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Transarterial chemoembolisation (TACE) is used for unresectable hepatocellular carcinoma (HCC) treatment, but TACE-induced hypoxia leads to poor prognosis. The anti-cancer effects of soybean isoflavones daidzein derivatives 7,3',4'-trihydroxyisoflavone (734THIF) and 7,8,4'-trihydroxyisoflavone (784THIF) were evaluated under hypoxic microenvironments. Molecular docking of these isomers with cyclooxygenase-2 (COX-2) and vascular endothelial growth factor receptor 2 (VEGFR2) was assessed. About 40 μM of 734THIF and 784THIF have the best effect on inhibiting the proliferation of HepG2 cells under hypoxic conditions. At a concentration of 40 μM, 784THIF significantly inhibits COX-2 expression in pre-hypoxia conditions compared to 734THIF, with an inhibition rate of 67.73%. Additionally, 40 μM 784THIF downregulates the expression of hypoxic, inflammatory, and metastatic-related proteins, regulates oxidative stress, and inhibits the expression of anti-apoptotic proteins. The uptake by HepG2 confirmed higher 784THIF level and slower degradation characteristics under post- or pre-hypoxic conditions. In conclusion, our results showed that 784THIF had better anti-cancer effects and cellular uptake than 734THIF.

Background: Advanced hepatocellular carcinoma (HCC) can be treated with sorafenib, which is the primary choice for targeted therapy. Nevertheless, the effectiveness of sorafenib is greatly restricted due to resistance. Research has shown that exosomes and circular RNAs play a vital role in the cancer's malignant advancement. However, the significance of exosomal circular RNAs in the development of resistance to sorafenib in HCC remains uncertain., Methods: Ultracentrifugation was utilized to isolate exosomes (Exo-SR) from the sorafenib-resistant HCC cells' culture medium. Transcriptome sequencing and differential expression gene analysis were used to identify the targets of Exo-SR action in HCC cells. To identify the targets of Exo-SR action in HCC cells, transcriptome sequencing and analysis of differential expression genes were employed. To evaluate the impact of exosomal circUPF2 on resistance to sorafenib in HCC, experiments involving gain-of-function and loss-of-function were conducted. RNA pull-down assays and mass spectrometry analysis were performed to identify the RNA-binding proteins interacting with circUPF2. RNA immunoprecipitation (RIP), RNA pull-down, electrophoretic mobility shift assay (EMSA), immunofluorescence (IF) -fluorescence in situ hybridization (FISH), and rescue assays were used to validate the interactions among circUPF2, IGF2BP2 and SLC7A11. Finally, a tumor xenograft assay was used to examine the biological functions and underlying mechanisms of Exo-SR and circUPF2 in vivo., Results: A novel exosomal circRNA, circUPF2, was identified and revealed to be significantly enriched in Exo-SR. Exosomes with enriched circUPF2 enhanced sorafenib resistance by promoting SLC7A11 expression and suppressing ferroptosis in HCC cells. Mechanistically, circUPF2 acts as a framework to enhance the creation of the circUPF2-IGF2BP2-SLC7A11 ternary complex contributing to the stabilization of SLC7A11 mRNA. Consequently, exosomal circUPF2 promotes SLC7A11 expression and enhances the function of system Xc- in HCC cells, leading to decreased sensitivity to ferroptosis and resistance to sorafenib., Conclusions: The resistance to sorafenib in HCC is facilitated by the exosomal circUPF2, which promotes the formation of the circUPF2-IGF2BP2-SLC7A11 ternary complex and increases the stability of SLC7A11 mRNA. Focusing on exosomal circUPF2 could potentially be an innovative approach for HCC treatment., (© 2024. The Author(s).)

Exosomes contribute substantially to the communication between tumor cells and normal cells. Benefiting from the stable structure, circular RNAs (circRNAs) are believed to serve an important function in exosome-mediated intercellular communication. Here, we focused on circRNAs enriched in starvation-stressed hepatocytic exosomes and further investigated their function and mechanism in hepatocellular carcinoma (HCC) progression. Differentially expressed circRNAs in exosomes were identified by RNA sequencing, and circTGFBR2 was identified and chosen for further study. The molecular mechanism of circTGFBR2 in HCC was demonstrated by RNA pulldown, RIP, dual-luciferase reporter assays, rescue experiments and tumor xenograft assay both in vitro and vivo. We confirmed exosomes with enriched circTGFBR2 led to an upregulated resistance of HCC cells to starvation stress. Mechanistically, circTGFBR2 delivered into HCC cells via exosomes serves as a competing endogenous RNA by binding miR-205-5p to facilitate ATG5 expression and enhance autophagy in HCC cells, resulting in resistance to starvation. Thus, we revealed that circTGFBR2 is a novel tumor promoter circRNA in hepatocytic exosomes and promotes HCC progression by enhancing ATG5-mediated protective autophagy via the circTGFBR2/miR-205-5p/ATG5 axis, which may be a potential therapeutic target for HCC., (© 2023. The Author(s).)

Background: Colorectal liver metastases attached major intrahepatic vessels has been considered to be a risk factor for survival outcome after liver resection. The present study aimed to clarify the outcomes of R1 surgery (margin < 1 mm) in CRLM patients, distinguishing parenchymal margin R1 and attached to major intrahepatic vessels R1., Methods: In present study, 283 CRLM patients who were evaluated to be attached to major intrahepatic vessels initially and underwent liver resection following preoperative chemotherapy. They were assigned to two following groups: R0 (n = 167), R1 parenchymal (n = 58) and R1 vascular (n = 58). The survival outcomes and local recurrence rates were analyzed in each group., Results: Overall, 3- and 5-year overall survival rates after liver resection were 53.0% and 38.2% (median overall survival 37 months). Five-year overall survival was higher in patients with R0 than parenchymal R1 (44.9%% vs. 26.3%, p = 0.009), whereas there was no significant difference from patients with vascular R1 (34.3%, p = 0.752). In the multivariable analysis, preoperative chemotherapy > 4 cycles, clinical risk score 3-5, RAS mutation, parenchymal R1 and CA199 > 100 IU/ml were identified as independent predictive factors of overall survival (p < 0.05). There was no significant difference for local recurrence among three groups., Conclusion: Parenchymal R1 resection was independent risk factor for CRLM. Vascular R1 surgery achieved survival outcomes equivalent to R0 resection. Non-anatomic liver resection for CRLM attached to intrahepatic vessels might be pursued to increase patient resectability by preoperative chemotherapy., (© 2023. The Author(s).)

Background: Hepatocellular carcinoma (HCC) is a common malignancy with poor prognosis and high mortality. This study aimed to explore the oncogenic mechanisms of TRAF5 in HCC and provide a novel therapeutic strategy for HCC., Methods: Human HCC cell lines (HepG2, HuH7, SMMC-LM3, and Hep3B), normal adult liver epithelial cells (THLE-2), and human embryonic kidney cells (HEK293T) were utilized. Cell transfection was performed for functional investigation. qRT-PCR and western blotting were used to detect mRNA expression of TRAF5, LTBR, and NF-κB and protein expression of TRAF5, p-RIP1(S166)/RIP1, p-MLKL(S345)/MLKL, LTBR, and p-NF-κB/NF-κB. Cell viability, proliferation, migration, and invasion were evaluated using CCK-8, colony formation, wound healing, and Transwell assays. Cell survival, necrosis, and apoptosis were assessed using flow cytometry and Hoechst 33342/PI double staining. Co-immunoprecipitation and immunofluorescence were performed to determine the interaction between TRAF5 and LTBR. A xenograft model was established to validate the role of TRAF5 in HCC., Results: TRAF5 knockdown inhibited HCC cell viability, colony formation, migration, invasion, and survival but enhanced necroptosis. Additionally, TRAF5 is correlated with LTBR and TRAF5 silencing down-regulated LTBR in HCC cells. LTBR knockdown inhibited HCC cell viability, while LTBR overexpression eliminated the effects of TRAF5 deficiency on inhibiting HCC cell proliferation, migration, invasion, and survival. LTBR overexpression abolished the promotive function of TRAF5 knockdown on cell necroptosis. LTBR overexpression undid the suppressive effect of TRAF5 knockdown on NF-κB signaling in HCC cells. Moreover, TRAF5 knockdown suppressed xenograft tumor growth, inhibited cell proliferation, and promoted tumor cell apoptosis., Conclusions: TRAF5 deficiency facilitates necroptosis in HCC by suppressing LTBR-mediated NF-κB signaling., Competing Interests: The authors declare that they have no competing interests., (© 2023 Wu et al.)

Hepatocellular carcinoma (HCC) is a malignant tumor, which seriously threatens the life of patients. LncRNA SLC7A11-AS1 was reported to be abnormally expressed in HCC. Here, the functions and relative molecular regulatory mechanism of SLC7A11-AS1 in HCC were investigated. Nude mice and HCC cells were used as the experimental subjects. Knockdown or overexpression of exogenous genes was conducted in HCC cells. RT-qPCR, IHC, and western blot were employed to evaluate the abundance of genes and proteins. The malignant behaviors were evaluated using CCK-8, clone formation, wound-healing, and Transwell. The locations of SLC7A11-AS1 and KLF9 in cells were determined by FISH and IF assays. The total m6A level was evaluated by dot-blot assay. m6A modification of SLC7A11-AS1 was detected using RNA MeRIP. The interactions among molecules were validated by RIP, ChIP, dual luciferase reporter assay, and co-IP. SLC7A11-AS1 was elevated apparently in HCC cells and HCC tissues from mice. SLC7A11-AS1 silencing could suppress HCC progression, which was validated in in vivo and in vitro experiments. Furthermore, METTL3 mediated m6A modification of SLC7A11-AS1 to elevate its expression. In addition, SLC7A11-AS1 downregulated KLF9 expression by affecting STUB1-mediated ubiquitination degradation and KLF9 enhanced PHLPP2 expression to inactivate the AKT pathway. Eventually, rescue experiments revealed that KLF9 knockdown abolished SLC7A11-AS1 silencing-mediated suppression of HCC progression in vivo and in vitro. Our results unveiled that m6A-modified SLC7A11-AS1 promoted HCC progression by regulating the STUB1/KLF9/PHLPP2/AKT axis, indicating that targeting SLC7A11-AS1 might alleviate HCC progression.

Background: Multiple liver metastases is considered a risk factor for overall survival of colorectal liver metastases patients (CRLM) after curative resection. However, whether the prognostic factors were constant in patients with various liver metastases (LM) numbers has not been adequately investigated. This retrospective study aimed to evaluate the changing of prognostic factors on overall survival (OS) in CRLM patients with various LM after curative resection., Methods: Patients who underwent liver resection for CRLM between January 2000 and November 2020 were retrospectively studied. They were divided into three subgroups according to LM numbers by X-tile analysis. Multivariable analysis identified prognostic factors in each subgroup. Nomograms were built using different prognostic factors in three subgroups, respectively. Performance of the nomograms was assessed according to the concordance index (C-index) and calibration plots. The abilities of different scoring systems predicting OS were compared by calculating the area under the time-dependent receiver operating characteristic (ROC) curve (AUC)., Results: A total of 1095 patients were included. Multivariable analysis showed tumor number increasing was an independent risk factor. Patients were subsequently divided into 3 subgroups according to the number of LM by X-tile analysis, namely solitary (n = 375), 2-4 (n = 424), and ≥ 5 (n = 296). The 3-year and 5-year OS rates were 64.1% and 54.0% in solitary LM group, 58.1% and 41.7% in 2-4 LM group, and 50.9% and 32.0% in ≥ 5 LM group, respectively (p < 0.001). In multivariable analysis, RAS mutation was the only constant independent risk factor in all subgroups. The nomograms were built to predict survival based on independent factors in three subgroups. The C-index for OS prediction was 0.707 (95% CI 0.686-0.728) in the solitary LM group, 0.695 (95% CI 0.675-0.715) in the 2-4 LM group, and 0.687 (95% CI 0.664-0.710) in the ≥ 5 LM group. The time-dependent AUC values of nomograms developed using different risk factors after stratifying patients by tumor number were higher than the traditional scoring systems without patient stratification., Conclusions: The prognostic factors varied among CRLM patients with different LM numbers. RAS mutation was the only constant risk factor. Building prediction models based on different prognostic factors improve patient stratification., (© 2022. The Author(s).)

Purpose: Transcatheter arterial chemoembolization (TACE) is a common clinical treatment for hepatocellular carcinoma (HCC). However, hypoxia induction after treatment might trigger tumor invasiveness and metastasis. Although pterostilbene (PTS) has antitumor effects, its chemoprevention in HepG2 cells under hypoxia has not been investigated yet. In addition, the poor water solubility of raw PTS limits its clinical application. Here, we prepared nanoparticles of PTS (PSN) and compared their antihepatoma activities with those of raw PTS in HepG2 under hypoxic conditions., Materials and Methods: The PTS nanoparticle formulation was prepared by nanoprecipitation, using Eudragit ® e100 (EE) and polyvinyl alcohol (PVA) as carriers. We analyzed the physicochemical properties of raw PTS and PSN, including yield, encapsulation efficiency, water-solubility, particle size, morphology, crystalline-to-amorphous transformation, and molecular interaction between PTS and carriers. We also evaluated their antihepatoma activities under hypoxia treatment in HepG2 cells, including cell viability, hypoxia, and apoptosis., Results: The yield and encapsulation efficiency of PSN were 86.33% and >99%, respectively. The water solubility and drug release of PTS were effectively improved after nanoprecipitation corresponding to the reduction in particle size, amorphous transformation, and formation of hydrogen bonding with carriers. PSN had a better cytotoxic effect than raw PTS in HepG2 under pre- and post-hypoxia conditions. In addition, hypoxia- and apoptosis-related proteins in HepG2 cells under two different hypoxic conditions were significantly inhibited by PSN compared with the control group with hypoxia only, except for HIF-1α in the post-hypoxia group. PSN was also significantly better in inhibiting these proteins, except for Bcl2, under pre-hypoxic conditions., Conclusion: Our results suggested that PSN could improve the water solubility and drug release of PTS and enhance the efficacy of HCC treatment under hypoxic conditions., Competing Interests: The authors report no conflicts of interest in this work., (© 2021 Tzeng et al.)

Objective: To determine whether serum prealbumin levels are associated with long-term survival after hepatectomy in patients with primary hepatocellular carcinoma(HCC)., Methods: A consecutive sample of 526 patients with HCC who underwent potentially curative hepatectomy from August 2007 to August 2010 was retrospectively analyzed. Patients were classified as having normal or reduced serum prealbumin based on cut-off values of 200 or 182 mg/L., Results: Multivariate analysis identified the preoperative level of serum prealbumin as an independent prognostic factor of long-term survival (P < 0.05): Survival was significantly better for those with normal levels than for those with reduced levels, based on either cut-off value. Similar results were observed in subgroup analyses based on the degree of cirrhosis, level of ɑ-fetoprotein and Barcelona Clinic Liver Cancer stage., Conclusions: Preoperative level of serum prealbumin may be useful for predicting long-term survival in patients with HCC after hepatectomy., (© 2019 Wiley Periodicals, Inc.)

Purpose: To evaluate the effectiveness of contrast-enhanced susceptibility-weighted imaging with ultrasmall superparamagnetic iron oxide (USPIO-enhanced SWI) in the assessment of intratumoral vascularity in hepatocellular carcinoma (HCC)., Materials and Methods: Orthotopic xenograft HCC nude mouse models were established first and magnetic resonance imaging (MRI) examinations were performed on a 1.5T MR scanner 28 days later. Three groups of mice, 10 in each, were imaged using unenhanced and USPIO-enhanced SWI at doses of 4, 8, and 12 mg Fe/kg. Intratumoral susceptibility signal intensity (ITSS) was scored. ITSS-to-tumor contrast-to-noise ratio (ITSST-CNR) was measured. These measurements were compared between unenhanced and USPIO-enhanced SWI at each dose and differences in the measurements between different dose groups were estimated. Correlation between ITSS and tumor microvessel density (MVD) was analyzed., Results: Compared with unenhanced SWI, significantly higher ITSS was identified on USPIO-enhanced SWI at doses of 8 mg Fe/kg (Z = -2.000, P = 0.046) and 12 mg Fe/kg (Z = -2.333, P = 0.020). Significantly higher ITSST-CNR was found on USPIO-enhanced SWI than that on unenhanced SWI (P < 0.05). Significantly higher ITSST-CNR at a dose of 8 mg Fe/kg was observed than that at 4 mg Fe/kg (Z = -3.326, P = 0.001). Positive correlation between ITSS on USPIO-enhanced SWI at a dose of 8 mg Fe/kg and tumor MVD was demonstrated (r = 0.817, P = 0.004)., Conclusion: USPIO-enhanced SWI at a dose of 8 mg Fe/kg greatly improves the detection of intratumoral vascularity in a xenograft HCC model. J. Magn. Reson. Imaging 2016;44:288-295., (© 2016 Wiley Periodicals, Inc.)

Background: Hepatocellular carcinoma (HCC) is the most common liver cancer worldwide, with poor prognosis and resistance to chemotherapy. This gives novel cancer treatment methods an overwhelming significance. Natural products offer great resources of developing new and effective chemopreventive or chemotherapeutic agents. Artocarpus communis extracts and its active constituent, prenylated flavonoid artocarpin induce human hepatocellular carcinoma cell death. However, the poor water solubility drawbacks of artocarpin restrict its clinical application and bioavailability., Purpose: This study developed the artocarpin nanoparticle system to overcome the poor water solubility drawbacks and investigated the improvement of therapeutic efficacy of artocarpin by adopting novel nanoparticle delivery strategy., Methods: Antiproliferative activity of artocarpin was evaluated by MTT assay. Cell morphology observation by microscope, DNA fragmentation assay, cell cycle analysis, Annexin V apoptosis cell staining, monodansylcadaverine and acridine orange staining and immunoblot analysis were used to evaluate the induction of autophagy by artocarpin. The determination of particle size, amorphous transformation, hydrogen-bond formation, yield, encapsulation efficiency and the solubility study were used to investigate the solubility enhancement mechanism of artocarpin., Results: The present study demonstrates that the anticancer effect of artocarpin in HepG2 and PLC/PRF/5 hepatoma cells is mediated through the autophagic cell death mechanism. Results also demonstrated that artocarpin nanoparticles enhanced the solubility of artocarpin by reducing particle size, transforming high energy amorphous state, and forming hydrogen bond with excipients. Additionally, ArtN exhibited better autophagic cytotoxicity compared to free artocarpin., Conclusion: This work reveals the antihepatoma activity of artocarpin by inducing autophagic cell death and the improvement of therapeutic efficacy of artocarpin by adopting novel nanoparticle delivery strategy. The research provided a basis of ArtN could be explored as a low-dose alternative of artocarpin in anticancer treatment and research applications., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

For centuries, natural plant extracts have played an important role in traditional medicine for curing and preventing diseases. Studies have revealed that Artocarpus communis possess various bioactivities, such as anti-inflammation, anti-oxidant, and anticancer activities. A. communis offers economic value as a source of edible fruit, yields timber, and is widely used in folk medicines. However, little is known about its molecular mechanisms of anticancer activity. Here, we demonstrate the antiproliferative activity of A. communis methanol extract (AM) and its dichloromethane fraction (AD) in two human hepatocellular carcinoma (HCC) cell lines, HepG2 and PLC/PRF/5. Colony assay showed the long-term inhibitory effect of both extracts on cell growth. DNA laddering and immunoblotting analyses revealed that both extracts did not induce apoptosis in the hepatoma cell lines. AM and AD-treated cells demonstrated different cell cycle distribution compared to UV-treated cells, which presented apoptotic cell death with high sub-G1 ratio. Instead, acridine orange staining revealed that AM and AD triggered autophagosome accumulation. Immunoblotting showed a significant expression of autophagy-related proteins, which indicated the autophagic cell death (ACD) of hepatoma cell lines. This study therefore demonstrates that A. communis AM and its dichloromethane fraction can induce ACD in HCC cells and elucidates the potential of A. communis extracts for development as anti tumor therapeutic agents that utilize autophagy as mechanism in mediating cancer cell death.

In some cancer cells, translocation of cytochrome c (Cyt c) from mitochondria to the cytoplasma is inhibited. This inhibition prevents cells from undergoing apoptotic cell death and can lead to uncontrolled cell growth. Increasing cytoplasmic concentration of Cyt c can induce apoptosis in cancer cells as a strategy of cancer therapy. Here we proposed a galactosylated albumin based carrier for intracellular delivery of Cyt c to hepatocarcinoma cells. Galactosylated albumin is recognized by highly expressed asialoglycoprotein receptors (ASGPR) on hepatocarcinoma cells and is further internalized into cells via receptor mediated endocytosis. Cyt c was chemically conjugated to galactosylated albumin with a reducible disulfide linker in order to release Cyt c from the carrier inside cells. We tested cellular uptake and cytotoxicity of Cyt c conjugates in ASGPR positive and negative hepatocarcinoma cells. The results showed galatosylated albumin significantly increased cellular uptake in both cell types resulting in cytotoxicity in a dose dependent manner through the induction of apoptosis. The lack of ASGPR specific uptake might be due to other carbohydrate-recognizing receptors expressed on tumor cells. In general, our work has shown that intracellular delivery of Cyt c to tumor cells can be an alternative therapeutic approach and galactosylated albumin can be a protein drug carrier for intracellular delivery.

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

Background: Radiotherapy is an efficient remedy in the treatment of hepatocellular carcinoma (Hca); however, some cancer cells can still survive from the radiation and the therapeutic effect is to be improved. Regulatory T cell (Tregs)-induced tumor tolerance and Akt expression play important roles in the tumor survival. This study aims to elucidate the role of radiation induces Akt expression in Tregs., Methods: A rat Hca model was developed. Hca tissue was collected from the rats with or without radiotherapy. The frequency of Treg and apoptotic Treg in Hca tissue was assessed by flow cytometry. A cell culture model was used to investigate the mechanism by which the tumor-infiltrating Tregs survive from irradiation., Results: After radiotherapy, the frequency of Treg was increased in Hca, the frequency of apoptotic Tregs was decreased and the expression of Akt was increased in the remaining Tregs. The results were reproduced in vitro with a cell culture model. The addition of Akt inhibitor blocked the irradiation-induced Treg survival. Tregs with high levels of Akt still preserve the immune suppressor function., Conclusions: Akt plays an important role in the radiation-induced tumor-infiltrating Treg survival in Hca.

Peptide vaccine based on tumor-associated antigen (TAA), which usually belongs to self-antigen with poor immunogenicity, has been considered as an attractive option for treatment of malignant tumors. The ideal TAA epitopes should have stable affinity to major histocompatibility complex (MHC) molecules and elicit strong anti-tumor immune response. Although point-mutation technology of TAA peptide may increase the binding capability to MHC molecules, some previous studies have revealed that part of the variant peptides results in lymphocyte not to effectively cross-recognize and kill the target tumor expressed wild-type TAA. Here, we designed a novel HLA-A2-restricted mutated TAA Survivin epitope nonapeptide Sur79L2 (KLSSGCAFL) that showed higher binding ability compared to wild-type peptide Sur79 (KHSSGCAFL) in T2-binding assays. To investigate whether Sur79L2 can induce Survivin-specific anti-hepatocellular carcinoma (HCC) response, we stimulated tumor-associated lymphocytes from a HCC patient with Sur79L2 in vitro. IFN-γ release and cytotoxicity assays showed Sur79L2 could effectively cross-recognize and lysis T2 cell plus peptide Sur79 and HCC cell lines (expression of wild-type Survivin antigen) in an HLA-A2-restricted manner. In contrast, peptide Sur95 (ELTLGEFLKL) that has been reported as a very promising anti-tumor epitope in a variety of tumors except HCC were not able to generate detectable cytotoxic immune responses against HCC in this study. Our results suggest that point-mutated peptide Sur79L2 is a new HLA-A2-restricted CTL epitope and may be useful for the immunotherapy for patients with HCC.

Aim: To evaluate the clinical significance of CpG island methylator phenotype (CIMP) in plasma and its association with hepatocellular carcinoma (HCC) progress., Methods: CIMP status of 108 HCC patients was analyzed using a methylation marker panel in tumor tissues and plasma with methylation-specific polymerase chain reaction. Fifteen samples of non-neoplastic liver tissues and 60 of plasma from healthy persons were examined simultaneously. Examined genes included APC, WIF-1, RUNX-3, DLC-1, SFRP-1, DKK and E-cad., Results: The frequencies of high-level methylation in HCC tissue and plasma were at least 15% for the seven genes: APC, 48/108, 44.44% in tissue and 26/108, 24.07% in plasma; WIF-1, 53/108, 49.07% in tissue and 35/108, 32.41% in plasma; RUNX-3, 52/108, 48.14% in tissue and 42/108, 38.89% in plasma; DLC-1, 38/108, 35.18% in tissue and 23/108, 21.30% in plasma; SFRP-1, 40/108, 37.04% in tissue and 31/108, 28.7% in plasma; DKK, 39/108, 36.1% in tissue and 25/108, 23.14% in plasma; and E-cad, 37/108, 34.3% in tissue and 18/108, 16.67% in plasma. CIMP+ (≥ 3 methylated genes) was detected in 68 (60.2%) tumor tissue samples and 62 (57.4%) plasma samples. CIMP was not detected in non-neoplastic liver tissues or plasma of healthy persons. CIMP status in tumor tissues differed significantly in gender, hepatitis B surface antigen, alpha-fetoprotein, and tumor-node-metastasis stage (P < 0.05). Similar results were obtained with plasma samples (P < 0.05). There was no difference in CIMP status in age, presence of hepatitis C virus antibody, cirrhosis, number of nodes, number of tumors, tumor size, or Edmondson-Steiner stage. A one-year follow-up found that the metastatic rate and recurrence rate in the CIMP+ group were significantly higher than in the CIMP- group as assessed with plasma samples (P < 0.05)., Conclusion: Plasma DNA can be a reliable sample source for CIMP analysis. CIMP in plasma may serve as a molecular marker of late-stage and poor-prognosis HCC.

Aim: To investigate the effect of Hepatitis C virus (HCV) core protein on the expression of cyclooxygenase 2 (COX-2)., Methods: The genes encoded HCV core protein were amplified from plasmid containing full length genome of HCV strain H77 using PCR, and were cloned into eukaryotic expression vector pcDNA3.1. The recombinant HCV-C/pcDNA3.1 was transiently co-transfected into HepG2 cells with luciferase reporter vector containing COX-2 promotor (COX2pro1.5 kb/luc). The luciferase activity and COX-2 protein expression were detected., Results: The recombinants HCV-C/pcDNA3.1 have been constructed successfully. The luciferase activity of COX-2 promotor was activated by the expressed HCV core, and the increased protein expression of COX-2 in transfected HepG2 cells was detected by Western blot., Conclusion: HCV core protein can activate the COX-2 promotor and induce its expression, which provides a new experimental basis for further research on relationship between COX-2 and HCV pathogenesis.

Purpose: To evaluate ultrasonographically (US)-guided high-intensity focused ultrasound ablation combined with transcatheter arterial chemoembolization (TACE) in the treatment of stage IVA hepatocellular carcinoma (HCC)., Materials and Methods: Institutional review board approval and informed consent were obtained. From November 1998 to May 2000, 50 consecutive patients with stage IVA HCC (TNM classification, T4N0-1M0) were alternately enrolled in one of two treatment groups: group 1 (n = 26), in which TACE was performed alone, and group 2 (n = 24), in which transcutaneous ablation of HCC with high-intensity focused ultrasound was performed 2-4 weeks after TACE. The tumors were 4-14 cm in diameter (mean, 10.5 cm). Immediate therapeutic effects were assessed at follow-up with Doppler US and computed tomography or magnetic resonance imaging. All patients were followed up for 3-24 months (mean, 8 months) to observe long-term therapeutic effects and complications in both groups. Tumor reduction rates, median survival time, and cumulative survival rates in both groups were calculated by using the unpaired Student t test and Kaplan-Meier method., Results: No severe complication was observed after focused ultrasound ablation, and no unexpected side effects were noted after TACE. Follow-up images showed absence or reduction of blood supply in the lesions after focused ultrasound ablation when compared with blood supply after TACE alone. The median survival time was 11.3 months in group 2 and 4.0 months in group 1 (P = .004). The 6-month survival rate was 80.4%-85.4% in group 2 and 13.2% in group 1 (P = .002), and the 1-year survival rate was 42.9% and 0%, respectively. Median reductions in tumor size as a percentage of initial tumor volume at 1, 3, 6, and 12 months after treatment, respectively, were 28.6%, 35.0%, 50.0%, and 50.0% in group 2 and 4.8%, 7.7%, 10.0%, and 0% in group 1 (P < .01)., Conclusion: The combination of high-intensity focused ultrasound ablation and TACE is a promising approach in patients with advanced-stage HCC, but large-scale randomized clinical trials are necessary for confirmation., ((c) RSNA, 2005.)

Background: High intensity focused ultrasound (HIFU) is a noninvasive treatment modality that induces complete coagulative necrosis of a deep tumor through the intact skin. The current study was conducted to determine the safety, efficacy, and feasibility of extracorporeal HIFU in the treatment of patients with hepatocellular carcinoma (HCC)., Methods: A total of 55 patients with HCC with cirrhosis were enrolled in this prospective, nonrandomized clinical trial. Among them, 51 patients had unresectable HCC. Tumor size ranged from 4 to 14 cm in diameter with mean diameter of 8.14 cm. According to tumor, node, metastasis (TNM) classification, 15 patients corresponded to stage II, 16 to stage IIIA, and 24 to IIIC. All patients had HIFU, and the median number of HIFU session was 1.69. Safety and efficacy of HIFU were assessed in this trial., Results: No severe side effect was observed in the patients treated with HIFU. Follow-up imaging showed an absence of tumor vascular supply and the shrinkage of treated lesions. Serum alpha-fetoprotein returned to normal level in 34% of patients. The overall survival rates at 6, 12, and 18 months were 86.1%, 61.5%, and 35.3%, respectively. The survival rates were significantly higher in patients in stage II than those in stage IIIA (P = .0132) and in stage IIIC (P = .0265)., Conclusion: As a noninvasive therapy, HIFU appears to be effective, safe, and feasible in the treatment of patients with HCC. It may play an important role in the ablation of large tumors.

Aim: To study the expression and serum level of HBxAg, Fas and FasL in tissues of HCC patients, and to assess the relationship between HBxAg and Fas/FasL system., Methods: Tissues from 50 patients with HCC were tested for the expression of HBxAg, Fas and FasL by S-P immunohistochemistry. Serum levels of sFas/sFasL and HBsAg/HBeAg were measured by ELISA assay. HBV X gene was detected by PCR in serum and confirmed by automatic sequencing. Fifty cases of liver cirrhosis and 30 normal controls were involved in serum analysis., Results: The expression of HBxAg, Fas and FasL in carcinoma tissues was 96 %, 84 % and 98 %, respectively. Staining of HBxAg, Fas and FasL was observed predominately in cytoplasms, no significant difference was found in intensity between HBxAg, Fas and FasL (P>0.05). HBxAg, Fas and FasL might express in the same area of carcinoma tissues and this co-expression could be found in most patients with HCC. The mean levels of sFas in serum from HCC, cirrhosis and normal controls were 762.29 +/- 391.56 microg.L(-1), 835.36 +/- 407.33 microg.L(-1) and 238.27 +/- 135.29 microg.L(-1). The mean levels of sFasL in serum from HCC, cirrhosis and normal controls were 156.36 +/- 9.61 microg.L(-1), 173.63 +/- 18.74 microg.L(-1) and 121.96 +/- 7.83 microg.L(-1). Statistical analysis showed that both sFas and sFasL in HCC and cirrhosis patients were significantly higher than those in normal controls (P<0.01). Serum HBV X gene was found in 32% of HCC patients and 46% of cirrhotic patients. There was no significant relationship between serum level of sFas/sFasL and serum X gene detection (P>0.05). Eight percent of HCC patients with negative HBsAg and HBeAg in serum might have X gene in serum and HBxAg expression in carcinoma tissues., Conclusion: Our data suggest that HBxAg and Fas/FasL system plays an important role in the development of human HCC. Expression of HBxAg can leads to expression of Fas/FasL system which and reverse apoptosis of hepatocellular carcinoma induced by FasL.