Synthetic
triptycene analogs (TT code number) mimic the antitumor effects of
daunorubicin (DAU) in vitro, but have the advantage of blocking
nucleoside transport, inhibiting both
DNA topoisomerase I and II activities, and retaining their efficacy in multidrug-resistant (MDR)
tumor cells. Since TT bisquinones induce
poly(ADP-ribose) polymerase-1 (PARP-1) cleavage at 6 h and internucleosomal DNA fragmentation at 24 h, which are, respectively, early and late markers of apoptosis, these
antitumor drugs were tested for their ability to trigger the release of mitochondrial
cytochrome c (Cyt c) and the
caspase activation cascade in the HL-60 cell system. Based on their ability to reduce the viability of wild-type,
drug-sensitive HL-60-S cells in the nanomolar range, six lead antitumor TT bisquinones have been identified so far: TT2, TT13, TT16, TT19, TT24 and TT26. In accord with the fact that effector
caspase-3 is responsible for PARP-1 cleavage, 4 microM concentrations of DAU and these TT bisquinones all maximally induce
caspase-3 activity at 6 h in HL-60-S cells, an effect which persists when the drugs are removed after a 1-h pulse treatment. Since
caspase-3 may be activated by initiator
caspase-9 and -8, it is significant to show that such
caspase activation cascade is induced by 4 microM DAU and TT bisquinones at 6 h in HL-60-S cells. Although the relationship is not perfect, the ability of TT analogs to induce
caspase-3, -8 and -9 activities may be linked to their
quinone functionality and cytotoxicity. Interestingly, 4 microM concentrations of TT bisquinones retain their ability to induce
caspase-3, -8 and -9 activities at 6 h in the MDR HL-60-RV cell line where 4 microM DAU becomes totally ineffective. The release of mitochondrial Cyt c is also detected within 6 h in HL-60-S cells treated with 4 microM DAU or TT bisquinones, a finding consistent with the fact that Cyt c is the apoptotic trigger that activates
caspase-9.
Caspase-2 and -8 may both act upstream of mitochondria to promote Cyt c release, but
caspase-2 is already maximally activated 6 h after 4 microM DAU or TT13 treatments, whereas DAU- or TT-induced
caspase-8 and -9 activities peak at 9 h. Pre-treatments with 15 microM of the
caspase-2 inhibitor benzyloxycarbonyl (z)-Val-Asp-Val-
Ala-Asp (VDVAD)-fluoromethyl
ketone (fmk) totally block DAU- and TT13-induced
caspase-2, -8 and -9 activities, whereas pre-treatments with 15 microM of the
caspase-8 inhibitor z-Ile-Glu-Thr-Asp (
IETD)-fmk prevent DAU and TT13 from inducing
caspase-8 activities without affecting their caspase-2- and -9-inducing activities, suggesting that the induction of apical
caspase-2 activity by these drugs may be a critical upstream event required for the activation of other downstream
caspases, including
caspase-9 and the mitochondrial amplification loop through
caspase-8. However, the mechanisms by which DAU and TT13 induce the release of mitochondrial Cyt c appear to be
caspase-independent since they are both insensitive to similar pre-treatments with 100 microM of these specific
caspase-2 and -8 inhibitors. Moreover, pre-treatments with 10 microg/ml of the antagonistic anti-Fas DX2 and ZB4
monoclonal antibodies (mAbs), and the neutralizing anti-
Fas ligand (FasL) NOK-1 mAb are all unable to prevent DAU and TT13 from inducing Cyt c release and
caspase-2, -8 and -9 activities, suggesting that the Fas-FasL signaling pathway is not involved in the mechanism by which these
quinone antitumor drugs trigger apoptosis in HL-60 cells.