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Nutrition/Metabolism Laboratory, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215
2To whom correspondence should be addressed. E-mail: jrzhou{at}caregroup.harvard.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-dihydrotestosterone (DHT) induced expression of PART-1 transcript in androgen-sensitive LNCaP, but not in androgen-independent DU 145 or PC-3 human prostate cancer cells. The soy isoflavones genistein and daidzein dose-dependently inhibited DHT-induced expression of the PART-1 transcript. Genistein at 50 µmol/L completely inhibited expression of the PART-1 transcript in LNCaP cells induced by DHT at 0.1 and 1.0 nmol/L. Daidzein was less potent than genistein, whereas glycitein at the same levels as genistein or daidzein did not inhibit DHT-induced PART-1 transcript expression. Our studies suggest that use of the PART-1 gene as a biomarker for evaluating the efficacy of soy isoflavones on androgen-dependent prostate cancer warrants further investigation.
KEY WORDS: • prostate cancer • isoflavones • prostate androgen-regulated transcript-1
Prostate cancer accounts for 29% of all the estimated new cancer cases diagnosed in American men. It is the second leading cause of death for men, with one in every five developing invasive cancer (1
). Latent prostate tumors are commonly found with similar frequency in many countries and ethnic groups (2
). In contrast, aggressive prostate cancer is significantly less prevalent among Asian men (3
,4
), whose intake of soy products is very high. That Asian men who adopt a Western lifestyle show higher rates of clinically significant disease indicates that the difference is due largely to environmental factors such as diet and nutrition (5
–7
). Among dietary factors, greater consumption of soybean products in the Asian diet has been found to reduce the risks of prostate cancer progression and mortality (4
,8
).
Experimental studies have identified soy isoflavones, which include genistein, daidzein and glycitein, as a major group of bioactive components in soy. There has been considerable attention focused on the antiestrogenic and anticarcinogenic properties of genistein. However, much less is known about the effect of daidzein, which is the other major isoflavone present in soy food and which has been shown to be the more bioavailable isoflavone (9
). Genistein inhibits growth of prostate cancer cells in vitro (10
–12
) and inhibits prostate cancer incidence and/or growth in vivo (12
–14
). Mechanistic studies have shown that genistein is a potent protein tyrosine kinase inhibitor (15
), inhibits proliferation of prostate cancer cells (10
–12
), induces prostate cancer cell apoptosis (12
,16
,17
), and inhibits angiogenesis (12
,17
–19
).
In contrast to genistein, the anticancer activities of daidzein have not been well studied. Previous studies showed that daidzein inhibits the growth of prostate cancer cells in vitro (12
,20
,21
) and prostate tumors in vivo (13
,22
), with less potent antiprostate cancer activity than genistein.
Prostate cancer cells rely on androgen for growth. Genes regulated by androgenic hormones are of critical importance for the normal physiologic function of the human prostate gland, and they contribute to the development and progression of prostate cancer. Recent studies have identified a novel androgen-responsive gene, prostrate androgen-regulated transcript-1 (PART-1)3 (23
,24
). PART-1 is expressed predominantly in the prostate and is transcriptionally regulated by androgens in human prostate cancer cells (23
,24
), suggesting that PART-1 may represent a novel androgen-dependent prostate cancer tumor marker (24
).
Studies designed to identify new genes that are responsive to androgens and are sensitive to chemoprevention or dietary and nutritional prevention of androgen-dependent prostate cancer have become a research priority. The objective of this study was to investigate the effect of soybean isoflavones on the expression of the PART-1 transcript in human prostate cancer cells in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Genistein, daidzein and glycitein were purchased from LC Laboratory (Woburn, MA) and dissolved in dimethyl sulfoxide (DMSO, Sigma Chemical, St. Louis, MO). 5-Dihydrotestosterone (DHT) was purchased from Sigma and dissolved in ethanol (17.4mol/L). Charcoal-striped fetal calf serum (FCS) was purchased from HyClone Laboratories (Logan, UT).
Prostate cancer cell culture studies.
Three human prostate cancer cell lines, LNCaP, DU 145, and PC-3 (American Type Culture Collection, Rockville, MD) were used for the studies. Human prostate cancer cell lines were maintained as monolayer cultures in Dulbecco’s modification of Eagle’s medium supplemented with FCS (100 mL/L), 100 kU/L of penicillin, 100 mg/L of streptomycin and 200 mmol/L of L-glutamine, in a humidified incubator with 5% CO2 and 95% air at 37°C. For in vitro experiments, prostate cancer cells were cultured in FCS-containing culture medium (100 mL/L) for 24 h, switched to the medium with charcoal-striped FCS (100mL/L), treated with soy isoflavones and/or DHT, or vehicles (DMSO and ethanol), and cultured for another 24 h. Each treatment group contained the same amount of the vehicles. Cells were then harvested and total RNA was isolated for analysis. The experiments were performed at least three times.
Reverse transcription-polymerase chain reaction (RT-PCR).
Total RNA was extracted from prostate cancer cells using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. Total RNA (2 µg) was reverse-transcripted into first strand cDNA using Read-To Go, You-Prime First-Strand Beads (Amersham Pharmacia, Piscataway, NJ) and oligo dT (Life Technologies, Frederick, MD) for 1 h at 37°C in a PTC-200 Peltier thermal cycler (MJ Research, Waltham, MA). Thermocycling for each reaction was done in final volume of 50 µL containing 2 µL cDNA sample, 2 µL deoxynucleotide triphosphate (dNTP), 5 µL reaction buffer, 3.5 µL MgCl2, 2 µL primer forward and 2 µL primer reverse, 0.5 µL Taq DNA polymerase and 33 µL RNase-free water. The cycling conditions of 35 cycles consisted of denaturation at 94°C for 45 s, annealing at 62°C for 1 min, and elongation at 72°C for 1 min after an initial denaturation at 95°C for 5 min, and a final extension at 72°C for 10 min. The primer pairs for PART-1 (5'-AAG GCC GTG TCA GAA CTC AA-3' and 5'-GTT TTC CAT CTC AGC CTG GA-3') (24
) and for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5'-CAAAGT TGT CAT GGA TGA CC-3' and 5'-CCA TGG AGA AGG CTG GGG-3') were purchased from Gibco BRL (Life Technologies). After PCR, each sample was run in agarose gel (15.0 g/L) electrophoresis to ensure that a right-size product was amplified in the reaction. Bands were visualized using ethidium bromide and the gels were photographed under UV light using Bio-Rad Gel Doc 1000 (Bio-Rad Laboratory, Hercules, CA). RT-PCR was repeated for each sample.
The intensity of the band was quantitated using Quantity One software (Bio-Rad) and was expressed as arbitrary units. The relative intensity of each band was normalized against GAPDH, and expressed as the percentage of the control.
Statistical analysis.
The relative intensity of PART-1 expression in each group from three repeated experiments was expressed as the mean ± SD. Data were statistically analyzed to calculate two-sided comparisons among groups through initial ANOVA followed by Fisher’s protected least-significant difference (25
). The StatView 5.0 program (SAS Institute, Cary, NC) was used to perform statistical analysis. Differences with a P-value of <0.05 were considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In an androgen-depleted culture system (with charcoal-striped FCS), none of the cell lines expressed detectable levels of PART-1 (Fig. 1A
). DHT (50 nmol/L) induced expression of the PART-1 transcript in androgen-sensitive LNCaP cells but not in androgen-independent DU 145 or PC-3 cells (Fig. 1
A).
|
B). DHT at 1, 10 and 50 nmol/L induced expression of the PART-1 transcript by 9.0 ± 0.3, (P < 0.005), 8.2 ± 1.4, (P < 0.0001), and 18.6 ± 3.5 fold (P < 0.0001), respectively, compared with that induced by 0.1 nmol/L of DHT. The induction of PART-1 gene expression was saturated by 100 nmol/L DHT (Fig. 1
B). DHT at 10 nmol/L was then used in the following experiments. Inhibition of DHT-induced expression of the PART-1 transcript by genistein.
To determine whether genistein inhibits androgen-induced expression of the PART-1 gene, genistein at different concentrations was added to LNCaP cells induced with 10 nmol/L DHT and cells were incubated for 24 h. Genistein dose-dependently inhibited DHT-induced expression of the PART-1 transcript (Fig. 2A
). Genistein at 12.5, 25, 50 and 100 µmol/L inhibited PART-1 gene expression induced by 10 nmol/L DHT by 24.3% (P < 0.0001), 47.1% (P < 0.0001), 83.3% (P < 0.0001) and 100% (P < 0.0001), respectively, compared with the control (Table 1
). The various doses also differed significantly from one another. Genistein at 50 µmol/L completely inhibited PART-1 gene expression induced by DHT at 0.1 and 1 nmol/L (Fig. 2
B).
|
|
Similar to genistein, daidzein also dose- dependently inhibited the expression of the PART-1 transcript induced by DHT, but its effect was less dramatic than that of genistein (not shown). At 12.5, 25, 50 and 100 µmol/L, daidzein inhibited DHT-induced (10 nmol/L) PART-1 gene expression by 15.5% (P < 0.01), 27.4% (P < 0.0005), 54.5% (P < 0.0001) and 55.0% (P < 0.0001), respectively, compared with the control (Table 1)
.
In contrast to genistein and daidzein, glycitein did not inhibit DHT-induced expression of the PART-I transcript (data not shown).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
). Previous studies have demonstrated that soy products or soy phytochemicals, including genistein, suppress carcinogenesis and/or growth of prostate tumors in vivo (12
–14
, 17
, 27
–29
). These laboratory studies, together with epidemiologic investigations, underscore the benefits of utilizing soy phytochemical components as dietary prevention agents to prostate cancer.
Surrogate end point biomarkers are required in clinical chemoprevention and intervention trials to avoid the excessively long study periods and high costs associated with the use of cancer incidence reduction as an end point, particularly with relatively slow-growing tumors such as prostate adenocarcinoma. Although prostate-specific antigen (PSA) is the most valuable tool available for the diagnosis and management of prostate cancer, it is insufficiently sensitive and specific for early detection or staging of the malignancy (30
), and additional biomarkers are required.
PART-1 is a newly discovered androgen-regulated gene expressed mainly in prostate tissue (23
,24
). PART-1 locates to human chromosome 5q12 and encodes for a 60-amino acid protein (23
). PART-1 is frequently present at higher levels in malignant than in benign tissue (24
), suggesting that PART-1 expression is altered during cancer and that its regulation follows different mechanisms than PSA, which is usually found in higher levels in benign tissue than cancerous tissue (31
). PART-1 may be a secreted protein and its overexpression in cancer suggests that it may have value as an additional circulating biomarker of prostate cancer (24
).
In this study, we found that PART-1 expression was upregulated by androgens in androgen-sensitive prostate cancer cells, but not in androgen-independent prostate cancer cells (Fig. 1)
, consistent with a previous finding that PART-1 expression is associated with androgen dependence (23
). This study also investigated for the first time the effect of soybean isoflavones on the expression of the PART-1 gene in prostate cancer cells and found that genistein and daidzein significantly downregulated androgen-induced PART-1 gene expression in androgen-sensitive LNCaP cells (Figs. 1
, 2)
. Our findings suggest that PART-1 may serve as a candidate biomarker for evaluating the efficacy of soy products on androgen-dependent prostate cancer prevention. This possibility merits further investigation.
We selected genistein and daidzein concentrations of 0–100 µmol/L because these compounds exert antigrowth effects on LNCaP cells at a 50% inhibitory concentration of 50–100 µmol/L (12
). On the other hand, in vivo studies indicate that blood levels of isoflavones after soy isoflavone or genistein supplementation can reach only a few µmol/L (17
), levels at which genistein or soy isoflavones significantly inhibited the growth and metastasis of LNCaP tumors (17
). The higher levels of genistein required in vitro than in vivo suggest a difference between in vitro and in vivo systems and care must be taken in extrapolating in vitro findings to in vivo/human situations.
It has been suggested that soy isoflavones, especially genistein, have weak estrogenic activities and may function as weak estrogens/antiestrogens (32
). However, the possible antiandrogen activities of soy isoflavones have not been well studied. In this study, we found that genistein and daidzein dose-dependently inhibited the androgen-induced expression of the PART-1 transcript, suggesting that soy isoflavones may have direct antiandrogen activities. The mechanism by which genistein and daidzein downregulate androgen-induced expression of the PART-1 transcript is currently unknown. Further in vitro and in vivo studies will help elucidate the mechanisms involved.
In summary, our in vitro results suggest that PART-1 may be a useful tumor marker for evaluating the effect of soy products in prostate cancer prevention. Further studies, especially animal studies, are necessary to determine whether soy isoflavones or soy products inhibit prostate tumor growth associated with inhibited PART-1 expression in vivo.
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FOOTNOTES |
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3 Abbreviations used: DHT, 5-dihydrotestosterone; DMSO, dimethyl sulfoxide; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PART-1, prostate androgen-regulated transcript-1; PSA, prostate-specific antigen; RT-PCR, reverse transcription-polymerase chain reaction.
Manuscript received 6 September 2002. Initial review completed 3 October 2002. Revision accepted 17 October 2002.
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