acyclovir and Carcinoma--Hepatocellular

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Although hepatectomy and transplantation have significantly improved survival, there is no effective chemotherapeutic treatment for HCC and its prognosis remains poor. Sustained activation of telomerase is essential for the growth and progression of HCC, suggesting that telomerase is a rational target for HCC therapy. Therefore, we developed a thymidine analogue pro-drug, acycloguanosyl-5'-thymidyltriphosphate (ACV-TP-T), which is specifically activated by telomerase in HCC cells and investigated its anti-tumour efficacy.. First, we verified in vitro whether ACV-TP-T was a telomerase substrate. Second, we evaluated proliferation and apoptosis in murine (Hepa1-6) and human (Hep3B, HuH7, HepG2) hepatic cancer cells treated with ACV-TP-T. Next, we tested the in vivo treatment efficacy in HBV transgenic mice that spontaneously develop hepatic tumours, and in a syngeneic orthotopic murine model where HCC cells were implanted directly in the liver.. In vitro characterization provided direct evidence that the pro-drug was actively metabolized in liver cancer cells by telomerase to release the active form of acyclovir. Alterations in cell cycle and apoptosis were observed following in vitro treatment with ACV-TP-T. In the transgenic and orthotopic mouse models, treatment with ACV-TP-T reduced tumour growth, increased apoptosis, and reduced the proliferation of tumour cells.. ACV-TP-T is activated by telomerase in HCC cells and releases active acyclovir that reduces proliferation and induces apoptosis in human and murine liver cancer cells. This pro-drug holds a great promise for the treatment of HCC.

Topics: Acyclovir; Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Guanosine; Hep G2 Cells; Hepatitis B virus; Humans; Liver Neoplasms; Liver Neoplasms, Experimental; Mice; Mice, Transgenic; Prodrugs; Telomerase; Thymine Nucleotides

To study the induction of sensitivity to ganciclovir (GCV) or acyclovir (ACV) in human hepatocellular carcinoma (HCC) cell line transferred by an Epstein-Barr virus (EBV)-based replicon expression vector carrying the herpes simplex virus thymidine kinase (HSV-tk) gene, including killing and "bystander" effect, and also the gene delivery procedure and route of gene therapy in vivo for HCC.. Liposome-entrapped plasmid pDR2/tk was transferred into HCC cells, and then different concentrations of GCV or ACV were added. The transferred cells were mixed with untransferred HCC cells in different proportion and 200 micromol/L GCV was then added into each well. After 72 hours, all samples were measured by MTT colorimetric assay. An EBV-based plasmid eukarotic expression vector carrying IL-2 cDNA was used. Three models of gene direct injection in the local liver, injection through the portal vein, and injection through the embolized hepatic artery were established in closed Wister rats. For each model, two subgroups, injected either naked plasmid DNA or lipofectin-plasmid complex were included. The expression of the IL-2 gene was regularly examined immunohistochemically.. GCV or ACV could apparently kill the transferred HCC cells at a concentration of 0.2 micromol/L. The inhibition rate was changed with different drug concentrations. The "bystander" effect was obviously induced at a transferred to untransferred HCC cells ratio of 1:5. IL-2 gene expression was observed in liver cells of all animals on day 3, which reached peak within 3-7 days, and declined after day 7. Injection of naked plasmid DNA through the hepatic artery plus embolization obtained a best expression.. EBV-based vector is suitable for carrying suicide gene therapy for hepatocellular carcinoma. Gene direct delivery in vivo combined with interventional surgery can be used to treat hepatocellular carcinoma.

Topics: Acyclovir; Animals; Antiviral Agents; Carcinoma, Hepatocellular; Cell Death; Cell Line, Tumor; Dose-Response Relationship, Drug; Ganciclovir; Gene Transfer Techniques; Genes, Transgenic, Suicide; Genetic Therapy; Genetic Vectors; Herpesvirus 4, Human; Humans; Liver Neoplasms; Protein-Tyrosine Kinases; Rats; Rats, Wistar; Simplexvirus

The delivery of "suicide" herpes simplex virus type-1 thymidine kinase gene (tk) into tumor cells, followed by treatment with synthetic nucleotide analogues (gancyclovir, acyclovir), is a perspective approach to cancer therapy. Serious limitations in employment of the existing means of gene delivery into target cells constitute the main obstacle for cancer gene therapy development. In the present work a possibility to use a nonviral gene delivery system is shown based on the employment of lysine rich peptide K8 and amphipathic peptide JTS-1 for transferring tk gene into human hepatoma HepG2 cells. Cationic peptide K8 forms compact complexes with plasmid DNA, and JTS-1 acts as a pH-dependent endosomal releasing agent. Transfection of HepG2 cells by tk expression vector coupled with K8/JTS-1 peptides, followed by acyclovir administration (50-100 micrograms/ml) for 24 h leads to cell cycle arrest in the G1/S checkpoint of some cells, which eventually die through apoptosis. Treatment of HepG2 cells with higher acyclovir concentration (200 micrograms/ml) additionally results in a nonspecific toxic effect. The above results demonstrate the efficacy of K8/JTS-1 delivery system for the "suicide" cancer gene therapy, and may be regarded as a basis for further elaboration of "suicide" cancer approaches in vivo.

Topics: Acyclovir; Antineoplastic Agents; Antiviral Agents; Basic-Leucine Zipper Transcription Factors; Carcinoma, Hepatocellular; Carrier Proteins; Ganciclovir; Gene Expression; Gene Transfer Techniques; Genes, Viral; Genetic Therapy; Genetic Vectors; Herpesvirus 1, Human; Humans; Liver Neoplasms; Repressor Proteins; Thymidine Kinase; Transfection; Tumor Cells, Cultured; Viral Proteins

In this work, we investigated the anti-hepatitis B virus (HBV) activity of lamivudine, adefovir, tenofovir, penciclovir and lobucavir after short-term (i.e. 24 or 48 h) or continuous (9 days) exposure of the HBV-containing cell line, HepG2 2.2.15, to these drugs. Lamivudine maintained significant anti-HBV activity when added for only 24 or 48 h to the cell cultures compared to when the drug was present for the whole period (9 days) on the cells, i.e. 50% effective concentration (EC50) values for the inhibition of HBV DNA synthesis were 0.07 +/- 0.02 microgram ml-1 after 24 h of incubation, 0.02 +/- 0.01 microgram ml(-1) after 48 h of incubation and 0.0016 +/- 0.001 microgram ml(-1) after 9 days of incubation. Similarly, the nucleoside phosphonate analogues, adefovir and tenofovir, retained significant anti-HBV activity when added for only a short period of time to the cells. The EC50 values were 12 +/- 1 microgram ml(-1) (24 h) and 1.0 +/- 0.2 microgram ml(-1) (48 h) vs 0.003 +/- 0.001 microgram ml(-1) (9 days) for adefovir, and 6.5 +/- 1.1 microgram ml(-1) (24 h) and 0.8 +/- 0.1 microgram ml(-1) (48 h) vs 0.03 +/- 0.02 microgram ml(-1) (9 days) for tenofovir. In contrast, penciclovir and lobucavir lost most of their anti-viral activity when present on the cells for 48 h or less.

Topics: Acyclovir; Adenine; Antiviral Agents; Carcinoma, Hepatocellular; DNA, Viral; Guanine; Hepatitis B virus; Lamivudine; Organophosphonates; Organophosphorus Compounds; Tenofovir; Tumor Cells, Cultured

L-Nucleoside analogs are new therapeutic agents for treatment of chronic hepatitis B. However, their clinical application was limited by the emergence of viral resistance. It is important to develop a new system to evaluate drug cross-resistance and to test new agents that may overcome resistant virus. In this report, three cell lines HepG2-WT10, HepG2-SM1, and HepG2-DM2 are presented; these cell lines were established by transfection of HepG2 cells with unique fully functional 1.1x hepatitis B virus (HBV) genomes: wild-type HBV-adr and its L526M and L526MM550V variants, respectively. We have demonstrated that these genomes have different susceptibilities to lamivudine [L(-)SddC] and penciclovir (PCV). By examining HBV RNA transcription, antigen expression, progeny DNA replication, and viral susceptibilities to L(-)SddC, PCV, and other nucleoside analogs, it is concluded that the cell lines are able to stably produce L(-)SddC- and PCV-sensitive and -resistant HBV virions. In addition, the relative susceptibilities of the wild-type and mutant HBV produced from the stably transfected cell lines to several anti-HBV nucleoside analogs were also examined and found to be about the same as those found by using a transient infection system. PMEA [9-(2-phosphonylmethoxytehyl)-adenine] and QYL685 are able to suppress L(-)SddC- and PCV-resistant HBV. In conclusion, this cell culture system is a novel and useful tool for evaluating anti-HBV compounds and biologics.

Topics: Acyclovir; Antigens, Viral; Antiviral Agents; Carcinoma, Hepatocellular; Drug Resistance, Microbial; Guanine; Hepatitis B virus; Humans; Lamivudine; Liver Neoplasms; Microbial Sensitivity Tests; Time Factors; Transfection; Tumor Cells, Cultured; Virus Replication

The emergence of resistant hepatitis B virus (HBV), with mutations in the YMDD motif of the polymerase gene after treatment with lamivudine, is becoming an important clinical problem. In this study, susceptibility of wild-type and lamivudine-resistant HBV M552I, M552V, and L528M/M552V mutants to other reverse transcriptase inhibitors was investigated by transient transfection of full-length HBV DNA into human hepatoma cells. HBV DNA replication was monitored by Southern blot hybridization, which showed the presence of a single-stranded band (representative of the HBV replicative intermediates) in the drug-free, wild-type HBV-transfected cells. This band was diminished in the samples of wild-type HBV DNA treated with either lamivudine, adefovir, or lobucavir. The band intensities from the lamivudine-resistant mutants were not decreased by treatment with lamivudine, but were decreased by the treatments with adefovir or lobucavir. In contrast, penciclovir and nevirapine did not diminish the intensity of the single-stranded band of wild-type HBV or the lamivudine-resistant mutants. These results demonstrate that lamivudine-resistant HBV is susceptible to adefovir and lobucavir. Lamivudine-resistant HBV should be treated with adefovir or lobucavir, and combination therapy with lamivudine and adefovir/lobucavir may prevent the emergence of lamivudine-resistant HBV.

Topics: Acyclovir; Adenine; Carcinoma, Hepatocellular; Drug Resistance, Microbial; Guanine; Hepatitis B virus; Humans; Lamivudine; Mutagenesis, Site-Directed; Nevirapine; Organophosphonates; Reverse Transcriptase Inhibitors; Transfection; Tumor Cells, Cultured

The alpha-fetoprotein (AFP) gene is normally expressed in fetal liver and is transcriptionally silent in adult liver but is reactivated in hepatocellular carcinoma. It has been shown that the positive and negative transcriptionally regulatory elements of the human AFP gene, which play an important role in its developmental regulation, exist over the quite extended region (4 kb). We constructed a hybrid gene consisting of herpes simplex virus thymidine kinase (HSV-tk) gene under the control of the 0.3-kb human AFP gene promoter and inserted it into a retroviral vector. When AFP-producing hepatoma cells were infected with this recombinant retrovirus (LNAF0.3TK virus), the cells expressed HSV-tk gene and exhibited increased sensitivity to ganciclovir parallel with the ability of AFP production. On the other hand, the retroviral infection had little effect on ganciclovir-mediated cytotoxicity in AFP-nonproducing hepatoma or non-hepatoma cells. Moreover, the addition of dexamethasone increased the cytotoxicity of aciclovir to the virus-infected, AFP-producing cells through a glucocorticoid-responsive element in the AFP promoter, although aciclovir, by itself, had little cytotoxicity. These results demonstrate that the AFP promoter sequence alone can provide enough tumor-specific activity for therapeutic gene expression and induce selective growth inhibition by ganciclovir in the virus-infected, AFP-producing human hepatoma cells. In addition, it is possible that expression of the therapeutic gene is modulated by administration of dexamethasone or other agents that alter AFP promoter activity after gene transduction.

Topics: 3T3 Cells; Acyclovir; alpha-Fetoproteins; Animals; Base Sequence; Binding Sites; Carcinoma, Hepatocellular; Cell Division; Dexamethasone; Drug Interactions; Ganciclovir; Gene Expression; Genetic Therapy; Genetic Vectors; HeLa Cells; Humans; Mice; Molecular Sequence Data; Promoter Regions, Genetic; Retroviridae; Simplexvirus; Thymidine Kinase; Tumor Cells, Cultured

HLA class I display on hepatitis B virus (HBV)-infected hepatocytes is important for limiting HBV infection. However, the effect of HBV replication on HLA class I expression on host cells has not been determined. Since acyclovir is known to inhibit HBV replication of the novel cell line HB611, which was transfected with HBV genome using human hepatoblastoma cells as the recipient and continuously replicates HBV DNA, we analyzed HLA class I expression on acyclovir-treated HB611 by quantitative flow cytometry. The results demonstrated that acyclovir treatment clearly increases the level of HLA class I on HB611, and suggested that HBV replication inhibits expression of HLA class I on infected hepatocytes. This effect of HBV replication on the host cell may be a means by which HBV evades immune surveillance to maintain chronic infection.

Topics: Acyclovir; Antibodies, Monoclonal; Carcinoma, Hepatocellular; Cell Line; Dose-Response Relationship, Drug; Fluorescent Antibody Technique; Hepatitis B virus; Histocompatibility Antigens Class I; Humans; Interferon alpha-2; Interferon-alpha; Interferon-beta; Interferon-gamma; Kinetics; Liver Neoplasms; Recombinant Proteins; Virus Replication

A human hepatoblastoma cell line (HB 611) that continuously synthesizes hepatitis B viral (HBV) DNA was grown in the presence of various inhibitors of DNA synthesis, and the DNA from the cells was analyzed by the Southern blotting method to examine selective inhibition of the viral DNA synthesis. Among those that showed selective inhibition, and interferons alpha and beta, acyclovir, and dideoxy cytidine were effective. This system should be useful for screening new antiviral agents against HBV.

Topics: Acyclovir; Adenine; Antiviral Agents; Blotting, Southern; Carcinoma, Hepatocellular; Cytarabine; Dideoxyadenosine; Dideoxynucleosides; DNA Replication; DNA, Viral; Hepatitis B virus; Humans; Interferons; Liver Neoplasms; Tumor Cells, Cultured; Vidarabine; Virus Replication; Zalcitabine; Zidovudine