Severity of cirrhosis should determine the operative modality for patients with early hepatocellular carcinoma and compensated liver function.

BACKGROUND: The optimum operative treatment for early hepatocellular carcinoma (HCC) in patients with compensated liver function remains controversial. This study aimed to assess the impact of the severity of cirrhosis on survival after liver resection (LR) and to determine the importance of the severity of cirrhosis in operative decision-making for early HCC. METHODS: The records of 307 patients with HCC with a solitary tumor ≤5 cm undergoing either LR or liver transplantation (LT) were reviewed retrospectively. The Child-Pugh class A patients in the LR group were stratified according to the severity of cirrhosis. Survival of each subgroup was compared with that of the LT group. RESULTS: Both the recurrence-free survival (RFS) and disease-specific survival (DSS) in the LR group were worse than those in the LT group. Stratification of the Child A patients in the LR group yielded 5-year RFS and DSS rates of 71% and 86%, respectively, for the cirrhosis-free subgroup, 58% and 79% for the mild cirrhosis subgroup, and 25% and 45% for the moderate/severe cirrhosis subgroup. There were no differences in the rates of RFS and DSS between either the cirrhosis-free or mild cirrhosis subgroup and the LT group, whereas the subgroup with moderate/severe cirrhosis had poorer RFS and DSS rates than the LT group. CONCLUSION: LR is the best treatment for early HCC in patients without cirrhosis or with mild cirrhosis and compensated liver function, whereas LT is recommended for those with moderate/severe cirrhosis, even if their liver function is well compensated.

Can serum glypican-3 be a biomarker for effective diagnosis of hepatocellular carcinoma? A meta-analysis of the literature.

OBJECTIVE: This review is to evaluate the diagnostic value of serum GPC3 for hepatocellular carcinoma (HCC) due to conflicting results reported. METHODS: NCBI PubMed and Embase were comprehensively searched for studies that have used serum GPC3 level as a diagnostic index for HCC. The quality of the included studies was assessed. Subgroup analyses were conducted to evaluate the sensitivity and specificity of GPC3 as a HCC marker. Statistical analysis was performed with the software STATA version 12.0. RESULTS: A total of 22 studies were included. The qualities of included studies were relatively poor. Among them, 18 studies have shown that serum GPC3 is a specific biomarker for HCC, and the pooled sensitivity and specificity of these studies were 69 and 93%, respectively. The other 4 studies have reported conflicting results, which were not caused by races, infection status of HBV and HCV, or assay reagents but due to one common experimental design of enrolling liver cirrhosis patients as control subjects. CONCLUSIONS: This meta-analysis indicates that serum GPC3 is elevated in HCC patients compared with healthy individuals, but more studies are needed to evaluate its effectiveness to differentially diagnose HCC and liver cirrhosis.

HBx protein promotes oval cell proliferation by up-regulation of cyclin D1 via activation of the MEK/ERK and PI3K/Akt pathways.

Growing evidence has shown that hepatic oval cells, also named liver progenitor cells, play an important role in the process of liver regeneration in various liver diseases. Oval cell proliferation has been reported in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) and chronic liver disease. Studies have found expression of HBV surface and core antigens in oval cells in the livers of patients with HCC, suggesting that HBV infection of oval cells could be a mechanism of human hepatocarcinogenesis. In addition, there is evidence of multiplication of HBV in oval cell culture. However, little research has been performed to explore the role of HBV-encoded proteins in the proliferation of hepatic oval cells. Previously, we successfully transfected the HBV x (HBx) gene, one of the four genes in the HBV genome, into a rat LE/6 oval cell line. In this study, we tested whether or not the transfected HBx gene could affect oval cell proliferation in vitro. Our results show that overexpression of HBx promotes the proliferation of oval cells and increases cyclin D1 expression, assessed at both the mRNA and protein levels. We also found that HBx activated the PI-3K/Akt and MEK/ERK1/2 pathways in HBx-transfected oval cells. Furthermore, the HBx-induced increases in cyclin D1 expression and oval cell proliferation were completely abolished by treatment with either MEK inhibitor PD184352 or PI-3K inhibitor LY294002. These results demonstrated that HBx has the ability to promote oval cell proliferation in vitro, and its stimulatory effects on cell proliferation and expression of cyclin D1 depend on the activation of the MEK/ERK and PI3K/Akt signaling pathways in cultured oval cells.

The niche of hepatic cancer stem cell and cancer recurrence.

Currently, surgical resection is one of only a few options for treating hepatocellular carcinoma (HCC). Unfortunately, postoperative tumor recurrence remains almost inevitable despite additional radiation or chemotherapy treatment following radical resection. Clinical observations and a growing body of experimental evidence have led to speculation that there is a population of persistent hepatic cancer stem cells (HCSCs), which are difficult to completely remove surgically. HCSCs are most often in a quiescent state and thought to reside in a specific microenvironment known as a niche that provides the cues necessary for HCSCs to maintain a balance of self-renewal and differentiation. Residual HCSCs following surgery may alter their fate by invading into the blood circulation. Furthermore, it remains to be determined if hepatectomy render the postoperative niche more favorable for the survival and growth of HCSCs, and therefore the recurrence of HCC. A better understanding of the mechanisms for HCSCs self-renewal, invasion and recurrence may provide new insights into curative strategies for treating HCC.

[The prognostic significance of Omi/HtrA2 expression, and correlation between Omi/HtrA2 and hypoxia-inducible factor-1α in primary hepatocellular carcinoma cells].

OBJECTIVES: To investigate the prognostic significance of Omi/HtrA2 expression, and the correlation between Omi/HtrA2 and Hypoxia-inducible factor (HIF)-1α in primary hepatocellular carcinoma cells. METHODS: The expression of HIF-1α and Omi/HtrA2 in 43 cases of hepatic carcinoma tissues were detected immunohistochemically. Follow-up data were obtained to perform the Kaplan-Meier survival analysis. The change of Omi/HtrA2 expression in HepG2 cell was measured after HIF-1α expression of HepG2 in vitro was regulated by Tet-on expression system. RESULTS: Omi/HtrA2 expression was correlated with lymph node metastasis and recurring within liver during 2 years. Statistical analysis estimation showed the cumulative survival rate of post-hepatectomy for the patients with the positive expression of Omi/HtrA2 was higher than that for other patients with the negative expression of Omi/HtrA2 (χ(2) = 6.13, P = 0.013). In the common paraffin-embedded specimen of hepatocellular carcinoma, most of the samples showing negative or weak positive HIF-1α immunopositivity showed moderate positive or strong positive Omi/HtrA2 immunopositivity, while most of the samples showing moderate positive or strong positive HIF-1α immunopositivity showed negative or weak positive Omi/HtrA2 immunopositivity. The mRNA expression intensity of Omi/HtrA2 was decreasing with the HIF-1α expression increasing, and the difference was statistically significant(F = 106.766, P < 0.01). CONCLUSIONS: Omi/HtrA2 may be an important prognostic marker for primary hepatocellular carcinoma. Omi/HtrA2 expression is reversely correlated with HIF-1α expression in hepatocellular carcinoma. During the apoptotic process Omi/HtrA2 participating in hepatocellular carcinoma cells, HIF-1α is involved in the controlling and regulating of Omi/HtrA2 expression.

Oval cell response is attenuated by depletion of liver resident macrophages in the 2-AAF/partial hepatectomy rat.

BACKGROUND/AIMS: Macrophages are known to play an important role in hepatocyte mediated liver regeneration by secreting inflammatory mediators. However, there is little information available on the role of resident macrophages in oval cell mediated liver regeneration. In the present study we aimed to investigate the role of macrophages in oval cell expansion induced by 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH) in rats. METHODOLOGY/PRINCIPAL FINDINGS: We depleted macrophages in the liver of 2-AAF/PH treated rats by injecting liposome encapsulated clodronate 48 hours before PH. Regeneration of remnant liver mass, as well as proliferation and differentiation of oval cells were measured. We found that macrophage-depleted rats suffered higher mortality and liver transaminase levels. We also showed that depletion of macrophages yielded a significant decrease of EPCAM and PCK positive oval cells in immunohistochemical stained liver sections 9 days after PH. Meanwhile, oval cell differentiation was also attenuated as a result of macrophage depletion, as large foci of small basophilic hepatocytes were observed by day 9 following hepatectomy in control rats whereas they were almost absent in macrophage depleted rats. Accordingly, real-time polymerase chain reaction analysis showed lower expression of albumin mRNA in macrophage depleted livers. Then we assessed whether macrophage depletion may affect hepatic production of stimulating cytokines for liver regeneration. We showed that macrophage-depletion significantly inhibited hepatic expression of tumor necrosis factor-α and interleukin-6, along with a lack of signal transducer and activator of transcription 3 phosphorylation during the early period following hepatectomy. CONCLUSIONS: These data indicate that macrophages play an important role in oval cell mediated liver regeneration in the 2-AAF/PH model.

Hepatic oval cell lines generate hepatocellular carcinoma following transfection with HBx gene and treatment with aflatoxin B1 in vivo.

Hepatic oval cells (HOC) are considered to be the stem cells of the liver and have been linked to the development of hepatic malignancies. Studies have demonstrated that chronic hepatitis B virus (HBV) infection and dietary aflatoxin B1 (AFB1) exposure are among the most important risk factors for the development of hepatocellular carcinoma (HCC). However, little research has been done to evaluate the role of oval cells in these two environmental factors on hepatocarcinogenesis. In this study, partial transformation of rat HOC (LE/6) were accomplished by transfected HBV x gene (HBx), and then transfected cells were implanted both intra-hepatically and subcutaneously into nude mice treated with AFB1 in vivo. We found the oval cells produced tumors (4/24 of the animals) in liver following transfection with HBx gene and treatment with AFB1. These intrahepatic tumors included HCC cells (immunopositive for HepParl, ALB, CK8 and AFP) and mesenchymal cells (immunopositive for Vimentin and SMA). Whereas mesenchymal tumors were observed at the subcutaneous tissue with a similar rate in all controls treated with cell lines (10/24 in HBx-oval cells/AFB1 group, 8/20 in HBx-oval cells/non-AFB1 group, 10/20 in non-HBx/AFB1 group; 9/20 in non-HBx/non-AFB1 group). Conversely, none of the controls developed intrahepatic tumors. These results provide an evidence that oval cells have the capacity to generate HCC through the combined effects of the HBx and AFB1 in the liver microenvironment.

[The hyposensitization of oval cell to antiproliferative effect of transforming growth factor ß1 by hepatitis B virus X gene].

OBJECTIVE: To determine whether hepatitis B virus X (HBX) protein expression affect the oval cells' response to anti-proliferative effect of transforming growth factor ß1 (TGFß1) in oval cells. METHODS: Real-time PCR, Western blot analysis were performed to detect the expression of TGFßRII in HBX-transfected oval cells named HBX-EGFP-LE/6, and EGFP-LE/6, LE/6 control cells. In addition, exogenous TGFß1 was added into all three oval cell lines, MTT assay was preformed to clarify different responses to the anti-proliferative effect of TGFß1. RESULTS: The TGFßRII mRNA levels in LE/6 and EGFP-LE/6 cells were (10.2 ± 1.8) and (8.8 ± 0.9) folds of those in HBX-EGFP-LE/6 cells, the difference was significant (P < 0.05). HBX protein expression also reduced the protein levels of TGFßRII in HBX-EGFP-LE/6 oval cells, compared to the control cells. The MTT results exhibited that, after TGFß1 addition, proliferative inhibition rate in the HBX-EGFP-LE/6 cells was 18.1% ± 1.5% while those in control cells were 42.2% ± 2.8% and 41.9% ± 5.0%, the difference was significant (P < 0.01). CONCLUSION: HBX protein expression affects TGFßRII transcriptional activity and protein synthesis, and insensitive oval cells to anti-proliferative effect of TGFß1.

Initial fuel temperature effects on burning rate of pool fire.

The influence of the initial fuel temperature on the burning behavior of n-heptane pool fire was experimentally studied at the State Key Laboratory of Fire Science (SKLFS) large test hall. Circular pool fires with diameters of 100mm, 141 mm, and 200 mm were considered with initial fuel temperatures ranging from 290 K to 363 K. Burning rate and temperature distributions in fuel and vessel wall were recorded during the combustion. The burning rate exhibited five typical stages: initial development, steady burning, transition, bulk boiling burning, and decay. The burning rate during the steady burning stage was observed to be relatively independent of the initial fuel temperature. In contrast, the burning rate of the bulk boiling burning stage increases with increased initial fuel temperature. It was also observed that increased initial fuel temperature decreases the duration of steady burning stage. When the initial temperature approaches the boiling point, the steady burning stage nearly disappears and the burning rate moves directly from the initial development stage to the transition stage. The fuel surface temperature increases to its boiling point at the steady burning stage, shortly after ignition, and the bulk liquid reaches boiling temperature at the bulk boiling burning stage. No distinguished cold zone is formed in the fuel bed. However, boiling zone is observed and the thickness increases to its maximum value when the bulk boiling phenomena occurs.

Recovery of aniline from wastewater by nitrobenzene extraction enhanced with salting-out effect.

OBJECTIVE: Nitrobenzene extraction enhanced by salting-out effect was employed to recover aniline from wastewater at 25 degrees C. METHOD: Batchwise experiments were conducted to elucidate the influence of various operating variables on the extracting performance, including acidity of wastewater, initial aniline concentration, ratios of solvent to wastewater, extraction stages, concentrations and different types of inorganic salts, such as NaCl, KCl, Na(2)SO(4), CaCl(2) and K(2)SO(4). RESULTS: Nitrobenzene with a concentration of 20% and a pH value of 9.1 at the temperature of 25 degrees C together with NaCl of a concentration of 14 wt.% realized nearly 100% aniline recovery at the fifth stage of wastewater treatment. CONCLUSIONS: High pH values and volume ratios of nitrobenzene/wastewater are more suitable for recovery of aniline. In addition, recovery of aniline is significantly elevated with increase of the concentration of salts, whose promoting effects are in the following order: NaCl>Na(2)SO(4)>K(2)SO(4)>CaCl(2)>KCl on the weight basis of wastewater. Furthermore, aniline in wastewater can be almost completely recovered by five-stage sequential nitrobenzene extraction, which is promoted continuously by the salting-out effect.

[The studies of hepatitis B virus X gene induces oval cell malignant transformation].

OBJECTIVE: To find out the mechanisms of HBx gene inducing oval cell malignant transformation into hepatoma carcinoma cell. METHODS: The changes of morphology, cell cycle, differentiated markers, c-myc and TGF-alpha in pEGFP-HBx oval cells strain, which stably expressed HBx gene, were studied by inversion phase contrast microscope and transmission electron microscopy, flow cytometry, periodic acid-schiff (PAS) staining, soft agar growth assay, real-time PCR, immunocytochemistry. pEGFP-oval cells and LE/6 oval cells were used as control groups. RESULTS: (1) The pEGFP-HBx oval cells showed bigger in size with malformed nucleus as compared with control groups. (2) Flow cytometry showed that, in contrast with the control groups, the proportion of pEGFP-HBx oval cells arrested in G0/G1 phase decreased but in S or G2/M phase rose. Moreover, the population of aneuploid cells increased obviously. (3) PAS staining showed that there were many glycogen granules in the cytoplasm of pEGFP-HBx oval cell. (4) The pEGFP-HBx oval cell formed colonies in the soft agar. (5) Compared with the control groups, the expression of HNF-4 alpha, AFP, c-myc and TGF-alpha rose obviously, whereas the expression of CK-7 and CK-19 decreased. And the expression of cps1 mRNA was not in the extent of detection. CONCLUSIONS: The HBx gene can provoke abnormal differentiation of oval cell and induce oval cell malignant transformation.

[Construction of pEGFP-HBx gene recombinant plasmid and establishment of stable transfected HBx gene liver oval cell strain].

OBJECTIVE: To construct pEGFP-HBx eukaryotic expression plasmid and establish stable and effective transfected rat oval cell (LE/6) strain expressing EGFP-HBx fusion protein to explore the roles of HBx gene and oval cell in carcinogenesis of hepatocellular carcinoma (HCC). METHODS: HBx gene with EcoRI and Hind III endo-enzyme sites was obtained by using PCR from plasmid pcDNA3.1-HBx. The purified HBx gene fragment was inserted into pEGFP-N1 expression vector, and the recombinant plasmid pEGFP-HBx was identified by restriction endonuclease and DNA sequencing analysis. LE/6 cells were transfected with recombinant pEGFP-HBx by lipofectamine reagent. Resistant to G418 clones were selected, and expression of EGFP-HBx fusion protein in clones were examined directly with fluorescence microscope, and these clones were isolated and proliferated. The expression of HBx was detected by RT-PCR analysis and immunocytochemistry. RESULTS: Plasmid pEGFP-HBx has whole HBx gene base and correct reading frame as indicated by restriction endonuclease and DNA sequencing analysis. After transfecting with pEGFP-HBx plasmid, LE/6 cell clones expressing EGFP-HBx fusion protein were obtained. RT-PCR analysis and immunocytochemistry showed that HBx gene was only expression in transfected pEGFP-HBx cells. CONCLUSIONS: The pEGFP-HBx recombinant expression vector was successfully constructed, and the stable transfected LE/6 strain expressing EGFP-HBx fusion protein was successfully established. It will be helpful in the further study on the roles of HBx and liver oval cell in carcinogenesis of HCC.

Inhibiting survivin expression enhances TRAIL-induced tumoricidal activity in human hepatocellular carcinoma via cell cycle arrest.

Human Hepatocellular carcinoma (HCC) cell types exhibit a major resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death, and the key determinants of mechanisms accounting for TRAIL susceptibility, still remain controversial. Our previous studies showed that overexpression of survivin reduced sensitivity of HCC cells to TRAIL. The aim of this study is to investigate how tumor cells escape TRAIL-mediated surveillance through survivin expression and how to reverse the resistance of TRAIL-inducing apoptosis. Seven tumor cell lines were treated with or without TRAIL protein and antisense oligodeoxynucleotides (ODNs) against survivin in culture. HepG(2) and SMMC7721 cells were treated with mimosine, thymidine or nocodazole to synchronize their cell cycle phases and then used to test their sensitivity to TRAIL. In vivo effects of TRAIL plasmid alone or in combination with survivin antisense ODNs on tumor growth were evaluated in a nude mouse hepatoma model of HepG(2) cell grafts. Varied levels of survivin mRNA in various cell lines were evaluated and negatively correlated to TRAIL-induced apoptosis. Hepatoma HepG(2) and SMMC7721 cells in G (1) or S phase are more sensitive to TRAIL than those in G(2) phase. Treatment with survivin antisense ODNscaused S phase arrest and significantly enhanced TRAIL-induced apoptosis. TRAIL protein caused G(2)/M arrest and resulted in an increase of survivin in HepG(2) cells. Combined TRAIL plasmid and survivin antisense ODNs significantly supressed the growth of tumor xenografts as compared to TRAIL plamid or antisense ODNs alone during four weeks of observation. The findings indicate that survivin may play a role in tumor cell resistance to TRAIL-induced apoptosis, at least in part, through cell cycle regulation. Manipulation of survivin expression levels may sensitizes tumor cells to TRAIL-induced apoptosis.

Removal of 5-amino-2-chlorotoluene-4-sulfonic and chlorhydric acids from wastewater by weakly basic resin: equilibrium and kinetics.

OBJECTIVE: To study the adsorption of 5-Amino-2-chlorotoluene-4-sulfonic (CLT) and chlorhydric (HC1) acids from wastewater by weakly basic resin. METHODS: The kinetics and isotherm were studied. Thermodynamic parameters for the adsorption of acids were calculated and discussed. RESULTS: The adsorption of CLT and HC1 acids followed Langmuir isotherm and the first-order kinetics model. CONCLUSION: The adsorptive affinity of the two acids on D301R is in the order of CLT acid > HCl acid. CLT and HCl acids can be separated.

[Expression of Twist gene in human hepatocellular carcinoma and its clinicopathological significance].

OBJECTIVE: To investigate the expression and clinicopathological significance of transcription factor Twist in hepatocellular carcinoma (HCC), paraneoplastic and cirrhotic tissues. METHOD: Immunohistochemistry was used to detect the expression of the Twist protein in 26 cases of HCC and paraneoplastic tissue and 10 cases of cirrhotic tissue. Meanwhile, the Twist mRNA and its protein were detected in 10 HCC tissues and 10 paraneoplastic tissues by RT-PCR and Western blot. And the expression differences and clinicopathological significances of the expression of Twist gene and its protein were analyzed. RESULTS: The positive rates of the Twist protein in HCC, paraneoplastic and cirrhotic tissues were 84.6%, 19.2 % and 20.0 %, respectively. The positive rate of Twist in HCC was higher than that in paraneoplastic or cirrhosis tissues (P < 0.05). Compared with in paraneoplastic tissues, the mRNA and protein expression of Twist were up-regulated in HCC by 2.52 and 2.13 times, respectively. CONCLUSION: Twist has an inappropriate expression in hepatocellular carcinoma and it may play an important role in the tumorigenesis and progression of HCC.

Adsorption of beta-naphthalenesulfonic acid/sulfuric acid from their solution by weakly basic resin: equilibrium.

Experiments for single and bisolute competitive adsorption were carried out to investigate the adsorption behavior of beta-naphthalenesulfonic acid (NSA) and sulfuric acid (H2SO4) from their solution at 25 degrees C onto weakly basic resin D301R. Adsorption affinity of sulfuric acid on D301R was found to be much higher than that of NSA. The data of single-solute adsorption were fitted to the Langmuir model and the Freundlich adsorption model. The ideal adsorbed solution theory (IAST) coupled with the single-solute adsorption models were used to predict the bisolute competitive adsorption equilibria. The IAST coupled with the Langmuir and the Freundlich model for sulfuric acid and NSA, respectively, yields the favorable representation of the bisolute competitive adsorption behavior.

[Chinese medicine Extractum trametes robiniophila murr augment tumor necrosis factor related apoptosis-inducing ligand induced apoptosis in human hepatic cancer cell lines].

OBJECTIVE: To investigate the effects of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) combined with Chinese medicine, Extractum trametes robiniophila murr in human hepatic cancer cells. METHODS: HepG2 cell line resistant to adriamycin (HepG2/ADR) was induced step by step. The effects of TRAIL (100 ng/L) combined with Extractum trametes robiniophila murr (1.0 g/L) promoting apoptosis in HepG2 or HepG2/ADM were analyzed. The proliferation was observed by MTT assay and the apoptosis of cells was also observed by flow cytometry. RESULTS: HepG2/ADM was confirmed resisting to ADM. The treatment of TRAIL (100 ng/L) combined with Extractum trametes robiniophila murr (1.0 g/L) showed significant inhibitory effects on the growth of both HepG2 and HepG2/ADM, and the percentage of apoptosis was increased compared with other groups within 24 to 72 h. CONCLUSIONS: Extractum trametes robiniophila murr dramatically augmented the sensitivity of both HepG2 and HepG2/ADM to TRAIL, but only has slightly killing effects on L02. TRAIL combined with Chinese medicine treatment could be a safe and attractive strategy to drug-resistant/TRAIL-resistant tumor cells.

UV-catalytic treatment of spent caustic from ethene plant with hydrogen peroxide and ozone oxidation.

The performance of UV/H2O2, UV/O3 and UV/H2O2/O3 oxidation systems for treating spent caustic from an ethylene plant was investigated. In UV/H2O2 system, with the increase of H2O2 dosage, removal efficiencies of COD and the ratio of biochemical oxygen demand (BOD) to chemical oxygen demand (COD) of the effluent were increased and a better performance was obtained than the H2O2 system alone. In UV/H2O2 system, removal efficiency of COD reach 68% under the optimum condition, and BOD/COD ratio was significantly increased from 0.22 to 0.52. In UV/O3 system, with the increase of O3 dosage, removal efficiency of COD and BOD/COD ratio were increased, and a better performance was obtained than the O3 system alone. Under the optimum condition, removal efficiency of COD was 54%, and BOD/COD ratio was significantly increased from 0.22 to 0.48. In UV/H2O2/O3 system, COD removal efficiency was found to be 22.0% higher than UV/O3 system.