Mixed function oxidase activities of established human colon carcinoma cell lines in the activation of cyclophosphamide.
Three established human colon carcinoma cell lines (LoVo, SW620, and SW403) with different degrees of phenotype differentiation were investigated for their sensitivity to the cytotoxic effects of cyclophosphamide (CP) and to its active metabolite, 4-hydroxycyclophosphamide (4-OH-CP), and for their mixed function oxidase (MFO) activities. None of the cell lines showed sensitivity to CP as determined by the inhibition of colony formation assay, even after continuous drug treatment at high concentrations (200 microgram/ml) for up to 72 h. CP also had no effect on the cellular doubling time or on the incorporation of [3H]-thymidine. Pretreatment with phenobarbital (PB) plus hydrocortisone (HC) was unable to induce CP cytotoxicity. In contrast, 4-OH-CP, the major metabolite formed from CP by MFO, was highly toxic to the cells. About 90% cell kill was obtained at drug concentrations of 17.5 microgram/ml (LoVo), 15 microgram/ml (SW620), and 55 microgram/ml (SW403) after 1-h incubation at 37 degrees. MFO activities were determined by measuring p-nitroanisole demethylase (PNAD) and arylhydrocarbon hydroxylase (AHH) in microsomes prepared from noninduced cells or from cells treated with benzanthracene or PB plus HC. Intrinsic AHH activities were below the level of detection for all cell lines [less than 1 pmol of 3-hydroxybenzo(a)pyrene (3-OH-BP) formed per min per mg of protein]. Treatment with benzanthracene resulted in AHH activities of 12 to 15 pmol of 3-OH-BP per min per mg of protein, but treatment with PB plus HC failed to induce significant AHH activities. PNAD activities in noninduced cells as well as in cells treated with benzanthracene were 0.05 to 0.08 nmol of p-nitrophenol formed per min per mg of protein; treatment with PB plus HC increased PNAD activities by only 1.5-fold. Thus, in contrast to reports for rat colon and for a single human colon cancer cell line, CP is inactive when applied directly to several other human colon carcinoma cell lines. Because these cells have minimally detectable intrinsic and induced MFO activities, we conclude that CP cannot be successfully metabolized into 4-OH-CP to induce a significant degree of cell kill.
D001189:Aryl Hydrocarbon Hydroxylases / Q000032:analysis; D001711:Biotransformation; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000201:enzymology*; D003520:Cyclophosphamide / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000032:analysis*; D013936:Thymidine / Q000378:metabolism; D014316:Tritium
D014316:Tritium; D003520:Cyclophosphamide; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; D013936:Thymidine