Zhang Wanguang

The inhibition of apoptosis of hepatoma cells induced by HBx is mediated by up-regulation of survivin expression

SummaryTo investigate the effect of HBx on expression of survivin in hepatoma cells and mechanisms of inhibition of apoptosis on hepatoma cells induced by HBx, the expression plasmid pHAHBx encoding full length of HBx was transfected into HepG2 cells and the transformed cells were identified by RT-PCR. The expression of survivin both in HepG2 cells and HBx-transfected cells was examined with RT-PCR. The nude mice model of hepatoma was established by injecting HepG2 cells and HBx-transfected cells into the flank of nude mice subcutaneously. The expression level of survivin both in HepG2 formed tumors and HBx-transfected cell-formed tumors in nude mice was examined with Western-blot. The TUNEL assay was used to detect the apoptotic cells of tumor tissues in nude mice after intraabdominal chemotherapy with adriamycin. The results indicated that the amplification of survivin in HBx-transfected HepG2 cells was up-regulated when compared with that in non-transfected cells. Western-blot showed that the tumor cells expressing HBx in nude mice had a positive band of survivin expression and the tumor cells without HBx expression had no positive band. The result of TUNEL assay showed that there were less apoptotic cells in tumor tissues expressing HBx than that in control group cells. It was concluded that HBx could upregulate the expression of survivin in hepatic carcinoma cells which can inhibit apoptosis of hepatic carcinoma cells induced by adriamycin.

An antisense plasmid targeting survivin expression induces apoptosis and sensitizes hepatocarcinoma cells to chemotherapy

SummaryTo explore the change of sensitivity to chemotherapy of antisense RNA targeting survivin on hepatocarcinoma carcinoma cellsin vitro. Survivin mRNA structure region was amplified by RT-PCR and inserted inversely into eukaryotic expression vector pcDNA3. The antisense expression plasmid pcDNA3/survivin was transfected into HepG2 with lipofectAMINETM, 2000 (FL2000), with low concentration of 5-fluorouracil (5-Fu) added. Survivin protein was detected by westernblot, the growth activity was measured by MTT, and apoptosis was detected by Flow cytometry 12 h, 24 h, 48 h after transfection. The activity of caspase-3 was found by quantitative assay 48 h after transfection. The construction of antisense RNA vector pcDNA3/survivin was verified by restricted endonuclease digestion and nucleotide sequencing. Compared with normal group, 5-Fu and antisense survivin group, the cells growth inhibition, apoptosis index, and caspase-3 activity were increased in antisense survivin transfected + 5-Fu group. The threshold of apoptosis was decreased after survivin was silenced, and the sensitivity to chemotherapy was increased. These findings suggest the existence of a potential new target for gene therapy.

Influence of ciglitazone on A549 cells growthin vitro andin vivo and mechanism

SummaryThe effect and mechanism of the ciglitazone on lung cancer cells A549 growthin vitro andin vivo were studied. Various concentrations of ciglitazone were added to the cultured A549 line, and the proliferation and differentiation of A549 cells were examined by MTT and cytometry analysis. A549 cells (1×106/mouse) were inoculated subcutaneously into 20 nude mice, which were randomly divided into two groups: the control group, the ciglitazone treated group. The weights of subcutaneous tumors were measured. The expression of cyclin D1 and P21 in the lung was detected by immohistochemistry and Western blot respectively. The results showed that the proliferation of A549 was inhibited significantly by ciglitazone in a dose- and time-dependent manner. There were more cells arrested in G1/G0 phase and the expression of PPARγ was markedly up-regulated in ciglitazone-treated group. Direct injection of ciglitazone into A549-induced tumors could suppress tumor growth in nude mice and the growth inhibitory rate was 36%. The expression of cyclin D1 was decreased and P21 increased significantly in ciglitazone-treated group as compared with control group. It was concluded that ciglitazone could inhibit A549 proliferation dose-dependently and time-dependently and induce differentiation, which might be related to the modulation of cell cycle interfered by PPARγ.

KLF6mRNA expression in primary hepatocellular carcinoma

SummaryTo investigate the expression of KLF6mRNA in primary hepatocellular carcinoma (HCC), nomal liver tissues and the tissues adjacent to the cancers, reverse-transcription polymerase chain reaction (RT-PCR) was employed to investigate the expression of the KLF6 gene in HCC, the corresponding adjacent non-cancerous tissues and normal liver tissue. Our results showed that an amplified fragment of 127 bp DNA was detected in 18 of 19 (94.7%) adjacent non-cancerous tissues and normal liver tissue, and in 12 (85.7%) of 11 HCC. There were no significant differences in the levels of KLF6 mRNA between normal liver and liver tumors (P>0.05). It is concluded that KLF6 mRNA is generally expressed in HCC.

Hypoxia-Inducible Factor-1alpha Suppressing Apoptosis and Increasing Tolerance of Lung Cancer Cells to Chemotherapy

In order to construct plasmid of hypoxia-inducible factor-lalpha (HIF-1α), and transfect into human lung cancer cells A549, the change in sensitivity of lung cancer cells A549 to chemotherapy was observed. HIF-1α mRNA structure region was amplified by RT-PCR and inserted into plasmid pcDNA3. The expression plasmid pcDNA3/HIF-1α was transfected into A549 with Lipofec-tAMINE™2000. The expression of HIF-1α protein was detected by Western blot. After A549 cells were transfected with HIF-1α prior to addition of 5-Fu, the growth activity was measured by growth curve, apoptosis was detected by flow cytometry at 48 h, and the levels of caspase3 and MDR-1 were determined by Western blot. The results showed that the constructed expression plasmid was analyzed with restriction enzymes and gel electrophoresis. Two DNA lanes at 2.55 kb and 5.4 kb respectively were found, which were consistent with that expected. The growth rate in 5-Fu group was significantly inhibited, and the apoptosis index and caspase3 activity were increased significantly as compared with control group. After HIF-1α being transfected into A549, the activity of MDR-1 was increased and the effect of 5-Fu was weakened. In conclusion, HIF-1α can promote chemoresistance by increasing the activation of MDR1 and suppressing apoptosis during lung cancer cells A549 induced with 5-Fu.

Antitumor Effects of Soluble TRAIL in Human Hepatocellular Carcinoma

SummaryThe therapeutic potential of soluble TRAIL (sTRAIL) in hepatocellular carcinoma (HCC) was studied. The expression of TRAIL receptors was detected in 60 HCC tissues, 20 normal liver samples and 2 HCC cell lines (HepG2 and SMMC-7721) byin situ hybridization. Before and after HepG2 and SMMC-7721 were treated with sTRAIL protein with various concentrations, the apoptosis rate was observed by using flow cytometry andin situ terminal deoxynucleotidyl tranferase (TdT) labeling. The results showed death receptor 4 (DR4) and DR5 were expressed in 60 HCC tissues and 20 normal liver samples, while the expression intensity of DR in HCC tissues was stronger than in normal liver samples. DcR1 and DcR2 were not detectable in 54 (90%) and 25 (41.7%) HCC tissues, while in 20 normal liver samples. The expression of DR5, DR4 and DcR2 in both HCC cell lines was detectable, but the expression of DcR1 was not detectable. The expression of DR in HCC tissues was related to the differentiation and grades of HCC. In the poor differentiated HCC, the expression of DR was decreased (P<0.01). The expression of DR in III/IV grades was significantly lower than that in I/II grades (P<0.05). The expression of DR was not related to gender, age, HBsAg, AFP, tumor size and metastasis. The expression of DR in the HCC drugresistant lines was decreased. After treatment with TRAIL (100 ng/ml) for 24 h, the apoptosis rate of HCC cells, Jurkat cells and human cholangiocarcinoma cell line QBC939 was 10%, 70%, 50% respectively. It was suggested that the TRAILR expression is prevalent in HCC with different expression patterns of different receptor types. HCC is resistant to TRAIL-mediated apoptosis. The treatment of TRAIL alone has a limited effect on inducing apoptosis of HepG2 and SMMC-7721.

Inhibition of the VEGF expression and cell growth in hepatocellular carcinoma by blocking HIF-1α and Smad3 binding site in VEGF promoter

SummaryIn order to investigate the inhibitory effects on the vascular endothelial growth factor (VEGF) expression and cell growth in hapatocellular carcinoma (HCC) by blocking HIF-1α and Smad3 binding site in the VEGF promoter, antisense oligodeoxynucleotides (ASODN) were designed to block HIF-1α and Smad3 binding site in the VEGF promoter. Different concentrations of ASODN and ODN were transfected into HCC cells respectively. The expression of VEGF mRNA and protein was detected by SABC, Western blot and RT-PCR techniques and the inhibitory effects on the expression of VEGF and cell growth of the HCC cells stimulated by the supernatants were determined by using MTT method. Immunohistochestry revealed that after co-inoculation of hepatocellular carcinoma cells with different concentrations of ODN and ASODN for 48 h, there was no significant difference in the expression of VEGF protein between ODN group and control group (P>0.05), but there was significant difference between ASODN, the difference was very significant (P<0.01). Western blot and RT-PCR revealed that, after treatment for 48 h at a concentration of 10 μmol/L, the integral gray levels and RNA odds were 59743.2±10412.5 and 0.783±0.032 in ODN group, and 38694.5±10925.1 and 0.468±0.015 in ASODN group, respectively, with the difference being very significant (P<0.01). Antisense ODN could inhibit the growth of HCC cells in a concentration-dependent manner. It was concluded that anti-gene technique of aiming at HIF-1α action site in the VEGF promoter could suppress the VEGF expression and inhibit HCC cell growth, and it is promising that anti-gene technique works as a new gene therapeutic tool for anti-angiogenesis of HCC.In order to investigate the inhibitory effects on the vascular endothelial growth factor (VEGF) expression and cell growth in hapatocellular carcinoma (HCC) by blocking HIF-1alpha and Smad3 binding site in the VEGF promoter, antisense oligodeoxynucleotides (ASODN) were designed to block HIF-1alpha and Smad3 binding site in the VEGF promoter. Different concentrations of ASODN and ODN were transfected into HCC cells respectively. The expression of VEGF mRNA and protein was detected by SABC, Western blot and RT-PCR techniques and the inhibitory effects on the expression of VEGF and cell growth of the HCC cells stimulated by the supernatants were determined by using MTT method. Immunohistochestry revealed that after co-inoculation of hepatocellular carcinoma cells with different concentrations of ODN and ASODN for 48 h, there was no significant difference in the expression of VEGF protein between ODN group and control group (P > 0.05), but there was significant difference between ASODN group and control group (P < 0.05). At a concentration of 10 micromol/L ASODN, the difference was very significant (P < 0.01). Western blot and RT-PCR revealed that, after treatment for 48 h at a concentration of 10 micromol/L. the integral gray levels and RNA odds were 59743.2 +/- 10412.5 and 0.783 +/- 0.032 in ODN group, and 38694.5 +/- 10925.1 and 0.468 +/- 0.015 in ASODN group, respectively, with the difference being very significant (P < 0.01). Antisense ODN could inhibit the growth of HCC cells in a concentration-dependent manner. It was concluded that anti-gene technique of aiming at HIF-1alpha action site in the VEGF promoter could suppress the VEGF expression and inhibit HCC cell growth, and it is promising that anti-gene technique works as a new gene therapeutic tool for anti-angiogenesis of HCC.

New concepts and techniques of hepato-pancreato-biliary surgery

Surgery is the most effective and preferred treatment for hepato-pancreato-biliary (HPB) tumors. The improvement of HPB surgery technique mainly focus on reducing the amount of bleeding, improving the safety of operation, simplifying the operation, reducing the complications and mortality all the time. As one of the largest HPB centers in China, hepatic center of Tongji hospital has set up a series of new concepts and new techniques of HPB surgery since 1980s. We proposed a new classification standard of hepatocellular carcinoma (HCC) to guide the surgical procedures and evaluation of prognosis. According to this method, HCCs lager than 5 cm were defined as lager (>5 cm and ³ 10 cm). Traditionally, the large/huge HCC patients were thought can’t tolerate hepatectomy because of the insufficient residual liver volume. However, we confirmed the feasibility and safety of the resection of large/huge HCC and applied it in clinic concurrently in 1990s, which greatly extended the inclusion criteria for operation. Serious intraoperative bleeding is another constraint for resection of large/huge HCC. In order to reduce the intraoperative bleeding and increase the safety of major hepatectomies, three new bleeding control techniques for hepatectomy were established: Infrahepatic inferior vena cava clamping combine with occlusion of the portal triad; tying up of inflow and outflow vessels without dissecting the hilus of the liver; and implementation of the liver double-hanging maneuver through the retrohepatic avascular tunnel on the right side of the inferior vena cava. According to the AASLD and the EASL guidelines for HCC management, patients with portal vein tumor thrombosis (PVTT) are excluded from either surgical treatment or TACE, and only palliative therapies are recommended. However, our studies confirmed treating PVTT patient by indicated surgical protocol according to the location and extension of PVTT is safety and effective. Based on the guideline set by western countries, liver resection is contraindicated in patients with portal hypertension. But in China, 85%–90% patients with HCC showed various degrees of liver cirrhosis, and a large proportion of these patients presented clinical signs of portal hypertension. Thus, we set up a surgical treatment strategy (limited liver resection plus splenectomy with or without devascularization of gastroesophageal varices) for HCC combined with portal hypertension. There are still controversies on the optimal extent of hepaticresection to achieve a high percentage of R0 resection for hilarcholangiocarcinoma. We proposed a new concept that treats hilar cholangiocarcinoma using minor liver resection which showed lower operative morbidity and good prognosis. We also created new surgical procedures such as new intrahepatic cholangiojujunostomy for multiple intrahepatic biliary ductal openings and the inserting bilio- jejunostomy for iatrogenic bile duct injury were all invented and proved to be available in clinic. In order to diminish pancreatic leakage, we established Chen’s U-suture technique for end-to-end invaginated pancreaticojejunostomy following pancreaticoduodenectomy. Many end-stage liver disease patients died due to severe shortage of organ donors. To address the shortage, we are the first to establish auxiliary partial orthotopic liver transplantation technique in the world and applied it in clinic successfully.

Expression profiles of TRAIL receptors and their clinical significance in human hepatocellular carcinoma

ObjectiveTo investigate the expression profiles and their clinical significance of TRAIL receptors (TRAILR) in human hepatocellular carcinoma (HCC).MethodsThe expression profiles of TRAILR were determined in 60 samples from hepatocellular carcinoma, 20 from normal liver tissue and two HCC cell lines HepG2, SMMC-7721 by in situ hybridization.ResultsBoth 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 (higher DR expression level and lower DcR expression level) were quite different from those in normal tissue. DR5, DR4, and DcR2 expressed in both cell lines, while no DcR1 expression was detected. The expression level of DR was correlated with HCC differentiation and stage. The weaker expression was more commonly found in HCC with poor differentiation and late stage, while the stronger expression was more common in HCC with middle to high-differentiation and early stage. No relationship was found between DR and gender, age, negative or positive HBsAg, tumor size, grade or metastasis. Multidrug resistance cell lines expressed lower level DR.ConclusionTRAILR expression was prevalent and discrepancy of receptor types was exited in HCC. Loss of DcR1 may contribute for TRAIL therapy for HCC.