Tocotrienol-rich fraction of palm oil induces cell cycle arrest and apoptosis selectively in human prostate cancer cells

BBRC Biochemical and Biophysical Research Communications 346 (2006) 447–453 www.elsevier.com/locate/ybbrc

Tocotrienol-rich fraction of palm oil induces cell cycle arrest and apoptosis selectively in human prostate cancer cells q,qq Janmejai K. Srivastava

a,b

, Sanjay Gupta

a,b,c,*

a

Department of Urology, Case Western Reserve University, Cleveland, OH 44106, USA Department of Urology, University Hospitals of Cleveland, Cleveland, OH 44106, USA Department of Urology, Case Comprehensive Cancer Center, Cleveland, OH 44106, USA

b c

Received 16 May 2006 Available online 2 June 2006

Abstract One of the requisite of cancer chemopreventive agent is elimination of damaged or malignant cells through cell cycle inhibition or induction of apoptosis without affecting normal cells. In this study, employing normal human prostate epithelial cells (PrEC), virally transformed normal human prostate epithelial cells (PZ-HPV-7), and human prostate cancer cells (LNCaP, DU145, and PC-3), we evaluated the growth-inhibitory and apoptotic effects of tocotrienol-rich fraction (TRF) extracted from palm oil. TRF treatment to PrEC and PZ-HPV-7 resulted in almost identical growth-inhibitory responses of low magnitude. In sharp contrast, TRF treatment resulted in significant decreases in cell viability and colony formation in all three prostate cancer cell lines. The IC50 values after 24 h TRF treatment in LNCaP, PC-3, and DU145 cells were in the order 16.5, 17.5, and 22.0 lg/ml. TRF treatment resulted in significant apoptosis in all the cell lines as evident from (i) DNA fragmentation, (ii) fluorescence microscopy, and (iii) cell death detection ELISA, whereas the PrEC and PZ-HPV-7 cells did not undergo apoptosis, but showed modestly decreased cell viability only at a high dose of 80 lg/ml. In cell cycle analysis, TRF (10–40 lg/ml) resulted in a dose-dependent G0/G1 phase arrest and sub G1 accumulation in all three cancer cell lines but not in PZ-HPV-7 cells. These results suggest that the palm oil derivative TRF is capable of selectively inhibiting cellular proliferation and accelerating apoptotic events in prostate cancer cells. TRF offers significant promise as a chemopreventive and/or therapeutic agent against prostate cancer.  2006 Elsevier Inc. All rights reserved. Keywords: Prostate cancer; Cancer chemoprevention; Tocotrienol-rich fraction; Palm oil; Cell cycle arrest; Apoptosis

An effective chemoprevention strategy relies on agents that can selectively eliminate proliferating cells via programmed cell death and spare quiescent or terminally differentiated cells [1,2]. Cancer chemoprevention has emerged as an important means of cancer control by using dietary agents or synthetic compounds that are capable of blocking neoplastic inception or delaying disease progression [1–5]. Research indicates that this strategy is promis-

q

Abbreviations: PrEC, normal human prostate epithelial cells; NF-jB, nuclear factor-jB; TRF, tocotrienols-rich fraction of palm oil. qq This work was supported by grants from United States Public Health Services RO1 CA108512, RO1 AT002709, R21 CA109424 and generous support of funds from Jim and Eillen Dicke. * Corresponding author. Fax: +1 216 368 0213. E-mail address: [email protected] (S. Gupta). 0006-291X/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.05.147

ing with respect to reducing the incidence of cancer in well-defined high-risk groups and in the general population [6,7]. In recent years considerable attention has been focused on the role of nutrients as chemopreventive agents. The concept of using micronutrients for the chemoprevention of cancer is based on evidence obtained from human epidemiologic studies, clinical trials, and studies of the cancer inhibiting potential of these agents on animal carcinogenesis models [8]. Basic research has identified a number of nutrients, some of which inhibit mutagenesis or hyperproliferation, and others that induce apoptosis or differentiation as critical characteristics of chemoprevention, regardless of their specific molecular targets [8–10]. Prostate cancer, because of its prolonged latency and high incidence, is an ideal target for the development of chemo-protective agents that could effectively reduce the

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incidence of this disease [9,10]. Use of naturally occurring compounds for chemoprevention, ones that are present in dietary sources, is considered to be a practical approach for the prevention of prostate cancer. Vitamin E, a fat soluble vitamin, is well known for its cellular antioxidant and lipid lowering properties [11–13]. The term vitamin-E has traditionally been applied to alpha (a)-tocopherol. It is now been increasingly accepted as a generic term for tocotrienols and tocopherols. Although both classes of compounds share the same aromatic chromanol ‘head,’ the two isoforms of vitamin E differ in their side chains. Tocopherols possess saturated phytyl sidechain while tocotrienols have an unsaturated isoprenoid chain along their ‘‘tails.’’ Both tocopherols and tocotrienols exist in four isomeric forms: alpha (a), beta (b), gamma (c), and delta (d). Each of these isomeric forms can be distinguished by the differing locations of methyl groups on their aromatic rings (Fig. 1). Accumulating evidence has suggested that tocotrienols possess cancer chemopreventive properties [14,15]. Tocotrienols extracted from crude palm oil consist mainly of a mixture of a-, c-, and d-tocotrienols and some a-tocopherols, referred to as tocotrienol-rich fraction (TRF). Tocotrienols have been shown to possess anti-oxidant, anti-inflammatory, anti-angiogenic, and anti-proliferative properties [16,17]. Tocotrienol-rich fraction from palm oil has been shown to inhibit proliferation and growth of human breast cancer cells [18,19]. Studies from our group have shown that TRF activates p53, modulates Bax/Bcl-2 ratio and induces apoptosis in human colon cancer cells [20]. More recent studies have shown that c-tocotrienols inhibits cell proliferation by decreasing Akt and activation of NF-jB, implicated in the regulation of cell growth, cell cycle, and apoptosis [21]. Dietary intake of palm oil, in contrast to other high-fat diets, suppressed carcinogen-induced mammary tumorigenesis in experimental animals [22–25]. Dietary supplementation of TRF has been shown to inhibit growth of estrogen-receptor positive human breast cancer MCF-7 cells in athymic nude mice [26]. Furthermore, stripping of tocotrienols from palm oil promoted mammary carcinogenesis, evidence supporting the anti-cancer attributes of tocotrienols [27]. At present information concerning the effects of TRF in prostate cancer is limited. This study provides the first evidence that TRF imparts differential anti-proliferative and apoptotic effects in human prostate cancer cells versus normal cells.

Materials and methods Cell lines. Androgen-responsive human prostate cancer cells, LNCaP (originally obtained from metastatic prostate cancer in a supraclavicular lymph node), androgen-refractory PC-3 (originally obtained from metastatic prostate cancer in bone), and DU145 (originally obtained from metastatic prostate cancer in brain), belonging to different metastatic sites, and virally transformed PZ-HPV-7 cells, derived from normal tissue of the peripheral zone of the prostate and immortalized by transfection with HPV 18 virus, were obtained from American Type Culture Collection (Manassas, VA). Normal human prostate epithelial cells (PrEC) were purchased from Clonetics and were propagated in the recommended medium. The LNCaP cells were cultured in RPMI 1640 medium with 10% FBS and 1% penicillin–streptomycin cocktail; DU145 and PC3 cells were cultured in RPMI 1640 media with 5% FBS and 1% penicillin–streptomycin cocktail (Cellgro, Mediatech, Inc., Herndon, VA) at 37 C in a humidified atmosphere of 5% CO2. The PZ-HPV-7 cells were cultured in keratinocyte serum-free medium supplemented with 5 ng/ml human recombinant EGF and 0.05 mg/ml bovine pituitary extract (Gibco). Tocotrienol-rich fraction (TRF) of palm oil. TRF was kindly provided by Abdul Gapor, Malaysian Palm Oil Board, Kuala Lumpur, Malaysia. The composition of TRF is as follows: a-tocotrienol 14.77%, c-tocotrienol 33.97%, d-tocotrienol 26.11%, a-tocopherol 18.10%, and other c-tocopherol, and tocotrienol-like compounds 7.05%, respectively. Proliferation assay. The effect of TRF on the viability of cells was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide) assay. Briefly, the cells were plated at 1 · 104 cells per well in 200 ll of complete culture medium containing 5, 10, 20, 40, and 80 lg/ ml concentrations of TRF in 96-well microtiter plates. TRF stock solutions were prepared in dimethyl sulfoxide (DMSO) at 100 lg/ml concentration and mixed with fresh medium to achieve the desired final concentration. Each concentration of TRF was repeated in ten wells. After incubation at 24 h at 37 C in a humidified incubator, cell viability was determined. Fifty microliters MTT (5 mg/ml in phosphate-buffered saline stock, diluted to working strength 1 mg/ml with media) was added to each well and incubated for 2 h after which the plate was centrifuged at 600g for 5 min at 4 C. The MTT solution was removed from the wells by aspiration. After careful removal of the medium, 0.1 ml of buffered DMSO was added to each well and plates were shaken. The absorbance was recorded on a microplate reader at the wavelength of 540 nm. The effect of TRF on growth inhibition was assessed as percent cell viability where vehicle-treated cells were taken as 100% viable. Colony formation assay. For anchorage-independent cell growth, soft agar colony formation assay was performed in a six-well plate (CostarCorning Incorporated, New York). Each well contained 2 ml of 0.5% agar in medium as the bottom layer, 1 ml of 0.38% soft agar (Sigma) in medium, and 2000 cells at the feeder layer treated with various concentrations of TRF (5–20 lg/ml in medium). Cultures were maintained at 37 C in a humidified 5% CO2 atmosphere. The number of colonies were determined after 2 weeks by counting them under an inverted phasecontrast microscope at 400· magnification and a group of 20 cells were counted as a colony. Cell cycle analysis. Asynchronized (70–80%) confluent cells were treated with 10, 20, and 40 lg/ml TRF for 24 h. After treatment cells were collected, washed twice with chilled PBS, and spun in a cold centrifuge at

Tocotrienol isoprenoid side chain

Tocotrienol isoforms

Tocopherol phytylside chain Fig. 1. Structure of vitamin E stereoisomers. Tocotrienols consist of a chromanol nucleus and a lipophilic isoprenoid chain. Tocopherols have phytyl side chain. Methyl group placement in the naturally occurring isoforms a, b, c, and d is as indicated in the box.

J.K. Srivastava, S. Gupta / Biochemical and Biophysical Research Communications 346 (2006) 447–453 600g for 10 min. The pellet was fixed by re-suspending in 50 ll PBS and 450 ll chilled methanol for 1 h at 4 C. The cells were washed twice with PBS at 600g for 5 min and again suspended in 500 ll PBS, and incubated with 5 ll RNAse (20 lg/ml final concentration) for 30 min at 37 C. The cells were chilled over ice for 10 min, stained with propidium iodide (50 lg/ml final concentration) for 1 h, analyzed by flow cytometry, and evaluated using Cell Quest & ModFit cell cycle analysis software. DNA fragmentation assay. Fragmentation of chromatin to units of single or multiple nucleosomes that form the nucleosomal DNA ladder in agarose gel is an established hallmark of programmed cell death or apoptosis [28]. Briefly, the cells were grown to about 70% confluence and treated with 20 lg/ml TRF for 48 h. Following these treatments, the cells were washed twice with phosphate-buffered saline (10 mM Tris, pH 7.5, 150 mM NaCl, 5 mM MgCl2, and 0.5% Triton X-100), left on ice for 15 min, and pelleted by centrifugation (14,000g) at 4 C. The pellet was incubated with DNA lysis buffer (10 mM Tris, pH 7.5, 400 mM NaCl, 1 mM EDTA, and 1% Triton X-100) for 30 min on ice and then centrifuged at 14,000g at 4 C. The supernatant obtained was incubated overnight with RNAse (0.2 mg/ml) at room temperature and then with Proteinase K (0.1 mg/ml) for 2 h at 37 C. DNA was extracted using phenol:chloroform (1:1) and precipitated with 95% ethanol for 2 h at 80 C. The DNA precipitate was centrifuged at 14,000g at 4 C for 15 min and the pellet was air-dried and dissolved in 20 ll of Tris–EDTA buffer (10 mM Tris–HCl, pH 8.0, and 1 mM EDTA). Total amount of DNA was resolved over 1.5% agarose gel, containing 0.3 mg/ml ethidium bromide in 1· TBE buffer (89 mM Tris, pH 8.3, 89 mM boric acid, and 2 mM EDTA) (BioWittaker, Inc., Walkersville, MD).The bands were visualized under UV transilluminator followed by digital photography. Cell death detection assay. Following TRF treatment, the extent of apoptosis was determined by Cell Death Detection ELISAPLUS assay (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s protocol. Briefly, the cells were harvested after TRF treatment at 10, and 20 lg/ml dose for 24 h, and incubated on ice for 30 min in Tris lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 20 mM NaF, 0.5% NP-40, and 1% Triton X-100) containing fresh protease inhibitors (5 lg/mL aprotinin, 10 lg /mL phenylmethylsulfonyl fluoride, and 10 lg/ml sodium vanadate), and then centrifuged at 14,000g for 10 min at 4 C. The total cell lysate was used for protein determination by the DC Bio-Rad protein assay. The lysate (30 lg of total protein) was added to lysis buffer and pipetted on a streptavidin-coated 96-well microtiter plate to which immunoreagent mix was added and incubated for 2 h at room temperature with continuous shaking at 600g. The wells were then washed with washing buffer, the substrate solution added, and the color developed (10–20 min) was read at 405-nm against the blank, reference wavelength of 490-nm. The enrichment factor (total amount of apoptosis) was calculated by dividing the absorbance of the sample (A405 nm) by the absorbance of the controls without treatment (A490 nm). Detection of apoptosis by fluorescence microscopy. Cells grown in eight chambered slides (Nunc-Labtek, Nunc, Naperville, IL) and were treated with 10 and 20 lg/ml for 24 h TRF. After 24 h cells were washed with PBS and processed for apoptosis detection by using Cytokeratin 18 (CK18) monoclonal antibody, which is an early marker for apoptosis. The fluorescence analysis was performed under the BX51 Olympus microscope by using M 30 Assay Kit (Roche Applied Sciences, Mannheim, Germany) according to vendor’s protocol. Statistical analysis. The values are expressed as means ± SE. The significance between the control and treated groups was performed by Student’s t test and p values less than 0.05 were taken as significant in all the experiments.

Results and discussion Tocotrienols are the primary form of vitamin E, a biologic agent that is commonly present in the seed endosperm of most monocots and certain dicots, including wheat, rice

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and barley. The highest fraction of tocotrienols is found in crude palm oil, in concentration of up to 800 mg/kg; this is known as the tocotrienol-rich fraction (TRF) [29,30]. The anti-cancer properties of TRF are currently under investigation [31]. Tocotrienols are believed to block or delay the malignant progression of transformed cells by modulating cell proliferation or differentiation [32,33]. These cytostatic effects could be attained by the ingestion of tocotrienols in the diet of healthy individuals who are at increased risk of developing prostate cancer. Cytostatic effects may also be beneficial by limiting the possibility of developing drug resistance and drug-induced toxicity. Therefore, we studied whether TRF has potential to slow the proliferation rate and eliminate prostate cancer cells via programmed cell death. In this study, we evaluated the growth-inhibitory effects of TRF in normal human prostate epithelial PrEC cells, virally transformed normal human prostate epithelial PZHPV-7 cells and human prostate carcinoma cells viz. LNCaP (androgen-responsive), DU145 (androgen-refractory), and PC-3 (androgen-refractory) cells, representing various stages in the biological spectrum of prostate cancer. We first assessed the effect of TRF on the viability of PrEC and PZ-HPV-7 cells. Exposure of PrEC cells to TRF resulted in a slight decrease in cell viability at the highest concentration of 80 lg/ml (data not shown). Exposure of PZ-HPV-7 cells to TRF showed similar findings, with a modest decrease in cell viability at the highest concentration of TRF exposure. In sharp contrast, all three cancer cell lines tested were more sensitive to TRF-mediated loss of viability, which occurred at lower doses and was much more pronounced than in the PrEC and PZ-HPV-7 cells. As shown in Fig. 2A, treatment of LNCaP cells with 5, 10, 20, 40, and 80-lg/ml concentrations of TRF for 24 h, resulted in 28.6%, 45.6%, 63.7%, 86.2%, and 87.8% inhibition of cell growth, compared to vehicle treated control. Exposure of DU145 cells to TRF resulted in 17.9%, 20.1%, 46.5%, 78.3%, and 79.8% inhibition of cell growth whereas PC-3 cells exhibited 18.5%, 43.3%, 54.5%, 85.8%, and 86.8% cell growth inhibition, respectively, after TRF exposure. Treatment of prostate cancer cells to 20 lg/ml concentration of TRF also resulted in time-dependent inhibition of cell growth in all three cell types and the effect was more pronounced at 48 h post-treatment (Fig. 2B). A modest time-dependent cell growth-inhibitory effect was observed in PZ-HPV-7 cells after TRF exposure. The IC50 value of TRF was estimated to be 16.5, 17.5, and 22.0 lg/ml for LNCaP, PC-3, and DU145 cells, respectively; however, for PZ-HPV-7 cells the value appears to be >80 lg/ml. These observations suggest that prostate cancer cells respond differentially to TRF exposure than their normal counterparts. We next investigated the effect of TRF on anchorage-independent growth by soft agar colony formation. Anchorage-independent growth is one of the hallmarks of malignancy, and is considered to be a direct in vitro assay for detection of malignant transformation of cells [34]. Tumor cells attain this capability via a series of genetic

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Fig. 2. Effect of TRF on cell viability (A) dose-dependent, and (B) time-dependent in virally transformed normal human prostate epithelial PZ-HPV-7 cells, and human prostate cancer LNCaP, DU145, and PC-3 cells. The cells were exposed to the specified concentration of TRF for 24 h, and viability of the cells was determined by MTT assay. For time-dependent assay cells were exposed to 20 lg/ml TRF for indicated times. Cell viabilities are depicted as percentages; vehicle-treated cells were regarded as 100% viable. The data represent the means of three experiments done in triplicate. Details are described in Materials and methods.

and epigenetic alterations. As shown in Fig. 2 compared with vehicle treated controls (22 colonies/field), exposure of LNCaP cells to TRF resulted in a significant decrease in anchorage-independent growth and colony formation to 11, 5, and 2 colonies/field at 5, 10, and 20 lg/ml concentration. In DU145 cells, TRF exposure at 5, 10, and 20 lg/ml concentrations resulted in 14, 12, and 7 colonies/field compared to the 35 colonies/field that were observed in vehicle treated controls. Similar observations were noted in PC-3 cells in which exposure to TRF resulted in 24, 16, and 8 colonies/field compared to the 44 colonies/field observed in vehicle treated control (Fig. 3). To establish a broad spectrum anticancer activity of TRF, we next evaluated the anti-proliferative effects on various human cancer cell lines viz. HeLa (cervical adenocarcinoma), MCF-7 (breast carcinoma), RKO (colon carci-

Fig. 3. Effect of TRF on anchorage-independent growth assay estimated by soft agar colony formation in human prostate cancer LNCaP, DU145, and PC-3 cells. The cells were grown over 0.38% agar medium along with vehicle (DMSO) only or specified concentration of TRF. The number of colonies was recorded after 14 days of treatment. Each bar represents number of colonies on soft agar + SE of three different assays. *p < 0.05, **p < 0.001. Details are described in Materials and methods.

noma), and HT1080 (fibrosarcoma) cells. TRF exposure at 20 lg/ml for 24 h resulted in 21.3% inhibition in HeLa cells, 30.2% inhibition in MCF-7 cells, 24.4% inhibition in RKO cells, and 17.1% inhibition in HT1080 cells, which was more pronounced at 72 h of TRF treatment (data not shown). These results suggest that TRF possess a broad spectrum anti-proliferative potential and its response is not limited to specific cell type. Manipulation of a number of biological pathways can be exploited for chemoprevention. Apoptosis is a physiological process for the elimination of redundant or damaged cells [35,36]. Apoptosis induction is arguably the most potent defense against cancer progression as most of the current-used chemotherapeutic drugs inhibit cancer cell proliferation by inducing apoptosis [37,38]. Unfortunately, chemotherapeutic agents are not designed to selectively target cancer cells. Consequently, chemotherapeutic agents typically damage normal cells, an adverse effect that hampers this therapeutic modality. Therefore, agents capable of preferentially eliminating cancer cells without affecting normal cells offer the safest modality for cancer treatment. We next investigated whether TRF-mediated loss of cell viability in human prostate cancer cells viz. LNCaP, DU145, PC-3, and PZ-HPV-7 cells is a consequence of apoptosis. We first evaluated the induction of apoptosis by TRF via classical DNA ladder assay. Compared to vehicle treated controls, exposure of LNCaP, DU145, and PC-3 cells to 20 lg/ml TRF for 48 h resulted in induction of apoptosis, as evidenced by the formation of internucleosomal DNA fragments (Fig. 4A). Importantly, it was noted that TRF exposure did not result in the formation of a DNA ladder in PZ-HPV-7 cells, exhibiting a selective dose–response effect compared to prostate cancer cells (Fig. 4B). Similar effects were observed with PrEC cells (data not shown). To quantitate and further support the finding that TRF exposure causes apoptosis in cancer cells we performed cell death detection by ELISA. Compared to

J.K. Srivastava, S. Gupta / Biochemical and Biophysical Research Communications 346 (2006) 447–453

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Fig. 4. Effect of TRF on induction of apoptosis in virally transformed normal human prostate epithelial PZ-HPV-7 cells, and human prostate cancer LNCaP, DU145, and PC-3 cells. (A) DNA fragmentation assay. The cells were treated with vehicle or 20 lg/ml concentration of TRF for 48 h, collected for DNA isolation and subjected to agarose gel electrophoresis, followed by visualization of bands and polaroid photography. The PZ-HPV-7 cells did not exhibit TRF-induced DNA fragmentation. (B) Apoptosis determined by Cell Death ELISA as per vendor’s protocol. Data are expressed as enrichment factor. Values represent mean ± SE of three different assays in duplicate, **p < 0.001. (C) Immunofluorescence detection of apoptosis by M30 CytoDEATH antibody that binds to caspase-cleaved epitope of the cytokeratin 18 cytoskeletal protein, a marker of apoptosis. A marked increase in M30 fluorescence was observed in the cells exposed to TRF. Representative figure from each group at 80· magnification is shown here. Details are described in Materials and methods.

vehicle treated controls, exposure of LNCaP cells to TRF at 10, and 20 lg/ml concentrations resulted in 4.5- and 5.8fold increases in induction of apoptosis. In DU145 cells, 3.9- and 5.1-fold increases in apoptosis induction were noted; and in PC-3 cells, 4.2- and 5.5-fold increases in apoptosis induction were noted at 10, and 20 lg/ml concentrations of TRF exposure, compared to vehicle treated controls (Fig. 4B). To further investigate the induction of apoptosis by TRF, fluorescence microscopy was employed after labeling the cells with M30 CytoDEATH, an antibody that binds to a caspase-cleaved epitope of the cytokeratin 18 cytoskeletal protein and functions as a marker for apoptosis. As shown in Fig. 4C, 20 lg/ml of TRF exposure resulted in apoptosis in all three prostate cancer cell lines. In contrast, no apoptotic morphology was observed in PZ-HPV-7 cells, after 20 lg/ml TRF concentration (data not shown). In recent years, many chemotherapeutic and chemopreventive agents have been shown to impart anti-proliferative effects via arrest of cell division at certain checkpoints in the cell cycle [39,40]. The concept of ‘cell cycle-mediated apop-

tosis’ has gained increasing attention as this pathway may provide minimal opportunity for acquired drug resistance, decreased mutagenesis, and reduced toxicity [41]. Furthermore, there is increasing interest in identifying natural dietary agents capable of selective/preferential elimination of cancer cells by influencing cell cycle regulation and/or causing apoptosis [8,10]. We evaluated the possibility that TRF influences the cell cycle in various prostate cancer cell lines. Compared to vehicle treated controls, TRF resulted in an appreciable arrest of cancer cells in the G0/G1 phase of the cell cycle after 24 h of exposure. The exposure caused an arrest of 74.1% cells in the G0/G1 phase of the cell cycle at a concentration of 10 lg/ml,, an effect that further increased to 77.2% at a concentration of 20 lg/ml and 75.3% at the highest exposure of 40 lg/ml in LNCaP cells. Similar observations were recorded in DU145 and PC-3 cell lines: exposure of these cells to TRF concentrations of 10, 20, and 40 lg/ml resulted in 56.9%, 61.4%, and 68.2% arrest in the G0/G1 phase in DU145 cells and 51.7%, 59.4%, and 62.5% arrest in PC-3 cells, respectively (Fig. 5). These

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LNCaP Cells

G0-G1: 68.2 S: 25.5 G2-M: 6.3 Sub G: 2.9

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Fig. 5. Effect of TRF on DNA cell cycle in asynchronously growing human prostate cancer LNCaP, DU145, and PC-3 cells. Log phase growing cells were exposed to increasing concentrations of TRF in complete medium for 24 h, stained with PI (50 lg/ml) and analyzed by flow cytometry. Percentages of cells in sub G1, G0/G1, S, and G2-M phase were calculated using Cell Quest & ModFit cell cycle analysis software, represented in the right side of the histogram. Data shown here are from a representative experiment repeated three times with similar results.

increases in the percentage of cells in the G0/G1 phase were accompanied with a concomitant decrease of cell populations in the S-phase and G2-M phases of the cell cycle in all three cell lines. In addition, cell exposure to TRF significantly increased the sub G1 accumulation of cells in a dosedependent manner in the order LNCaP > PC-3 > DU145, a finding that is considered to be a hallmark of apoptotic cell death. This analysis provided additional evidence that exposure of prostate cancer cells to TRF resulted in dosedependent apoptosis, which is consistent with the fluorescent microscopy data (Fig. 4C). Prostate cancer, because it is highly prevalent and is presumed to have a long latency period between its induction and the development of clinically evident disease, is viewed as an excellent candidate for cancer chemopreventive strategies [9,10]. Numerous epidemiologic studies have provided a basis for the development cancer chemoprevention protocols using bio-active dietary agents capable of eliminating pre-malignant or malignant cells. The results of this study suggest that TRF has the potential to affect the

steady state cell population and thus may be a good candidate for development as a chemopreventive and/or therapeutic agent against prostate cancer. To our knowledge this is the first report showing the selective effects of tocotrienol-rich fraction of palm oil in causing cell cycle arrest and apoptosis in prostate cancer cells without affecting normal cells. However, more detailed studies are required to determine the exact mechanism(s) of action of TRF, specifically evaluating its effects on epigenetic and signal transduction pathways. Based on previous investigations, the p53 and Akt/NF-jB signaling pathways may be involved in TRF-mediated cell cycle perturbations and apoptosis in prostate cancer cells. Further studies are needed to verify our data and to evaluate the effectiveness of TRF in preclinical models of prostate cancer. Acknowledgments The authors thank Mr. A. Gapor, Malaysian Palm Oil Board, Kuala Lumpur, Malaysia for kindly providing

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