Anti-steroidogenic effects of cholesterol hydroperoxide trafficking in MA-10 Leydig cells: Role of mitochondrial lipid peroxidation and inhibition thereof by selenoperoxidase GPx4.

Steroid hormone synthesis in steroidogenic cells requires cholesterol (Ch) delivery to/into mitochondria via StAR family trafficking proteins. In previous work, we discovered that 7-OOH, an oxidative stress-induced cholesterol hydroperoxide, can be co-trafficked with Ch, thereby causing mitochondrial redox damage/dysfunction. We now report that exposing MA-10 Leydig cells to Ch/7-OOH-containing liposomes (SUVs) results in (i) a progressive increase in fluorescence probe-detected lipid peroxidation in mitochondrial membranes, (ii) a reciprocal decrease in immunoassay-detected progesterone generation, and ultimately (iii) loss of cell viability with increasing 7-OOH concentration. No significant effects were observed with a phospholipid hydroperoxide over the same concentration range. Glutathione peroxidase GPx4, which can catalyze lipid hydroperoxide detoxification, was detected in mitochondria of MA-10 cells. Mitochondrial lipid peroxidation and progesterone shortfall were exacerbated when MA-10 cells were treated with Ch/7-OOH in the presence of RSL3, a GPx4 inhibitor. However, Ebselen, a selenoperoxidase mimetic, substantially reduced RSL3's negative effects, thereby partially rescuing the cells from peroxidative damage. These findings demonstrate that co-trafficking of oxidative stress-induced 7-OOH can disable steroidogenesis, and that GPx4 can significantly protect against this.

The Ability of Functionalized Fullerenes and Surface-Modified TiO2 Nanoparticles to Photosensitize Peroxidation of Lipids in Selected Model Systems.

Photochemical properties of a new class of inorganic nanoparticles, namely a cationic C60 fullerene substituted with three quaternary pyrrolidinium groups (BB6) and a surface-modified nanocrystalline TiO2 with bromopyrogallol red (Brp@TiO2 ) were examined for their effectiveness in photogenerating singlet oxygen and free radicals. In particular, their ability to photosensitize peroxidation of unsaturated lipids was analyzed in POPC:cholesterol liposomes and B16 mouse melanoma cells employing a range of spectroscopic and analytical methods. Because melanoma cells typically are pigmented, we examined the effect of melanin on the photosensitized peroxidation of lipids in liposomes and B16 melanoma cells, mediated by BB6 and Brp@TiO2 nanoparticles. The obtained results suggest that peroxidation of unsaturated lipids, photosensitized by BB6 occurs mainly, although not exclusively, via Type II mechanism involving singlet oxygen. On the other hand, if surface-modified TiO2 is used as a photosensitizer, Type I mechanism of lipid peroxidation dominates, as indicated by the predominant formation of the free radical-dependent cholesterol oxidation products. The protective effect of melanin was particularly evident when BB6 was used as a photosensitizer, suggesting that melanin could efficiently interfere with Type II processes.

GD2 ganglioside-binding antibody 14G2a and specific aurora A kinase inhibitor MK-5108 induce autophagy in IMR-32 neuroblastoma cells.

The process of autophagy and its role in survival of human neuroblastoma cell cultures was studied upon addition of an anti-GD2 ganglioside (GD2) 14G2a mouse monoclonal antibody (14G2a mAb) and an aurora A kinase specific inhibitor, MK-5108. It was recently shown that combination of these agents significantly potentiates cytotoxicity against IMR-32 and CHP-134 neuroblastoma cells in vitro, as compared to the inhibitor used alone. In this study we gained mechanistic insights on autophagy in the observed cytotoxic effects exerted by both agents using cytotoxicity assays, RT-qPCR, immunoblotting, and autophagy detection methods. Enhancement of the autophagy process in the 14G2a mAb- and MK-5108-treated IMR-32 cells was documented by assessing autophagic flux. Application of a lysosomotropic agent-chloroquine (CQ) affected the 14G2a mAb- and MK-5108-stimulated autophagic flux. It is our conclusion that the 14G2a mAb (40 µg/ml) and MK-5108 inhibitor (0.1 µM) induce autophagy in IMR-32 cells. Moreover, the combinatorial treatment of IMR-32 cells with the 14G2a mAb and CQ significantly potentiates cytotoxic effect, as compared to CQ used alone. Most importantly, we showed that interfering with autophagy at its early and late step augments the 14G2a mAb-induced apoptosis, therefore we can conclude that inhibition of autophagy is the primary mechanism of the CQ-mediated sensitization to the 14G2a mAb-induced apoptosis. Although, there was no virtual stimulation of autophagy in the 14G2a mAb-treated CHP-134 neuroblastoma cells, we were able to show that PHLDA1 protein positively regulates autophagy and this process exists in a mutually exclusive manner with apoptosis in PHLDA1-silenced CHP-134 cells.

Impairment of Macrophage Cholesterol Efflux by Cholesterol Hydroperoxide Trafficking: Implications for Atherogenesis Under Oxidative Stress.

OBJECTIVE: Oxidative stress associated with cardiovascular disease can produce various oxidized lipids, including cholesterol oxides, such as 7-hydroperoxide (7-OOH), 7-hydroxide (7-OH), and 7-ketone (7=O). Unlike 7=O and 7-OH, 7-OOH is redox active, giving rise to the others via potentially toxic-free radical reactions. We tested the novel hypothesis that under oxidative stress conditions, steroidogenic acute regulatory (StAR) family proteins not only deliver cholesterol to/into mitochondria of vascular macrophages, but also 7-OOH, which induces peroxidative damage that impairs early stage reverse cholesterol transport. APPROACH AND RESULTS: Stimulation of human monocyte-derived THP-1 macrophages with dibutyryl-cAMP resulted in substantial upregulation of StarD1 and ATP-binding cassette (ABC) transporter, ABCA1. Small interfering RNA-induced StarD1 knockdown before stimulation had no effect on StarD4, but reduced ABCA1 upregulation, linking the latter to StarD1 functionality. Mitochondria in stimulated StarD1-knockdown cells internalized 7-OOH slower than nonstimulated controls and underwent less 7-OOH-induced lipid peroxidation and membrane depolarization, as probed with C11-BODIPY (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-inda-cene-3-undecanoic acid) and JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide), respectively. Major functional consequences of 7-OOH exposure were (1) loss of mitochondrial CYP27A1 activity, (2) reduced 27-hydroxycholesterol (27-OH) output, and (3) downregulation of cholesterol-exporting ABCA1 and ABCG1. Consistently, 7-OOH-challenged macrophages exported less cholesterol to apoA-I or high-density lipoprotein than did nonchallenged controls. StarD1-mediated 7-OOH transport was also found to be highly cytotoxic, whereas 7=O and 7-OH were minimally toxic. CONCLUSIONS: This study describes a previously unrecognized mechanism by which macrophage cholesterol efflux can be incapacitated under oxidative stress-linked disorders, such as chronic obesity and hypertension. Our findings provide new insights into the role of macrophage redox damage/dysfunction in atherogenesis.

Macrophage mitochondrial damage from StAR transport of 7-hydroperoxycholesterol: implications for oxidative stress-impaired reverse cholesterol transport.

StAR family proteins in vascular macrophages participate in reverse cholesterol transport (RCT). We hypothesize that under pathophysiological oxidative stress, StARs will transport not only cholesterol to macrophage mitochondria, but also pro-oxidant cholesterol hydroperoxides (7-OOHs), thereby impairing early-stage RCT. Upon stimulation with dibutyryl-cAMP, RAW264.7 macrophages exhibited a strong time-dependent induction of mitochondrial StarD1 and plasma membrane ABCA1, which exports cholesterol. 7α-OOH uptake by stimulated RAW cell mitochondria (like cholesterol uptake) was strongly reduced by StarD1 knockdown, consistent with StarD1 involvement. Upon uptake by mitochondria, 7α-OOH (but not redox-inactive 7α-OH) triggered lipid peroxidation and membrane depolarization while reducing ABCA1 upregulation. These findings provide strong initial support for our hypothesis.