ABT-737 induces relatively large MOMP in cancer cell mitochondria.

<p>Isolated mitochondria from mouse liver, PC-3 and Jurkat cells were untreated (NT) or incubated either with alamethicin (Ala; 20 µg/ml; positive control), Bak BH3 peptide (10 µM), ABT-737 (1 µM) or recombinant t-Bid (1 nM) for 45 min. Mitochondrial supernatants were subjected to immunodetection of cytochrome c, Smac/DIABLO, Omi/Htra2 and AIF (Western blots are representative of 3 independent experiments). Note that cytochrome c (15 kDa), Smac/DIABLO (23 kDa), and Omi/Htra2 (37 kDa) but not AIF (56 kDa) are released from cancer cell mitochondria.</p

Multiparametric screen of known mitochondria-targeting molecules.

<p><b>A.</b> Mitochondria isolated from mouse liver, human non-cancerous (HME-1) and cancerous (PC-3) cells were treated with increasing concentrations of t-Bid, Bak BH3, HA-14.1, YC-137, Chelerythrine, Gossypol, TW-37, EM20-25 and ABT-737 before evaluation of mitochondrial swelling and ΔΨ_m loss. Alternatively, mitochondrial supernatant was subjected to ELISA assays for quantification of cytochrome c release. Effective concentration inducing 50% of the maximal effect (EC₅₀) is given for swelling (100% of effect with 50 µM Ca²⁺), ΔΨ_m loss (100% of effect with 50 µM <i>m</i>ClCCP) and cytochrome c release (100% of effect with 20 µg/ml alamethicin) (n = 3 independent experiments). <b>B.</b> Mitochondria isolated from mouse liver or HME-1, PC-3, HCT-116, HT-29 and Jurkat cell lines were incubated for 45 min at 30°C with increasing concentrations of ABT-737 and the supernatants were subjected to cytochrome c immunoblot (NT: untreated; Ala: alamethicin 20 µg/ml).</p

ABT-737 induces Bax and Bak liberation from Bcl-2 and Bcl-xL.

<p>Mitochondria isolated from PC-3 (<b>A</b>), HT-29 (<b>B</b>) and Jurkat (<b>C</b>) cells were untreated (NT) or treated with t-Bid (Bid; 2 nM) or ABT-737 (ABT; 1 µM) before to be immunoprecipitated by the antibodies directed against the Bcl-2, Bcl-xL and Mcl-1 anti-apoptotic proteins. Mitochondrial total extracts (TE; positive control of immunoblot; 25 µg) were used as control while a mitochondrial lysate was subjected to immunoprecipitation process without antibody (C; negative control of immunoprecipitation). Thus Western blot analysis was performed to determine bindings between anti-apoptotic proteins and pro-apoptotic Bax and Bak proteins (representative Western Blots of 3 independent experiments).</p

Isolation and functional characterization of mouse liver and human tumor cell line mitochondria.

<p><b>A.</b> Ultrastructural analysis of isolated mitochondria and their ability to swell. Electron micrographs were obtained after incubation of mitochondria isolated from healthy mouse liver, or PC-3 tumor cell lines untreated (NT) or treated with Ca²⁺ (50 µM) or with a 5 min-preincubation with cyclosporin A (CsA; 10 µM) or ruthenium red (RR; 1 µM) before calcium addition. The percentage of swollen mitochondria was <10% in the control and >80% 30 min after Ca²⁺ addition (n = 3). Scale bars 1 µm. <b>B.</b> Oxidative properties of isolated liver and PC-3 mitochondria. Traces represent oxygen consumption by isolated mitochondria (100 µg) after addition of the indicated reagents. Numbers along the trace are nmoles of O₂ consumed per minute per milligram of protein. The respiratory control index (RCI) is calculated for each type of mitochondria as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009924#s4" target="_blank">Materials and Methods</a>. <b>C.</b> To evaluate mitochondrial swelling and ΔΨ_m loss, mitochondria isolated from healthy mice liver or PC-3 cell line were distributed in 96-well microplates and incubated for 30 min either with Ca²⁺ (100 µM) in presence (yellow) or absence (pink) of CsA (10 µM), with <i>m</i>ClCCP (turquoise; 50 µM) or with t-Bid (purple; 1 nM). For quantitation of cytochrome c release, isolated mitochondria were treated with increasing concentrations of the t-Bid recombinant protein and mitochondrial supernatant was subjected to ELISA assays, given in percentage of release compared to 20 µg/ml alamethicin (Ala; 100% of cytochrome c release) (n = 3 independent experiments).</p

ABT-737 induces a Bax/Bak-dependent cytochrome c release.

<p><b>A.</b> Total cell extracts from HCT-116 Bax +/-, Bax -/-, Bak -/- and Bax/Bak -/- (DKO) cell lines were subjected to Bax and Bak immunoblot to control their Bax and Bak content. <b>B.</b> Mitochondria isolated from HCT-116 Bax +/-, Bax -/-, Bak -/- and Bax/Bak -/- (DKO) cell lines were incubated with increasing concentrations of ABT-737 and the supernatant was subjected to immunoblot detection of cytochrome c (NT: untreated; Ala: alamethicin 20 µg/ml). <b>C.</b> Cytochrome c release induced by t-Bid and ABT-737 is inhibited by an excess of recombinant Bcl-xL. PC-3 mitochondria were incubated with ABT-737 (1 µM) or t-Bid (1 nM) for 45 min after a 5 min-pretreatment with recombinant Bcl-xL (100 to 400 nM) and the supernatant was subjected to anti-cytochrome c immunoblot (NT: untreated; Ala: alamethicin 20 µg/ml). Note that Bcl-xL strongly reduces both t-Bid and ABT-737-induced cytochrome c release (n = 2 independent experiments).</p

Pro- and anti-apoptotic protein pattern of isolated mitochondria.

<p>Total cell extracts (TE) and mitochondrial extracts (M) from PC-3, HT-29, Jurkat and HCT-116 cancer cell lines or from healthy HME-1 cell line and mouse liver were analyzed by Western blot for detection of the anti-apoptotic (<b>A</b>) Bcl-2, Bcl-xL, Bcl-w, Mcl-1L and A1 proteins and the pro-apoptotic (<b>B</b>) Bak, Bax, Bim, Bad and Mcl-1S proteins.</p