Effect of curcumin on glutathione (GSH) level of 1321 N1 and JIMT-1 cells.

<p>Astrocytoma 1321N1 (A) and JIMT-1 (B) cells were cultured for 24 h at 37°C and 5% CO₂ in the absence or presence of curcumin. Cells were harvested and GSH concentration was determined in the cytosolic extract. 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

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

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 on apoptosis/necrosis of tumor and normal cells.

<p>Apoptosis/necrosis was evaluated using annexin V-5-fluorescein isothiocyanate (FITC)/propidium iodine (PI) staining followed by flow cytometric analysis. (A) Representative plots of showing annexin V-FITC/PI staining of Astrocytoma 1321N1 cells cultured in the presence of curcumin. The proportion of dead cells (a: annexin V-FITC⁻/PI⁺), late apoptotic/necrotic cells (b: annexin V-FITC⁺/PI⁺), non-apoptotic cells (c: annexin V-FITC⁻/PI⁻) and early apoptotic cells (d: annexin V-FITC⁺/PI⁻). (B) Results of 3 independent experiments. (C) Vitality response of primary human hepatocytes to 24-h incubation with curcumin as assessed by WST-1 test. Data represent the mean±S.D. of three independent experiments.</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

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

Keto-Enol Tautomeric forms of curcumin and transition-state of glutathione (GSH) and methylglyoxal (MGO).

<p>(A) (<i>1E,6E</i>)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione. (B) (<i>1E,4Z,6E</i>)-5-hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,4,6-triene-3-one. (C) Transition-state compound of reaction between GSH and MGO.</p