23-hydroxybetulinic acid reduces tumorigenesis, metastasis and immunosuppression in a mouse model of hepatocellular carcinoma via disruption of the MAPK signaling pathway.

Hepatocellular carcinoma (HCC) shows recurrence and lung metastasis even after treatment. 23-hydroxybetulinic acid (23-HBA), a major active constituent of Pulsatilla chinensis, exhibits potent antitumor activities. We herein investigate the biological effect of 23-HBA on metastasis and immunosuppression in a mouse model of HCC. Microarray-based gene expression profiling was employed to identify the target genes of 23-HBA in the treatment of HCC. The effect of 23-HBA on the progression of HCC was evaluated by in-vitro cell function measurements along with in-vivo xenograft implantation, lung metastasis and CD11b+Gr1+ staining experiments. The potential mechanism involving target signaling pathway was investigated by western blot analysis. Bioinformatics analysis revealed that matrix metalloproteinase 2 (MMP2) was a key target gene mediated by 23-HBA in HCC, whereas Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis demonstrated that MMP2 mainly affects the development and metastasis of HCC. 23-HBA significantly reduced cell malignant functions in vitro while delaying the HCC growth and metastasis in vivo. In addition, the number of myeloid-derived suppressor cells was shown to be reduced following administration of 23-HBA in mice. Mechanistic analysis indicated that these effects of 23-HBA during HCC were involved with the mitogen-activated protein kinase (MAPK) signaling pathway inactivation and resulted in decreased phosphorylation of both mitogen-activated protein kinases 1/2 and extracellular signal-regulated kinase 1/2. Our study reveals that 23-HBA acts as a tumor suppressor agent and suppresses HCC tumorigenesis, metastasis and immunosuppression via blockade of the MAPK signaling pathway, suggesting that 23-HBA may serve as a promising drug target to treat HCC.

Notch1-Snail1-E-cadherin pathway in metastatic hepatocellular carcinoma.

Notch signaling, a critical pathway for tissue development, also contributes to tumorigenesis in many cancers, but its pathological function in liver cancer is not well defined. In our study, Notch1 expression and its clinicopathological parameters were evaluated in 82 human hepatocellular carcinoma (HCC) patients. Plasmid-based siNotch1 shRNA was transiently or stably transfected into metastatic HCC cells and subsequently evaluated for the effects on orthotopic liver tumor metastasis in a mouse model as well as the effects on downstream pathways. Aberrant high expression of Notch1 was significantly associated with metastatic disease parameters in HCC patients, such as tumor-node-metastasis Stages III-IV and tumor venous invasion. Knocking-down Notch1 reduced cell motility in vitro and orthotopic tumor metastasis from the liver to the lung in vivo in a mouse model. In metastatic HCC cells, abnormal expression of Notch1 was associated with increased expression of Snail1 and repressed expression of E-cadherin; the Notch1-Snail1-E-cadherin association can also be found in HCC patient tumors. Inhibition of Notch1 by shRNA abolished Snail1 expression, which further resulted in the re-establishment of repressed E-cadherin in metastatic HCC cells. Thus, abnormal Notch1 expression was strongly associated with HCC metastatic disease, which might be mediated through the Notch1-Snail1-E-cadherin pathway. Knock-down of Notch1 reversed HCC tumor metastasis in a mouse model. Therefore, these data suggest that effective targeting of Notch signaling might also inhibit tumor metastasis.

LncRNA ZNF674-AS1 Hinders Proliferation and Invasion of Hepatic Carcinoma Cells through the Glycolysis Pathway.

Purpose: Long noncoding RNAs (lncRNAs) play important roles in regulating various functions of cells at the levels of transcription and posttranscription. Extensive investigations have illustrated that lncRNAs are critical in the glucose metabolism of tumor cells, but their mechanisms of action need to be further explored. This study evaluates the role of lncRNA ZNF674-AS1 on the apoptosis and proliferation of human hepatic carcinoma cells in vitro through the glucose metabolism and its related mechanisms. Methods: Real-time quantitative PCR was employed for detecting the level of expressions for lncRNA ZNF674-AS1 in liver cancer tissues (25 cases), paracancerous tissues, and liver cancer cell lines. The lncRNA ZNF674-AS1 high expression cell strain was constructed by the lentiviral overexpression vector. CCK-8, plate colony formation, transwell assay, lactate production, glucose consumption, and ATP levels were used to detect the change of cell proliferation, colony formation, migration, and invasion, as well as glycolytic capability. Western blot was carried out to detect the expression of HK2, PFKL, PKM2, GLUT1, and PKM1, which are the key proteins of glycolysis in cells. Result: The lncRNA ZNF674-AS1 was undesirably expressed in liver cancer cell lines and tissues. Cell function assessments showed that compared with the blank control group (vector), overexpression of lncRNA ZNF674-AS1 could substantially hinder the proliferation, colony formation, migration, and invasion capability of liver cancer cells. Furthermore, overexpression of lncRNA ZNF674-AS1 could inhibit cell glycolysis (inhibit glucose consumption and reduce intracellular lactate and ATP levels) by inhibiting the expression of key proteins (such as PFKL, HK2, PKM2, and GLUT1) in the process of glycolysis. Conclusion: As a tumor repressor gene, lncRNA ZNF674-AS1 inhibits the expression of key proteins in glycolysis to inhibit glycolysis level, thereby inhibiting cell migration and proliferation. Therefore, lncRNA ZNF674-AS1 could be a potent therapeutic target or a novel diagnostic molecule for patients suffering from liver cancer.

A Five-Gene-Based Prognostic Signature for Hepatocellular Carcinoma.

Objective: This study intends to identify potential prognostic marker genes associated with the prognosis of patients suffering from hepatocellular carcinoma (HCC) based on TCGA and GEO analysis. Methods: TCGA-LIHC cohort was downloaded and the data related to HCC were extracted from The Cancer Genome Atlas (TCGA) database and subjected to differential analysis. HCC-related gene expression datasets were retrieved from the GEO database, followed by differential analysis. After intersection of the results of TCGA and GEO databases, gene interaction analysis was performed to obtain the core genes. To identify the genes related to the prognosis of HCC patients, we conducted univariate and multivariate Cox analyses. Results: Based on differential analysis of TCGA database, 854 genes were differentially expressed in HCC, any of which might link to the occurrence and progression of HCC. Meanwhile, joint analysis of HCC-related gene expression datasets in the GEO database screened 214 genes. Five core genes CDC20, TOP2A, RRM2, UBE2C and AOX1 were significantly associated with the prognosis of HCC patients and the risk model based on these five genes effectively predicted the prognosis of HCC patients. Conclusion: Collectively, our data suggest that CDC20, TOP2A, RRM2, UBE2C and AOX1 may be the key genes affecting the prognosis of patients with HCC. The five-gene signature could accurately predict the prognosis of HCC patients.

Feasibility of using a liver infected with Clonorchis sinensis for liver transplantation: fourteen cases.

Use of livers infected with Clonorchis sinensis as donor organs for transplantation is controversial because of the potential associated risks. The low availability of donor livers at Tianjin First Center Hospital since 2003 prompted us to undertake cadaveric liver transplantation in 14 patients using donor livers infected with C. sinensis. None of the donors had been diagnosed with liver fluke infection before organ procurement, and in none of them was there laboratory evidence of abnormal liver function. After livers had been harvested and preserved, dead liver flukes were found in the bile of each donor; subsequent pathological examination of the flukes confirmed the diagnosis of clonorchiasis. Conventional orthotopic liver transplantation, with insertion of a T- tube, was undertaken in all 14 patients. Praziquantel, 25 mg/kg three times daily for two days, was administrated to the recipients starting on postoperative day 2. Results of tests of liver function improved rapidly after the operation in all of the patients. The median duration of follow-up was 31 months. The 1- and 3-year survival rates of the grafts were 85.7% and 78.6%, respectively. Postoperative biliary complications occurred in 2 patients (14.3%). No ova were detected in the bile or feces of any of the patients postoperatively. These findings suggest that livers infested with C. sinensis can be used as donor organs for liver transplantation. Further studies are required to establish definitive criteria for determining whether such donor organs may be used in a liver transplantation program.

Identification of the Pathogenic Biomarkers for Hepatocellular Carcinoma Based on RNA-seq Analyses.

The purpose of this study was to explore potential biomarkers in the diagnosis of hepatocellular carcinoma (HCC) based on RNA-seq. The microarray data GSE98269 were downloaded from the GEO database, including the miRNA, mRNA and lncRNA expression profiles of 3 HCC tissues and 3 normal liver tissues from 3 HCC patients. The limma package was used to identify the differentially expressed miRNAs (DEMs) and the differentially expressed lncRNAs in HCC tissues compared with normal liver tissues. Database of DAVID, KEGG PATHWAY and Reactome were used to perform the functional and pathway enrichment. Putative targets for DEMs, and the miRNA-gene pairs were predicted via the miRWalk V2.0 database. The protein-protein pairs of DEGs were screened via String software. The expression features of the differentially expressed lncRNAs were analyzed. The regulated network of DEGs and DEMs were constructed, and related genes and miRNAs were detected in the HCC tissues and normal liver samples with Q-PCR. A total of 678 DEGs, 32 DEMs and 411 differential expressed lncRNAs were identified. The DEGs were enriched in 196 GO terms and 79 pathways. 38 negative regulation miRNA-gene pairs and 1205 protein-protein interactions were screened out, and the regulated network was constructed based on them. KNG1, CDK1, EHHADH, CYP3A4, hsa-miR-199a-5p and hsa-miR-455-3p might be biomarkers in the occurrence of HCC.

Liver retransplantation for ischemic-type biliary lesions after orthotopic liver transplantation: a clinical report of 66 cases.

BACKGROUND: Ischemic-type biliary lesions (ITBLs) play an extremely important role in influencing the long-term survival and quality of life of recipients after orthotopic liver transplantation (OLT). Some patients can be cured by interventional therapies, however others lose their grafts at last and receive liver retransplantation (re-OLT). The aim of this study was to analyze the data of 66 patients who had received re-OLT at our center because of ITBL and to discuss the treatment of ITBL after OLT. METHODS: We retrospectively analyzed 66 re-OLT cases due to ITBL from September 2001 to February 2007 at our center. The Kaplan-Meier method and the Cox-Mantel test were used to identify factors associated with mortality for univariate analysis and multivariate analysis, respectively. RESULTS: Fifty-five of 66 ITBL cases underwent interventional therapies before re-OLT. The actuarial survival at 1 month and 1 year for these patients was 83% and 74%, respectively. Prognostic factors for mortality in univariate analysis were model of end-stage liver disease score (MELD) >16.5 (Chi(2)=5.856, P=0.016), cold ischemia time >8 hours (Chi(2)=6.539, P=0.011), infections (Chi(2)=5.550, P=0.018) and complications (Chi(2)=12.168, P=0.002) after re-OLT. In the multivariate analysis (Cox regression), the risk factors independently associated with mortality were MELD score >16.5 (RR: 3.140; P=0.035), cold ischemia time >8.2 hours (RR: 0.192; P=0.016) and complications (RR: 3.896, P=0.003). CONCLUSIONS: The incidence of ITBL in China is higher than in other countries. Based on our experience, MELD score, cold ischemia time and complications after re-OLT are risk factors independently associated with mortality in retransplanted ITBL patients.

[Long-term complications after liver transplantation].

OBJECTIVE: To investigate the long-term complications after liver transplantation. METHODS: Totally 85 living patients who received liver transplantation from December 30th 1998 to May 28th 2002 in Tianjin First Central Hospital were followed up till October 2007. Liver and kidney functions, blood drug levels, blood pressure, blood sugar, and blood fat were recorded and ultrasound imaging was performed during follow-up. RESULTS: At the end of the study, most patients had experienced one or more complications of prolonged immunosuppressant treatment, including posttransplantation diabetes mellitus (21.18%, 18/85), hypertension (10.59%, 9/85), renal impairment (8.24%, 7/85), hyperlipemia (7.06%, 6/85), hyperuricaemia (7.06%, 6/85), denovo malignancy (2.35%, 2/85), new-onset hepatitis C (7.06%, 6/ 85), recurrent hepatitis B (5.56%, 4/72). CONCLUSION: Recipients of liver transportation often suffers long-term complications, which should be carefully managed to improve their quality of life.

Icotinib inhibits the proliferation of hepatocellular carcinoma cells in vitro and in vivo dependently on EGFR activation and PDL1 expression.

PURPOSE: Hepatocellular carcinoma (HCC) accounts for one of the most prevalent tumor types in the world and still lacks an effective treatment regimen. The EGFR tyrosine-kinase inhibitor icotinib is capable of inhibiting proliferation of several kinds of cancer cells, but its anticancer effect in HCC is still not verified. METHODS: In the current study, ten HCC cell lines were selected to test their original EGFR-activation status and PDL1 protein level, and in vitro antiproliferation assays were also conducted to analyze the IC50 and further investigate the correlation between IC50 and protein level of phosphorylated EGFR and PDL1. A in vivo nude mouse xenograft animal model was used as well to analyze its anticancer effect. RESULTS: Icotinib showed significant inhibitory effects only on HCC cell lines that had both higher p-EGFR and PDL1 protein level. This specific HCC cell line was subcutaneously injected to establish the in vivo xenograft tumor model, and icotinib reduced tumor weight remarkably and growth dose dependently. Molecular mechanism study revealed that icotinib inhibited the phosphorylation of EGFR and PDL1 expression in cancer cells and activated apoptosis. Knocking down PDL1 significantly reduced the inhibitory effect of icotinib on HCC, and knocking in PDL1 increased the sensitivity of icotinib in HCC. CONCLUSION: The current research suggests that icotinib has an inhibitory effect on a subgroup of HCC cells that have both higher p-EGFR and PDL1. This hints at the potential clinical usage of icotinib in HCC based on PDL1-biomarker examination.

Impact of non-oncological factors on tumor recurrence after liver transplantation in hepatocellular carcinoma patients.

Hepatocellular carcinoma (HCC) is the most common primary neoplasm of the liver and is one of the leading causes of cancer-related death worldwide. Liver transplantation (LT) has become one of the best curative therapeutic options for patients with HCC, although tumor recurrence after LT is a major and unaddressed cause of mortality. Furthermore, the factors that are associated with recurrence are not fully understood, and most previous studies have focused on the biological properties of HCC, such as the number and size of the HCC nodules, the degree of differentiation, the presence of hepatic vascular invasion, elevated serum levels of alpha-fetoprotein, and the tumor stage outside of the Milan criteria. Thus, little attention has been given to factors that are not directly related to HCC (i.e., "non-oncological factors"), which have emerged as predictors of tumor recurrence. This review was performed to assess the effects of non-oncological factors on tumor recurrence after LT. The identification of these factors may provide new research directions and clinical strategies for the prophylaxis and surveillance of tumor recurrence after LT, which can help reduce recurrence and improve patient survival.

Liver transplantation for hepatocellular carcinoma.

Marked improvement in the prognosis for patients with liver cancer who undergo liver transplantation has been achieved as a result of advances in liver transplantation techniques. Given the current shortage of organs in China, a favorable long-term survival rate might be achieved with rigorous selection of suitable patients and therefore benefit society the most. Further study of the mechanism of cancer recurrence following liver transplantation, continuing to optimize pretreatment strategies prior to liver transplantation, and paying closer attention to the prevention and treatment of cancer recurrence following liver transplantation are important steps to improve the long-term clinical benefit of liver transplantation for patients with hepatocellular carcinoma. Perfecting the techniques of liver transplantation using a marginal donor liver is the main way to solve the current problem of an organ shortage for patients with liver cancer.