tiazofurin and Ovarian-Neoplasms

Inosine 5 -monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme for the synthesis of GTP and dGTP. Two isoforms of IMPDH have been identified. IMPDH Type I is ubiquitous and predominantly present in normal cells, whereas IMPDH Type II is predominant in malignant cells. IMPDH plays an important role in the expression of cellular genes, such as p53, c-myc and Ki-ras. IMPDH activity is transformation and progression linked in cancer cells. IMPDH inhibitors, tiazofurin, selenazofurin, and benzamide riboside share similar mechanism of action and are metabolized to their respective NAD analogues to exert antitumor activity. Tiazofurin exhibits clinical responses in patients with acute myeloid leukemia and chronic myeloid leukemia in blast crisis. These responses relate to the level of the NAD analogue formed in the leukemic cells. Resistance to tiazofurin and related IMPDH inhibitors relate mainly to a decrease in NMN adenylyltransferase activity. IMPDH inhbitors induce apoptosis. IMPDH inhitors are valuable probes for examining biochemical functions of GTP as they selectively reduce guanylate concentration. Incomplete depletion of cellular GTP level seems to down-regulate G-protein function, thereby inhibit cell growth or induce apoptosis. Inosine 5'-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) catalyzes the dehydrogenation of IMP to XMP utilizing NAD as the proton acceptor. Studies have demonstrated that IMPDH is a rate-limiting step in the de novo synthesis of guanylates, including GTP and dGTP. The importance of IMPDH is central because dGTP is required for the DNA synthesis and GTP plays a major role not only for the cellular activity but also for cellular regulation. Two isoforms of IMPDH have been demonstrated. IMPDH Type I is ubiquitous and predominately present in normal cells, whereas the IMPDH Type II enzyme is predominant in malignant cells. Although guanylates could be salvaged from guanine by the enzyme hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8), the level of circulating guanine is low in dividing cells and this route is probably insufficient to satisfy the needs of guanylates in the cells.

Topics: Antineoplastic Agents; Apoptosis; cdc25 Phosphatases; Clinical Trials as Topic; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Drug Resistance, Neoplasm; Enzyme Inhibitors; Female; Guanosine Triphosphate; HL-60 Cells; Humans; IMP Dehydrogenase; Leukemia, Myeloid; Neoplasms; Nucleosides; Organoselenium Compounds; Ovarian Neoplasms; Protein Tyrosine Phosphatases; Ribavirin; Ribonucleosides; RNA, Messenger; Time Factors; Tumor Cells, Cultured

The purpose was to identify novel targets for the chemotherapy of ovarian carcinoma.. Assays were worked out to measure the activities of P1 kinase, PIP kinase and PLC in ovarian carcinoma samples and in OVCAR-5 cells and to compare the activities to those in normal ovaries. A method was also designed for measuring the concentration of the end product of signal transduction, IP3.. Signal transduction activity was markedly increased in ovarian cancer cells as shown by the increased steady-state activities of the three enzymes and the elevated concentrations of IP3. Inhibitors blocked activities of PI kinase (quercetin), PIP kinase (genistein), and lowered GTP concentration required for PLC (tiazofurin). Combinations of tiazofurin with quercetin, tiazofurin with genistein, and quercetin with genistein yielded a synergistic kill of ovarian cancer cells. Tiazofurin, quercetin and genistein are in various stages of clinical trials.. The increased signal transduction activity provides novel, sensitive targets to chemotherapy in ovarian cancer cells.

Topics: Cell Survival; Drug Synergism; Female; Genistein; Humans; Ovarian Neoplasms; Quercetin; Ribavirin; Signal Transduction

Ovarian carcinoma is one of the most common causes of cancer death in women. Tiazofurin, a C-nucleoside, arrests the cell cycle at S phase and reduces the activities of PI (phosphatidylinositol) utilizing enzymes in signal transduction by depleting cellular GTP concentration. Quercetin (QN), a flavonoid, attacks the cell cycle at the G1 and S phase boundary and mainly inhibits PI kinase (1-phosphatidylinositol 4-kinase, EC 2.7.1.67) activity in the signal transduction pathway. Because tiazofurin and QN attack different biochemical targets and arrest different phases of the cell cycle, we tested the hypothesis that the two drugs might be synergistic against human carcinoma cells. In human ovarian carcinoma OVCAR-5 cells in growth inhibition assay, the IC50s (drug concentration that inhibits 50% of cell proliferation) for tiazofurin and QN were (mean +/- SE) 13 +/- 1.2 and 66 +/- 3.0 microM; in clonogenic assays they were 6 +/- 0.5 and 15 +/- 1.2 microM, respectively. When tiazofurin was added to cells followed 12 h later by QN, synergism was observed in both growth inhibition and clonogenic assays. The combination also yielded synergistic reduction of IP3 (inositol 1,4,5-trisphosphate) concentration in the cells which may explain, at least in part, the synergistic action of tiazofurin and QN in OVCAR-5 cells. The protocols yielding synergism may have implications in the clinical treatment of human ovarian carcinoma.

Topics: Antineoplastic Agents; Cell Cycle; Down-Regulation; Drug Synergism; Female; Humans; Inositol 1,4,5-Trisphosphate; Ovarian Neoplasms; Quercetin; Ribavirin; Signal Transduction; Tumor Cells, Cultured

Recent work in this Laboratory showed increased activity of PI 4-kinase, PIP kinase and PLC in various cancer cells, indicating a stepped-up capacity for signal transduction. This elevated potential was paralleled with increased concentration of the end product of signal transduction, IP3. Current investigations showed that in normal cells the activities of the specific phosphatases (which degrade PIP2 and PIP and oppose those of the synthetic enzymes) were 4 to 5 orders of magnitude higher than those of the synthetic kinases. In hepatoma cells the specific phosphatase activities markedly decreased. Thus, in cancer cells the marked elevations in activities of the synthetic enzymes were opposed by a reduction in the activities of the degradative specific phosphatases. This enzymic imbalance is responsible, in part at least, for the elevated capacity of signal transduction and IP3 concentration. Since the enzymic activities measured were proportionate with time elapsed and amount of enzyme added, the alterations in activities should reflect changes in enzyme amounts. These alterations indicate a reprogramming of gene expression which should confer selective advantages to the cancer cells, marking out the elevated synthetic enzyme activities as potentially sensitive targets for drug treatment. We showed earlier that tiazofurin, which curtailed the biosynthesis of enzymes with short half-lives such as PI and PIP kinases, down-regulated signal transduction and brought down IP3 concentration. Quercetin and genistein chiefly inhibited PI-4 kinase and PIP kinase, respectively, and as a result reduced IP3 concentration in cancer cells. Current studies reveal that tiazofurin with quercetin, tiazofurin with genistein, and quercetin with genistein were synergistic in killing human cancer cells and in reducing signal transduction activity. In estrogen receptor-negative MDA-MB-435 human breast carcinoma cells which have elevated signal transduction activity, tamoxifen caused IC50S for growth inhibition and cytotoxicity of 12 and 0.7 microM, respectively. When tiazofurin was added to breast carcinoma cells, followed 12 hr later by tamoxifen, synergism was observed in growth inhibition, in clonogenic assays and in the reduction of IP3 concentration. The synergistic action of tiazofurin and tamoxifen and the other synergistic drug interactions outlined above may have implications in the clinical treatment of neoplasias.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Differentiation; Cell Division; Colonic Neoplasms; Down-Regulation; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Genistein; Humans; Inositol Phosphates; Liver Neoplasms, Experimental; Neoplasms; Ovarian Neoplasms; Phosphoric Monoester Hydrolases; Rats; Ribavirin; Signal Transduction; Tamoxifen; Tumor Cells, Cultured

Tiazofurin, an oncolytic drug, reduces PI kinase activity and arrests chiefly in S phase. Genistein, an inhibitor of PIP kinase, tyrosine kinase, and topoisomerase-II, induces arrest in G2 and/or early M phase in most carcinoma cells. Both tiazofurin and genistein reduce second messenger IP3 concentration in ovarian carcinoma cells. Because genistein and tiazofurin attack different enzymic targets and arrest the cell cycle at different phases, we tested the hypothesis that tiazofurin might be synergistic with genistein. Human ovarian carcinoma OVCAR-5 cells were grown in flasks in monolayers. In growth inhibition assay for tiazofurin and genistein the IC50s were 26 and 18 microM, respectively, and in clonogenic assays the LC50s were 17 and 4 microM, respectively. Various combinations of the two drugs were tested. The best protocol took into consideration that tiazofurin decreased GTP concentration in cells by 50% at 12 h after administration. Tiazofurin (20 microM) and genistein (20 microM) as single agents reduced cell counts to 60% and 50%, respectively. The predicted value, as a sum of the effect of two drugs, would have been 30% of controls. However, genistein added 12 h after tiazofurin decreased cell counts to 8%, showing synergistic action of the two drugs for growth inhibition. Similar results were observed in the clonogenic assays, which also revealed synergistic cytotoxicity. The protocol yielding synergism might be of value in the clinical treatment of human ovarian carcinoma.

Topics: Antineoplastic Agents; Carcinoma; Drug Screening Assays, Antitumor; Drug Synergism; Female; Genistein; Growth Inhibitors; Humans; Ovarian Neoplasms; Ribavirin; Tumor Cells, Cultured

The steady-state activities of the first two enzymes of the phosphatidylinositol (PI) phosphorylation pathway, PI 4-kinase, EC 2.7.1.67 (PI kinase) and PI 4-phosphate 5-kinase, EC 2.7.1.68 (PIP kinase) as compared to human normal ovary are elevated in human ovarian carcinomas (4.1- and 2.7-fold) and in human OVCAR-5 cells in tissue culture (31.2- and 8.9-fold). Compared to normal human breast parenchymal cells. PI kinase and PIP kinase activities were increased in breast carcinoma MDA-MB-435 cells grown in nude mice as solid tumors (7.3- and 2.3-fold, respectively) and in MDA-MB-435 cells grown in tissue culture (95.8- and 15.5-fold, respectively). When the human carcinoma cells were plated and expressed their neoplastic proliferative program in the log phase, in the MDA-MB-435 breast carcinoma cells the PI and PIP kinase activities coordinately increased 11-fold; in ovarian carcinoma OVCAR-5 cells 5.8- and 4.5-fold, respectively. These studies provide the first evidence in human cancer cells of an increased capacity for the operation of signal transduction. This is indicated by the markedly elevated activities of PI and PIP kinases in the phosphatidylinositol phosphorylation sequence which leads to production of second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG).

Topics: 1-Phosphatidylinositol 4-Kinase; Animals; Antineoplastic Agents; Breast; Breast Neoplasms; Cell Division; Depression, Chemical; Down-Regulation; Female; Humans; Liver; Liver Neoplasms, Experimental; Male; Mice; Mice, Hairless; Mice, Nude; Neoplasm Transplantation; Ovarian Neoplasms; Ovary; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Inbred ACI; Ribavirin; Signal Transduction; Tumor Cells, Cultured

Since taxol (NSC 125975) and tiazofurin (NSC 286193) attack at two different sites in microtubular synthetic processes, we tested the rationale that the two drugs might be synergistic in human ovarian (OVCAR-5), pancreatic (PANC-1) and lung carcinoma (H-125) cells and in rat hepatoma 3924A cells. In human OVCAR-5, PANC-1, H-125 and rat 3924A cells, for taxol the anti-proliferative IC50 was 0.05, 0.06, 0.03 and 0.04 microM, respectively; for tiazofurin IC50 = 8.3, 2.3, 1.8 and 6.9 microM. Thus, the concentrations for taxol required for IC50 for inhibiting cell proliferation were 166-, 38-, 60- and 173-fold lower than those for tiazofurin. Taxol and tiazofurin proved synergistic in all four cell lines tested. The synergism of taxol with tiazofurin should have implications in the clinical treatment of human solid tumors with particular relevance to ovarian, pancreatic, lung and hepatocellular carcinomas.

Topics: Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Carcinoma; Carcinoma, Adenosquamous; Cell Division; Drug Screening Assays, Antitumor; Drug Synergism; Female; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Liver Neoplasms, Experimental; Lung Neoplasms; Ovarian Neoplasms; Paclitaxel; Pancreatic Neoplasms; Rats; Ribavirin; Spindle Apparatus; Tumor Cells, Cultured

Tiazofurin (TR), an inhibitor of IMP dehydrogenase, causes remissions and induced differentiation in human leukemia through lowering the concentrations of GTP and dGTP. A deoxycytidine analog, difluorodeoxycytidine (DFDC), is an anti-tumor agent phosphorylated by deoxycytidine kinase, resulting in decreased concentration of dCTP, leading to inhibition of DNA synthesis. In HL-60 cells DFDC induced differentiation and inhibited proliferation in a dose-dependent manner (IC50 = 4 nM); TR provided synergism with DFDC. DFDC inhibited proliferation in OVCAR-5 human ovarian carcinoma cells (IC50 = 25 nM) and colony formation in PANC-1 human pancreatic carcinoma cells (IC50 = 2 nM) and rat hepatoma 3924A cells (IC50 = 22 nM). TR and DFDC are synergistically cytotoxic in hepatoma cells and additive in PANC-1 cells. The two drugs together should be helpful in treating leukemias and solid tumors in humans.

Topics: Animals; Antimetabolites, Antineoplastic; Cell Differentiation; Cell Line; Cell Survival; Deoxycytidine; Dose-Response Relationship, Drug; Drug Synergism; Female; Gemcitabine; Humans; IMP Dehydrogenase; Kinetics; Leukemia, Promyelocytic, Acute; Liver Neoplasms, Experimental; Models, Biological; Ovarian Neoplasms; Pancreatic Neoplasms; Rats; Ribavirin; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Stem Cell Assay

Cancer cells have an increased ability to synthesize GTP (guanosine triphosphate) because of increased activity of IMP DH (inosine 5'-phosphate dehydrogenase, EC 1.1.1.205). Because IMP DH activity is rate limiting for de novo biosynthesis of GTP, this enzyme was suggested as a sensitive target for chemotherapy. Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) is converted in the cells into the active metabolite, TAD, (thiazole-4-carboxamide adenine dinucleotide) which potently inhibits IMP DH activity. By adding TAD to tissue extracts one can determine the extent of inhibition of IMP DH. We applied the IMP DH assay method to extracts of normal ovaries (N = 11) and epithelial ovarian carcinomas (N = 10). The IMP DH activity (mean +/- SE) in ovarian carcinoma was 21.1 +/- 5.8 which was markedly higher than that observed in normal ovaries (2.9 +/- 0.7 nmol/hr/mg protein) (P < 0.05%). The inhibition by TAD of IMP DH activity in ovarian carcinomas (N = 4) was 81%. The results indicate that IMP DH activity is elevated sevenfold in ovarian carcinomas as compared to normal ovary and can be inhibited by exposure to tiazofurin (TAD). Similar high IMP DH activity and inhibition of the activity by TAD was observed in patients with chronic granulocytic leukemia in blast crisis among whom 70 to 80% remissions were reported. Since there is increased IMP DH activity in human ovarian carcinomas and in OVCAR-5 cells and tiazofurin and TAD inhibit IMP DH activity of these cells and the proliferation of human ovarian carcinoma xenografts in the mouse, tiazofurin may merit serious consideration for a Phase II trial for patients with recurrent/refractory epithelial ovarian carcinoma.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Female; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Middle Aged; Ovarian Neoplasms; Ovary; Ribavirin; Tissue Extracts

The activity of 2-beta-D-ribofuranosylthiazole-4-carboxamide (tiazofurin) was determined in nine untreated human ovarian cancer specimens, using the murine subrenal capsule xenograft assay. Tumor cytotoxic effect was demonstrated in seven out of the nine tumors implanted. The drug was well tolerated by the test animals. Tiazofurin may prove to be an effective chemotherapeutic agent in the treatment of ovarian cancer.

Topics: Animals; Antineoplastic Agents; Female; Mice; Mice, Inbred Strains; Neoplasm Transplantation; Ovarian Neoplasms; Ribavirin; Ribonucleosides