Influence of gallate esterification on the activity of procyanidin B2 in androgen-dependent human prostate carcinoma LNCaP cells

Abstract

Purpose: Present study assessed the influence of gallate esterification on the anti-cancer activity of procyanidin B2 (B2) in androgen-dependent human prostate carcinoma LNCaP cells employing B2-3,3'-di-O-gallate (B2-G(2)), two mono-gallate esters B2-3-O-gallate (B2-3G) and B2-3'-O-gallate (B2-3'G) and the parent compound B2, all isolated from grape seed extract (GSE).

Materials and methods: Study compounds were isolated from GSE by several chromatographic steps and structures determined by a combination of enzymatic hydrolysis, mass spectrometry and comparisons with standards. Cells, treated with these compounds, were assessed for viability and apoptosis and examined by western blotting.

Results: Gallate esters B2-G(2), B2-3G and B2-3'G significantly decreased LNCaP cell viability; however, B2 and gallic acid were ineffective. Furthermore, only B2-G(2) also significantly decreased cell growth. Decreases in cell viability were largely due to apoptosis induction with B2-G(2) and B2-3'G exhibiting comparable effects, whereas B2-3G was less effective. In mechanistic studies, B2-G(2) and B2-3'G treatments caused caspases-9 and -3 and PARP cleavage, and down-regulated Bcl-2, Bcl-Xl and androgen receptor levels.

Conclusion: Together, our findings demonstrate anti-PCA efficacy of B2-G(2) and suggest that a gallate ester moiety at 3' position of procyanidin B2 contributes more extensively toward the biological activity of the di-gallate ester than esterification of position 3.

Fig. 1

Structures and abbreviations of the procyanidin B2 dimer and gallate esters discussed in the text.

Fig. 2

Negative ion LC-MS studies of hydrolysis products formed from B2-G₂. A) Total ion current chromatogram of products from the partial enzymatic hydrolysis of B2-G₂. Peaks 1, 2 and 5 correspond to gallic acid, B2 and B2G₂, respectively, by comparison with authentic standards. B) MS spectrum of peak 3 and C) MS spectrum of peak 4, both demonstrating a deprotonated molecular ion (M – H)⁻ at 729 Da. D) MS/MS spectrum of the 729 ion from peak 3. E) MS/MS spectrum of the 729 ion from peak 4. The unique ion at 441 Da corresponds to interflavin bond cleavage and charge retention the lower unit as shown.

Fig. 3

Comparative effect of procyanidins B2-3G, B2-3’G and B2-G₂ on the growth and viability of human prostate carcinoma LNCaP cells in vitro. LNCaP cells were cultured in 60 mm culture plates for 36 h and were then treated with 0–50 µM concentrations of B2-3G (A), B2-3’G (B) and B2-G₂ (C) in DMSO or DMSO alone for 12 h. At the end of treatment times, cell viability was measured by Trypan blue dye exclusion assay as described under Materials and Methods section. Data indicate mean ± SD, n=3. *, P<0.05; $, P<0.001 compared with control.

Fig. 4

Apoptosis induction in human prostate carcinoma LNCaP cells by procyanidins B2-3G, B2-3’G and B2-G₂ in vitro. LNCaP cells were plated for 36 h at a density of 5000 cells per cm² in 60 mm culture dishes. A) Cells were treated with 10, 25 and 50 µM concentrations of B2-3G, B2-3’G or B2-G₂ for 12 h. At the end of treatment time, cells were collected and stained with annexin V-PI followed by flow cytometric analysis. Bars indicate mean ± SD, n=3. $, P<0.001 represents statistical significance of differences between control and test compound treated groups. Representative flow cytometric profiles are also shown below the bars for control and lower-dose agent treated cells. B) Cells were pretreated with pan caspase inhibitor (CI) Z-VAD.fmk (50 µM) for 2 h followed by treatment with 25 µM of either B2-G₂ or B2-3’G for 6 h. Cells were also treated with DMSO, Z-VAD.fmk, B2-G₂ or B2-3’G individually for 6 h. At the end of treatment time, the extent of apoptotic cell death was measured by using Cell Death ELISA kit as described in methods. Bars indicate mean ± SD, n=3.

Fig. 5

Apoptosis induction by procyanidins B2-G₂ and B2-3’G is associated with caspase-9 and -3 and PARP cleavage and modulation in the levels of Bcl-2 family members. LNCaP cells were plated, left overnight and then treated with 0–50 µM of either B2-G₂ or B2-3’G for 12 h. Total cell lysates were prepared and analyzed by western immunoblotting for cleaved caspase-9, -3, cleaved PARP, Bcl-xl, Bcl-2 and AR. Membranes were stripped and reprobed with β-actin as a loading control. Densitometric value shown below each band represents fold-change as compared to control after normalization with respective loading controls.