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    • Among steroid saponins ginsenosides are the active component

    2023-01-28

    Among steroid saponins, ginsenosides are the active components of ginseng, a well-known chinese medicinal plant. More than hundreds of different ginsenosides have been isolated from ginseng and have shown in the past to be membrane active substances and to influence the membrane by modulating lipid membrane dynamics (Qi et al., 2011; Tsuchiya, 2015; Kwon et al., 2008). In this study, we focus on the ginsenoside Rh2 (Fig. 1), phytosterol from Panax ginseng. To the best of our knowledge, only two studies have reported the disruption of lipid rafts by Rh2 leading to apoptosis, via either the FAS oligomerization in Hela MPC 6827 hydrochloride (Yi et al., 2009) or inactivation of Akt in human epidermoid carcinoma A431 cells and in human breast cancer MBA-MB-231 cells (Park et al., 2010). Taken together, these data suggest that rafts could be involved in Rh2-induced apoptosis. However, mechanistic understanding of the mode of interactions between Rh2 and membrane lipids and the exact subsequent apoptotic pathways are still poorly understood. Since lipid rafts are enriched in cholesterol and since some saponins have been shown to interact with cholesterol, the aim of the present study was to explore the role of membrane cholesterol in the cytotoxic activity of Rh2. To this end, we used three cell lines exhibiting differential membrane cholesterol level: carcinomic human alveolar basal epithelial A549 > human monocytic leukemia THP-1 > U937 cells. We demonstrated that Rh2 induced apoptosis in a concentration- and time-dependent manner in the three cell lines. More importantly, A549, THP-1 and U937 cells can be classified from the more resistant to the more susceptible to the Rh2-induced apoptosis. In addition, upon cholesterol depletion via methyl-β-cyclodextrin (MβCD), those three cell lines became more sensitive to Rh2-induced apoptosis. To explore the mechanistic behind this observation, we then focused on the cholesterol-auxotroph U937 cell line (Billheimer et al., 1987). We showed that Rh2 altered plasma membrane fluidity, induced Akt dephosphorylation and the activation of the intrinsic pathway of apoptosis. Fluidity changes, Akt dephosphorylation and apoptosis appeared faster in cholesterol-depleted cells, which could be explained by a faster cell accumulation of Rh2 in these conditions.
    Materials and methods
    Results In a first series of experiments, we examined the time and concentration dependence of the Rh2-induced apoptosis in A549, THP-1 and U937 cells. To this aim, cells were treated with increasing concentrations of Rh2 (20, 40 and 60 μM) for increasing periods of time (1 h to 24 h) and tested for apoptosis by DAPI staining. As shown in the left panel of Fig. 2, the Rh2-induced apoptosis extent was Rh2 concentration- and incubation time- as well as cell line-dependent. For instance, the U937 cell line (C) was more susceptible to the Rh2-induced apoptosis than THP-1 cells (B), themselves more susceptible than A549 cells (A). After 6 h of incubation, 60 μM Rh2 induced ~50% of fragmented nuclei in U937, ~15% in THP-1 without any effect in A549 cells. Since U937, THP-1 and A549 cells exhibited increasing membrane cholesterol (Table 1), those results suggested an inversed correlation between susceptibility to Rh2 and membrane cholesterol content: higher the cholesterol content, lower the Rh2-induced apoptosis. To further test this hypothesis, the same experiment was performed in cells depleted in cholesterol (right panel of Fig. 2). For this purpose, THP-1 were pretreated with 3 mM MβCD, whereas A549 and U937 cells were pretreated with 5 mM MβCD, a cholesterol-sequestering agent (Zidovetzki and Levitan, 2007). Those non-cytotoxic MβCD concentrations reduced by ~50% the ratio of cholesterol/protein in the three cell lines (Table 1). As shown in the right panel of Fig. 2, in all the conditions investigated, cholesterol-depleted cells were more sensitive to Rh2-induced apoptosis, corroborating the idea that a decrease in cholesterol content correlated with an increased number of apoptotic cells induced by Rh2.