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

Abstract

Etoposide (VP-16) is known to promote cell apoptosis either in cancer or in normal cells as a side effect. This fact is preceded by the induction of several mitochondrial events, including increase in Bax/Bcl-2 ratio followed by cytochrome c release and consequent activation of caspase-9 and -3, reduction of ATP levels, depolarization of membrane potential (Dc) and rupture of the outer membrane. These events are apoptotic factors essentially associated with the induction of the mitochondrial permeability transition (MPT). VP-16 has been shown to stimulate the Ca 2 + -dependent MPT induction similarly to prooxidants and to promote apoptosis by oxidative stress mechanisms, which is prevented by glutathione (GSH) and N-acetylcysteine (NAC). Therefore, the aim of this work was to study the effects of antioxidants and thiol protecting agents on MPT promoted by VP-16, (2002) 169-184 170 attempting to identify the underlying mechanisms on VP-16-induced apoptosis. The increased sensitivity of isolated mitochondria to Ca 2 + -induced swelling, Ca 2 + release, depolarization of Dc and uncoupling of respiration promoted by VP-16, which are prevented by cyclosporine A proving that VP-16 induces the MPT, are also efficiently prevented by ascorbate, the primary reductant of the phenoxyl radicals produced by VP-16. The thiol reagents GSH, dithiothreitol and N-ethylmaleimide, which have been reported to prevent the MPT induction, also protect this event promoted by VP-16. The inhibition of the VP-16-induced MPT by antioxidants agrees with the prevention of etoposide-induced apoptosis by GSH and NAC and suggests the generation of oxidant species as a potential mechanism underlying the MPT that may trigger the release of mitochondrial apoptogenic factors responsible for apoptotic cascade activation.

The effect of intracellular ascorbate on the susceptibility of HL60 and Jurkat cells to chemotherapy agents

“…Several studies have also suggested that this drug may have redox properties [36,37]. Etoposide contains a phenolic ring that can participate in radical reactions, especially in the presence of myeloperoxidase, an enzyme that is abundant in myeloid cells.…”

Antitumor triptycene analogs induce a rapid collapse of mitochondrial transmembrane potential in HL-60 cells and isolated mitochondria

“…The reports that mitochondria isolated from cells or tissue previously treated for 12-48 h with high μM concentrations of DOX or other DNA-damaging drugs are severely impaired are totally different and suggest that, in contrast to TT analogs, indirect and delayed damage to mitochondria in situ is the consequence rather than the cause of DOXinduced cytotoxicity (9,26,27). Similarly, huge 500-μM concentrations of VP-16 must be used to detect rapid MPT events in isolated mitochondria, whereas lower concentrations of VP-16 require 24-48 h to promote cellular apoptosis (10). The TT2-induced ↓Δ"m in isolated mitochondria is blocked or inhibited by CsA, BA and d-Ub, suggesting that antitumor TT bisquinones directly interact with components of the PTP complex to trigger this marker of MPT.…”

“…Disrupted cells were centrifuged (1,000 g x 10 min) in a 50-ml conical polypropylene tube to precipitate unlysed cells, nuclei and large membrane fragments. The supernatant was decanted and then recentrifuged (10,000 g x 10 min) in 15-ml Corex borosilicate glass tubes to collect the mitochondrial pellets, which were washed, pooled and resuspended at a final concentration of 50 mg protein/ml of mitochondrial storage buffer, containing 10 mM HEPES, pH 7.2, 225 mM mannitol and 75 mM sucrose (9,10). The protein concentrations of the mitochondrial samples were determined using the BCA Protein Assay Kit (Pierce, Rockford, IL).…”

The antiestrogen 4-hydroxytamoxifen protects against isotretinoin-induced permeability transition and bioenergetic dysfunction of liver mitochondria: comparison with tamoxifen

“…Δ= was estimated from the decrease of TPP + concentration in the reaction medium. The Δ= associated to MPT induction was determined with mitochondria (0.5 mg/ml) incubated in 2 ml of MPT reaction medium (200 mM sucrose, 10 mM Tris-Mops, 1 mM KH 2 PO 4 and 10 μM EGTA, pH 7.4) supplemented with 4 μM TPP + and 2 μM rotenone in the absence and in the presence of 1 μM cyclosporine A (CyA), thiol protecting and antioxidant agents [1 mM dithiothreitol (DTT), 50 μM (Custodio et al 2002). CyA, TAM and OHTAM were also added during the Δ= depolarization as indicated in the figure legends.…”