brivudine and Pancreatic-Neoplasms

Several reports describe the importance of the chaperone HSP27 (HSPB1) in cancer progression, and the demand for drugs that modulate HSPB1-activity is increasing rapidly. We reported earlier that RP101 (Bromovinyldeoxyuridine, BVDU, Brivudine) improves the efficacy of chemotherapy in pancreatic cancer.. Chemistry: Binding of RP101 and HSPB1 was discovered by affinity chromatography. Molecular and cell biology: HSPB1 in vitro transcription/translation (TNT), Pull down using RP101-coupled magnetic beads, Immuno Co-precipitations, Structural modeling of HSP27 (HSPB1), Introduction of point mutations into linear expression templates by PCR, Heat shock, Tumor Invasion. Animal experiments: Treatment of AH13r Sarcomas in SD-rats. Clinical Studies with late-stage pancreatic cancer patients: Pilot study, Dose finding study, Phase II study (NCT00550004).. Here, we report that RP101 binds in vitro to the heat shock protein HSPB1 and inhibits interaction with its binding partners. As a result, more activated CASP9 was detected in RP101-treated cancer cells. We modeled HSPB1-structure and identified the RP101 binding site. When we tested RP101 as an anti-cancer drug in a rat model, we found that it improved chemotherapy. In clinical studies with late-stage pancreatic cancer patients, the dose of 500 mg/day was safe and efficient, but 760 mg/day turned out to be too high for lightweight patients.. The development of RP101 as a cancer drug represents a truly novel approach for prevention of chemoresistance and enhancement of chemosensitivity.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Bromodeoxyuridine; Disease Models, Animal; Dose-Response Relationship, Drug; Double-Blind Method; Drug Evaluation, Preclinical; Female; Heat-Shock Proteins; HSP27 Heat-Shock Proteins; Humans; Male; Middle Aged; Models, Molecular; Molecular Chaperones; Pancreatic Neoplasms; Pilot Projects; Placebos; Rats; Rats, Sprague-Dawley; Sarcoma; Survival Analysis

RP101 [(E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU)], which supports apoptosis and prevents the acquisition of chemoresistance, was tested in cultured human pancreatic tumor cells. RP101 downregulated uridine phosphorylase, a marker of poor prognosis, and APEX1, which is involved in DNA repair, and repressed Stat3 and its target vascular endothelial growth factor. Furthermore, RP101 activated antitumor immunity as demonstrated by enhanced cytolytic activity of NK-92 natural killer cells. This was concomitant with an enhanced expression of lymphotoxins alpha and beta, natural killer cell transcript 4, tumor necrosis factor LIGHT/TNFSF-14, and intercellular adhesion molecule-1 in pancreas carcinoma cells. These results encouraged us to investigate the effect of RP101 in pancreas cancer patients. Here, we present data from two RP101 combination therapy schemes. In a first pilot study, 13 patients in stage III and VI of the disease were treated with gemcitabine +cisplatin+RP101. RP101 co-treatment enhanced remissions, survival and time to progression. Seventy-seven percent of the patients lived or have lived longer than 1 year, and 23% have lived more than 2 years. Median survival was 447 days, time to progression 280 days and the response rate 33%. A second study with 21 patients in similar stages of disease, treated with RP101+gemcitabine alone, confirmed the results of the pilot study. Eighty-three percent of the presently evaluable patients live or lived 0.5 years or longer and 33% 1 year or longer. Considering both studies, the tumor control was 94%. The data indicate that acquisition of chemoresistance was prevented and the antitumor efficacy of standard chemotherapy was improved. To our knowledge, RP101 co-treatment is more efficient than any other regimen published.

Topics: Adult; Aged; Bromodeoxyuridine; Cell Line, Tumor; Cisplatin; Deoxycytidine; DNA-(Apurinic or Apyrimidinic Site) Lyase; Gemcitabine; Humans; Killer Cells, Natural; Middle Aged; Pancreatic Neoplasms

Herpesvirus saimiri (HVS) has the capacity to incorporate large amounts of heterologous DNA and can infect many different human cell types. To develop its potential as a gene therapy vector, we cloned herpes simplex virus thymidine kinase (TK) gene into the HVS genome in the form of an enhanced green fluorescent protein (EGFP) fusion protein, using a cosmid-based approach. At multiplicity of infection = 100 over 90% of human leukemic K562 and Jurkat cells were transduced with HVS/EGFP-TK. Conditions of no selective pressure expression were maintained at > 92% per cell division. Expression of the EGFP-TK fusion protein rendered transfected leukaemic cells sensitive to cytotoxic treatment with the prodrugs ganciclovir (GCV) and (E)-5-(2-bromovinyl)-2'deoxyuridine (BVDU) at concentrations as low as 10 ng/ml. The viral vector was also screened against a panel of colorectal and pancreatic carcinoma cell lines. All cell lines were transduced but showed a range of sensitivity to infection. Three of the most easily transduced cell lines: Mia PaCa, HCT116 and SW948 transduced with HVS/EGFP-TK were effectively ablated by subsequent treatment with GCV or BVDU. Our results show that in its current form HVS/EGFP-TK could be utilized as an antitumour agent, or it could be developed further by inclusion of a therapeutic gene, with TK presence ensuring a mechanism of controlled removal of modified cells when no longer necessary. These results suggest that HVS/EGFP-TK has a great potential for a number of gene therapy applications.

Topics: Animals; Antiviral Agents; Aotidae; Base Sequence; Bromodeoxyuridine; Cells, Cultured; Colorectal Neoplasms; Ganciclovir; Genetic Vectors; Green Fluorescent Proteins; Humans; Jurkat Cells; K562 Cells; Kidney; Leukemia; Molecular Sequence Data; Pancreatic Neoplasms; Recombinant Fusion Proteins; Simplexvirus; Thymidine Kinase; Transfection

Nucleoside kinases from several species are investigated as "suicide genes" for treatment of malignant tumors by combined gene/chemotherapy. We have recently cloned a multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK), and we have shown that the enzyme phosphorylates cytotoxic pyrimidine and purine nucleoside analogs. The broad substrate specificity of the enzyme, as well as its very high catalytic rate, makes it a unique member of the nucleoside kinase enzyme family. In the present study, we evaluated Dm-dNK as a suicide gene by constructing a replication-deficient retroviral vector that expresses the enzyme. The human pancreatic adenocarcinoma cell line MIA PaCa-2 and a thymidine kinase-deficient osteosarcoma cell line were transduced with the recombinant virus. We showed that Dm-dNK can be expressed in human cells, that the enzyme retained its enzymatic activity, and that it is localized in the cell nuclei due to a nuclear localization signal in its C-terminal region. The cells expressing Dm-dNK exhibited increased sensitivity to several cytotoxic nucleoside analogs, such as 1-beta-d-arabinofuranosylcytosine, 1-beta-d-arabinofuranosylthymine, (E)-5-(2-bromovinyl)-2'-deoxyuridine, 2-chloro-2'-deoxyadenosine, and 2',2'-difluorodeoxycytidine. These findings suggest that Dm-dNK may be used as a suicide gene in combined gene/chemotherapy of cancer.

Topics: Adenocarcinoma; Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antiviral Agents; Arabinonucleosides; Bromodeoxyuridine; Catalysis; Cell Division; Cell Nucleus; Cladribine; Cytarabine; Deoxycytidine; Drosophila melanogaster; Gemcitabine; Humans; Inhibitory Concentration 50; Kinetics; Osteosarcoma; Pancreatic Neoplasms; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Promoter Regions, Genetic; Retroviridae; Substrate Specificity; Thymidine; Thymidine Kinase; Transduction, Genetic; Tumor Cells, Cultured