Inhibition of apoptosis by ascorbic and dehydroascorbic acids in Xenopus egg extracts

Abstract

Purpose The viability of mammalian eggs after ovulation is reported to be improved by the presence of ascorbic acid in the culture medium. However, the pro-survival mechanisms of ascorbic acid are poorly understood. The molecular pathways of apoptosis are evolutionarily conserved among animal species, and Xenopus eggs are technically and ethically more suitable for biochemical analyses than mammalian eggs. We used Xenopus egg cytoplasmic extracts to examine the direct intracellular effects of ascorbic acid. Methods Incubation of egg extracts for more than 4 h induces the spontaneous release of cytochrome c from mitochondria. This event triggers the activation of caspases, cleavage of substrate proteins, and execution of apoptosis. Multiple signal transduction pathways including proteolysis and protein phosphorylation are also involved in this process. We examined whether any of these events might be inhibited by the addition of ascorbic acid. Results Ascorbic acid showed no effect against cytochrome c release, but prevented caspase activation and substrate cleavage. Ascorbic acid also blocked the proteolysis of apoptosis inhibitor proteins and the dephosphorylation of p42 MAP kinase. However, dehydroascorbic acid (oxidized form of ascorbic acid) and acetate (unrelated acid) were equally effective, indicating that these effects were primarily due to their acidity. In addition, dehydroascorbic acid inhibited caspase activities directly in vitro. Conclusions The anti-apoptotic effect of ascorbic acid in Xenopus egg extracts is mainly due to cytoplasmic acidification rather than its intracellular antioxidant activity. Instead, oxidative conversion of ascorbic acid into dehydroascorbic acid may inhibit apoptosis through the inhibition of caspases.

Anti-apoptotic Activity and Proteasome-mediated Degradation of Xenopus Mcl-1 Protein in Egg Extracts

“…Cytostatic factor (CSF) 2 -arrested metaphase egg extracts were prepared as described previously (17,33,36,39). Interphase egg extracts were prepared by the addition of 0.4 mM CaCl 2 and 0.1 mg/ml cycloheximide.…”

“…35 S-Radiolabeled recombinant N-terminally His 6 -tagged proteins were produced using TNT T7 Quick rabbit reticulocyte lysates (RRL; Promega). RRL were mixed with interphase extracts containing cycloheximide at 1:9 as described previously (17,33,36,39).…”

“…Polyclonal antibody against actin was purchased from Sigma. Western blot was carried out as described previously (17,33,36,39).…”

“…20 and 21). Egg extracts, as well as the mitochondria isolated from them, are frequently used for biochemical experiments of apoptosis (17,(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). Apoptosis in egg extracts is triggered solely by the cytoplasmic release of cytochrome c from mitochondria, and neither damage-inducing treatment nor death receptor-ligand signaling is required.…”

“…For this reason, endogenous Bcl-2 family proteins present in Xenopus egg extracts or mitochondria are still poorly characterized. We have been studying the functions and contributions of apoptosis regulators present in Xenopus egg extracts (17,33,36,39). We recently reported the pro-apoptotic activity of endogenous Xenopus Bid (xBid), a BH3-only member of the Bcl-2 family of proteins, in egg extracts (17).…”