Resveratrol-induced cell growth inhibition and apoptosis is associated with modulation of phosphoglycerate mutase B in human prostate cancer cells: two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry evaluation
Introduction
Prostate cancer (PCa) is the most frequently diagnosed malignancy and the second leading cause of death from cancer among men in the United States [1] and in other Western countries. The incidence of clinically detected prostate cancer is rising with more frequent diagnosis in younger patients [2]. Detection of early prenoplastic markers is therefore an important aim; in this context identification of agent-specific biomarkers is equally essential for evaluating the efficacy of chemopreventive agents [3], [4]. Advanced molecular approaches, such as genomic and proteomic analysis would clearly enhance the detection of new biomarkers in response to several phenolic antioxidants [5], [6]. Resveratrol (trans-3,5,4′-trihydroxystilbene) (Fig. 1), a potential chemopreventive agent found in grapes and other fruit, is an antioxidant and anti-mutagen that induces phase II drug-metabolizing enzymes (anti-initiation); it mediates anti-inflammatory effects, and it also inhibits cyclooxygenase and hydroperoxidase activity (anti-promotion activity) [7], [8], [9].
Using human cDNA microarray analyses, we have demonstrated earlier that a resveratrol-induced apoptosis is mediated through global changes in the expression of genes involved in cell cycle regulation, apoptosis, androgen-receptor (AR), and prostate-specific antigen (PSA) regulation [9]. We report here for the first time on the differential expression of genes involved in glycolysis. In normal cells, after glucose is broken down to pyruvic acid, it is then carried into the mitochondria and totally combusted by some 12 enzymatic reactions into carbon dioxide and water. Normal cells derive approximately 70% of their total energy needs from the breakdown of pyruvic acid and fats through the Krebs cycle. However, cancer cells may have a defective Krebs cycle that promotes cell proliferation. In this study, using genomic and proteomic approaches, we identified a surprising target of resveratrol, namely phosphoglycerate mutase B. This enzyme is involved in the isomerization of 2- and 3-phosphoglycerates that are essential for glucose metabolism. Our findings by real-time PCR analysis indicate that the enzyme is highly expressed in three human prostate cancer cells LNCaP, PC-3, and DU145 and that it is down regulated by resveratrol in LNCaP cells.
The modulating effect of phosphoglycerate mutase B in prostate cancer cells implies an effect on glycolysis. Higher expression of glycolytic enzymes in neoplastic cells is not uncommon as described in recent literature [10], [11]. Parallel findings from other studies also place importance on glycolysis-mediated tumor cell proliferation [12], [13]. We conclude that the chemopreventive effect mediated by resveratrol may in part be due to crippling of the glycolytic pathway in highly proliferating cancer cells without affecting normal cells.
Section snippets
Cell culture and treatment
Human prostate cancer cells LNCaP, PC-3, and DU145 were grown at 37 °C in RPMI, GIBCO brand (Invitrogen Life Technologies, Inc., Carlsbad, CA) supplemented with l-glutamine, 7.5% fetal bovine serum (FBS), and antibiotics in a water-saturated atmosphere of 5% CO2. Resveratrol was obtained from Calbiochem (La Jolla, CA), and was re-crystallized from ethanol until a purity of 99% was demonstrated in high performance liquid chromatography (HPLC). After the HPLC purification, aliquots of resveratrol
Effect of resveratrol on cell viability
The first set of experiments was performed to examine the effects of resveratrol (10−5 M) in all three human prostate cancer cells LNCaP, PC-3, and DU145 after exposing the cells for 0, 12, 24, and 48 h. As shown in Fig. 2, a linearity in the time effect was observed on the total number of viable cells determined with the Trypan blue exclusion assay. A decline in cell viability was significant after 24 and 48 h in all three cell lines with a percentage of growth inhibition of 70, 67, and 68% for
Discussion
We have previously identified the transcriptional profiles of genes involved in cell cycle regulation and apoptosis in prostate cancer cells exposed to resveratrol [9]. Knowing the potential role of several of these genes is crucial for cancer preventive effects; therefore, it is imperative to investigate the level of expression of their corresponding proteins. Towards this end, in the present study we used a two-pronged approach to examine the effect of resveratrol on metabolism-related
Acknowledgements
This study was presented at the American Association for Cancer Research 94th Annual Meeting (Abstract #R1830) 2003 held in Washington, DC. This work was supported by the NCI Cancer Center Support Grant: CA-17613, and AICR grant: 01A015. We thank Dominic Nargi for his technical assistance. We are also grateful for the mass-spectral analysis performed by Mary Ann Gawinowicz, PhD, Columbia University Protein Core Facility, New York, NY, and thank Ilse Hoffmann for editing the manuscript.
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