Dunaliella salina Exhibits an Antileukemic Immunity in a Mouse Model

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Article pubs.acs.org/JAFC

Dunaliella salina Exhibits an Antileukemic Immunity in a Mouse Model of WEHI‑3 Leukemia Cells Wen-Chen Chuang,†,§ Yung-Chyuan Ho,⊗ Jiunn-Wang Liao,*,‡ and Fung-Jou Lu*,§,⊗,○ †

Department of Veterinary Medicine and ‡Graduate Institute of Veterinary Pathobiology, National Chung Hsing University, Taichung 402, Taiwan § Institute of Medicine and ⊗School of Medical Applied Chemistry, Chung Shan Medical University, Taichung 402, Taiwan ○ Department of Medical Research, Chung Shan Medical University Hospital, Taichung 402, Taiwan ABSTRACT: Dunaliella salina has been shown to have antioxidant property and induce apoptotic cell death of human cancer cells in vitro. However, there is no information available on D. salina showing an antileukemia effect or immunomodulatory activity in vivo. This study applied D. salina to syngeneic leukemia-implanted mice (BALB/c and WEHI-3) to investigate its immunological and antileukemia properties. Oral administration of D. salina (184.5, 369, and 922.5 mg/kg) inhibited spleen metastasis and prolonged the survival in BALB/c mice that had received an intravenous injection of WEHI-3 cells. The results revealed that D. salina had reduced spleen enlargement in murine leukemia. It had also increased the population and proliferation of T-cells (CD3) and B-cells (CD19) following Con A/LPS treatment on flow cytometry and MTT assay, respectively. Furthermore, D. salina increased the phagocytosis of macrophages and enhanced the cytotoxicity of natural killer cells on flow cytometry and LDH assay. Moreover, D. salina enhanced the levels of interferon-γ and interleukin 2 (IL-2) but reduced the levels of IL-4 and IL-10 in leukemic mice. In conclusion, these results demonstrated that the application of D. salina had beneficial effects on WEHI-3 leukemic mice by prolonging survival via modulating the immune responses. KEYWORDS: Dunaliella salina, antileukemia, immune responses

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INTRODUCTION Algae have been widely consumed for a long time as nutritional or pharmaceutical agents because of their biofunctional substance. Dunaliella salina is a halophic unicellular microalga from the Chlorophyceae class. Significant amounts of three valuable products, glycerol, β-carotene, and proteins, are accumulated when the alga experiences high salinity or high intensity of light stress. As D. salina lacks a cell wall, it is easily digested.1−3 According to mutigenerational and toxicological studies, D. salina is safe for human consumption.3,4 As D. salina contains abundant β-carotene, it has been utilized as a functional food (i.e., pro-vitamin A supplement and hepatoprotective food).1,5−7 β-Carotene has been investigated for its anticancer properties, both in vitro and in vivo.8−12 As cis bonds are more reactive than trans bonds, the 9-cis isomer has been shown to be more antioxidative. In contrast, all-trans-βcarotene and α-carotene are more easily absorbed than the 9-cis β-isomer.13,14 Our previous analysis showed that the major carotenoids in D. salina were all-trans-β-carotene and 9- or 9′cis-β-carotene.5,15 When the extract of D. salina was compared with pure all-trans-β-carotene, α-carotene, lutein, and zeaxanthin, the extract of D. salina showed the highest antioxidant activity.5 We also showed that D. salina suppressed lipopolysaccharide-stimulated inflammation in vitro15 and ameliorated carbon tetrachloride or UV-B-induced oxidative stress in vivo.6,16 Immune responses to tumors include humoral and cellular immunity.17−19 B-cell-mediated humoral immune defense is a specific antigen antibody reaction. The reaction can neutralize toxins and protect against pathogen-induced infection. T-cells also release many regulatory factors including lymphotoxin, © XXXX American Chemical Society

macrophage mobile factor, interferon, transfer factor, etc. These factors can promote differentiation and proliferation of immune cells, activity of natural killer cells (NK cells), and phagocytosis of macrophages. Thus, both antibody- and cell-mediated immune defense contribute to anticancer potential.19 Recently, the anticancer activity of D. salina had been investigated in various cancer cells including lung,9 skin, and prostate cancer cell lines.8 We hypothesized that D. salina might induce apoptosis, differentiation, and immunomodulation activities against leukemia. As there had been no report on the effects of D. salina on immunomodulatory responses or on antileukemia activity in vivo, our study focused on these aspects of D. salina. The syngeneic leukemia-implanted mice (BALB/c versus WEHI-3) had been used for evaluating the antileukemia activity of drugs and natural products for many years. In this study, D. salina has been shown to have antileukemia properties via increasing the immunity in WEHI-3 leukemic mice.

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MATERIALS AND METHODS

Materials and Reagents. Commercially available spray-dried preparations of D. salina were cultured under light and 20% salt cultivation in the outdoor cultivation pool at GONG BIH Enterprise Co., Ltd. (Yunlin City, Taiwan, ROC). The contents of water, protein, fat, ash, sugar, and dietary fiber were 40, 466, 220, 57, 130, and 87 mg/ g algae, respectively. The quality and authenticity of D. salina powder were described and provided by the company. The carotenoid Received: July 28, 2014 Revised: November 5, 2014 Accepted: November 7, 2014

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dx.doi.org/10.1021/jf503564b | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

Figure 1. Effects of D. salina on survival rate and body weight. BALB/c mice were iv injected with WEHI-3 cells (1 × 105 cells/mice) and treated with D. salina by oral administration for 4 weeks. (n = 10, mean ± SD.) ∗, ∗∗, and ∗∗∗ indicate significant difference between two groups at p < 0.05, p < 0.01, and p < 0.001, respectively. contents in the D. salina of all-trans-β-carotene, 9- or 9′-cis-β-carotene, all-trans-zeaxanthin, all-trans-lutein, 13- or 13′-cis-β-carotene, all-transα-carotene, and 9- or 9′-cis-β-carotene were 138.25, 124.65, 11.27, 6.55, 4.95, 2.69, and 2.41 mg/g algae, respectively, measured by HPLC as described previously.5,15 The ratio of all-trans- and cis-β-carotene, the major carotenoids in the algae, was 53:47. RPMI-1640 medium was obtained from Hyclone (South Logan, UT, USA). Fetal bovine serum (FBS) was obtained from Biological Industries (Kibbutz Beit Haemek, Israel). Penicillin−streptomycin and glutamine were obtain from Gibco BRL (Grrand Island, NY, USA). β-Carotene was obtained from Sigma (St. Louis, MO, USA). Animals. A total of 140 male BALB/c mice, each at the age of 6 weeks and about 22−28 g in weight, were purchased from the Animal Department of BioLASCO Taiwan Co., Ltd. (Taipei City, Taiwan, ROC). The animals were maintained under a 12 h light/dark cycle in a humidity- and temperature-controlled room under standard laboratory conditions. Mice were housed five in each cage and allowed food and water ad libitum. They were acclimated and quarantined for a week prior to experimentation. The protocol for the animal study was approved by the Institutional Animal Care and Committee (IACUC no. 1074), and all mice were cared for following the institutional ethical guideline.6,16 Cell Culture. The WEHI-3 murine myelomonocytic leukemia cell line and YAC-1 murine lymphoma cell line were purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan). The cells were cultured onto 10 cm2 dishes and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 μg/mL streptomycin, 100 U/mL penicillin, and 2 mM L-glutamine at 37 °C in a 5% CO2 incubator. Experimental Designs. The experiment was made up of two parts. In part I, over a period of 4 weeks, the effect of D. salina on the survival rate in WEHI-3 leukemic mice was investigated. In part II, over a period of 2 weeks, the immunomodulatory effects of D. salina on WEHI-3 leukemic mice were investigated. Seventy BALB/c mice were randomly divided into 7 groups with 10 animals in each group. D. salina was suspended in distilled water before use and administered by oral gauge. The dosages of D. salina used in this study were calculated from the commercial product’s suggestion for daily intake (i.e., 15 mg/ kg bw/human equals 184.5 mg/kg bw/mice). Group I was the control and group II was treated with D. salina only. Groups III−VII were intravenously injected via the tail vein with WEHI-3 cells (1 × 105 cells/100 μL PBS) only. Group IV−VI were cotreated with D. salina at doses of 184.5, 369, and 922.5 mg/kg, respectively. Group VII was cotreated with 922.5 mg/kg β-carotene as the positive control. Cell Surface Markers Examination. The mice were euthanized by CO2, and whole blood was collected via heart puncture after injection of WEHI-3 cells for 2 weeks. The blood samples were

immediately lysed with BD Pharm Lyse (BD Biosciences) and then centrifuged at 1500 rpm (1000g) for 15 min at 4 °C. The collected white blood cells evaluated for cell surface markers including T-cells, B-cells, monocytes, and macrophages were respectively stained with anti-CD3-FITC, anti-CD19-PE, anti-CD11b-PE, and anti-Mac-3-FITC antibodies (SouthernBiotech Inc.) and then were analyzed by flow cytometry (FACS Calibur, Becton Dickinson, NJ, USA) as previously described.20 Organ Weight and Pathological Examination. A necropsy was performed on each mouse, and the spleen was weighed after dissection. Then, it was examined grossly and fixed in 10% formalin buffer. A transverse section in the hilus was made at the largest extension of the spleen; red and white pulps of spleen were trimmed and embedded in paraffin, sectioned to a thickness of 2 μm, and stained with hematoxylin and eosin for the microscopic examination. For semiquantitative grading, the severity of the lesions was graded on the basis of the criteria of Shackelford et al.21 with five grades of severity of lesions specified: 1 = minimal (