n-3 Polyunsaturated fatty acids induce acute myeloid leukemia cell death associated with mitochondrial glycolytic switch and Nrf2 pathway activation

Abstract

Acute Myeloid Leukemia (AML) remains a therapeutic challenge and improvements in chemotherapy are needed. n-3 polyunsaturated fatty acids (PUFAs), present in fish oil (FO) at high concentrations, have antitumoral properties in various cancer models. We investigated the effects of two n-3 PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), in AML cell lines and primary AML blasts. EPA and DHA induced a dose-dependent decrease in cell viability in five AML cell lines, which was also observed with FO, but not SO (devoid of n-3 PUFAs) in cell lines and primary leucoblasts. Mitochondrial energy metabolism shifted from oxidative respiration to glycolytic metabolism in the U937, MOLM-13, and HL-60 cell lines. This phenomenon was associated with major disorganization of the mitochondrial network and mitochondrial swelling. Transcriptomic analysis after 6 h and 24 h of exposure to FO revealed a Nrf2 activation signature, which was confirmed by evidence of Nrf2 nuclear translocation in response to oxidative stress, but insufficient to prevent cell death following prolonged exposure. Apoptosis studies showed consistent phosphatidylserine exposition among the AML cell lines tested and a reduced mitochondrial membrane potential. The cell-killing effect of FO was additive with that of cytarabine (AraC), by the Chou and Talalay method, and this combination effect could be reproduced in primary AML blasts. Altogether, our results show deleterious effects of n-3 PUFAs on mitochondrial metabolism of AML cells, associated with oxidative stress and Nrf2 response, leading to cell death. These observations support further investigation of n-3 PUFA addition to standard chemotherapy in AML.

Introduction

Acute Myeloid Leukemia(AML) is a heterogeneous group of hematological malignancies for which improvements in therapy are needed, mostly in the adult setting. Upfront standard care for fit patients younger than 60 years is a combination of anthracyclines with cytarabine (AraC), except for specific molecular subgroups, such as FLT3-ITD, for which targeted therapy may be added [1]. Other treatment combinations with anthracyclines and AraC have shown a survival benefit in adult AML, such as gemtuzumab ozogamicin [2] or norethandrolone [3]. Nevertheless, relapses still occur, even with allogeneic transplantation consolidation. AML thus remains an unmet medical need, particularly in the elderly population.

cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) and all-cis-5,8,11,14,17-eicosapentaenoic acid (EPA) are long-chain n-3 polyunsaturated fatty acids (PUFAs) which are found at high levels in fish oil (FO) [4]. Low n-3 PUFA intake has been associated with an increased risk of breast cancer [5], and higher consumption of n-3 PUFAs with lower pancreatic cancer risk [6]. Furthermore, a high level of whole blood n-3 PUFAs was associated with lower prostate cancer risk in 476 men participating in a Scottish study [7]. These beneficial effects of n-3 PUFAs in cancer prevention suggest a potential role for these compounds in cancer therapy.

Preclinical data show that n-3 PUFAs reduce the IC₅₀ of chemotherapy agents in breast cancer cell lines [8,9], sensitize murine mammary cancers to anthracyclines [10], and trigger cytotoxicity in numerous solid cancer models, such as ovarian cancer [11] and colon cancer [12]. Several mechanisms of action of n-3 PUFAs have been described in solid tumor models. Structurally, EPA and DHA incorporate into cancer cell membranes [13] and modify lipid raft composition [14]. They have been shown to inhibit COX-2 and PGE2 pro-inflammatory activity and to be associated with reduced cancer invasion [15], and n-3 PUFAs can inhibit TLR-induced MHC class II upregulation and cytokine production [16]. DHA can also impede I-κB phosphorylation, thus suppressing NF-κB transcription factor activity [17]. The PI3K/AKT signaling pathway is inhibited by DHA in the MDA-MB-231 cell line engrafted to BALB/c nude mice fed a high DHA diet [18]. Evidence of cell arrest in G1-phase has been observed in breast cancer [19]. An anti-angiogenic effect has also been described through the attenuation of VEGF-induced angiogenesis [20].

In acute leukemia, it has been reported that DHA can induce direct in vitro cytotoxicity in the KG1a cell line [21], and cycle arrest in S-phase of the Jurkat cell line [22]. Hallmarks of apoptosis, such as nuclear fragmentation, have been demonstrated in the U937 cell line exposed to 100 μM DHA [21], as well as upregulation of Bax mRNA in the HL-60 cell line [23]. Exposure of HL-60 cells to EPA induces increased fluorescence of H2-DCFDA, suggesting the involvement of reactive oxygen species (ROS) in triggering cell death [24]. Mechanisms involving cytoplasmic calcium release, associated with cell death, have also been demonstrated in U937 cells [25,26]. A ROS-induced mechanism for the sensitization of HL-60 cells to arsenic trioxide by DHA has been described [27] and lymphoid cell lines may be sensitized to other anticancer agents, with an increase in lipid peroxide formation [28].

Whether n-3 PUFAs can induce mitochondrial impairment leading to cell death in AML, is still unclear, and the potential synergism of n-3 PUFAs with chemotherapy has not been yet evaluated. Here, we show that EPA, DHA and clinical-grade FO can induce in vitro cell death of AML cells, associated with phosphatidylserine exposure and a decrease in mitochondrial membrane potential. We demonstrate a mitochondrial metabolic shift and mitochondrial genomic damage, as well as an oxidative stress-related Nrf2 pathway activation, unable to avoid cell death. Furthermore, these effects of FO were additive with the cytotoxicity of AraC.