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Chemistry 6: Natural Products

Dietary flavonoids cause profound cell cycle arrest, and alterations in cell cycle regulatory proteins in prostate cancer cells in vitro

Ahmed Haddad, Vasundara Venkateswaran, Laurence Klotz and Neil Fleshner
Ahmed Haddad
Sunnybrook & Women’s College Health Sciences Centre, Toronto, ON, Canada and Princess Margaret Hospital, Toronto, ON, Canada
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Vasundara Venkateswaran
Sunnybrook & Women’s College Health Sciences Centre, Toronto, ON, Canada and Princess Margaret Hospital, Toronto, ON, Canada
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Laurence Klotz
Sunnybrook & Women’s College Health Sciences Centre, Toronto, ON, Canada and Princess Margaret Hospital, Toronto, ON, Canada
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Neil Fleshner
Sunnybrook & Women’s College Health Sciences Centre, Toronto, ON, Canada and Princess Margaret Hospital, Toronto, ON, Canada
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DOI:  Published May 2005
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Proc Amer Assoc Cancer Res, Volume 46, 2005

Abstract

3073

Introduction: Flavonoids are a group of over 4000 dietary polyphenols that have been shown to possess various biological properties. We have studied the effects of a representative subgroup of 30 flavonoids on prostate cancer cell lines in vitro. Methods: Human prostate cancer cells (LNCaP and PC3) were cultured in 96 well plates and treated with various flavonoids for 72 hours. The effect of flavonoids on cell proliferation was determined using a DNA binding fluorescent assay. In order to evaluate alterations in cell cycle, PC3 and LNCaP cells were exposed to flavonoid for 24, 48, and 72 hours, and flow cytometric analysis carried out. Asynchronously growing cells were pulse labeled with 10 mM Bromodeoxyuridine (BrdU) for 2 hours with or without prior treatment with flavonoids. Cells were stained with anti-BrdU-conjugated FITC antibody and counter stained with propidium iodide and analyzed on a dual channel flow cytometer. Changes in cell cycle regulatory proteins were further investigated using Western blotting. Cell lysates were collected from LNCaP and PC3 cells treated with flavonoid for 24, 48 and 72 hours. Proteins were separated on SDS polyacrylamide gel, and probed for cyclins, associated kinases and inhibitors (A, D1, D3, cdk2, p21, p27 and p53). Results: The flavonoids 2,2-dihydroxychalcone, Fisetin, 5-methoxyflavone, and baicalin had the greatest growth inhibitory effect on LNCaP and PC3 cells (IC50 values of 20, 20, 25 and 30 μM respectively). 2,2-dihydroxychalcone at 20μM caused a G2/M arrest in LNCaP and PC3 cells with a 95% reduction in cells in the synthesis (S) phase. This effect was evident within 24 hours of treatment. Fisetin (20μM) caused a G2/M arrest in PC3 cells, and a G1 arrest in LNCaP cells with a 90% and 85% reduction in S phase respectively. Cell cycle arrest was also demonstrated with 5-methoxyflavone (G1 arrest), baicalin (G2/M arrest), luteolin (G1 arrest), and quercetin (G1 arrest). Western blotting on 2,2-dihydroxyflavone treated LNCaP cells showed a dramatic reduction in the levels of expression of cdk2 and cyclin A at 72 hours. No such alteration was observed for the Fisetin treated cells. In addition, levels of cyclin D1, D3, p21, p27 and p53 remained unaltered in both Fisetin and 2,2-dihydroxflavone treated cells. Conclusions: The flavonoids 2,2-dihydroxychalcone and Fisetin have a potent inhibitory effect on proliferation and induce cell cycle arrest with a dramatic reduction in S-phase cell numbers. The reduction in cell cycle proteins cyclin A and cdk2 as demonstrated for 2,2- dihydroxychalcone, are consistent with the G2/M arrest caused by this flavonoid. As naturally occurring dietary constituents, flavonoids hold great promise as cancer preventative agents.

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Cancer Research: 65 (9 Supplement)
May 2005
Volume 65, Issue 9 Supplement
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Dietary flavonoids cause profound cell cycle arrest, and alterations in cell cycle regulatory proteins in prostate cancer cells in vitro
Ahmed Haddad, Vasundara Venkateswaran, Laurence Klotz and Neil Fleshner
Cancer Res May 1 2005 (65) (9 Supplement) 722;

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Dietary flavonoids cause profound cell cycle arrest, and alterations in cell cycle regulatory proteins in prostate cancer cells in vitro
Ahmed Haddad, Vasundara Venkateswaran, Laurence Klotz and Neil Fleshner
Cancer Res May 1 2005 (65) (9 Supplement) 722;
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