Azidothymidine-induced cytotoxicity and incorporation into DNA in the human colon tumor cell line HCT-8 is enhanced by methotrexate in vitro and in vivo.
We have reported that 5-fluorouracil can increase the cytotoxic and antineoplastic activity of 3'-azido-3'-deoxythymidine (AZT). To further evaluate the antineoplastic utility of AZT we now have assessed its effect in combination with methotrexate (MTX) in the human colon tumor model HCT-8. Incubation of these cells for 5 days in AZT and MTX caused a reduction in the 50% inhibitory concentration of AZT and isobologram analysis revealed additive effects which were reversed by the addition of 50 microM thymidine to the incubation media. This enhanced cytotoxicity appeared not to be related to an effect of AZT on MTX activity; in whole-cell assays the ability of MTX to inhibit de novo dTMP synthesis and deplete intracellular pools of dTTP was not affected by AZT. In contrast, although MTX did not alter AZT triphosphate production, it did affect AZT triphosphate utilization in DNA synthesis. Incubation of cells for 24 h in [3H]AZT alone (5 microM, 3 microCi/ml) resulted in 6.6 pmol AZT incorporated into cellular DNA/10(6) cells. Coincubation of these cells in [3H]AZT (5 microM) plus 5 or 15 nM MTX increased AZT incorporation into DNA to 8.0 and 20.5 pmol/10(6) cells, respectively. Biochemically, this effect appeared to correlate with the concentration-dependent ability of 5 or 15 nM MTX to deplete intracellular dTTP pools, which were reduced by 25 and 49%, respectively. Further evidence of the relationship between intracellular dTTP pools and AZT cytotoxicity was that, in the presence of both MTX and 50 microM thymidine, intracellular dTTP pools remained near normal levels and the incorporation of 5 microM AZT into DNA was not enhanced. Therapeutically, studies conducted in athymic (nude) mice bearing HCT-8 xenografts that received six weekly cycles of MTX (87.5 mg/kg) and AZT (300 mg/kg) revealed that the two-drug regimen exerted superior antineoplastic effects compared to either drug alone (treated versus control approximately 0.9 for AZT or MTX and approximately 0.3 for MTX plus AZT). In addition, the combination did not increase toxicity compared to therapy with MTX alone. These findings are discussed in light of their biochemical and clinical implications.
Tosi
P
P
Department of Medicine, Brown University, Providence, Rhode Island.
Calabresi
P
P
Goulette
F A
FA
Renaud
C A
CA
Darnowski
J W
JW
D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D001711:Biotransformation; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D004273:DNA, Neoplasm / Q000378:metabolism*; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology* / Q000627:therapeutic use; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D013940:Thymidylate Synthase / Q000378:metabolism; D014183:Transplantation, Heterologous; D015215:Zidovudine / Q000378:metabolism* / Q000494:pharmacology* / Q000627:therapeutic use
D004273:DNA, Neoplasm; D015215:Zidovudine; D013940:Thymidylate Synthase; D008727:Methotrexate