title: Use of human cancer cell lines mitochondria to explore the mechanisms of BH3 peptides and ABT-737-induced mitochondrial membrane permeabilization.
abstract: Current limitations of chemotherapy include toxicity on healthy tissues and multidrug resistance of malignant cells. A number of recent anti-cancer strategies aim at targeting the mitochondrial apoptotic machinery to induce tumor cell death. In this study, we set up protocols to purify functional mitochondria from various human cell lines to analyze the effect of peptidic and xenobiotic compounds described to harbour either Bcl-2 inhibition properties or toxic effects related to mitochondria. Mitochondrial inner and outer membrane permeabilization were systematically investigated in cancer cell mitochondria versus non-cancerous mitochondria. The truncated (t-) Bid protein, synthetic BH3 peptides from Bim and Bak, and the small molecule ABT-737 induced a tumor-specific and OMP-restricted mitochondrio-toxicity, while compounds like HA-14.1, YC-137, Chelerythrine, Gossypol, TW-37 or EM20-25 did not. We found that ABT-737 can induce the Bax-dependent release of apoptotic proteins (cytochrome c, Smac/Diablo and Omi/HtrA2 but not AIF) from various but not all cancer cell mitochondria. Furthermore, ABT-737 addition to isolated cancer cell mitochondria induced oligomerization of Bax and/or Bak monomers already inserted in the mitochondrial membrane. Finally immunoprecipatations indicated that ABT-737 induces Bax, Bak and Bim desequestration from Bcl-2 and Bcl-xL but not from Mcl-1L. This study investigates for the first time the mechanism of action of ABT-737 as a single agent on isolated cancer cell mitochondria. Hence, this method based on MOMP (mitochondrial outer membrane permeabilization) is an interesting screening tool, tailored for identifying Bcl-2 antagonists with selective toxicity profile against cancer cell mitochondria but devoid of toxicity against healthy mitochondria.
authors.affiliation: THERAPTOSIS SA, Oncology Department, Biocitech Technology Park, Romainville, France.
authors.forename: Nelly
authors.initials: N
authors.lastname: Buron
authors.forename: Mathieu
authors.initials: M
authors.lastname: Porceddu
authors.forename: Magali
authors.initials: M
authors.lastname: Brabant
authors.forename: Diana
authors.initials: D
authors.lastname: Desgué
authors.forename: Cindy
authors.initials: C
authors.lastname: Racoeur
authors.forename: Myriam
authors.initials: M
authors.lastname: Lassalle
authors.forename: Christine
authors.initials: C
authors.lastname: Péchoux
authors.forename: Pierre
authors.initials: P
authors.lastname: Rustin
authors.forename: Etienne
authors.initials: E
authors.lastname: Jacotot
authors.forename: Annie
authors.initials: A
authors.lastname: Borgne-Sanchez
mesh_terms: D000818:Animals; D001713:Biphenyl Compounds / Q000494:pharmacology*; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000494:pharmacology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008564:Membrane Potentials; D051379:Mice; D008807:Mice, Inbred BALB C; D008928:Mitochondria / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000187:drug effects*; D009596:Nitrophenols / Q000494:pharmacology*; D010446:Peptide Fragments / Q000737:chemistry*; D010539:Permeability; D010879:Piperazines / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology*
chemical_list: C501332:ABT-737; C474007:Bax protein (53-86); D001713:Biphenyl Compounds; D003432:Cross-Linking Reagents; D009596:Nitrophenols; D010446:Peptide Fragments; D010879:Piperazines; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D013449:Sulfonamides