tiazofurin and Breast-Neoplasms

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

Breast carcinoma is a leading cause of cancer death in women in the US. Tamoxifen (TAM), an antiestrogen, is used as a chemopreventive and chemotherapeutic compound against human breast carcinoma. Tiazofurin (TR), an oncolytic C-nucleoside, inhibits IMP dehydrogenase activity, decreases cellular GTP pools, and downregulates ras gene expression. MDA-MB-435 cells are estrogen receptor negative human breast carcinoma cells that have elevated signal transduction activity. Because TR and TAM decrease signal transduction enzyme activity and inositol 1,4,5-trisphosphate (IP3) concentration via different mechanisms, we tested the hypothesis that the two compounds may be synergistic in human breast carcinoma cells. In MDA-MB-435 cells in growth inhibition assay, the IC50s for TR and TAM were (mean +/- SE) 17 +/- 1.2 and 12 +/- 1.1 microM; in clonogenic assays they were 4 +/- 0.3 and 0.7 +/- 0.3 microM, respectively. When TR was added to MDA-MB-435 cells, followed 12 h later by TAM, synergism was observed in growth inhibition and clonogenic assays and in the reduction of IP3 concentration. The latter may explain, at least in part, the synergistic action of TR and TAM in these cells. The synergistic action of TR and TAM may have implication in the clinical treatment of human breast carcinoma.

Topics: Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Down-Regulation; Drug Synergism; Female; Humans; Inhibitory Concentration 50; Inositol 1,4,5-Trisphosphate; Ribavirin; Signal Transduction; Tamoxifen; Tumor Cells, Cultured

Brefeldin A (NSC 89671), a macrocyclic lactone, blocks cellular protein transport by disturbing the association and dissociation of the Golgi apparatus with a 110-kD protein which is regulated by GTP. Brefeldin also induces retrograde transport from the Golgi membrane to the endoplasmic reticulum, which is mediated by microtubules which also require GTP for their biosynthesis. The anti-cancer action of taxol is exerted by enhancing tubulin polymerization in microtubule assembly; tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide, NSC 28693) acts through decreasing cellular GTP concentrations. Therefore, we tested the hypothesis that taxol (paclitaxel, NSC 125975) or tiazofurin might provide synergism with brefeldin. In human breast carcinoma MDA-MB-435 cells in the growth inhibition assays for brefeldin, taxol and tiazofurin, the IC50s were 41 nM, 6 nM and 13 microM, respectively. When brefeldin and taxol were given simultaneously, addition (brefeldin 10 nM with taxol 2 to 8 nM) or synergism (brefeldin 30 nM with taxol 2 to 8 nM) was observed. When brefeldin and tiazofurin were given simultaneously, or tiazofurin was followed 12 h later by brefeldin, addition was observed. The protocols yielding synergism and addition should be of value in the design of clinical trials for breast carcinoma.

Topics: Anti-Bacterial Agents; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Brefeldin A; Cell Division; Cyclopentanes; Drug Synergism; Humans; Macrolides; Paclitaxel; 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

The effect of the nucleoside anti-metabolite tiazofurin (TR) was examined on the growth and phenotypic alterations of MCF-7 breast cancer and HBL-100 normal breast cell lines. TR was shown to inhibit MCF-7 cell growth. This inhibition could be reversed by exogenous addition of guanosine. The anti-proliferative effect of TR is accompanied by phenotypic alterations that include lipid accumulation and an increase in alkaline phosphatase activity. In contrast to MCF-7 cells, the HBL-100 breast milk derived cell line is relatively resistant to inhibition by TR. Alkaline phosphatase is not affected by TR and untreated cells accumulate lipid droplets, similar to TR-treated MCF-7 cells. Determination of GTP and ATP pools in both cell lines revealed that TR markedly reduces GTP content in MCF-7 cells. In HBL-100 cells, TR induces only a small decrease in GTP and does not affect ATP levels. The prototypic IMP dehydrogenase inhibitor, mycophenolic acid (MA), markedly inhibits HBL-100 cell growth, similarly to its effect on MCF-7 breast cancer cells. These findings may suggest differential metabolism of TR in MCF-7 and HBL-100 cells.

Topics: Adenosine Triphosphate; Antimetabolites, Antineoplastic; Breast Neoplasms; Cell Division; Cell Line; Female; Guanosine Triphosphate; Humans; Milk, Human; Mycophenolic Acid; Ribavirin; Ribonucleosides; Tumor Cells, Cultured