A0, a Cu(II) thioxotriazole complex, produces severe cytotoxic effects on HT1080 human
fibrosarcoma cells with a potency comparable to that exhibited by
cisplatin. A0 induced a characteristic series of changes, hallmarked by the formation of
eosin- and
Sudan Black-B-negative vacuoles. No evidence of nuclear fragmentation or
caspase-3 activation was detected in cells treated with A0 which, rather, inhibited
cisplatin-stimulated
caspase-3 activity. Membrane functional integrity, assessed with
calcein and
propidium iodide, was spared until the late stages of the death process induced by the
copper complex. Vacuoles were negative to the autophagy marker
monodansylcadaverine and their formation was not blocked by
3-methyladenine, an inhibitor of autophagic processes. Negativity to the extracellular marker
pyranine excluded vacuole derivation from the extracellular fluid. Ultrastructural analysis indicated that A0 caused the appearance of many electronlight cytoplasmic vesicles, possibly related to the endoplasmic reticulum, which progressively enlarge and coalesce to form large vacuolar structures that eventually fill the cytoplasm. It is concluded that A0 triggers a non-apoptotic, type 3B programmed cell death (Clarke in Anat Embryol (Berl) 181:195-213, 1990), characterized by an extensive cytoplasmic vacuolization. This peculiar cytotoxicity pattern may render the employment of A0 to be of particular interest in apoptosis-resistant cell models.