Treatment regimens for acute myeloid leukemia (AML) continue to offer weak clinical outcomes. Through a high-throughput cell-based screen, we identified avocatin B, a lipid derived from avocado fruit, as a novel compound with cytotoxic activity in AML. Avocatin B reduced human primary AML cell viability without effect on normal peripheral blood stem cells. Functional stem cell assays demonstrated selectivity toward AML progenitor and stem cells without effects on normal hematopoietic stem cells. Mechanistic investigations indicated that cytotoxicity relied on mitochondrial localization, as cells lacking functional mitochondria or CPT1, the enzyme that facilitates mitochondria lipid transport, were insensitive to avocatin B. Furthermore, avocatin B inhibited fatty acid oxidation and decreased NADPH levels, resulting in ROS-dependent leukemia cell death characterized by the release of mitochondrial proteins, apoptosis-inducing factor, and cytochrome c. This study reveals a novel strategy for selective leukemia cell eradication based on a specific difference in mitochondrial function. Cancer Res; 75(12); 2478-88. Ó2015 AACR.
Acute myeloid leukemia (AML) is a devastating disease characterized by poor patient outcome and suboptimal chemotherapeutics. Here, a high-throughput screen identified diosmetin, a citrus flavonoid, with anti-AML activity. Diosmetin imparted selective toxicity against leukemia and leukemia stem cells and with no effect on normal hematopoietic stem cells. Mechanistically, we demonstrated that diosmetin targets estrogen receptor (ER) β. ERβ expression conferred cell sensitivity, as patient-derived AML cells with high levels of ERβ were sensitive, whereas cells with low ERβ were insensitive to diosmetin. Knockdown of ERβ confirmed resistance, whereas overexpression enhanced sensitivity to diosmetin, which was demonstrated to be mediated by reactive oxygen species signaling. In summary, these studies highlight targeting of ERβ with diosmetin as a potential novel therapeutic strategy for the treatment of AML. .
Acute myeloid leukemia (AML) is an aggressive malignant disease characterized by poor patient outcome and suboptimal front-line chemotherapy. To identify novel anti-AML compounds, we performed a high-throughput screen of a natural products library (n=800). This screen was performed against the AML cell line (TEX), which has several properties of leukemia stem cells, the cells responsible for disease pathophysiology and patient relapse. Here, avocatin B was identified as a potent and novel anti-leukemia agent. Avocatin B, at concentrations as high as 20µM, had no effect on normal peripheral blood stem cell viability. In contrast, it induced death of primary AML cells with an EC50 of 1.5-5.0 µM. Selective toxicity towards a functionally defined subset of primitive leukemia cells was also demonstrated. Avocatin B (3µM) reduced clonogenic growth of AML progenitor cells with no effect on clonogenic growth of normal hematopoietic stem cells. Further, treatment of primary AML cells with avocatin B (3µM) diminished their ability to engraft into the bone marrow of pre-conditioned, NOD/SCID mice (t18=6.5; p<0.001). Together, these results confirm that avocatin B is a novel anti-AML agent with selective toxicity toward leukemia and leukemia stem cells. Mechanistically, avocatin B-induced reactive oxygen species (ROS)-dependent leukemia cell apoptosis that was characterized by the release of mitochondrial proteins, cytochrome c and apoptosis inducing factor (AIF). Cytochrome c and AIF were detected in the cytosol of avocatin B treated TEX cells by flow cytometry. Avocatin B-induced apoptosis, as measured by the Annexin V/Propidium iodide assay, DNA fragmentation and PARP cleavage, was abolished in the presence of anti-oxidants confirming the functional importance of ROS. Next, we further evaluated the role of mitochondria in avocatin B-induced apoptosis. First, we generated leukemia cells lacking mitochondria by successive culturing in media containing ethidium bromide. The drastic (>80%) reduction in mitochondria were confirmed by nonyl acridine orange staining and flow cytometry and a near absence of the mitochondria specific proteins ANT and ND1, as measured by Western blotting. Avocatin B’s activity was abolished in leukemia cells lacking mitochondria. Next, using lentiviral knockdown, we generated leukemia cells lacking CPT1, the enzyme that facilitates transport of 16-20 carbon lipids into mitochondria. Avocatin B’s activity was abolished in cells with reduced CPT1 expression (>70% as measured by qPCR analysis). To further confirm the importance of CPT1 in avocatin B-induced death, we chemically inhibited CPT1 with etomoxir. Avocatin B’s activity was blocked in the presence of etomoxir, further demonstrating that avocatin B accumulates in mitochondria. Since avocatin B is a lipid that targets mitochondria and that mitochondria can oxidize fatty acids for energy, we next assessed the impact of avocatin B on fatty acid oxidation, using the Seahorse Bioanalyzer. Avocatin B inhibited leukemia cell fatty acid oxidation (>40% reduction in oxygen consumption at 10µM) and this occurred at a 10-fold less concentration than etomoxir, the standard experimental molecule used to probe this pathway. Further, avocatin B resulted in a 50% reduction in levels of NADPH, an important co-factor generated during fatty acid oxidation that participates in catabolic processes during cell proliferation. These results show that avocatin B accumulates in mitochondria to inhibit fatty acid oxidation and reduce NADPH to result in ROS-mediated leukemia cell apoptosis. This highlights a novel AML-therapeutic strategy by which mitochondria are targeted to impair cellular metabolism leading directly to AML cell death. Disclosures No relevant conflicts of interest to declare.
<p>Supplementary Tables S1-S3. Summary of EC50 values for avocatin B in a panel of AML cell lines (S1); AML patient sample details used for annexin/PI (S2); AML patient sample details used for colony formation assays (S3).</p>
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.