Expression of PADI4 in hepatocellular carcinoma

Objective： The aim of the research was to study peptidylarginine deiminase type 4 （PAD4/PADI4） expression and its tumodgenic mechanism in hepatocellular carcinomas. Methods： Expressions of PADI4 and p53 were investigated in tumors and non-tumor tissues by Western blot in patients with hepatocellular carcinomas. We constructed plasmid of PADI4-Flag and transfected it in Hela cells to investigate the mechanism. Results： Western blot analysis showed higher PADI4 expression in hepatocellular carcinomas than in the surrounding healthy tissues. Furthermore, by Western blot, we detected decreased p53 levels in the tumor tissues of patients with hepatocellular carcinomas compared to surrounding healthy tissues. In Hela cells transfected with PcDNA3.0-Flag-PADI4 plasmid, the expression of p53 decreased obviously. Conclusion： Our results suggest that PADI4 elevated in the tissues of hepatocellular carcinomas and induced tumorigenic by down-regulating p53 expression.     

Detection of PRL-2 gene expression in hepatocellular carcinoma by real-time fluorescence quantitative PCR

Objective： To quantitatively detect the expression level of PRL-2 in primary hepatocellular carcinoma using realtime fluorescence quantitative PCR. Methods： Total RNA isolated from human HCC and liver tissue adjacent to the tumor was reversely transcribed into cDNA. Real-time fluorescence quantitative PCR （Q-PCR） method was used to analyze the expression level of PRL-2 gene. Results： The Q-PCR method was performed successfully to precisely detect RNA level. PRL-2 was expressed in all portal vein tumor thrombosis （PVTT） and HCC, but only in some paratumor tissue. The highest expression level of PRL-2 gene was recorded in PVTT; meanwhile expression level of PRL-2 was higher than that in paratumor liver tissues and in HCC （P 〈 0.01）, and it was higher in HCC than that in paratumor liver tissues. Conclusion： The Q-PCR may be the most precise method to quantitatively detect RNA level and can be used in gene expression changes. The PRL- 2 gene has higher expression in PVTT than that in HCC and in paratumor liver tissue cells, indicating that it plays an important role in the development and metastasis of the HCC.     

The difference between multi-drug resistant cell line A549/Gem and its parental cell A549

Objective： To discuss the difference between multi-drug resistant cell line A549/Gem and its parental cell A549 on the basis of establishment of human gemcitabine-resistant cell line A549/Gem so as to elaborate the possible mechanisms of gemcitabine resistance. Methods： Human gemcitabine-resistant non-small cell lung cancer cell line A549/Gem was established by the method of repeated clinical serous peak concentration plus gradually increasing concentration of gemcitabine from its parental cell human lung adenocarcinoma cell line A549 which was sensitive to gemcitabine. During the course of inducement, we had monitored their morphology, checked their resistance indexes and resistant pedigree by MTT method, gathered their growth curves and calculated their doubling time, examined their DNA contents and cell cycles by FCM; at the same time, we had measured their expressions of P53, EGFR, Cerb-B-2, PTEN, PCNA, c-myc, VEGF, MDR-1, Bcl-2, nm23, MMP-9, TIMP-1, and CD44v6 proteins via immunocytochemistry staining, RRM1 and ERCC1 mRNA by real-time fluorescent quantitative-PCR. Results： The resistance index of A549/Gem＇ cells （the deputy of cells in the process of inducement） to gemcitabine was 163.228, and the cell line also exhibited cross-resistance to vinorelbine, taxotere, fluorouraci, etoposide and cisplatin, but kept sensitivity to paclitaxol and oxaliplatin. The doubling time of A549/Gem＇ was shorter and figures in G0-G1 phases were increased than A549 cells. Compared with A549 cells, A549/Gem＇ cells achieved EGFR and c-myc proteins expressions, nm23 protein expression enhanced, P53, Cerb-B-2 and Bcl-2 proteins expressions reduced, PTEN, PCNA and MDR-1 proteins expressions vanished, but those of MMP-9, VEGF, CD44v6 and TIMP-1 proteins changed trivially. Meanwhile, expressions of RRM1 and ERCC1 mRNA were augmented markedly. The resistance index of A549/Gem cells to gemcitabine was 129.783, and the cell line also held cross-resistance to vinorelbine, taxotere, etoposide, cisplatin and sensitivity to     

Expression of HIF-la in hepatocellular carcinoma and its relationship with vasculogenic mimicry and clinical pathology

Objective： The aim of this study was to discuss HIF-la expression and vasculogenic mimicry （VM） in hepatocel- lular carcinoma （HCC） and their relationship with the clinical pathological features and clinical significance. Methods： Two hundred and seven specimens from patients in The Affiliated Hospital of North Sichuan Medical College who received hepatic cell carcinoma resection were tested by immunohistochemistry and double staining of CD31 and PSA. Then detected the expression of HIF-la, VM, and analysed the relationship between clinical pathology. Results： The HIF-la positive rate was 71.01% and its expression was associated with liver cirrhosis, tumor size and TNM stage （P 〈 0.05）. HIF-la protein expres- sion was positively associated with the VM （y = 0.1988, P = 0.0041）. Conclusion： Hypoxia may be the reason for VM in high invasive HCC, regulating the tumor microenvironment may have great significance in inhibiting invasion and metastasis of HCC.     

Efficacy of percutaneous ethanol injection in the adjuvant treatment of hepatocellular carcinoma after TACE

Objective： To evaluate the efficacy of percutaneous ethanol injection （PEI） in the adjuvant treatment of hepatocellular carcinoma （HCC） after transcatheter arterial chemoembolization （TACE） by primary end points of time to progress （TTP）. Methods： The study population consisted of 73 consecutive patients with inoperable HCC （China Classification System IIN liB）. Among them, 22 patients were treated with TACE and PEI （experimental group）, and the rest 51 were treated only with TACE （control group）, and then the time to progress （TTP） and overall survival （OS） of these two groups were analyzed. Results： The median TTP was 10 months [95% confidence interval （CI）, 7.9-12.1 months] in experimental group and 6 months （95% CI, 4.7-7.3 months） in control group. The 3-month,6-month, and 1-year Progression Free Survival （PFS） rates were respectively 77.3%, 63.6%, and 48.1% in experimental group, and 76.5%, 42.15%, and 24.8% in control group. The TTP of experimental group was significantly longer than that of control group （P 〈 0.05）. The median survival period was 17 months [95% confidence interval （CI）, 11-23 months] of experimental group and 12 months （95% CI, 10-14 months） of control group （P 〉 0.05）. Conclusion： Compared with single TACE, the combination of TACE and PEI can obviously postpone disease progress and prolong survival of HCC patients.     

Effects of monoclonal antibodies against human stathmin I combined paclitaxel on proliferation of human hepatocellular carcinoma cell lines

Objective： We investigated the effects of monoclonal antibodies against stathmin 1 combined paclitaxel on the proliferation of HCC cells. Methods： HepG2 cells were treated with monoclonal antibodies against stathmin 1, paclitaxel alone or their combination, with the untreated cells used as the control, 24, 48, 72, 96 h later, the cell growth condition was observed by invert microscope and inhabitation rate was studied by MTT assay; The apoptosis was analyzed by flow cytometry with Annexin V/PI. Results： The population decreased and shape, size changed after treating with different concentration of experimental groups. Monoclonal antibodies against stathmin 1 and paclitaxel used alone or in combination both inhibited the proliferation of HepG2 cells, the inhibition ratio of their combination was more higher （P 〈 0.05）, and a synergistic effect of the two agents was noted in their combined action （P 〈 0.05）. Combined treatment of the cells resulted in significantly higher apoptosis rate than that in the other groups （P 〈 0.05）. Conclusion： Monoclonal antibodies against stathmin 1 and paclitaxel used alone or in combination both can inhibit proliferation of HepG2 cells and induce apoptosis. A synergistic effect is obsewed between the monoclonal antibodies against stathmin 1 and paclitaxel in their inhibition of HepG2 cell proliferation.     

Biological effect of NK4 gene mediated by attenuated Salmonella typhimurium on hepatocellular carcinoma cell HepG2

Objective： The aim of the study was to construct a stable strain of recombined attenuated Salmonella typhimurium expressing NK4 gene, and observe the effect of the strain on the metastatic potentiality of HepG2 cells. Methods： The NK4 cDNA was isolated from PCAGGS/hNK4 plasmid by PCR, and subcloned into eukaryotic expression vector pcDNA4. The recombinant plasmid was electro-transferred into attenuated Salmonella typhimurium Ty21a to obtain the recombinant strain encoding NK4 gene （TPN）. Simultaneously, the recombinant attenuated Salmonella typhimurium carrying GFP gene （TPG） was also constructed. After the TPG and TPN were transferred into HepG2 cells, the transfection rate and the expression level of NK4 protein were detected by flow cytometry and ELISA, and the effects of expression product on the proliferation and migration of HepG2 and angiogenesis were observed. Results： The TPN and TPG were successfully constructed. Fortyeight hours after transfection with TPG, the infection rate was 82.58% ± 1.74%, and the expression level of NK4 protein in supernatant was （181.5 ± 11.7） ng/6 × 10^5 cells. The supematant had obviously depressant effect on the proliferative activity of HepG2 cells （P 〈 0.05）, and could obviously restrain the hepatocyte growth factor-mediated migration of tumor cells （P 〈 0.01）. The inhibitory effect of the expression product on the tumor angiopoiesis was obviously observed （P 〈 0.05）, without a dosage-effect relation. Conclusion： The TPN could effectively transfer tumor cells in vitro and express interest NK4 protein. The expression product could effectively inhibit the proliferation and migration of hepatocellular carcinoma cells and the tumor angiopoiesis.     

Relationship between p38MAPK activity and apoptosis during the drug resistance of breast carcinoma cell lines

Objective： The aim of this study was to investigate the relationship between p38MAPK activity and apoptosis during the drug resistance of breast carcinoma cell lines. Methods： Using p38MAPK special inhibitor SB203580 to analyze the effect on the cell apoptosis of MCF-7/ADM cell. Cell apoptosis was analysed by PI staining and flow cytometry （FCM） （F test）. 50% inhibition concentration （IC50） of adriamycin on MCF-7/ADM was determined by MTT method （F test） in vitro. MDR-1 mRNA expression was detected by RT-PCR （F test） and Western Blot （F test） respectively. Results： After SB203580 24 h action the MCF-7/ADM＇s apoptosis rate was 26.73±4.90%, higher than the control group and untreated group （F = 143.80, P 〈 0.001）. The sensitity to the ADM was improved significantly （F = 148927.1, P 〈 0.001）, and the reversal effect of treat SB203580 group was 68.45%. The P38MAPK protein （F= 685.419, P 〈 0.001） and MDR-1 mRNAexpression after SB203580 24 h action were lower than the control group and untreated group （F = 9139.24, P 〈 0.001）. Conclusion： P38MAPK signal way plays an important role in drug resistance of breast carcinoma ceil. p38MAPK can protect MCF-7/ADM cells from apoptosis, and blocking the p38MAPK signal way can increase the apoptosis for breast carcinoma drug resistance cell lines.     

The role and status of fuzheng in cancer prevention and treatment

Traditional Chinese medicine （TCM） treatment of cancer has a long history, and is an important part of cancer prevention and control in China. Fuzheng, also called reinforcing healthy qi and supplementing the root, is the most funda- mental principle of TCM in cancer prevention and control. In recent decades, this treatment has been thoroughly studied and widely applied, and played a crucial role in cancer prevention and treatment. With regard to the treatment of malignant tumors, Chinese medicine is mainly used in the following areas： improving symptoms, enhancing the quality of life, reducing postop- erative recurrence and metastasis, increasing efficacy and decreasing toxicity together with radiotherapy and chemotherapy treatments, and to some extent prolonging the survival of advanced tumors.     

The impact of Deta-elemene on Deta-tuDulJn of human hepatoma hepg2 cells

Objective： The aim of this study was to investigate the impact of beta-elemene injection on the growth and beta-tubulin of human hepatocarcinoma HepG2 cells. Methods： Cell proliferation was assessed by MTT assay. Cell cycle distribution was detected by flow cytometry （FCM）. The mRNA expression of beta-tubulin was measured by RT-PCR. West- ern blot analysis was used to determine protein expression of beta-tubulin and the polymerization of beta-tubulin. Results： Beta-elemene injection inhibited HepG2 cells proliferation in a dose- and time-dependent manner; FCM analysis indicated beta-elemene injection induced cell cycle arrested at S phase. RT-PCR and western-blot analysis showed that beta-elemene injection down-regulated beta-tubulin expression at both mRNA and protein levels, presenting a dose-dependent manner. Moreover, beta-elemene injection reduced the polymerization of microtubules in a dose-dependent manner. Conclusion： Beta-elemene injection can inhibit the proliferation of hepatoma HepG2 cells, the mechanism might be partly related to the down-regulation of beta -tubulin and inhibition of microtubular polymerization.