Curcumin Inhibits Glyoxalase 1 - a possible link to its anti-inflammatory and anti-tumor activity

BACKGROUND: Glyoxalases (Glo1 and Glo2) are involved in the glycolytic pathway by detoxifying the reactive methylglyoxal (MGO) into D-lactate in a two-step reaction using glutathione (GSH) as cofactor. Inhibitors of glyoxalases are considered as anti-inflammatory and anti-carcinogenic agents. The recent finding that various polyphenols modulate Glo1 activity has prompted us to assess curcumin's potency as an Glo1 inhibitor. METHODOLOGY/PRINCIPAL FINDINGS: Cultures of whole blood cells and tumor cell lines (PC-3, JIM-1, MDA-MD 231 and 1321N1) were set up to investigate the effect of selected polyphenols, including curcumin, on the LPS-induced cytokine production (cytometric bead-based array), cell proliferation (WST-1 assay), cytosolic Glo1 and Glo2 enzymatic activity, apoptosis/necrosis (annexin V-FITC/propidium iodide staining; flow cytometric analysis) as well as GSH and ATP content. Results of enzyme kinetics revealed that curcumin, compared to the polyphenols quercetin, myricetin, kaempferol, luteolin and rutin, elicited a stronger competitive inhibitory effect on Glo1 (K(i) = 5.1±1.4 µM). Applying a whole blood assay, IC(50) values of pro-inflammatory cytokine release (TNF-α, IL-6, IL-8, IL-1β) were found to be positively correlated with the K(i)-values of the aforementioned polyphenols. Moreover, whereas curcumin was found to hamper the growth of breast cancer (JIMT-1, MDA-MB-231), prostate cancer PC-3 and brain astrocytoma 1321N1 cells, no effect on growth or vitality of human primary hepatocytes was elucidated. Curcumin decreased D-lactate release by tumor cells, another clue for inhibition of intracellular Glo1. CONCLUSIONS/SIGNIFICANCE: The results described herein provide new insights into curcumin's biological activities as they indicate that inhibition of Glo1 by curcumin may result in non-tolerable levels of MGO and GSH, which, in turn, modulate various metabolic cellular pathways including depletion of cellular ATP and GSH content. This may account for curcumin's potency as an anti-inflammatory and anti-tumor agent. The findings support the use of curcumin as a potential therapeutic agent

Specific activity of Glo1¹, Glo2 and GSH² content in various tumor cells.

<p>Cytosolic extracts were prepared from cultured tumor cells and specific activity of enzymes and GSH was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003508#s4" target="_blank">Materials and Methods</a>. The values represent means±S.D. determined in triplicates (n = 6).</p>1<p>glyoxalase 1 & glyoxalase 2.</p>2<p>glutathione.</p

Effect of curcumin and methylglyoxal (MGO) at the ATP level in tumor cells.

<p>Astrocytoma 1321N1 cells (A) and JIMT-1cells (B) were seeded (5000 cells/well) and cultured in the presence of curcumin (0–100 µM) or MGO (0–1000 µM) for 6 h. Cellular ATP content was determined using the CellTiter-Glo® Luminescent Cell Viability Assay. Data represent the mean±S.D. of independent experiments (n = 12).</p

Effect of polyphenols on IL-1β release from LPS-stimulated blood cells.

<p>Heparinized whole blood was stimulated by LPS in the absence or presence of polyphenols at increasing concentrations and incubated for 6 h at 37°C with 5% CO₂. (A) Released IL-1β as measured in cell supernatants. Samples without additives but LPS were set at 100%. (B) The calculated LD₅₀ values of the respective polyphenols. Data represent mean±S.D. of independent experiments (n = 6).</p

Cytosolic activity and protein content of glyoxalase 1 upon treatment of JIMT-1 cells with curcumin.

<p>(A) JIMT-1 cells were treated with curcumin at 37°C and 5% CO₂ for 24 h. Cells were harvested and the specific activity of Glo1 and LDH was determined in the cytosolic extract. (B) Semi-quantitative analysis of protein content as performed by Western blot using specific antibodies against β-actin and Glo1. The blot was developed by chemoluminescense detection. (C) D-lactate release as determined in supernatants of astrocytoma 1321N1 cells following 24-h incubation with curcumin. Data represent the mean±S.D. of independent experiments (n = 6).</p

Release of L-lactate dehydrogenase (L-LDH) and vitality of 1321N1 cells upon incubation with 50 µM curcumin.

<p>Tumor cells were cultured and treated as mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003508#pone-0003508-g003" target="_blank">Fig. 3</a>. (A) LDH activity was measured in cell supernatants. (B) Cell vitality as assayed by the trypan blue exclusion test. Data represent the mean±S.D. of independent experiments (n = 6).</p

Inhibition of glyoxalase 1 activity by polyphenolic compounds.

<p>Purified Glo1 (70 mU) was incubated with increasing concentrations of different polyphenols at three substrate concentrations; and enzyme activity was recorded (n = 3). The inhibitor concentration was plotted vs. the reciprocal of enzyme activity (1/v) in Dixon plots.</p