MiR-124 affects the apoptosis of brain vascular endothelial cells and ROS production through regulating PI3K/AKT signaling pathway.

The article "MiR-124 affects the apoptosis of brain vascular endothelial cells and ROS production through regulating PI3K/AKT signaling pathway, by S.-W. Wang, L.-X. Deng, H.-Y. Chen, Z.-Q. Su, S.-L. Ye, W.-Y. Xu, published in Eur Rev Med Pharmacol Sci 2018; 22 (2): 498-505-DOI: 10.26355/eurrev_201801_14201-PMID: 29424909" has been withdrawn from the authors due to inaccuracies (there are some errors and incorrect data). The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/14201.

MiR-124 affects the apoptosis of brain vascular endothelial cells and ROS production through regulating PI3K/AKT signaling pathway.

OBJECTIVE: The apoptosis of vascular endothelial cells (VEC) is related to ischemic stroke. Phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT/PKB) signaling pathway can upregulate Bcl-2 expression, reduce reactive oxygen species (ROS) production, and induce apoptosis. The level of miR-124 was significantly increased after cerebral ischemia. This study aimed to investigate the role of miR-124 in regulating PI3K expression, brain VEC apoptosis, and ROS production. MATERIALS AND METHODS: The expressions of miR-124, PI3K, p-AKT, and Bcl-2 in brain VEC of rats from the sham group and middle cerebral artery occlusion (MCAO) group were tested. Bioinformatics analysis showed the complementary binding site between miR-124 and PI3K mRNA. ROS content and cell apoptosis were detected by flow cytometry. Rat brain VEC were cultured in vitro and treated by oxygen-glucose deprivation (OGD) for 6 h. VECs were divided into four groups, including miR-NC, miR-124 inhibitor, pIRES2-blank, and pIRES2-PI3K groups, and were further treated by OGD. RESULTS: MiR-124 expression, ROS content, and cell apoptosis were markedly increased, whereas the levels of PI3K, p-AKT, and Bcl-2 were markedly reduced in rat VECs from MCAO group compared with that in the sham group. OGD treatment significantly induced VECs apoptosis, upregulated miR-124 expression and ROS content, and down-regulated the levels of PI3K, p-AKT, and Bcl-2. MiR-124 inhibitor or transfection of pIRES2-PI3K plasmid apparently enhanced PI3K, p-AKT, and Bcl-2 expressions, alleviated cell apoptosis and decreased ROS content in VECs induced by OGD. CONCLUSIONS: Our data demonstrated that miR-124 induced the apoptosis of brain vascular endothelial cells via the down-regulation of PI3K/AKT signaling pathway and promotion of ROS production.

Blood service in the Tibetan regions of Garzê and Aba, China: a longitudinal survey.

BACKGROUND: Garzê and Aba form the second largest Tibetan-inhabited area of China. Blood services have never been reported for this region before. OBJECTIVE: To assess the current situation and analyse whether a safe and adequate blood supply has been developed in both Garzê and Aba. METHODS: We conducted a longitudinal survey covering the period 2011-2016. The subjects of interest were recruited from non-remunerated voluntary donation, blood testing, clinical transfusion practices and infrastructure of local blood service systems. RESULTS: The donation rate and blood collection volume were below the average levels of both the Sichuan Province and mainland China. Component therapy was widely used, but inappropriate usage of whole blood existed. A lack of national specific standards for people on the plateaus led to local blood transfusions being conducted without full clinical assessment. Endemic and frequently occurring disease, such as hydatid disease and gastrointestinal disease, were inevitable risks for blood utilisation and safety. The potential influence of religious belief and traditions, like 'male-leaving marriages', of Tibetans on donor recruitment and blood safety requires further research. CONCLUSIONS: A relatively safe and complete blood service system has been developed in this region. However, there is still an urgent need for comprehensive and effective support from the government in terms of policies and finance. As an epidemic area of hydatid disease and sexually transmitted disease, this region needs to emphasise public health measures, such as blood safety and inappropriate usage of blood products.

Cohn process influences the functional anticoagulant activity of human protein C.

Cohn fraction IV (CFIV) is a byproduct of a plasma fractionation process known as the Cohn process. It is an inexpensive source of protein C, retaining about 90% of protein C (PC) in human plasma. We investigated whether PC is affected during the Cohn process and evaluated correlations among coagulant activity, amidolytic activity and PC antigen during the Cohn process. CFIV was redissolved with citrate-buffered saline for 5 h at 4°C, and then centrifuged at 3500 g for 40 min at 4°C. Functional anticoagulant activity was measured with a one-stage coagulation method based on activated partial thromboplastin time. The functional amidolytic activity of PC was determined using chromogenic substrate assay, and measurement of PC antigen was performed by ELISA. In CFIV, anticoagulant activity declined significantly, with a loss of >80%, while amidolytic activity was not significantly altered, compared to PC antigen. Prior to the Cohn process, high-rank correlations were observed in cryosupernatant, with rs = 0.921 for anticoagulant and amidolytic activities (P = 0.009), 0.896 for anticoagulant activity and antigen (P = 0.014) and 0.832 for amidolytic activity and antigen (P = 0.031). After the Cohn process in CFIV, there was also a high correlation between amidolytic activity and antigen (rs = 0.782, P = 0.038). There were no significant correlations between anticoagulant activity and antigen (rs = 0.223, P = 0.653), or anticoagulant and amidolytic activity (rs = 0.236, P = 0.675). We conclude that the Cohn process significantly influences the anticoagulant activity of PC. Compared to the antigen, PC lost greater than 80% of its anticoagulant activity, but retained its amidolytic activity, during the Cohn process.

Transmission of avian influenza A(H7N9) virus from father to child: a report of limited person-to-person transmission, Guangzhou, China, January 2014.

We investigated a possible person-to-person transmission within a family cluster of two confirmed influenza A(H7N9) patients in Guangzhou, China. The index case, a man in his late twenties, worked in a wet market that was confirmed to be contaminated by the influenza A(H7N9) virus. He developed a consistent fever and severe pneumonia after 4 January 2014. In contrast, the second case, his five-year-old child, who only developed a mild disease 10 days after disease onset of the index case, did not have any contact with poultry and birds but had unprotected and very close contact with the index case. The sequences of the haemagglutinin (HA) genes of the virus stains isolated from the two cases were 100% identical. These findings strongly suggest that the second case might have acquired the infection via transmission of the virus from the sick father. Fortunately, all 40 close contacts, including the other four family members who also had unprotected and very close contact with the cases, did not acquire influenza A(H7N9) virus infection, indicating that the person-to-person transmissibility of the virus remained limited. Our finding underlines the importance of carefully, thoroughly and punctually following-up close contacts of influenza A(H7N9) cases to allow detection of any secondary cases, as these may constitute an early warning signal of the virus's increasing ability to transmit from person-to-person.

GIDEON (Global Investigation of therapeutic DEcisions in hepatocellular carcinoma and Of its treatment with sorafeNib): second interim analysis.

BACKGROUND: GIDEON (Global Investigation of therapeutic DEcisions in hepatocellular carcinoma [HCC] and Of its treatment with sorafeNib) is a global, prospective, non-interventional study undertaken to evaluate the safety of sorafenib in patients with unresectable HCC in real-life practice, including Child-Pugh B patients who were excluded from clinical trials. METHODS: Patients with unresectable HCC, for whom the decision to treat with sorafenib, based on the approved label and prescribing guidelines, had been taken by their physician, were eligible for inclusion. Demographic data and disease/medical history were recorded at entry. Sorafenib dosing and adverse events (AEs) were collected at follow-up visits. The second interim analysis was undertaken when ~1500 treated patients were followed up for ≥ 4 months. RESULTS: Of the 1571 patients evaluable for safety, 61% had Child-Pugh A status and 23% Child-Pugh B. The majority of patients (74%) received the approved 800 mg initial sorafenib dose, regardless of Child-Pugh status; however, median duration of therapy was shorter in Child-Pugh B patients. The majority of drug-related AEs were grade 1 or 2, and the most commonly reported were consistent with previous reports. The incidence and nature of drug-related AEs were broadly similar across Child-Pugh, Barcelona Clinic Liver Cancer (BCLC) and initial dosing subgroups, and consistent with the overall population. CONCLUSIONS: Consistent with the first interim analysis, overall safety profile and dosing strategy are similar across Child-Pugh subgroups. Safety findings also appear comparable irrespective of initial sorafenib dose or BCLC stage. Final analyses in > 3000 patients are ongoing.

First interim analysis of the GIDEON (Global Investigation of therapeutic decisions in hepatocellular carcinoma and of its treatment with sorafeNib) non-interventional study.

AIMS: Global Investigation of therapeutic DEcisions in hepatocellular carcinoma and Of its treatment with sorafeNib (GIDEON), a global, non-interventional, surveillance study, aims to evaluate the safety of sorafenib in all patients with unresectable hepatocellular carcinoma (uHCC) under real-life practice conditions, particularly Child-Pugh B patients, who were not well represented in clinical trials. METHODS: Treatment decisions are determined by each physician according to local prescribing guidelines and clinical practice. Patients with uHCC who are candidates for systemic therapy, and for whom a decision has been made to treat with sorafenib, are eligible for inclusion. Demographic data and medical and disease history are recorded at entry. Sorafenib dosing and adverse events (AEs) are collected throughout the study. RESULTS: From January 2009 to April 2011, >3000 patients from 39 countries were enrolled. The prespecified first interim analysis was conducted when the initial approximately 500 treated patients had been followed up for ≥4 months; 479 were valid for safety evaluation. Preplanned subgroup analyses indicate differences in patient characteristics, disease aetiology and previous treatments by region. Variation in sorafenib dosing by specialty are also observed; Child-Pugh status did not appear to influence the starting dose of sorafenib. The type and incidence of AEs was consistent with findings from previous clinical studies. AE profiles were comparable between Child-Pugh subgroups. DISCUSSION: The GIDEON study is generating a large, robust database from a broad population of patients with uHCC. First interim analyses have shown global and regional differences in patient characteristics, disease aetiology and practice patterns. Subsequent planned analyses will allow further evaluation of early trends.

High expression of CXCR7 combined with Alpha fetoprotein in hepatocellular carcinoma correlates with extra-hepatic metastasis to lung after hepatectomy.

The lung is the most frequent metastatic site of hepatocellular carcinoma (HCC), negatively impacting on survival rates. In this study, we evaluated the prognostic role of the chemokine receptor CXCR7 in lung metastasis of HCC after hepatectomy, using immunohistochemical detection on tissue microarrays of HCCs, with and without lung metastasis. Using three categories based on staining characteristics, patients with high CXCR7 expression demonstrated a shorter time to development of lung metastasis compared with patients with low CXCR7 expression (log-rank test) with no effet on overall survival. Analysis of tissue adjacent to tumor showed patients with microvascular invasion to have higher CXCR7. Stratification based on alpha fetoprotein level >20 ng/ml also showed high expression of CXCR7 to be a strong independent prognostic factor. These findings suggest that high expression of CXCR7 in HCCs with elevated alpha fetoprotein levels correlates with metastasis to lung and poor survival after hepatectomy, indicating potential use as a prognostic factor.

Design and rationale for the non-interventional Global Investigation of Therapeutic DEcisions in Hepatocellular Carcinoma and Of its Treatment with Sorafenib (GIDEON) study.

BACKGROUND: Hepatocellular carcinoma (HCC) is a complicated condition influenced by multiple confounding factors, making optimum patient management extremely challenging. Ethnicity, stage at diagnosis, comorbidities and tumour morphology affect outcomes and vary from region to region, and there is no common language to assess patient prognosis and make treatment recommendations. Despite recent efforts to reduce the incidence of HCC, most patients present with unresectable disease. Non-surgical treatments include ablation, transarterial chemoembolisation and the multikinase inhibitor, sorafenib, but their effects in all patient subgroups are not known and further information is needed to optimise the use of these treatments. AIMS: The Global Investigation of Therapeutic DEcisions in Hepatocellular Carcinoma and Of its Treatment with SorafeNib (GIDEON) study (ClinicalTrials.gov identifier NCT00812175; http://clinicaltrials.gov/) is an ongoing global, prospective, non-interventional study of patients with unresectable HCC who are eligible for systemic therapy and for whom the decision has been taken to treat with sorafenib under real-life practice conditions. The aim of this study is to evaluate the safety and efficacy of sorafenib in different subgroups, especially Child-Pugh B where data are limited. DISCUSSION: This study will recruit 3000 patients from > 40 countries and follow them for approximately 5 years to compile a large and robust database of information that will be used to analyse local, regional and global differences in baseline characteristics, disease aetiology, treatment practice patterns and treatment outcomes, with a view to improve the knowledge base used to guide physician treatment decisions and to improve patient outcomes.

Non-invasive foetal RHD genotyping via real-time PCR of foetal DNA from Chinese RhD-negative maternal plasma.

BACKGROUND: A majority of studies predicting the foetal RhD blood group in free foetal DNA from RhD-negative maternal plasma have been conducted in Caucasian populations, whereas limited data have been accumulated for Asian populations. In this study, we assessed the feasibility of prenatal genotyping of RHD in RhD-negative Chinese pregnant women. MATERIALS AND METHODS: Cell-free plasma DNA was extracted from 78 RhD-negative Chinese women carrying a singleton foetus (gestation between 14 and 40 weeks). Foetal DNA was confirmed by testing SRY or nine different polymorphic STR loci in the maternal plasma and buffy coat. Foetal RHD exons 5, 7 and 10 and intron 4 were successfully amplified with RQ-PCR. The RHD1227A allele was examined in all RhD-positive individuals. The foetal RHD genotyping results were compared with the infant cord blood serological analysis. RESULTS: Among the 78 specimens, RHD genotyping results of 70 cases were in complete concordance with serological results from foetal umbilical cord blood. Sixty of these cases were identified as RhD-positive, and 10 cases were typed as RhD-negative. In addition, five cases were 'false-positives', while three cases were considered inconclusive. The detection rate was 89.7% (70/78). In four of the five 'false-positive' cases, the RhDel phenotype was assessed by detecting the RHD1227A allele. Thus, this method yielded a 94.9% (74/78) accuracy rate. CONCLUSIONS: The correct foetal RhD phenotype may be accurately predicted from RhD-negative maternal plasma in Chinese subjects. The RHD1227A allele proved to be an important genetic marker in the RhDel Chinese population.

A tumor-specific conditionally replicative adenovirus vector expressing TRAIL for gene therapy of hepatocellular carcinoma.

We constructed a novel hepatocellular carcinoma-specific conditionally replicative adenovirus (CRAd). This adenovirus, designated Ad.HS4.AFP.E1A/TRAIL, expresses E1A to mediate viral replication and TRAIL to enhance HCC-killing efficacy under the control of a modified AFP promoter. An insulator HS-4 was placed in front of the AFP promoter to enhance the fidelity of the heterologous promoter. This virus was shown to have specific cytolytic activity in AFP-expressing HCC cells in vitro. Furthermore, the replication efficiency of Ad.HS4.AFP.E1A/TRAIL correlated well with AFP expression of the host cells, showing a 100-fold and 1 000 000-fold decrease in the low-and non-AFP-expressing HCC cells, respectively, compared to the high AFP-expressing HCC cells. An increase in mRNA of TRAIL and the elevated Caspase-3 activity were also observed in Ad.HS4.AFP.E1A/TRAIL-infected HCC cells. These results indicated that TRAIL expression from the viral vector activated the Caspase-3 enzymatic capacity and the HCC cells were sensitive to TRAIL. In vivo, Ad.HS4.AFP.E1A/TRAIL effectively prevented the growth of low AFP-expressing BEL-7404 xenografts. These results indicate that Ad.HS4.AFP.E1A/TRAIL could provide a new strategy of gene therapy for HCC.

Combined IL-12 and GM-CSF gene therapy for murine hepatocellular carcinoma.

Among various immunotherapeutic approaches, interleukin-12 (IL-12) is particularly appealing because of its superior antitumor effects, which have been demonstrated in preclinical as well as clinical studies. However, IL-12 therapy was often accompanied by severe side effects due mainly to the supranormal induction of interferon-gamma. To optimize the therapeutic efficacy and lower the side effects of IL-12, we have investigated the antitumor activity of combined IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene therapy in a highly malignant and poorly immunogenic murine hepatocellular carcinoma model. Using a versatile hydrodynamics-based DNA delivery method, we showed that the combined gene delivery of IL-12 and GM-CSF induced very strong antitumor cellular immunity and achieved significant therapeutic efficacy, whereas each cytokine gene alone yielded appreciable but less effects. We also observed that the combined therapy induced lower levels of interferon-gamma than did IL-12 alone. These results suggest that combined IL-12 and GM-CSF therapy can render a stronger antitumor effect as well as lowering potential side effects.

The potential of plasma thrombomodulin as a biomarker of portal vein tumor thrombus in hepatocellular carcinoma.

PURPOSE: To study the relationship between thrombomodulin (TM) plasma levels and the formation of portal vein tumor thrombus (PVTT) in patients with hepatocellular carcinoma (HCC). METHODS: Pre- and-postoperative plasma TM levels of 45 patients with HCC and six patients with benign liver-occupying lesion were measured by enzyme-linked immunosorbent assay (ELISA), and the expression of TM in human HCC tissues was determined by immunohistochemistry assay. RESULTS: The preoperative plasma TM level of patients with HCC (10.2+/-5.7 ng/ml) was significantly higher than that of those patients with benign liver-occupying lesion (6.1+/-2.2 ng/ml) and that of normal controls (5.7+/-1.0 ng/ml), respectively (P<0.05). The postoperative TM level of 40 patients with HCC whose tumors had been removed decreased significantly than the preoperative TM level (10.8+/-5.3 ng/ml versus 7.6+/-4.2 ng/ ml, P < 0.05), whereas there was no significant difference between the preoperative and postoperative TM level of six patients with benign liver-occupying lesion (6.1+/-2.2 ng/ml versus 5.9+/-1.8 ng/ml, P>0.05). The preoperative plasma TM level of patients with single HCC (11.5+/-5.9 ng/ml) or no PVTT (11.4+/-5.6 ng/ml) was significantly higher than that of those patients with multiple HCC (8.1+/-4.6 ng/ml) or PVTT (6.9+/-4.5 ng/ ml), respectively (P<0.05). The preoperative plasma TM level of the patients with HCC tissue that stained positive for TM was significantly higher than those with tissue that stained negative for TM (12.2+/-6.5 ng/ ml versus 8.7+/-4.6 ng/ml, P<0.05). The postoperative plasma TM level showed no difference between the patients with HCC tissue stained positive and negative for TM (8.3+/-4.1 ng/ml versus 7.6+/-4.4 ng/ml, P>0.05). There was also no significant difference between the plasma TM level and other clinicopathological futures. CONCLUSIONS: Plasma TM increases in patients with HCC and can be a biomarker of the formation of PVTT.

Chromosome 8p deletion is associated with metastasis of human hepatocellular carcinoma when high and low metastatic models are compared.

Recently, we found that chromosome 8p deletion might be associated with hepatocellular carcinoma (HCC) metastasis by analyzing the differences in chromosomal alterations between primary tumors and their matched metastatic lesions of HCC with comparative genomic hybridization (CGH) (Qin et al. 1999). To further confirm this interesting finding, the genomic changes of two models bearing human HCC with different metastatic potentials (LCI-D20 and LCI-D35), and the new human HCC cell line with high metastatic potential (MHCC97) were analyzed by CGH. Gains on 1q, 6q, 7p, and 8q, and losses on 13p, 14p, 19p, 21, and 22 were detected in both LCI-D20 and LCI-D35 models. However, high copy number amplification of a minimum region at 1q12-q22 and 12q, and deletions on 1p32-pter, 3p21-pter, 8p, 9p, 10q, 14q, and 15p were detected only in the LCI-D20 model. Gains on 1p21-p32, 2p13-p21, 6p12-pter, 9p, 15q, and 16q11-q21, and losses on 2p23-pter, 4q24-qter, 7q31-qter, 12q, 17p, and 18 were detected only in the LCI-D35 model. The chromosomal aberration patterns in the MHCC97 cell line were similar to its parent LCI-D20 model, except that gains on 19q and losses on 4, 5, 10q, and 13q were found only in the cell line. These results provide some indirect clues to the metastasis-related chromosomal aberrations of HCC and further support the finding that 8p deletion is associated with HCC metastasis. 1q12-22 and 12q might harbor a novel oncogene(s) that contributes to the development and progression of HCC. Amplification on 8q and deletions on 4q and 17p may be not necessary for HCC metastasis.

Experience of 1000 patients who underwent hepatectomy for small hepatocellular carcinoma.

BACKGROUND: Recently, the implementation of screening programs using alpha-fetoprotein (AFP) and ultrasonography in high risk populations has identified increasing numbers of patients with small hepatocellular carcinoma (small HCC). The aim of this study was to summarize the authors' experience in patients who underwent hepatectomy for small HCC and the factors that influence or improve long term survival. METHODS: The study included 1000 patients who underwent hepatectomy for small HCC (< or = 5 cm) and compared them with 1366 patients who underwent hepatectomy for large HCC (> 5 cm) during the same period. A Cox proportional-hazards model was used for multivariate analysis of prognostic factors. RESULTS: Comparison between patients with small HCC (n = 1000 patients) and patients with large HCC (n = 1366 patients) revealed that those with small HCC had a higher resection rate (93.6% [1000 of 1068 patients] vs. 55.7% [1366 of 2451 patients]; P < 0.01), a higher curative resection rate (80.5% [805 of 1000 patients] vs. 60.7% [829 of 1366 patients]; P < 0.01), a lower operative mortality rate (1.5% [15 of 1000 patients] vs. 3.7% [50 of 1366 patients]; P < 0.01), better differentiation of tumor cells (Edmondson Grade 3-4; 14.9% vs. 20.1%; P < 0.01), a higher incidence of single nodule tumors (82.6% vs. 64.4%; P < 0.01), a higher proportion of well encapsulated tumors (73.3% vs. 46.3%; P < 0.01), a lower incidence of tumor emboli in the portal vein (4.9% vs. 20.8%; P < 0.01), and higher survival rates after undergoing resection (5 years: 62.7% vs. 37.1%; P < 0.01; 10 years: 46.3% vs. 29.2%; P < 0.01). No significant difference was found between survival after undergoing minor resection (n = 949 patients) or lobectomy (n = 51 patients) in patients with small HCC (P > 0.05). Reresection for subclinical recurrence or solitary pulmonary metastasis after small HCC resection was undertaken in 84 patients. CONCLUSIONS: Resection is still the modality of first choice for the treatment of patients with small HCC. Minor resection instead of lobectomy was the key to increasing resectability and decreasing operative mortality, and reresection for subclinical recurrence or solitary pulmonary metastasis was important approach to prolonging survival further.

Multimodality treatment in hepatocellular carcinoma patients with tumor thrombi in portal vein.

AIM: To compare the therapeutic effect and significances of multimodality treatment for hepatocellular carcinoma (HCC) with tumor thrombi in portal vein (PVTT). METHODS: HCC patients (n=147) with tumor thrombi in the main portal vein or the first branch of portal vein were divided into four groups by the several therapeutic methods. There were conservative treatment group in 18 out of patients (group A); and hepatic artery ligation(HAL) and/or hepatic artery infusion (HAI) group in 18 patients (group B), in whom postoperative chemoembolization was done periodically; group of removal of HCC with PVTT in 79 (group C) and group of transcatheter hepatic arterial chemoembolization (TACE) or HAI and/or portal vein infusion (PVI) after operation in 32 (group D). RESULTS: The median survival period was 12 months in our series and the 1-,3-, and 5-year survival rates were 44.3%, 24.5% and 15.2%, respectively. The median survival times were 2, 5, 12 and 16 months in group A, B, C and D, respectively. The 1-, 3- and 5-year survival rates were 5.6%, 0% and 0% in group A; 22.2%, 5.6% and 0% in group B; 53.9%, 26.9% and 16.6% in group C; 79.3%, 38.9% and 26.8% in group D, respectively. Significant difference appeared in the survival rates among the groups (P < 0.05). CONCLUSION: Hepatic resection with removal of tumor thrombi and HCC should increase the curative effects and be encouraged for the prolongation of life span and quality of life for HCC patients with PVTT, whereas the best therapeutic method for HCC with PVTT is with regional hepatic chemotherapy or chemoembolization after hepatic resection with removal of tumor thrombi.

Metastatic human hepatocellular carcinoma models in nude mice and cell line with metastatic potential.

Metastatic human HCC model is needed for the studies on mechanism and intervention of metastatic recurrence. By using orthotopic implantation of histologically intact tissues of 30 surgical specimens, a patient-like metastatic model of human HCC in nude mice (LCI-D20) and a low metastatic model of human HCC in nude mice (LCI-D35) have been established. All mice with transplanted LCI-D20 tumors exhibited extremely high metastatic ability including spontaneous metastasis to liver, lungs, lymph nodes and peritoneal seeding. Remarkable difference was also found in expression of some of the invasiveness related genes and growth factors between the LCI-D20 and LCI-D35 tumors. PAI-1 increased gradually following tumor progression in LCI-D20 model, and correlated with tumor size and AFP level. Phasic expression of tissue intercellular adhesion molecule-1 in this model was also observed. Using corneal micropocket model, it was demonstrated that the vascular response induced by LCI-D20 tumor was stronger than that induced by LCI-D35 tumor. Similar report on metastatic human HCC model in nude mice and human HCC cell line with metastatic potential was rarely found in the literature. This LCI-D20 model has been widely used for the studies on intervention of metastasis, including anti-angiogenesis,antisense approach, metalloproteinase inhibitor, differentiation inducer, etc. It is concluded that the establishment of metastatic human HCC model in nude mice and human HCC cell line with metastatic potential will provide important models for the in vitro and in vitro study of HCC invasiveness, angiogenesis as well as intervention of HCC recurrence.

Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97.

AIM: To establish clone cells with different metastatic potential for the study of metastasis-related mechanisms. METHODS: Cloning procedure was performed on parental hepatocellular carcinoma (HCC) cell line MHCC97, and biological characteristics of the target clones selected by in vivo screening were studied. RESULTS: Two clones with high (MHCC97-H) and low (MHCC97-L) metastatic potential were isolated from the parent cell line. Compared with MHCC97-L, MHCC97-H had smaller cell size (average cell diameter 43 microm vs 50 microm) and faster in vitro and in vivo growth rate (tumor cell doubling time was 34.2h vs 60.0h). The main ranges of chromosomes were 55-58 in MHCC97-H and 57-62 in MHCC97-L. Boyden chamber in vitro invasion assay demonstrated that the number of penetrating cells through the artificial basement membrane was (37.5 +/- 11.0) cells/field for MHCC97-H vs (17.7 +/- 6.3)/field for MHCC97-L. The proportions of cells in G0-G1 phase, S phase, and G2-M phase for MHCC97-H/MHCC97-L were 0.56/0.65, 0.28/0.25 and 0.16/0.10, respectively, as measured by flow cytometry. The serum AFP levels in nude mice 5wk after orthotopic implantation of tumor tissue were (246 +/- 66) microg.L(-1) for MHCC97-H and (91 +/- 66) microg.L(-1) for MHCC97-L. The pulmonary metastatic rate was 100% (10/10) vs 40% (4/10). CONCLUSION: Two clones of the same genetic background but with different biological behaviors were established, which could be valuable models for investigation on HCC metastasis.

The targeted expression of interleukin-2 in human hepatocellular carcinoma cells.

To improve the safety and efficiency of human hepatocellular carcinoma (HCC) gene therapy, we explored the use of a liver-specific promoter and a tumor-specific enhancer to achieve regular IL-2 gene expression for treatment of HCC. The human alpha-fetoprotein (AFP) enhancer [E(AFP)] and the albumin promoter [P(ALB)] were amplified from human genomic DNA. We used eukaryotic expression vector pcDNA-3 for the delivery of the IL-2 gene because this plasmid is a non-transient, fast-selection expression vector. A recombinant plasmid was constructed including the selectable marker neoR gene and the human IL-2 gene derived by the E(AFP) - P(ALB). The liver-predominant expression pattern of the IL-2 gene was observed in the medium of the transfected cells. When human HCC cell lines displaying different levels of AFP and non-hepatocyte tumor cell lines were transfected with the recombinant plasmid, IL-2 was expressed highly in AFP and albumin-positive HCC cells, but low in nonhepatocyte tumor cells. Moreover, the expression level of IL-2 gene was positively proportional to the level of AFP expression in the transfected cells.

High-dose and long-term therapy with interferon-alfa inhibits tumor growth and recurrence in nude mice bearing human hepatocellular carcinoma xenografts with high metastatic potential.

Postoperative recurrence of human hepatocellular carcinoma (HCC) is the major issue that must be addressed to further improve prognosis. This study was undertaken to investigate the effects of interferon-alfa-1b (IFN-alpha-1b) on recurrent tumor and metastasis after curative resection in nude mice bearing an HCC xenograft with high metastatic potential. Tumor tissues from LCI-D20, a metastatic model of HCC in nude mice, were orthotopically implanted in 105 nude mice. Eleven days later, 64 mice underwent curative resection of liver tumors. IFN-alpha at different doses was administered subcutaneously to mice with or without resection. In mice without resection, when comparison was made among control, IFN 7.5 x 10(6) U/kg/day, 1.5 x 10(7) U/kg/day for treated groups, and 3 x 10(7) U/kg/day; tumor volume was 8,475 mm(3) +/- 2,636 mm(3), 7,963 mm(3) +/- 3,214 mm(3), 769 mm(3) +/- 287 mm(3), and 13 mm(3) +/- 9 mm(3); incidence of lung metastasis being 100%, 80%, 40%, and 0%; life span was 45 +/- 4 days, 53 +/- 8 days, 81 +/- 6 days, and 105 +/- 24 days, respectively. In mice with curative resection, when comparison was made among control, IFN 5 x 10(5) U/kg/day, 1 x 10(6) U/kg/day, 4 x 10(6) U/kg/day, 7.5 x 10(6) U/kg/day, 1.5 x 10(7) U/kg/day, and 3 x 10(7) U/kg/day for treated groups; incidence of recurrent tumor was 100%, 100%, 87.5%, 100%, 87.5%, 62.5%, and 12.5%; lung metastasis being 100%, 75%, 87.5%, 50%, 62.5%, 0%, and 0%, respectively. IFN-alpha inhibited neovascularization induced by LCI-D20 tumor specimens implanted into the micropocket of nude mice corneas. In conclusion, high-dose and long-term therapy with IFN-alpha dose-dependently inhibits tumor growth and recurrence after resection of HCC. The effect of IFN-alpha may be attributed to antiangiogenesis in this experiment. These results provide potential clinical implication, particularly for the prevention of recurrence after curative resection of HCC.