Limits...
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.

Show MeSH

Related in: MedlinePlus

Metabolites of curcuminoids in different cell lines.(A) CCF-STTG1 cells were incubated with media containing 47 µM curcuminoids (SOLID+DMSO) for 24 h. During reversed phase chromatography of media samples, the elution profiles were monitored at wavelengths 280 nm (hatched lines), and 310 nm (solid lines). Prominent absorption peaks representing hexa- and octahydrocurcuminoids were detected at 280 nm wavelength. (B) Primarily hexahydrocurcuminoids were produced with similarly incubated HeLa cells, in addition to smaller amounts of curcuminoid reduction products C*, D*, and B*. (C). In contrast, NT2/D1 cells generated only reduction products C*, D*, and B*.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3383725&req=5

pone-0039568-g010: Metabolites of curcuminoids in different cell lines.(A) CCF-STTG1 cells were incubated with media containing 47 µM curcuminoids (SOLID+DMSO) for 24 h. During reversed phase chromatography of media samples, the elution profiles were monitored at wavelengths 280 nm (hatched lines), and 310 nm (solid lines). Prominent absorption peaks representing hexa- and octahydrocurcuminoids were detected at 280 nm wavelength. (B) Primarily hexahydrocurcuminoids were produced with similarly incubated HeLa cells, in addition to smaller amounts of curcuminoid reduction products C*, D*, and B*. (C). In contrast, NT2/D1 cells generated only reduction products C*, D*, and B*.

Mentions: The faster elution times and the loss of absorption at longer wavelengths compared to the parent compounds suggested reductive modifications within the conjugated keto-enol linker region of the curcuminoids. For structural assessment, the peak corresponding to the metabolic product of CUR was collected and further analyzed by mass spectrometry. This generated a compound with a molecular mass of 371, which was identical to that of tetrahydrocurcumin. However, a direct comparison with tetrahydrocurcumin revealed that the mass spectra of the two compounds were different although they shared some overlapping features. Furthermore, the spectral absorption of the two compounds differed. While tetrahydrocurcumin only absorbed at 280 nm, the curcuminoid metabolic products showed a higher absorption at 310 nm wavelength. More importantly, the elution peaks of the two compounds did not overlap. This indicated that the metabolic product identified here was different from tetrahydrocurcumin. It should here be noted that the elution peaks produced by hexahydrocurcuminoids, overlap extensively with the alternative metabolic products described here (Fig. 10). However, the absorption maxima do not align perfectly and hexahydrocurcuminoids do not absorb at 310 nm wavelengths. Based on the observations from mass spectrometry and the absorption characteristics of the compounds, the metabolic product of curcumin (C*) was tentatively assigned the structure 1,7-bis(4-hydroxy-3-methoxyphenyl)-5-hydroxy-1-heptene-3-one (Fig. 9C). The remaining carbon-carbon double bond within the aliphatic linker thus forms a more extended conjugated double bond system with the remaining keto-group than is the case in tetrahydrocurcumin, and the compound is therefore expected to absorb at longer wavelengths. However, the ultimate structure assignment would need to be confirmed by NMR.


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

Quitschke WW - PLoS ONE (2012)

Metabolites of curcuminoids in different cell lines.(A) CCF-STTG1 cells were incubated with media containing 47 µM curcuminoids (SOLID+DMSO) for 24 h. During reversed phase chromatography of media samples, the elution profiles were monitored at wavelengths 280 nm (hatched lines), and 310 nm (solid lines). Prominent absorption peaks representing hexa- and octahydrocurcuminoids were detected at 280 nm wavelength. (B) Primarily hexahydrocurcuminoids were produced with similarly incubated HeLa cells, in addition to smaller amounts of curcuminoid reduction products C*, D*, and B*. (C). In contrast, NT2/D1 cells generated only reduction products C*, D*, and B*.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0039568-g010: Metabolites of curcuminoids in different cell lines.(A) CCF-STTG1 cells were incubated with media containing 47 µM curcuminoids (SOLID+DMSO) for 24 h. During reversed phase chromatography of media samples, the elution profiles were monitored at wavelengths 280 nm (hatched lines), and 310 nm (solid lines). Prominent absorption peaks representing hexa- and octahydrocurcuminoids were detected at 280 nm wavelength. (B) Primarily hexahydrocurcuminoids were produced with similarly incubated HeLa cells, in addition to smaller amounts of curcuminoid reduction products C*, D*, and B*. (C). In contrast, NT2/D1 cells generated only reduction products C*, D*, and B*.
Mentions: The faster elution times and the loss of absorption at longer wavelengths compared to the parent compounds suggested reductive modifications within the conjugated keto-enol linker region of the curcuminoids. For structural assessment, the peak corresponding to the metabolic product of CUR was collected and further analyzed by mass spectrometry. This generated a compound with a molecular mass of 371, which was identical to that of tetrahydrocurcumin. However, a direct comparison with tetrahydrocurcumin revealed that the mass spectra of the two compounds were different although they shared some overlapping features. Furthermore, the spectral absorption of the two compounds differed. While tetrahydrocurcumin only absorbed at 280 nm, the curcuminoid metabolic products showed a higher absorption at 310 nm wavelength. More importantly, the elution peaks of the two compounds did not overlap. This indicated that the metabolic product identified here was different from tetrahydrocurcumin. It should here be noted that the elution peaks produced by hexahydrocurcuminoids, overlap extensively with the alternative metabolic products described here (Fig. 10). However, the absorption maxima do not align perfectly and hexahydrocurcuminoids do not absorb at 310 nm wavelengths. Based on the observations from mass spectrometry and the absorption characteristics of the compounds, the metabolic product of curcumin (C*) was tentatively assigned the structure 1,7-bis(4-hydroxy-3-methoxyphenyl)-5-hydroxy-1-heptene-3-one (Fig. 9C). The remaining carbon-carbon double bond within the aliphatic linker thus forms a more extended conjugated double bond system with the remaining keto-group than is the case in tetrahydrocurcumin, and the compound is therefore expected to absorb at longer wavelengths. However, the ultimate structure assignment would need to be confirmed by NMR.

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