novobiocin and Colonic-Neoplasms

We tested the potential impact of tyrosine phosphorylation on the expression of the c-myc gene in two colon cancer cell lines, HCT8 and SW837. We found that the protein tyrosine kinase inhibitor genistein causes a decrease in the abundance of c-myc RNA and an inhibition of proliferation with a similar dose response. Geldanamycin, a mechanistically different tyrosine kinase inhibitor, also causes a decrease in both the expression of c-myc RNA and proliferation. Genistein has also been found to inhibit topoisomerase II, but the topoisomerase II inhibitor novobiocin did not lower the expression of c-myc. The most likely interpretation is that inhibition of protein tyrosine kinase activity caused a decrease in c-myc expression in these cells. The impact of tyrosine phosphorylation on the expression of the c-myc gene is further supported by the finding that inhibition of phosphotyrosine phosphatase using orthovanadate causes an increase in the level of c-myc RNA. The effect of genistein on HCT8 cells is not dependent on the synthesis of new protein and does not involve an alteration in the stability of the message. Analysis of transcription in the c-myc gene reveals a more complicated picture with a decrease in initiation and an increase in elongation but no net change in transcription. We speculate that the genistein induced reduction in myc expression is the result of a posttranscriptional intranuclear event(s).

Topics: Benzoquinones; Cell Count; Cell Line; Colonic Neoplasms; Cycloheximide; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Genes, myc; Genistein; Half-Life; Humans; Isoflavones; Lactams, Macrocyclic; Novobiocin; Phosphorylation; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Quinones; RNA, Messenger; Topoisomerase II Inhibitors; Tyrosine; Vanadates

Previous reports from this laboratory have demonstrated that novobiocin produces supraadditive cytotoxicity and increases the formation of drug-stabilized topoisomerase II-DNA covalent complexes in WEHI-3B myelomonocytic leukemia and A549 lung carcinoma cells when combined with etoposide (VP-16). Inhibition of the efflux of VP-16 by novobiocin is responsible for the increase in VP-16 accumulation, which in turn leads to increased formation of VP-16-stabilized topoisomerase II-DNA covalent complexes and increased cytotoxicity. We now report that novobiocin synergistically enhanced the sensitivity of the multidrug resistant variants, WEHI-3B/NOVO and A549(VP)28, to VP-16, causing almost complete reversal of the resistance to the epipodophyllotoxin. These two tumor cell variants are resistant to several topoisomerase II-targeted drugs, particularly VP-16, but not to Vinca alkaloids; this finding corresponds to the fact that they do not overexpress the P-glycoprotein. The effects of novobiocin in these resistant sublines are mediated through the intracellular accumulation of VP-16, resulting in an increase in the formation of lethal VP-16-induced topoisomerase II-DNA covalent complexes. In the P-glycoprotein expressing multidrug resistant HCT116(VM)34 colon carcinoma and L1210/VMDRC0.06 leukemia cell lines, the latter being transfected with the human mdr-1 gene, novobiocin did not potentiate the cytotoxic activity of VP-16 nor increase the intracellular accumulation of VP-16 and the formation of covalent complexes, whereas their normal counterparts were sensitive to the potentiating activity of novobiocin when used in combination with VP-16. These results indicate that the action of novobiocin on the intracellular transport of VP-16 is not directed at the level of the P-glycoprotein, but that the action of novobiocin is antagonized by the presence of the P-glycoprotein. Since novobiocin is a clinically available antibiotic, has numerous structural analogues available for comparative studies, and has a relatively low toxicity profile, this drug, as well as structurally related agents, would appear to have significant clinical potential in combination with an epipodophyllotoxin for the treatment of non-P-glycoprotein expressing multidrug resistant tumors.

Topics: Adenocarcinoma; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carrier Proteins; Colonic Neoplasms; DNA Topoisomerases, Type II; DNA, Neoplasm; Drug Resistance; Etoposide; Flow Cytometry; Humans; Leukemia L1210; Lung Neoplasms; Membrane Glycoproteins; Mice; Novobiocin; Tumor Cells, Cultured

Topics: Cell Line; Colonic Neoplasms; Cytarabine; Dideoxynucleosides; DNA Polymerase I; DNA Polymerase II; DNA Repair; Humans; Kinetics; Nalidixic Acid; Novobiocin; Nucleic Acid Synthesis Inhibitors; Thymidine; Topoisomerase I Inhibitors