Evidence for enzymatic activation and oxygen involvement in cytotoxicity and antitumor activity of N,N',N''-triethylenethiophosphoramide.
The cytotoxicity of N,N',N''-triethylenethiophosphoramide (thiotepa) was studied in vitro in the MCF-7 human breast carcinoma cell line and in vivo using the EMT6 mouse mammary tumor model, under various conditions of oxygenation and in the presence and absence of Aroclor 1254-induced liver preparations. The cytotoxicity of thiotepa toward exponentially growing MCF-7 cells was markedly dependent on the presence of oxygen during the period of drug exposure, with 3 log greater cell kill at 500 microM thiotepa being observed when the cells were normally oxygenated compared with hypoxic cells. Incubation of thiotepa with an Aroclor 1254-induced rat liver S-9 homogenate, in the presence of a NADPH-regenerating system, resulted in an 8-fold increase in cytotoxicity towards the MCF-7 cells over a wide range of drug concentrations. Thiotepa was shown to be metabolized under these conditions in a NADPH- and O2-dependent reaction that was catalyzed by one or more microsomal cytochrome P-450 enzymes that were present in the S-9 fraction. The thiotepa metabolite triethylene phosphoramide, which hydrolyzes significantly faster than thiotepa, was significantly less cytotoxic toward the MCF-7 cells than was thiotepa itself, suggesting that it is unlikely to be the S-9 metabolite responsible for the observed increase in drug cytotoxicity. Moreover, triethylene phosphoramide cytotoxicity was only partially O2 dependent and was largely unaffected by incubation in the presence of the S-9 preparation, indicating a mechanism of action distinct from that of thiotepa. Tumor cell survival experiments with the EMT6 mouse mammary carcinoma system revealed that a 3.6-fold increase in thiotepa cytotoxicity was obtained by prior administration of the liver inducer Aroclor 1254 to the tumor-bearing animals, 5 days before drug treatment. Finally, the therapeutic effectiveness of thiotepa was significantly enhanced (3- to 5.8-fold increase in tumor growth delay) when an increase in oxygenation was achieved, by carbogen breathing, in animals given the perfluorochemical emulsion Fluosol-DA. These findings establish that the cytotoxic effects of thiotepa are oxygen dependent and may involve, at least in part, metabolic processes catalyzed by cytochrome P-450 enzymes.
Teicher
B A
BA
Division of Cancer Pharmacology, Dana-Farber Cancer Institute, Boston, Massachusetts.
Waxman
D J
DJ
Holden
S A
SA
Wang
Y Y
YY
Clarke
L
L
Alvarez Sotomayor
E
E
Jones
S M
SM
Frei
E
E
D000818:Animals; D001711:Biotransformation; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D005260:Female; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008325:Mammary Neoplasms, Experimental / Q000188:drug therapy / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008862:Microsomes, Liver / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D013852:Thiotepa / Q000378:metabolism* / Q000494:pharmacology / Q000627:therapeutic use; D014407:Tumor Cells, Cultured / Q000187:drug effects
D009243:NAD; D009249:NADP; D013852:Thiotepa