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Nutrition and Cancer

Dietary Genistein Improves Survival and Reduces Expression of Osteopontin in the Prostate of Transgenic Mice with Prostatic Adenocarcinoma (TRAMP)¹

Roycelynn Mentor-Marcel, Coral A. Lamartiniere, Isam A. Eltoum^*, Norman M. Greenberg and Ada Elgavish^**,2

Departments of Pharmacology/Toxicology, ^* Pathology, and ^** Genetics, University of Alabama at Birmingham School of Medicine, Birmingham, AL 35294 and Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109

²To whom correspondence should be addressed. E-mail: aelgavis{at}uab.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Studies in vitro suggest that osteopontin (OPN), an extracellular matrix protein secreted by macrophages infiltrating prostate tumors, and by tumor cells, may have a role in the transition from clinically insignificant tumors to metastatic prostate cancer (PC). Latent PC occurs at equal rates in Western and Asian men, but the incidence of advanced PC is many-fold higher in Western men. Our earlier studies in TRAnsgenic Mouse Prostate adenocarcinoma (TRAMP) mice showed that genistein, an isoflavone found in soybeans, lowered the incidence of advanced PC. This suggested that lower intake of dietary soy may be one possible cause for higher incidence of advanced PC in Western men. The objective of the present study was to test the hypothesis that genistein may exert its preventive effect by inhibiting OPN expression. From 5 to 28 wk of age, 80, 68, and 30 TRAMP mice were fed AIN-76A diet containing 0, 250, or 500 mg genistein/kg body weight, respectively. Organ weights were measured. The steady-state level of OPN mRNA was evaluated by RT-PCR in a longitudinal study in 74 TRAMP and 32 nontransgenic litter mates (NTM). Administration of 250 and 500 mg genistein/kg AIN-76A improved survival (P = 0.008 and P = 0.005, respectively) and reduced mean weight of prostates with poorly differentiated cancer (PD) (P < 0.001), as well as the mean weight of periaortic lymph nodes (LN), although the latter was not significant. OPN was upregulated 10-fold in PD compared with prostates with a lower pathological score from TRAMP or NTM of any age (P = 0.003). OPN mRNA levels in the dorsolateral prostate and metastasis to LN were significantly correlated (r = 0.643; P = 0.00006). Genistein had a dose-dependent, significant inhibitory effect on OPN transcript levels in prostates displaying advanced prostate cancer (PD; score 6; P = 0.05). Studies are consistent with the possibility that dietary genistein may delay the progression from benign to malignant tumors by inhibiting OPN expression.

KEY WORDS: • genistein • prostate • osteopontin • TRAMP • cancer progression

Prostate cancer (PC)³ is currently recognized as the most frequent form of cancer in men (1). Although localized PC may be cured, 70% of patients with metastases will die of cancer rather than of an unrelated cause (2). The molecular events involved in the transition from slow-growing prostate tumors to metastatic PC are poorly understood.

International epidemiologic studies suggest that the environment may have a role in the progression of PC to the metastatic stage (3). Thus, the incidence of noninvasive PC is the same in U.S. and Asian men. However, the incidence of metastatic PC, although lower in Asian men (4), increases as they emigrate to the West (5). Change in the diet, from one high in soy to one low in soy, is one possible cause (6). This is supported by studies showing that dietary soy inhibits the growth of LNCaP xenografts in immunodeficient SCID mice (7).

Several studies, in vitro and in vivo, support the possibility that genistein (5,7,4'-trihydroxyisoflavone), the most abundant isoflavone component in soy, may have a chemopreventive role in PC progression in carcinogen-triggered rat models of PC (6,8). We showed that dietary genistein reduces the incidence of advanced PC in TRAnsgenic Mouse Prostate adenocarcinoma (TRAMP) mice (9), a transgenic mouse model that develops PC spontaneously (10).

The mechanisms that may mediate the preventive effect of genistein are not well understood. We postulated that because metastasis involves detachment of tumor cells from the prostate and migration to distant sites (11,12), genistein may prevent the progression to metastatic growth by affecting the expression of cell adhesion genes. This hypothesis is supported by studies in vitro showing that genistein increases cell adhesion (13) and inhibits in vitro invasion of PC cells (14). On the basis of our earlier studies in vitro (15), we postulated that genistein may act by inhibiting the expression of osteopontin.

Osteopontin (OPN) is an extracellular matrix protein secreted by macrophages infiltrating prostate tumors (16) and by tumor cells themselves (17,18). Human prostate tumor cells that have a propensity to metastasize and human PC tissue specimens with increased Gleason scores synthesize, secrete, and/or deposit large amounts of matrix proteins such as OPN, osteocalcin (OC), and bone sialoprotein (19). There are also reports of increased tumorigenicity and metastatic ability of tumor cells with higher expression of OPN (20). Transfection of cells with OPN increases their malignant phenotype (21), whereas transfection with antisense oligonucleotides yields populations with reduced malignant potential (22). OPN increases anchorage-independent growth of prostate cancer cells (17), as well as proliferation of normal prostate basal cells (15). Many of the studies suggesting that OPN might have an important role in the progression to metastatic growth were carried out in cell cultures of human prostate cells (11,15,17,23,24) or xenografts of human prostate tumor cells in severely immunodeficient mice (16). Demonstration of the role of OPN in PC progression in vivo has been delayed by a lack of animal models that develop PC spontaneously.

TRAMP mice were generated with the SV40 small and large T antigen (Tag) regulated by the rat probasin gene promote. Because probasin is a prostate-specific protein, this provides targeted expression of Tag in the prostate, resulting in transgene expression spatially restricted to the prostate. Expression correlates with sexual maturity and is hormonally regulated by androgens. TRAMP mice display progressive forms of PC, with the development of premalignant lesions, carcinoma in situ, local advancement into lymphatics, metastatic disease, and androgen receptor resistance (25).

We postulated that progression to advanced prostate cancer and subsequent metastasis to lymph nodes might be due to upregulation of OPN expression in the prostate of TRAMP mice. One objective of the present longitudinal study was to test this hypothesis. The second objective was to test the hypothesis that genistein, which we had found to inhibit progression to advanced PC (9), may inhibit the expression of this cell adhesion gene, delaying progression and improving survival.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Materials. Genistein was generously provided by Hoffman-LaRoche. Its purity was assessed by HPLC (98.5% pure, 1.5% methanol) as previously described (26).

    Transgenic mice. TRAMP mice on a pure C57BL/6 background were bred in our colony at the University of Alabama at Birmingham (UAB), as reported previously (9). Breeders were fed standard pellet mouse feed (Harlan Teklad 7012). Diet and water were consumed ad libitum.

Transgenic males and nontransgenic male litter mates (NTM), entering the study at 5 wk of age, were fed powdered AIN-76A (Harlan Teklad), a semipurified diet containing no detectable phytoestrogens (limit of detection, 5 nmol/L) until they were 12, 18, 24, or 28 wk old. These longitudinal studies were carried out in 74 TRAMP mice (12, 5, 7, and 50 TRAMP mice of age 12, 18, 24, and 28 wk, respectively) and 32 NTM (14, 7, 2, and 9 mice aged 12, 18, 24, and 28 wk, respectively). Mortality was monitored throughout the study.

To explore the effect of dietary genistein on OPN expression, transgenic males, entered the study at 5 wk of age and were fed phytoestrogen-free powdered AIN-76A diet containing varying levels of genistein, 0 (n = 80) 250 (n = 68) or 500 mg (n = 30) per kg AIN-76A. Mice were fed this diet until the completion of the study. Each mouse was weighed once a week. At 28 wk of age, mice were killed using a lethal dose of ketamine.

Animal care and treatments were conducted in accordance with established guidelines and protocols approved by the University of Alabama Animal Care Committee.

    Preparation and analysis of tissues. At necropsy, the dorsolateral prostate (DLP), periaortic lymph nodes (LN), and kidneys were weighed. A portion of the DLP was rapidly frozen in liquid nitrogen and stored at –80°C until further processing for mRNA analysis by RT-PCR. The rest of the DLP was fixed in an acid alcohol solution containing 96% ethanol, 1% glacial acetic acid, and 3% distilled water, as described by Folkvord et al. (27). LN was fixed in 10% neutral buffered formalin phosphate. Fixed tissues were embedded in paraffin and 4- to 5-µm sections were stained with hematoxylin and eosin (LN) or with Gomori trichrome staining (DLP) (28). Prostate lesions were scored using the 1–6 scale established for TRAMP mice (9,29).

    Analysis of relative steady-state mRNA levels of cell adhesion genes by RT-PCR. Total RNA was isolated from each mouse using the Rneasy Mini kit from QIAGEN. With total RNA as the starting material, AMV reverse transcriptase and the cDNA Cycle kit from InVitrogen were used to generate cDNA.

cDNA was amplified by PCR using the following primers (30): OPN-forward: AGG CAT TCT CGG AGG AAA C; and OPN-reverse: TCA TCA GAC TCA TCC GAA TG. cDNA for the housekeeping gene ß-actin was amplified in the same PCR reaction, using the following primers (31): ß-actin-forward: CTT TGC AGC TCC TTC GTT G; and ß-actin-reverse: TGC CAA TAG TGA TGA CCT G. In some of the studies, the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (32) was amplified in the same PCR reaction instead of ß-actin, using the following primers: GAPDH-forward: TAC ATG TTC CAG TAT GAC TCC ACT CAC GG; and GAPDH-reverse: GGT GGA AGA GTG GGA GTT GCT GTT G. PCR was performed using an initial denaturing step at 94°C (3 min), followed by 34 cycles consisting of a denaturing step at 94°C (1 min), an annealing step at 60°C (30 s), an extension step at 72°C (30 s), and, finally, one last step at 72°C (5 min). PCR products were separated on a 1.5% agarose gel and visualized by staining with ethidium bromide. Expected fragment sizes were 399 bp for OPN, 749 bp for GAPDH, and 799 bp for ß-actin. The intensity of the band of each PCR product was measured by densitometry, using the Bio-Rad Fluor-S MultiImager and the software QuantityOne, version 4.1.0. The relative steady-state level of OPN was expressed as a ratio of the intensity of the PCR product for OPN and the housekeeping gene, both amplified in the same PCR reaction.

    Data analysis. Data were analyzed using SigmaStat, version 2.03 (SPSS). When 2 groups were compared, Student’s t test was used if data in the 2 groups were normally distributed with equal variance. When the effect of 2 variables was examined, 2-way ANOVA was performed. To isolate the group or groups that differed from the others, an all pairwise multiple comparison procedure (Tukey’s test) was carried out. When means were compared, results were presented as means ± SD. ² analysis was used to compare frequencies in multiple groups. Pearson’s correlation coefficient r was calculated to evaluate relations. P-values for the appropriate tests are given with the results.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Dietary genistein improves survival. Mice were observed daily and mortality was recorded. The percentage of mice that survived in each of the experimental groups was measured as a function of age (Fig. 1). ² analysis showed that survival was significantly improved in TRAMP administered 250 (P = 0.008) or 500 (P = 0.005) mg genistein/kg AIN-76A diet compared with TRAMP fed AIN-76A alone. In the control group, 100% of the mice survived until 19 wk of age; 100% of mice fed 250 or 500 mg genistein/kg AIN-76A survived until 21 and 23 wks of age, respectively. We postulated that genistein improves survival by reducing tumor burden. This possibility was examined next.

View larger version (23K): [in this window] [in a new window]   FIGURE 1 Dietary genistein improves TRAMP survival. Mice were fed a phytoestrogen-free diet containing 0 (n = 80), 250 (n = 68) or 500 (n = 30) mg genistein/kg AIN-76A starting at 5 wk of age until they were 28 wk of age. Mice were observed every day and mortality was recorded. The percentage of mice that survived was calculated once a week. The y-axis (% Mice Survived) is the number of mice that survived to the respective age, as a percentage of all mice in the respective experimental group.

View larger version (15K): [in this window] [in a new window]   FIGURE 2 Organ weights in 28-wk old TRAMP with poorly differentiated prostate cancer (score 6) and age-matched TRAMP with prostate scores < 6. DLPs (Panel A), LN (Panel B), and kidneys (Panel C) were weighed at necropsy. Values are means ± SD, and describe organ weights measured in the 28-wk-old TRAMP mice in Fig. 1 who were fed the AIN-76A diet without genistein. *Different from prostates with a score < 6, P < 0.001.

Using 2-way ANOVA, followed by a pairwise multicomparison test, we determined that dietary genistein significantly inhibited mean PD (score 6) weight (P = 0.05), but had no marked effect on the weight of DLPs with lower scores (Fig. 3A). Using the same statistical approach, genistein had no effect on LN from TRAMP with DLPs with scores < 6 (Fig. 3B). In contrast, genistein inhibited LN weight in a dose-dependent manner in mice with PD (score 6), but the effect was not significant (Fig. 3B).

View larger version (19K): [in this window] [in a new window]   FIGURE 3 The effect of dietary genistein on DLP (Panel A) and LN (Panel B) weights at necropsy in 28-wk old TRAMP mice in Fig. 1. Values are means ± SD, of DLP and LN weights from TRAMP with a prostate score < 6 or 6, in groups fed diets containing varying levels of genistein. *Different from control TRAMP fed diet without genistein, P = 0.05.

    OPN transcript levels are elevated in PD of 28-wk-old ("middle aged") TRAMP mice. Our studies focused on transcript levels because the expression of the OPN gene is regulated at the transcript level (24). The optimal conditions for quantitative RT-PCR were determined first. cDNA was prepared by RT from each mouse DLP sample. Before routine amplification by PCR, conditions under which band intensity determined by densitometry was a linear function of primer concentrations, number of PCR cycles, and template dilution were determined. The relative steady-state level of OPN transcript was expressed as a ratio of the band intensity of the OPN PCR product and a housekeeping gene, both amplified in the same PCR reaction. In initial studies, OPN expression was evaluated relative to ß-actin, as well as GAPDH. Finally, to ensure reproducible, quantitative data, the PCR amplification step was carried out several times for every mouse DLP cDNA, each time with serial dilutions of the template.

The steady-state level of OPN was higher in DLPs with a score of 6 (PD) compared with DLPs with a lower score, whether expressed relative to ß-actin (Fig. 4A, P = 0.003) or GAPDH (Fig. 4B, P = 0.006). Given the slightly better P-value of data obtained using the housekeeping gene ß-actin, only the latter was used in the rest of the studies. Expression of OPN transcripts was very low in prostates with scores from 1 to 5, in 12-, 18-, 24-, or 28-wk-old transgenic or NTM (Fig. 5). In this study, PD was never found in TRAMP mice < 24 wk old. OPN transcript levels were 10-fold higher in TRAMP PD (score 6) compared with levels in any other experimental group (Fig. 5) (P < 0.001).

View larger version (12K): [in this window] [in a new window]   FIGURE 4 Relative steady-state levels of OPN mRNA expression are higher in PD (score 6) compared with those in DLPs with a lower pathological score, regardless of the housekeeping gene used. Steady-state mRNA levels of OPN were determined by RT-PCR in the 28-wk old TRAMP fed AIN-76A diet without genistein in Figure 1. Values are means ± SD, of the relative steady-state level of OPN, expressed as a ratio of the intensity of the PCR product band for OPN and the intensity of the PCR product band for the housekeeping gene (Panel A, ß-actin; Panel B, GAPDH), both amplified in the same PCR reaction. *Different from DLPs with a score < 6, P = 0.003; **different from DLPs with a score < 6, P = 0.006.

View larger version (18K): [in this window] [in a new window]   FIGURE 5 Relative steady-state levels of OPN mRNA expression as a function of age in TRAMP mice and NTM. These longitudinal studies were carried out in 74 TRAMP mice (12, 5, 7, and 50 TRAMP mice of age 12, 18, 24, and 28 wk, respectively) and 32 NTM (14, 7, 2, and 9 mice aged 12, 18, 24, and 28 wk, respectively) fed AIN-76A diet. Steady-state mRNA levels of OPN were determined by RT-PCR. Values are means ± SD, of the relative steady-state level of OPN mRNA, expressed as a ratio of the band intensity of the PCR product for OPN and the band intensity of the PCR product for the housekeeping gene ß-actin, both amplified in the same PCR reaction. *Different from levels found at any age in TRAMP DLPs with a score < 6, or nontransgenic DLP, P < 0.001.

View larger version (19K): [in this window] [in a new window]   FIGURE 6 The effect of dietary genistein on steady-state mRNA levels of OPN measured in DLPs from each 28-wk-old mouse in Fig. 1 using RT-PCR. Values are means ± SD, of the relative steady-state level of OPN mRNA in each experimental group, expressed as a ratio of the band intensity of the PCR product for OPN and the band intensity of the PCR product for the housekeeping gene ß-actin, both amplified in the same PCR reaction. *Different from control TRAMP fed the diet without genistein, P = 0.05.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Another possibility is that genistein may improve survival by reducing metastasis. Because LNs are the first organ invaded by metastatic tumor cells (9,25), we postulated that genistein might inhibit metastatic spread to LN. Because metastatic invasion in LN and LN weight were correlated in TRAMP mice (r = 0.623; P = 0.0004), we examined the effect of dietary genistein on LN weight. As was reported previously for R-Flurbiprofen (33), dietary genistein reduced LN weight in TRAMP with advanced PC (Fig. 3B), consistent with reduced metastatic invasion.

Earlier studies showed that human PC cell lines that have a propensity to metastasize and prostate cancer tissue specimens with increased Gleason scores synthesize, secrete, and/or deposit large amounts of noncollagenous bone matrix proteins, e.g., OPN (17,36). Therefore, we tested the hypothesis that OPN may be upregulated in advanced TRAMP prostate tumors (score 6) and that genistein may exert its chemopreventive potential by reducing expression of OPN.

    OPN transcript level in TRAMP mice is 10-fold elevated in PD. In a longitudinal study in vivo, we showed that the steady-state level of OPN transcripts was 10-fold higher in the prostate of 28-wk-old TRAMP with PD (score 6) compared with that in age-matched TRAMP with a lower pathological score (Fig. 5) or age-matched NTM (Fig. 5). This excluded the possibility that high OPN levels observed in prostates with a score of 6 may simply be a result of the aging process.

It was postulated previously that OPN overexpression, probably secondary to transformation with oncogenes (37), may have a role in metastasis. Several mechanisms by which this may occur are possible. There is evidence that OPN induces immune cell (e.g., macrophage) migration to sites of inflammation resulting from prostate growth (38). Moreover, tumor cells producing elevated levels of OPN may suppress the oxidative burst, inhibit NO production, and thus protect themselves from killing by specific host cell types, e.g., macrophages (39). OPN was also shown to mediate neovascularization (40), stimulate proliferation (15), and inhibit apoptosis (17). Further studies will be necessary for definitive distinction between the possibilities that OPN overexpression in the TRAMP prostate is a by-product of progression to metastatic stage or an active contribution to this process. However, the present study suggests that OPN expression is involved in progression, as supported by the fact that LN metastasis and OPN mRNA levels in DLP were correlated (r = 0.643; P = 0.00006). Although the expression of the OPN gene is regulated at the transcript level (24), additional studies will be necessary to confirm that the changes in OPN transcript level in PD are translated into similar changes in OPN protein expression.

    Dietary genistein reduces OPN transcript level in the TRAMP prostate. Lifelong dietary genistein reduced OPN transcript levels in PD (score 6) (Fig. 6). Because OPN is elevated in inflamed tissues (24), one possibility is that genistein may exert its preventive effect indirectly, by reducing inflammation in the enlarged PD.

An alternative possibility is that genistein may act by reducing serum levels of estrogen and testosterone, as supported by the low serum levels in Japanese men consuming a high-soy diet (41). Because the OPN promoter contains estrogen response elements (42), genistein-inhibited levels of estrogens may cause lower OPN expression.

The OPN promoter also contains activator protein (AP)-1 binding sites (21). The estrogen receptor mediates gene transcription from AP-1 enhancer elements as well as estrogen-responsive elements (43). Earlier (43) and recent studies (44) indicate opposite effects of estrogen and environmental estrogenic chemicals, depending on whether they act via AP-1–mediated transactivation and estrogen receptor or AP-1–mediated suppression and estrogen receptor ß. Therefore, genistein, a phytoestrogen, may inhibit directly the expression of OPN. This possibility is supported by studies showing that genistein binds selectively to estrogen receptor ß (45).

Another possibility is that genistein alters OPN transcription indirectly, by affecting expression or function of growth factors. Although genistein increases mammary cell transforming growth factor-ß (TGF-ß) expression in vitro (46), recent studies showed that genistein decreases TGF-ß expression in vivo (47). Therefore, in vivo, genistein may affect OPN transcript levels via TGF-ß–mediated mechanisms. Because genistein inhibits epidermal growth factor (EGF) expression (38), it is possible that, as in rat mammary gland terminal ducts, genistein effects on OPN expression in TRAMP PD may also be mediated by EGF.

In conclusion, the products of oncogenes act in signal transduction pathways associated with growth factors. Their gain-of-function mutations cause uncontrolled cell cycle progression. Various growth factors also induce the transcription of genes for migration and invasion. Their dysregulation in cancer mediates the constitutive expression of metastasis genes, thus inducing a malignant phenotype. This connection distinguishes malignant tumors from benign tumors. It is possible that prevention of OPN induction may delay the progression from benign to malignant tumors (24). In the TRAMP mouse prostate, expression of the transgene (an oncogene), may act to trigger overexpression of OPN (a "metastasis gene"). Inhibiting induction of OPN, e.g., by genistein, may delay progression of benign prostate tumors to metastatic growth.

	ACKNOWLEDGMENTS

 
We thank Douglas Hamm for his help with the design of several of the primers used in the RT-PCR studies. We are also thankful to Marilyn Shackelford and Jane Hosmer for their expert preparation of prostate tissue for pathological evaluation.

	FOOTNOTES

 
¹ Supported by The American Institute for Cancer Research (AICR 00A023, to A.E.), the Department of Defense (DAMD 17–98-1–85-82, to C.A.L.), the National Cancer Institute (NCI CA 84926, to N.M.G.) and by fellowships (to R.M.) from the UAB Comprehensive Minority Faculty and Student Development Program and the NCI Cancer Prevention and Control Training Program (CPCTP).

³ Abbreviations used: AP-1, activator protein-1; DLP, dorsolateral prostate; EGF, epidermal growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LN, periaortic lymph node; MD, moderately differentiated prostate adenocarcinoma; NTM, nontransgenic litter mates; OC, osteocalcin; OPN, osteopontin; PC, prostate cancer; PD, poorly differentiated prostate adenocarcinoma; PIN, prostate intraepithelial neoplasia; Tag, T-antigen; TGF-ß, transforming growth factor-ß; TRAMP, transgenic mouse with prostate adenocarcinoma; WD, well-differentiated prostate adenocarcinoma.

Manuscript received 5 November 2004. Initial review completed 7 December 2004. Revision accepted 4 February 2005.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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