N(1),N(11)-Diethylnorspermine (
DENSPM) is a
polyamine analogue with clinicalrelevance as an experimental
anticancer agent and the ability to elicit a profound apoptotic response in certain cell types. Here, we characterize the
polyamine effects and apoptotic signaling events initiated by treatment of SK-MEL-28 human
melanoma with 10 microM
DENSPM. Maximal induction of the
polyamine catabolic
enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) and
polyamine pool depletion were seen by 16 h, whereas early apoptosis was first apparent at 36 h. Intermediate events related to apoptotic signaling were sought between 16 and 36 h. A loss of mitochondrial transmembrane potential (Deltapsi(m)) beginning at 24 h was followed by the release of
cytochrome c into the cytosol at 30 h. Loss of mitochondrial integrity was accompanied by
caspase-3 activation and
poly(ADP-ribose) polymerase digestion from 30 to 36 h. The
caspase inhibitor Z-Asp-2,6-dichlorobenzoyloxymethylketone rendered cells resistant to analogue-induced
caspase-3 activation and reduced the apoptotic response in a dose-dependent manner. Because
polyamine reduction achieved by inhibitors of
polyamine biosynthesis inhibited growth but did not cause apoptosis, we looked for alternative
polyamine-related events, focusing on induction of SSAT. Three
DENSPM analogues that differentially induced SSAT activity but similarly depleted
polyamine pools revealed a close correlation between enzyme induction and
cytochrome c release,
caspase activation, and apoptosis. Dose-dependent inhibition of
polyamine oxidase, an
enzyme that oxidizes acetylated
polyamines generated by SSAT and releases toxic by-products such as H(2)O(2) and
aldehydes, prevented
cytochrome c release,
caspase activation, and apoptosis. Taken together, the findings indicate that
DENSPM-induced apoptosis is at least partially initiated via massive induction of SSAT and related oxidative events and subsequently mediated by the mitochondrial apoptotic signaling pathway as indicated by
cytochrome c release and
caspase activation.