Activity and Cytotoxicity of Algae Extracts On Normal and Leukemic Hematopoietic Cells.

Abstract 4815

Algae preparations are commonly used in complementary and alternative medicine for presumed anti-oxidant, anti-inflammatory, and anti-cancer properties. In this study, we have examined the effects of extracts from algae and algae components on the proliferation of normal hematopoietic and leukemia cells. To prepare extracts, 1 gram of Dunaliella salina (Dun), Astaxanthin (Ast), Spirulina (C-phycocyanin) (Spir), or Aphanizomenon flos-aquae (AFA) were added to 10 ml of 70% ethanol and incubated at 4°C on a shaker for 24 hours. The slurry was centrifuged at 400 g for 10 minutes at 4°C, and the supernatant was filtered through 413-grade filter paper. Leukemia cell lines were purchased from ATCC and blood or marrow aspirates from normal subjects or patients with leukemia were subjected to Ficoll-Hypaque density gradient centrifugation. CD34+ cells were isolated using Miltenyi Biotec MiniMACS magnetic bead cell separation columns. To determine effects on cell proliferation, increasing concentrations of algae extracts were added in fresh medium to plated cells, and MTT reagent was added followed by detergent and absorbance readings were recorded at 570 nm. Leukemic cell lines such as HL60 and MV4-11 were significantly inhibited by Ast and AFA at concentration of 0.8 mg/mL of extract (p<0.05 compared with control conditions by two-tailed t-tests). Dun and Spir did not significantly inhibit proliferation of these cell lines. The PC3 prostate cell line and the MCF-7 breast cancer cell line were inhibited only by AFA at 1.5 to 2.0 mg/ml extract at 24 hours as assessed in an MTT assay. Through use of an Annexin V assay to determine effects on apoptosis, enriched primary AML blasts demonstrated increased apoptosis in the presence of AFA at 0.8 and 1.5 mg/ml. Primary CLL cells demonstrated increased apoptosis after 24 hour exposure to 1.5mg/mL Dun, AST, Spir, and AFA (p<0.05) by two-tailed t-tests. Western blots confirmed that apoptosis was mediated in part by increase in cleaved Caspase 3. Only AFA decreased the viability of normal light density marrow cells at 2.0 mg/ml, and normal CD34+ cells were inhibited by 0.8mg/ml Dun and at higher concentrations by Ast, Spir, and AFA. When effect of the extracts on outgrowth of BFU-E and CFU-GM was determined, only Dun and AFA inhibited BFU-E, but all 4 extracts were inhibitory to CFU-GM at 0.8 or 1.5 mg/ml (p<0.05; n=3). AFA was significantly more inhibitory than AST. Cell cycle analysis of AML cell lines exposed to increasing concentrations of all extracts showed G0/G1 arrest. No increase in reactive oxygen species was found using dihydrorhodamine 123. These data suggest that extracts from algae utilized as food supplements have the ability to inhibit AML cell lines and primary leukemia blasts but also demonstrate inhibitory effects on normal hematopoiesis. Each algae extract demonstrated a different pattern of inhibition, and whether these extracts are inhibitory at a stem cell level or would have differential effects on normal or leukemic cell growth in vivo remains to be determined.