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

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 construct a recombinant retrovirus vector expressing small interfering RNA (siRNA) targeting human telomerase reverse transcriptase (hTERT), and assess its effect on proliferation and apoptosis of human hepatocellular carcinoma cells.

METHODSThe sequence of the siRNA targeting hTERT, U6 promoter and EGFP gene were amplified by PCR and inserted into the mammalian retroviral expression vector pLXSN to construct the recombinant retroviral vector pLXSN-EGFP-U6-siTERT. The vector was then used to infect human hepatocellular carcinoma cell HepG2. The telomerase activity of the infected cells was detected by telomerase repeat amplification protocol-silver staining, and the cell apoptosis was examined using flow cytometry. The inhibition rate of HepG2 cell proliferation was analyzed by MTT assay.

RESULTSSequence analysis and restriction enzyme digestion showed confirmed successful construction of the recombinant expression vector pLXSN-EGFP-U6-siTERT. The telomerase activity of the infected HepG2 cells was reduced by 23.84%, 58.03% and 85.01% at 24, 48 and 72 h after the infection, respectively (P<0.05). The cell apoptosis rate of the infected cells was 29.05% at 24 h after the infection. The cell proliferation was markedly inhibited by the infection with the vector in comparison to that of the control group.

CONCLUSIONhTERT siRNA can effectively silence hTERT gene and suppress the telomerase activity and proliferation of HepG2 cells.

Apoptosis ; Carcinoma, Hepatocellular ; enzymology ; genetics ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; Genetic Vectors ; genetics ; Humans ; Liver Neoplasms ; enzymology ; genetics ; pathology ; RNA Interference ; RNA, Small Interfering ; genetics ; Retroviridae ; genetics ; Telomerase ; genetics ; metabolism ; Transfection

OBJECTIVETo investigate the effect of lidamycin (LDM) on telomerase activity in human hepatoma BEL-7402 cells under the condition of LDM inducing mitotic cell death and senescence.

METHODSChromatin condensation was detected by co-staining with Hoechst 33342 and PI. Cell multinucleation was observed by Giemsa staining and genomic DNA was separated by agarose gel electrophoresis. Fluorescent intensity of Rho123 was determined for mitochondrial membrane potential. MTT assay and SA-beta-gal staining were employed to analyze the senescence-like phenotype. The expression of proteins was analyzed by Western blot. Telomerase activity was assayed by telomerase PCR-ELISA.

RESULTSMitotic cell death occurred in LDM-treated cells characterized by unique and atypical chromatin condensation, multinucleation and increased mitochondrial membrane potential. However, no apoptotic bodies or DNA ladders were found. In addition, apoptosis-related proteins remained nearly unaltered. Senescence-like phenotype was identified by increased and elongated size of cells, growth retardation, enhanced SA-beta-gal activity and the changes of senescence-related protein expression. Telomerase activity markedly decreased (P<0.01) in LDM-treated hepatoma BEL-7402 cells.

CONCLUSIONMitotic cell death and senescence could be triggered simultaneously or sequentially after exposure of hepatoma BEL-7402 cells to LDM. The decrease in telomerase activity may play a key role in the defective mitosis and aging morphology. Further investigation of detailed mechanism is needed.

Aminoglycosides ; pharmacology ; Antibiotics, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Azure Stains ; Benzimidazoles ; Carcinoma, Hepatocellular ; enzymology ; pathology ; Cell Nucleus ; drug effects ; metabolism ; Cellular Senescence ; drug effects ; Chromatin ; metabolism ; DNA, Neoplasm ; analysis ; Dose-Response Relationship, Drug ; Enediynes ; pharmacology ; Genome, Human ; genetics ; Humans ; Liver Neoplasms ; enzymology ; pathology ; Membrane Potential, Mitochondrial ; drug effects ; Mitosis ; drug effects ; Phenotype ; Propidium ; Telomerase ; metabolism ; Time Factors ; beta-Galactosidase ; metabolism

OBJECTIVETo study the role of inhibition FAK expression by FAK siRNA in liver cancer cell (MHCC97-H) adhesion, invasion and cytoskeleton rearrangement.

METHODSFAK siRNA was transfected into MHCC97-H cell by Lipofectamine 2000; then, FAK expression was detected by Western blot analysis. The change of cell adhesive and invasive ability after RNAi was checked by cell adhesive assay and cell invasive assay respectively. Meanwhile, matrix metalloproteinase-2 secretion was checked by gelatin zymography. Cytoskeleton rearrangement labeled by immunofluorescence antibody was examined by confocal laser scanning microscope.

RESULTSFAK expression in MHCC97-H cell was obviously inhibited by specific FAK siRNA; However, it was not inhibited by negative siRNA. Adhesion rate between MHCC97-H cell and extracellular matrix decreased from 57.3% to 35.8% after RNA interference (P < 0.05). Compared with untreated group, the number of cell penetrating matrigel also decreased from 31.3 +/- 2.6 to 14.5 +/- 3.1 after transfection (P < 0.05). Besides, matrix metalloproteinase-2 secretion was significantly reduced for FAK expression inhibited by FAK siRNA. FAK inhibition influenced Vinculin rearrangement, blocked the formation of lamellipodium, delayed the time of focal adhesion formation.

CONCLUSIONDown-regulation the expression of FAK can reduce adhesive rate and invasive number of MHCC97-H cell by influencing cytoskeleton rearrangement and decreasing matrix metalloproteinase-2 secretion.

Blotting, Western ; Carcinoma, Hepatocellular ; enzymology ; genetics ; pathology ; Cell Adhesion ; Cell Line, Tumor ; Cell Movement ; Cytoskeleton ; metabolism ; Fluorescent Antibody Technique ; Focal Adhesion Kinase 1 ; genetics ; metabolism ; Humans ; Liver Neoplasms ; enzymology ; genetics ; pathology ; Matrix Metalloproteinase 2 ; metabolism ; Microscopy, Confocal ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection ; methods

OBJECTIVETo investigate the effects of cyclooxygenase inhibitor diclofenac on the proliferation and cyclooxygenase-2 (COX-2) mRNA expression of cultured hepatocellular carcinoma cell lines HepG2, Hep3B and human hepatocellular cell line QSG-7701.

METHODSAfter exposure to diclofenac at various concentrations (10-200 micromol/L) for 24, 48 and 72 h, the cell proliferation was analyzed by Cell Counting Kit-8 (CCK-8) assay and mRNA expression determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSDiclofenac exposure for 24, 48 and 72 h significantly inhibited HepG2 and Hep3B cell proliferation in a concentration-dependent manner, with inhibition rate of 40.47% and 54.49% after 48 h exposure to 50 micromol/L diclofenac and IC50 of 70.54 and 48.39 micromol/L, respectively. A much weaker antiproliferative effect on QSG-7701 cells was shown, with IC50 of 189.91 micromol/L after 48-hour exposure to diclofenac. RT-PCR detected COX-2 mRNA in HepG2 and Hep3B cells, but hardly in QSG-7701 cells. Treatment with diclofenac or 5-Fu resulted in elevated COX-2 mRNA expression both in HepG2 and Hep3B cells.

CONCLUSIONDiclofenac can specifically inhibit the proliferation of COX-2-expressing HepG2 and Hep3B cells, and induce up-regulation of COX-2 mRNA expression, which indicates the important role of COX-2 in the proliferation of hepatoma cells.

Carcinoma, Hepatocellular ; enzymology ; genetics ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclooxygenase 2 ; genetics ; Cyclooxygenase Inhibitors ; pharmacology ; Diclofenac ; pharmacology ; Gene Expression Regulation, Enzymologic ; drug effects ; Humans ; Liver Neoplasms ; enzymology ; genetics ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

Apoptosis ; physiology ; Carcinoma, Hepatocellular ; enzymology ; pathology ; Carrier Proteins ; biosynthesis ; genetics ; DNA-Binding Proteins ; metabolism ; Gene Targeting ; Humans ; Liver Neoplasms ; enzymology ; pathology ; RNA Interference ; Telomerase ; metabolism ; Tumor Cells, Cultured