Effects of diallyl trisulfide on induction of apoptotic death in murine
leukemia WEHI-3 cells in vitro and alterations of the immune
responses in normal and leukemic mice in vivo
Fang-Ming Hung1,#, Hsu-Feng Lu2,3#, Nou-Ying Tang4, Jen-Jyh Lin5,6, Kung-Wen
Lu7, Jing-Pin Lin8, Yang-Ching Ko9, Chien-Chih Yu10, Jung-Chi Liao11,
Hai-Lung Wang11,* and Jing-Gung Chung12,13,*
1Department of Surgical Intensive Care Unit, Far Eastern Memorial Hospital, New
Taipei 220, Taiwan, 2Department of Clinical Pathology, Cheng Hsin General
Hospital, Taipei 112, Taiwan, 3Department of Restaurant, Hotel and Institutional
Management, Fu-Jen Catholic University, New Taipei 242, Taiwan, 4School of
Chinese Medicine, China Medical University, Taichung 404, Taiwan, 5Graduate
Institute of Chinese Medicine, China Medical University, Taichung 404, Taiwan,
6Division of Cardiology, Department of Medicine, China Medical University
Hospital, Taichung 404, Taiwan, 7School of Post-Baccalaureate Chinese Medicine,
China Medical University, Taichung 404, Taiwan, 8School of Chinese Medicine,
China Medical University, Taichung 404, Taiwan, 9Division of Pulmonary and
Critical Care Medicine, Department of Internal Medicine, St. Martin De Porres Hospital, Chiayi 600, Taiwan, 10School of Pharmacy, China Medical University,
Taichung 404, Taiwan, 11Department of Medical Laboratory Science and
Biotechnology, Yuanpei University, Hsinchu 300, Taiwan, 12Department of Biological
Science and Technology, China Medical University, Taichung 404, Taiwan,
13Department of Biotechnology, Asia University, Taichung 413, Taiwan.
Running title: DATS affects leukemia cells in vivo and in vitro.
Correspondence to: Jing-Gung Chung, Ph.D., Department of Biological Science and Technology, China Medical University. No 91, Hsueh-Shih Road, Taichung 40402, Taiwan.
E-mail: [email protected]
Hai-Lung Wang, Ph.D., Department of Medical Laboratory Science and Biotechnology, Yuanpei University. No 306, Yuanpei St., Hsinchu 30015, Taiwan. E-mail: ha i [email protected]
Abstract. Diallyl trisulfide (DATS), a chemopreventive dietary constituent and
extracted from garlic, has been shown to against cultured many types of human cancer cell liens but the fate of apoptosis in murine leukemia cells in vitro and immune responses in leukemic mice remain elusive. Herein, we clarified the actions of DATS on growth inhibition of murine leukemia WEHI-3 cells in vitro and used WEHI-3 cells to generate leukemic mice in vivo, following to investigate the effects of DATS in animal model. In in vitro study, DATS induced apoptosis of WEHI-3 cells through the G0/G1 phase arrest and induction of caspase-3 activation. In in
vivo study DATS decreased the weight of spleen of leukemia mice but did not affect
the spleen weight of normal mice. DATS promoted the immune responses such as promotions of the macrophage phagocytosis and NK cell activities in WEHI-3 leukemic and normal mice. However, DATS only promotes NK cell activities in normal mice. DATS increases the surface markers of CD11b and Mac-3 in leukemia mice but only promoted CD3 in normal mice. In conclusion, the present study indicates that DATS induces cell death through induction of apoptosis in mice leukemia WHEI-3 cells. DATS also promotes immune responses in leukemia and normal mice in vivo.
Key words: DATS, WEHI-3 leukemia cells, caspase-3, apoptosis, immune responses,
Introduction
Leukemia is one of the caused deaths of the malignancies in the human population . Numerous efforts towards the development of adjuvant and neoadjuvant therapies have been made to improve survival rate . Leukemia generally responds poorly to conventional treatment modalities such as chemotherapy and radiation therapy . The toxicity and inherent resistance of chemotherapeutic agent are may present, and there is no consensus regarding optimal therapeutic agents in leukemia. Thus, the development of novel approaches to prevent and treat leukemia is an important mission. Epidemiological studies have demonstrated that diet rich in fruits, vegetables and some spices may be protective against various human malignancies .
It was reported that consuming total Allium vegetables in men has significantly lower the risk of prostate cancer and the anti-carcinogenic effect of Allium vegetables is via the organosulfur compounds (OSCs) that are generated upon processing from these vegetables . Furthermore, animal models have been shown that diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS) are the garlic-derived organosulfur compounds which offer significant protection against cancer, resulting from induction of a variety of chemical carcinogens . DATS has been recognized as an antioxidant that has anti-proliferative and anti-carcinogenic properties . DATS-induced cell-cycle arrest is mediated by reactive oxygen species (ROS) and activation of checkpoint kinase 1 (Chk1) which is associated with c-Jun
N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK)-regulated
phosphorylation of Bcl-2 in human prostate cancer cells (PC-3 and DU145).
DATS has been reported to increase histone acetylation and p21WAF1/CIP1
expression in human colon tumor cell lines and to increase the effectiveness of TNF-related apoptosis-inducing ligand (TRAIL) death receptor, and it suppresses
prostate cancer growth in an orthotopic model . DATS inhibits cyclooxygenase-2 gene expression in HEK 293T cells . DATS can augment the activation of T cells and enhance the antitumor function of macrophage and it was suggested that DATS may be potentially useful in tumor therapy . However, there is no available information to show that whether DATS provokes apoptosis in murine leukemia cells and promotes immune responses in leukemic mice in vivo. The goal of the present study is investigated the cytotoxic effects of DATS on murine myelomonocytic leukemia WEHI-3 cells in vitro and examined the influences of DATS on immune responses in normal and leukemia mice in vivo. And we found that DATS induced cell cycle arrest and triggered apoptosis in WEHI-3 cells, and it promotes immune responses in leukemic and normal BALB/c mice in vivo.
Materials and methods
Materials and reagents. Diallyl Trisulfides (DATS), dimethyl sulfoxide (DMSO),
propidium iodide (PI), triton X-100 and trypan blue were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). RPMI-1640 medium, fetal bovine serum (FBS), penicillin-streptomycin and L-glutamine were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA).
Murine WEHI-3 leukemia cell line. The murine myelomonocytic leukemia cell line
(WEHI-3) was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were maintained in RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 Units/ml penicillin and 100 μg/ml streptomycin in
culture plastic flasks (75-cm2) at 37°C under a humidified 5% CO
2 atmosphere. The
Assessment of cell viability of WEHI-3 cells. A total of 2×105 cells/well of WEHI-3
cells were cultured in 12-well plates and incubated at 37°C for 24 h before each well were treated with 0, 1, 2, 3, 4 and 5 μM DATS for 24 h. DMSO (0.1% solvent for DATS) was used for the control regimen. At the end of incubations, cells were harvested from each treatment and then analyzed immediately by a PI exclusion method and using a FACSCalibur cytometer (Becton Dickinson, San Jose, CA, USA) as previously described .
Determinations of sub-G1 phase (apoptosis) and DNA content by flow cytometry.
Approximately 2×105 cells/well of WEHI-3 cells were cultured in 12-well plates and
exposed to 0, 1, 2 and 3 μM DATS for 24 h. At the end of treatment, cells were collected by centrifugation for 5 min at 4°C, at 1000 rpm, washed twice with ice-cold PBS and then fixed in ice-cold 75% ethanol overnight at -20°C. Next, cells were centrifuged for 5 min at 4°C at 1000 rpm, washed and resuspended in 1 ml of DNA staining solution (20 μg/ml of PI, 0.1% Triton-X 100 and 100 μg/ml of RNase A in PBS) for 30 min in the dark. Then cells were analyzed sub-G1 and cell cycle distribution by flow cytometric protocol . Cell-cycle distributions were calculated on DNA plots by ModFit LT software (Verity Software House Inc., Topsham, ME, USA). All experiments were performed at least three times.
Assay for caspae-3 activity and its specific inhibitor for cell viability. Approximately
2×105 cells/ml of WEHI-3 cells were pretreated with or without the caspase-3
inhibitor (Z-IETD-FMK) before cells were treated with 2 μM DATS for 12, 24 or 48 h. WEHI-3 cells from each treatment were harvested for determination of caspase-3
activity according to the manufacturer’s guideline (PhiPhiLuxTM-G
OncoImmunin, Inc. Gaithersburg, MD, USA) and viability in DATS-treated cells as described elsewhere . Data were analyzed using the CellQuest Pro software (BD Biosciences) and the MFI been used to correspond to the relative level of caspase-3 activity .
Establish leukemic BALB/c mice and normal mice. All male BALB/c mice 4 weeks of
age and approximately 22-28 g in weight were obtained from the National Laboratory Animal Center (Taipei, Taiwan). Fifty animals were randomly divided into 5 groups to receive different treatments. Then thirty mice from randomly 3 groups were
intraperitoneally (i.p.) injected with 1×105 WEHI-3 cells for 2 weeks and then were
randomly separated into 3 groups as a model of leukemia. Group I served as a control (10 animals). Group II mice were treated with olive oil as vehicle. Group III animals were treated with olive oil after intraperitoneal injection WEHI-3 cells. Group IV and V mice were treated with DATS (1 and 10 mg/kg, respectively) in olive oil after intraperitoneal injection of WEHI-3 cells. DATS was administered by oral gavage to the treatment groups the above dose daily for up to 2 weeks before being weighed .
For the normal mice experiment, as above designs, forty mice were divided into 4 groups (10 animals per group) without injections with WEHI-3 cells and orally administered with or without 1 and 10 mg/kg of DATS, respectively . Mice were treated daily for 14 days before being weighed and sacrificed.
Body, spleen and liver tissues weights. At the end of treatment, all animals were
weighed and blood withdrawn. Spleen and liver samples were isolated and weighed individually .
treatment, blood samples of 1 ml from all animals were collected before mice were sacrificed. Collected blood sample were individually exposed to Pharm Lyse lysing buffer (BD Biosciences, San Jose, CA, USA) for lysing of the red blood cells and then all samples were centrifuged for 15 min at 1500 rpm at 4°C. The isolated white blood cells from each group were stained by the fluorescein isothiocyanate (FITC) anti-mouse CD3, phycoerythrin (PE) anti-mouse CD19, FITC anti-mouse CD11b and PE anti-mouse Mac-3 antibodies (BD Pharmingen Inc, San Diego, CA, USA) before being analyzed to determine the levels of cell marker by flow cytometry as previously described .
Quantification of phagocytic activity of macrophages. Macrophage phagocytosis was
used the PHAGOTEST kit (Glycotope Biotechnology GmbH, Heidelberg, Germany) as previously described . In brief, a total of 1×105 leukocytes in 100 μl whole blood
from each animal of each group was incubated for 1 h at 37°C with FITC-labeled
Escherichia coli (E. coli) (2×107 bacteria in 20 μl 1× solution from the kit). The
quenching solution (100 μl) was added according to the manufacturer’s instruction. After the completion of phagocytosis by monocytes/macrophages, DNA was stained according to the manufacturer’s protocol. Peritoneal macrophages from each mouse in DATS-treated or un-treated groups were isolated as described elsewhere . Cells from each animal were analyzed by flow cytometery as previously describe . Fluorescence data were collected on 10,000 cells and analyzed using the BD CellQuest Pro software.
Assay for natural killer (NK) cell cytotoxicity. A total of 1×105 leukocytes from the
spleens of all individual animals in 1 ml of RPMI-1640 medium were individually
cells, a target cells of NK cells, were cultured in 15-mL tubes with serum-free RPMI-1640 medium and then PKH-67/Dilunt C buffer (Sigma-Aldrich, St Louis, MO, USA) was added to the cells, mixed thoroughly for 2 min at 25˚C then 2 ml PBS was added for 1 min. RPMI-1640 medium at 4 ml was added for a 10 min-incubation then were followed by centrifugation at 1200 rpm of 25˚C. YAC-1 cells in 100 μl were placed on 96-well plates before the addition of the leukocytes from each treatment to the wells for 12 h and determination of NK cell activity by flow cytometry and a PI exclusion assay as previously described .
Statistics analysis. The results were expressed as mean ± S.D. and the difference
between DATS-treated and control groups was analyzed by one-way ANOVA followed by Dunnett's multiple comparison test and Student’s t test. A p value <0.05 was considered as a significant difference.
Results
DATS treatment induced cell death, caused apoptosis and promoted G0/G1 phase arrest in murine leukemia WEHI-3 cells. To address the effects of DATS on the cell
viability and cell cycle distribution of WEHI-3 cells, we used WEHI-3 cells as models and employed a PI exclusion method as an assay system to analyze the effects by flow cytometric assay. Our results shown in Figure 1 indicated that DATS exhibited the killing effect in a dose dependent efficiency in suppressing the survival rates (Figure 1A). On the basis of the growth inhibitory effect of DATS in WEHI-3 cells, we next examined its effect on cell cycle using various concentrations (0, 1, 2, and 3 μM) of DATS (Figure 1B). As summarized in Figure 1C, WEHI-3 cells displayed G0/G1 phase arrest at 24 h after exposure to DATS. After 24 h of treatment, DATS at 3 μM exposure induced 18% G0/G1 percentage in WEHI-3 cells when compared to that of
control (0 μM). In addition, DATS increased the sub-G1 population (apoptosis) throughout the dose-points we examined (Figure 1D). DATS caused G0/G1 phase arrest approximately increased by 0.48-fold (40% to 59.2%) in WEHI-3 cells after a 24-h treatment. We also proceeded to characterize pro-apoptotic effects of DATS using 4’-6-diamidino-2-phenylindole (DAPI) staining (data not shown). We found that WEHI-3 cell were treated with DATS for 24 h s resulted in enrichment of apoptotic cells with condensed chromatin, which indicated the reaction of apoptotic cells increased upon treatment with DATS in a concentration- dependent manner in WEHI-3 cells.
DATS promoted caspase-3 activity and Z-DEVD-FMK increased the viability of WEHI-3 cells after exposure to DATS. Cells after treatment with DATS for 12, 24 and
48 h were harvested for determining caspase-3 activity and for percentage of viability determinations as can be seen in Figure 2. The results indicated that DATS promoted the activity of caspase-3 (Figures 2A and B) and these effects are time-dependently. WEHI-3 cells were pretreated with Z-DEVD-FMK (a caspase-3 inhibitor) and exposed to 2 μM DATS for a 24-h incubation. Cells were then harvested and determined, and the percentage of viable WEHI-3 cells was measured. Figure 3C indicates that DATS-treated cells before Z-DEVD-FMK pretreatment increased the viable cells when compared to DATS-treated cells only. These observations indicated that DATS induced cell apoptosis via caspase-3-dependent signaling in WEHI-3 cells.
DATS affected the spleen weight in WEHI-3 leukemic BALB/c mice. At the end of
DATS treatment, body weights of each animal from each group were weighed then spleen or liver tissues were isolated and were weighed. Results shown in Table 1 indicated that DATS did not affect the mice and there is no significant increase in the
body weights of each treatment as compared to the leukemia control. Importantly, DATS significantly decreased the spleen weight in comparison to leukemic mice group but there was no significant difference (p>0.05) in liver weight between leukemic and DATS-treated mice as seen in Table 1.
DATS affected on specific surface markers of leukocytes from WEHI-3 leukemic BALB/c mice. In order to investigate whether DATS affects the levels of cell surface
marker, leukocytes from DATS-treated or untreated groups were isolated and the levels of CD3, CD19, CD11b and Mac-3, respectively, were measured using flow cytometric analysis. The data for cell markers in Table 1 indicated that DATS did not significantly affect the levels of CD3 and CD19 in leukemic mice. However, DATS promoted CD11b (1 mg/kg/day and 10 mg/kg/day: 11.5% and 19.3%) (Table 1) in both examined doses and it also promoted Mac-3 (10 mg/kg/day: 8.9%) when compared to the WEHI-3 only treated mice group.
DATS promoted the phagocytosis by macrophages from peripheral blood mononuclear cell (PBMC) and peritoneal cavity in WEHI-3 leukemic BALB/c mice.
To clarify if whether or not DATS alters phagocytosis in leukemic mice, the leukocytes from DATS-treated or olive oil-treated groups were isolated and phagocytic activity was measured. Results are shown in Table 1, which indicated that DATS (1 and 10 mg/kg/day) promoted the activity of phagocytosis from PMBC (1 mg/kg/day: 9.4%; 10 mg/kg/day: 8.9%) and peritoneal cavity (10 mg/kg/day: 7.1%) from leukemic mice.
DATS stimulated NK cell cytotoxicity and affected T- and B-cell proliferation from splenocytes in WEHI-3 leukemic BALB/c mice. The leukocytes from DATS-treated or
untreated groups were isolated and NK cell activity was determined as can be seen in Figure 3A. Results indicated that the YAC-1 target cells were killed by NK cells from DATS-treated group when compared to the untreated groups at 10 mg/kg/day at target cells ratio of 25:1 (Figure 3A), but it did not show any significant difference at 50:1 (Figure 3A). This dose (DATS 10 mg/kg per day) was effective at 25:1 target ratio and increased the activity of NK cells (10 mg/kg/day, 13.4% at target cells ratio of 25:1). We also investigated whether or not DATS affects B- and T-cell proliferation in leukemic mice. Results shown in Figure 3B indicated that DATS at high dose treatment (10 mg/kg/day) inhibited the B cell proliferation, but both doses did not affect T cell proliferation (p>0.05).
DATS affected on T cells, macrophage phagocytosis and NK cell cytotoxicity from normal BALB/c mice. To explore whether DATS influences the levels of cell surface
marker from normal animals, leukocytes from DATS-treated or untreated groups were isolated and levels of CD3, CD19, CD11b and Mac-3 were measured and the data indicated that DATS only promoted the level of CD3 (Figure 4A) at 1 and 10 mg/kg/day treatment, but it did not significant affect the levels of CD19, CD11b and Mac-3 (data not shown).
Moreover, DATS affected phagocytosis in DATS-treated or un-treated mice, and the leukocytes were isolated from PBMC and peritoneal cavity to measure phagocytic activity. Results shown in Figure 8B and C indicated that DATS (1 and 10 mg/kg/day) did not promoted macrophage phagocytosis from PMBC (Figure 4B) and peritoneal cavity (Figure 4C) from normal mice. The results from NK cell cytotoxicity are shown in Figure 4D and indicated that the YAC-1 target cells were killed by NK cells from DATS-treated group when compared to the untreated groups
at 1 and 10 mg/kg/day at target cells ratio of 25:1 and 50:1 (Figure 4D). Both doses were effective at target ratio and increased the activity of NK cell cytotoxicity at ratio of 25:1 and 50:1 as shown in Figure 4D. For B- and T- cell proliferation examination, DATS did not show significant responses in B- and T-cell proliferation between DATS-treated and un-treated normal mice (data not shown).
Discussion
It is well documented that the suppression of cancer cell growth by many natural products, such as phenylethyl isothiocyanate, benzyl isothiocyanate and sulforaphane from cruciferous vegetable , paclitaxel from Taxus brevifolia and traditional oriental medicine constituents that induced apoptosis of cancer cells . Garlic has been used as vegetable and s supplement in many kind of food of human for long time ago . Diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS) are major components from garlic and many reports have been shown those compounds can induce cytotoxic effects on cancer cells .
Numerous evidences have been shown that DATS act as an anticancer agents and it can induced apoptosis in many human cancer cell lines . Herein, we also found that DATS induced cytotoxic effects through the induction of G0/G1 phase arrest and apoptosis in mice leukemia WEHI-3 cells (Figure 1). Furthermore, DATS induced apoptosis in WEHI-3 cells are via caspase-3 activation in vitro (Figure 2). Our earlier reports also show that DAS and DADS can inhibits murine WEHI-3 leukemia cells in BALB/c mice in vitro and in vivo through modulating immune responses . Other investigators in earlier reports DATS can augment the activation of T cells and enhance the antitumor function of macrophage . However, there is no available information to address the effects of DATS on induction of apoptosis of murine WEHI-3 leukemia cells in vitro and the immune responses in leukemia mice in vivo.
In the present study, we established leukemic BALB/c mice through the injection of WEHI-3 cells , and then chronically treated mice with DATS. Our findings indicated that DATS decreased the weight of spleen, but it did not significantly affect the weights of animal body and liver in leukemic mice when compared to the leukemic mice without treatment with DATS (control) (Table 1).
Results from the examination of cell markers from leukemic mice after oral treatment of DATS indicated that the percentages of CD11b (monocytes) and Mac-3 (macrophages) were significantly increased in DATS-treated leukemic mice, but the population of CD3 (T cells) and CD19 (B cells) were not significantly affected (p>0.05) (Table 1). However, in normal mice after treatment with DATS, only CD3 (T cells) level was increased (Figure 4A) and other types of cell surface markers did not alter (data not shown) between DATS-treated and un-treated normal mice. The results suggested that DATS inhibited leukemia-related spleen growth in leukemic mice in vivo.
Herein, we also found that DATS promoted the phagocytosis of macrophages (Table 1) and elevated the NK cell cytotoxicity (Figure 3A) in leukemic mice. Furthermore, DATS could not only promote the immune response (promoted NK cell activities), but also increase the cell population of monocytes and macrophages. Instead, DATS did not decrease the T- and B-cell (Table 1) population in the leukemic mice. Numerous evidences demonstrated that macrophage phagocytosis and NK cell activities both are played major roles for immune responses after animals were exposed to antigen . These results may suggest that DATS is able to increase the immune response and might via increasing the activities of macrophages and NK cells against leukemia in general. These observations are in agreement with the results from normal mice showing that oral treatment of DATS also promoted immune responses
(Figure 4). Alternatively, DATS did not influence the body, spleen and liver weights in normal BALB/c mice (data not shown)
In this study, we investigated that DATS induced cytotoxic cell death through the induction of G0/G1 phase arrest and apoptosis through the caspase-3 activation in mouse leukemia WEHI-3 cells in vitro. Furthermore, we also found that DATS could promote immune responses in BALB/c leukemic and normal mice in vivo. DATS acts as a potent immunological adjuvant in vivo and anticancer agent, and its application provides an effective strategy to improve the efficacy of immune responses in vivo in the future.
Acknowledgements
This study was supported by the grant DOH101-TD-C-111-005 from the Taiwan Department of Health, China Medical University Hospital Cancer Research Center of Excellence and by the grant CMU97-127 from China Medical University, Taichung, Taiwan.
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Figure legends
Figure 1. DATS decreased the percentage of viability, induced cell cycle arrest and
sub-G1 population in WEHI-3 cells. Cells were cultured in RPMI-1640 + 10% FBS with 0, 1, 2, 3, 4, and 5 μM DATS for 24 h. The percentages of viable WEHI-3 cells (A) were determined, and DNA content (B and C) and sub-G1 group (D) were analyzed by flow cytometry as described in the Materials and Methods. Each point is mean ± S.D. of three experiments. *p < 0.05, significantly different when compared with DMSO-treated control (0 μM) and DATS treatment samples.
Figure 2. DATS stimulated the caspase-3 activity of WEHI-3 cells. Cells were
pretreated with or without a caspase-3 inhibitor (Z-DEVD-FMK) and then exposed to 2 μM DATS for 0, 12, 24 and 48 h. The cells were harvested, washed with PBS and then were measured the caspase-3 activity as described in the Materials and Methods. (A) Representative profile of caspase-3 activities; (B) quantative data of caspase-3 activity; (C) the percentage of viable cells after WEHI-3 cells preincubation with Z-DEVD-FMK. Each experiment was done with triple sets (Mean ± S.D.): *p < 0.05 shows a significant difference in DATS-treated groups.
Figure 3. DATS affected the NK cell cytotoxicity and cell proliferation from
leukemic BALB/c mice. The animals were intraperitoneally injected with WEHI-3 cells for 2 weeks and were orally treated with or without DATS for 2 weeks. The YAC-1 target cells were killed by NK cells of splenocytes from the mice after treatment with DATS by oral administration at 1 and 10 mg/kg/day in target cells ratio of 25:1 and 50:1 (A). The cell proliferation (B) was performed as described in the Materials and Methods. Each point is mean ± S.D. *p<0.05 was considered
significantwhen compared with the WEHI-3 leukemia mice (n=10).
Figure 4. DATS affected the levels of T-cell marker and NK cell activities from
normal mice. The animals were orally treated with or without DATS for 2 weeks. Blood was collected from each group animal and was analyzed for CD3 cell markers (A: percentage of CD3 levels) by flow cytometry. The percentages of phagocytosis with phagocyte green fluorescent particles (FITC-E. coli.) at DATS oral treatment were determined by flow cytometric analysis (B: macrophages phagocytosis from PBMC. C: macrophages phagocytosis from peritoneal cavity.). The YAC-1 target cells were killed by NK cells of splenocytes from the mice after treatment with DATS by oral administration at 1 and 10 mg/kg/day in target cells ratio of 25:1 and 50:1 (D: NK cell cytotoxicity.) as described in the Materials and Methods. Each point is mean ± S.D.*p<0.05 was considered significant when compared with the normal mice group (n=10).
