OBJECTIVETo investigate the effect of enoyl coenzyme A hydratase-1 (Ech1) on the proliferation and invasion ability of mouse hepatocarcinoma Hca-P cells in vitro.

METHODSRecombinant pcDNA3.1(+)-Ech1 gene and pcDNA3.1(+) were transfected into Hca-P cells by cationic liposomes introduction. Clone of PEch1 cells that stably expressing Ech1 and clone of control Pvector cells were screened by G418. The Ech1 expression was identified subsequently by reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The malignant behaviors of the cell lines were compared by proliferation, invasion and migration test.

RESULTSThe cell line Hca-P cells stably expressing Ech1 gene was constructed. The relative expression of Ech1 mRNA in the PEch1 group was 3.21 ± 0.43 and in the Pvector group was 1.44 ± 0.03, with a significant difference between the two groups (P = 0.029). The results of ELISA revealed that the expression of Ech1 protein was 0.140 ± 0.005 in the PEch1 group, 0.088 ± 0.003 in the Pvector group, and 0.078 ± 0.006 in the Hca-P group, showing a significant difference between the PEch1 group and the Pvector and Hca-P groups (P < 0.05). Transwell migration test showed that the number of penetrated cells in the PEch1 group was 143.00 ± 7.25 cells, significantly higher than that of the Pvector group (95.73 ± 3.88 cells) and un-treated Hca-1 group (106.67 ± 3.54 cells, both P < 0.05). The Transwell invasion assay showed that the number of penetrated cells was 77.20 ± 5.46 cells in the PEch1 group, significantly higher than 46.34 ± 4.35 cells in the Pvector group and 49.80 ± 5.21 cells in the un-treated Hca-1 group (both P < 0.05).

CONCLUSIONSThe results showed that overexpressed Ech1 in Hca-P cells may significantly increase the cell proliferation in a time-dependent manner. The up-regulation of Ech1 may increase to some extent the migration and invasion capacity of Hca-P cells. The efforts aiming at up-regulation of Ech1 expression may become a therapeutic target in the treatment of hepatocarcinoma.

Animals ; Carbon-Carbon Double Bond Isomerases ; genetics ; metabolism ; Cell Movement ; Cell Proliferation ; Liver Neoplasms, Experimental ; enzymology ; pathology ; Mice ; Neoplasm Invasiveness ; Plasmids ; RNA, Messenger ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Transfection ; Tumor Cells, Cultured ; Up-Regulation

NM23 is a family of structurally and functionally conserved proteins known as nucleoside diphosphate kinases (NDPK). There is abundant mRNA expression of NM23-H1, NM23-H2, or a read through transcript (NM23-LV) in the primary sites of hepatocellular carcinoma (HCC). Although the NM23-H1 protein is implicated as a metastasis suppressor, the role of NM23-H2 appears to be less understood. Thus, the aim of this study was to examine whether NM23-H2 is associated with hepatocarcinogenesis. The level of NM23-H2 expression in tumor tissues and the surrounding matrix appeared to be independent of etiology and tumor differentiation. Its subcellular localization was confined to mainly the cytoplasm and to a lesser extent in the nucleus. Ectopic expression of NM23-H2 in NIH3T3 fibroblasts and HLK3 hepatocytes showed a transformed morphology, enhanced focus formation, and allowed anchorage-independent growth. Finally, NIH3T3 fibroblasts and HLK3 hepatocytes stably expressing NM23-H2 produced tumors in athymic mice and showed c-Myc over-expression. In addition, NF-kappaB and cyclin D1 expression were also increased by NM23-H2. Lentiviral delivery of NM23-H2 shRNA inhibited tumor growth of xenotransplanted tumors produced from HLK3 cells stably expressing NM23-H2. Collectively, these results indicate that NM23-H2 may be pro-oncogenic in hepatocarcinogenesis.
Animals ; Carcinoma, Hepatocellular/*enzymology/genetics/pathology ; Cell Line ; Cell Line, Tumor ; *Gene Expression Regulation, Neoplastic ; Humans ; Liver/*enzymology/metabolism/pathology ; Liver Neoplasms/*enzymology/genetics/pathology ; Mice ; Mice, Nude ; NIH 3T3 Cells ; NM23 Nucleoside Diphosphate Kinases/*genetics/metabolism

To examine the differential expression pattern of Ech1 protein in mouse Hca-F and Hca-P hepatocarcinoma cell lines with high and low rates of lymphatic metastasis, respectively, and to investigate the relationships between Ech1 expression and adhesion of Hca-F cells. Fluorescence two-dimensional difference in-gel electrophoresis (2D DIGE) and mass spectrometry were used to detect Ech1 expression. Ech1 gene silencing was achieved by stable transfection of Hca-F cells with a plasmid vector harboring short hairpin RNA (shRNA) targeting Ech1, pGPU6/GFP/Neo-shRNA-Ech1. Ech1 mRNA and protein expressions were detected by real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting analysis, respectively. Adhesive properties of cells were assessed by hematoxylin-eosin staining and fluorimetric detection of extracellular matrix (ECM) proteins. Endogenous Ech1 protein level was remarkably higher in the highly metastatic Hca-F cell line than in the Hca-P cell line (2.7-fold by 2D DIGE; 1.5-fold by Western blotting). shRNA-induced silencing of Ech1 significantly reduced the adhesion ability of Hca-F cells, as evidenced by decreased absorbance values of fibronectin and collagen I (Hca-F cells vs. pGPU6/GFP/Neo-shRNA-Ech1 cells: 1.42+/-0.26 vs. 1.01+/-0.27 and 1.14+/-0.07 vs. 0.90+/-0.09, respectively; P less than 0.05). Down-regulation of Ech1 can inhibit the adhesive capacity of metastatic Hca-F cells.
Animals ; Carbon-Carbon Double Bond Isomerases ; genetics ; metabolism ; Carcinoma, Hepatocellular ; enzymology ; genetics ; pathology ; Cell Adhesion ; Cell Line, Tumor ; Down-Regulation ; Electrophoresis, Gel, Two-Dimensional ; Gene Expression Regulation, Neoplastic ; Liver Neoplasms ; enzymology ; genetics ; pathology ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; Mice ; Plasmids ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; pharmacology ; Transfection

BACKGROUNDPrimary liver cancer (PLC) is a common malignant tumor. Over the past decade, although farnesyltransferase (FTase) has emerged as a significant target for anticancer therapies and has become a hotspot of cancer research, its exact mechanism of action remains unknown. The aim of this study was to investigate the expression of FTase in PLC and its role in the development of PLC.

METHODSExpression of FTase was detected by real-time fluorescent quantitative-polymerase chain reaction (FQ-PCR) in cancer and surrounding normal tissues from 32 patients with PLC.

RESULTSExpression of FTase mRNA in PLC was significantly higher than that in normal hepatic tissues (P < 0.001). Overexpression of FTase was as high as 87.5%. The positive rate for FTase mRNA in the high tendency to metastatic recurrence group was obviously higher than that in the low tendency to metastatic recurrence group (P = 0.02). The positive rate for FTase mRNA in patients with metastatic recurrence during postoperative follow-up was also significantly higher than that in those without metastatic recurrence (P = 0.01).

CONCLUSIONSThe level of FTase mRNA expression in cancer tissues is much higher than in normal tissues. FTase may play an important role in the genesis and development of PLC and may be one of the reliable markers for the metastatic activity gained by liver tumor cells. FTase could be used clinically in predicting metastatic recurrence of PLC.

Adult ; Aged ; Farnesyltranstransferase ; genetics ; Female ; Humans ; In Vitro Techniques ; Liver Neoplasms ; enzymology ; pathology ; Male ; Middle Aged ; Polymerase Chain Reaction ; RNA, Messenger ; Young Adult

OBJECTIVETo study the expression of enoyl CoA hydratase 1 (ECH1) and the effect when down-regulation of ECH1 gene expression in mouse hepatocarcinoma cell.

METHODSImmunofluorescence was used for detecting the expression of ECH1, and stably transfected Hca-F cells with pGPU6/GFP/Neo-shRNA-ECH1 expression plasmids. Cell proliferation was assessed by Cell counting kit-8 (CCK8) assay. The Boyden-transwell assay (8 µm pore size) was performed to analyze the inhibitory effect of shRNA on Hca-F cell migration and invasion.

RESULTSECH1 expression was obtained in the cytoplasm and upregulated expression in Hca-F cells than that in Hca-P cells. The down-regulation of ECH1 could inhibit the cell proliferation of Hca-F cells, decrease the number of cell pass through Transwell (27.07 ± 17.49) compared with scramble-negative (72.38 ± 18.83) and Hca-F controls (59.06 ± 30.33), decrease the migration capacities of Hca-F cells, increase the ratio of Hca-F cells in S phase (86.1%) compared with scramble-negative (75.8%) and Hca-F controls (66.2%) and decrease the ratio of G(1) phase (9.4%) compared with scramble-negative (24.2%) and Hca-F controls (30.3%).

CONCLUSIONECH1 serves as a potential critical factor attributes to tumor lymphatic metastasis.

Animals ; Cell Cycle ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cytoplasm ; enzymology ; Down-Regulation ; Enoyl-CoA Hydratase ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Liver Neoplasms, Experimental ; enzymology ; pathology ; Lymphatic Metastasis ; Mice ; Plasmids ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo investigate the effects of silencing heparanase (HPA) on growth, angiogenesis and metastasis of human gastric carcinoma transplanted in nude mice.

METHODSHuman gastric carcinoma SGC-7901 cells and those cells with silenced HPA (gastric carcinoma SGC-7901-HPA(-)) were separately transplanted subcutaneously in 6 nude mice. The time, size and speed of tumor growth were recorded. RT-PCR and Western-blot were used to detect the expression of HPA mRNA and protein in the subcutaneous tumors of the two groups. Immunohistochemical staining was used to detect microvessel density (MVD) in the subcutaneous tumors of the two groups. Cells of the subcutaneous transplanted tumors of the two groups were separately injected into the peritoneal cavity of nude mice, 6 mice each. The growth of metastatic tumors in nude mice was observed.

RESULTSHuman gastric carcinoma SGC-7901 cells and SGC-7901-HPA(-) cells were subcutaneously inoculated in nude mice, and tumors appeared at 4 days and 7 days after inoculation, respectively. The MVD was (20.69 ± 1.20)/HP and (11.35 ± 1.94)/HP, respectively (P < 0.05). The expressions of HPA mRNA and protein of the subcutaneously transplanted SGC-7901-HPA(-) tumor were decreased. Four voluminous metastatic tumors caused by SGC-7901 cells occurred in 3 mice in the liver, right kidney, omentum and intestine. Two smaller abdominal metastatic tumors of SGC-7901-HPA(-) cells were found in the liver and right kidney.

CONCLUSIONSilencing HPA can inhibit the tumor growth, angiogenesis and metastasis of human gastric cancer in nude mice. It suggests that HPA might become a new target for prevention and treatment of gastric cancer.

Adenocarcinoma ; blood supply ; enzymology ; genetics ; pathology ; secondary ; Animals ; Cell Line, Tumor ; Gene Silencing ; Glucuronidase ; biosynthesis ; genetics ; physiology ; Humans ; Kidney Neoplasms ; secondary ; Liver Neoplasms ; secondary ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Microvessels ; pathology ; Neoplasm Transplantation ; Neovascularization, Pathologic ; RNA, Messenger ; metabolism ; Stomach Neoplasms ; blood supply ; enzymology ; genetics ; pathology

The scaffold protein IQGAP1 shows elevated levels in several cancer types, but its expression in hepatocellular carcinoma is unknown. We found that 58% of human hepatocellular carcinoma tissue samples had increased IQGAP1 expression compared to adjacent normal tissue. Overexpressing IQGAP1 raised the in vivo tumorigenicity of hepatocellular carcinoma cells, and forced overexpression of IQGAP1 in vitro stimulated cell proliferation. Cell growth was reduced by knockdown or mutation of IQGAP1, or by treatment of cells with a phosphotidylinositol 3-kinase inhibitor. To determine the mechanism by which IQGAP1 overexpression affected hepatocellular carcinoma cells, we confirmed its interaction in these cells with mammalian target of rapamycin (mTOR), a serine/threonine kinase that integrates signals about nutrient and energy status with downstream effectors that influence cell division. In addition, we discovered a new interaction involving IQGAP1, mTOR and Akt, which is a downstream target of mTOR. Akt phosphorylation on Ser-473, which is catalyzed by mTOR and required for Akt activation, increased with increasing amounts of IQGAP1, and decreased with IQGAP1 mutation. We hypothesize that IQGAP1 is a scaffold that facilitates mTOR and Akt interaction.
Animals ; Carcinoma, Hepatocellular/*enzymology/*pathology ; Cell Proliferation ; Enzyme Activation ; *Gene Expression Regulation, Neoplastic ; Hep G2 Cells ; Humans ; Liver Neoplasms/*enzymology/*pathology ; Mice ; Phosphatidylinositol 3-Kinases/metabolism ; Protein Binding ; Proto-Oncogene Proteins c-akt/*metabolism ; TOR Serine-Threonine Kinases/metabolism ; Up-Regulation ; ras GTPase-Activating Proteins/genetics/*metabolism

OBJECTIVETo study focal adhesion kinase (FAK) expression in hypoxia-stressed SMMC-7721 cells and the role of FAK expression in the hypoxia-induced invasion of SMMC-7721 cells.

METHODSSMMC-7721 cells were cultured in 21% O2 or 1% O2. FAK expression was determined by Western blot. The siRNA expression vector pshRNA-FAK targeting to FAK and the control vector pGensil-2 were transfected into SMMC-7721 cells. The hypoxia-induced migration and invasion ability of SMMC-7721 cells transfected with pshRNA-FAK were analyzed. In normoxia, invasion of SMMC-7721 cells transfected with pcDNA3-FAK was analyzed.

RESULTSThe expression of FAK was increased significantly in SMMC-7721 cells 24 h after hypoxia stress (P<0.01). The level of FAK protein was decreased by 74.6%+/-5.1% after the pshRNA-FAK transfection in normoxia and hypoxia. The migration and invasion of SMMC-7721 cells was increased in 1% O2 (P<0.01). However, the migration and invasion of SMMC-7721 cells transfected with pshRNA-FAK was decreased in 1% O2 (P<0.05). Overexpression of FAK significantly stimulated the invasion of SMMC-7721 cells.

CONCLUSIONUp-regulation of FAK may play an important role in the invasion of SMMC-7721 cells induced by hypoxia.

Blotting, Western ; Carcinoma, Hepatocellular ; enzymology ; genetics ; pathology ; Cell Line, Tumor ; Cell Movement ; Focal Adhesion Protein-Tyrosine Kinases ; genetics ; metabolism ; Gene Expression Regulation, Enzymologic ; Humans ; Hypoxia ; Liver Neoplasms ; enzymology ; genetics ; pathology ; Neoplasm Invasiveness ; Plasmids ; genetics ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo examine how the thymidine phosphorylase (TP) gene expression is upregulated by interferon-alpha (IFN-alpha) in human hepatocellular carcinoma SMMC-7721 cells.

METHODSTP mRNA levels were determined by RT-PCR. Whether the JAK-STAT cascade mediates IFN-alpha-induced TP mRNA expression was studied by pretreatment with Janus Kinase (JAK) inhibitor, AG-490. Effects of IFN-alpha on TP mRNA stability were detected with additional actinomycin D.

RESULTSThe expression of TP mRNA was induced by IFN-alpha in a dose- and time-dependent manner in SMMC-7721 (human hepatocellular carcinoma) cells. TP mRNA levels rose at 8 h, reached the peak value at 12 h, and remained at a high level up to 72 h in SMMC-7721 cells treated with IFN-alpha 10000 U/ml. IFN-alpha at a dose of 5000 or 10000 U/ml up-regulated TP expression about 3 fold compared with that of non-treated cells (P < 0.05). Induction of TP mRNA expression by IFN-alpha was significantly inhibited in SMMC-7721 cells by pretreatment with AG-490, in comparison with that treated with IFN-alpha alone. Pretreatment of SMMC-7721 cells with IFN-alpha 10000 U/ml for 24 h caused a substantial stabilization of TP mRNA, with a half-live of 35.8 h, compared with 8.5 hr in the control SMMC-7721 cells.

CONCLUSIONIFN-alpha at certain doses upregulates TP mRNA expression via both JAK-STAT transcriptional activation and post-transcriptional mRNA stabilization in human hepatocellular carcinoma SMMC-7721 cells.

Carcinoma, Hepatocellular ; enzymology ; pathology ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Gene Expression Regulation, Neoplastic ; Humans ; Interferon-alpha ; administration & dosage ; pharmacology ; Janus Kinases ; metabolism ; Liver Neoplasms ; enzymology ; pathology ; RNA, Messenger ; metabolism ; STAT1 Transcription Factor ; metabolism ; Thymidine Phosphorylase ; biosynthesis ; genetics ; Transcriptional Activation ; drug effects ; Tyrphostins ; pharmacology

OBJECTIVETo study location of MT1-MMP and effect of its change in expression on rabbit VX2 tumor tissues after transarterial embolization with hydroxyapatite nanoparticles loaded with lipiodol.

METHODSSixty rabbits implanted with tumor tissue of cell line VX2 were divided into three groups (control group, lipiodol group, hydroxyapatite nanoparticles loaded with lipiodol group). The transarterial embolization was performed super-selectively via gastro- duodenal artery of rabbits, each rabbit in control group was inserted with 1 ml normal saline,that in lipiodol group was inserted with 0.3 lipiodol ml/kg, also 0.3 ml hydroxyapatite nanoparticles loaded with lipiodol per kg for that in the last group. Results of embolization were detected by using CT scanning 3 days after operation. After two weeks, all tumors were took out as specimens to investigate location of MT1-MMP in VX2 tumor tissues,and also to determine the change of its expression in tumor tissues after embolization with different medicines, with three-step immunohistochemical technique (S-P). MT1-MMP mRNA was measured by RT-PCR to determine whether there were differences in three groups. Western blot technique was performed to determine difference of MT1-MMP protein expression of in three groups.

RESULTSImmunohistochemical results exposed that MT1-MMP was expressed on membrane of tumor cells and in extracellular matrix of tumor cells. Comparison of MT1-MMP expression in control group with that in other two groups, showed a significant lower level in control group (P < 0.05). There was no difference in MT1-MMP expression between lipiodol group, hydroxyapatite nanoparticles loaded with lipiodol group (P > 0.05). Western blot supported this conclusion. RT-PCR detecting MT1-MMP mRNA was found no differences among three groups (P > 0.05).

CONCLUSIONSMT1-MMP was mainly expressed on membrane of tumor cells and in extracellular matrix of tumor cells. There was an increasing tendency on expression of MT1-MMP in tumor tissues and extracellular matrix after transarterial embolization with hydroxyapatite nanoparticles loaded with lipiodol,it might be one of important mechanisms provoking high recurrence rate for hepatocellular carcinoma after treatment embolization.

Animals ; Durapatite ; Embolization, Therapeutic ; Iodized Oil ; Liver Neoplasms, Experimental ; enzymology ; pathology ; therapy ; Matrix Metalloproteinase 14 ; genetics ; metabolism ; Nanoparticles ; RNA, Messenger ; genetics ; Rabbits