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 of the 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 the capacity 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 ginseng root. 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).
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 as well 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.
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.