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    2019-09-23

    r> 2.5. Statistical analysis
    Data are expressed as the mean ± SD of at least three independent experiments. Means were compared with the Mann-Whitney U test provided in GraphPad Prism (GraphPad Software, CA). P-values ≤ 0.05 were considered significant.
    3. Results and discussion
    Bioactive lipids are known to regulate several cellular processes [29–32], including cell growth, proliferation, differentiation, and death [33,34]. Among the bioactive lipids, oxysterols are potent, biologically active molecules, involved in several cell functions, including inhibiting cell proliferation and promoting cell death [35,36]. The most studied oxysterols, at least in terms of toxicity, are 25-hydroxycholesterol, 7β-hydroxycholesterol, and 7-ketocholesterol. The cytotoxic effects of these oxysterols have been demonstrated in several cell lines [16,22,35,37–42].
    Here, as expected, 7-KC, triol, and diol reduced the number of cells. As described previously, apoptosis was involved as a cause of cell death [24]. We explored the mechanisms of apoptosis promoted by 7-KC, triol, and diol by evaluating the effects of subtoxic doses (30 μM) on the sonic hedgehog (SHh) pathway and liver X receptor alpha (LXRα). SHh can cause different effects on Oxidopamine hydrochloride at different concentrations. The SHh pathway is activated when SHh binds to its receptor, the transmem-brane protein, Patched (PTCH) [43]. PTCH proteins prevent down-stream signaling by attenuating Smoothened (SMO) activity [44]. However, when SHh binds to PTCH, it removes the repression of SMO, which then activates a signal transduction pathway in the cytoplasm [45]. Recently, it was shown that oxysterols could allosterically activate SMO by binding to its extracellular cysteine-rich domain [46].
    Here, we evaluated SHh with immunofluorescence. None of the oxy-sterols or cholesterol (as control) changed SHh protein expression (Fig. 1A). The effect of oxysterols on SMO was evaluated by assessing fluorescence intensity in the membrane/cytoplasm and in the nucleus.
    Fig. 1. Immunofluorescence detection of sonic hedgehog (SHh) and smoothened (SMO) expression in MDA-MB-231 cell line after 24 h incubation with 30 µM oxysterols. A: SHh expression; B: SMO expression in the membrane/cytoplasm;C: SMO expression in the nucleus. The intensity of fluorescence was quantified with MetaXpress software. Abbreviations: 7KC: 7-ketocholesterol; triol: cholestan-3α-5β-6α-triol; diol: 5α-cholestane-3β,6β-diol/5α-cholestane-3β,6α-diol. Cholesterol was used as control. Data are presented as the mean ± SEM from three independent experiments performed in triplicate. *p < 0.005 compared to control.
    Fig. 2. Immunofluorescence detection of LXRα expression in the MDA-MB-231 cell line after 24 h incubation with 30 µM of oxysterols. The intensity of fluorescence was quantified with MetaXpress software. Representative images of immunofluorescence after incubations with A: 7-ketocholesterol (7KC); B: cholestan-3α-5β-6α-triol (triol); C: 5α-cholestane-3β,6β-diol/5α-cholestane-3β,6α-diol (diol); or D: cholesterol (control); E: quantification of LXRα expression. Data are presented as the mean ± SEM from three independent experiments performed in triplicate. *p < 0.005 compared to control.
    Cells expressed SMO protein. Neither oxysterols nor cholesterol changed SMO expression in the membrane/cytoplasm (Fig. 1B). On the other hand, SMO expression in the nucleus increased lines after treatment with 7-KC and triol (Fig. 1C). Cholesterol had no effect on nuclear SMO levels. Therefore, these oxysterols did not appear to act on SMO by changing SHh expression, but a possible direct action on SMO should be considered.
    LXRs are nuclear receptors with important roles in the transcrip-tional control of lipid metabolism. They were initially described as orphan receptors, but later, oxysterols were identified as their natural ligands. Activated LXRs form heterodimeric complexes with retinoic acid receptors (RXRs) [47]. LXRs exert important effects, including control of transcription factors and gene regulation. The genes targeted