Differential solubility of curcuminoids in serum and albumin solutions: implications for analytical and therapeutic applications

<p>Abstract</p> <p>Background</p> <p>Commercially available curcumin preparations contain a mixture of related polyphenols, collectively referred to as curcuminoids. These encompass the primary component curcumin along with its co-purified derivatives demethoxycurcumin and bisdemethoxycurcumin. Curcuminoids have numerous biological activities, including inhibition of cancer related cell proliferation and reduction of amyloid plaque formation associated with Alzheimer disease. Unfortunately, the solubility of curcuminoids in aqueous solutions is exceedingly low. This restricts their systemic availability in orally administered formulations and limits their therapeutic potential.</p> <p>Results</p> <p>Methods are described that achieve high concentrations of soluble curcuminoids in serum. Solid curcuminoids were either mixed directly with serum, or they were predissolved in dimethyl sulfoxide and added as aliquots to serum. Both methods resulted in high levels of curcuminoid-solubility in mammalian sera from different species. However, adding aliquots of dimethyl sulfoxide-dissolved curcuminoids to serum proved to be more efficient, producing soluble curcuminoid concentrations of at least 3 mM in human serum. The methods also resulted in the differential solubility of individual curcuminoids in serum. The addition of dimethyl sulfoxide-dissolved curcuminoids to serum preferentially solubilized curcumin, whereas adding solid curcuminoids predominantly solubilized bisdemethoxycurcumin. Either method of solubilization was equally effective in inhibiting dose-dependent HeLa cell proliferation in culture. The maximum concentration of curcuminoids achieved in serum was at least 100-fold higher than that required for inhibiting cell proliferation in culture and 1000-fold higher than the concentration that has been reported to prevent amyloid plaque formation associated with Alzheimer disease. Curcuminoids were also highly soluble in solutions of purified albumin, a major component of serum.</p> <p>Conclusion</p> <p>These results suggest the possibility of alternative therapeutic approaches by injection or infusion of relatively small amounts of curcuminoid-enriched serum. They also provide tools to reproducibly solubilize curcuminoids for analysis in cell culture applications. The differential solubility of curcuminoids achieved by different methods of solubilization offers convenient alternatives to assess the diverse biological effects contributed by curcumin and its derivatives.</p

Two adjacent nuclear factor-binding domains activate expression from the human PRNP promoter

<p>Abstract</p> <p>Background</p> <p>The transmissible spongiform encephalopathies (TSEs) comprise a group of fatal degenerative neurological diseases in humans and other mammals. After infection, the cellular prion protein isoform PrP^Cis converted to the pathological PrP^SCscrapie isoform. The continued conversion of PrP^Cto PrP^SCrequires <it>de novo </it>endogenous PrP synthesis for disease progression. The human prion protein gene (<it>PRNP</it>) promoter was therefore investigated to identify regulatory elements that could serve as targets for therapeutic intervention.</p> <p>Findings</p> <p>The human prion protein gene (<it>PRNP</it>) promoter from position -1593 to +134 relative to the putative transcriptional start site (+1) was analyzed by transient transfection in HeLa cells. Deletions from the 5' end between positions -1593 and -232 yielded little change in activity. A further 5' deletion at position -90 resulted in a decline in activity to a level of about 30% of the full-length value. DNase I footprinting of the region between positions -259 and +2 identified two adjacent protected domains designated as prpA (-116 to -143) and prpB (-147 to -186). Internal deletions combined with mobility shift electrophoresis and methylation interference assays indicated the presence of sequence specific nuclear factor complexes that bind to the prpA and prpB domains and activate expression from the human <it>PRNP </it>promoter in an additive fashion.</p> <p>Conclusion</p> <p>Results from transient transfection, DNase I footprinting, mobility shift electrophoresis, and methylation interference experiments suggest that two DNase I protected domains designated as prpA and prpB are binding sites for as yet unidentified regulatory factors that independently activate expression from the <it>PRNP </it>promoter.</p

Curcuminoid Binding to Embryonal Carcinoma Cells: Reductive Metabolism, Induction of Apoptosis, Senescence, and Inhibition of Cell Proliferation

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

The Structural Complexity of the Human BORIS Gene in Gametogenesis and Cancer

BORIS/CTCFL is a paralogue of CTCF, the major epigenetic regulator of vertebrate genomes. BORIS is normally expressed only in germ cells but is aberrantly activated in numerous cancers. While recent studies demonstrated that BORIS is a transcriptional activator of testis-specific genes, little is generally known about its biological and molecular functions.Here we show that BORIS is expressed as 23 isoforms in germline and cancer cells. The isoforms are comprised of alternative N- and C-termini combined with varying numbers of zinc fingers (ZF) in the DNA binding domain. The patterns of BORIS isoform expression are distinct in germ and cancer cells. Isoform expression is activated by downregulation of CTCF, upregulated by reduction in CpG methylation caused by inactivation of DNMT1 or DNMT3b, and repressed by activation of p53. Studies of ectopically expressed isoforms showed that all are translated and localized to the nucleus. Using the testis-specific cerebroside sulfotransferase (CST) promoter and the IGF2/H19 imprinting control region (ICR), it was shown that binding of BORIS isoforms to DNA targets in vitro is methylation-sensitive and depends on the number and specific composition of ZF. The ability to bind target DNA and the presence of a specific long amino terminus (N258) in different isoforms are necessary and sufficient to activate CST transcription. Comparative sequence analyses revealed an evolutionary burst in mammals with strong conservation of BORIS isoproteins among primates.The extensive repertoire of spliced BORIS variants in humans that confer distinct DNA binding and transcriptional activation properties, and their differential patterns of expression among germ cells and neoplastic cells suggest that the gene is involved in a range of functionally important aspects of both normal gametogenesis and cancer development. In addition, a burst in isoform diversification may be evolutionarily tied to unique aspects of primate speciation

The dose-dependent binding of curcuminoids to NT2/D1 cells.

<p>(A) Binding dose curves with variable serum and curcuminoid concentrations in media containing FCS with saturated SOLID(S)-[•, short dash] and DMSO(D)-solubilized [▪, long dash] curcuminoids, and a 1∶1 mixture of the two (S+D) [▾, solid line]. The data points represent values from three independent experiments normalized to cell number. The following additional statistical parameters were calculated: R²-values: 0.99 (S), 0.98 (D), and 0.99 (S+D); K_Ds: 6.27±1.03 µM (S), 7.23±1.54 µM (D), 10.24±1.36 µM (S+D); and the slope m (see equation in text) indicating degree of non-specific binding: 1.9×10⁻³ (S), 2.9×10⁻³ (D), and 3.9×10⁻³ (S+D). (B) Binding dose curves with variable curcuminoid concentrations (S+D) at three constant total serum concentrations (5%, 50%, and 100%). (C) Binding dose curve with constant 47 µM curcuminoid concentration (S+D) and variable serum concentration.</p

Effect of incubating NT2/D1 cells for short time periods with high concentrations of curcuminoids.

<p>(A) Cells were either incubated with standard medium (•) or with medium containing 47 µM curcuminoids (SOLID+DMSO) for 0.5 h (○), 1 h (▪), 2 h (□), or 4 h (▴). The media were then removed, cells washed, and replenished with standard medium. Cells were photographed and counted daily. (B) Phase contrast micrographs of NT2/D1 cells before (day 0) and 2, 5, 6, 7, and 8 days after incubation with 47 µM curcuminoids for 4 h. Arrows point to larger dividing cells and arrowheads to cells reminiscent of a senescent phenotype. Brackets delineate groups of cells that have reverted to the original phenotype (compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039568#pone-0039568-g003" target="_blank">Fig. 3</a>).</p

Curcuminoids bound to NT2/D1 cells and their concentration in media as a function of incubation time.

<p>(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%).</p

Cellular senescence in response to curcuminoids.

<p>Phase contrast micrographs of NT2/D1 cells incubated in media without curcuminoids (A and D) or with 6 µM SOLID-solubilized curcuminoids in FCS for 4 (B and E) and 12 (C and F) days. Panels D, E, and F show cells stained for SA-β-galactosidase activity at pH 6 (blue color).</p