Acute Myeloid Leukemia (AML) represents a life-threatening myeloid malignancy for which standard chemotherapy remains the cornerstone of therapy. Chemosensitizing agents are needed to improve the therapeutic results of chemotherapy. Both Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) belong to the n-3 polyunsaturated fatty acid (n-3-PUFA) family and display chemosensitizing properties in solid tumors both in vitro and in vivo. AIMS: To explore the effect of the combination of DHA with standard chemotherapy in vitro, and the effect of n-3-PUFA- containing fish oil emulsion on AML cell lines and primary blasts in vitro. METHODS: Two parenteral nutrition lipidic emulsions, OMEGAVEN (OM, containing a high level of n-3 PUFAs) and INTRALIPIDE (IL, containing a very low level of n-3 PUFAs, used as a control) were purchased from Fresenius Kabi GMBH. After 24h and 48h of culture, viable cells were counted with Trypan blue exclusion dye by an automated cell counter (TC10TM, BIO-RAD, Hercules, CA, USA). Assessement of phosphatidylserine exposure was performed with AnnexinV-FITC/7-AAD Kit (PN IM3614, Beckman Coulter), and mitochondrial membrane depolarization by flow cytometry DioC6(3) cellular uptake quantification. p62 and GRP98 expression were assessed by Western Blotting. Primary blasts were retrieved from the blood of patients with hyperleucocytic refractory AML, with informed consent. RESULTS: OM, DHA, and EPA consistently inhibited cell growth in U937, ML-2, HL-60, and THP-1 cell lines in a dose-dependent manner, whereas oleic acid (OA) or IL controls did not interfere with cell growth. OM also displayed a dose-dependent proliferation inhibition on AML cell lines, but did not interfere with normal granulopoiesis in vitro. U937 cell killing with OM was associated with p62 and GRP98 overexpression, suggesting the involvement of autophagy and endoplasmic reticulum stress, and apoptosis mechanisms. The cell-kiling effect of OM on AML primary blasts was associated with increased phosphatidylserine exposure and mitochondrial membrane depolarization. The supplementation of IL with DHA and EPA reproduced the effect of OM on the U937 cell line. At therapeutic concentrations of daunorubicin, cytarabine, or both, OM addition induced significant cytotoxity compared to IL in vitro. The isobologram assay displayed a pattern consistent with an additivity between OM and cyarabine (AraC) in vitro. DISCUSSION: This study confirms previous findings of n-3 PUFAs on AML cell line cytotoxicity both in murine and human models. We show for the first time that this effect is observed with primary blasts, is additive with chemotherapy, and can be obtained with commercially available fish oil emulsions. CONCLUSION: Altogether, these results indicate that DHA and EPA are the contributors of the cytotoxicity of a commercially available n-3 PUFA emulsion and provide a strong rationale for exploring the combination of parenteral n-3 fatty acid supplementation during AML chemotherapy.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.