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Article

ß-Carotene Modulates Human Prostate Cancer Cell Growth and May Undergo Intracellular Metabolism to Retinol^{1 ,2 ,3}

Alexa W. Williams^*, Thomas W.-M. Boileau^*, Jin Rong Zhou, Steven K. Clinton^** and John W. Erdman, Jr.^*4

^* Division of Nutritional Sciences and Department of Food Science and Human Nutrition, University of Illinois, Urbana-Champaign, IL 61801; Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; and ^** Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, Division of Hematology and Oncology, The Ohio State University, Columbus, OH 43210-1240

⁴To whom correspondence should be addressed.

	ABSTRACT

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Epidemiologic and animal studies provide support for a relationship between high intakes of carotenoids from fruits and vegetables with reduced risk of several malignancies including prostate cancer. The highly controlled environments of in vitro systems provide an opportunity to investigate the cellular and molecular effects of carotenoids. The effects of ß-carotene (BC) on in vitro growth rates, p21^WAF1 and p53 gene expression, as well as the conversion of BC to retinol were investigated in three human prostate adenocarcinoma cell lines: PC-3, DU 145 and LNCaP. In these experiments, media concentrations of 30 µmol BC/L for 72 h significantly (P < 0.05) slowed in vitro growth rates in all three cell lines, independently of p53 or p21^WAF1 status or expression. ¹⁴C-labeled retinol was detected in prostate tumor cells incubated with ¹⁴C-labeled BC, suggesting metabolic conversion of BC to retinol. Conversely, no ¹⁴C-labeled retinol was detected in media incubated without prostate cancer cells. These studies support a hypothesis that in vitro biological effects of BC on prostate cells may result in part from the conversion of BC to retinol or other metabolites. The possibility that prostate cancer cells in vivo locally metabolize provitamin A carotenoids to retinol and other related metabolites may have implications for our understanding of prostate cancer etiology and the design of future prevention studies.

KEY WORDS: • prostate cancer • ß-carotene • vitamin A • retinoids • cell culture

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Evidence from epidemiologic and laboratory studies suggests that carotenoids and/or vitamin A modulate risk of prostate cancer (Clinton and Giovannucci 1998 ). However, a unifying hypothesis of how carotenoids, vitamin A or synthetic retinoids influence the initiation and progression of prostate cancer has not emerged. Interest in the provitamin A carotenoid, ß-carotene (BC)⁵ , has been stimulated by results from the Physicians’ Health Study, reporting that men with low baseline blood BC concentrations at the beginning of the study experience a decreased risk of developing prostate cancer when supplemented every other day with 50 mg BC (Cook et al. 1999 ). Since some carotenoids, such as BC, can be metabolized by mammals to retinol, it is necessary to consider this relationship when investigating the role of provitamin A carotenoids in prostate carcinogenesis. Many questions still remain regarding the provitamin A activity of BC, including identification of cell types and tissues capable of metabolic conversion, and the regulatory effects of vitamin A status, general health or other dietary components on conversion efficiency (Castenmiller and West 1998 ).

Prostate cell culture models allow researchers to examine the molecular, biochemical and cellular processes regulated or modulated by carotenoids and/or retinoids under precisely controlled conditions. Published data concerning the effects of carotenoids on cultured prostate tumor cells are limited. The inhibition of DU 145 human prostate adenocarcinoma cell proliferation was reported after incubation with canthaxanthin at concentrations of 10^-10-10^-8 mol/L, with less potent effects noted for BC, lycopene, retinoic acid, cryptoxanthin and zeaxanthin (Hall 1996 ). Although canthaxanthin is not a pro-vitamin A carotenoid, its conversion to the biologically active retinoid, 4-oxo-retinoic acid, has been shown (Hanusch et al. 1995 ).

We hypothesize that BC inhibits in vitro growth of three human prostate cancer cell lines (DU 145, PC-3, LNCaP) at media concentrations achieved in human serum by dietary BC supplementation (8–13 µmol BC/L) (Prince and Frisoli 1993 ). To further characterize potential mechanisms whereby BC may act, we measured the effects of BC on the expression of two genes known to influence cell cycle control (p53 and p21^WAF1) in prostate cell lines (Kinzler and Vogelstein 1996 , Norimura et al. 1996 , Xiong et al. 1993 ).

The in vitro conversion of BC to retinol has been demonstrated in hepatocytes (Blaner and Olson 1994 ), human colon cancer cells (During et al. 1998 ), human lung fibroblasts (Scita et al. 1992 ) and human skin fibroblasts (Wei et al. 1998 ). We hypothesize that BC and other provitamin A carotenoids may provide tissues, such as the prostate, a mechanism whereby local retinol concentrations may be increased even though serum vitamin A is tightly controlled. Conversion of BC to retinol by prostate cells may influence the concentration of intracellular ligands for retinoid receptors, subsequently influencing gene expression and biological effects regulated by the steroid receptor superfamily. To obtain additional information relative to this hypothesis, our studies also examine the ability of prostate cancer cells to convert ¹⁴C labeled BC to retinol in vitro.

	MATERIALS AND METHODS

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Cell culture methods.

Low passage (<10) PC-3, DU 145 and LNCaP human prostate adenocarcinoma cells were obtained from the American Type Culture Collection (Rockville, MD), and cell culture supplies were purchased from Sigma Chemical Company (St. Louis, MO). Cell lines were maintained at 37°C with 5% CO₂ in Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 10% fetal calf serum (catalog #2442), 60,000 U penicillin, 60 mg streptomycin (catalog #P0781) and 2.4 mmol L-glutamine (catalog #G7513) per 500 mL DMEM. BC was delivered to cells by one of two methods described below: tetrahydrofuran (THF) or water dispersible BC beadlets (beadlets). Media and cell pellets were stored in polypropylene tubes at -20°C overnight prior to extraction and HPLC analysis.

THF.

Delivery of carotenoids by THF to cells in culture was performed as previously described. (Bertram et al. 1991 ). Crystalline BC was solubilized in 10 mL THF containing 10 g/L BHT. Serial dilutions of the initial THF/BC solution were made to achieve lower concentrations. THF/BC solutions (100 µL) were used per 20 mL of prostate media for a final THF concentration of 0.5%, which is not cytotoxic (Bertram et al. 1991 ). Control media was prepared with 0.5% THF plus BHT. Fresh HPLC-grade THF was necessary since non-HPLC grade was found to be more toxic to cells in culture. BC dissolved in THF will adhere to plastic surfaces; therefore media was prepared and delivered in glass containers.

Beadlets.

Water-dispersible BC beadlets were provided as gifts from Hoffmann-LaRoche Inc. (Nutley, NJ). A known quantity of BC beadlets containing 10% BC and 1% -tocopherol was dissolved by sonication and vortex mixing in 10 mL DMEM and placed in a bath sonicator for 5 min. Serial dilutions using DMEM were made for less concentrated BC media. BC beadlet solutions (100 µL) were added per 20 mL media (Wamer et al. 1993 ).

In vitro growth assays.

Prostate cell lines were maintained as monolayer cultures in DMEM medium supplemented with 10% fetal bovine serum, 2 mmol L-glutamine, 1X10⁵ U penicillin/L and 100 g streptomycin/L in a 95% air/5% CO₂ water-saturated atmosphere. The in vitro growth studies were completed with 5 x 10⁴ cells plated into 96-well microplates, treated with BC beadlets and incubated for 72 h. In vitro growth rates were quantitated by the sodium 3'-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate (XTT) assay (Roehm et al. 1991 ). XTT is an indirect measure of cell number that is a variation of the MTT assay. This assay measures the production of a highly colored formazan product produced by the conversion of XTT by dehydrogenase enzymes of metabolically active cells. The colored product of XTT is water-soluble, eliminating the need for formazan crystal solubilization as is the case with the MTT assay (Roehm et al. 1991 ). The in vitro growth assay was completed in quadruplicate, the experiment replicated and statistically analyzed. The results of the XTT assay were confirmed by direct cell counting using a hemocytometer (n = 3) with trypan blue exclusion to assay for cell viability.

p53 and p21^WAF1 expression.

Western blotting was performed to evaluate the effects of BC on the expression of p53 and p21^WAF1, critical regulators of cell cycle progression and apoptotic pathways (Kinzler and Vogelstein 1996 , Norimura et al. 1996 , Xiong et al. 1993 ). Prostate cancer cells (LNCaP, PC-3, DU 145) were treated with BC beadlets at media concentrations of 0, 5, 10 and 20 µmol/L for 3 d. Cells were harvested and debris removed by brief centrifugation. Cells were disrupted in ice-cold lysis buffer (PBS, pH 7.4, 10 g/L NP-40, 5 g/L sodium deoxycholate, 1 g/L SDS) with freshly-added proteinase inhibitors (10 mmol/L N-ethylmaleimide, 10 g/L aprotinin, 2 g/L pepstatin A, 10 g/L leupeptin, 2 mmol/L phenylmethylsulfonyl fluoride, 1.0 mmol/L NaVO₄, 10 mmol/L NaF), followed by centrifugation (14,000 x g for 30 min). Protein concentrations of cell lysates were determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Richmond, CA). Proteins (20 mg) were separated by SDS-PAGE and transferred onto the membranes using standard procedures. After blocking nonspecific binding sites (5% nonfat dry milk in PBS overnight), the membrane was incubated with primary antibodies against p53 (1:1000 dilution, mouse antihuman monoclonal antibody; Santa Cruz Biotechnologies, Santa Cruz, CA) or p21^WAF1 (1:200, mouse antihuman monoclonal antibody; Oncogene Research Products, Cambridge, MA) for 30–60 min, washed three times with PBST (0.1% Tween 20 in PBS), incubated with HRP-conjugated secondary antibody (1:2000; Amersham Life Science, Arlington Heights, IL) for 30 min, and washed four times with PBST. The expression was detected by incubating the membrane with the chemiluminescent reagent according to the manufacturer’s recommendations (ECL, Amersham Life Science) for 1 min, followed by exposure to X-ray film.

Evaluation of BC conversion to retinol.

¹⁴C-labeled BC (gift of Hoffmann-LaRoche, Inc.) was purified by HPLC separation and peak collection on the HPLC system described below. The HPLC eluent was evaporated using the Savant AS 160 Speedvac (Farmington, NY), and the labeled BC was stored in hexane at -20°C overnight. ¹⁴C carbons were located at 10, 11, 10', 11' of the BC chain, resulting in a specific activity of 43 Bq/mmol BC. Immediately prior to media preparation, the labeled BC solution was aliquoted into Eppendorf tubes to be used in either prostate media or conditioned media from each of the three cell lines. The labeled BC was incorporated into the media using THF with a final solvent concentration of 0.5%. Each flask of cells and conditioned media contained ~0.19 µmol/L ¹⁴C labeled and 0.20 µmol/L unlabeled BC.

Three flasks (75 cm²) per cell line (DU 145, PC-3 and LNCaP) were plated at ~1 x 10⁹ cells/L and incubated for 2 d. On d 3, the conditioned medium was removed and fresh medium containing both radiolabeled and unlabeled BC (~0.4 µmol/L total BC) was added to the cells. Radiolabeled BC was also added to the conditioned medium from each of the three cell lines. Cells with fresh medium and conditioned medium without cells were incubated for an additional 48 h. At harvest, medium was collected from all flasks, cells were rinsed with DMEM, trypsinized and pelleted. Cells and medium samples were extracted as described below. The extracts were separated by HPLC using the Supelcosil C18 column (Supelco, Bellefonte, PA) with the 47:47:6 methanol/acetonitrile/chloroform mobile phase and a Waters M991 photodiode array system (Milford, MA). Eluent fractions were collected in glass scintillation vials every 30 s and evaluated for radioactivity using a Beckman LS900 Scintillation Counter (Fullerton, CA). The UV-visible spectra of retinol at 325 nm were confirmed on the photodiode array system.

Extraction and HPLC analysis of cells and medium.

The cells were transferred from the polypropylene centrifuge tubes in which they were frozen to glass tubes by rinsing with 0.5 mL distilled water three times. Aliquots for protein assays were removed and analyzed by the BCA method (Sigma Kit TPRO-562). The centrifuge tubes were further rinsed into the glass tubes with 1 mL of 100% ethanol (containing 1 g/L BHT) twice. Saturated KOH (200 µL) was added and the mixture was vortexed and saponified for 20 min at 60°C. After cooling, 200 µL distilled water and an internal standard, echinenone (Hoffmann-La Roche), were added. After adding equal volumes of hexane, the samples were vortexed thoroughly and allowed to separate on ice. The hexane layer which contained the carotenoids and retinoids was removed, and the hexane addition and extraction were repeated. The samples were evaporated with a Savant AS 160 Speedvac (Farmingdale, NY) and stored at -20°C under argon gas before analysis which was completed within 48 h of the extraction. Reconstitution for reverse-phase HPLC analysis was in methylene chloride. Typically, 100 µL of media was extracted by adding 100 µL ethanol/BHT solution and echinenone. Without saponification, the extraction procedure proceeded with the addition of equal volumes of hexane as stated above.

A Vydac 201TP54 C18 reverse-phase column (The Separations Group; Hesperia, CA) was used for carotenoid analysis and ¹⁴C-BC purification (2 mL/min) with mobile phase of 88% methanol, 9% acetonitrile, 2% water with the addition of 1% 2,2,4-trimethyl pentane as a solvent modifier. Detection at 450 nm and integration utilized a Bio-Rad model 170 UV-VIS detector and a Shimadzu CR601 Chromatopac integrator (Kyoto, Japan). The appearance of retinol or polar metabolites was monitored at 325 nm with a Waters 486 uv-vis detector from a Supelcosil C18 column with a mobile phase of 47% acetonitrile, 47% methanol and 6% chloroform (1.5 mL/min). All HPLC solvents were from Fisher Scientific (Pittsburgh, PA).

Statistics.

For in vitro growth assays, groups were compared using one-way ANOVA. Significant tests (P < 0.05) were further analyzed using the post-hoc Fisher’s Protected Least Squares Difference test (Statview, Brain Power, Calabasas, CA).

	RESULTS

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BC and in vitro growth of prostate cancer cells.

Figure 1 shows the in vitro growth of PC-3, DU 145 and LNCaP cells treated with BC solubilized in THF for 72 h as measured by the XTT assay. Medium concentrations of BC > 30 µmol/L significantly (P < 0.05) slowed growth of all three cell lines after 3 d. Due to insufficient sample size and carotenoid detection limits, cellular BC concentrations are not available for the 96-well plate experiments.

View larger version (23K): [in this window] [in a new window]   Figure 1. Growth inhibition of three human prostate cancer cell lines by ß-carotene (BC) using the sodium 3'-[1-(phenylaminio)-carbonyl-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sulfoxic acid hydrate (XTT) assay. Prostate cancer cell lines were plated at d 0 and treated with various concentrations of BC at 0, 5, 10, 40 or 80 µmol/L final concentrations. Cell numbers were quantitated after 72 h, and the data are presented as the percentage of vehicle-treated controls for each of the BC-treated cell lines. Data represents the means of two separate experiments with four wells (n = 8/point). Standard error for each point is <7%.

The prostate cell lines showed the expected pattern of p53 and p21^WAF1 expression based upon status of the genomic DNA. No p53 was noted in the PC-3 cell line since both genes are deleted. The DU 145 cell line has a mutant p53 gene which leads to a decreased rate of degradation and accumulation of the protein. LNCaP cells exhibit normal expression of p53. Furthermore, in p53 expressing LNCaP and DU 145 cells, no change in expression was noted with variations in media BC. The LNCaP cell line was the only cell line to express detectable p21^WAF1. BC had no effect on p21^WAF1 expression. These studies show that the ability of BC to inhibit the in vitro growth of prostate cancer cells is independent of p53 and p21^WAF1 status.

¹⁴C radiolabeled BC conversion to retinol.

A polar metabolite tentatively identified as retinol was detected in culture media in proportion to media BC (see Tables 1 2 3 ). Based upon retention time and UV-VIS spectra of this polar metabolite, we tentatively identified this peak as retinol. We therefore hypothesize that prostate cells can convert BC to retinol in vitro. ¹⁴C-BC incubated for 3 d in conditioned medium without cells degraded into a diverse array of compounds (Figs. 2A and B ), but none consistent with retinol. In contrast, HPLC analysis of ¹⁴C-BC incubated with cells for 3 d resulted in the appearance of two major compounds, at about 3- and 15-min retention times (Fig. 2C) . The retention time and UV spectra of the radiolabeled compound in the 3-min peak are characteristic of retinol, while the 15-min peak is BC. Similar results were obtained from PC-3, DU 145 and LNCaP cell lines. Additional characterization of BC cleavage products or quantitative differences among the three prostate cell lines were not possible due to limited quantities of ¹⁴C-BC.

View larger version (20K): [in this window] [in a new window]   Figure 2. Evidence for the in vitro conversion of ß-carotene (BC) to retinol by PC-3 human prostate cancer cells. (A) 14C-BC conditioned media before incubation, (B) 14C-BC conditioned media after incubation for 3 d without cells and (C) PC-3 cells incubated with 0.4 µmol/L BC media for 3 d (0.19 µmol/L 14C-labeled BC and 0.2 µmol/L unlabeled BC). Extracts from media and cells were separated by HPLC, collected in 30 s aliquots and analyzed for radioactivity using a liquid scintillation counter. The radioactive peak at 3 min is tentatively identified as retinol by methods described in text. Figures are representative of results observed in triplicate in DU 145, PC-3 and LNCaP cell lines.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

BC and prostate cancer cell growth.

Without dietary supplementation, serum BC concentrations of humans vary markedly but are typically between 0.25 and 1.0 µmol/L. However, following consumption of carotenoid supplements or carotenoid-rich fruits and vegetables, serum BC concentrations between 8 and 13 µmol/L can be achieved (Cook et al. 1999 , Prince and Frisoli 1993 ). The media BC concentrations employed in our cell culture studies include and exceed the range of serum concentrations observed in humans. We observed a slight nonsignificant inhibition of prostate tumor cell growth in vitro in the range of BC concentration achieved with dietary supplementation. However, we are skeptical that in vitro results can directly predict in vivo activity. For example, the optimal oxygenation and nutrient status achieved in vitro is not typical of the in vivo tumor microenvironment (Kinzler and Vogelstein 1996 ).

The inhibition of growth curves by BC in vitro for the PC-3, DU 145 and LNCaP cell lines also appears to be independent of p53 and p21^WAF1 status (data not shown). Interestingly, these three cell lines show different patterns of p53 and p21^WAF1 expression. A protein product of a tumor suppressor gene, p53, acts as a transcription factor, the targets of which include genes controlling apoptosis, cell cycle progression, genomic integrity and metastases (Kinzler and Vogelstein 1996 , Norimura et al. 1996 , Ruley 1996 ). p21^WAF1 is a cyclin kinase inhibitor and previous studies have suggested that increased expression may be associated with an inhibition of cell cycle progression (Xiong et al. 1993 ). Our observations indicate that BC acts to inhibit in vitro growth of human prostate cancer cells in a manner that does not significantly alter p53 or p21^WAF1 expression or depend upon their presence.

Hypothesized mechanisms whereby carotenoids inhibit growth of malignant cells in vitro are numerous and remain speculative. Increased cellular differentiation (Rock et al. 1995 ), down-regulation of epidermal growth factor receptors (Muto et al. 1995 ), reduced adenyl cyclase activity (Hazuka et al. 1990 ), suppression of insulin-like growth factor bioactivity (Levy et al. 1995 ), enhanced expression of gap junctional proteins (Zhang et al. 1992 ) and protection against oxidative damage (Martin et al. 1996 ) have all been proposed.

Readers should be cognizant of the potential instability of carotenoids in cell culture media and the possibility that biological and molecular events detected in vitro may in fact be the result of carotenoid degradation produced under cell culture conditions (Williams et al. 2000 ). For example, two oxidation products of canthaxanthin, all-trans and 13-cis 4-oxo-retinoic acid, have been shown to influence biological responses in vitro (Hanusch et al. 1995 ). All-trans 4-oxo-retinoic acid binds the retinoid ß receptor with similar affinity as all-trans retinoic acid and is therefore a potential modulator of gene expression. Clearly, additional studies are needed to determine optimal approaches for the incorporation of carotenoids into in vitro cell culture systems to define biologically relevant mechanisms of action.

Conversion of BC to retinol in prostate tumor cells.

Our studies provide evidence that retinol is produced in vitro from the incubation of BC with prostate cancer cells. The mechanism by which BC is converted to retinol in vitro in the presence of prostate cells is unknown. Prostate cells may express a specific enzyme which cleaves BC as has been suggested for hepatocytes (Blaner and Olson 1994 ), human colon cancer cells (During et al. 1998 ), human lung fibroblasts (Scita et al. 1992 ) and human skin fibroblasts (Wei et al. 1998 ). It may be equally intriguing to consider the impact dietary carotenoids could have on prostate biology and disease risk if local cleavage to retinol or other metabolites such as retinoic acid does occur in vivo. Retinoic acid (10^-5 mol/L) has been found to repress androgen-stimulated cell growth of LNCaP cells (Young et al. 1994 ). Decreased cellular replication has also been observed in PC-3 prostate carcinoma cells incubated with 4-HPR (10 µmol/L), a synthetic retinoid (Igawa et al. 1994 ). Since serum concentrations of vitamin A are tightly regulated in vivo, the conversion of provitamin A carotenoids to retinol in the prostate could serve as a "by-pass" mechanism to increase local retinol concentrations.

In summary, we have demonstrated that BC inhibits in vitro growth of three different human prostate cancer cell lines without influencing p53 or p21^WAF1 expression. Investigators should consider several possible mechanisms whereby BC may influence cellular and molecular events in vitro: direct effects of BC on cellular processes controlling cell growth, secondary effects related to conversion of BC to retinol and other retinoids and the effects of carotenoid metabolites or degradation products unique to cell culture. The last possibility suggests a mechanism whereby in vitro studies could provide artifactual results that have little in vivo relevance. A combination of several in vitro and in vivo systems is recommended to provide the best possible data for characterizing how provitamin A carotenoids may influence prostate carcinogenesis.

	FOOTNOTES

 
¹ Supported by the Public Health Service, National Institutes of Health, National Cancer Institute: KO7 CA01680 and RO1 CA72482 to S. K. Clinton and NRI-U.S. Department of Agriculture program agreement #95–37200 to J. W. Erdman, Jr.

² Supported by the Comprehensive Cancer Center, The Ohio State University Grant P30CA16058, National Cancer Institute.

³ The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked as an "advertisement" in accordance with 18 USC 734 solely to indicate this fact.

⁵ Abbreviations used: BC, ß-carotene; DMEM, Dulbecco’s Modified Eagle’s Media; HPR, N-(4-hydroxyphenyl) retinamide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; THF, tetrahydrofuran; XTT, sodium 3'-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis (4-methoxy-6-nitro)benzene-sulfonic acid hydrate.

Manuscript received August 16, 1999. Initial review completed September 17, 1999. Revision accepted December 6, 1999.

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