Since synthetic analogs of triptycene (TT code number), such as bisquinones TT2 and TT13, can trigger cytochrome c release without caspase activation and retain their ability to induce apoptosis in multidrug-resistant (MDR) tumor cells, fluorescent probes of transmembrane potential have been used to determine whether these antitumor compounds might directly target mitochondria in cell and cell-free systems to cause the collapse of mitochondrial membrane potential (↓Δψm) that is linked to permeability transition pore (PTP) opening. Using JC-1 dye, the abilities of various TT analogs to induce the ↓Δψm in wild-type and MDR HL-60 cells are rapid (within 5-20 min), irreversible after drug removal, concentration dependent in the 0.64-25 µM range, and generally related to their antitumor activities in vitro. The ↓Δψm caused by TT2 and TT13, which are more potent than mitoxantrone, staurosporine and the reference depolarizing agent, carbonyl cyanide m-chlorophenylhydrazone (CCCP), in HL-60 cells, are not prevented by caspase-2 or -8 inhibitors, suggesting that activation of these apical caspases upstream of mitochondria is not involved in this process. Antitumor TT analogs (0.64-25 µM) also mimic the abilities of the known depolarizing agents, CCCP, alamethicin, gramicidin A and 100 µM CaCl2, to directly induce within 20 min the ↓Δψm in isolated mitochondria prepared from mouse liver and loaded with rhodamine 123 dye. The fact that 20 µM Ca2+, which is insufficient to trigger depolarization on its own, is required to reveal the depolarizing effect of TT2 in isolated mitochondria suggests that antitumor TT analogs might interact with the PTP to alter its conformation and increase its Ca2+ sensitivity. Indeed, such Ca2+-dependent ↓Δψm of isolated mitochondria treated with 25 µM TT2 or 100 µM Ca2+ are blocked by ruthenium red. Daunorubicin (DAU) is unable to mimic the rapid ↓Δψm caused by antitumor TT bisquinones within 5-40 min of treatment in HL-60 cells or isolated mitochondria. Moreover, the ↓Δψm caused by 25 µM TT2 or 100 µM Ca2+ in isolated mitochondria are similarly blocked by cyclosporin A (CsA), bongkrekic acid and decylubiquinone, which prevent PTP opening, suggesting that, in contrast to DAU, antitumor TT analogs that directly target mitochondria to trigger the Ca2+-dependent and CsA-sensitive ↓Δψm, might induce PTP opening and the mitochondrial pathway of apoptosis even in the absence of nuclear signals.

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