Mitochondrial permeability transition induced by the anticancer drug etoposide

Abstract

Etoposide (VP-16) is widely used for the treatment of several forms of cancer. The cytotoxicity of VP-16 has been assigned to the induction of apoptotic cell death but the signaling pathway for VP-16-induced apoptosis is essentially unknown. There is some evidence that this process depends on events associated with the loss of mitochondrial membrane potential (ÁÉ) and/or release of apoptogenic factors, putatively as a consequence of mitochondrial permeability transition (MPT) induction. This work evaluates the interference of VP-16 with MPT in vitro, which is characterized by the Ca 2+ -dependent depolarization of ÁÉ, the release of matrix Ca 2+ and by extensive swelling of mitochondria. ÁÉ depolarization and Ca 2+ release were measured with ion-selective electrodes, and mitochondrial swelling was monitored spectrophotometrically. Incubation of rat liver mitochondria with VP-16 results in a concentration-dependent induction of MPT, evidenced by an increased sensitivity to Ca 2+ -induced swelling, depolarization of ÁÉ, Ca 2+ release by mitochondria and stimulation of state 4 oxygen consumption. All of these effects are prevented by preincubating the mitochondria with cyclosporine A, a potent and specific inhibitor of the MPT. Therefore, VP-16 increases the sensitivity of isolated mitochondria to the Ca 2+ -dependent induction of the MPT. Together, these data provide a possible mechanistic explanation for the previously reported effects of VP-16 on apoptosis induction. #

Thiol protecting agents and antioxidants inhibit the mitochondrial permeability transition promoted by etoposide: implications in the prevention of etoposide-induced apoptosis

“…2, trace 1) and accumulation of the added Ca 2 + (Fig. 3, trace 1), as we have previously reported [5]. However, mitochondria pre-incubated for 5 min with VP-16 undergo complete uncoupling of respiration after addition of Ca 2 + (Fig.…”

“…2, trace 2), Ca 2 + promotes a gradual and irreversible depolarization of Dc, which is completely prevented by CyA (Fig. 2, trace 3) added to mitochondria before VP-16 incubation, as we have previously reported [5]. Similarly, the addition of either Asc (Fig.…”

“…1. As we have previously reported [5], VP-16 (1 mmol/mg protein-500 mM) increases the sensitivity of mitochondria to undergo a rapid and large decrease in the absorbance due to MPT induced by Ca 2 + (140 nmol/mg protein) (Fig. 1, trace 1) since it is prevented by CyA (1 mM) (Fig.…”

“…Inhibitor effects of Asc, glutathione (GSH) and NEM on VP-16-induced Ca 2 + -dependent mitochondrial swelling. Mitochondria (0.5 mg protein/ml) incubated at 30°C in 2 ml of standard reaction medium, supplemented with 2 mM rotenone and 0.5 mg oligomycin/ml, were energized with 5 mM succinate after addition of either VP-16 (1 mmol/mg protein) (6), Ca 2 + (140 nmol/mg protein) (5) or VP-16 plus Ca 2 + (1). Asc (1 mM) (4), GSH (1 mM) (2) and NEM (40 mM) (3) were pre-incubated with mitochondria before addition of VP-16 and their protective effects were compared to that afforded by CyA (1 mM) (7).…”

“…Recently, it has been reported that its cytotoxicity is mediated by triggering mitochondrial events that seem to be obligatory steps of early (pre-nuclear) apoptosis. Such events include the release of cytochrome c from mitochondria and disruption of outer membrane due to mitochondrial permeability transition (MPT) induction [4,5], which precedes the activation of caspase 9 and 3 [6,7], the reduction of mitochondrial membrane potential (Dc) and the formation of ultracondensed mitochondria [8]. The destructive effects on the outer membrane integrity, with subsequent release of the intermembrane apoptogenic factors from mitochondria, could also suggest the involvement of VP-16-induced MPT on the activation of apoptotic cascade [9,10].…”