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Departments of
*
Cancer Biology,
Pathology and
**
Urology, Wake Forest University School of Medicine, Winston-Salem, NC 27157
2To whom correspondence should be addressed. E-mail: scramer{at}wfubmc.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,25-dihydroxycholecalciferol, synergistically inhibited the growth of primary human prostatic epithelial cells (HPEC) and prostate cancer cells. Synergistic growth inhibition of HPEC was also observed between genistein and the low-calcemic vitamin D compound 25-hydroxycholecalciferol. Flow cytometry with HPEC indicated that genistein induced arrest in the G2M phase, whereas 1,25-dihydroxycholecalciferol or 25-hydroxycholecalciferol induced arrest in the G1/0 phase of the cell cycle. Combining genistein with either vitamin D compound resulted in both G2M and G1/0 arrest in HPEC. In contrast, flow cytometry of prostate cancer cells indicated that both genistein and 1,25-dihydroxycholecalciferol induced a G1/0 arrest either alone or in combination. These are the first studies that demonstrate synergism between the prostatic cell growth inhibition elicited by genistein and that elicited by vitamin D compounds.
KEY WORDS: • synergism • genistein • vitamin D • prostatic epithelial cells • chemoprevention
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), there is wide variation worldwide in the distribution of clinical prostate cancer. Rates are much lower in the East (China and Japan) than in the United States (2
). Interestingly, when Japanese men migrate to the U.S. rates of clinical prostate cancer increase (3
,4
), suggesting an environmental effect on prostate cancer incidence (5
). Epidemiological studies demonstrate an inverse correlation with prostate cancer incidence and increased consumption of soy products (4
) as well as increased exposure to vitamin D (6
,7
). Soy is a rich source of isoflavones such as genistein and daidzein, which are known to have antiproliferative effects on a number of tissues, including the prostate (reviewed in Ref. 8
). Schwartz et al. (6
) hypothesized that vitamin D deficiency increases the risk of prostate cancer. This hypothesis is supported by the demonstration that vitamin D exerts an antiproliferative as well as a prodifferentiating influence on prostatic cells in vitro (reviewed in Ref. 9
). Taken together, epidemiological and dietary data suggest that both soy and vitamin D play important roles in protecting against prostate cancer (8
).
Genistein is a dietary-derived isoflavonoid found in high concentrations in serum after ingestion of soy-rich meals (10
,11
). Rats fed a genistein-rich diet show selective uptake of genistein in the prostate (12
). Growth inhibition of prostatic cell lines by genistein occurs by cell cycle arrest at low doses and by apoptosis at high doses. Various mechanisms of growth arrest by genistein have been identified, including tyrosine kinase inhibition, topoisomerase II inhibition, DNA damage and anti-estrogenic effects. Because of its growth inhibitory properties in prostate cancer cell lines and the apparent lack of associated side effects, genistein is being developed as a chemopreventative agent for prostate cancer (9
,13
).
Vitamin D is a hormone that can be obtained in the diet or produced endogenously by a series of reactions that culminates in the most active metabolite of vitamin D, 1,25-dihydroxycholecalciferol [1,25(OH)2D3]. 1,25(OH)2D3 exerts strong antiproliferative actions on a number of tissues, including normal prostatic cells (14
) and prostate cancer cell lines (15
,16
). 1,25(OH)2D3 exerts its growth inhibitory properties via a G1/0 block in the cell cycle or, in some instances, by apoptosis (9
). Growth inhibition is mediated through the nuclear vitamin D receptor (VDR), a ligand-regulated transcription factor in the steroid receptor superfamily. 1,25(OH)2D3 has 
500–1000 times greater affinity for the VDR than does its relatively inactive precursor 25-hydroxycholecalciferol (25-OHD3) (17
). The conversion of 25-OHD3 to 1,25(OH)2D3 is mediated by 25-hydroxyvitamin D 1 hydroxylase (1OHase) (18
). 1OHase activity has been demonstrated in prostatic cells (19
), and we showed that 25-OHD3 inhibits prostatic cell growth nearly as effectively as 1,25(OH)2D3 (20
). Epidemiological studies have shown a strong association with decreased circulating levels of 25-OHD3 and increased prostate cancer risk (21
,22
). However, therapeutic use of 1,25(OH)2D3 has been limited because of the associated calcemic side effects of high-dose therapy with 1,25(OH)2D3 and the difficulty in maintaining sufficient serum concentrations of the hormone to inhibit prostatic growth (23
,24
).
In this report we demonstrate that genistein and vitamin D compounds [1,25(OH)2D3 or 25-OHD3] can synergistically inhibit both benign and malignant prostatic cell growth via cell cycle arrest. Our data suggest that dietary supplementation with genistein and vitamin D may be an effective strategy for prostate cancer chemoprevention and possibly chemotherapy.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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MCDB 105 was obtained from Sigma-Aldrich (St. Louis, MO) and RPMI 1640 was obtained from Invitrogen (Carlsbad, CA). MCDB 105 Complete was MCDB 105 supplemented with growth factors and hormones as described previously (20
). Genistein (4,5,7-trihydroxyisoflavone) was obtained from Sigma-Aldrich and stored at -20°C in the dark as single-use aliquots of concentrated stock in dimethylsulfoxide (DMSO). 1,25(OH)2D3 and 25-OHD3 were obtained from Biomol (Plymouth Meeting, PA) and stored at -80°C in the dark as concentrated stocks in ethanol.
Tissue culture
LNCaP cells were obtained from American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 supplemented with 100 mL/L fetal bovine serum (FBS). HPEC were isolated from prostatectomy specimens at Wake Forest University School of Medicine (Winston-Salem, NC) using previously described protocols (20
). Nomenclature for epithelial cell strains is "WFU," followed by the strain number and then the histology of origin (PZ, benign peripheral zone) (e.g., WFU7PZ).
Growth assays
HPEC were inoculated at 2 x 104 cells per 60-mm collagen-coated plate in MCDB 105 Complete. LNCaP cells were inoculated at 2 x 105 cells per 60-mm plate in RPMI 1640/FBS. Twenty-four to 48 h after inoculation, cells were treated with experimental media for 3–4 d as indicated. Media were changed every 24 h. At the end of the incubation period cells were washed two to three times with HEPES-buffered saline and detached by light trypsinization, and the number of viable cells was determined by staining with trypan blue and counting cells. For each dose response, at least five doses of a particular treatment were used. For 1,25(OH)2D3 and 25-OHD3, this range was between 0.01 and 100 nmol/L. For genistein, this range was between 0.1 and 50 µmol/L.
Flow cytometry
HPEC. HPEC were inoculated at 1 x 105 cells per 10-cm collagen-coated plate. At semiconfluence, the cells were treated with experimental media for 48 h with one media change at 24 h. At the end of the incubation period cells were trypsinized and fixed in ice-cold 70% ethanol at 4°C for at least 24 h. After fixation the cells were pelleted at 1000 x g, washed with phosphate-buffered saline and treated with 0.5 g/L ribonuclease A (Sigma-Aldrich) at 37°C for 4–6 h. Cells were then pelleted by centrifugation at 1000 x g, resuspended in 1 mL propidium iodide stain (50 µg/mL propidium iodide, 600 µL/L Nonidet P-40) and stored in stain at 4°C overnight. Cells were then analyzed using a FACStar Plus flow cytometer (BD Biosciences, Mansfield, MA). Between 10,000 and 20,000 events were acquired for every sample and analyzed using the CellQuest program (BD Biosciences). The percentage of cells in each phase of the cell cycle was then determined using the software program ModFit LT v. 2.0 (Verity Software House, Topsham, MN). Each experimental treatment was performed in duplicate.
LNCaP cells. LNCaP cells were inoculated at 7.5 x 105 cells per 10-cm culture dish. At 60–70% confluence cells were treated with experimental media for 24 h. After incubation cells were harvested by trypsinization and fixed in 70% ethanol at 4°C for at least 24 h. Fixed cells were resuspended in 700 µL propidium iodide stain prepared as above with the addition of 37 µg/mL ribonuclease A. Flow cytometry was performed immediately on resuspended cells as described above for HPEC.
Statistical analysis
Synergism was assessed using the Chou-Talalay method (25
) and CalcuSyn software (Biosoft, Ferguson, MO). The dose effect for each drug alone was determined based on the experimental observations using the median effect principle: the combination index (CI) for each combination was calculated according to the following equation: CI = [(D)1/(Dx)1] + [(D)2/(Dx)2] + [(D)1(D)2/(Dx)1(Dx)2], where (D)1 and (D)2 are the doses of drug that have x effect when used in combination and (Dx)1 and (Dx)2 are the doses of drug 1 and drug 2 that have the same x effect when used alone. CI = 1 represents the conservation isobologram and indicates additive effects. CI values < 1 indicate a more than expected additive effect (synergism).
All other statistical analyses were performed using the statistical software package NCSS 2002 (Number Cruncher Statistical Systems, Kaysville, UT). Differences in growth data were determined by two-way ANOVA controlling for vitamin D or genistein dose with post hoc analysis by Fisher’s test. Cell cycle distributions were compared by ANOVA with post hoc analysis by Fisher’s test. In all cases, P 
0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We compared the action of genistein on HPEC versus LNCaP and PC-3 cells and determined that HPEC were more sensitive than prostate cancer cell lines to the growth inhibitory effects of genistein. Half maximal growth inhibition of HPEC occurred between 1 and 4 µmol/L, whereas in LNCaP and PC-3 cells half maximal growth inhibition occurred at 20 and 50 µmol/L genistein, respectively (data not shown). To test if the combination of genistein and vitamin D would synergistically inhibit prostatic cell growth we treated HPEC (WFU55PZ) with increasing doses of genistein, 1,25(OH)2D3 or combinations of the two compounds and assessed the viable cell number. Treatment of cells with 0.5 µmol/L genistein, 0.1 nmol/L 1,25(OH)2D3 or 0.5 nmol/L 1,25(OH)2D3 alone had no significant effect on growth (Fig. 1A
). However, treatment of cells with a combination of 0.1 nmol/L 1,25(OH)2D3 and 0.5 µmol/L genistein significantly decreased viable cell number. The combination of 0.5 nmol/L 1,25(OH)2D3 and 0.5 µmol/L genistein also significantly decreased viable cell number (Fig. 1
A). Isobolographic analysis of the entire growth data set (data not shown) determined that these interactions were synergistic (CI 0.7). In this same experiment we also observed synergism with 0.1 or 1 µmol/L genistein when combined with 0.1 or 0.5 nmol/L 1,25(OH)2D3 (data not shown). This experiment was repeated with another HPEC strain (WFU58PZ) with similar results (data not shown). We performed similar experiments in LNCaP human prostate cancer cells and found synergism between 10 nmol/L 1,25(OH)2D3 and 15 µmol/L genistein (Fig. 1
B). Treatment of PC3 cells with the combination of genistein and 1,25(OH)2D3 had similar synergistic growth inhibitory effects (data not shown).
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C). In this same experiment we also observed synergism when 1 µmol/L genistein was combined with these same doses of 25-OHD3 (data not shown). This experiment was repeated with another HPEC strain (WFU55PZ) with similar results (data not shown). Cell cycle arrest by genistein and 1,25(OH)2D3
A total of 5 µmol/L genistein, 50 nmol/L 1,25(OH)2D3 or the combination of the two did not cause apoptosis as evidenced by a complete lack of a sub-G1 peak in flow cytometric analysis and the absence of membrane blebs or apoptotic bodies on microscopic observation (data not shown). A total of 50 nmol/L 1,25(OH)2D3 alone induced a G1/0 arrest of WFU7PZ whereas 5 µmol/L of genistein alone caused a G2M arrest (Fig. 2A
). When both genistein and 1,25(OH)2D3 were used in combination there was a G1/0 arrest as well as a G2M arrest. The combination reduced the percentage of cycling cells (S phase) from 33% in control treated cells to 16.5% in cells treated with both genistein and 1,25(OH)2D3 (Fig. 2
A, inset). A separate HPEC strain (WFU55PZ) showed similar results (data not shown). Additional experiments with 25-OHD3 indicated that 25-OHD3 alone induced a G0/1 arrest, and in combination with genistein there was both a G0/1 and G2M arrest (data not shown). In LNCaP cells, genistein and 1,25(OH)2D3 both independently or in combination caused a block in G1/0 of the cell cycle with an associated reduction in S-phase cells (Fig. 2
B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,13
,23
,24
). In addition, soy protein extract (with high isoflavone content) and cholecalciferol (vitamin D-3) are both available as over-the-counter formulations that are used by an increasing number of people as dietary supplements for alternative medicine purposes, including for prostate cancer treatment and prevention. Given that men with normal prostatic tissue may be (or are currently) exposed to dietary-derived genistein, and that the effects of genistein on benign prostatic tissue are unknown, we evaluated the effects of genistein alone or in combination with vitamin D compounds on the proliferation of benign and tumor-derived prostatic cells. We found that genistein inhibited HPEC growth more potently than prostate cancer cell growth. We determined that genistein and 1,25(OH)2D3 have a potent synergistic effect on HPEC growth inhibition. The combination of 1,25(OH)2D3 and genistein also synergistically inhibited LNCaP cell growth, although the effects were not as robust as with HPEC.
The synergism results with 1,25(OH)2D3 and genistein on HPEC show promise for the development of an effective chemopreventative strategy for prostate cancer. The proliferative index of normal or benign prostatic cells may increase the probability of the acquisition of oncogenic mutations in somatic cells (26
). Indeed, the tumor-promoting properties of androgens in the prostate are thought to be the result of increased proliferative capacity of normal prostate (27
). Due to the calcium-mobilizing effects of 1,25(OH)2D3, it is unlikely that 1,25(OH)2D3 will be used as a chemopreventative for prostate cancer. However, recent data have demonstrated that prostatic cells express 1OHase and this sensitizes them to the antiproliferative effects of 25-OHD3. The data presented here demonstrating that 25-OHD3 can also act synergistically with genistein to inhibit benign prostatic cell proliferation support the exploration of dietary supplementation of vitamin D and soy products for prostate cancer chemoprevention.
Having demonstrated synergism between vitamin D compounds and genistein in HPEC and LNCaP cells, we sought to explore the mechanism. 1,25(OH)2D3 and its analogs have been reported to inhibit the proliferation of a number of cell lines from various tissues by blocking cells in G1/0 of the cell cycle and/or by induction of apoptosis (reviewed in Ref. 8
). Various mechanisms have been proposed for genistein that are apparently dose and cell type dependent. Our data indicate that synergistic growth inhibition in HPEC may be mediated by blocking cells in both G1/0 and G2M phases of the cell cycle. In contrast, LNCaP cells were blocked only in G1/0. The lack of a G2M block by genistein in tumor cells may contribute to the reduced magnitude of synergism observed for human prostate cancer cell lines compared with HPEC.
In summary, we demonstrate that vitamin D can synergize with genistein to inhibit prostatic cell growth. Our data support the use of a combination of these two agents for both prevention and treatment of prostate cancer.
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FOOTNOTES |
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3 Abbreviations used: 1,25(OH)2D3, 1,25-dihydroxycholecalciferol; 1Ohase, 25-hydroxyvitamin D 1 hydroxylase; 25-OHD3, 25-hydroxycholecalciferol; CI, combination index; DMSO, dimethylsulfoxide; FBS, fetal bovine serum; HPEC, primary human prostatic epithelial cell; PZ, benign peripheral zone; VDR, vitamin D receptor.
Manuscript received 1 May 2002. Initial review completed 4 June 2002. Revision accepted 5 July 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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