Abstract
Bisindolylpyrrole at 0.1 μM is cytoprotective in 2% FBS that is counteracted by cyclosporin-A (CsA), an inhibitor of cyclophilin-D (CypD). We hypothesized that the cytoprotective effect might be due to transient mitochondrial permeability transition (tPT). This study tested the hypothesis that bisindolylpyrrole can trigger tPT extensively, thereby leading to cell death under certain conditions. Indeed, CsA-sensitive tPT-mediated apoptosis could be induced by bisindolylpyrrole at > 5 μM in HeLa cells cultured in 0.1% FBS, depending on CypD and VDAC1/2, as shown by siRNA knockdown experiments. Rat liver mitochondria also underwent swelling in response to bisindolylpyrrole, which proceeded at a slower rate than Ca2+-induced swelling, and which was blocked by the VDAC inhibitor tubulin and the ANT inhibitor bongkrekate, indicating the involvement of the ANT-associated, smaller pore. We examined why 0.1% FBS is a prerequisite for apoptosis and found that apoptosis is blocked by PKC activation, which is counteracted by the overexpressed defective PKCε. In mitochondrial suspensions, bisindolylpyrrole triggered CsA-sensitive swelling, which was suppressed selectively by pretreatment with PKCε, but not in the co-presence of tubulin. These data suggest that upon PKC inactivation the cytoprotective compound bisindolylpyrrole can induce prolonged tPT causing apoptosis in a CypD-dependent manner through the VDAC1/2-regulated ANT-associated pore.
Highlights
The mitochondrial permeability transition (PT) occurs in response to high matrix Ca2+ and reactive oxygen species
The present study showed that BP can induce cytochrome c mediated apoptosis at concentrations of 5−10 μM in low FBS conditions (< 0.1%) and that the apoptosis is regulated by CypD, and mediated by the prolonged induction of transient mitochondrial permeability transition (tPT) (Fig. 1)
The tPT may be mediated by the voltage-dependent anion channel (VDAC) (Fig. 3) which is linked to the adenine nucleotide translocase (ANT) (Fig. 4B) regulated by CypD activity (Fig. 1B)
Summary
The mitochondrial permeability transition (PT) occurs in response to high matrix Ca2+ and reactive oxygen species. The PT occurs C a2+-independently and CsA/CypD-insensitively This phenomenon was hypothesized to take place when misfolded mitochondrial membrane proteins, responsible for pore formation, exceed CypD16. A large body of evidence shows that VDAC proteins have a pro-apoptotic activity or pro-survival activity, depending on their binding partners, which influence the VDAC c onductance[27]. Quantification of calcein and TMRM signals over 3 h (initial fluorescence intensities were normalized for comparative purposes; *p < 0.005 compared with BP treatment at 3 h; + p < 0.001 compared with control at the same time point by Welch’s t-test with Bonferroni’s correction; n = 100 cells) (ii). One of the PKCε targets may be VDAC1, since this isoform exists as a complex with VDAC1 and can prevent C a2+-induced mitochondrial swelling through VDAC1 phosphorylation[33]
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