Therapeutic Effect of Blueberry Extracts for Acute Myeloid Leukemia

Abstract

Acute myeloid leukemia (AML) is an aggressive hematological malignancy with high incidence in the aging population. In addition, AML is one of the more common pediatric malignancies. Unfortunately, both of these patient groups are quite sensitive to chemotherapy toxicities. Investigation of blueberries specifically as an anti-AML agent has been limited, despite being a prominent natural product with no reported toxicity. In this study, blueberry extracts are reported for the first time to exert a dietary therapeutic effect in animal models of AML. Furthermore, in vitro studies revealed that blueberry extracts exerted anti-AML efficacy against myeloid leukemia cell lines as well as against primary AML, and specifically provoked Erk and Akt regulation within the leukemia stem cell subpopulation. This study provides evidence that blueberries may be unique sources for anti-AML biopharmaceutical compound discovery, further warranting fractionation of this natural product. More so, blueberries themselves may provide an intriguing dietary option to enhance the anti-AML efficacy of traditional therapy for subsets of patients that otherwise may not tolerate rigorous combinations of therapeutics.

Keywords: acute myeloid leukemia; blueberry; mouse models.

Figure 1. In vitro anti-AML therapeutic effect of blueberry extracts

(A) Cellular viability was determined by MTS assay following 48-hour exposure of AML cell lines (dashed line with circles: C1498, solid line with squares: HL-60/vcr, dashed line with upside down triangles: 32D-FLT3-ITD) and a CML cell line (solid line with right side up triangles: 32D-p210-GFP) to crude blueberry extract. (B) Colony forming capacity of a poor prognosis patient AML case #329 was determined in the presence of 500 μg/ml crude blueberry extract (t-test, p=0.0245, n≥3). (C) Ficol-prepared WBC fractions of patient AML (case #651, #652, #657, #658, and #661) and patient CML (case #653) were exposed to 500 μg/ml crude blueberry extracts for 48 hours followed by flow cytometry to determine percent apoptosis (Annexin V+) in the CD45-gated blast population (t-test, p=0.0098, n=6).

Figure 2. Blueberry extracts regulate Erk and Akt phosphorylation in leukemia stem cells

(A) HL-60/vcr cells were exposed overnight (12 hours) to blueberry extracts and phosphorylation of Akt and Erk were monitored by Phosflow analysis (n=3 per data point). Antibodies were used to define leukemia stem cells as CD34+CD38− or CD34+CD38−CD96+. (B) Phosflow analysis was similarly used to evaluate cells exposed overnight to blueberry extracts from AML patient case #329 (poor prognosis) (n=3 per data point).

Figure 3. Survival extended in mice with AML following dietary blueberry extract supplementation

(A) NOD-Scid mice were xenografted with the human AML cell line HL-60/vcr (2×10⁶ cells/mouse by tail vein injection) and survival was monitored after mice were given crude blueberry extract (1% w/v) (dark line with diamonds) ad libitum in the drinking water, or control water (light gray line with squares), starting one week following engraftment (Mantel-Cox Logrank test, p=0.0017, n=7/group). (B) NOD-Scid mice were xenografted with the human AML cell line KG-1 (2.5×10⁶ cells/mouse by tail vein injection) and survival was monitored after mice were given crude blueberry extract (1% w/v) (dark line with diamonds) ad libitum in the drinking water, or control water (light gray line with squares), starting one week following engraftment (Mantel-Cox Logrank test, p=0.0382, n=7/group).

Figure 4. Dietary blueberry extract supplementation in combination with chloroquine extended the survival of mice with AML

C57BL/6J mice were engrafted with the murine AML cell line C1498 (1×10⁶ cells/mouse by retro-orbital injection) and survival was monitored after mice were given a crude blueberry preparation to eat (25 mg/mouse/week) (line with upside down triangles), chloroquine in the drinking water (25 μM sweetened with 1% w/v glucose to alleviate bitterness) ad libitum (line with right side up triangles), or a combination of both (line with circles), or control water alone (line with squares), starting one week following engraftment (Mantel-Cox Logrank tests: Males & Females together, p=0.0263, n≥14/group; Males only, not significant at p=0.1225, n≥7/group; Females only, p=0.0479, n≥7/group).