CoCl(2) induces apoptosis via the 18 kDa translocator protein in U118MG human glioblastoma cells.
The 18 kDa translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, has been reported to be closely associated with the mitochondrial permeability transition pore (MPTP). TSPO is believed to exert pro-apoptotic functions via modulation of MPTP opening. Cobalt chloride (CoCl(2)), which is sometimes used as a hypoxia mimicking agent, is also known to be able to induce apoptosis. One of our questions was whether CoCl(2) may induce apoptosis via the TSPO. To address this question, we used the U118MG human glioblastoma cell line. We applied the specific TSPO ligand, PK 11195, as well as TSPO knockdown with siRNA and studied their influence on the effects of CoCl(2) on cell death, including activation of the mitochondrial apoptosis pathway. To assay TSPO expression, we applied binding assays and Western blotting to whole cell homogenates and mitochondrial fractions. To assay activation of the mitochondrial apoptosis pathway, including some of the cellular mechanisms involved, we determined the incidence of collapse of the mitochondrial membrane potential (Deltapsi(m)) and cardiolipin oxidation and measured the level of DNA fragmentation to assay apoptotic rates. We found that the TSPO ligand, PK 11195, significantly counteracted induction of cell death by 0.4 mM CoCl(2), including apoptosis, collapse of the Deltapsi(m), and cardiolipin oxidation. Moreover, we found that TSPO knockdown with siRNA fully protected against mentioned cell death mechanisms. Thus, we found that the TSPO is required for cell death induction by CoCl(2), including apoptosis. In conclusion, our studies show that activation of TSPO by CoCl(2) application is required for ROS generation, leading to cardiolipin oxidation, and collapse of the Deltapsi(m), as induced by CoCl(2).
Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Department of Molecular Pharmacology, Ephron Street, P.O. Box 9649, Bat-Galim, Haifa 31096, Israel.
Sivan
S
Zeno
Menashe
M
Zaaroor
Svetlana
S
Leschiner
Leo
L
Veenman
Moshe
M
Gavish
D017209:Apoptosis / Q000187:drug effects*; D002308:Cardiolipins / Q000378:metabolism; D045744:Cell Line, Tumor; D003035:Cobalt / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D055785:Gene Knockdown Techniques; D005909:Glioblastoma / Q000473:pathology*; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D008024:Ligands; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism; D000083162:Mitochondrial Permeability Transition Pore; D010084:Oxidation-Reduction; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D018079:Receptors, GABA / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
D002308:Cardiolipins; D007546:Isoquinolines; D008024:Ligands; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D018079:Receptors, GABA; C499229:TSPO protein, human; D003035:Cobalt; C018021:cobaltous chloride; C037850:PK 11195