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Curcuminoid binding to embryonal carcinoma cells: reductive metabolism, induction of apoptosis, senescence, and inhibition of cell proliferation.

Quitschke WW - PLoS ONE (2012)

Bottom Line: Cellular binding was overwhelmingly associated with membrane fractions and bound curcuminoids were metabolized in NT2/D1 cells via a previously unidentified reduction pathway.Both the binding affinities for curcuminoids and their reductive metabolic pathways varied in other cell lines.The dose-dependent effects of these responses further imply that distinct cellular pathways are sequentially activated and that this activation is dependent on the affinity of curcuminoids for the respective binding sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry and Behavioral Science, State University of New York at Stony Brook, Stony Brook, New York, United States of America. wolfgang.quitschke@sbumed.org

ABSTRACT
Curcumin preparations typically contain a mixture of polyphenols, collectively referred to as curcuminoids. In addition to the primary component curcumin, they also contain smaller amounts of the co-extracted derivatives demethoxycurcumin and bisdemethoxycurcumin. Curcuminoids can be differentially solubilized in serum, which allows for the systematic analysis of concentration-dependent cellular binding, biological effects, and metabolism. Technical grade curcumin was solubilized in fetal calf serum by two alternative methods yielding saturated preparations containing either predominantly curcumin (60%) or bisdemethoxycurcumin (55%). Continual exposure of NT2/D1 cells for 4-6 days to either preparation in cell culture media reduced cell division (1-5 µM), induced senescence (6-7 µM) or comprehensive cell death (8-10 µM) in a concentration-dependent manner. Some of these effects could also be elicited in cells transiently exposed to higher concentrations of curcuminoids (47 µM) for 0.5-4 h. Curcuminoids induced apoptosis by generalized activation of caspases but without nucleosomal fragmentation. The equilibrium binding of serum-solubilized curcuminoids to NT2/D1 cells incubated with increasing amounts of curcuminoid-saturated serum occurred with apparent overall dissociation constants in the 6-10 µM range. However, the presence of excess free serum decreased cellular binding in a hyperbolic manner. Cellular binding was overwhelmingly associated with membrane fractions and bound curcuminoids were metabolized in NT2/D1 cells via a previously unidentified reduction pathway. Both the binding affinities for curcuminoids and their reductive metabolic pathways varied in other cell lines. These results suggest that curcuminoids interact with cellular binding sites, thereby activating signal transduction pathways that initiate a variety of biological responses. The dose-dependent effects of these responses further imply that distinct cellular pathways are sequentially activated and that this activation is dependent on the affinity of curcuminoids for the respective binding sites. Defined serum-solubilized curcuminoids used in cell culture media are thus suitable for further investigating the differential activation of signal transduction pathways.

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Curcuminoids bound to NT2/D1 cells and their concentration in media as a function of incubation time.(A) Cells were incubated with media containing 27 µM starting concentrations (0 min) of curcuminoids (SOLID+DMSO). The amount of curcuminoids bound to cells (▪, right axis) and their concentration in media (•, left axis) during 5 h of incubation. (B) Cells incubated for 24 h with media containing a 27 µM starting concentration (100%) of curcuminoids (SOLID+DMSO). Left panel: The relative concentrations (%) of curcuminoids in the media after 0, 6, 12, 18, and 24 h of incubation either in the presence (black bars) or absence (white bars) of cells. Right panel: The amounts of curcuminoids bound to cells after 1, 6, 12, 18, and 24 h of incubation relative to the amount of curcuminoids bound to cells after 1 h of incubation (100%). (C) Representative chromatograms showing the elution profiles of curcuminoids in media before incubation (0 h, left panel) and after 24 h of incubation without cells (center panel) or with cells (right panel). The integrated and adjusted (ε) values under each individual peak are expressed relative to the respective starting concentrations (100%). (D) Curcuminoid binding to cells after 1 h (left panel) and 24 h (right panel). Curcuminoids bound after 24 h of incubation are expressed relative to the amount bound after 1 h (100%).
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pone-0039568-g005: Curcuminoids bound to NT2/D1 cells and their concentration in media as a function of incubation time.(A) Cells were incubated with media containing 27 µM starting concentrations (0 min) of curcuminoids (SOLID+DMSO). The amount of curcuminoids bound to cells (▪, right axis) and their concentration in media (•, left axis) during 5 h of incubation. (B) Cells incubated for 24 h with media containing a 27 µM starting concentration (100%) of curcuminoids (SOLID+DMSO). Left panel: The relative concentrations (%) of curcuminoids in the media after 0, 6, 12, 18, and 24 h of incubation either in the presence (black bars) or absence (white bars) of cells. Right panel: The amounts of curcuminoids bound to cells after 1, 6, 12, 18, and 24 h of incubation relative to the amount of curcuminoids bound to cells after 1 h of incubation (100%). (C) Representative chromatograms showing the elution profiles of curcuminoids in media before incubation (0 h, left panel) and after 24 h of incubation without cells (center panel) or with cells (right panel). The integrated and adjusted (ε) values under each individual peak are expressed relative to the respective starting concentrations (100%). (D) Curcuminoid binding to cells after 1 h (left panel) and 24 h (right panel). Curcuminoids bound after 24 h of incubation are expressed relative to the amount bound after 1 h (100%).

Mentions: Within 5 h of incubation, the curcuminoid concentration in the media had decreased to about 59% of the original value. About half of that decrease occurred during the first 0.5 h of incubation, which was followed by a more gradual decline for the remaining period. The initial media depletion of curcuminoids coincided with the establishment of maximum cellular binding, which was attained after about 0.5 h. Thereafter, the levels of cellular-bound curcuminoids effectively remained unchanged for the remaining 5 h incubation period (Fig. 5A). These cellular binding data are similar to those observed in other studies [22], [23].


Curcuminoid binding to embryonal carcinoma cells: reductive metabolism, induction of apoptosis, senescence, and inhibition of cell proliferation.

Quitschke WW - PLoS ONE (2012)

Curcuminoids bound to NT2/D1 cells and their concentration in media as a function of incubation time.(A) Cells were incubated with media containing 27 µM starting concentrations (0 min) of curcuminoids (SOLID+DMSO). The amount of curcuminoids bound to cells (▪, right axis) and their concentration in media (•, left axis) during 5 h of incubation. (B) Cells incubated for 24 h with media containing a 27 µM starting concentration (100%) of curcuminoids (SOLID+DMSO). Left panel: The relative concentrations (%) of curcuminoids in the media after 0, 6, 12, 18, and 24 h of incubation either in the presence (black bars) or absence (white bars) of cells. Right panel: The amounts of curcuminoids bound to cells after 1, 6, 12, 18, and 24 h of incubation relative to the amount of curcuminoids bound to cells after 1 h of incubation (100%). (C) Representative chromatograms showing the elution profiles of curcuminoids in media before incubation (0 h, left panel) and after 24 h of incubation without cells (center panel) or with cells (right panel). The integrated and adjusted (ε) values under each individual peak are expressed relative to the respective starting concentrations (100%). (D) Curcuminoid binding to cells after 1 h (left panel) and 24 h (right panel). Curcuminoids bound after 24 h of incubation are expressed relative to the amount bound after 1 h (100%).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3383725&req=5

pone-0039568-g005: Curcuminoids bound to NT2/D1 cells and their concentration in media as a function of incubation time.(A) Cells were incubated with media containing 27 µM starting concentrations (0 min) of curcuminoids (SOLID+DMSO). The amount of curcuminoids bound to cells (▪, right axis) and their concentration in media (•, left axis) during 5 h of incubation. (B) Cells incubated for 24 h with media containing a 27 µM starting concentration (100%) of curcuminoids (SOLID+DMSO). Left panel: The relative concentrations (%) of curcuminoids in the media after 0, 6, 12, 18, and 24 h of incubation either in the presence (black bars) or absence (white bars) of cells. Right panel: The amounts of curcuminoids bound to cells after 1, 6, 12, 18, and 24 h of incubation relative to the amount of curcuminoids bound to cells after 1 h of incubation (100%). (C) Representative chromatograms showing the elution profiles of curcuminoids in media before incubation (0 h, left panel) and after 24 h of incubation without cells (center panel) or with cells (right panel). The integrated and adjusted (ε) values under each individual peak are expressed relative to the respective starting concentrations (100%). (D) Curcuminoid binding to cells after 1 h (left panel) and 24 h (right panel). Curcuminoids bound after 24 h of incubation are expressed relative to the amount bound after 1 h (100%).
Mentions: Within 5 h of incubation, the curcuminoid concentration in the media had decreased to about 59% of the original value. About half of that decrease occurred during the first 0.5 h of incubation, which was followed by a more gradual decline for the remaining period. The initial media depletion of curcuminoids coincided with the establishment of maximum cellular binding, which was attained after about 0.5 h. Thereafter, the levels of cellular-bound curcuminoids effectively remained unchanged for the remaining 5 h incubation period (Fig. 5A). These cellular binding data are similar to those observed in other studies [22], [23].

Bottom Line: Cellular binding was overwhelmingly associated with membrane fractions and bound curcuminoids were metabolized in NT2/D1 cells via a previously unidentified reduction pathway.Both the binding affinities for curcuminoids and their reductive metabolic pathways varied in other cell lines.The dose-dependent effects of these responses further imply that distinct cellular pathways are sequentially activated and that this activation is dependent on the affinity of curcuminoids for the respective binding sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry and Behavioral Science, State University of New York at Stony Brook, Stony Brook, New York, United States of America. wolfgang.quitschke@sbumed.org

ABSTRACT
Curcumin preparations typically contain a mixture of polyphenols, collectively referred to as curcuminoids. In addition to the primary component curcumin, they also contain smaller amounts of the co-extracted derivatives demethoxycurcumin and bisdemethoxycurcumin. Curcuminoids can be differentially solubilized in serum, which allows for the systematic analysis of concentration-dependent cellular binding, biological effects, and metabolism. Technical grade curcumin was solubilized in fetal calf serum by two alternative methods yielding saturated preparations containing either predominantly curcumin (60%) or bisdemethoxycurcumin (55%). Continual exposure of NT2/D1 cells for 4-6 days to either preparation in cell culture media reduced cell division (1-5 µM), induced senescence (6-7 µM) or comprehensive cell death (8-10 µM) in a concentration-dependent manner. Some of these effects could also be elicited in cells transiently exposed to higher concentrations of curcuminoids (47 µM) for 0.5-4 h. Curcuminoids induced apoptosis by generalized activation of caspases but without nucleosomal fragmentation. The equilibrium binding of serum-solubilized curcuminoids to NT2/D1 cells incubated with increasing amounts of curcuminoid-saturated serum occurred with apparent overall dissociation constants in the 6-10 µM range. However, the presence of excess free serum decreased cellular binding in a hyperbolic manner. Cellular binding was overwhelmingly associated with membrane fractions and bound curcuminoids were metabolized in NT2/D1 cells via a previously unidentified reduction pathway. Both the binding affinities for curcuminoids and their reductive metabolic pathways varied in other cell lines. These results suggest that curcuminoids interact with cellular binding sites, thereby activating signal transduction pathways that initiate a variety of biological responses. The dose-dependent effects of these responses further imply that distinct cellular pathways are sequentially activated and that this activation is dependent on the affinity of curcuminoids for the respective binding sites. Defined serum-solubilized curcuminoids used in cell culture media are thus suitable for further investigating the differential activation of signal transduction pathways.

Show MeSH
Related in: MedlinePlus