The existence of clusters of proteins and lipids and especially, the transient nanometric cholesterol-and sphingolipid-enriched domains, called rafts,are described as signaling platforms for a wide range of cellular responses to stimuli including reactive oxygen species (ROS) generation, inflammatory cytokines expression and cell death. we explored the role of cholesterol and cholesterol-enriched domains for cellular toxicity ofthe potential anticancer drug, the ginsenoside Rh2 and the anti-inflammatory complex budesonide: HPβCD.
Taking benefit from our previous studies investigating the mechanisms involved in nephrotoxicity induced by aminoglycoside antibiotics, we explored thecapacity of new antibiotics to accumulate within the cells and to induce accumulation of undigested lipids within the lysosomes. More recently, we started to explore the mitochondrial alterations induced by oxazolidinone antibiotics.
Pursuing our studies on the molecular mechanism involved in necrosis and apoptosis in leukemic monocytes induced by saponins (α-hederin, a monodesmosidic triterpenoid) and especially the critical role of cholesterol and cholesterol-enriched domains, we extend to ginsenoside Rh2, a steroid saponin (protopanaxatriol) known as one of the active principles of Panax ginsengroot. This work is performed in close collaboration with J. Leclercq’s team.
We demonstrated that membrane cholesterol could delay the activity of ginsenoside Rh2, renewing the idea that saponin cytotoxicity is ascribed to an interaction with membrane cholesterol.
The cytotoxic activity of Rh2 is accelerated in human leukemic U937 cell lines upon cholesterol depletion via the pretreatment with methyl-β-cyclodextrin, a cholesterol-sequestering agent. Mechanistically, Rh2 alters plasma membrane fluidity by compacting the hydrophobic core of lipid bilayer (DPH anisotropy) and relaxing the interfacial packaging of the polar head of phospholipids (TMA-DPH anisotropy). The treatment with Rh2 consequently conducts to the dephosphorylation of Akt and the activation of the intrinsic pathway of apoptosis (loss of mitochondrial membrane potential, caspase-9 and -3 activation).
Rh2 decreases the phosphorylation of Akt faster upon cholesterol depletion. Cells depleted in cholesterol with 5 mM MβCD for 2 h, or not, were incubated for the indicated times with 60 μM Rh2 or with 0.1% DMSO (vehicle). Equal amounts of cell extracts were subjected to western-blot analysis for pAkt, Akt and β-actin protein (Verstraeten et al, 2018, Toxicol. Appl. Pharmacol.)
All these features are induced faster in cholesterol-depleted cells, which could be explained by faster cell accumulation of Rh2 in these conditions.
Budesonide (BUD), a poorly soluble anti-inflammatory drug, is used to treat patients suffering from asthma and COPD (Chronic Obstructive Pulmonary Disease). Hydroxypropyl-β-cyclodextrin (HPβCD), a biocompatible cyclodextrin known to interact with cholesterol, is used as a drug-solubilizing agent in pharmaceutical formulations. Budesonide administered as an inclusion complex within HPβCD (BUD:HPβCD) required a quarter of the nominal dose of the suspension formulation and significantly reduced neutrophil-induced inflammation in a COPD mouse model exceeding the effect of each molecule administered individually. This suggests the role of lipid domains enriched in cholesterol for inflammatory signaling activation.
We first showed that BUD:HPβCD induced an increase in membrane fluidity and permeability induced by BUD:HPβCD in vesicles containing cholesterol. We also demonstrated on giant unilamellar vesicles (GUVs) and lipid monolayers, the disruption of cholesterol-enriched raft-like liquid ordered domains aswell as changes in lipid packing and lipid desorption from the cholesterol monolayers, respectively. Except for membrane fluidity, all these effects were enhanced when HPβCD was complexed with budesonide as compared with HPβCD.
Confocal fluorescence microscopy imaging of membrane phase separation in GUVs upon incubation with BUD:HPβCD, and HPβCD. Imaging of membrane domains in GUVs composed of (left) DOPC:pSM (1:1) and (right) DOPC:pSM:Chol (1:1:3) before (top, control) and after (descending) 5, min with the BUD:HPβCD complex or HPβCD. DOPC:pSM vesicles were labeled with Rho-DOPE (red channel) to visualize the liquid disordered (ld)/solid ordered (so) phase separation in red/dark. DOPC:pSM:Chol were labelled with Rho-DOPE (red channel) and NBD-PE (greenchannel) to visualize the liquid disordered (ld)/liquid ordered (lo) phase separation in red/green channels, respectively. Dos Santos et al, 2017, Biochim. Biophys. Acta. Biomembranes
Since changes in biophysical membrane properties have been linked to membrane signaling including pathways involved in inflammation processes, we moved on cellular models (A549) and demonstrated that BUD:HPβCD could limit (i) hydrogen peroxide-and lipopolysaccharide-induced ROS generation, (ii) alveolar cell death mainly due to HPβCD, and (iii) CXCL8/interleukine-8 expression mainly due to BUD. Our results suggest that BUD:HPβCD would potentially be more beneficial than BUD to deal with COPD-related inflammation.
Oxazolidinones extert their antibacterial effect by inhibitingprotein synthesis in bacteria. We evidenced a specific inhibition of the synthesis of protein encoded by the mitochondrial genome accompanied by an inhibition of the respiratory function and morphological alterations (swelling of mitochondria and disappearance of cristae). We are now exploring whether these changes may contribute to explain the thrombocytopenia and anemia reported in patients treated by these drugs.Our current data suggest that oxazolidinones prevent the maturation of platelet precursors.
Left: Influence of increasing concentrations of linezolid (LZD) and tedizolid (TZD) on CYTox I expression in HL-60 promyelocytes incubated for 120 h in the presence of increasing concentrations of these drugs.Western blots of CYTox I (protein encoded by the mitochondrial genome) and of Tom 20 (encoded by the nuclear genome) of mitochondrial protein fractions. Right: electron microscopy images of mitochondria from HL-60 cells exposed to 15 mg/L linezolid or 3 mg/L of tedizolid.Milosevic et al,2018, Antimicrobial Agents and Chemotherapy