CoCl₂Induces Apoptosis via the 18 kDa Translocator Protein in U118MG Human Glioblastoma Cells

Abstract

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).

TSPO interacts with VDAC1 and triggers a ROS-mediated inhibition of mitochondrial quality control

“…20, 21 We here demonstrate that when the ratio of TSPO to VDAC1 increases, mitochondrial production of ATP is limited and ROS levels are increased (Fig. 6), evidence that is consistent with previous publications.…”

“…6), evidence that is consistent with previous publications. 10,20,21 By influencing the cellular redox system, TSPO blocks the ubiquitination of mitochondrial proteins, essential for effective recruitment of the autophagosomal machinery via SQSTM1. 52 Cells largely devoid of TSPO show indeed an upregulation of mitochondrial ubiquitination with the same being observed in PRKCE ¡/¡ MEFs (Fig.…”

“…10 A role for TSPO in the execution of apoptosis has long been proposed and its natural and synthetic ligands i) endozepine, 11,12 ii) PK11195 [13][14][15][16] and iii) Ro-54864 17 are widely exploited as chemotherapy co-adjuvants. Other functions linked to TSPO include: i) the regulation of mitochondrial ATP production, 18,19 ii) the generation of reactive oxygen species, 20,21 and iii) the modulation of Ca 2C signaling. 22,23 Notably, its precise role in cell physiology has received relatively little attention, leaving its contribution to mitochondrial function largely illdefined.…”

Effect of Sodium Thiosulfate on Isolated Cardiac Interfibrillar and Subsarcolemmal Mitochondria

“…Recently, its cardioprotective effect was documented through its ability to release hydrogen sulfide in the mitochondria [12] . CoCl 2 is effective in inducing ROS via mitochondria [32] and our results with STS on this model demonstrate that the drug has the potential to scavenge ROS effectively, when it was given along with cobalt chloride. Interestingly, the absence of difference between the subpopulation in the ROS scavenging ability emphasizes the relationship between the functional difference of IFM and SSM with respect to their location.…”

The 18 kDa Translocator Protein and Atherosclerosis in Mice Lacking Apolipoprotein E

“…Notably, wild type rats are not prone to develop atherosclerosis even when subjected to HFHC diet (Dimitrova-Shumkovska et al, 2010a). We have shown that reduction of TSPO expression by genetic manipulation in vitro in cell culture reduces mitochondrial ROS generation (Veenman et al, 2008Zeno et al, 2009). We have discussed previously that the reduced TSPO levels accompanying atherogenic challenges may be a compensatory mechanism to counteract oxidative stress in the aorta and liver (Dimitrova-Shumkovska et al, 2010 a, b, c).…”