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    • To further investigate the role

    2024-03-28

    To further investigate the role of cholesterol in the Rh2-induced cytotoxicity, we then focused on the U937 cell line, which is cholesterol auxotroph and considered as a valuable model to study the importance of cholesterol in membrane structure and function (Billheimer et al., 1987), and used the effective concentration of 60 μM Rh2. We started by analyzing the effect of Rh2 on membrane permeability and cell death via trypan blue assay. Depletion of cholesterol by MβCD effectively increased and accelerated the Rh2-induced cell death, confirming the results obtained with DAPI staining (Fig. 3A). Noteworthy, the apoptosis was observed before the appearance of cell death in non-depleted cells. Acridine orange/ethidium bromide staining was also performed to visualize membrane permeabilization in parallel with nucleus morphology changes (Lorent et al., 2016). The treatment with Rh2 conducted first to apoptosis observed by nuclear fragmentation followed in a second step by the loss of plasma membrane integrity and necrosis (Fig. 3B). >45% of the AS 602801 lost their membrane integrity after 3 h of incubation with 60 μM of Rh2 in cholesterol-depleted cells while this phenomenon was not yet observed in non-depleted cells. In all the conditions investigated, depletion of cholesterol accelerated the cytotoxic effect of Rh2. To confirm the protective role of membrane cholesterol in the Rh2-induced apoptosis, U937 cells were pretreated with 0 to 7 mM MβCD. Upon treatment with 5 mM and 7 mM MβCD, the cholesterol/phospholipid ratio was reduced to ~75% and 60% of that of control cells, respectively (Fig. 4A). Cells with varying contents of cholesterol were then treated with 60 μM Rh2 for 2 h and DAPI assay was performed to determine the percentage of fragmented nuclei (Fig. 4B). As expected, the increase of membrane cholesterol depletion via the pretreatment with raising concentrations of MβCD correlated with an increased number of apoptotic cells following Rh2 treatment (Fig. 4C). These data confirm that lower the cholesterol level is, higher the Rh2-induced apoptosis is. To determine whether those effects are specific to membrane cholesterol depletion, cells were incubated with 0 to 60 mU/ml Bacillus cereus sphingomyelinase (SMase) for sphingomyelin depletion. In these conditions, the reduction of sphingomyelin content reached ~50% of that of control cells (Fig. 4D) without inducing any cell death (data not shown). As shown in Fig. 4E, F, the increase of sphingomyelin depletion via the pretreatment with raising concentration of SMase correlated with a decreased number of apoptotic cells following Rh2 treatment. Therefore, in contrast to cholesterol removal, sphingomyelin depletion confers resistance towards Rh2-induced apoptosis, indicating that the cytotoxic activity of Rh2 depends on the membrane lipid nature. To investigate whether the faster cytotoxic effect of Rh2 upon cholesterol depletion could result from a difference in Rh2 cell accumulation, the time course of Rh2 uptake was investigated in cells depleted or not in cholesterol. Cells were incubated with Rh2 over time and the amount of Rh2 in cells was measured by HPLC MS/MS (Fig. 5). After 10 min of treatment, cholesterol-depleted cells accumulated two-fold more Rh2 as compared to non-depleted cells and this difference was maintained until 45 min. These results indicated faster cellular uptake of Rh2 upon cholesterol depletion. To next ask whether Rh2 could affect plasma membrane biophysical properties, we measured plasma membrane fluidity, a highly regulated property, including by cholesterol. To this aim, we used DPH and TMA-DPH that respectively monitor the fluidity in the hydrophobic core and in the interfacial region of the lipid bilayer (do Canto et al., 2016). In the absence of Rh2, whereas no significant difference was observed for TMA-DPH anisotropy, DPH anisotropy value (r) of cholesterol-depleted cells was significantly higher than in non-depleted cells (Fig. 6), suggesting a higher rigidity in the lipid core region. Rh2 significantly increased the fluorescence polarization of DPH within 5 min in cholesterol-depleted cells but only after 30 min in non-depleted cells as compared with respective controls, suggesting that Rh2 compacted faster the lipid core region of cholesterol-depleted membranes. In contrast, Rh2 decreased the anisotropy value of TMA-DPH within 10 min in cholesterol-depleted cells and after 30 min in non-depleted cells, suggesting that Rh2 relaxed earlier the interfacial region of the lipid bilayer upon cholesterol depletion. Based on all these results, we propose that Rh2 affected faster the physical state of lipid bilayers in cholesterol-depleted cells than in non-depleted cells.