Cloning and sequence analysis of the DHBV genome of the brown ducks in Guilin region and establishment of the quantitative method for detecting DHBV / 病毒学报

Brown ducks carrying DHBV were widely used as hepatitis B animal model in the research of the activity and toxicity of anti-HBV dugs. Studies showed that the ratio of DHBV carriers in the brown ducks in Guilin region was relatively high. Nevertheless, the characters of the DHBV genome of Guilin brown duck remain unknown. Here we report the cloning of the genome of Guilin brown duck DHBV and the sequence analysis of the genome. The full length of the DHBV genome of Guilin brown duck was 3 027bp. Analysis using ORF finder found that there was an ORF for an unknown peptide other than S-ORF, PORF and C-ORF in the genome of the DHBV. Vector NTI 8. 0 analysis revealed that the unknown peptide contained a motif which binded to HLA * 0201. Aligning with the DHBV sequences from different countries and regions indicated that there were no obvious differences of regional distribution among the sequences. A fluorescence quantitative PCR for detecting DHBV was establishment based on the recombinant plasmid pGEM-DHBV-S constructed. This study laid the groundwork for using Guilin brown duck as a hepatitis B animal model.

Chinese medicine Extractum trametes robiniophila murr augment tumor necrosis factor related apoptosis-inducing ligand induced apoptosis in human hepatic cancer cell lines / 中华外科杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Tumor necrosis factor related apoptosis-inducing ligand induces apoptosis and sensitizes resistant hepatic cancer cells to chemotherapy / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effective of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) combination with chemotherapeutic agent inducing apoptosis in resistant hepatic cancer cells.</p><p><b>METHODS</b>HepG2 cells resistant to Adriamycin (HepG2/ADR) were induced stepwise. The effects of TRAIL in combination with ADM (0.1 mg/L) on promoting apoptosis in HepG2/ADR were analyzed, the proliferation was observed by MTT assay, the apoptosis of cells was also observed by flow cytometry and TUNEL method.</p><p><b>RESULTS</b>HepG2/ADR was confirmed resisting to ADM. Treated with TRAIL combination with ADM (0.1 mg/L), it showed significant inhibitory effect on the growth of HepG2/ADM, the percentage of apoptosis was increased as comparison with control at 24 h.</p><p><b>CONCLUSION</b>MDR1 might not take part in resistance to TRAIL-induced apoptosis. TRAIL dramatically augmented the sensitivity to chemotherapeutic agents in HepG2/ADR. Combined TRAIL with chemotherapeutic agents treatment could be a novel and attractive strategy to drug-resistant/ TRAIL-resistant tumor cells.</p>

Interleukin-12 enhanced tumor necrosis factor related apoptosis-inducing ligand TRAIL-induced apoptosis in human hepatocellular carcinoma by inhibiting expression of survivin / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate therapeutic potential of TRAIL in hepatocellular carcinoma (HCC) and the mechanism of sTRAIL resistance and to reverse the resistance to sTRAIL-inducing apoptosis.</p><p><b>METHODS</b>The expression profiles of TRAILR were determined 60 HCC samples, in 20 normal liver tissues and 2 HCC cell lines HepG2 and SMMC-7721 by in situ hybridization. Cellular effects of sTRAIL in promoting apoptosis on HCC cell lines HepG2 and SMMC-7721 were analyzed after exposure to recombinant protein and after transfection with a cDNA expression construct. In vivo effects of sTRAIL on tumor growth were investigated using a nude mice HCC model of hepG2. Furthermore, the expression of survivin in HCC was detected, and treatment with antisence oligonucleotide was accepted. Finally, therapeutic effect on HCC by combining sTRAIL and interleukin-12 (IL-12) was detected.</p><p><b>RESULTS</b>Both DR4 and DR5 were present in all HCC tissues as well as normal hepatic tissues. In contrast, 54 HCC tissues did not express DcR1 and 25 did not express DcR2. But both DcR were detectable in all of the normal liver tissues. The expression patterns of DR and DcR in HCC samples were quite different from those in normal tissue. DR5, DR4, and DcR2 expressed in both cell lines, while no DcR1 expression was detected. Recombinant sTRAIL alone was found to have a slight activity as it killed a maximum of 15% of HCC cells within 24 h while killing over 70% of Jurkat cells. In vivo administration of the TRAIL gene couldn't inhibit tumor growth in a nude mice HCC model. Mostly, HCC tissue and both HCC cell lines expressed survivin, whereas normal liver tissue did not express survivin. Treatment with antisence oligonucleotide enhanced sTRAIL-inducing apoptosis. IL-12 significantly augmented sTRAIL-inducing apoptosis and inhibited survivin expression.</p><p><b>CONCLUSIONS</b>HCC cells are insensitive towards TRAIL-mediated apoptosis. Survivin may play a role in resistance to TRAIL-induced apoptosis in HCC, and antisence oligonucleotide could partly reverse the resistance to TRAIL-inducing apoptosis. IL-12 may sensitize HCC cells to TRAIL-induced apoptosis by preventing survivin. Combining gene therapy strategy such as combining gene therapy of TRAIL with IL-12 may be a promising maneuver to HCC.</p>

Hepatitis B x protein activated vascular endothelial growth factor expression through hypoxia inducible factor-1 pathway / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate whether hepatitis B x protein (HBx) stimulates vascular endothelial growth factor (VEGF) through hypoxia inducible factor-1 (HIF-1 alpha) pathway.</p><p><b>METHODS</b>Two plasmids including pIRES-EGFP-HBx and pTK-Hyg were co-transfected to a hepatocellular carcinoma cell line SMMC-7721. With fluorescence-positive and fluorescence-negative hygromycin-resistant colonies selected, expressions of VEGF and HIF-1 alpha in protein or/and mRNA level were detected.</p><p><b>RESULTS</b>Fluorescence-positive cells were stably integrated with HBx, in which expression of HIF-1 alpha and VEGF were upregulated. Fluorescence-negative cells did not express HBx, VEGF or HIF-1 alpha.</p><p><b>CONCLUSION</b>HBx can activate VEGF through HIF-1 alpha pathway.</p>

Antitumor effects of soluble TRAIL in human hepatocellular carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate therapeutic potential of soluble TRAIL (sTRAIL) in hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>Expression of TRAILR was determined by in situ hybridization in 60 samples of resected hepatocellular carcinoma, 20 samples of normal liver tissue near the margin of benign tumor and 2 HCC cell lines of HepG2 and SMMC-7721. The clinical data of the patients were analyzed as well as cellular effects of sTRAIL in promoting apoptosis on HCC cell lines HepG2 and SMMC-7721 (p53 gene mutated) after exposure to different concentrations of recombinant protein.</p><p><b>RESULTS</b>High death receptor (DR) expression and low DcR expression in HCC tissue differed from low DR expression and high DcR expression in the normal hepatic tissue with statistical significance. DR5, DR4, and DcR2 but not DcR1 were expressed in both cell lines. The expression of DR was closely correlated with HCC differentiation, with the weak expression in poor differentiation. The positive rate of DR expression in 32 cases of grade III-IV was significantly lower than that in 28 cases of grade I-II (P < 0.05). Cell apoptosis rates were 10%, 70% and 50% of HCC cells, Jurkat cells and human cholangiocarcinoma cell line QBC939 24 h after recombinant of TRAIL alone.</p><p><b>CONCLUSION</b>TRAILR expression is prevalent in HCC, with different receptor types existing. HCC is resistant to TRAIL-mediated apoptosis. The treatment of TRAIL alone only has a limited effect on inducing apoptosis on HCC cell lines of HepG2 and SMMC-7721.</p>