Abstract A03: Procyanidin B2 3,3”-di-O-gallate from grape seed causes metabolic alterations in prostate cancer cells: 1H-, 13C- and 31P-NMRS-based metabolomics study
Authors: Raina Komal , Kumar Dileep , Agarwal Rajesh , Agarwal Chapla 
Published: May 2016

Abstract
Herein, we assessed the efficacy of procyanidins from grape seed against a panel of human prostate cancer (PCa) cell lines which ranged from ‘classical’ cell lines to the new variants that differed in their androgen responsiveness, castration resistance, and metastatic potential. The classical cell lines chosen were PC3, DU145 and LNCaP. Of these cell lines: PC3 and DU145 do not need androgens for growth, i.e, these are androgen independent (AI); they also lack androgen receptors (AR). The LNCaP cell line on the other hand, demonstrates androgen sensitivity (AS) and also requires androgens for its growth; while it harbors a mutated AR. Of the new variants of PCa cell lines available, we further chose a castration resistant variant of LNCaP, C4-2B, derived from the xenografts of castration resistant LNCaP subline-C4 in castrated mice. C4-2B thus represents castration resistant PCa (CRPC) cell line; while it does not require androgen for growth it demonstrates androgen sensitivity due to the presence of AR and is thus categorized as CRPC (AI/ AS) variant. Another PCa cell line, 22Rv1, which was chosen, does not require androgens for growth but is androgen responsive and like C4-2B is categorized as CRPC (AI/ AS).Trypan Blue dye exclusion assays utilizing total procyanidins from grape seed or its active constituent procyanidin B2 3,3”-di-O-gallate (B2G2) were performed to determine procyanidin effects, as a function of time, on the viability of the selected human PCa cell lines. Results indicated that the androgen dependent LNCaP cell line and its castration resistant variant, C4-2B, were more sensitive to procyanidins treatment at lower doses; importantly, another castration resistant cell line, 22Rv1, also showed significant decrease in cell viability at lower doses. While at these lower doses significant cell death was observed even at time points as early as 12 h in these cell lines; the classical AI cell lines PC3 and DU145 were most resistant to procyanidin treatment at lower doses and showed sensitivity (growth inhibition and cell death) only at higher doses after 72 h of procyanidins exposure. On similar lines, cancer stem cells (CD44+alpha2beta1high cells) isolated from PC3 and DU145 cells were also more resistant to treatment with procyanidins in prostasphere formation assays compared to other PCa cell lines. To understand the differential efficacy of procyanidins or its active constituent B2G2 towards different PCa cell lines, we carried out a metabolomics study utilizing quantitative high-resolution nuclear magnetic resonance spectroscopy (1H-, 13C- and 31P-NMR) to assess the metabolic profile and energy state of the B2G2 treated PCa cells. We examined global metabolic profile, including glucose metabolism, energy state, and lipid metabolism in these cells after B2G2 treatment. The time-course of B2G2 effects on glucose uptake and lactate release was also studied in the media of B2G2-treated PCa cells from 4-72 hrs of treatment. Results indicated that there was differential effect of B2G2 on mitochondrial glucose metabolism in these cells. While glucose uptake was markedly reduced as a function of time, there was also a significant decrease in extracellular lactate export with increased B2G2 exposure-time. Importantly, B2G2 preserved citrate concentration in the PCa cells as indicated by increase in citrate levels after treatments; citrate generation is indicative of normal secretion functions of the prostate epithelial cells. The fact that the citrate was not further utilized for cholesterol synthesis was also confirmed by decrease in cholesterol levels in these cells, indicating decreased cholesterogenesis by B2G2. Taken together these studies show that the procyanidins from grape seed differentially induce metabolic alterations in various PCa cell lines; these modulatory effects can be correlated with the effects on cell growth, proliferation and death, as well as the androgen dependency/ or lack of it.
Citation Format: Komal Raina, Dileep Kumar, Rajesh Agarwal, Chapla Agarwal. Procyanidin B2 3,3”-di-O-gallate from grape seed causes metabolic alterations in prostate cancer cells: 1H-, 13C- and 31P-NMRS-based metabolomics study. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A03.