Acute myeloid leukemia (AML) is a malignant disease of the hematopoietic cells characterized by impaired differentiation and uncontrolled clonal expansion of myeloid progenitors/precursors, resulting in bone marrow (BM) failure and impaired normal hematopoiesis [1
]. Currently, 40–50% of AML patients can be cured using conventional chemotherapy [2
]. However, the incidence of AML increases exponentially with age, and the cure rate is significantly lower in older adults [3
]. Although high levels of supportive care have improved patient outcomes, a high cure rate in AML has not yet been achieved [2
Hematopoietic stem cells (HSCs) reside in the BM niche, a specialized microenvironment that contains complex and diverse stromal cell populations, mainly adipocyte cells and osteocyte [4
]. Mesenchymal stem cells (MSCs) are multipotent cells that are also found in the niche and generate most BM stromal cell lineages. MSCs can lose their unspecialized or undifferentiated states and transform into other mesenchymal lineages [5
], generating osteoblasts, chondrocytes, fibroblasts, adipocytes, endothelial cells, and myocytes [6
]. The self-renewal property is essential for expansion of the stem cell pool during fetal development, as well as maintenance of the stem cell pool throughout the lifespan of the organism [7
MSCs contribute substantially to the creation of a hematopoietic niche [8
]; they can secrete several cytokines/growth factors that can modulate the immune response and increase the potential for the expansion and differentiation of host cells [10
]. Furthermore, in the BM they provide a niche for the growth, differentiation, and survival of normal and malignant hematopoietic cells, playing an essential role in normal hematopoiesis by regulating HSC proliferation and differentiation [11
]. Mutations in stromal cells from the tumor microenvironment have been reported to be involved in malignant transformation in breast and colon cancer, as well as myelodysplastic syndromes (MDSs) and AML [13
]. Recently, an inherent defect in AML mesenchymal stem cells (LMSCs) was demonstrated to lead to dysfunctional crosstalk between the BM niche and HSCs, potentially resulting in AML initiation or propagation [14
]. Specific changes in MSCs have also been demonstrated to initiate leukemia in mice [15
]. In addition, it is well accepted that BM stromal cells, including MSCs, promote leukemia cell resistance to chemotherapy [12
Cholesterol is essential for cell function and viability; it is a component of the plasma membrane and lipid rafts, and is a precursor of several compounds, including bile acids and steroid hormones [18
]. Oxidation and peroxidation are important features of lipid metabolism [19
]. Cholesterol is also susceptible to cellular oxidation induced by enzymes or reactive oxygen species (ROS), generating several oxidized derivatives [21
]. Several oxysterols are biologically active as regulatory molecules and are involved in the regulation of sterol and lipid metabolism, the modulation of signaling pathways, and influencing cell proliferation and differentiation [22
]. A number of important roles have been ascribed to oxysterols, such as cholesterol turnover, atherosclerosis, apoptosis, necrosis, inflammation, and immunosuppression [23
7-Ketocholesterol (7-KC) is an oxysterol that differs from free cholesterol by the addition of a functional ketone group at C7 [24
]. It is a biologically active molecule capable of promoting, among numerous cell types, an apoptotic mode of cell death associated with autophagy (oxiapoptophagy) [25
] and characterized by reduced cell proliferation, increased membrane permeability, nucleus condensation, decreased production of nitric oxide, and oxidative damage to the DNA [31
7-KC has also been shown to induce cell death in MSCs isolated from adipose tissue, as well as cell apoptosis through mitochondrial and nuclear damage [30
]. However, the actions of 7-KC in the MSCs of other organs and tissues are not very well-known, and even less so in those affected by disease. Since MSCs are essential for the maintenance of the bone marrow niche and consequently hematopoiesis, this work was designed to evaluate the action of 7-KC in causing cell death in the bone marrow MSCs of patients with AML, including the type of death and possible mechanisms.
Our finds are summarized in Scheme 1
. Human MSCs were isolated from the BM of five patients with AML. They exhibited all of the characteristics of MSCs: adherence to plastic, fibroblast-like morphology, membrane markers CD29+, CD44+, CD90+, and CD105+, lack of membrane markers CD34, CD11b, CD45, and HLA-DR, and the capacity for cell differentiation per The International Society for Cellular Therapy [44
]. All LMSC samples were similar in terms of phenotype and differentiation capacity. The main obstacle for MSC isolation and characterization is their low number in fresh tissue, especially BM. Their numbers have been estimated to be approximately 0.01% of nucleated BM cells, declining with age [45
]. Corradi et al. [11
] were unable to isolate MSCs from the marrow of a substantial fraction (25%) of AML patients, demonstrating the difficulty in isolating this type of cell. In support of their findings, we were unable to obtain LMSCs from the BM of 36% of patients with AML (data not shown).
Ex vivo-expanded MSCs are usually considered to exhibit six biological functions of therapeutic interest: proliferation, multipotency, migration/homing, trophic capacity, immunosuppression, and cell death modulation, often examined independently of one another [46
]. Cell death is also characterized by morphological alterations. According to the recommendations of the Nomenclature Committee on Cell Death in 2018 [47
], cell death is classified into three types: type I cell death or apoptosis; type II cell death or autophagy; and type III cell death or necrosis.
Most investigations have reported that oxysterols are potent inducers of cell death [48
]. Although more than 60 different oxysterols have been described, only some of them are cytotoxic. One of the most studied oxysterols is 7-KC, which has been described as one of the most toxic and predominant element components of oxidized low-density lipoprotein (oxLDL) [49
]. Oxysterols such as 7β-hydroxycholesterol and 7-KC are potent inducers of apoptosis in tumor and normal cells [50
]. Furthermore, 7-KC induces a particular mode of cell death, oxiapoptophagy [54
Reports on the effects of oxysterols on MSCs are relatively scarce, and most are related to the osteogenic effects of some types of oxysterols. Some oxysterols are osteoinductive molecules that can induce the lineage-specific differentiation of cells into osteoblasts [57
]. Our previous data suggested that 7-KC induces short-term apoptosis in MSCs from adipose tissue, at least in part by direct action on mitochondria [30
]. However, other types of oxysterols promote a complex mode of cell death in MSCs, including apoptosis and autophagy, in addition to decreased cell proliferation [36
Here, we described the early effects of 7-KC on BM MSCs from patients with AML. The cytotoxic effect of 7-KC was tested with the PI method for detecting cell death. The IC50 of 7-KC in LMSCs was 81.1 µM, higher than that described in normal human adipose tissue-derived MSCs (59.54 µM) [30
], normal human fibroblasts (47.66 μM) [27
], and other cancer cell lines [59
]. It is tempting to postulate that LMSCs are less susceptible to the death-promoting effect of 7-KC than other cell types.
Several in vitro studies have described the pro-apoptotic effect of 7-KC in several cell systems [23
]. In the extrinsic pathway of apoptosis, caspase-8 and caspase-3/7 are activated. The intrinsic signaling pathway of apoptosis involves a diverse array of non-receptor-mediated stimuli that produce intracellular signals that act directly on targets within the cell. The resulting mitochondrial-initiated events lead to mitochondrial dysfunction and subsequent release of cytochrome c and activation of caspase-9 and caspase-3 [60
]. Oxysterol-induced apoptosis has been shown to be mediated by both intrinsic mitochondrial pathways and an extrinsic death receptor-dependent pathway [22
Here, 7-KC exposure triggered the extrinsic pathway of apoptosis as shown by the increase in activated caspase-8. Caspase-8 induces the cleavage and activation of Bid protein, which results in activation of Bax and direct activation of caspase-3 [62
]. 7-KC was able to increase the percentage of LMSCs expressing caspase-3 activity. After the signaling pathway is triggered via caspase-3 activation by caspase-8, the death process cannot be stopped, even if negative regulators are involved in the pathway [63
To further elucidate the 7-KC mode of action in LMSCs, the status of caspase activation was determined using Z-VAD-FMK, a cell-permeant pan-caspase inhibitor [64
]. We have demonstrated that Z-VAD-FMK only partially reverses 7-KC-promoting cell death at high concentrations. This suggests that additional mechanisms other than caspase-dependent pathways are involved.
The pro-apoptotic effect of oxysterols has also been associated with overproduction of ROS, changes in intracellular calcium levels, or modification of the mitochondrial membrane associated with the intrinsic pathway [4
]. 7-KC is known to induce O2−
] and has been shown to activate monocytes/macrophages, resulting in hydrogen peroxide (H2
) and superoxide anion (O2−
) overproduction, lipid peroxidation, plasma membrane alteration, organelle dysfunction, DNA damage, and cytokine secretion, leading to cell death [25
7-KC increased ROS generation by LMSCs. This increase was related to the decrease in cell viability, as the death of LMSCs was partially reversed in the presence of NAC, a ROS inhibitor. Mitochondria are widely accepted to play a key role in the regulation of apoptosis. Changes in the mitochondrial transmembrane potential in response to various stimuli lead to ROS production and mitochondrial membrane permeabilization (MMP) [23
], causing a loss of mitochondrial membrane potential (ΔΨm), with consequent release of pro-apoptotic proteins [40
]. In our study, the decrease in ΔΨm in LMSCs in response to 7-KC is evidence of mitochondrial dysfunction.
Although the 7-KC impact mitochondria, their effect on other organelles such as lysosomes could be responsible for the observed effects. The autophagy-lysosomal pathway is a degradation process of dysfunctional organelles that has been associated with several pathophysiological processes [65
]. The decrease of lysosomal function, including the autophagy-lysosomal pathway, leads to acceleration of ROS generation. Although we did not have investigated the mechanisms of ROS generation, the above observations may further support the interpretation of increased ROS generation promoted by 7-KC in LMSCs.
Components of the cytoskeletal network, such as the actin microfilament meshwork or intermediate filaments, are known targets of oxidative and thiol-depleting agents that induce cell injury [67
]. Treatment with oxysterols has been described to cause progressive disruption of actin microfilaments and to induce cell detachment in several cell lines [30
]. We have also observed that higher concentrations of 7-KC lead to gradual changes in the morphology of LMSCs, with disruption of the cytoskeleton.
A number of studies have shown that oxysterols affect the cell proliferation capacity and cell cycle. HepG2 cells accumulate in G2/M phase upon incubation with 25-hydroxycholesterol [70
]. Similar observations have been made in other cell types [71
], but 7-KC does not change the cell cycle in adipose tissue MSCs [36
]. Our present data show that the anti-proliferative effects of 7-KC occur in LMSCs through blockage at S phase. We measured an increase in the number of cells in S phase and a decrease in the number of cells in the G1/S transition. The mechanisms responsible for these effects in LMSCs and other cell types remain to be investigated.
It is clear that, despite autophagy being considered a cell survival mechanism, its excessive increase, in certain conditions, may lead to a caspase-independent non-apoptotic type of cell death (type II cell death) [74
]. Autophagy plays a consistent role in the modulation of cell proliferation, differentiation, and stemness in a wide variety of cell types, including MSCs [75
]. In addition, autophagy has been reported to play an important role in the maintenance and survival of cancer stem cells or tumor-initiating cells in breast and pancreatic carcinomas [76
]. Oxidized lipids have been reported to stimulate autophagy in advanced human atherosclerotic plaques and cultured vascular smooth muscle cells [77
]. 7-KC also regulates the expression of autophagic genes [78
] and has been shown to trigger autophagy in vascular smooth muscle cells and coronary arterial myocytes [79
]. Here, we described the accumulation of autophagosomes using fluorescence microscopy with the LC3B-GFP sensor in the presence of high concentrations of 7-KC. In our model system, inhibition of autophagy by the administration of 3-MA in LMSCs did not change the percentage of dead cells after treatment with 7-KC. 3-MA produces a metabolic effect that is not related to the inhibition of autophagy, raising questions about its specificity as an autophagy inhibitor [79
]. Therefore, more studies are needed to better understand autophagy in LMSCs after treatment with 7-KC.
Programmed necrosis, also called necroptosis (type III cell death), is a caspase-independent RIP3-mediated form of cell death that was recently identified as a novel mechanism of cell death [80
]. Activation of RIP3 is regulated by the kinase RIP1 [81
], a key player in the modulation of cell fate in response to different stimuli [82
]. Here, we showed, using necrostatin-1, a blocker of both RIPK1 and indoleamine 2,3-dioxygenase (IDO) [83
], that necroptosis is not the type of cell death caused by 7-KC.
The Hedgehog (Hh) signaling pathway plays an important role in adult stem cell function and/or progenitor cells in several tissues [84
]. Hh signaling occurs through interaction of the Hh ligand with its receptors Ptc and SMO. In the absence of Hh, Ptc inhibits SMO and, through suppression of Gli represses downstream gene expression [80
]. When Hh is present, it binds to Ptc and releases SMO, which activates the Hh signaling pathway [66
]. Mutations in Hh pathway components, including Ptc1 and SMO, that lead to pathway activation have been linked to basal cell carcinoma and medulloblastoma [85
]. The constitutively activated SHh signaling pathway has been shown to be important for growth or progression of liver, lung, stomach, breast, leukemia, and esophageal cancers [85
]. Hh signals play a critical role in the survival and expansion of cancer stem cells in hematological malignancies [87
We evaluated the effects of subtoxic doses (70–30 μM) of 7-KC on the SHh pathway and demonstrated that 7-KC is able to downregulate the SHh protein in LMSCs. However, 24 h incubation with 7-KC did not change the expression of SMO. Our previous results with a breast cancer cell line showed no alteration in SHh expression as promoted by different oxysterols; however, 7-KC and cholestan-3α-5β-6α-triol were able to increase the expression of SMO in the nucleus [38
]. Selective inhibition of AML cell growth by a SHh pathway antagonist has been shown, with no interference in the growth of normal stem cells [88
There are some limitations in this study. One is the high concentrations of 7-KC used; in most cases, effects were observed only at the concentration of 100 µM, higher than the IC50 (81 µM). 7-KC studies are usually made with doses much higher than those found physiologically. Even the concentration of 7-KC found in oxidized LDLs is lower than that commonly used in experimental studies. Thus, the biological relevance of 7-KC at high concentrations lies in the perspective of use as a chemotherapeutic adjuvant. Also, activation of caspase 9 has not been studied; nevertheless, other mechanisms participating in this pathway were described, such as mitochondrial alterations and the presence of ROS. Finally, another limitation is the lack of time course experiments, because some effects of 7-KC in low concentration could potentially cause some physiological rather than pharmacological effects after longer periods of time.
In conclusion, taken together, our data demonstrate that oxiapoptophagy, including ROS overproduction, apoptosis, and autophagy, could be a particular type of cell death activated by 7-KC in MSCs from patients with AML. This is the first report showing that oxiapoptophagy can occur in cells other than nerve cells. Although many more studies are needed to better understand the role of oxiapoptophagy caused by 7-KC in LMSCs, it is tempting to postulate that 7-KC, present in oxidized LDL or acting as a pharmacological agent, could modulate leukemic cell proliferation and death by acting in LMSCs.