Phenethyl Isothiocyanate Promotes Immune Responses in Normal BALB/c Mice, Inhibits Murine Leukemia WEHI-3 Cells, and Stimulates Immunomodulations In Vivo
Mei-Fen Tsou,1 Ni Tien,1 Chi-Cheng Lu,2 Jo-Hua Chiang,2 Jai-Sing Yang,3 Jing-Pin Lin,4 Ming-Jen Fan,5 Jang-Jih Lu,1,6 Su-Peng Yeh,7,8 Jing-Gung Chung5,9
1Department of Laboratory Medicine, China Medical University Hospital, Taichung 404, Taiwan
2Department of Life Sciences, National Chung Hsing University, Taichung 402, Taiwan
3Department of Pharmacology, China Medical University, Taichung 404, Taiwan
4School of Chinese Medicine, China Medical University, Taichung 404, Taiwan
5Department of Biotechnology, Asia University 413, Taichung, Taiwan
6Graduate Institute of Clinical Medical Science, China Medical University, Taichung 404, Taiwan
7Division of Hematology and Oncology, China Medical University Hospital, Taichung 404, Taiwan
8School of Medicine, China Medical University, Taichung 404, Taiwan
9Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan
ABSTRACT: Enhanced cruciferous vegetable consumption is associated with the reduction of cancer incidence as shown in epidemiological studies. Phenethyl isothiocyanate (PEITC), one of the important compounds in cruciferous vegetables, has been shown to induce apoptosis in many types of human can- cer cell lines, but there is no available information addressing the effects on normal and leukemia mice in vivo. The purpose of this study is to focus on the in vivo effects of PEITC on immune responses of normal and WEHI-3 leukemia BALB/c mice in vivo. Influences of PEITC on BALB/c mice after intraperitoneal (i.p.) injection with WEHI-3 cells and normal mice were investigated. In normal BALB/c mice, PEITC did not affect the body weight when compared to the olive oil treated animals. Moreover, PEITC promoted phago- cytosis by macrophages from peripheral blood mononuclear cells (PBMC)
and peritoneal cavity, increased the levels of CD11b and Mac-3, decreased the level of CD19 and promoted natural killer (NK) cell cytotoxic activity, but it did not alter the level of CD3. Also, PEITC enhanced T cell proliferation after concanavalin A (Con A) stimulation. Otherwise, PEITC increased the body weight, but decreased the weight of liver and spleen as compared to the olive oil-treated WEHI-3 leukemia mice. PEITC also increased the level of CD19, decreased the levels of CD3 and Mac-3 rather than influence in the level of CD11b, suggesting that the differentiation of the precursor of macrophages and T cells was inhibited, but the differentiation of the precursor of B cells was promoted in leukemia mice. Furthermore, PEITC enhanced phagocytosis by monocytes and macrophages from PBMC and peritoneal cavity, and also pro- moted the NK cell cytotoxic activity in comparison with the group of leukemia mice. Based on these observations, the biological properties of PEITC can promote immune responses in normal and WEHI-3 leukemia mice in vivo.
Keywords: isothiocyanates; PEITC; immune responses; murine leukemia WEHI-3 cells; leukemia mice
INTRODUCTION
Numerous studies have been shown that the antitumor agents can exhibit the functions for rapid elimination of tu- mor cells through the induction of cancer cell apoptosis (Guchelaar et al., 1997; Jaffrezou et al., 1998; Debatin,
2000; Woynarowska and Woynarowski, 2002; Chiang et al.,
2011). Apoptosis is a programmed cell death process for re- moval of unwanted cells of the development and homeosta- sis of multicellular organisms (Arends and Wyllie, 1991). It is well-documented that the characters of apoptosis include membrane blebbing, translocation of phosphatidylserine of the plasma membrane from the inner to the outer leaflet, nu- clear and cytoplasmic shrinkage, DNA fragmentation occur, and activation of a family of caspase (Earnshaw et al., 1999; Strasser et al., 2000). The caspase activation is considered to be a key hallmark of apoptosis (Earnshaw et al., 1999; Lu et al., 2010). In addition, apoptotic cell death is also accom- panied by a loss of mitochondrial membrane potential (DCm) before induction of cytochrome c release and activa- tion of caspase-3 (Kluza et al., 2003; Lim et al., 2003). In Taiwan, leukemia is the twelfth most common cancer and about 4 persons per 100 thousand died annually from leuke- mia based on the reports from Department of Health, R.O.C. (Taiwan) in 2009 (Lai et al., 2010b). Although many types of treatments, including radiation, chemotherapy, or combi- nation of radiotherapy with chemotherapy have been used in leukemia patients, it is still
unsatisfying.
Conaway et al. demonstrated that dietary intake of cru- ciferous vegetables may decrease the risk of various types of malignancies (Conaway et al., 2002). The organic iso- thiocyanates (ITCs) one of the components in these vegeta- bles (Conaway et al., 2002), including phenethyl ITC (PEITC), allyl ITC (AITC), and benzyl ITC (BITC) (Kumar and Sabbioni, 2010). PEITC exhibits cancer che- mopreventive activity in rat (Stoner et al., 1991). PEITC induces apoptosis in HT-29 cells through the inhibition of cytochrome P450 (CYP) enzymes and the induction of phase II detoxification enzymes (12). Also, PEITC suppresses 4-(methylnitrosamino)-1-(3-pyridyl)-1-butone-induced pulmonary neoplasia in A/J mouse lung (Morse et al., 1989) and reduces azoxymethane-induced colonic aberrant crypt foci formation (Zhang et al., 1994). Many reports have shown that PEITC induced apoptosis in many types of tumor cell lines (Chen et al., 2002; Kuang and Chen, 2004; Rose et al., 2005; Wu et al., 2005; Hwang and Lee, 2010). Our previous studies also showed that PEITC inhibited migration and invasion of human gastric cancer AGS cells (Yang et al., 2010b) and colon cancer HT29 cells (Lai et al., 2010a), but there are no reports to show that PEITC promoted immune responses in normal and WEHI-3 leukemia mice in vivo. Therefore, the purpose of the present study was to investigate the hypothesis of PEITC-affecting the immune responses in normal and leu- kemia mice in vivo.
MATERIALS AND METHODS Materials and Reagents
PEITC and olive oil were obtained from Sigma-Aldrich (St. Louis, MO). RPMI 1640 medium, fetal bovine serum (FBS), penicillin-streptomycin, and L-glutamine were obtained from Invitrogen Life Technologies (Carlsbad, CA).
BALB/c Mice
Ninety male BALB/c mice 8 weeks of age (rv22–28 g) were purchased from the Laboratory Animal Center, College of Medicine, National Taiwan University (Taipei, Taiwan).
WEHI-3 Cells
The murine myelomonocytic leukemia cell line (WEHI-3) was purchased from the Food Industry Research and Devel- opment Institute (Hsinchu, Taiwan). Cells were maintained onto 75 cm2 cell culture flasks with RPMI 1640 medium containing 100 U mL21 penicillin, 100 lg mL21 strepto- mycin, 2 mM L-glutamine and 10% FBS at 378C under a humidified 5% CO2 and one atmosphere (Yu et al., 2010).
Normal Mice Studies PEITC Treatment
Forty BALB/c mice were divided into four groups (10 ani- mals per group). Group I was control and group II was only treated with olive oil orally. Group III was orally treated with PEITC (40 mg kg21) in olive oil. Group IV was treated with PEITC (80 mg kg21) in olive oil by oral administration (Xiao and Singh, 2010). Mice were treated daily for 30 days before being weighed and sacrificed (Yang et al., 2010a). WEHI-3 Leukemia Mice Studies
Establishment of Leukemia Mice and PEITC Treatment
Fifty BALB/c mice were divided into five groups (10 ani- mals per group). Group I was control and group II was only orally treated with olive oil. Group III was intraperitoneally (i.p.) injected with WEHI-3 cells (1 3 105 cells/mice) in phosphate buffered saline (PBS) only. Group IV and V was intraperitoneally injected with WEHI-3 cells (1 3 105 cells/mice) and then treated with PEITC (80 and 160 mg kg21, respectively) in olive oil. After WEHI-3 cells intra- peritoneal injection for 2-week incubation, animals were orally treated daily for 2 weeks before being weighed and sacrificed (Yang et al., 2010a; Yu et al., 2010).
Blood Collections and Immunofluorescence Staining
All mice were treated as described above, and 1 mL of whole blood was collected from all animals (normal and WEHI-3 cells-injected mice). The blood sample was immediately exposed to 13 Pharm LyseTM lysing buffer (BD Biosciences, San Jose, CA) for lysing of the red blood cells and then was centrifuged at 1500 rpm at 48C for 15 min as previously added to stop the reaction. Phagocytosis by monocytes and macrophages was determined, and the analysis of DNA content was measured for viable cells as previously described (Chang et al., 2009). Fluorescent data were col-lected and analyzed by flow cytometery. All data were cal- culated by using BD CellQuestTM Pro Software (Becton Dickinson) (Su et al., 2008; Tsou et al., 2009). Natural Killer (NK) Cell Cytotoxic Activity
Tissues of spleen were isolated from each mouse of both studies and then were prepared for the splenocytes as previ- ously described (Chang et al., 2009; Lin et al., 2010). The rv1 3 105 splenocytes in 1 mL of RPMI 1640 medium were cultured in each well of 96-well plates. YAC-1 (2.5 3 107 cells) target cells in 15 mL tubes with serum-free RPMI 1640 medium and the PKH-67/Dil. C buffer (Sigma-Aldrich Corp.) was added to the cells then mixed thoroughly for 2 min at 258C and then 2 mL PBS was added for 1 min. Four milliliter medium was added for a 10-min incubation and then centrifuged at 1200 rpm and 258C. YAC-1 cells (2.5 3 106) were placed into
96-well plates for 100 lL before the addition of the leukocytes to the 96-well for 6 h and determina- tion of the NK cell cytotoxic activity by flow cytometry as described elsewhere (Chang et al., 2009; Lin et al., 2010).
Determinations for T and B Cell Proliferation Spleen tissues from mice in each group were isolated and described (Lin et al., 2009) for isolation of white blood cells the splenocytes (1 3 10 cells/well) were isolated from the (WBC). Isolated WBC were measured for cell surface markers of T-cell (CD3), B-cell (CD19), monocyte, and macrophage (CD11b and Mac-3) (WEHI-3 is a myelomonocytic leukemia spleens of each mouse. One hundred microliters RPMI 1640 medium was added, and splenocytes were placed in 96-well plates and stimulated with concanavalin A (Con A, cell line) by using anti-CD3-FITC, CD11b-FITC, CD19-PE, for 3 days and lipopolysaccharide (LPS, and Mac-3-PE antibodies (BD Pharmingen, San Diego, CA) for determining the levels of cell marker by flow cytometry (FACS CaliburTM, Becton Dickinson, Franklin Lakes, NJ) as described elsewhere (Hsu et al., 2011; Yang et al., 2010a).
Body Weight, Liver, and Spleen Tissues
All mice were weighed before blood was drawn for the other tests. Liver and spleen were isolated and also weighed for individual mice from both study animals (Su et al., 2008).
Phagocytic Activity of Macrophages
The level of phagocytosis by macrophages was measured by using the PHAGOTEST kit (Glycotope Biotechnology, Heidelberg, Germany). Cells were isolated from PBMC and peritoneum in experimental animals of both studies. Isolated cells from each mice were individually incubated for 1 h at 378C with opsonised fluorescein isothiocyanate (FITC)-labeled E. coli (20 lL) according to the manufac- turer’s protocols. The quenching solution (100 lL)) for 5 days. Splenocytes were collected and the cells were also determined for the cell proliferation by using CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI) as previously described (Chang et al., 2009; Yang et al., 2010a).
Statistical Analysis
All data were expressed as mean 6 S.D. and differences between control and PEITC-experimental groups were analyzed by one-way ANOVA followed by Dunnett’s test or Student’s t test. *P less than 0.05 was used as the level of significance.
RESULTS
PEITC Affected the Body Weight, Spleen, and Liver from Normal or WEHI-3 Cells-Injected Mice
The effects of PEITC on the body weights of normal and WEHI-3 cells-injected leukemia mice are presented in Figure 1(A,B), which indicated that PEITC did not influ- ence the body weight in normal mice [Fig. 1(A)], but it can increase the body weight of leukemia mice [Fig. 1(B)] as compared with WEHI-3 only injected-mice. Tissues of spleen and liver were isolated from leukemia mice and weighed individually. The effects of PEITC on the weights of spleen and liver from WEHI-3 injected mice are shown in Figure 1(C,D), indicating that PEITC decreased the weight of spleen [Fig. 1(C)] and liver [Fig. 1(D)]. These effects are dose-dependent manners. PEITC Affected the Phagocytotic Activity of Normal BALB/c and WEHI-3 Leukemia Mice
Changes of macrophages with phagocytosed green fluores- cent (fluorescein isothiocyanate, FITC) particles from PBMC and peritoneal cavity of control and PEITC-treated groups from both studies are presented in Figure 2(A–D). In normal mice, PEITC promoted the phagocytosis by mac- rophages from PBMC [Fig. 2(A)] and peritoneal cavity [Fig. 2(B)]. In WEHI-3 leukemia mice groups, PEITC also enhanced the phagocytotic activity in macrophages from PBMC [Fig. 2(C)] and peritoneal cavity [Fig. 2(D)]. Base on these results, PEITC induced significant differences in phagocytotic activity by macrophages isolated from PBMC and peritoneal cavity as compared with the control or WEHI-3 leukemia groups.
PEITC Affected the Whole Blood Cell Surface Markers of Normal BALB/c Mice and Leukemia Mice
Alterations of cell markers in white blood cells from nor- mal BALB/c mice and mice were injected with WEHI-3 cells after treatment with PEITC or olive oil treatment only are shown in Figures 3 and 4, respectively. In normal mice, PEITC increased the levels of CD11b and Mac-3 [Fig. 3(C,D)] and decreased the level of CD19 [Fig. 3(B)], but it did not affect the level of CD3 [Fig. 3(A)]. In WEHI-3 leu- kemia mice, PEITC promoted the level of CD19 [Fig. 4(B)], reduced the levels of CD3 and Mac-3 [Fig. 4(A,D)], but it did not alter the level of CD11b [Fig. 4(C)], suggest- ing that the differentiation of the precursor of macrophage and T cells was inhibited, but the differentiation of the B cells was promoted.
PEITC Affected the Cytotoxic Activity of Natural Killer Cells from BALB/c Mice and Mice were Injected with WEHI-3 Cells
The YAC-1 target cells killed by NK cells were isolated from spleens in mice from both studies after exposure to PEITC in target cells ratio of 25:1, 50:1, and 100:1. Results can be seen in Figure 5(A,B). In normal mice, PEITC (80 mg kg21) treatment stimulated significant differences between control and the tested agent treatment in target cells ratio of 25:1 and 50:1 [Fig. 5(A)]. In mice after WEHI-3 cells injection, PEITC (80 mg kg21) treatment promoted significant differences between the WEHI-3 con- PEITC Affected the Cytotoxic Activity of Natural Killer Cells from BALB/c Mice and Mice were Injected with WEHI-3 Cells
The YAC-1 target cells killed by NK cells were isolated from spleens in mice from both studies after exposure to PEITC in target cells ratio of 25:1, 50:1, and 100:1. Results can be seen in Figure 5(A,B). In normal mice, PEITC (80 mg kg21) treatment stimulated significant differences between control and the tested agent treatment in target cells ratio of 25:1 and 50:1 [Fig. 5(A)]. In mice after WEHI-3 cells injection, PEITC (80 mg kg21) treatment promoted significant differences between the WEHI-3 con-(1) induction of apoptotic cell death in different human can- cer cell lines (Zhang et al., 2000; Nguyen et al., 2004; Granado-Serrano et al., 2006; Lee et al., 2006; VijayabaBu et al., 2006); (2) effects on cytokine and growth factor- secretion (Huang et al., 1999; Lee et al., 2004) and; (3) in- hibition of migration and invasion of human hepatoma SK- HEP-1 cells and gastric cancer AGS cells (Hwang and Lee,
2006; Yang et al., 2010b). However, the roles of PEITC on normal and leukemia mice in vivo are not being fully inves- tigated. Our primary experiments also demonstrated that PEITC-induced cytotoxicity and triggered apoptosis in human gastric cancer cells (Yang et al., 2010b). Therefore, this raises the possibility that PEITC could affect normal and leukemia mice in vivo. The present study indicated that PEITC effectively suppressed leukemia BALB/c mice injection with WEHI-3 cells. Furthermore, PEITC pro- moted the phagocytosis by macrophages and NK cell cyto- toxic activity in normal and leukemia WEHI-3 cells- injected mice in vivo. In this study, we investigated
WEHI-3 cells that were intraperitoneally injected to the BALB/c mice and set up leukemia animal model. The major reason is that the WEHI-3 was a mouse leukemia cell line (a mu- rine myelomonocytic leukemia) (Abe et al., 1986; Lai et al., 2010b) and it also has a low cost and needs short peri- ods of time to easily develop leukemia mice in vivo. This leukemia model has been used for potential anticancer agents (Yang et
al., 2006, 2010a; Wen et al., 2010; Yu et al., 2010).
Our data indicated that PEITC did not affect the body weight of normal mice [Fig. 1(A)]. However, PEITC signif- icantly decreased the averaged body weight [Fig. 1(B)] and weights of liver [Fig. 1(C)] and spleen [Fig. 1(D)] of BALB/c mice intraperitoneally injected with leukemia WEHI-3 cells when compared with the olive oil-treated only WEHI-3 group [Fig. 1(B–D)]. Also, PEITC increased phagocytosis by macrophages from isolations of PBMC [Fig. 2(A,C)] and peritoneal cavity [Fig. 2(B,D)] of both examined mice. Moreover, PEITC promoted NK cell cyto- toxic activity from both groups of study mice (Fig. 5).
Our results also indicated that PEITC increased the lev- els of CD11b and Mac-3 [Fig. 3(C,D)] and decreased the level of CD19 [Fig. 3(A)], but it did not alter the levels of CD3 [Fig. 3(A)] in normal mice. In leukemia animal groups, however, PEITC stimulated the level of CD19 [Fig. 3(B)] and attenuated the levels of CD3 and Mac-3 [Fig. 3(A,D)], but PEITC has no effect on the level of CD11b [Fig. 4(C)], suggesting that the differentiation of the precur- sor of macrophage and T cells was inhibited, but the differ- entiation of B cells was promoted. The B cell population in normal mice compared with PEITC treatment was slightly reduced based on the level of CD19 (B-cell surface marker) as can be seen in Figure 3(B). CD19 is a 95-kDa transmem- brane protein expressed from the early stage of B cell de- velopment up to the stage of plasma cell differentiation. It is well-known as an essential downstream element of B cell receptor signaling required for B cell maturation and acti- vation, T cell dependent, antigen-specific antibody responses, and germinal center formation (Otero et al.,
2003; Otero and Rickert, 2003). In normal mice, our results also revealed that PEITC promoted the proliferation of T cells after Con A stimulation [Fig. 5(A)], but it had no sig- nificant effects in B cell proliferation even LPS exposure.
The proportions of NK cell cytotoxic activity between with or without PEITC treatment from normal and leuke- mia WEHI-3 cells-injected mice were much lower in absent PEITC-treated group than present exposure mice in YAC-1 target cells ratio of 50:1 (Fig. 5) (NK cell cytotoxicity,
21.6% vs. 47.7% from the normal mice group; NK cell activities, 27.3% vs. 56.4% in the leukemia mice group). It is known that NK cells are an early source of IFN-c and, in general, to promote a type 2 response (Godfrey et al., 2000; Stein-Streilein et al., 2000). Therefore, the increase in NK cell cytotoxic activity could be responsible in part for the increased immune response of infections (Moretta, 2007).
In addition to the lymphocyte population, PEITC also
regulated the generation and differentiation of CD11b posi- tive cells (monocytes) and promoted the function of phago- cytosis by macrophages and monocytes (Fig.
2). The pro- portion of CD11b positive in neonatal mice was 25% of the adult level [Fig. 3(C)]. The proportions of phagocytosis by macrophages between present or absent PEITC treatment from normal and leukemia mice were significantly different (Fig. 2). It is well-documented that macrophages are major contributors to innate immunity (Bu et al., 2006; Mantovani and Sica, 2010). Although the percentage of CD11b posi- tive cells in leukemia mice was not significantly affected by PEITC; however, the functions of phagocytosis by mac- rophages are promoted by PEITC in groups of animals.
Overall, PEITC can promote the phagocytosis by macro- phages and NK cell cytotoxic activity from normal and leu- kemia mice, indicating that PEITC treatment significantly increased a wide spectrum of immune cells in mice in vivo. The regulation of immune cell populations and function by PEITC might directly affect functional outcomes in BALB/ c mice in vivo.
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Fig. 1. PEITC affected the body weight, spleen, and liver from normal and WEHI-3 cells- injected leukemia mice. Animals including normal and leukemia BALB/c mice were treated with or without PEITC for 30 days or 2 weeks, respectively. Blood samples were isolated from each mouse; body weight from normal mice (A) and WEHI-3 cells leukemia mice (B) was measured and then were sacrificed for examinations of tissues of spleen (C) and liver (D) and were individually weighed. Each point is mean 6 S.D. (n 5 10) and the difference is considered statistically significant at P values \0.05.
Fig. 2. PEITC affected the phagocytostic activity by macrophages from normal BALB/c mice and WEHI-3 cells-injected leukemia mice. Mice were intraperitoneally (i.p.) cells-injected without or with WEHI-3 cells (1 3 105 cells/mice) in PBS for 2 weeks and then orally treated with present or absent PEITC for 2 weeks. Cells were
collected from animals of PBMC and peritoneal cavity, and they were analyzed for phagocytotic activity and macro- phages by flow cytometry as described in ‘‘Materials and Methods.’’ Each point is mean 6 S.D. (n 5 10) and P values less than 0.05 are considered significant. A: PBMC from normal mice; B: macrophages from peritoneal cavity of normal mice; C: PBMC from WEHI-3 cells-injected mice; D: macrophages from peritoneal cavity of leukemia mice
Fig. 3. PEITC affected the whole blood cell surface markers of normal BALB/c mice. The animals were orally treated with or without PEITC (40 and 80 mg kg21) for 30 days. Blood was collected from individually animals and analyzed for cell markers including CD3 (A), CD19 (B), CD11b (C), and Mac-3 (D) by flow cytometry as described in ‘‘Materials and Methods.’’ Each point is mean 6 S.D. of three experiments (n 5 10). A P value less than 0.05 was considered significant and N.S. 5 not significant.
Fig. 4. PEITC affected cells’ markers of white blood cells from leukemia mice. The ani- mals were intraperitoneally injected with WEHI-3 cells (1 3 105 cells/mice) for 2 weeks and treated without or with PEITC by dietary oral gavage administration for 2 weeks. Blood was collected from individual animals and analyzed for cell markers such as CD3 (A), CD19 (B), Mac-3 (C), and CD11b (D) by flow cytometry as described in ‘‘Materials and Methods.’’ Each point is mean 6 S.D. (n 5 10). *P \ 0.05 was considered statistically sig- nificant and N.S. 5 not significant.
Fig. 5. PEITC affected the natural killer (NK) cell cytotoxic activity from BALB/c normal mice and leukemia mice. The effects of PEITC on the YAC-1 target cells which were killed by NK cells from the mice after treatment with or without PEITC at 40, 80, or 160 mg kg21 in target cells ratio of 25:1, 50:1, or 100:1. Groups of normal (A) and leukemia (B) mice were analyzed for NK cell cytotoxic activity by flow cytometry as described in ‘‘Materials and Methods.’’ Each point is mean 6 S.D. of three experiments (n 5 10). There was a sig- nificant difference when P value less than 0.05.
Fig. 6. PEITC affected the proliferation of T cells (Con A- stimulated splenocytes) from normal BALB/c mice. Spleno- cytes were isolated from each group of BALB/c mice after oral treatment with PEITC and then Con A (A) stimulation for 3 days or LPS (B) stimulation for 5 days for examinations of T and B cell proliferation respectively as described in ‘‘Mate- rials and Methods.’’ Each point is mean 6 S.D (n 5 10). The analysis reveals a significant difference when P \ 0.05 and N.S. 5 not significant.
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