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7-Ketocholesterol Promotes Oxiapoptophagy in Bone Marrow Mesenchymal Stem Cell from Patients with Acute Myeloid Leukemia.
Paz JL
,
Levy D
,
Oliveira BA
,
de Melo TC
,
de Freitas FA
,
Reichert CO
,
Rodrigues A
,
Pereira J
,
Bydlowski SP
.
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7-Ketocholesterol (7-KC) is a cholesterol oxidation product with several biological functions. 7-KC has the capacity to cause cell death depending on the concentration and specific cell type. Mesenchymal stem cells (MSCs) are multipotent cells with the ability to differentiate into various types of cells, such as osteoblasts and adipocytes, among others. MSCs contribute to the development of a suitable niche for hematopoietic stem cells, and are involved in the development of diseases, such as leukemia, to a yet unknown extent. Here, we describe the effect of 7-KC on the death of bone marrow MSCs from patients with acute myeloid leukemia (LMSCs). LMSCs were less susceptible to the death-promoting effect of 7-KC than other cell types. 7-KC exposure triggered the extrinsic pathway of apoptosis with an increase in activated caspase-8 and caspase-3 activity. Mechanisms other than caspase-dependent pathways were involved. 7-KC increased ROS generation by LMSCs, which was related to decreased cell viability. 7-KC also led to disruption of the cytoskeleton of LMSCs, increased the number of cells in S phase, and decreased the number of cells in the G1/S transition. Autophagosome accumulation was also observed. 7-KC downregulated the SHh protein in LMSCs but did not change the expression of SMO. In conclusion, oxiapoptophagy (OXIdative stress + APOPTOsis + autophagy) seems to be activated by 7-KC in LMSCs. More studies are needed to better understand the role of 7-KC in the death of LMSCs and the possible effects on the SHh pathway.
Figure 2. Caspase-3/7 and caspase-8 activities, cell cycle phases, mitochondrial transmembrane potential, and effects of reactive oxygen species (ROS) on bone marrow mesenchymal stem cells from patients with acute myeloid leukemia after 24 h 7-KC treatment. A: Percentage of cells with caspase-3/7 activity measured by CellEvent Caspase-3/7 Green. B: Percentage of cells with caspase-8 activity determined by the Vybrant FAM Caspase-8 Assay Kit. C: Cell cycle phases determined by Hoechst 33342 staining. D: Mitochondrial transmembrane potential evaluated by TMRE. E–I: Representative mitochondrial staining (E: 10 µM 7-KC; F: 25 µM 7-KC; G: 50 µM 7-KC; H: 100 µM 7-KC; and I: control). J: Cell death and relationship with ROS. Cells were treated with or without N-acetyl-l-cysteine (NAC) for 4 h following by incubation with 7-KC. Cytotoxicity was evaluated by Hoechst 33342/propidium iodide staining. K: Percentage of cells producing ROS as evaluated by DCFH-DA. L–P: Representative figures of ROS-producing AML mesenchymal stem cells (LMSCs) (L: 10 µM 7-KC; M: 25 µM 7-KC; N: 50 µM 7-KC; O: 100 µM 7-KC; and P: control). Data are mean ± SEM from three independent experiments in duplicate. * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bar, 100 µM.
Figure 3. Sonic Hedgehog (SHh), smoothened (SMO), and changes in actin organization in bone marrow mesenchymal stem cells from patients with acute myeloid leukemia after 24 h 7-KC treatment. A–D: Representative figures of SHh expression measured by immunofluorescence (A: 30 µM 7-KC; B: 50 µM 7-KC; C: 70 µM 7-KC; and D: control). E: Percentage of cells expressing SHh. F: Mean fluorescence intensity of SHh expression. G: Mean fluorescence intensity of SMO expression in the nucleus of LMSCs. H: Mean fluorescence intensity of SMO expression in the membrane and cytoplasm. I–L: Representative figures of SMO expression measured by immunofluorescence (I: 30 µM 7-KC; J: 50 µM 7-KC; K: 70 µM 7-KC; and L: control). M–Q: Actin organization evaluated by Alexa Fluor 532 phalloidin staining (M: 10 µM; N: 25 µM 7-KC; O: 50 µM 7-KC; P: 100 µM 7-KC; and Q: control). White arrow: enhanced fluorescence in the membrane edges. Data are mean ± SEM from three independent experiments in duplicate. ** p < 0.01, *** p < 0.001. Scale bar, 100 µM.
Scheme 1. Scheme of cell death promoted by 7-KC in LMSC.
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