The two most common edible cyanobacteria include Spirulina and AFA , both of which contain phycocyanin, a molecule shown to induce apoptosis in the chronic myeloid leukemia cell line, K562  and other types of cancer [7–9]. Spirulina has been taken as a nutritional supplement for many years and has shown no undesirable side effects . It has been dubbed nature's richest and most complete source of nutrition . Orally administered spirulina extract has been shown to enhance tumoricidal NK activation through the MyD88 pathway, and spirulina exerted a synergistic antitumor activity with BCG-cell wall skeleton when used as immunotherapy of melanoma . Our studies exposing cells to Spir extract demonstrated little effect on the growth or viability of either cancer cell lines or normal hematopoietic or stem cells at the doses tested. The only significant effects observed were a decrease in MV-4-11 cell viability at high doses (20 μl/ml) and an inhibition of CFU-GM.
Hart et al. showed that the extract of A. flos-aquae (AFA) is a potent in vitro activator of NK cells, which are capable of killing some tumor cells without prior sensitization to antigen. However, this effect appears to be dependent on accessory cells as activation was not observed on isolated NK cells . Pugh and Pasco demonstrated that AFA extract activated the monocyte cell line THP-1 . Increased levels of both IL-1β and TNF-α in cells exposed to 0.5 pg/ml of extract were detected.
AFA is known to produce several toxins including the hepatotoxin cylindrospermopsin (CYN) , and paralytic shellfish poisons, a potent neurotoxin [15, 16]. In contrast to our findings with Spir, our studies show that AFA extract alone increased apoptosis in all of the cancer cell lines we tested. In addition, significant toxicity was observed for normal bone marrow cells as well as for erythroid and granulocyte-macrophage progenitors.
Dunaliella (Dun) provides a plenteous source of natural carotenoids that are in high demand due to applications in nutrition and medicine by consumers today. Dunaliella carotene, extracted from Dunaliella alga, showed no mortality or treatment-related adverse clinical effects throughout a 90-day subchronic toxicity study performed in F344 rats . Murthy et al. showed a beneficial effect of dunaliella carotenoid compared to synthetic carotene as an antioxidant through decreased antioxidant enzymes catalase, superoxide dismutase, and peroxidase compared to controls . Our studies showed a significant effect on apoptosis (Annexin V staining) by exposure of leukemic cell lines or primary tumor cells to Dun extract. We also observed that higher doses of Dun were associated with decreased viability of CD34+ stem cells. In addition Dun exposure also induced a significant decrease in both CFU-GM and BFU-E colony formation.
The astaxanthin (AST) rich H. pluvialis extract has inhibited cell growth in a dose- and time-dependent manner, by arresting cell-cycle progression and by promoting apoptosis in the HCT-116 colon cancer line . Apoptosis was upregulated by modifying the ratio of Bax/Bcl-2 and Bcl-XL, and the phosphorylation of p38, JNK, and ERK1/2 was increased . Other carotenoids derived from algae induce cell-cycle arrest through alteration in cyclin D2, D2, CDK4, and CDK6, and they may alter the expression of Bcl-1, XIAP, and survivin. Caspase activation has also been reported . Ishikawa et al. showed Ast had mild inhibitory effects on HTLV-1-infected T-cell lines, a model system for adult T-cell leukemia . It has been suggested that astaxanthin may improve antitumor immune responses by inhibiting lipid peroxidation induced by stress. Astaxanthin has been shown to protect several cell types from oxidative damage including neuronal cells , cervical cancer cells , and human umbilical-vein endothelial cells [23–27]. The ultimate effect of algal compounds and their metabolites on oxidative status is often indeterminate. Induction of apopotosis via generation of reactive oxygen species in human leukemia cell lines has been demonstrated , but other enzymatic extracts from algae have been found to protect against DNA damage induced by H2O2  or to demonstrate chemopreventive properties through inhibition of nitric oxide and other inflammatory mediators such as TNF-alpha and COX-2 . To address cellular ROS in our model, we treated primary AML blasts with algal extracts using dihydrorhodamine 123 as an indicator of peroxynitrite formation. We did not observe a cellular increase in reactive oxygen species caused by exposure to any of the algae extracts at a two-hour time point, suggesting that apoptosis was not induced by this pathway of reactive oxygen generation (Figure 7). Similar results were obtained for the cell lines HL-60 and MV-4-11 (Data not shown).
In our studies, astaxanthin (AST) exposure resulted in no decrease in viability of normal light density marrow cells, or of normal CD34+ cells and no inhibitory effect on BFU-E colony formation or primary AML blasts. However, significant reductions in viability were observed in the leukemic cell lines MV-4-11 and HL-60, as well as primary chronic lymphocytic leukemia cells.
The primary algae samples used were consistent throughout this study; however, because the constituents are largely unknown, we expect considerable variability between batches and manufacturers within the health food industry. This lack of uniformity compounded with several unknown bioactive components is a significant concern for health care providers, where patients take various herbal or algal supplements. To address this aspect of herbal pharmocology, we have used algae readily available to the general public, sold by the health food industry. Furthermore, use of extracts does not allow for determination of components or secondary metabolites, which might be responsible for the effects seen. For example, secondary metabolites of various algae have been found to affect cell adhesion . In other cases, polysaccharide or deacetylated components have been responsible for activities reported .
In the present study, we demonstrated that ethanol extracts from astaxanthin (AST), Dunaliella salina (Dun), and aphanizomenon flos-aquae (AFA) inhibit both HL-60 and MV4-11 leukemic cell lines. Using primary patient samples of AML and CLL, we identified doses of AFA that significantly reduced viability of malignant cells in both types of leukemia. Cells from chronic lymphocytic leukemia patients were sensitive to all of the extracts tested except Spirulina. High doses of Dun, Ast, and AFA significantly reduced the viability of CLL cells in culture.
While some cyano- and carotenoid-algae strongly inhibit the AML cell lines HL-60 and MV-4-11 as well as several primary leukemia blasts, we also observed cytotoxic effects on normal hematopoietic cells as demonstrated by Annexin V staining of light density peripheral blood cells. Of the four extracts we examined, each showed a pattern of inhibition that was different, indicating there are probably several compounds and mechanisms that are active against the malignant cells as well as normal light density marrow cells and hematopoietic progenitor cells. The role, if any, that these extracts have on normal or leukemic stem cells in vivo remains to be determined.
It is recognized that these in vitro studies of direct cytotoxicity are limited in that they are conducted apart from the tumor microenvironment and immunologic milieu of the host. Furthermore, these algal compounds are ingested in forms that do not allow measurement of feasible serum or tissue concentrations to compare with the in vitro concentrations employed here.