Epidemiologic studies have demonstrated an inverse association between flavonoid intake and prostate cancer (PCa) risk. The East Asian diet is very high in flavonoids and, correspondingly, men in China and Japan have the lowest incidence of PCa worldwide. There are thousands of different naturally occurring and synthetic flavonoids. However, only a few have been studied in PCa. Our aim was to identify novel flavonoids with antiproliferative effect in PCa cell lines, as well as determine their effects on cell cycle. We have screened a representative subgroup of 26 flavonoids for antiproliferative effect on the human PCa (LNCaP and PC3), breast cancer (MCF-7), and normal prostate stromal cell lines (PrSC). Using a fluorescence-based cell proliferation assay (Cyquant), we have identified five flavonoids, including the novel compounds 2,2′-dihydroxychalcone and fisetin, with antiproliferative and cell cycle arresting properties in human PCa in vitro. Most of the flavonoids tested exerted antiproliferative effect at lower doses in the PCa cell lines compared to the non-PCa cells. Flow cytometry was used as a means to determine the effects on cell cycle. PC3 cells were arrested in G2/M phase by flavonoids. LNCaP cells demonstrated different cell cycle profiles. Further studies are warranted to determine the molecular mechanism of action of 2,2′-DHC and fisetin in PCa, and to establish their effectiveness in vivo.
Diets rich in flavonoids have been associated with a reduced incidence and mortality of prostate cancer (PCa). The lowest incidence of PCa worldwide is seen in populations consuming the largest amount of flavonoids.1 In East Asian countries (China and Japan), diets are up to 100 times more abundant in flavonoids than in the West, due in part to the consumption of soy and green tea.2, 3 Correspondingly, the incidence of PCa in China and Japan is 60- to 80-fold lower than in North America.4 Studies on Japanese migrants to the United States have shown that migrants born in Japan and living in the United States have a higher incidence of PCa compared to men living in Japan.5, 6 The incidence rates for Japanese Americans born in the United States increases further, approaching that of American white men. Although these studies are not definitive, they emphasize the importance of environmental, lifestyle and dietary factors on PCa incidence. A number of case–control studies have correlated increased flavonoid intake with a reduced incidence of a number of malignancies including PCa.1, 7, 8, 9, 10
Flavonoids comprise over 4000 structurally related polyphenols,11 which are ubiquitous in plants, and ingested to varying degrees in the diet. The estimated average daily intake of flavonoids is up to 1 g.12 This by far exceeds the intake of other antioxidants such as vitamin E, and highlights the potential importance of flavonoids in the diet. Flavonoids have been shown to possess a wide range of biological activity , including antioxidant (greater than vitamin C),13 anti-inflammatory, antithrombogenic, and antiangiogenic activity.14 The anticancer properties of flavonoids have been demonstrated in a variety of cell types in vitro and in vivo.15 Despite the large number of flavonoids, studies have focused only on a select few. The flavonoids most intensely studied in PCa to date are the soy isoflavones (genestein/daidzein),16, 17 the green tea catechins (EGCG-epigallocatechin-3-gallate)18, 19 and the milk thistle flavonones (silibinin/silymarin).20, 21 Little is known of the biological effect of most other flavonoids.22, 23
In an attempt to identify novel flavonoids with growth-arresting properties in PCa cells, we have screened a number of compounds from each of the major flavonoid subgroups (Table 1). We have examined their antiproliferative effect on the PCa cell lines PC3 (androgen independent) and LNCaP (androgen dependent), MCF-7 breast cancer cell line and a nonmalignant prostate stromal cell line (PrSC). We have identified a number of novel flavonoids with antiproliferative and cell cycle effects in human PCa cells in vitro. The compound identified with the greatest antiproliferative effect was the synthetic flavonoid 2,2′-dihydroxychalcone (2,2′-DHC).
Materials and methods
The flavonoids, which have been included in this study, are presented in Table 1. Quercetin, pinostrobin, kaempferol, pelargonidin, galangin, formononetin, prunetin, 5-methoxyflavone, acacetin, morin, 6-aminoflavone, 7,8-benzoflavone, epigallocatechin and epicatechin gallate were purchased from Sigma Aldrich (St Louis, MO, USA). All other flavonoids were procured from Indofine Chemical Co. (Hillsborough, NJ, USA). Green tea catechin-epigallocatechin (EGC) was dissolved in water. All other flavonoids were dissolved in dimethylsulphoxide (DMSO) to a stock concentration of 100 mM. Working standards were made up in serum containing media. The final concentration of DMSO in culture did not exceed 0.2%. Flavonoids that were poorly soluble in DMSO were not studied further. These included the flavonoids prunetin, acacetin, formononetin, diosmin and karanjin.
The human PCa cell lines, LNCaP (mutated androgen receptor (AR), p53 wild-type), PC3 (AR null, p53 null), and the estrogen receptor positive breast cancer cell line MCF-7 were obtained from the American Type Tissue Collection (ATCC), Rockville, MD, USA. The nonmalignant prostate stromal cell line, PrSC, was obtained from Cambrex, NJ, USA. LNCaP cells were cultured in RPMI 1640 medium (Gibco, New York) supplemented with 10% foetal bovine serum (FBS) and 100 IU/ml penicillin and 100 μg/ml streptomycin. PC3 cells were cultured in DMEM/F12 medium with 10% FBS and antibiotics. MCF-7 cells were cultured in IMEM media supplemented with 5% FBS, antibiotics, and insulin. PrSC medium consisted of stromal cell basal media, and the manufacturer's growth factor supplements (Cambrex, NJ, USA). All cells were cultured at 37°C with 5% CO2.
Cell proliferation assay
Proliferation was assessed using the CyQuant cell proliferation assay (Molecular Probes, OR, USA). In this assay, the proprietary CyQuant dye binds to DNA, and the fluorescence emitted by the dye is linearly proportional to the number of cells in the well. Cells were plated in 96-well black fluorescence micro-titre plates, at a density of 4000 cells/well. At 24 h after plating, triplicate wells were treated with the appropriate flavonoid at a concentration of 10–150 μ M. Control wells were treated with vehicle alone (DMSO 0.2%). After 72 h of treatment at 37°C, the media was discarded, and the plates frozen at −80°C until use. On the day of the analysis, the plates with the adherent cells were thawed and incubated with the CyQuant dye for 5 min in the dark. Fluorescence was measured on an FL600 fluorescence micro-plate reader (Bio-Tek, VT) with filters set at 480 nm excitation and 520 nm emission. The IC50 for each flavonoid was determined in the four cell lines tested (Table 2). Each experiment was performed independently at least three times.
Flow cytometry and cell cycle analysis
Cell cycle arrest pattern and S phase enumeration were determined by flow cytometry on cells labelled with anti-BrdU FITC and propidium iodide. Asynchronously growing cells (5 × 105 cells/plate) were plated in 10 cm dishes and treated for 72 h with the flavonoid at the IC50 concentration as determined earlier. Control plates were treated with vehicle alone (DMSO 0.2%). The cells were pulse labelled with bromodeoxyuridine (BrdU) for 2 h prior to harvesting. As a negative control, a no-BrdU control was also included. Cells were trypsinized, fixed in ice cold 70% ethanol and stored at −20°C until further analysis. Cells were then washed in buffer (PBS and 0.5% Tween-20) and treated with 2 N HCl for 20 min to expose labelled DNA. Cells were incubated for 1 h on ice with anti-BrdU conjugated fluorescein isothiocyanate (Becton Dickinson, San Jose, CA, USA). Cells were washed, centrifuged, and resuspended in 10 μg/ml propidium iodide, and allowed to incubate for 30 min on ice. Samples were filtered through a nylon mesh and cell cycle analysis performed on the FACSCalibur flow cytometer using the Cell Quest Pro software package (Becton Dickinson, CA, USA).
Cell proliferation was determined by Cyquant fluorescence and expressed as a percentage of untreated control. The percentage value for each treatment was obtained from three replicate experiments. The mean of the three experiments was plotted on a concentration–response curve. A best-fit regression curve was determined by polynomial third-order equation, and performed individually for each flavonoid (GraphPad Prism version 4.03 for Windows, GraphPad Software, San Diego, CA, USA). We next determined (from the regression curves) the concentration of flavonoid at which there was a 50% reduction in cell number compared to the control (IC50).
The unpaired t-test (two-tailed) was performed to determine statistical significance of S-phase alterations in flavonoid treated and control cells (GraphPad Prism version 4.03 for Windows).
Flavonoids exert an antiproliferative effect on human PCa cells in vitro
The antiproliferative effects of flavonoids were assessed in LNCaP and PC3 cells in vitro. A subset of flavonoids with antiproliferative effect were also tested in the MCF-7 breast cancer cell line, and the nonmalignant prostate stromal cell line PrSC. Proliferation studies were initially performed by the more commonly used MTT assay. As many flavonoids are coloured when in solution, the MTT assay was unsuitable as this interfered with the calorimetric nature of this assay. In addition, flavonoids are known to reduce the tetrazolium in the MTT solution, even in the absence of cells.24 The CyQUANT assay overcame these limitations and was used for the high throughput screening of flavonoids in all further experiments.
Concentration–response curves were generated for each flavonoid. We determined the concentration at which flavonoids caused 50% growth inhibition compared to control (IC50) (Table 2). The five flavonoids with the greatest antiproliferative effect in LNCaP were 2,2′-dihyroxychalcone, baicalein, isoliquiritigenin (ISLQ), luteolin and quercetin. In PC3, the most potent compounds were 2,2′-DHC, luteolin, fisetin, quercetin and ISLQ. 2,2′-DHC caused growth inhibition at low doses in all four cell lines tested, the lowest IC50 observed in PC3 and LNCaP (10.26 and 10.8 μ M, respectively), and the highest in MCF7 (23 μ M) (Table 2). Fisetin had an IC50 of 22.65 μ M in LNCaP and 32.5 μ M in PC3. In total, 50% reduction in cell number at the maximum concentration was not achieved in MCF-7 and PrSC. Similarly, ISLQ had greater antiproliferative effect in PCa compared to non-PCa cell lines (Table 2). Luteolin demonstrated antiproliferative activity in LNCaP (IC50 18.22 μ M), PC3 (IC50 28.84 μ M) and MCF-7 cells (IC50 29.13 μ M) and to a lesser degree on PrSC (IC50 68.37 μ M). Quercetin had IC50 of 33.41 and 19.44 μ M in PC3 and LNCaP, with 50% reduction in cell numbers compared to control not reached in the prostatic stromal or breast cancer cell lines.
Other flavonoids shown to inhibit proliferation included 5-methoxyflavone, baicalin, baicalein, chrysin and kaempferol as detailed in Table 2. A greater effect on proliferation of LNCaP than PC3 cells were observed with 5-methoxyflavone (IC50 25.22 and 97.31 μ M, respectively). Baicalein had a lower IC50 than the glycosylated baicalin when tested in LNCaP, while baicalin seemed to be more potent in PC3 than LNCaP. Kaempferol and chrysin had an IC50 above 40 μ M in LNCaP and PC3 cells, a concentration unlikely to be achieved physiologically. Galangin inhibited proliferation in LNCaP only at very high concentrations, and the remainder of the flavonoids (6-aminoflavone, EGC, geraldol, gossypin, morin, myricetin, pinostrobin, and pelargonidin) did not inhibit proliferation at the doses tested.
Flavonoids cause alteration in cell cycle regulation in human PCa cells in vitro
Having identified the flavonoids that possessed the greatest effect on proliferation, we next examined the effect of these compounds on cell cycle profiles in LNCaP and PC3 cells. Cells were treated with flavonoids for 72 h in vitro, and flow cytometry performed with dual labelling of cells with PI and anti-BrdU (Figures 1 and 2). All five flavonoids (2,2′-DHC, fisetin, ISLQ, luteolin, and quercetin) caused cell cycle arrest in PC3 and LNCaP cells. Interestingly, all of these flavonoids caused a similar pattern of cell cycle arrest (G2/M) in PC3 cells (Figure 1), while LNCaP cells were arrested in both G1 and G2/M (Figure 2). All of the flavonoids tested caused a reduction in the proportion of cells in the synthesizing (S phase) of the cell cycle in both cell lines (Table 3). Time course studies performed demonstrated that the effect of flavonoid treatment on the cell cycle was observed as early as 24 h of treatment (data not shown).
There was up to a three-fold increase in cells in the G2/M phase in PC3 cells with flavonoid treatment compared to control cultures. Fisetin caused the greatest accumulation of cells in G2/M, with 66.5% (±12.6) of cells in G2/M compared to 13.0% (±2.8) in the control. A corresponding reduction of S and G1 phase cells was seen in PC3 cells. In terms of S-phase reduction in PC3 cells, the greatest effect was seen with quercetin, fisetin and luteolin (81.7, 68.3 and 61.2% reduction, respectively). 2,2′-DHC, the flavonoid with the greatest antiproliferative effect, caused a 53.4% reduction in S phase, and a three-fold rise in G2/M phase cells in PC3 (Figure 1). ISLQ caused a smaller reduction in S-phase cell numbers (42.9%) (Table 3).
A different pattern of cell cycle arrest was observed in LNCaP (Figure 2). Although LNCaP cells treated with flavonoid demonstrated an increase in G2/M phase cell numbers, indicative of a G2/M arrest, the percentage of cells in G1 phase did not decrease. This suggests that cells were being partially arrested in G1 phase. Compared to PC3, the increase in the proportion of G2/M cells was smaller in LNCaP, with most flavonoids causing around a two-fold increase in G2/M phase distribution. However, as in PC3, all flavonoids caused a reduction in percentage of cells in S phase in LNCaP cells. Fisetin, at a concentration of 25 μ M, caused the greatest reduction of cells in S phase in LNCaP cells (76.6% reduction) with a concomitant rise in the proportion of cells in G2 phase (19.4%±4.9 compared to 8.7%±0.63 in control) and a nonsignificant increase in G1 phase (71.2%±6.2 in fisetin vs 69%±4.3 in control) (Table 3). LNCaP cells treated with ISLQ, quercetin, luteolin and 2,2′-DHC also caused reductions in the percentage of cells in the S phase compared to vehicle only control (71.1, 63.2, 57.3 and 47.2%, respectively). In general, the proportion of cells in S phase in flavonoid treated groups was significantly lower than the DMSO control, with few exceptions (Table 3).
In this study, we have screened a diverse group of flavonoids for their antiproliferative effect on PCa, breast cancer and normal prostate stromal cells in vitro. Most of the flavonoids screened are novel compounds as they that have not been previously evaluated. We have identified a number of flavonoids that cause growth arrest at low concentrations in PCa, and appear to be less effective on non-PCa cell lines (MCF-7 and PrSC). We have also demonstrated that these flavonoids caused cell cycle arrest (G1 and G2/M), with a reduction in the number of cells in the S phase (81% reduction) (Figures 1 and 2).
As evident from Table 2, the flavonoid with the lowest IC50 concentration was 2,2′-DHC. This is a synthetic flavonoid belonging to the chalcone subgroup of flavonoids that are precursors in the flavone synthesis pathway in plants.25 The growth inhibitory effect of 2,2′-DHC was observed as early as 24 h following treatment (data not shown). Whereas most of the flavonoids tested were minimally cytotoxic to the PrSC and MCF-7 cell lines, 2,2′-DHC was unique in that it was effective at low concentrations on PrSC and MCF-7 cells (IC50 17.47 and 22.99 μ M respectively). The apparent resistance of MCF-7 and PrSC cells to most flavonoids may be a true effect; however, this is difficult to ascertain since all cell lines have been cultured in cell-specific media, and differences in growth media differences may account for some of the observations. Furthermore, it is not possible to reach conclusions on the cell specificity of flavonoids based on the limited number of cell lines that we have used and this would have to be carried out on a battery of cell lines. In this study, the PrSC stromal cell line was preferred to the PREC benign epithelial cell line, due to the slow growth kinetics and difficult culture conditions of the latter.
Other flavonoids identified with antiproliferative effect were fisetin, ISLQ, luteolin, quercetin, baicalein and 5-methoxyflavone (Table 2). Fisetin, isolated from the bark of Rhus cotinus,26 demonstrated cytotoxicity in both PC3 and LNCaP cells. Fisetin was less cytotoxic in MCF-7 and PrSC cells (IC50>80 μ M). ISLQ is a chalcone found in liquorice root and is a constituent of a some herbal remedies.27 Luteolin is a flavone widely distributed in nature, and found in sources such as parsley, artichoke, and celery.28 Quercetin is the most prevalent flavonoid in the Western diet,29 and is ubiquitous in plants. It is the most widely studied flavonoid and has been shown to exert antiproliferative effect on a number of cell lines.30, 31, 32 ISLQ, luteolin and quercetin appeared to have greater effect on proliferation in LNCaP than in PC3 cells. In addition, two other flavonoids, 5-methoxyflavone (synthetic) and baicalein (component of PCSPES herbal remedy), had low IC50 in LNCaP but not PC3 (Table 2). A number of differences between PC3 and LNCaP cells may account for this difference in sensitivity. PC3 is a more aggressive cell line, and is null for p53 and androgen receptor (AR). LNCaP, a much less aggressive cell line possesses a wild-type p53 and a mutated (AR). ISLQ and quercetin have less antiproliferative effect on MCF-7 and PrSC cells. Luteolin has a low IC50 in the MCF-7 breast cancer cell line, a property that merits further investigation (Table 2).
Despite the close similarities in the structure of the flavonoids, we observed a marked difference in their antiproliferative activities (Table 1). For example, quercetin and gossypin differ by a single hydroxyl substitution at the C-8 position. While quercetin caused growth arrest in LNCaP and PC3 cells, gossypin had no such action despite high concentrations. Microscopically, at the highest doses tested (100–150 μ M), some of the flavonoids such as EGC caused minimal cell toxicity leading to a high rate of cell survival. Treatment at high concentration with the more toxic flavonoids such as 2,2′-DHC resulted in morphological changes (viz, rounding and cell detachment). However, even with doses as high as 500 μ M of 2,2′-DHC, a small number of cells (500–600) remained adherent as detected by the CyQuant assay (data not shown).
Several flavonoids such as myricetin, pelargonidin and epigallocatechin (EGC) previously identified in epidemiological studies (as being linked to reduced incidence of PCa)19, 33, 34, 35, 36, 37, 38 did not display antiproliferative activity in our experiments. These studies, however, were based on retrospective dietary assessments and numerous problems arise while attempting to examine the effect of flavonoids by this methodology. The in vivo anticancer properties of flavonoids cannot be accurately determined from epidemiologic studies partly due to the confounding effects of the hundreds of other flavonoids present in the diet. Flavonoids examined in previous in vitro studies include EGC (IC50 88 μ M in DU145 prostate cancer cell line) and ISLQ (IC50 13 μ M in DU145). In our study, EGC did not inhibit cell growth (50% growth inhibition) in LNCaP or PC3 cells, at concentration up to 150 μ M.
In previous studies, flavonoids have been shown to cause alterations in cell cycle regulation in a number of cell lines. We performed FACS analysis to confirm the alterations in cell cycle regulatory properties of the key flavonoids identified in this study (Figures 1 and 2). Results indicate that 2,2′-DHC, fisetin, ISLQ, luteolin and quercetin all caused a G2/M arrest in both LNCaP and PC3 cells. The percentage of cells in G2 increased with corresponding decrease in the percentage of cells in the S phase cells for all flavonoids in both LNCaP and PC3 cells. In PC3 cells, the percentage of cells in the G1 phase decreased, while in LNCaP, the proportion of cells in G1 remained unchanged compared to the control. The p53 status may partly explain the different cell cycle arrest pattern observed between cell lines. As mentioned earlier, PC3 cells are p53 null, and as a result may be defective in G1 checkpoint control, explaining the lack of a G1 arrest in this cell line. This hypothesis remains to be proven. Previous studies have shown that quercetin and ISLQ cause cell cycle arrest in PCa cell lines.39, 40, 41 However, there have been no reports to date on the cell cycle regulatory effects of 2,2′-DHC, fisetin and luteolin.
The precise mechanism by which the flavonoids identified in this study exert their action remains to be determined. Flavonoids have been shown to alter a number of key proteins implicated in growth and differentiation. These include induction of cyclin-dependent kinase (cdk) inhibitors p21Cip1/WAF1 and p27Kip1,39, 42 inhibition of phosphorylation of retinoblastoma (Rb) protein,43 decrease in levels of cyclins B, D and E and cdk 2,4 and 6,44, 45, 46 induction of apoptosis,47, 48 inhibition of topoisomerase II, and alterations to the MAPkinase pathway.49 Genestein, a soy isoflavone, enhances the antiproliferative effects of vitamin D by upregulating vitamin D receptor and p21Cip1/WAF1 protein levels.50 This synergistic effect with vitamin D may contribute to the reduced incidence of PCa associated with diets that are rich in both vitamin D and flavonoids, such as the traditional Asian diet. In the present study, antiproliferative effects were seen in both AR dependent and independent human PCa cell lines, suggesting that the AR is not a critical component for mediating the growth-arresting properties of flavonoids.
Preliminary animal studies investigating the in vivo effects of flavonoids have been promising, with a number of flavonoids having demonstrated anti-PCa activity.51, 52 For example, TRAMP mice (transgenic adenocarcinoma of the mouse prostate) fed genestein showed a reduction in the incidence prostate carcinomas.51 Gupta et al.52 have shown that TRAMP mice placed on a green tea catechin rich extract had reduced liver metastases. Other investigators have confirmed the effect of selected flavonoids in PCa employing xenograft models.21, 53, 54 In these experiments, there have been no reports of flavonoid induced systemic toxicity (a desirable property of an anticancer agent). Since we have shown 2,2′-DHC, fisetin, luteolin and quercetin to have greater in vitro activity than genestein and catechins, we feel it would be necessary to examine the effect of these compounds in vivo. 2,2′-DHC and fisetin have demonstrated no toxicity at the concentrations used in this experiment (12–14 μ M) when tested on isolated rat hepatocytes,55, 56 and minimal toxicity would be expected in vivo.
Flavonoids account for many of the beneficial effects observed with diets rich in fruits and vegetables. We have identified several antiproliferative flavonoids that cause cell cycle arrest in PCa in vitro. Further studies are underway to explore the molecular mechanisms of action of the novel flavonoids identified in this study, and to determine their properties in vivo.
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This work was supported by grant from the Canadian Prostate Cancer Research Initiative, and the Canadian Prostate Cancer Research Bionet.
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Haddad, A., Venkateswaran, V., Viswanathan, L. et al. Novel antiproliferative flavonoids induce cell cycle arrest in human prostate cancer cell lines. Prostate Cancer Prostatic Dis 9, 68–76 (2006). https://doi.org/10.1038/sj.pcan.4500845
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