TYRPHOSTIN AG17, I-TERT-BUTYL-4-HYDROXYBENZYLIDENE)-MALONONITRILE], INHIBITS CELL-GROWTH BY DISRUPTING MITOCHONDRIA

Di-tert-butyl-4-hydroxybenzylidene)-malononitrile] (AG17), ''tyrphostt n'' tyrosine kinase antagonist, was found to inhibit tumor cell growth with 50% growth inhibition ranging from 0.7 to 4.0 mu M in a panel of 13 human tumor cell lines, as evaluated by tetrazolium dye reduction and inhibition of precursor incorporation into macromolecules. The pro myelocytic leukemia cell Line HL-60(TB), was the most sensitive with i rreversible total growth inhibition after 12 h of exposure to 1.5 mu M drug. Antiproliferative effects of AG17 in HL-60(TB) cells were tempo rally related to disruption of mitochondrial function, which occurred within I h after drug exposure as demonstrated by a significantly decr eased mass of ATP in drug-treated cells, loss of the fluorescent mitoc hondrial membrane potential probe rhodamine 123, and ultrastructural e xamination of mitochondria using fluorescence and electron microscopy. Specific decreases of total or tyrosine-phosphorylated substrate at c oncentrations of the drug not affecting ATP levels were not detected. These data raise the possibility that AG17 may act in part by altering mitochondrial function and/or structure, and that impairment of mitoc hondrial function may be exploitable as a potentially useful mechanism to modulate tumor cell proliferation. This study also emphasizes the importance of evaluating carefully the effects of potential protein ki nase antagonists, since these structures have effects in intact cells in addition to what might be expected from in vitro enzyme assays.