To elucidate whether membrane fluidity changes could impact

To elucidate whether membrane fluidity changes could impact on signaling pathways, we examined the effects of Rh2 on the activation of Akt, a lipid raft-associated protein kinase, which promotes cell survival and blocks the apoptotic pathways. Upon pretreatment with 5 mM MβCD and in the absence of Rh2, the levels of phospho-Akt and Akt were similar between Cy5 EGFP mRNA depleted or not in cholesterol (Fig. 7). This observation could be at a first glance surprising since several papers show the dephosphorylation of Akt after cholesterol depletion via MβCD treatment (Calay et al., 2010; Gotoh et al., 2014). However, upon treatment with 5 mM MβCD (same concentration than our experimental condition) for 4 h, Upadhyay AK. et al. do not observe any effect neither on the Akt phosphorylation nor on the apoptosis in human breast cancer lines MCF-7 and MDA-MB-231 (Upadhyay et al., 2006). Therefore, we propose that differential incubation conditions with MβCD, i.e. dose and incubation time, could reconcile the different observations in distinct cell models. Additionally, the treatment with Rh2 decreased the level of the phosphorylated form of Akt (Ser473) earlier in cholesterol-depleted cells. Regarding the disappearance of total Akt amount after 2 h of treatment with Rh2, different non-mutually exclusive explanations can be provided. Firstly, at this time, we have shown that Rh2 activated caspase-3 and could lead to the cleavage of Akt protein due to its critical role in the cell growth and its anti-apoptotic signaling properties, as suggested by Widmann C et al. (Widmann et al., 1998). Secondly, a recent study demonstrated that 60 μM Rh2 reduces Akt expression and its phosphorylation in glioma cell lines A172 without affecting the β-actin level (Li et al., 2018). Finally, it was also reported that Akt molecules can be degraded by macroautophagy (Calay et al., 2010).The dephosphorylation of Akt in a time-dependent manner resulting in the loss of its enzymatic activity could suggest the inhibition of Akt-dependent survival signaling pathways and the activation of the intrinsic apoptotic pathway by Rh2 (Franke et al., 2003). To test for this hypothesis, apoptosis markers including loss of mitochondrial membrane potential (ΔΨ) and caspase-9 and -3 activations were investigated. ΔΨ was analyzed using the JC-1 dye. As shown in Fig. 8A, the treatment with Rh2 led to the early increase of the fluorescence intensity ratio at 525 nm (green) over 590 nm (red) upon cholesterol depletion. This result evidenced that Rh2 induced mitochondrial membrane depolarization faster in cholesterol-depleted than in non-depleted cells. Further downstream in the intrinsic apoptotic pathway, caspase-9 activity was monitored by detecting the cleavage of a specific caspase-9 substrate (Ac-LEHD-pNA). Rh2 activated faster caspase-9 upon cholesterol depletion (Fig. 8B). To further address the caspase-9 involvement, cells were pretreated with specific caspase-9 inhibitor (Z-LEHD-FMK) followed by Rh2 treatment. The fragmented nuclei percentage was decreased more significantly by caspase-9 inhibitor in cholesterol-depleted cells as compared to non-depleted cells (Fig. 8C). Moreover, throughout the treatment with Rh2, the caspase-3 was progressively cleaved into two fragments resulting to its activation. As shown in Fig. 8D, the cleavage fragment (19 kDa) was detected after 2 h in non-depleted cells and only 1 h in cholesterol-depleted cells. As the caspase-9 activity, the caspase-3 activation was faster in cholesterol-depleted cells. Those data reported faster activation of the intrinsic apoptotic pathway by Rh2 observed via the depolarization of ΔΨ and the activation of caspase-9 and -3.
Discussion Although many studies have investigated the Rh2-induced apoptosis in cancer cells (Shi et al., 2016; Lv et al., 2016; Choi et al., 2011), the role of membrane cholesterol in this process remains largely unclear. We here showed that cholesterol depletion enhanced the cellular accumulation of Rh2 in U937 cells that could result into faster Rh2-induced cytotoxicity. Based on the observation by Lorent et al. that α-hederin-induced apoptosis is reduced in cholesterol-depleted human leukemic U937 (Lorent et al., 2016), we suggest that the faster Rh2-induced cytotoxicity in cholesterol-depleted cells does not result from an unspecific mechanism due to pretreatment with MβCD. We also excluded the possibility that the observed effect could be cell-dependent, as revealed by a similar protective role of cholesterol in the Rh2-induced apoptosis in A549 and THP-1 cells.