Identification of agents that are nontoxic but can delay onset and/or progression of prostate cancer, which is the second leading cause of cancer-related deaths among men in the United States, is highly desirable. We now show that p.o. gavage of garlic constituent diallyl trisulfide (DATS; 1 and 2 mg/day, thrice/week for 13 weeks beginning at age 8 weeks) significantly inhibits progression to poorly differentiated prostate carcinoma and pulmonary metastasis multiplicity in transgenic adenocarcinoma of mouse prostate (TRAMP) mice without any side effects. There was a trend of a decrease in average wet weights of the urogenital tract and prostate gland in 1 and 2 mg DATS–treated mice compared with controls (∼25–46% decrease in DATS-treated mice compared with controls). The incidence and the area of the dorsolateral prostate occupied by the poorly differentiated carcinoma were significantly lower in both 1 and 2 mg DATS–treated mice compared with control mice. In addition, DATS administration resulted in a statistically significant decrease in pulmonary metastasis multiplicity compared with controls (P = 0.002). The dorsolateral prostate from DATS-treated TRAMP mice exhibited decreased cellular proliferation in association with induction of cyclinB1 and securin protein levels, and suppression of the expression of neuroendocrine marker synaptophysin. However, DATS administration did not have any appreciable effect on apoptosis induction, angiogenesis, or natural killer and dendritic cell function. In conclusion, the results of the present study show, for the first time, that DATS administration prevents progression to invasive carcinoma and lung metastasis in TRAMP mice. [Cancer Res 2008;68(22):9503–11]
Prostate cancer is one of the most commonly diagnosed visceral malignancies and the second leading cause of cancer-related deaths among men in the United States (1). Prostate carcinogenesis is characterized by gradual transformation of normal prostate epithelium to prostatic intraepithelial neoplasia (PIN), localized tumor to advanced and metastatic disease (2). Prostate cancer is usually diagnosed in the sixth or seventh decade of life providing a large window of opportunity for intervention to prevent or slow progression of the disease. Consequently, identification of novel agents that are nontoxic but can delay onset and/or progression of human prostate cancer is highly desirable. Natural products have received increasing attention in recent years for the discovery of novel cancer preventive and therapeutic agents (3).
Epidemiologic studies continue to support the premise that dietary intake of Allium vegetables (e.g., garlic) may lower the risk of various types of malignancies including cancer of the prostate (4–6). For example, the risk of prostate cancer was shown to be significantly lower in men consuming >10 g/day of total Allium vegetables than in men with total Allium vegetable intake of <2.2 g/day in a population-based, case-control study (6). The anticarcinogenic effect of Allium vegetables is attributable to organosulfur compounds (OSC) including diallyl sulfide, diallyl disulfide (DADS), and diallyl trisulfide (DATS), which are generated upon processing (e.g., cutting or chewing) of these vegetables (7). The OSCs have been shown to afford significant protection against chemically induced cancers in animal models, including benzo(a)pyrene-induced forestomach and pulmonary carcinogenesis in mice, N-nitrosomethylbenzylamine–induced esophageal cancer in rats, and azoxymethane-induced colon carcinogenesis in rats (8–11). Initial studies indicated that the OSC-mediated prevention of chemically induced cancer correlated with induction of phase 2 carcinogen inactivating enzymes and inhibition of cytochrome P450-dependent monooxygenases (12–14).
More recent studies including those from our laboratory have revealed that certain garlic-derived OSCs are effective in suppressing proliferation of human cancer cells (15–25). The OSC-mediated suppression of cancer cell growth in association with G2-M phase cell cycle arrest and/or apoptosis induction was documented for DADS and DATS in human colon, neuroblastoma, and prostate cancer cells (15–20). Studies from our laboratory have revealed that DATS is a much more potent suppressor of proliferation of human prostate cancer cells compared with either diallyl sulfide or DADS (18). Interestingly, a normal prostate epithelial cell line is significantly more resistant to G2-M phase cell cycle arrest by DATS compared with prostate cancer cells (19), a feature highly desirable for cancer chemopreventive agents. Mechanistic studies have revealed that DATS treatment not only negatively regulates signaling pathways implicated in cell proliferation (e.g., Akt) and angiogenesis (vascular endothelial growth factor signaling axis) but also causes G2 and prometaphase arrest and mitochondria-mediated apoptotic cell death (18, 19, 21–26). Additionally, the growth of PC-3 human prostate cancer cells s.c. implanted in athymic mice was significantly retarded by p.o. administration of DATS (27).
Demonstration of in vivo efficacy of potential cancer chemopreventive agents in suitable animal models is a prerequisite for their further clinical development. The present study was undertaken to test whether DATS administration offers protection against prostate carcinogenesis in a transgenic mouse model of prostate cancer (transgenic adenocarcinoma of mouse prostate; hereafter abbreviated as TRAMP). We now show that p.o. gavage of DATS significantly prevents development of invasive prostate carcinoma and pulmonary metastasis multiplicity in TRAMP[C57BL/6×FVB]F1 mice without causing weight loss or affecting T-antigen expression.
Materials and Methods
Reagents. DATS (purity ∼99%) was purchased from LKT Laboratories. Antibodies against CD31 (platelet/endothelial cell adhesion molecule 1; PECAM-1) and cyclinB1 were purchased from Santa Cruz Biotechnology; the anti–E-cadherin antibody was from BD Transduction Laboratories; the antibodies against proliferating cell nuclear antigen (PCNA) and Ki-67 were from DakoCytomation; the anti-securin antibody was purchased from MBL; and the antibodies against T-antigen and synaptophysin were from BD Biosciences. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was performed using the In Situ Apoptosis Detection kit from Chemicon International.
Animal model and DATS treatment. Various mouse models of prostate cancer have been generated in recent years [e.g., TRAMP, LADY (12T-10) transgenic mice, PTEN conditional knockout mice, etc.], which develop lesions analogous to those observed in human prostate cancer (reviewed in ref. 28). Our choice to use TRAMP mice for the present study was based on the following considerations: (a) a rather well-defined course of disease progression analogous to the human prostate carcinogenesis from histologic PIN to invasive carcinoma with distant site metastasis coupled with the overall high tumor incidence renders TRAMP mice suitable for chemoprevention studies (29, 30), (b) the chemoprevention studies in TRAMP mice can be completed in a reasonable time frame (29, 30), and (c) TRAMP model has been used successfully to test chemopreventive efficacy of several synthetic and natural agents (31–33). We opted to use TRAMP[C57BL/6×FVB]F1 progeny because these mice tend to form well-defined tumor nodules and the frequency of phylloides-like structures, which are rare in human prostate cancers, is relatively higher in C57BL/6 pure transgenic mice (28–30). Male TRAMP[C57BL/6×FVB]F1 hybrid mice were generated by breeding female TRAMP in C57BL/6 background with nontransgenic FVB male mice. Transgene verification was performed using DNA obtained from tail clippings at 14 to 17 d of life as described by Greenberg and colleagues (29). Use of mice for the studies described herein was approved by the Institutional Animal Care and Use Committee. After transgene verification, male TRAMP mice were maintained in a climate-controlled environment with a 12-h light/12-h dark cycle. The mice were fed food and water ad libitum. At age 8 wk, mice were randomized into three groups. Mice in the control group (n = 16) received 0.1 mL PBS by p.o. gavage thrice per week (Monday, Wednesday, and Friday), whereas the experimental groups of mice (n = 19) received either 1 mg DATS/d or 2 mg DATS/d in 0.1 mL PBS by p.o. gavage thrice per week (Monday, Wednesday, and Friday). Body weights of the control and DATS-treated mice were recorded once each week beginning at the onset of the study. After 13 wk of treatment, the animals were sacrificed 24 h after the last administration of the vehicle or DATS by CO2 inhalation followed by cervical dislocation. The weights of the vital organs, urogenital tract, and prostate were determined. A portion of the prostate/tumor tissue was placed in 10% neutral buffered formalin and paraffin embedded. Tissues were sectioned at 4- to 5-μm thickness for H&E staining, TUNEL assay, and immunohistochemical analyses.
Pathologic evaluation and scoring of tumor stage and metastasis. Ten randomly selected fields on H&E-stained sections of the dorsolateral prostate of individual mouse of control and DATS-treated groups were independently scored by two investigators for incidence and percentage of the area corresponding to each pathologic stage. Pathologic grading was performed as described by Greenberg and colleagues (29, 30). Tissue histology was classified as normal prostate gland with open ducts lined with tall secretory epithelial cells surrounded by a closely associated thin sheet of smooth muscle cells; PIN with piling up of the epithelium, changes in the nuclear to cytoplasmic ratio, elongation of the nucleus and an increase in epithelial stratification, and formation of cribriform structures; well-differentiated (WD) carcinoma with clear invasion of the epithelial cells into the stroma; and poorly differentiated (PD) carcinoma with sheets of anaplastic cells with little or no glandular structures. Lung, kidney, liver, and pelvic lymph nodes were evaluated for the presence of metastasis by two independent investigators. The incidence of metastasis (percentage of mice with metastatic lesions) in each tissue and pulmonary metastasis multiplicity (number of pulmonary metastatic lesions/mouse) were computed for the control and DATS-treated groups.
Immunohistochemical analyses. Deparaffinized and rehydrated sections were quenched with 3% hydrogen peroxide and blocked with normal serum. The sections were then incubated with the desired primary antibody (anti–T-antigen, anti-PCNA, anti–Ki-67, anti–E-cadherin, anti-synaptophysin, anti-CD31, anti-cyclinB1, or anti-securin antibody) and washed with TBS followed by incubation with appropriate biotinylated secondary antibody. Characteristic brown color was developed by incubation with 3,3-diaminobenzidine. The sections were counterstained with Meyers Hematoxylin (Sigma) and examined under a Leica microscope. At least three nonoverlapping representative images of each tissue were captured from each section using a camera mounted onto the microscope. The images were analyzed using Image ProPlus 5.0 software (Media Cybernetics) for quantitation of PCNA, Ki-67, E-cadherin, T-antigen, cyclinB1, and securin expression and analysis of microvessel number and vessel diameter (CD31 staining). Immunoblotting for T-antigen expression using prostate/tumor tissue supernatants from control and DATS-treated mice was performed as previously described by us for other proteins (27).
Detection of apoptotic bodies by TUNEL staining. The paraffin-embedded tissue sections were deparaffinized, rehydrated, and then used to visualize apoptotic bodies by TUNEL staining using the ApopTag Plus Peroxidase In Situ Apoptosis kit and following the manufacturer's protocol. Apoptosis was quantified by counting the number of TUNEL-positive cells in at least three randomly selected, nonoverlapping high-power fields.
Cytotoxicity of natural killer and dendritic cells. The natural killer (NK) cells were isolated from the spleen of control and DATS-treated mice using MACS beads (Myltenyi Biotech) as described by Giezeman-Smith and colleagues (34) and cultured in interleukin-2–supplemented (1,000 units/mL) medium. The DCs were isolated from the bone marrow of control and DATS-treated mice using the adhesion selection method (35) and cultured in medium supplemented with interleukin-4 (500 ng/mL), granulocyte macrophage colony-stimulating factor (500 ng/mL), and Flt-3 (25 ng/mL). After 5 d of culture, NK cells were plated in 96-well plates alone or cocultured with dendritic cells (DC; day 5) at different ratios (10:0, 10:4, or 10:8). After 24 h, TRAMP-C1 target cells were added to the wells at specified ratio. After 24 h, plates were spun down and supernatant was collected for analysis of cytotoxicity using the Cytotox 96 nonradioactive assay (Promega), which determines release of lactate dehydrogenase. Monolayer cultures of TRAMP-C1 cells, a generous gift from Dr. Barbara Foster (Roswell Park Cancer Institute, Buffalo, NY), were maintained as described by us previously (36).
Statistical analysis. Statistical significance of difference in mean urogenital and prostate weights and metastasis multiplicity between groups was assessed by Wilcoxon test. The difference in metastasis incidence between groups was assessed by Fisher's exact test. Differences were considered significant at P value of ≤0.05.
P.o. administration of DATS prevented development of PD prostate carcinoma in TRAMP mice. A preliminary dose-finding study involving 8-week-old nontransgenic male [C57BL/6×FVB]F1 littermates (5 mice per group) revealed that p.o. gavage of 1 and 2 mg DATS, thrice per week, was well-tolerated by the mice. The nontransgenic mice treated with 1 and 2 mg DATS seemed healthy and did not exhibit weight loss or signs of distress. All vital organs (liver, lung, kidney, heart, and spleen) and prostate glands of 1 and 2 mg DATS-treated nontransgenic mice were also normal as judged by histology and wet organ weight measurements (data not shown). The DATS concentrations used in the present study (1 and 2 mg DATS) are within the range that can be generated through dietary intake of garlic (37). As can be seen in Fig. 1A, the average wet weight of the urogenital tract in mice given 1 and 2 mg DATS was ∼26% and 35% lower, respectively, compared with control mice. The average wet weight of the prostate gland in 1 and 2 mg DATS-treated mice was also lower by ∼32% and 46%, respectively, compared with control mice. The differences in urogenital and prostate weights between control and DATS-treated mice did not reach statistical significance due to large data scatter. However, there was a trend of a dose-dependent decrease in average wet weights of the urogenital tract and prostate in DATS-treated mice compared with controls (Fig. 1A). As shown in Fig. 1B, the initial and final body weights of the control and DATS-treated mice did not differ significantly.
The histologic grades (normal prostate, PIN, WD carcinoma, and PD carcinoma) in dorsolateral prostate of control and DATS-treated mice with prostate weight of <1g were independently scored by two investigators. Prostate sections from control as well as DATS-treated mice with prostate weight of >1 g were excluded because the majority of the prostate gland in these mice was occupied by PD carcinomas. Representative H&E staining in dorsolateral prostate sections from two different control and DATS-treated mice are depicted in Fig. 2A. The dorsolateral prostate of the vehicle-treated control TRAMP mice exhibited low- and high-grade PIN, WD carcinoma, and PD carcinomas along with areas consistent with normal prostate. The incidence of the PD carcinoma in the dorsolateral prostate of mice treated with 1 and 2 mg DATS was lower by 34% (P = 0.0147) and 41% (P = 0.035), respectively, in comparison with control mice (Fig. 2B). Moreover, the area occupied by the PD carcinoma in the dorsolateral prostate of DATS-treated mice was statistically significantly lower compared with that in vehicle-treated control mice. For example, the percentage of the area occupied by the PD carcinoma in dorsolateral prostate of mice given 1 and 2 mg DATS was lower by ∼64% (P = 0.0484) and 76% (P = 0.0189), respectively, compared with control mice (Fig. 2C). The DATS administration also resulted in a modest increase in the area occupied by the PIN and WD carcinomas compared with control mice (Fig. 2C). For example, the area occupied by the PIN in dorsolateral prostate of mice treated with 1 mg DATS and 2 mg DATS was higher by ∼57% (P = 0.0692 by Wilcoxon rank-sum test) and 60% (P = 0.0277 by Wilcoxon rank-sum test), respectively, compared with vehicle-treated control mice (Fig. 2C). Likewise, the area occupied by the WD carcinoma in the dorsolateral prostate of mice treated with 2 mg DATS was higher by ∼45% compared with control mice (P = 0.0986; Fig. 2C). These results indicated that DATS administration significantly inhibited progression from PIN/WD to PD carcinoma in TRAMP mice, which was independently verified by two investigators. These results are significant because the mortality in prostate cancer patients is mainly attributable to advanced and metastatic disease (2).
Prostate carcinogenesis in TRAMP mice is driven by the expression of the viral large T and small t antigen in the secretory epithelial cells of the prostate under the control of the minimal rat probasin promoter (29). We considered the possibility that the DATS-mediated prevention of PD carcinoma incidence/burden in TRAMP mice was due to the suppression of the transgene expression. We tested this possibility by determining the expression of T-antigen in the prostate of control and 2 mg DATS–treated mice by immunohistochemistry and immunoblotting. Fig. 3A depicts immunohistochemical staining for the T-antigen expression in the prostate of a representative mouse of both control and DATS-treated group. As can be seen in Fig. 3B and C, the DATS-mediated inhibition of prostate cancer development in TRAMP mice was not due to the suppression of the T-antigen expression.
DATS administration reduced pulmonary metastasis multiplicity. Because DATS administration significantly retarded the development of PD carcinoma, we proceeded to determine the incidence and multiplicity of pulmonary and pelvic lymph node metastasis. Figure 4A depicts H&E staining in a lung section of a representative mouse of both the control group and 2 mg DATS–treated group. The incidence of pulmonary metastasis was ∼88% in vehicle-treated control mice, which was reduced to ∼79% and 68% in mice given with 1 and 2 mg DATS, respectively (Fig. 4B). Likewise, the incidence of metastasis to the pelvic lymph nodes was ∼1.5- to 1.9-fold higher in the vehicle-treated control mice compared with the DATS-treated mice (Fig. 4B). Majority of the control mice exhibited multiple pulmonary metastatic lesions. The multiplicity of the pulmonary metastasis in mice given 1 and 2 mg DATS was lower by ∼49% and 55% (P = 0.002) compared with control mice (Fig. 4C). Together, these results indicated that DATS administration delayed development of metastatic lesions especially pulmonary metastasis multiplicity in comparison with the vehicle-treated control mice.
DATS administration decreased cellular proliferation and synaptophysin expression in the dorsolateral prostate. Because studies in cultured human prostate cancer cells (18, 25) and PC-3 xenografts (27) have shown that DATS treatment reduces cell proliferation, we performed immunohistochemistry for well-known proliferation marker PCNA (38) to determine the effect of DATS treatment on proliferation index in the dorsolateral prostate of TRAMP mice. Immunohistochemical comparisons for PCNA expression in prostate of the control and DATS-treated mice were carried out using size-matched tissues. Immunohistochemical staining for PCNA expression in representative prostate of a vehicle-treated control mouse and a 2 mg DATS–treated mouse is shown in Fig. 5A. The PCNA expression was ∼46% lower in the dorsolateral prostate of mice given 2 mg DATS compared with that of control mice (P = 0.035). Inhibitory effect of DATS administration on proliferation index was confirmed by immunohistochemical analysis of Ki-67 expression (Fig. 5B), which is another widely used marker for cellular proliferation (39). The PD carcinomas in TRAMP mice exhibit neuroendocrine (NE) differentiation (40, 41). Because DATS administration inhibited incidence and burden of PD carcinoma (Fig. 2B and C), we raised the question of whether DATS treatment affected fraction of NE cells. We addressed this question by determining the expression of synaptophysin in the prostate of control and 2 mg DATS–treated mice. Synaptophysin is a membrane-associated glycoprotein and well-accepted marker of NE cells (41). The expression of synaptophysin in tissue sections from control mice was predominant in the membrane (Fig. 5C). The fraction of synaptophysin-expressing NE cells was significantly lower in the prostate of 2 mg DATS–treated mice compared with that of control mice. Collectively, these results indicated that the DATS-mediated prevention of PD carcinoma development in TRAMP mice correlated with reduced cellular proliferation and suppression of NE differentiation.
Our previous studies in cultured human prostate cancer cells have revealed that DATS treatment causes prometaphase arrest that is characterized by accumulation of cyclinB1 and securin proteins (21, 24). Consistent with the cellular results (21, 24), the dorsolateral prostate of 2 mg DATS–treated mice exhibited increased expression of cyclinB1 and securin proteins compared with the control mice (Fig. 5D).
DATS administration failed to cause apoptosis or alter E-cadherin expression. We have shown previously that DATS treatment causes apoptosis in cultured human prostate cancer cells (18, 22, 25). We performed TUNEL assay using prostate sections from control and treated mice to test whether DATS-mediated inhibition of prostate cancer development in TRAMP mice was due to increased apoptosis. Although the average number of TUNEL-positive apoptotic bodies was slightly higher in the prostate of 2 mg DATS–treated mice compared with control mice, the difference did not reach statistical significance (Fig. 6A).
E-cadherin is considered to be a suppressor of invasion and growth of many epithelial cancers (42). Some anticancer agents function by causing up-regulation of E-cadherin expression (32). We therefore compared expression of E-cadherin in prostate of the control and 2 mg DATS–treated mice. Expression of E-cadherin did not differ in the dorsolateral prostate between control and 2 mg DATS groups (Fig. 6B). Collectively, these results indicated that the DATS-mediated prevention of PD carcinoma development in TRAMP mice was not due to increased apoptosis or altered expression of E-cadherin.
DATS administration failed to inhibit neovascularization. We have shown previously that DATS treatment inhibits in vitro angiogenic features (e.g., formation of capillary-like tube structures and/or migration) in human umbilical vein endothelial cells and prostate cancer cells (26). To test whether DATS administration caused suppression of neovascularization in vivo, the dorsolateral prostate sections from the vehicle-treated control and 2 mg DATS–treated mice were stained for angiogenic marker CD31 (also known as PECAM-1). The average number of vessels as well as the mean vessel diameter did not differ significantly in the dorsolateral prostate between the vehicle-treated control and the 2 mg DATS–treated mice (data not shown). These results indicated that the DATS-mediated suppression of angiogenesis in vitro was not translated into inhibition of neovascularization in vivo, at least with the DATS dosing regimen used in the present study.
Cytotoxicity of NK and dendritic cells isolated from control and DATS-treated mice against TRAMP-C1 target cells. Experimental data exist to support the hypothesis that NK and DC play an important role in immune surveillance during tumorigenesis (43, 44). Moreover, garlic compounds have been shown to modulate T-cell function (45). To test whether DATS-mediated suppression of PD carcinoma development in TRAMP mice was accompanied by boosting of NK/DC function, the cytotoxicity of these cells isolated from control and 2 mg DATS–treated mice was determined against TRAMP-C1 as a target cell line. The DATS administration did not have any appreciable effect on cytotoxic effects of NK cell alone or NK/DC cell cocultures against TRAMP-C1 cells (data not shown).
The long latency of prostate carcinogenesis renders this disease highly amenable to chemoprevention. Accordingly, identification and preclinical evaluation of novel agents potentially useful for chemoprevention of human prostate cancer is highly desirable and could have a significant effect on disease-related cost, morbidity, and mortality for a large segment of population. Guided by the results of population-based case-control studies (6), we have devoted considerable effort toward identification and preclinical evaluation of Allium vegetable–derived sulfur compounds for their efficacy against human prostate cancer cells (18, 19, 21–27). Our previous studies have revealed that garlic constituent DATS is highly effective in suppressing growth of human prostate cancer cells in culture as well as in vivo in xenograft model (18, 19, 21–27). The present study builds upon these observations and shows that DATS administration prevents development of PD carcinoma and multiplicity of lung metastasis in male TRAMP mice without causing weight loss or affecting the T-antigen expression. The incidence and the area occupied by the PD carcinoma were statistically significantly lower in the dorsolateral prostate of DATS-treated TRAMP mice compared with control mice. The DATS concentrations effective against development of PD carcinoma and pulmonary metastasis multiplicity are within the range that can be generated through dietary intake of garlic (37). It is important to point out that DATS has been administered to humans at a dose of 200 mg in combination with 100 μg selenium every other day for 1 month without any harmful side effects (46).
We have shown previously that DATS treatment suppresses growth of cultured human prostate cancer cells by causing G2 and M phase cell cycle arrest (19, 21, 23, 24). Exposure of PC-3, DU145, and/or LNCaP human prostate cancer cells to growth suppressive concentrations of DATS results in accumulation of G2 and prometaphase cells (19, 21). The DATS-mediated G2 phase cell cycle arrest in human prostate cancer cells is transient and correlates with down-regulation and increased Ser216 phosphorylation of cell division cycle 25C (19).4
S.V. Singh, unpublished results.
We have shown previously that apoptosis induction is an equally important mechanism in antiproliferative effect of DATS against human prostate cancer cells (18, 25). It is interesting to note that the number of apoptotic bodies is comparable in the dorsolateral prostate of control and DATS-treated mice. Several possibilities exist to explain discrepancies in the results between cultured human prostate cancer cells and TRAMP model in vivo. One possibility relates to the frequency and dose of DATS administration. A more intensive dosing regimen, such as higher dose and/or daily administration of DATS, may be required to elicit apoptotic response in the dorsolateral prostate of TRAMP mice in vivo. Likewise, the possibility that earlier treatment with DATS (e.g., starting at age 4 weeks) leads to increased apoptosis as well as even greater protection against prostate carcinogenesis in TRAMP mice cannot be ignored. Additional work is needed to systematically explore these possibilities.
Metastasis is the major cause of death in prostate cancer patients (2). The pathogenesis of metastasis is dynamic and complex involving a series of molecular events including synthesis and secretion of several angiogenic factors to promote neovascularization, motility and local invasion of the host stroma, entry into the circulation, detachment, and extravasation (47). Proliferation of the tumor cells within vasculature or the organ parenchyma is necessary for the completion of metastasis process (47). Steps leading to metastasis are complex and regulated by multiple molecules including growth factors, matrix metalloproteinases, and cell adhesion molecules (47). For example, loss of expression of cell adhesion molecules especially E-cadherin is believed to be important for development of metastatic lesions (42). Inhibition of prostate carcinogenesis and metastasis by plant flavonoids apigenin in TRAMP mice was shown to correlate with retained expression of E-cadherin (32). The present study reveals that the DATS administration inhibits pulmonary metastasis multiplicity in TRAMP mice. However, the inhibitory effect of DATS administration against pulmonary metastasis multiplicity seems independent of changes in E-cadherin expression or inhibition of angiogenesis. Determination of the precise mechanisms by which DATS administration inhibits pulmonary metastatic multiplicity requires additional work.
The TRAMP model shares many features important in human prostate cancer progression, including metastasis to distant sites, progression to androgen independence, and NE differentiation (48). The number of NE cells correlates with stage, Gleason Grade, and survival in castration-recurrent prostate cancers (48–50). The PD tumors and lymph node metastases in C57/BL6×FVB TRAMP mice express NE marker synaptophysin (40). We found that the fraction of synaptophysin-expressing NE cells is significantly lower in the prostate of 2 mg DATS–treated mice compared with control mice. These results indicate that DATS administration suppresses NE cells in TRAMP mice, which is consistent with inhibition of PD carcinoma development.
In conclusion, the results of the present study indicate that p.o. administration of DATS prevents development of PD carcinoma and multiplicity of pulmonary metastatic lesions in TRAMP mice without causing weight loss or affecting T-antigen expression. The DATS-mediated prevention of prostate cancer development correlates with reduced cell proliferation as evidenced by suppression of PCNA and Ki-67 expression and accumulation of cyclinB1 and securin proteins.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Grant support: National Cancer Institute grant CA113363. We thank Dr. Barbara Foster for the generous gift of TRAMP-C1 cells.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.