Synergistic cytotoxicity of the ribonucleotide reductase inhibitor didox (3,4-dihydroxy-benzohydroxamic acid) and the alkylating agent carmustine (BCNU) in 9L rat gliosarcoma cells and DAOY human medulloblastoma cells.
OBJECTIVE
Ribonucleotide reductase (RR) is the rate-limiting enzyme of de novo DNA synthesis and has been shown to be upregulated linked with proliferation and malignant transformation. It was therefore identified as an excellent target for antitumor therapy. In the present study we investigated the biochemical and cytotoxic effects of didox, an inhibitor of RR, as a single agent and in combination with BCNU, an alkylating anticancer drug, in 9L rat gliosarcoma cells and DAOY human medulloblastoma cells.


METHODS
The effect of didox on the intracellular concentrations of deoxynucleosidetriphosphates (dNTPs) was studied in 9L cells. Pool sizes were determined by HPLC. In addition, the cytotoxic effects of didox and BCNU as single drugs and in equimolar combination were tested in 9L and in DAOY cells. Combination effects were determined according to the equation of Chou and Talalay. The expression of DNA repair-related genes was determined after exposure of 9L cells to BCNU, didox and a combination of the two compounds, using a cDNA array.


RESULTS
Incubation of 9L cells with 30 microM didox for 24 h significantly decreased the intracellular concentrations of the DNA precursors dCTP (61% of control) and dGTP (17% of control), and significantly increased the concentration of dATP (155% of control). This dNTP imbalance compromised DNA synthesis and repair and might therefore have been, at least in part, responsible for the highly synergistic cytotoxic effects seen when BCNU was used simultaneously with didox in 9L and in DAOY cells. With almost all combinations tested, highly synergistic effects were seen, as indicated by combination indices of <1 according to the equation of Chou and Talalay. In 9L cells, BCNU upregulated the expression of DNA repair-associated genes, whereas coincubation of the cells with didox reduced overexpression of some of these repair-related genes.


CONCLUSIONS
A combination of BCNU and didox was proven to act in a synergistic manner in two cell lines, 9L rat gliosarcoma and DAOY human medulloblastoma cells. Further in vivo tests using these two compounds systemically and/or locally at the tumor site might be warranted.
Horvath
Zsuzsanna
Z
General Hospital of Vienna, Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, 5H Waehringer Guertel 18-20, 1090, Austria.
Höchtl
Thomas
T
Bauer
Wolfgang
W
Fritzer-Szekeres
Monika
M
Elford
Howard L
HL
Szekeres
Thomas
T
Tihan
Tarik
T
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000633:toxicity*; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology* / Q000633:toxicity*; D001932:Brain Neoplasms / Q000473:pathology*; D002330:Carmustine / Q000494:pharmacology* / Q000633:toxicity*; D002528:Cerebellar Neoplasms / Q000473:pathology*; D004347:Drug Interactions; D018316:Gliosarcoma / Q000473:pathology*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology* / Q000633:toxicity*; D008527:Medulloblastoma / Q000473:pathology*; D051381:Rats; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D018906:Antineoplastic Agents, Alkylating; D006877:Hydroxamic Acids; C035419:3,4-dihydroxybenzohydroxamic acid; D002330:Carmustine