCC cell lines SMMC-7721 and SK-Hep1 by activating transcription factor FOXO1. Interestingly, the combination of MDC and sorafenib significantly inhibited the invasion and metastasis of HCC cell lines SMMC-7721 and SK-Hep1 compared with the single drug treatment via the activated transcription factor FOXO1. Our work revealed that MDC obviously inhibited the invasion and metastasis of HCC cells, and suggested that MDC could be a potential candidate molecule against the invasion and metastasis of HCC., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide due to a high rate of tumour metastasis and disease recurrence. In physiological conditions, tetraspanins interact with specific partner proteins in tetraspanin-enriched microdomains and regulate their subcellular localization and function. However, the function of Tspan5 in pathological processes, particularly in cancer biology and its clinical significance, are still unclear. Here, we describe that a high expression of Tspan5 is significantly associated with some clinicopathological features including invasive length, vascular invasion, clinical stage and poor overall survival of HCC patients. Alterations of Tspan5 expression by lentivirus transductions in HCC cells demonstrated that Tspan5 promotes wound healing and cell migration in vitro and tumour metastasis of HCC cells in vivo. Mechanistic studies revealed that Tspan5 promoted cell migration and tumour metastasis by increasing the enzymatic maturation of ADAM10 and activating Notch signalling via the increase of the cleavage of the Notch1 receptor catalysed by the γ-secretase complex. Activation of Notch signalling by Tspan5 was shown further to enhance the epithelial-mesenchymal transition (EMT) and actin skeleton rearrangement of tumour cells. In clinical HCC samples, Tspan5 expression is strongly correlated with many key molecules acting in Notch signalling and EMT, highlighting the role of Tspan5 in the regulation of Notch signalling, EMT and tumour metastasis of HCC. Our findings provide new insights into the mechanism of tumour metastasis and disease progression of HCC and may facilitate the development of novel clinical intervention strategies against HCC., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The incidence and mortality of hepatocellular carcinoma (HCC) are gradually increasing during the past years. Recently, some studies have reported that malic enzyme (ME) plays an important role in cancer development, while the involvement of ME2 in HCC remains still undetermined. Here, we demonstrated that ME2 played an oncogenic role in HCC. ME2 was overexpressed in HCC tissues. TCGA database showed that the ME2 transcript level was inversely associated with the survival of HCC patients. Loss-of-function and gain-of-function assays showed that ME2 promoted HCC cell growth and migration. Furthermore, the xenografted tumorigenesis of MHCC97H cells was retarded by ME2 knockdown. ME2 silencing also suppressed the cell cycle process and induced apoptosis. Mechanistically, ME2 potentiated triglyceride synthesis, inhibition of which suppressed the proliferation and migration. We propose that ME2 promotes HCC progression by increasing triglyceride production., (© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: Many molecular alterations are shared by embryonic liver development and hepatocellular carcinoma (HCC). Identifying the common molecular events would provide a novel prognostic biomarker and therapeutic target for HCC., Methods: Expression levels and clinical relevancies of SLC38A4 and HMGCS2 were investigated by qRT-PCR, western blot, TCGA and GEO datasets. The biological roles of SLC38A4 were investigated by functional assays. The downstream signalling pathway of SLC38A4 was investigated by qRT-PCR, western blot, immunofluorescence, luciferase reporter assay, TCGA and GEO datasets., Results: SLC38A4 silencing was identified as an oncofetal molecular event. DNA hypermethylation contributed to the downregulations of Slc38a4/SLC38A4 in the foetal liver and HCC. Low expression of SLC38A4 was associated with poor prognosis of HCC patients. Functional assays demonstrated that SLC38A4 depletion promoted HCC cellular proliferation, stemness and migration, and inhibited HCC cellular apoptosis in vitro, and further repressed HCC tumorigenesis in vivo. HMGCS2 was identified as a critical downstream target of SLC38A4. SLC38A4 increased HMGCS2 expression via upregulating AXIN1 and repressing Wnt/β-catenin/MYC axis. Functional rescue assays showed that HMGCS2 overexpression reversed the oncogenic roles of SLC38A4 depletion in HCC., Conclusions: SLC38A4 downregulation was identified as a novel oncofetal event, and SLC38A4 was identified as a novel tumour suppressor in HCC., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Objective To observe the effect of cryptotanshinone on the ferroptosis of human liver cancer HepG2 cells. Methods The viability of the HepG2 cells cultured in vitro was determined using the Cell Counting Kit-8(CCK-8),and the half maximal inhibitory concentration(IC 50 )was calculated.The cell morphology was observed using an inverted microscope.The reactive oxygen species(ROS)level was detected with the 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA)probe.The glutathione(GSH)assay kit was used to determine the GSH level.Western blot analysis was employed to detect the expression of cystine/glutamate antiporter system light chain(xCT)and glutathione peroxidase 4(GPX4),two marker proteins in ferroptosis.Additionally,the cell viability,ROS level,GSH level,and the expression levels of xCT and GPX4 were detected for the cells treated with the ferroptosis inhibitor ferrostain-1(Fer-1),the iron chelator deferoxamine(DFO),and the ROS scavenger N-acetylcysteine(NAC).Results Cryptotanshinone significantly inhibited the cell viability of HepG2 cells with an IC 50 of 93.73 μmol/L,and caused the morphological changes and death of the cells.It could significantly induce ROS accumulation,reduce GSH level,and down-regulate the expression of xCT and GPX4 in HepG2 cells.Fer-1,DFO,and NAC can remedy the cryptotanshinone-caused decrease in the cell viability of HepG2 cells.Fer-1 could inhibit cryptotanshinone-induced ROS accumulation,restore GSH level,and recover the expression of xCT and GPX4. Conclusion Cryptotanshinone may increase the accumulation of ROS by inhibiting the expression of xCT and GPX4 to induce the ferroptosis of HepG2 cells.

The rapid development of CRISPR/Cas9 systems has opened up tantalizing prospects to sensitize cancers to chemotherapy using efficient targeted genome editing, but safety concerns and possible off-target effects of viral vectors remain a major obstacle for clinical application. Thus, the construction of novel nonviral tumor-targeting nanodelivery systems has great potential for the safe application of CRISPR/Cas9 systems for gene-chemo-combination therapy. Here, we report a polyamidoamine-aptamer-coated hollow mesoporous silica nanoparticle for the co-delivery of sorafenib and CRISPR/Cas9. The core-shell nanoparticles had good stability, enabled ultrahigh drug loading, targeted delivery, and controlled-release of the gene-drug combination. The nanocomplex showed >60% EGFR-editing efficiency without off-target effects in all nine similar sites, regulating the EGFR-PI3K-Akt pathway to inhibit angiogenesis, and exhibited a synergistic effect on cell proliferation. Importantly, the co-delivery nanosystem achieved efficient EGFR gene therapy and caused 85% tumor inhibition in a mouse model. Furthermore, the nanocomplex showed high accumulation at the tumor site in vivo and exhibited good safety with no damage to major organs. Due to these properties, the nanocomplex provides a versatile delivery approach for efficient co-loading of gene-drug combinations, allowing for precise gene editing and synergistic inhibition of tumor growth without apparent side effects on normal tissues.

Cadmium (Cd) is a toxic heavy metal and widespread in environment and food, which is adverse to human and animal health. Food intervention is a hot topic because it has no side effects. Selenium (Se) is an essential trace element, found in various fruits and vegetables. Many previous papers have described that Se showed ameliorative effects against Cd. However, the underlying mechanism of antagonistic effect of Se against Cd-induced cytotoxicity in avian leghorn male hepatoma (LMH) cells is unknown, the molecular mechanism of Se antagonistic effect on Cd-induced and calcium (Ca 2+ ) homeostasis disorder and crosstalk of ER stress and autophagy remain to be explored. In order to confirm the antagonistic effect of Se on Cd-induced LMH cell toxicity, LMH cells were treated with CdCl 2 (2.5 μM) and Na 2 SeO 3 (1.25 and 2.5 μM) for 24 h. In this study, Cd exposure induced cell death, disrupted intracellular Ca 2+ homeostasis and Ca 2+ homeostasis related regulatory factors, interfered with the cycle of cadherin (CNX)/calreticulin (CRT), and triggered ER stress and autophagy. Se intervention inhibited Cd-induced LDH release and crosstalk of ER stress and autophagy via regulating intracellular Ca 2+ homeostasis. Moreover, Se mitigated Cd-induced Intracellular Ca 2+ overload by Ca 2+ /calmodulin (CaM)/calmodulin kinase IV (CaMK-IV) signaling pathway. Herein, CNX/CRT cycle played a critical role for the protective effect of Se on Cd-induced hepatotoxicity. Based on these findings, we demonstrated that the application of Se is beneficial for prevention and alleviation of Cd toxicity., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of 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 © 2020 Elsevier Ltd. All rights reserved.)

Background: Mounting evidence suggests that solid tumors display the features of collective invasion, however, the molecular mechanisms are far from clear. This study aims to verify the role and the underlying mechanisms of CD147 in collective invasion in hepatocellular carcinoma., Methods: Immunostaining was used to analyze human hepatocellular carcinoma specimens and three-dimensional cultures. Three-dimensional invasion model was established to mimic in vivo invasion. RNA-sequencing was used to identify downstream effectors., Results: Human hepatocellular carcinoma underwent collective invasion and CD147 was observed to be upregulated at the invasive front of tumor cell groups. CD147 was demonstrated to promote collective invasion using the modified three-dimensional invasion model, which recapitulated the main features of collective invasion. Through transcriptome analysis and enzyme activity assay, we found that CD147 enhanced cathepsin B expression and activity. Upregulated cathepsin B in hepatocellular carcinoma cells facilitated migration and invasion, which mediated CD147-induced invasive phenotype in hepatocellular carcinoma. In terms of mechanism, we found that CD147 promoted cathepsin B transcription by activating β-catenin signaling as a result of reduced GSK-3β expression. Furthermore, we found that elevated expression of CD147 as well as cathepsin B were correlated with poor prognosis in patients with hepatocellular carcinoma., Conclusions: CD147 promotes hepatocellular carcinoma cells collective invasion via upregulating cathepsin B expression and targeting CD147 would be valuable for the development of novel therapeutic modalities against invasion and metastasis of cancer.

Purpose: We aimed to explore the influence of tube voltage, current and iterative reconstruction (IR) in computed tomography perfusion imaging (CTPI) and to compare CTPI parameters with microvessel density (MVD)., Methods: Hepatic CTPI with three CTPI protocols (protocol A, tube voltage/current 80 kV/40 mAs; protocol B, tube voltage/current 80 kV/80 mAs; protocol C: tube voltage/current 100 kV/80 mAs) were performed in 25 rabbit liver VX2 tumor models, and filtered back projection (FBP) and IR were used for reconstruction of raw data. Hepatic arterial perfusion (HAP), hepatic portal perfusion (HPP), total perfusion (TP), hepatic arterial perfusion index (HPI), blood flow (BF) and blood volume (BV) of VX2 tumor and normal hepatic parenchyma were measured. Image noise, signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) were quantified and radiation dose was recorded. MVD was counted using CD34 stain and compared with CTPI parameters., Results: The highest radiation dose was found in protocol C, followed by protocols B and A. IR lowered image noise and improved SNR and CNR in all three protocols. There was no statistical difference between HAP, HPP, TP, HPI, BF and BV of VX2 tumor and normal hepatic parenchyma among the three protocols (P > 0.05) with FBP or IR reconstruction, and no statistical difference between IR and FBP reconstruction (P > 0.05) in either protocol. MVD had a positive linear correlation with HAP, TP, BF, with best correlation observed with HAP; MVD of VX2 tumor showed no or poor correlation with HPI and BV., Conclusion: CTPI parameters are not affected by tube voltage, current or reconstruction algorithm; HAP can best reflect MVD, but no correlation exists between BV and MVD.

Ginkgo biloba extract (GBE) is used in traditional Chinese medicine as a herbal supplement for improving memory. Exposure of B6C3F1/N mice to GBE in a 2-year National Toxicology Program (NTP) bioassay resulted in a dose-dependent increase in hepatocellular carcinomas (HCC). To identify key microRNAs that modulate GBE-induced hepatocarcinogenesis, we compared the global miRNA expression profiles in GBE-exposed HCC (GBE-HCC) and spontaneous HCC (SPNT-HCC) with age-matched vehicle control normal livers (CNTL) from B6C3F1/N mice. The number of differentially altered miRNAs in GBE-HCC and SPNT-HCC was 74 (52 up and 22 down) and 33 (15 up and 18 down), respectively. Among the uniquely differentially altered miRNAs in GBE-HCC, miR-31 and one of its predicted targets, Cdk1 were selected for functional validation. A potential miRNA response element (MRE) in the 3'-untranslated regions (3'-UTR) of Cdk1 mRNA was revealed by in silico analysis and confirmed by luciferase assays. In mouse hepatoma cell line HEPA-1 cells, we demonstrated an inverse correlation between miR-31 and CDK1 protein levels, but no change in Cdk1 mRNA levels, suggesting a post-transcriptional effect. Additionally, a set of miRNAs (miRs-411, 300, 127, 134, 409-3p, and 433-3p) that were altered in the GBE-HCCs were also altered in non-tumor liver samples from the 90-day GBE-exposed group compared to the vehicle control group, suggesting that some of these miRNAs could serve as potential biomarkers for GBE exposure or hepatocellular carcinogenesis. These data increase our understanding of miRNA-mediated epigenetic regulation of GBE-mediated hepatocellular carcinogenesis in B6C3F1/N mice.

Background: Transcription factor 21 (TCF21) is identified as a tumor suppressor in a variety of human tumors. The purpose of the study was to examine its expression tendency and prognostic value in hepatocellular carcinoma (HCC)., Methods: Relative expression of TCF21 mRNA in tissue samples from HCC patients and healthy volunteers were detected through quantitative real-time polymerase chain reaction (qRT-PCR) while its protein level was examined via immunohistochemistry analysis. Chi-square test was adopted to assess the association of TCF21 expression with the clinicopathological characteristic of the patients. Then Kaplan-Meier analysis was employed to analyze the function of TCF21 expression on overall survival among HCC patients., Results: Both the mRNA and protein levels of TCF21 were significantly reduced in HCC tissue samples compared with healthy controls (p < .05). Also, its expression was obviously affected by the classification of tissue pathology, metastasis, T stage, N stage and pathological grading. According to Kaplan-Meier analysis, patients with higher expression of TCF21 experienced dramatically longer overall survival time than those with lower expression (log rank test, p < .001)., Conclusions: TCF21 expression was decreased in HCC patients and it could act as a prognostic marker.

Gamma-glutamyl transpeptidase-to-platelet ratio (GPR) and fibrosis-4 (FIB-4) index have been reported to be useful predictors in predicting hepatocellular carcinoma (HCC) development in chronic hepatitis B (CHB) patients. However, their predictive performances on HCC development have not been validated in elderly patients. Thus, the aim of this study was to evaluate the predictive values of the GPR and FIB-4 index on HCC in elderly CHB patients with in China.Between January 2007 and December 2016, 1011 CHB patients older than 60 years were enrolled in the study, and their data were retrospectively analyzed. Receiver-operating characteristic (ROC) curve analysis was used to determine the optimal cutoff points of GPR and the FIB-4 index. Cumulative HCC incidence rates were calculated by the Kaplan-Meier method and compared by the log-rank test. Univariate and multivariate analyses were performed to detect risk factors for HCC development. The prediction performances of GPR and FIB-4 index were compared based on time-dependent ROC analyses.After a median follow-up of 6.8 (interquartile range 3.9-8.4) years, 39 (3.9%) patients developed HCC. The ROC analysis of GPR and the FIB-4 index at the 5-year time point revealed that the optimal cutoff point was 0.23 for GPR and 4.15 for the FIB-4 index. When stratified by low and high GPR values and FIB-4 indices, the patients' subgroups showed significantly different cumulative incidences of HCC. The multivariate analysis revealed that high GPR (hazard ratio [HR] 4.224; 95% confidence interval [CI] 1.891-9.434, P < .001) was an independent risk factor for HCC development, whereas a high FIB-4 index was not (HR 0.470; 95% CI 0.212-1.043; P = .063). In the time-dependent ROC analysis, GPR showed higher area under curve (AUC) values than the FIB-4 index did at all time points and reached statistical significance at the 5-, 7-, and 10-year time points (GPR vs FIB-4 index, AUC 0.725 vs 0.549 at 5 years, P = .005; GPR vs FIB-4 index, AUC 0.733 vs 0.578 at 7 years, P = .001; GPR vs FIB-4 index, AUC 0.837 vs 0.475 at 10 years, P < .001).In conclusion, our study suggests GPR is superior to the FIB-4 index in predicting HCC development in elderly CHB patients in China.

Objective: The clinical significance of coexistence of HBsAg/anti-HBs in chronic hepatitis B (CHB) patients remains controversial. This study was aimed to assess the association of this serological pattern with hepatocellular carcinoma (HCC) in patients with CHB., Methods: In this cross-section study, 206 CHB patients with coexistence of HBsAg/anti-HBs and 206 CHB patients with HBsAg alone were included to evaluate the risk of HCC development by logistic regression analysis. In addition, a retrospective cohort of 260 patients with CHB was recruited to estimate the cumulative incidence of HCC by Kaplan-Meier analysis., Results: The serological pattern of coexistence of HBsAg/anti-HBs, with high levels of ("High") HBsAg/low levels of ("Low") anti-HBs, were considered as independent risk factors for HCC. In particular, patients with "High" HBsAg/"High" anti-HBs [odds ratio (OR), 4.295; 95% confidence interval (CI), 1.104-16.699; p = 0.035] and "Low" HBsAg/ "High" anti-HBs (OR, 3.207; 95%CI, 1.299-7.919; p = 0.012) exhibited significantly higher risk for HCC development. However, only "Low" HBsAg /"High" anti-HBs might increase risk of HCC in CHB patients with high HBV load (logrank p < 0.001) in our cohort study., Conclusion: The coexistence of "Low" HBsAg /"High" anti-HBs might increase the risk of HCC development in CHB patients with high HBV load, which reflected that the long-term interaction between immune response and virus might lead to the development of HCC. The identification of the patients with poor prognosis will help clinicians to refine the therapeutic decisions and individualize follow-up strategies., (Copyright © 2019 Sociedade Brasileira de Infectologia. Published by Elsevier España, S.L.U. All rights reserved.)

Purpose: CD147 is a tumor-associated antigen that plays a key regulatory role in tumor invasion and distant metastasis. However, the exact role of CD147 phosphorylation, which is deregulated during cancer progression, is unknown. Here, the effects of CD147 phosphorylation on the malignant behavior of hepatocellular carcinoma (HCC) cells and its possible underlying mechanisms are explored., Methods: An in situ Duolink-proximity ligation assay (PLA) was used to detect CD147 phosphorylation. Tandem mass spectrometry was employed to identify the phosphorylation sites of CD147. The effects of CD147 phosphorylation on the malignant behavior of HCC cells were evaluated using scratch wound healing assays, transwell invasion assays and cell cycle assays. The genes regulated by CD147 phosphorylation were detected by RNA sequencing., Results: We identified phosphorylated serine-246 in the C terminus of CD147 in primary HCC tissues, whereas serine to alanine substitution mutation analysis suggested that CD147 is phosphorylated mainly at serine-252 in HCC-derived Huh-7 cells. Recovery expression of S246A/S252A mutants in CD147 knockout cells revealed significantly increased migration and invasion capacities compared to wildtype CD147 expressing cells. Cyclophilin A (CyPA) treatment decreased the phosphorylation level of CD147, whereas NIMA-related kinase 6 (NEK6) increased the CD147 phosphorylation level. Moreover, the CD147 phosphorylation level was found to be dramatically decreased in HCC tissues in patients with distant metastases, and a low phosphorylation level of CD147 was found to be associated with a high serum AFP level, recurrence and a poor overall survival., Conclusions: From our data we conclude that hypo-phosphorylated CD147 promotes the migration and invasion of HCC cells and correlates with an unfavorable prognosis in HCC patients, indicating that targeting the aberrantly hypo-phosphorylated form of CD147 may be instrumental for the development of novel therapeutic modalities directed against HCC metastasis.

Background: Sorafenib is the only approved first line systemic therapy for advanced hepatocellular carcinoma (HCC) in the last decade. Tumour resistance to sorafenib has been of major obstacles to improve HCC patient survival., Methods: We polarised THP-1 cells to M1 and M2 macrophages, performed various in vitro assays and developed sorafenib-resistant xenograft models to investigate the role of tumour-associated macrophages (TAM)-secreted molecules in HCC resistance to the targeted therapy., Results: We demonstrated M2, but not M1, macrophages not only promote proliferation, colony formation and migration of hepatoma cells but also significantly confer tumour resistance to sorafenib via sustaining tumour growth and metastasis by secreting hepatocyte growth factor (HGF). HGF activates HGF/c-Met, ERK1/2/MAPK and PI3K/AKT pathways in tumour cells. Tumour-associated M2 macrophages were accumulated in sorafenib-resistance tumours more than in sorafenib-sensitive tumours in vivo and produced abundant HGF. HGF chemoattracts more macrophages migrated from surrounding area, regulates the distribution of M2 macrophages and increases hepatoma resistance to sorafenib in a feed-forward manner., Conclusions: Our results provide new insights into the mechanisms of sorafenib resistance in HCC and rationale for developing new trials by combining sorafenib with a potent HGF inhibitor such as cabozantinib to improve the first line systemic therapeutic efficacy.

Background: Several studies have focused on the association between KIF1B rs17401966 polymorphism and susceptibility to hepatitis B virus-related (HBV-related) hepatocellular carcinoma (HCC), but the conclusions have been inconsistent. We have conducted this updated meta-analysis to explore the association between KIF1B rs17401966 polymorphism and HCC susceptibility., Methods: Eligible studies were identified through systematic searches in PubMed, OVID, ISI Web of Science, Chinese National Knowledge Infrastructure, and Wanfang databases. The quality of evidence was systematically assessed by use of the Newcastle-Ottawa Scale for case control studies in meta-analyses., Results: Ten studies containing 18 independent case-control studies were included. The results revealed a significant association between KIF1B rs17401966 polymorphism and susceptibility to HCC under a random-effect allelic model (OR = 0.85, 95% CI 0.76-0.94, P = 0.003); HBV-positive subgroup (OR = 0.82, 95% CI 0.72-0.95, P = 0.007); and Chinese-subgroup (OR = 0.82, 95% CI 0.72-0.93, P = 0.002)., Conclusions: G-allele appears to be a protective allele of KIF1B for HCC, especially in HBV-positive and Chinese populations. More well-designed studies with larger sample size and various ethnic groups and risk factors are needed to establish that KIF1B rs17401966 polymorphism is significantly associated with risk of HCC.

Objective: Accumulating evidence suggests that pseudogenes play potential roles in the regulation of their cognate wild-type genes, oncogenes, and tumor suppressor genes. ANXA2P2 (annexin A2 pseudogene 2) is one of three pseudogenes of annexin A2 that have recently been shown to be aberrantly transcribed in hepatocellular carcinoma (HCC) cells. However, its clinical meaning and biological function in HCC have remained unclear. Therefore, the present study was aimed at exploring the prognostic value of a high expression of ANXA2P2 in HCC tissue and at identifying whether it can affect the efficacy of targeted drugs (sorafenib, regorafenib, and lenvatinib)., Methods: We obtained ANXA2P2 mRNA expression levels from The Cancer Genome Atlas (TCGA) RNA sequence database. The expression levels of ANXA2P2 in 49 pairs of intratumoral and peritumoral liver tissues were examined by RT-PCR. Wound healing and transwell assays were performed to confirm the tumor-promoting properties of ANXA2P2 in HCC cells. CCK8 assay was conducted to identify whether ANXA2P2 can affect the growth of HCC cells when administered with targeted drugs (sorafenib, regorafenib, and lenvatinib)., Results: The expression of ANXA2P2 in HCC tissues was significantly higher than that in adjacent cancerous tissues from TCGA database and validation group. Additionally, patients with high ANXA2P2 expression in HCC tissue had a shorter overall survival, whereas no statistically significant correlation was found between ANXA2P2 expression and disease-free survival ( p = 0.08) as well as other clinical parameters, such as age, gender, histological grade, T classification, stage, albumin level, alpha-fetoprotein, and vascular invasion ( p = 0.7323, 0.8807, 0.5762, 0.8515, 0.7113, 0.242, 1.0000, and 0.7685, respectively). Furthermore, in vitro experiments showed that knockdown of ANXA2P2 inhibited migration and invasion of HCC cells but did not have an influence on the HCC cell proliferation when treated with targeted drugs (sorafenib, regorafenib, and lenvatinib)., Conclusion: Our study confirmed elevated ANXA2P2 expression levels in HCC tissue compared with adjacent noncancerous tissue and a worse prognosis of patients with high ANXA2P2 levels in the HCC tissue. The newly found properties of promoting migration and invasion of ANXA2P2 in HCC help to explain this phenomenon. ANXA2P2 could be a novel and suitable predicative biomarker for the risk assessment of recurrence or metastasis of HCC patients but may not be effective to predict the efficacy of targeted drugs.

Objective: To explore the role of hsa-microRNA-99b/let-7e/microRNA-125a cluster in the progression of liver cancer and its possible regulatory mechanisms., Patients and Methods: Ten liver cancer tissues were randomly selected and matched with normal liver tissue samples. The mRNA expression levels of miR-99b, let-7e and miR-125a were detected by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR). The three miRNA mimics were transfected alone or together into hepatoma cells SMMC-7721; at the same time, the knockdown of the three miRNAs was also performed. Then the cell invasive and migratory abilities were examined through transwell assay. Bioinformatics analysis was used to detect the potential transcription factors that bind to the hsa-microRNA-99b/let-7e/microRNA-125a promoter sequence, and the binding of the two was verified by the Luciferase reporting assay. The level of hsa-microRNA-99b/let-7e/microRNA-125a mature mRNA was detected after ZEB1 was inhibited by ZEB1 siRNA. Meanwhile, after interfering with Drosha and ZEB1, the expression level of hsa-microRNA-99b/let-7e/microRNA-125a of the primary transcript was examined. Rescue experiments were carried out to assess the role of hsa-microRNA-99b/let-7e/microRNA-125a in the ZEB1 regulation of invasive cell capacity., Results: The mRNA levels of microRNA-99b, let-7e, and microRNA-125a in 10 selected hepatocarcinoma tissues were significantly higher than those in the matched paracancerous tissues. After overexpressing the three miRNA mimics either alone or together, cell invasive and migratory abilities were extensively enhanced, and vice versa. It was found that there is a binding site in the upstream sequence of the promoter region of the hsa-microRNA-99b/let-7e/microRNA-125a cluster for ZEB1. The Luciferase reporter gene results showed an increase in the Luciferase activity of the cells transfected with E-box element wild-type sequence, while mutant E-box element group did not change. After knocking out ZEB1, the levels of mature microRNA-99b, let-7e and microRNA-125a were reduced. However, when DROSHA was knocked out, the levels of immature microRNA-99b, let-7e and microRNA-125a were increased, while simultaneous knocking out ZEB1 reversed this effect. Besides, the invasive ability of SMMC-7721 cells decreased after KEB1 was knocked down, while the opposite result was observed after transfection of hsa-microRNA-99b/let-7e/microRNA-125a alone or together., Conclusions: Hsa-microRNA-99b/let-7e/microRNA-125a cluster is highly-expressed in hepatocarcinoma, and its expression can be regulated by ZEB1. In addition, the overexpression of this cluster can promote the invasion of liver cancer cells and advance liver cancer progression.

The tyrosine phosphatases family member PTEN is a tumor suppressor which is widely expressed throughout the body and is involved in a variety of biological functions. PTEN is known to be frequently mutated or downregulated in human cancers. However, the underlying molecular mechanism remains largely unknown. Here, using a proteomic approach, we identified the E3 ubiquitin ligase HRD1, which was previously reported to be involved in endoplasmic reticulum associated degradation (ERAD), as one of the PTEN-interacting proteins. We also found that HRD1 promoted PTEN degradation by positively regulating its ubiquitination. In addition, suppression of HRD1 expression resulted in the inhibition of the growth, migration and invasion of hepatocellular carcinoma in vitro and in vivo. Finally, we detected a negative correlation between HRD1 and PTEN expression in human hepatocellular carcinoma. From these results we propose a novel molecular mechanism of HRD1 to promote hepatocellular tumorigenesis via PTEN inactivation. We conclude that targeting HRD1 may represent a new therapeutic strategy for PTEN-loss hepatocellular carcinoma., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Despite the fact that a large number of synthetic channels have been developed in the last three decades, few of them can function in mammalian cell membranes because of their weak membrane insertion abilities. This study describes a tubular molecule with terminal positively charged amino groups that displays a strong ability to insert into lipid bilayers composed of phosphatidylcholine and consequently forming unimolecular transmembrane channels. It has been demonstrated that the insertion of the channel into the phosphatidylcholine bilayers was driven by the electrostatic interaction between the positively charged amino groups of the channel molecules and the negatively charged phosphate groups of the lipid molecules. The high affinity of the channels for lipid bilayers led to efficient mammalian cell membrane insertion. The channels showed high effective activity against HepG2 cancer cells at concentrations above 5.1 μM.

Haishengsu (HSS) is an active natural extract isolated from Tegillarca granosa, which has previously been demonstrated to inhibit the proliferation of several types of cancer cells in vitro. Our previous study indicated that HSS may induce apoptosis to suppress growth of human hepatocellular carcinoma BEL‑7402 cells by activating Fas pathway. The present study demonstrated that HSS treatment induces the in vitro apoptosis of BEL‑7402 cells via the mitochondrial‑mediated apoptotic pathway detected by DNA fragmentation assay, caspase activity assay and transmission electron microscopy assay, and inhibits tumor xenograft growth in vivo. Alterations in apoptotic regulatory proteins were detected, including decreased expression of B‑cell lymphoma2 (Bcl‑2), upregulation of Bcl‑2‑associated X protein and mitochondrial cytochrome c release, and downstream activation of apoptotic signaling. Furthermore, apoptotic induction was caspase‑dependent, as indicated by cleavage of the caspase substrate, poly (ADP‑ribose) polymerase. Oral administration of 62.5‑250 mg/kg HSS markedly educed the growth of hepatocellular carcinoma tumor xenografts in nude mice. In addition, immunohistochemical staining for caspase‑3 protein and transmission electron microscopy further indicated the induction of apoptosis in these tumor tissues. Taken together, the present study demonstrated that HSS may effectively induce apoptosis to suppress the growth of BEL‑7402 cells in vitro and in vivo, and therefore may hold promise for further development as a novel cancer therapy.

Objective: To explore the effect of silencing lumican on the invasion and migration of liver cancer cells., Methods: Lumican was silenced by shRNA in liver cancer cells (HepG2 and MHCC97H). The mRNA levels of lumican were detected by qRT-PCR. Cell invasion was measured by Transwell. Cell migration was tested via wound healing. The protein levels of lumican,MMP-9,VEGF,ERK1,JNK,p-ERK1 and p-JNK were measured by Western blot., Results: Liver cancer cells (HepG2 and MHCC97H) had higher levels of mRNA and protein of lumican compared with normal hepatocyte L02 ( P <0.01). shRNA lowered the levels of mRNA and protein of lumican ( P <0.01),and weakened the invasion and migration of cancer cells ( P <0.01). The expressions of MMP-9 and VEGF decreased with the shRNA silence ( P <0.01). shRNA also reduced the protein level of p-ERK1 and p-JNK ( P <0.01)., Conclusion: Silencing lumican by shRNA attenuates the invasion and migration of liver cancer cells via inhibiting the activation of ERK1/JNK pathway., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Science Edition).)

Object: To explore microcirculation features with intravoxel incoherent motion (IVIM) and to compare IVIM with CT perfusion imaging (CTPI) and microvessel density (MVD)., Materials and Methods: Hepatic CTPI and IVIM were performed in 16 rabbit liver VX2 tumor models. Hepatic arterial perfusion (HAP), hepatic arterial perfusion index (HPI), Blood flow (BF), and blood volume (BV) from CTPI were measured. Apparent diffusion coefficient (ADC), true diffusion coefficient (D), perfusion fraction (f), and pseudo-diffusion coefficient (D*) from IVIM were measured. MVD was counted with CD34 stain. The microcirculation features with IVIM were compared with CTPI parameters and MVD., Results: Strong linear correlations were found between D value (0.89±0.21×10 -3 mm 2 /s) and HAP (15.83±6.97ml/min/100mg) (r=0.755, P=0.001) and between f value (12.64±6.66%) and BV (9.74±5.04ml/100mg) (r=0.693, P=0.004). Moderate linear correlations were observed between ADC (1.07±0.32×10 -3 mm 2 /s) and HAP (r=0.538, P=0.039), respectively; and between D value and MVD (9.31±2.57 vessels at 400×magnification) (r=0.509, P=0.044). No correlations were found between D* (119.90±37.67×10 -3 mm 2 /s) and HAP, HPI (68.34±12.91%), BF (4.95±2.16ml/min/100mg), BV., Conclusion: IVIM parameters can characterize microcirculation to certain extent and separate it from pure water molecular diffusion. There is fair correlation between D or ADC value and CTPI parameters or MVD, but no correlation between D* or f value and CTPI parameters or MVD except f value and BV, which is still unclear and need further clinical studies to validate., (Copyright © 2017 Elsevier Inc. All rights reserved.)

To compare the performance of contrast-enhanced ultrasound (CEUS) using high-frequency linear and convex probes in the detection of small colorectal liver metastases (CRLMs). A total of 85 patients with 143 small CRLMs were evaluated. High-frequency ultrasound (US) and CEUS detected significantly more superficial lesions within 60 mm below the skin than a convex probe (p <0.05). The detection rate decreased in the chemotherapy group, especially when using a convex probe for US (p <0.05). By combining convex and linear probes, detection rates of US and CEUS were significantly higher than that of a convex or a linear probe alone (p <0.05). High-frequency US and CEUS helped to improve detection of small CRLMs and reduce the influence of chemotherapy. For patients with a high risk of CRLMs and those after chemotherapy, we recommend first scanning the liver by using a convex probe and subsequently screening the surface area of the liver and suspicious small lesions by using a linear probe., (Copyright © 2017 World Federation for Ultrasound in Medicine and Biology. Published by Elsevier Inc. All rights reserved.)

Walsuronoid B is a limonoid compound extracted from Walsura robusta. Previous studies have shown that limonoid compounds possess anti-cancer potential, although the molecular mechanism of this activity remains elusive. In this study, we demonstrated for the first time that walsuronoid B inhibited cell proliferation in several human cancer lines. Liver cancer cells (HepG2 and Bel-7402) were chosen for their high sensitivity to walsuronoid B. Walsuronoid B induced cell death through G 2 /M phase arrest and apoptosis and induced the accumulation of autophagosomes through the suppression of mTOR signaling, which serves as a cell survival mechanism and prevents cell death. We further examined the molecular mechanisms and found that walsuronoid B-induced dysfunction of the mitochondria and lysosomes rather than the endoplasmic reticulum contributed to its cell death effect. Walsuronoid B enhanced the generation of hydrogen peroxide, nitric oxide and superoxide anion radical, resulting in elevated levels of reactive oxygen species (ROS). In addition, ROS induced by walsuronoid B upregulated p53 levels; conversely, p53 stimulated ROS. These results suggested that ROS and p53 reciprocally promoted each other's production and cooperated to induce liver cancer cell death. We found that the induction of ROS and p53 significantly triggered G 2 /M phase arrest and mitochondrial and lysosomal apoptosis. Finally, walsuronoid B suppressed tumor growth in vivo with few side effects. In summary, our findings demonstrated that walsuronoid B caused G 2 /M phase arrest and induced mitochondrial and lysosomal apoptosis through the ROS/p53 signaling pathway in human liver cancer cells in vitro and in vivo., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The aim of the study was to investigate the effects of arsenic trioxide (As2O3) treatment on the expression of ezrin and serum alpha-fetoprotein (AFP) levels in hepatocellular carcinoma (HCC).A total of 24 patients (20 males and 4 females) with resectable HCC were treated with venous injection of As2O3 for 14 days (10 mg/d) before surgery. The ezrin expression and serum AFP levels were assessed before and after treatment, respectively.The serum AFP levels were 325.5 ng/L before treatment and 278.6 ng/L after treatment, with statistical significant difference (Z = -2.360, P < .05). The expression of ezrin was negative, weak positive, and strong positive in 11, 7, and 6 cases, respectively, before As2O3 treatment, and 17, 5, 2 cases respectively after the treatment. The difference between the 2 groups was statistically significant (χ = 5.619, P < .05). Also, the results showed that there was a significant correlation between the high serum AFP level (AFP ≥ 500 ng/L) and high expression of ezrin (χ = 8.080, P < .05).As2O3 treatment can significantly downregulate the expression of ezrin in HCC.

Objective: To explore the related factors for primary hepatic carcinoma (PHC) caused by chronic hepatitis B (CHB) and hepatitis C (CHC). Methods: According to the principle of cross-sectional study, a cluster random sample method was used, a total of 366 chronic hepatitis patients in hospitals were recruited from three provincial tertiary hospitals in Shanxi, Henan and Jilin between July 2016 and October 2016, respectively. Using a self-designed unified questionnaire, face-to-face interviews was conducted on subjects, including sex, age, alcohol consumption, coffee consumption, green tea consumption, fish consumption, smoking, HBV/HCV diagnosis and treatment, diabetes mellitus, family history of PHC (whether PHC in first-degree relatives), etc . Multivariate unconditional logistic regression were performed to identify the related factors for PHC with CHB and CHC. According to the clinical diagnosis the patients were divided into a chronic hepatitis group (not developing to PHC) and a PHC group. Results: Among 366 cases patients, 287 (78.4%) cases were male, 79 cases were female (21.6%), average age was (52.7±9.3) years. 202 cases were chronic hepatitis group, 164 cases were PHC group. Multivariate unconditional logistics regression analysis indicated that alcohol consumption (odds ratio ( OR )=2.11, 95 %CI: 1.18-3.75), family history of PHC ( OR= 5.12, 95 %CI: 2.60-10.08) were positively correlated with the development of PHC in chronic b, green tea consumption ( OR= 0.45, 95 %CI: 0.23-0.88), antiviral treatment ( OR= 0.19, 95 %CI: 0.11-0.32) were negatively correlated. Alcohol consumption ( OR= 3.98, 95 %CI: 1.14-13.85) was positively correlated with the development of PHC in chronic c, antiviral treatment ( OR= 0.14, 95 %CI: 0.04-0.50) was negatively correlated. Conclusion: Alcohol consumption, family history of PHC, green tea consumption and antiviral treatment were the related factors for the development of PHC in chronic hepatitis b. Alcohol consumption and antiviral treatment were the related factors for the development of PHC in chronic hepatitis c.

Background: This study aimed to highlight the type of tumor thrombus and identify the prognostic factors influencing the long-term survival outcomes in patients with hepatocellular carcinoma (HCC) having a tumor thrombus. A tumor thrombus in HCC is associated with poor prognosis., Methods: Eighty patients diagnosed with HCC having a tumor thrombus between May 2006 and April 2014 were enrolled in this study. Age, gender, clinical characteristics, laboratory findings, Child-Pugh classification, performance status (ECOG), types of tumor thrombi, radiotherapy method, biologically effective dose (BED), and primary treatment method were analyzed to identify the prognostic factors associated with the overall survival (OS) rates. Statistical analyses were performed using SPSS version 19.0., Results: The median follow-up duration was 24 months (range 6-90). The 1-, 3-, and 5-year OS rates of the patients were 77.6%, 37.6%, and 18.8%, respectively. On univariate analysis, gender, radiotherapy method, BED, types of tumor thrombi, Child-Pugh classification, ECOG, and total bilirubin were associated with OS (P < 0.001, P = 0.001, P = 0.016, P = 0.003, P < 0.001, P < 0.001, P = 0.039, respectively). The prognostic factors for OS in multi-variate analyses were gender (P < 0.001), BED (P = 0.044), Child Pugh classification (P = 0.020), performance status (ECOG) (P = 0.004), and types of tumor thrombi (P = 0.001). The median OS for the high-BED group was better than that for the low-BED groups (42 months vs. 19 months, P = 0.016)., Conclusions: Gender, BED, performance status (ECOG), Child-Pugh classification, and types of tumor thrombi seemed to affect OS, and a stepwise decrease in survival was observed with the types of tumor thrombi ranging from I to IV. High-BED palliative radiotherapy might improve the long-term outcomes for patients with HCC having a tumor thrombus.

Krüppel-like factor 8 (KLF8) is highly expressed in hepatocellular carcinoma (HCC) and contributes to tumor initiation and progression by promoting HCC cell proliferation and invasion. However, the role of KLF8 in liver cancer stem cells (LCSCs) is not known. In the current study, we investigated the role of KLF8 in LCSCs to determine if KLF8 is a novel marker of these cells. We found that KLF8 was highly expressed in primary HCC tumors, distant migrated tissues, and LCSCs. Patients with high KLF8 expression had a poor prognosis. KLF8 promoted stem cell-like features through activation of the Wnt/β-catenin signaling pathway. Cell apoptosis was significantly increased in HCC cells with knockdown of KLF8 compared with the control cells when treated with the same doses of sorafenib or cisplatin. Taken together, our study shows that KLF8 plays a potent oncogenic role in HCC tumorigenesis by maintaining stem cell-like features through activation of the Wnt/β-catenin signaling pathway and promoting chemoresistance. Thus, targeting KLF8 may provide an effective therapeutic approach to suppress tumorigenicity of HCC. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Increasing evidence has demonstrated that Notch genes, including Notch1, Notch2, Notch3 and Notch4, are involved in carcinogenesis. However, the expression and regulation of Notch genes in hepatocellular carcinoma (HCC) tissues have not been fully investigated. In the present study, immunohistochemical and quantitative real-time PCR (qPCR) analyses were performed to examine the expression of Notch genes in normal human liver, HBV-related HCC and paired peritumoral tissues. Additionally, qPCR and western blotting were utilized to investigate the impact of hepatitis B virus X protein (HBx) and hypoxia‑inducible factor-1α (HIF-1α) on the regulation of Notch gene expression. The immunohistochemical and qPCR results showed that the expression levels of Notch1, Notch3 and Notch4 were significantly higher in HCC tissues than the levels in peritumoral and normal liver tissues. However, no significant difference in Notch2 expression was found between HCC and peritumoral tissues. Among the four Notch genes, immunohistochemical analyses found that only the increased level of Notch3 in HCC tissues was positively correlated with vascular invasion of liver cancer (P<0.05). Moreover, we found that overexpression of both HBx and HIF-1α increased the expression of Notch1, Notch3 and Notch4 in HepG2 and Bel-7404 cell lines. In summary, the present study demonstrated that Notch1, Notch3 and Notch4 were upregulated in HCC tissues and that HBx and HIF-1α may be the factors that cause the overexpression of Notch genes. Furthermore, the increased expression of Notch3 was closely related to the vascular invasiveness of HCC.

We investigated the levels of target lymphocyte subsets in peripheral blood lymphocyte samples from patients with hepatocellular carcinoma (HCC). A total of 715 high-risk patients with primary HCC were recruited in Guangxi, China as the case group. The control group included 100 patients who received health examinations at the same hospital during the same period. Fasting elbow venous blood (10 mL) was collected from each participant, and flow cytometry was used to detect the levels of NK cells and CD3+, CD4+ and CD19+ T cells in peripheral blood samples. All included patients with prmary HCC were treated by surgical resection, and followed up for one year. The levels of CD19+ and NK cells were lower in cases than in controls (both P < 0.05). In addition, the level of CD8+ cells was greater in the case group than in the control group (P < 0.05). In the high-HCC-risk population, CD8+, CD19+ and NK cell levels all differed between male and female patients, patients in TNM stages I-II and stages III-IV, patients with and without extrahepatic metastasis, and patients with and without HBV infection (all P < 0.05). After follow-up, detected recurrence and survival rate was 33.71% and 83.64%, respectively. CD8+ levels was reduced following surgical resection, whereas the levels of CD19+ and NK cells were increased (all P < 0.05). In conclusion, altered levels of CD8+, CD19+ and NK cell levels may be used as reference values for monitoring immune function in certain populations with high HCC risk, and as potential evidence for the clinical diagnosis and treatment of HCC.

Cluster of differentiation 147 (CD147), also known as extracellular matrix metalloproteinase inducer, is a transmembrane glycoprotein that mediates oncogenic processes partly through N-glycosylation modifications. N-glycosylation has been demonstrated to be instrumental for the regulation of CD147 function during malignant transformation. However, the role that site-specific glycosylation of CD147 plays in its defective function in hepatocellular carcinomacells needs to be determined. Here, we demonstrate that the modification of N-glycosylation at Asn152 on CD147 strongly promotes hepatocellular carcinoma (HCC) invasion and migration. After the removal of N-glycans at Asn152, CD147 was more susceptible to degradation by ER-localized ubiquitin ligase-mediated endoplasmic reticulum-associated degradation (ERAD). Furthermore, N-linked glycans at Asn152 were required for CD147 to acquire and maintain proper folding in the ER. Moreover, N-linked glycans at Asn152 functioned as a recognition motif that was directly mediated by the CNX quality control system. Two phases in the retention-based ER chaperones system drove ER-localized CD147 trafficking to degradation. Deletion of N-linked glycosylation at Asn152 on CD147 significantly suppressed in situ tumour metastasis. These data could potentially shed light on the molecular regulation of CD147 through glycosylation and provide a valuable means of developing drugs that target N-glycans at Asn152 on CD147.

Hepatocellular carcinoma (HCC) is currently the third most common cause of cancer-related death in the Asia-Pacific region. Our previous work showed that knockdown of CD98 significantly inhibits malignant HCC cell phenotypes in vitro and in vivo. The level of CD98 in the membrane is tightly regulated to mediate complex processes associated with cell-cell communication and intracellular signaling. In addition, the intracellular domain of CD98 (CD98-ICD) seems to be of vital importance for recycling CD98 to the membrane after it is endocytosed. The intracellular and transmembrane domains of CD98 associate with β-integrins (primarily β1 but also β3), and this association is essential for CD98 mediation of integrin-like signaling and complements dominant suppression of β1-integrin. We speculated that isolated CD98-ICD would similarly suppress β1-integrin activation and inhibit the malignant behaviors of cancer cells. In particular, the exact role of CD98-ICD has not been studied independently in HCC. In this study, we found that ectopic expression of CD98-ICD inhibited the malignant phenotypes of HCC cells, and the mechanism possibly involves β1-integrin suppression. Moreover, the expression levels of CD98, β1-integrin-A (the activated form of β1-integrin) and Ki-67 were significantly increased in HCC tissues relative to those of normal liver tissues. Therefore, our preliminary study indicates that ectopic CD98-ICD has an inhibitory role in the malignant development of HCC, and shows that CD98-ICD acts as a dominant negative mutant of CD98 that attenuates β1-integrin activation. CD98-ICD may emerge as a promising candidate for antitumor treatment., Competing Interests: The authors declare no conflict of interest.

Drug resistance is one characteristic of hepatocellular carcinoma (HCC) and can affect the prognosis of patients directly. To explore drug resistance well, we established an oxaliplatin (OXA)-resistant cell line Bel-7402/OXA by exposure to gradually increased concentration of OXA. Some biological characters, such as proliferation, migration, and invasion, were studied. Drug sensitivity and the mechanisms of drug resistance were also investigated. We found that the resistant index of Bel-7402/OXA was 8.3. In comparison with Bel-7402, the percentages of cells in S and G2/M phase were increased. The nature apoptosis rate and drug-after apoptosis rate were all decreased in Bel-7402/OXA, as compared to Bel-7402. Bel-7402/OXA acquired increased migration and invasion ability with epithelial-mesenchymal transition (EMT) phenotype. Knockdown of EMT transcription factor Snail could reverse EMT and sensitized Bel-7402/OXA cells to OXA. EMT was one mechanism of drug resistance and may be a novel target of treatment for drug resistance.

A growing number of studies suggest that the hepatitis B virus X protein (HBx) enhances the protein stability of the hypoxia-inducible factor-1α (HIF-1α). However, the relationship between hepatitis B virus (HBV), HBx and hypoxia-inducible factor-2α (HIF-2α) has not yet been fully elucidated. Immunohistochemical analysis was employed to detect the expression of HIF-2α in normal liver, HBV-related chronic hepatitis, and HBV-related and non-HBV-related hepatocellular carcinoma (HCC) tissues. Quantitative real‑time PCR (qPCR) and western blotting were used to investigate the impact of HBV and HBx on the expression of HIF‑2α. Immunoprecipitation and immunofluorescence were applied to explore the underlying mechanisms. The HIF‑2α expression was found to be higher in HBV‑related chronic hepatitis tissues than in normal liver tissues. Furthermore, it was higher in HBV‑related HCC tissues and HBV‑integrated HepG2 cells than in the corresponding non‑HBV‑related HCC tissues and HepG2 cells. Both HBV and HBx enhanced the protein stability of HIF‑2α. HBx‑mediated upregulation of HIF‑2α resulted mainly from an inhibition of the degradation of HIF‑2α due to the binding of HBx to the von Hippel‑Lindau protein (pVHL). In addition, HBx upregulated the expression of HIF‑2α by activating the NF‑κB signaling pathway. Thus, the present study identified that HBV induces the HIF‑2α expression through its encoded protein HBx. This upregulates the HIF-2α expression by binding to the pVHL activating the NF-κB signaling pathway.

CD147, a type I transmembrane glycoprotein, is highly expressed in various cancer types and plays important roles in tumor progression, especially by promoting the motility and invasion of hepatocellular carcinoma (HCC) cells. These crucial roles make CD147 an attractive target for therapeutic intervention in HCC, but no small-molecule inhibitors of CD147 have been developed to date. To identify a candidate inhibitor, we used a pharmacophore model derived from the structure of CD147 to virtually screen over 300,000 compounds. The 100 highest-ranked compounds were subjected to biological assays, and the most potent one, dubbed AC-73 (ID number: AN-465/42834501), was studied further. We confirmed that AC-73 targeted CD147 and further demonstrated it can specifically disrupt CD147 dimerization. Moreover, molecular docking and mutagenesis experiments showed that the possible binding sites of AC-73 on CD147 included Glu64 and Glu73 in the N-terminal IgC2 domain, which two residues are located in the dimer interface of CD147. Functional assays revealed that AC-73 inhibited the motility and invasion of typical HCC cells, but not HCC cells that lacked the CD147 gene, demonstrating on-target action. Further, AC-73 reduced HCC metastasis by suppressing matrix metalloproteinase (MMP)-2 via down-regulation of the CD147/ERK1/2/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Finally, AC-73 attenuated progression in an orthotopic nude mouse model of liver metastasis, suggesting that AC-73 or its derivatives have potential for use in HCC intervention. We conclude that the novel small-molecule inhibitor AC-73 inhibits HCC mobility and invasion, probably by disrupting CD147 dimerization and thereby mainly suppressing the CD147/ERK1/2/STAT3/MMP-2 pathways, which are crucial for cancer progression.

This study evaluated the factors impacting overall survival (OS) and time to progression (TTP) in patients with unresectable hepatocellular carcinoma (HCC) who received transarterial chemoembolization (TACE). HCC patients were grouped based on tumor vascularity and lipidiol deposition after TACE. Tumor vascularity was classified based on contrast enhancement on arterial phase baseline CT scans. Lipiodol deposition was evaluated using CT scans. The progression-free rate was significantly higher in patients with good blood supply + good lipiodol deposition compared to those with good blood supply + poor lipiodol deposition. In patients with poor lipidiol deposition, risk of death was significantly positively correlated with stage, and negatively correlated with number of TACE procedures and degree of lipidiol deposition after the first TACE. Risk of disease progression in these patients was positively correlated with tumor size, and negatively correlated with number of TACE procedures and degree of lipidiol deposition after the first TACE. Our data showed that tumor vascularity and lipiodol deposition can be used as early radiological markers to identify patients who do not respond to TACE, and who can be considered earlier for alternative combination treatment strategies. Our data also indicated that poor lipiodol retention may predict a poor TTP and OS despite the blood supply status.

Nemo-like kinase (NLK) is an evolutionarily conserved serine/threonine protein kinase and belongs to the extracellular signal-regulated kinases/microtubule-associated protein kinase families (Erks/MAPKs). Previous studies have indicated that abnormal expressions of NLK played critical roles in various types of human cancers. Recent studies suggested that NLK expression was significantly upregulated in the hepatocellular carcinoma (HCC) specimens. However, the clinical significance of NLK expression in HCC remains largely unknown. In this study, we focused on the clinical significance of NLK in HCC and found that high expression of NLK was significantly associated with Edmondson-Steiner grade (P = 0.002), tumor size (P = 0.022), and no. of tumor nodules (P < 0.001), and NLK was positively correlated with proliferation marker Ki-67 (P < 0.01). Univariate analysis suggested that NLK expression was associated with poor prognosis (P < 0.001). Multivariate analysis indicated that NLK expression was an independent prognostic indicator for HCC (P = 0.0370). In conclusion, NLK overexpression is associated with poor overall survival in patients with HCC, it might be an independent poor prognostic marker for HCC.

Oscillations in intracellular Ca2+ concentrations ([Ca2+]i) mediate various cellular function. Although it is known that [Ca2+]i oscillations are susceptible to dysregulation in tumors, the tumor-specific regulators of [Ca2+]i oscillations are poorly characterized. We discovered that CD147 promotes hepatocellular carcinoma (HCC) metastasis and proliferation by enhancing the amplitude and frequency of [Ca2+]i oscillations in HCC cells. CD147 activates two distinct signaling pathways to regulate [Ca2+]i oscillations. By activating FAK-Src-IP3R1 signaling pathway, CD147 promotes Ca2+ release from endoplasmic reticulum (ER) and enhances the amplitude of [Ca2+]i oscillations. Furthermore, CD147 accelerates ER Ca2+refilling and enhances the frequency of [Ca2+]i oscillations through activating CaMKP-PAK1-PP2A-PLB-SERCA signaling pathway. Besides, CD147-promoted ER Ca2+ release and refilling are tightly regulated by changing [Ca2+]i. CD147 may activate IP3R1 channel under low [Ca2+]i conditions and CD147 may activate SERCA pump under high [Ca2+]i conditions. CD147 deletion suppresses HCC tumorigenesis and increases the survival rate of liver-specific CD147 knockout mice by regulating [Ca2+]i oscillations in vivo. Together, these results reveal that CD147 functions as a critical regulator of ER-dependent [Ca2+]i oscillations to promote oncogenic progression in HCC.

Background: Dysregulated endocytosis of membrane proteins contributes significantly to several hallmarks of cancer. Basigin can enhance cancer progression, but its precise mechanism remains unclear. CD98 promotes cell spreading and tumorigenicity by triggering integrin clustering and enhancing cell adhesion to the extracellular matrix. The endocytosis and recyle of basigin and CD98 might play critical roles in cancer., Methods: The role of CD98 was confirmed in liver cancer cells by cell spreading in vitro and tumorigenicity by nude mice xenograft tumor assay in vivo; membrane expression of basigin and CD98 in SMMC-7721 was measured by FCAS; pull down and SPR analysis were uses to reveal the direct association between basigin and CD98; DsRed1 tagged CD98 was blocked in the cytoplasm in K7721 (whose basigin was knockn out) and had a well colocalization with ER and Rab5a positive recycling endosomes under co-focal; finally, by FRET imaging and FCAS we observed the internalization of basigin and CD98 was flotillin-1-regulated, and their recycle at early steps was Arf6-mediated., Results: Basigin and CD98 were highly expressed and co-localized on the human hepatocellular carcinoma (HCC) cell membrane; basigin can directly bind to CD98, mediating CD98 redistribution on the HCC cell membrane and activating the downstream integrin signaling pathway. Internalization of basigin and CD98 was flotillin-1 regulated the and their recycling was mediated by Arf6. This recycling process for basigin and CD98 promotes cell spreading and tumor growth in liver cancer xenografts., Conclusion: Basigin, as a redistribution chaperone of CD98, plays a critical role in promoting cell spreading and the progression of hepatocellular carcinoma.