OBJECTIVETo study the difference of the gene expression profile and to identify the different expression after transfection of the ARL-1 gene.

METHODSThe cDNA probes were synthesized from total RNA of study group and control group, which was differentially hybridized to cDNA chips and confirmed by a gene specific semiquantitative reverse transcription polymerase chain reaction (RT-PCR).

RESULTSSix kinds of gene expression were increased and 9 kinds of gene expression were decreased. The findings were correlated with protein metabolism, signal pathway, metastasis, and drug resistance.

CONCLUSIONScDNA chips showed that gene expression profile of liver carcinoma cell was changed after transfection of the ARL-1 gene. It is a useful method in understanding the mechanism of drug resistance.

Aldehyde Reductase ; genetics ; Drug Resistance, Neoplasm ; Gene Expression Profiling ; Humans ; Liver Neoplasms ; drug therapy ; genetics ; Oligonucleotide Array Sequence Analysis ; Transfection

Animals ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Humans ; Liver Cirrhosis ; drug therapy ; genetics ; metabolism ; Liver Neoplasms ; drug therapy ; genetics ; metabolism ; Phytotherapy ; Procollagen ; biosynthesis ; genetics ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics

BACKGROUNDHuman sulfatase-1 (Hsulf-1) is an endosulfatase that selectively removes sulfate groups from heparan sulfate proteoglycans (HSPGs), altering the binding of several growth factors and cytokines to HSPG to regulate cell proliferation, cell motility, and apoptosis. We investigated the role of combined cancer gene therapy with Hsulf-1 and cytosine deaminase/5-fluorocytosine (CD/5-FC) suicide gene on a hepatocellular carcinoma (HCC) cell line, HepG2, in vitro and in vivo.

METHODSReverse transcription polymerase chain reaction and immunohistochemistry were used to determine the expression of Hsulf-1 in HCC. Cell apoptosis was observed through flow cytometry instrument and mechanism of Hsulf-1 to enhance the cytotoxicity of 5-FC against HCC was analyzed in HCC by confocal microscopy. We also establish a nude mice model of HCC to address the effect of Hsulf-1 expression on the CD/5-FC suicide gene therapy in vivo.

RESULTSA significant decrease in HepG2 cell proliferation and an increase in HepG2 cell apoptosis were observed when Hsulf-1 expression was combined with the CD/5-FC gene suicide system. A noticeable bystander effect was observed when the Hsulf-1 and CD genes were co-expressed. Intracellular calcium was also increased after HepG2 cells were infected with the Hsulf-1 gene. In vivo studies showed that the suppression of tumor growth was more pronounced in animals treated with the Hsulf-1 plus CD than those treated with either gene therapy alone, and the combined treatment resulted in a significant increase in survival.

CONCLUSIONSHsulf-1 expression combined with the CD/5-FC gene suicide system could be an effective treatment approach for HCC.

Animals ; Apoptosis ; drug effects ; genetics ; Carcinoma, Hepatocellular ; enzymology ; metabolism ; Cell Movement ; drug effects ; genetics ; Cell Proliferation ; drug effects ; genetics ; Cytosine Deaminase ; genetics ; metabolism ; Flucytosine ; pharmacology ; Genetic Therapy ; Hep G2 Cells ; Humans ; Liver Neoplasms ; enzymology ; metabolism ; Sulfatases ; genetics ; metabolism

OBJECTIVESTo investigate the reversal effect of gene MDR1 and MRP with combinational antisense phosphorothioate oligonucleotide on Drug-resistant human hepatocellular carcinoma cells SMMC-7721/ADM.

METHODSSMMC-7721/ADM was transfected with synthetic antisense phosphorothioate oligonucleotides complementary to gene MDR1 and MRP mediated by Lipofectamine. Drug sensitivity was measured by MTT assay, Fluorescence intensity of cells was determined by flow cytometric analysis, RH123 and DNR retention was assayed by confocal scanning laser microscopy.

RESULTSASODN of MDR1+MRP increased the sensitivity of SMMC-7721/ADM to chemotherapeutic drug more significantly than that any of MDR1 and MRP did separately. But they did not enhance the inhibition expression of protein of p190 or p170.

CONCLUSIONDrug-resistance could be reversed significantly when antisense phosphorothioate oligonucleotide of MDR1+MRP were transfected into drug-resistant human hepatocellular carcinoma cells SMMC-7721/ADM together.

Carcinoma, Hepatocellular ; drug therapy ; genetics ; Cell Line, Tumor ; Daunorubicin ; metabolism ; pharmacology ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Genes, MDR ; Humans ; Liver Neoplasms ; drug therapy ; genetics ; Multidrug Resistance-Associated Proteins ; genetics ; Oligonucleotides, Antisense ; pharmacology ; Rhodamine 123 ; metabolism

Occult HBV infection is defined as the presence of HBV DNA in the liver (with or without detectable or undetectable HBV DNA in the serum) of individuals testing negative for HBsAg. Studies on occult HBV infection in hepatitis C patients have reported highly variable prevalence, because the prevalence of occult HBV infection varies depending on the hepatitis B risk factors and methodological approaches. The most reliable diagnostic approach for detecting occult HBV detection is through examination of liver DNA extracts. HCV has been suspected to strongly suppress HBV replication up to the point where it may be directly responsible for occult HBV infection development. However, more data are needed to arrive at a definitive conclusion regarding the role of HCV in inducing occult HBV infection. Occult HBV infection in chronic hepatitis C patients is a complex biological entity with possible relevant clinical implications. Influence of occult HBV infection on the clinical outcomes of chronic hepatitis C may be considered negative. However, recent studies have shown that occult HBV infection could be associated with the development of hepatocellular carcinoma and contribute to the worsening of the course of chronic liver disease over time in chronic hepatitis C patients. Nevertheless, the possible role of occult HBV infection in chronic hepatitis C is still unresolved and no firm conclusion has been made up until now. It still remains unclear how occult HBV infection affects the treatment of chronic hepatitis C. Therefore, in order to resolve current controversies and understand the pathogenic role and clinical impacts of occult HBV infection in chronic hepatitis C patients, well-designed clinical studies are needed.
Carcinoma, Hepatocellular/complications ; DNA, Viral/analysis ; Hepacivirus/genetics ; Hepatitis B/*complications/*diagnosis/drug therapy ; Hepatitis B virus/genetics ; Hepatitis C, Chronic/*complications/*diagnosis/drug therapy ; Humans ; Interferon-alpha/therapeutic use ; Liver/virology ; Liver Neoplasms/complications

Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and mortality globally. It accounts for the majority of primary liver cancer cases. Amyloid precursor protein (APP), a cell membrane protein, plays a vital role in the pathogenesis of Alzheimer's disease, and has been found to be implicated in tumor growth and metastasis. Therefore, to understand the relationship between APP and 5-fluorouracil (5-FU) resistance in liver cancer, Cell Counting Kit-8, apoptosis and cell cycle assays, western blotting, and reverse transcription-quantitative polymerase chain reaction (qPCR) analysis were performed. The results demonstrated that APP expression in Bel7402-5-FU cells was significantly up-regulated, as compared with that in Bel7402 cells. Through successful construction of APP-silenced (siAPP) and overexpressed (OE) Bel7402 cell lines, data revealed that the Bel7402-APP^751-OE cell line was insensitive, while the Bel7402-siAPP cell line was sensitive to 5-FU in comparison to the matched control group. Furthermore, APP overexpression decreased, while APP silencing increased 5-FU-induced apoptosis in Bel7402 cells. Mechanistically, APP overexpression and silencing can regulate the mitochondrial apoptotic pathway and the expression of apoptotic suppressor genes (B-cell lymphoma-2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xl)). Taken together, these results preliminarily revealed that APP overexpression contributes to the resistance of liver cancer cells to 5-FU, providing a new perspective for drug resistance.
Amyloid beta-Protein Precursor/physiology* ; Apoptosis/drug effects* ; Carcinoma, Hepatocellular/drug therapy* ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Fluorouracil/pharmacology* ; Humans ; Liver Neoplasms/drug therapy* ; Mitochondria/physiology* ; Proto-Oncogene Proteins c-bcl-2/genetics* ; bcl-X Protein/genetics*

Hepatocellular carcinoma (HCC) is a major cause of cancer-related mortality in part due to its high resistance to chemotherapeutic drugs. The anti-apoptotic Mcl-1 expression has been reported as a resistance factor in various types of tumors. Here, we investigated the expression of Mcl-1 in hepatoma cells and HCC tissues and its relationship with p53, and analyzed the possibility of the gene as a molecular target for HCC therapy. HCC specimens of 30 patients were examined by immunohistochemistry for Mcl-1 and p53 expression. Mcl-1 expression in hepatoma cell lines was measured by RT-PCR and Western blotting. The suppression of Mcl-1 by RNA interference or specific phosphatidylinositol-3 kinase (PI3K) inhibitor, LY294002, was evaluated as monotherapy, and it was combined with mitomycin C (MMC) in treating hepatoma cell line HepG2. Cell viability and apoptosis were assessed by MTT and FACS analysis. Finally, changes of Mcl-1 or p53 expression in various hepatoma cell lines were examined after transfection with Mcl-1 siRNA, the Mcl-1 expression plasmid, or the wide-type p53 expression plasmid, respectively. Mcl-1 protein was remarkably enhanced in HCC tissues as compared with adjacent non-tumor liver tissues. In addition, Mcl-1 was prominently expressed in HepG2 and Hep3B cells, weakly in SMMC7721 cells, and not in L02 cells. P53 protein was also overexpressed in HCC tissues and there was a significant correlation between the expression of p53 and Mcl-1. Silencing Mcl-1 by RNAi or LY294002 downregulated Mcl-1 expression and led to decreased cell viability and increased apoptosis. Combination of MMC and Mcl-1 RNAi or LY294002 exhibited a significant chemosensitizing effect. The expression of p53 was not influenced by Mcl-1 siRNA in HepG2 cells or transfection with the Mcl-1 expression plasmid in L02 cells. Furthermore, the expression of Mcl-1 in Hep3B cells was also not significantly changed after transfection with the wild-type p53 expression plasmid. It is concluded that Mcl-1 is overexpressed in HCC tissues. The mechanisms by which silencing Mcl-1 sensitizes hepatoma cells towards chemotherapy may be not attributed to the upregulated expression of p53 but the dysfunction of p53 through Mcl-1/p53 interaction. Mcl-1 may be a potential target of gene therapy for HCC.
Adenoma, Liver Cell ; drug therapy ; genetics ; pathology ; Apoptosis ; drug effects ; Biomarkers, Tumor ; biosynthesis ; genetics ; Chromones ; administration & dosage ; Gene Expression Regulation, Neoplastic ; drug effects ; Hep G2 Cells ; Humans ; Liver Neoplasms ; drug therapy ; genetics ; pathology ; Morpholines ; administration & dosage ; Myeloid Cell Leukemia Sequence 1 Protein ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Transfection ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics

OBJECTIVETo observe anti-cancer effects of Jianpi Jiedu Recipe (JJR) on liver cancer (LC) rats with Pi deficiency syndrome (PDS) and its relation with the third complementary-determining region gene spectratyping of TCRVβ-chain (TCRVβCDR3).

METHODSRats were divided into 8 groups according to random digit table, i.e., the blank control group (normal), the PDS group, the LC model group, the LC-PDS group, high, middle, and low dose JJR groups (75.00, 37.50, 18.75 g/kg, respectively by gastrogavage, once per day), the thymus pentapeptide group (5 mg/kg, intramuscular injection, twice per week), 8 in each group. Rats in the normal group were administered with physiological saline by gastrogavage once per day. PDS rat model was prepared by bitter-cold purgation. LC model was prepared by orthotopic transplantation method. Twenty gene subfamilies of TCRβCDR3 in the thymus, liver, and LC tissues were detected by Gene Scan.

RESULTSHigh and middle dose JJR could postpone the growth of LC volume (P < 0.05), with equivalent liver index and thymus index to those of the normal group (P > 0.05). In thymus and liver tissue of the normal group, the number of clones (20 and 19), gene fragment number (220 and 113), Quasi-Gaussian distribution ratio of TCRVβCDR3 gene repertoire (100.0% and 42.1%), and fragment fluorescence peak area (6,539 ± 2,325 and 1,238 ± 439) were at the highest level among the 8 groups. TCRVβCDR3 expressions in thymus and liver tissue of high and middle dose JJR groups were approximate to those of the normal group. They were in the middle of the thymus pentapeptide group, the PDS group, the LC model group, and poorest in the LC-PDS group. TCRVβCDR3 in liver tissue expressed the best in the thymus pentapeptide group.

CONCLUSIONJJR might inhibit the growth of LC cells, and its mechanism might be related to enhancing TCRVβCDR3 spectratype expression.

Animals ; Complementarity Determining Regions ; genetics ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Regulation, Neoplastic ; Genes, T-Cell Receptor beta ; Liver Neoplasms ; drug therapy ; genetics ; Random Allocation ; Rats

OBJECTIVETo investigate whether multidrug resistance gene 1(mdr1) could reverse multidrug resistance (MDR) in HepG2R cells.

METHODSAn adenovirus vector, Adeno-asmdr, containing the antisense RNA driven by AFP promoter, was construct. Adeno-EGFP was transfected into HepG2, an AFP producing cell line, L02, a normal human liver cell line, and HeLa, a human cervical cancer cell line, the EGFP transcription level was detected by RT-PCR. Adeno-asmdr was transfected into HepG2R cells, the expression of P-gp170 was detected by western blotting, apoptosis was detected using TUNEL and flow cytometry, cell cycle was analyzed by flow cytometry.

RESULTSEGFP was highly expressed in HepG2 cells, however, its expression in L02 or HeLa cells was very weak. Western blot showed that the P-gp170 was marked down-regulated 48h after transfection with Adeno-asmdr, and the expression of P-gp170 was detectable at least 7d post-transfection. Compared with control cells, Adeno-asmdr transfected HepG2R cells were more sensitive to different chemicals, as indicated by TUNEL staining and flow cytometry. Chemical treatment arrested the cells in S and G0/M phase.

CONCLUSIONThe recombinant adenoviral vector, Adeno-asmdr, can block the expression of mdr1, and reverse MDR in HepG2R cells.

ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Adenoviridae ; genetics ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; drug therapy ; genetics ; metabolism ; Cell Cycle ; drug effects ; Drug Resistance, Multiple ; genetics ; Drug Resistance, Neoplasm ; genetics ; Gene Expression Regulation, Neoplastic ; HeLa Cells ; Hep G2 Cells ; Humans ; Liver Neoplasms ; drug therapy ; genetics ; metabolism ; RNA, Antisense ; Transfection

OBJECTIVEThe X protein of the hepatitis virus B plays an important role in the development of hepatocellular carcinoma (HCC). In this experiment we studied the effects of lamivudine on replication, transcription and expression of the X gene.

METHODSThe replication of transfected X gene was measured by polymerase chain reaction. The mRNA of transfected X gene was analyzed by reverse transcriptase polymerase chain reaction. The expression of X protein was detected by biosensor. Moreover, we studied if these effects changed in a dose and time dependent manner.

RESULTSThe OD data of purpose band reflected the replication of X gene for control and lamivudine groups were 151.4+/-3.5 and 144.0+/-11.4, respectively. The transcription of mRNA for X gene could be inhibited by lamivudine at a concentration of 4.36 x 10(-4) mol/L for 24 h. The OD data of purpose band for control and treatment groups (16 h) were 243.9+/-9.0 and 133.2+/-7.8. The inhibition was enhanced with the increase of lamivudine concentration and reacting time. The expression of the X protein in HepG2x cells was suppressed by lamivudine at a concentration of 4.36 x 10(-4) mol/L for 16 hours. The resonance unit for expression of the X protein decreased from 353.3+/-15.9 to 252.3+/-18.8.

CONCLUSIONAlthough the replication of transfected X gene of HepG2x cells is not influenced by lamivudine, it may significantly inhibit the transcription and expression of the X protein.

Carcinoma, Hepatocellular ; drug therapy ; virology ; Female ; Gene Expression Regulation, Neoplastic ; Hepatitis B virus ; genetics ; Humans ; Lamivudine ; therapeutic use ; Liver Neoplasms ; drug therapy ; virology ; Male ; Reverse Transcriptase Inhibitors ; therapeutic use ; Trans-Activators ; biosynthesis ; genetics ; Transcription, Genetic ; drug effects