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Curcumin inhibits glyoxalase 1: a possible link to its anti-inflammatory and anti-tumor activity.

Santel T, Pflug G, Hemdan NY, Schäfer A, Hollenbach M, Buchold M, Hintersdorf A, Lindner I, Otto A, Bigl M, Oerlecke I, Hutschenreuther A, Hutschenreuter A, Sack U, Huse K, Groth M, Birkemeyer C, Schellenberger W, Gebhardt R, Platzer M, Weiss T, Vijayalakshmi MA, Krüger M, Birkenmeier G - PLoS ONE (2008)

Bottom Line: 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 microM).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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, University of Leipzig, Leipzig, Germany.

ABSTRACT

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 microM). Applying a whole blood assay, IC(50) values of pro-inflammatory cytokine release (TNF-alpha, IL-6, IL-8, IL-1beta) 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.

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Related in: MedlinePlus

Cytosolic activity and protein content of glyoxalase 1 upon treatment of JIMT-1 cells with curcumin.(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).
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pone-0003508-g005: Cytosolic activity and protein content of glyoxalase 1 upon treatment of JIMT-1 cells with curcumin.(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).

Mentions: To proof that inhibition of the Glo1activity accounts for cellular effects, we exposed JIMT-1 breast carcinoma cells to curcumin for 24 h. After cell harvesting and solubilisation, we found that specific Glo1 activity in treated cells was lower than in non-treated cells and decreased dose-dependently (Fig. 5A). As a control, we measured the LDH activity, another cytosolic enzyme, which we found to be increased at higher curcumin concentrations. This indicates the very selective effect of curcumin at the para-glycolytic enzyme Glo1. Again, a small augmentation of Glo1 activity was observed at 10 µM curcumin which may reflect the findings shown in figure 3. Estimating the protein level, Western blot analysis revealed no alteration of Glo1 content (Fig. 5B). This substantiates the assumption that curcumin acts as an enzyme inhibitor rather than through modulating the expression or degradation of Glo1. We found similar effects with other tumor cells. To corroborate the results, we measured the concentration of D-lactate in the medium of cultured 1321N1 cells incubated with increasing concentrations of curcumin for 24 h. D-lactate is produced in the MGO pathway as the end-product of the Glo2 enzymatic reaction. Inhibition of glyoxalases is known to result in a decrease in D-lactate release. Our results demonstrated that D-lactate decreased from 34.6±6 µM in the control cells to 22±1.9 µM at 20 µM curcumin.(Fig. 5C). This indicates that cytosolic Glo1 is inhibited by curcumin and thereby may result in accumulation of MGO.


Curcumin inhibits glyoxalase 1: a possible link to its anti-inflammatory and anti-tumor activity.

Santel T, Pflug G, Hemdan NY, Schäfer A, Hollenbach M, Buchold M, Hintersdorf A, Lindner I, Otto A, Bigl M, Oerlecke I, Hutschenreuther A, Hutschenreuter A, Sack U, Huse K, Groth M, Birkemeyer C, Schellenberger W, Gebhardt R, Platzer M, Weiss T, Vijayalakshmi MA, Krüger M, Birkenmeier G - PLoS ONE (2008)

Cytosolic activity and protein content of glyoxalase 1 upon treatment of JIMT-1 cells with curcumin.(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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2567432&req=5

pone-0003508-g005: Cytosolic activity and protein content of glyoxalase 1 upon treatment of JIMT-1 cells with curcumin.(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).
Mentions: To proof that inhibition of the Glo1activity accounts for cellular effects, we exposed JIMT-1 breast carcinoma cells to curcumin for 24 h. After cell harvesting and solubilisation, we found that specific Glo1 activity in treated cells was lower than in non-treated cells and decreased dose-dependently (Fig. 5A). As a control, we measured the LDH activity, another cytosolic enzyme, which we found to be increased at higher curcumin concentrations. This indicates the very selective effect of curcumin at the para-glycolytic enzyme Glo1. Again, a small augmentation of Glo1 activity was observed at 10 µM curcumin which may reflect the findings shown in figure 3. Estimating the protein level, Western blot analysis revealed no alteration of Glo1 content (Fig. 5B). This substantiates the assumption that curcumin acts as an enzyme inhibitor rather than through modulating the expression or degradation of Glo1. We found similar effects with other tumor cells. To corroborate the results, we measured the concentration of D-lactate in the medium of cultured 1321N1 cells incubated with increasing concentrations of curcumin for 24 h. D-lactate is produced in the MGO pathway as the end-product of the Glo2 enzymatic reaction. Inhibition of glyoxalases is known to result in a decrease in D-lactate release. Our results demonstrated that D-lactate decreased from 34.6±6 µM in the control cells to 22±1.9 µM at 20 µM curcumin.(Fig. 5C). This indicates that cytosolic Glo1 is inhibited by curcumin and thereby may result in accumulation of MGO.

Bottom Line: 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 microM).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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, University of Leipzig, Leipzig, Germany.

ABSTRACT

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 microM). Applying a whole blood assay, IC(50) values of pro-inflammatory cytokine release (TNF-alpha, IL-6, IL-8, IL-1beta) 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.

Show MeSH
Related in: MedlinePlus