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Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA 23298-0613;
*
Endocrine, Reproductive and Developmental Toxicology Program, CIIT Centers for Health Research, Research Triangle Park, NC 27709-2137; and
NIEHS, Research Triangle Park, NC 27709
3To whom correspondence should be addressed. E-mail: tlguo{at}hsc.vcu.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • genistein • cytotoxic T cell activity • natural killer cell activity • antibody forming cell responses • B16F10 tumor model
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–4
). The potential beneficial effects of GEN in the human population are supported by similar findings in experimental animals (5
–7
). Although GEN has been shown to inhibit the growth of a wide variety of tumor cells in culture by inhibiting the activity of some enzymes, e.g., protein tyrosine kinases and topoisomerase II, this effect of GEN has not been associated with antitumor effects in vivo (5
,8
,9
). In contrast, there is a large amount of evidence to demonstrate that GEN has weak estrogenic effect on the reproductive systems, especially at low concentrations (10
,11
). However, it is currently unclear how phytoestrogen GEN exerts its chemopreventive effect.
One possible mechanism for GEN to inhibit tumor development may involve its effect on the immune system. In addition to estrogen-dependent tumors, GEN has been shown to be effective in preventing the development of tumors for which there is no strong evidence supporting a requirement for estrogen (12
). Furthermore, in athymic mice, which lack the development of T cells, no inhibitory effect was observed on the growth of estrogen-dependent or estrogen-independent tumors when they were exposed to dietary GEN (13
,14
). Considering the importance of both innate immunity and acquired immunity in antitumor mechanisms, it was hypothesized that GEN could modulate immune responses in female B6C3F1 mice. To delineate the effect of GEN on differential immune functions in vivo, GEN was administered to adult female B6C3F1 mice for 28 d by gavage, a relevant route of exposure. A series of assays related to the cellular or humoral components of the immune system were performed, utilizing the splenocytes from mice that had been exposed to GEN. The results indicated that GEN could increase host resistance to B16F10 tumor challenge, and this enhancement was associated with an increase in cytotoxic T-cell and natural killer (NK) cell activities.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Female B6C3F1 mice were obtained from Charles River Breeding Laboratories (Portage, MI) or Taconic Farm (Germantown, NY). Mice arrived at 4–6 wk of age and were quarantined upon arrival. The mice were between 8 and 10 wk old at the beginning of the studies. Mice were housed four per cage in plastic shoe-box cages with hardwood chip bedding, and consumed Harlan Teklad Laboratory Diets (NIH 07; Madison, WI) and tap water from water bottles ad libitum. The diet contained crude protein (22.5%), crude fats (4.5%) and crude fiber (4.5%). The animal room was maintained at 21–24°C and the relative humidity between 40 and 70%. The light:dark cycle was maintained on 12-h intervals. All animal procedures were conducted under an animal protocol approved by the Virginia Commonwealth University Institutional Animal Care and Use Committee (IACUC).
Genistein (Sigma, St. Louis, MO) solutions were freshly prepared daily in 25 mmol/L Na2CO3 at concentrations of 0.2, 0.6 and 2 g/L (2
). The mice were administered these GEN solutions or the vehicle (25 mmol/L Na2CO3) for 28 d by gavage (0.1 mL/10 g body), and this treatment resulted in dose groups of 2, 6 and 20 mg/kg body, respectively.
B16F10 melanoma tumor model.
Host resistance to the B16F10 melanoma tumor was assayed as described (15
). Briefly, 1 d after the last exposure to vehicle or GEN, mice (12 mice/group) were challenged with 1 x 105 B16F10 melanoma cells in 0.2 mL PBS intravenously (iv). Eighteen days after tumor cell challenge, mice were killed by CO2 inhalation. The lungs were removed, placed in Bouin’s solution (Sigma) and counted for nodules.
In vitro proliferation of B16F10 tumor cells.
To determine whether GEN and/or its metabolites present in the sera of exposed mice had any effect on the proliferation of B16F10 melanoma cells, blood was collected from anesthetized mice by cardiac puncture. These mice had been exposed to the vehicle or GEN for 28 d as described. Serum was isolated and stored at -20°C until use. All procedures were performed in a sterile condition. For B16F10 cell proliferation assay, cells (1 x 104/100 µL) were cultured in the presence of 50% serum in complete RPMI 1640 medium supplemented with sodium bicarbonate, HEPES, L-glutamine, gentamicin and 2-mercaptoethanol. After 24 h of incubation at 37°C and 5% CO2, all wells were pulsed with 20 µL of 3H-thymidine (1:10 dilution). The plates were incubated for another 14–16 h before harvest. For harvesting, the supernatant was pipetted out of the wells and the cells in the wells were rinsed twice with Hank’s balanced salt solution (HBSS). Trypsin was added to all wells for 45 min to release the cells from the plates; all wells were then harvested using the Cambridge Cell Harvester (Cambridge, MA) with consistent mechanical scraping and agitation. The results were expressed as kBq/1 x 104 cells.
Cytotoxic T lymphocyte (CTL) activity.
The assay for CTL activity was performed as described (16
). The splenocytes from control and treated mice were washed once with HBSS, resuspended in Eagle’s minimal essential medium (E-MEM, Hazelton, Lenexa, KS). The cells were sensitized in vitro with mitomycin C–treated P815 mastocytoma cells at a responder:sensitizer ratio of 50:1 for 5 d at 37°C in 5% CO2. For the preparation of mitomycin C–treated P815 cells, cells were incubated in the dark with mitomycin C at a concentration of 50 µg/2 x 107 cells for 30 min at 37°C. Then the cells were washed four times. After the sensitization phase, cultured spleen cells were harvested and resuspended in E-MEM for determination of CTL activity. 51Cr-labeled P815 cells (2 x 104 cells/100 µL) were cocultured in duplicate with 100 µL of graded numbers of splenic effector cells in U-bottom microtiter culture plates to yield a serial half-dilution of effector:target ratios from 25:1 to 0.75:1. For the preparation of 51Cr-labeled P815 cells, 15 x 106 cells were incubated with 18.4 MBq of 51Cr for 60 min at 37°C with finger vortex every 15–20 min followed by four washes. After a 4-h incubation at 37°C and 5% CO2, the plates were centrifuged for 10 min at 250 x g. Supernatant from each well (100 µL) was removed and counted in a LKB gamma spectrophotometer (Gaithersburg, MD). Controls for spontaneous and maximum release were generated by culturing labeled target cells in the presence of either E-MEM medium or 0.1% Triton X-100, respectively.
Natural killer (NK) cell activity.
The activity of NK cells was assayed using the NK-sensitive target, Na51CrO4-labeled YAC-1 cells, as described with modification (17
). Briefly, the splenocytes at different dilutions were mixed with the target cells to obtain effector:target ratios of 200:1, 100:1, 50:1, 25:1, 12.5:1 and 6.25:1. The spontaneous release was determined by adding 100 µL of medium to 12 replicate cultures containing the targets. The maximum release was determined by adding 100 µL of the YAC-1 cells and 100 µL 0.1% Triton X-100 to each of 12 replicate wells. NK cell-specific lysis (%) of 51Cr-labeled YAC-1 cells was used as the endpoint of the assay. For interleukin (IL)-2 augmented NK assay, single cell suspensions of individual spleens were prepared and cultured with 5 x 106 IU/L recombinant IL-2 (Chiron, Emeryville, CA) overnight, and then assayed for NK cell activity using 51Cr-labeled YAC-1 cells as the target cells.
The activity of the mononuclear phagocyte system.
The functional activity of the mononuclear phagocyte system was measured as described (18
). The mice were injected iv with 51Cr-sheep red blood cells (sRBC, 0.10 hematocrit in PBS at 100 µL/10 g body). Blood samples were taken every 3 min from the tail of each mouse; blood (5 µL) was dispensed into a tube containing 1 mL of water. The clearance of the labeled sRBC from the blood was determined over the first 30 min and the half-life was calculated on the basis of the amount of radioactivity remaining in the blood at each time point. After 60 min, mice were killed, exsanguinated and different organs removed. The uptake of the labeled sRBC by various organs was determined by the amount of radioactivity present. Radioactivity was measured using a 
-counter (LKB).
Recruited peritoneal macrophage activity.
The activity of recruited peritoneal macrophages was determined as described previously by Geissler et al. (19
) with slight modification. Briefly, 5 d before killing (during the genistein exposure period), the mice were injected intraperitoneally with 1 mL of a 10% thioglycollate (Sigma). Mice were killed by a gentle cervical dislocation. The recruited peritoneal cells (PEC) were obtained by lavage using HBSS. Cells (1 x 105) from each mouse were dispensed to each well in 96-well plates and adhered for 2 h at 37°C in 5% CO2. The nonadherent cells were then rinsed off; the adherent cells (macrophages), unstimulated or stimulated with interferon- (IFN-; 1 x 104 U/L; PharMingen, San Diego, CA) plus lipopolysaccharide (LPS; 10 µg/L), were further cultured for 4 h. Plates were rinsed twice and incubated for 48 h with the addition of the B16F10 melanoma target cells (1 x 104/well). All wells were pulsed with 3H-thymidine 14–16 h before harvest. The procedures for harvesting were described above. Inhibition of B16F10 tumor cell proliferation was used as an indicator of recruited peritoneal macrophage activity.
Spleen immunoglobulin (Ig)M/IgG antibody response to the T-dependent antigen, sRBC.
The primary IgM response to sRBC was enumerated using a modified hemolytic plaque assay as described (20
). Mice receiving the appropriate exposure were sensitized (iv) with sRBC (7.5 x 107) on d 25 of exposure. On d 29, 1 d after the last exposure to GEN, mice were killed and spleen cells were prepared. An aliquot of cells was added to a test tube containing guinea pig complement, sRBC and warm agar. After thorough mixing, the test tube mixture was plated in a petri dish, covered with a microscope cover slip and incubated at 37°C for 3 h. The plaques developed were counted using a Bellco plaque viewer (Bellco Glass, Inc., Vineland, NJ). Cell counts were performed on the 3-mL sample and the number of cells/spleen, antibody-forming cells (AFC)/spleen and AFC/106 spleen cells determined.
For IgG AFC responses, an indirect IgG AFC measurement (the Jerne-Nordin method) was employed (21
). The first injection of sRBC was on d 14 of exposure, and the second injection was on d 25. The AFC response was performed on d 29. In addition to IgM AFC response, total AFC (both IgG and IgM) was determined using procedures described above except that an appropriate dilution of rabbit anti-mouse IgG (Organon Teknika, West Chester, PA) was added to the mixture. The IgG AFC response was determined by subtracting the IgM AFC from the total AFC response.
Flow cytometric analysis of lymphocytes and NK cells.
The percentages of lymphocytes and NK cells in the spleen were measured using flow cytometric analysis (22
). For NK cells (NK1.1+CD3-), spleen single-cell suspensions were dually labeled with fluorescein isothiocyanate (FITC) conjugated anti-mouse CD3 mAb (1:80; Becton Dickinson, San Jose, CA) and phycoerythrin conjugated anti-NK1.1 mAb (1:80; PharMingen) for 30 min on ice. The cells were washed and enumeration performed on a Becton Dickinson FACScan Flow Cytometer in which log fluorescence intensity was read gated on propidium iodide to eliminate dead cells and a forward scatter threshold high enough to eliminate RBC. The data were analyzed using the CellQuest software (Becton Dickinson). For B cells, the staining procedures were the same as those described for NK cells except that goat anti-mouse IgG (heavy and light chain specific) conjugated to FITC was used. Irrelevant, isotype matched antibodies were used as the control.
Mixed leukocyte response to DBA/2 spleen cells.
The assay was performed as described (23
). One hundred microliters of splenocytes (1 x 109/L) were added to each well of a U-bottom microtiter plate (Costar 3799, Cambridge, MA). DBA/2 spleen cells were used as the allogeneic cells (stimulator) for the B6C3F1 mice (responder). Stimulator cells were treated with mitomycin C to render them unable to proliferate; the ratio of stimulators to responders has previously been optimized to be 4:1. The cells were cultured for 5 d, during the last 18 h in the presence of 36.8 kBq 3H-thymidine. The cells were collected with a cell harvester and counted in a LKB liquid scintillation counter. The amount of 3H-thymidine incorporated into the proliferating responder cells was expressed as Bq/105 cells.
Anti-CD3 antibody-mediated spleen T-cell proliferation.
The proliferation of splenocytes in the presence of anti-CD3 antibody was performed as described below. A single spleen cell suspension was prepared and resuspended in RPMI medium supplemented with fetal bovine serum (10%), sodium bicarbonate, HEPES, L-glutamine, gentamicin and 2-mercaptoethanol. The splenocytes (5 x 105/well) were cultured in the microtiter wells coated with anti-CD3 antibody (1 mg/L; PharMingen) or in wells without antibody coating at 37°C at 5% CO2 and 95% humidity. Before harvest on d 3, the cells were pulsed with 3H-thymidine for 18–24 h. The incorporation of 3H-thymidine into the proliferating cells was expressed as kBq/5 x 105 cells.
Statistical analysis.
The data were analyzed as follows. Bartlett’s test for homogeneity was used to select the type of analysis to be conducted. Homogeneous data were analyzed using a one-way ANOVA; when significant, Dunnett’s t test was used to determine differences between the experimental and the control groups. For nonhomogeneous data, a nonparametric ANOVA was used; when significant, differences between the control group and the experimental groups were determined using the Wilcoxon Rank Test. Jonckheere’s Test was used to test for dose-related trends across the vehicle and the GEN exposure groups.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Exposure to GEN resulted in a dose-related decrease in pulmonary tumor formation. One possible explanation for GEN-enhanced host resistance to B16F10 tumor was that GEN or its metabolites might directly inhibit the proliferation of B16F10 tumor cells. Thus, B16F10 tumor cell proliferation was evaluated in the presence of sera collected from vehicle- or GEN-exposed mice. The proliferation of B16F10 tumor cells did not differ among vehicle control and GEN treatment groups when the sera (50%) from treated mice were used to culture the B16F10 tumor cells (Fig. 1B
).
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). When the data were expressed as specific activity, vehicle control mice had 45.9 lytic units (LU) in 107 spleen cells, and 1.2- and 1.6-fold increases were observed in the middle and high dose groups, respectively (Fig. 2A
). When the data were expressed as total activity, vehicle controls had 625.8 LU in spleen, and 1.2- and 1.6-fold increases were observed in the middle and high dose groups, respectively (Fig. 2B
). Na2CO3, the vehicle used in this study, did not affect the CTL activity compared with naïve controls. Exposure to GEN had no effect on the percentage of CD8+ T cells in the spleen (Table 1
).
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). However, exposure to GEN increased splenocyte basal proliferation. Similarly, an increase of splenocyte basal proliferation was observed in an additional experiment in which anti-CD3 antibody-mediated proliferation was assayed, although GEN did not affect the splenocyte proliferation in the presence of anti-CD3 antibody (Table 2)
. Exposure to GEN did not affect the total number of recovered splenocytes or the percentage of CD4+CD8- T cells (Table 1)
.
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) and LU/spleen (Fig. 3B
) were significantly increased. However, GEN did not affect basal NK cell activity (data not shown). Furthermore, exposure to GEN had no effect on the percentage of NK cells in the spleen (Table 1)
.
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). The kidneys are considered to be a nonphagocytic organ; therefore localization of 51Cr-sRBC in the kidneys was determined for control purposes. In vehicle control mice, the half-life (T1/2) of 51Cr-sRBC was 10.9 min; exposure to GEN did not affect the vascular clearance of 51Cr-sRBC. The percentage of uptakes of 51Cr-sRBC by liver, spleen, lungs and kidneys were 19.7, 13.3, 1.4 and 2.1%, respectively. Exposure to GEN had no effect on the uptake of 51Cr-sRBC by these organs when evaluated as either percentage of uptake or Bq/mg tissue (Table 3
). For thymus, the treatment groups did not differ from the vehicle control separately, but a significant decreasing trend was observed when the data were expressed as percentage of uptake and Bq/mg (P 
0.01 by Jonckheere’s Test). Furthermore, exposure to GEN produced an increasing trend in the terminal body weight and the weights of liver, spleen and lungs (P 0.05 by Jonckheere’s Test).
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and LPS (data not shown).
The IgM AFC response to the T-dependent antigen, sRBC, was also evaluated. Although GEN increased IgM AFC at the dose of 6 mg/kg (Table 4
), no effect was observed in the high dose group. Furthermore, exposure to GEN had no effect on IgG AFC response (Table 4)
. The splenic B cell percentage was not altered by GEN (Table 1)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–4
). To improve our understanding of GEN's protective effect, we examined its effects on immune responses in adult female B6C3F1 mice. Consistent with the tumor inhibitory effect of GEN reported in humans, increased host resistance in the B16F10 tumor model was observed after exposure of mice to GEN by gavage, a relevant route of exposure, for 28 d. This is also in agreement with previous studies that oral administration of GEN inhibited B16F10 lung-tumor nodule formation (24
) and decreased B16 tumor volume (6
). For a 4-mo-old infant who consumes soy formula as directed by the manufacturers, 
6–9 mg/kg body of isoflavones can be achieved (25
). A GEN concentration as high as 5 µmol/L in plasma can be reached in women who take tablets of soy extract (26
). In our study, physiologically relevant doses of GEN (2–20 mg/kg) were employed. At the dose levels of 6 and 20 mg/kg, we demonstrated that GEN significantly decreased B16F10 lung tumor nodule formation.
There are several possible mechanisms that may be responsible for the inhibitory effect of GEN on B16F10 tumor formation. Some in vitro studies have demonstrated that GEN can directly inhibit the proliferation of tumor cells (6
,27
). However, this mechanism may not play an important role in our in vivo tumor model. As demonstrated in our study, GEN and/or its metabolites present in the sera of GEN-exposed mice had no effect on B16F10 proliferation. This suggested that either the serum levels of GEN and/or its metabolites in our experimental animals were not high enough to directly inhibit the proliferation of B16F10 tumor cells or they were in a form (e.g., protein-bound) that had no direct effect on the proliferation. Because GEN concentration in plasma is higher than that in lungs after oral dosing of [14C]GEN (28
), the inhibitory effect of GEN on B16F10 lung tumor formation is unlikely due to a direct effect of GEN present in the lungs. It should be noted that GEN has been found to be an inhibitor of protein tyrosine kinases at high concentrations while acting as an estrogenic compound at low concentrations (4
,29
). Furthermore, there is evidence that inhibition of protein tyrosine phosphorylation prevented T cell–mediated cytotoxicity (30
) instead of enhancing CTL activity as demonstrated in our study. Therefore, the enhancing effect of GEN on host resistance in the B16F10 tumor model is likely due to its function as an estrogenic compound instead of an enzyme inhibitory agent.
In addition, the B16F10 tumor model requires the extravasation of melanoma cells from the cardiovascular system into the interstitum of the lungs by invading the subendothelial basement membrane (31
). Genistein has been shown, in vitro, to inhibit the invasion of the extracellular matrix by mammary carcinoma cells (32
), which may also play a role in our in vivo tumor model. However, this effect of GEN has been attributed to its inhibition of protein tyrosine kinases (31
). As indicated above, the lack of effect on the proliferation of B16F10 tumor cells by GEN and/or its metabolites present in the sera of GEN-exposed mice suggests that enhanced host resistance to B16F10 tumor model was not due to these enzyme inhibitory effects.
Cytotoxic T cells, NK cells and macrophages are suggested to be the major immune mechanisms responsible for clearance and growth inhibition of B16F10 melanoma tumor (33
–35
). In this study, we demonstrated that GEN significantly increased the activity of CTL at the dose levels of 6 and 20 mg/kg. However, whether increased CTL activity is responsible for enhanced host resistance to tumors is currently unknown. Moreover, the mechanism underlying GEN-enhanced CTL activity remains to be defined. The expression of estrogen receptor protein or transcript in thymus and peripheral T cells has been demonstrated (36
,37
). When C3H/HeJ mice were treated orally with tamoxifen (an estrogen antagonist), enhanced CTL activity to H2712 carcinoma was observed (38
). Genistein may function as an antiestrogen in a similar manner in our experimental model to enhance CTL activity. Although GEN had no effect on the MLR and anti-CD3 antibody-mediated splenocyte proliferation, an increased basal splenocyte proliferation was observed in both studies. What antigen and which type of cells contributes to this increase is currently unknown.
Lymphokine-activated killer cells have been shown to be successful in cancer immunotherapy (39
). In our study, in vitro IL-2–augmented NK cell activity but not basal NK cell activity was increased in the high GEN dose group. The IL-2–augmented NK cell response may be more physiologically relevant to what occurs in animals undergoing tumor proliferation and a better indicator of actual NK activity than the evaluation of basal NK activity. In the course of tumor development, the immune system is activated, and soluble factors such as cytokines are secreted to enhance the innate immunity. The importance of elevated cytokine-driven NK cell activity in GEN-mediated chemoprevention requires further investigation. Because estrogen has been shown to have an inhibitory effect on murine NK cell activity (40
,41
), GEN enhancement of NK cell activity lends additional support to the notion that GEN may be an estrogen antagonist in adult female B6C3F1 mice.
Although exposure to GEN did not affect the overall activities of the mononuclear phagocyte system and peritoneal macrophages, there was a slight decrease in the 51Cr-sRBC uptake by thymus, which is in contrast to the stimulatory effect of estrogenic compounds on the mononuclear phagocyte system (42
). However, this is in agreement with GEN's antiestrogen effect discussed above. Interestingly, daidzein, another phytoestrogen in soy products, also exerted a stimulatory effect on the phagocytic response of peritoneal macrophages at high doses (43
). Therefore, in future evaluations of the effects of soy and soy extract dietary supplements, it will be necessary to consider possible actions of other components of soy on immune function. Finally, slight increases in the weights of liver, spleen and lungs were observed in GEN-exposed mice, which may be related to a slight increase in the terminal body weight.
In summary, we observed in this study that exposure to GEN at physiologically relevant doses significantly increased host resistance in the B16F10 tumor model. Importantly, an association between enhanced host resistance and increased immune responses (enhanced CTL activity, increased IL-2 driven NK cell activity and increased basal splenocyte proliferation) was observed. Genistein modulation of immune responses may offer a possible explanation for the report that exposure to GEN had no effect on estrogen-dependent and -independent tumors in athymic mice (13
,14
). Further study of the molecular mechanisms underlying the potentiating effect of GEN on the immune responses will allow us to understand more about its chemopreventive function and the safety issue associated with ingestion of this compound.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by the funds provided from the Thomas F. Jeffress and Kate Miller Jeffress Memorial Trust and partially from NIEHS contract no. ES-05454.
4 Abbreviations used: AFC, antibody-forming cells; CTL, cytotoxic T lymphocyte; E-MEM, Eagle’s minimal essential medium; FITC, fluorescein isothiocyanate; GEN, genistein; HBSS, Hank’s balanced salt solution; IFN, interferon; Ig, immunoglobulin; IL, interleukin; iv, intravenously; LPS, lipopolysaccharide; LU, lytic units; MLR, mixed leukocyte response; NK, natural killer cells; PEC, peritoneal cells; sRBC, sheep red blood cells.
Manuscript received June 8, 2001. Initial review completed July 19, 2001. Revision accepted September 14, 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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