Inhibition of chemically-induced neoplastic transformation by a novel tetrasodium diphosphate astaxanthin derivative

Laura M. Hix 1, 2, 5, Dean A. Frey 3, Mark D. McLaws 3, Marianne Østerlie 4, Samuel F. Lockwood 2 and John S. Bertram 5, 6, *

1 Department of Cell and Molecular Biology, University of Hawaii at Manoa, Honolulu, HI 96822, USA, 2 Hawaii Biotech, Inc., Aiea, HI 96701, USA, 3 Chemical Development and Analytical Quality Services, Albany Molecular Research Inc., Albany, NY 12212, USA, 4 HIST, Department of Food Science, N-7004, Trondheim, Norway and 5 Cancer Research Center of Hawaii, University of Hawaii at Manoa, Honolulu, HI 96813, USA

* To whom correspondence should be addressed. Tel: +1 808 586 2957; Fax: +1 808 586 2984; Email: john{at}crch.hawaii.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Carotenoids have been implicated in numerous epidemiological studies as being protective against cancer at many sites, and their chemopreventive properties have been confirmed in laboratory studies. Astaxanthin (AST), primarily a carotenoid of marine origin, responsible for the pink coloration of salmon, shrimp and lobster, has received relatively little attention. As with other carotenoids, its highly lipophilic properties complicate delivery to model systems. To overcome this issue we have synthesized a novel tetrasodium diphosphate astaxanthin (pAST) derivative with aqueous dispersibility of 25.21 mg/ml. pAST was delivered to C3H/10T1/2 cells in an aqueous/ethanol solution and compared with non-esterified AST dissolved in tetrahydrofuran. We show pAST to (i) upregulate connexin 43 (Cx43) protein expression; (ii) increase the formation of Cx43 immunoreactive plaques; (iii) upregulate gap junctional intercellular communication (GJIC); and (iv) cause 100% inhibition of methylcholanthrene-induced neoplastic transformation at 10–6 M. In all these assays, pAST was superior to non-esterified AST itself; in fact, pAST exceeded the potency of all other previously tested carotenoids in this model system. Cleavage of pAST to non-esterified (free) AST and uptake into cells was also verified by HPLC; however, levels of free AST were ~100-fold lower than in cells treated with AST itself, suggesting that pAST possesses intrinsic activity. The dual properties of water dispersibility (enabling parenteral administration in vivo) and increased potency should prove extremely useful in the future development of cancer chemopreventive agents.

Abbreviations: AST, astaxanthin; CTX, canthaxanthin; Cx32, connexin 32; Cx43, connexin 43; dAST, disodium salt disuccinate ester of astaxanthin; GJIC, gap junctional intercellular communication; MCA, methylcholanthrene; pAST, tetrasodium diphosphate astaxanthin; THF, tetrahydrofuran; TTNPB, 5,6,7,8-tetrahydro-(5,5,8,8-tetramethyl-2-napthyl) propenyl benzoic acid.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cancer chemoprevention, defined as the reduction in cancer incidence through the use of natural or synthetic agents is potentially a powerful strategy in the fight against cancer. Potential targets for chemoprevention include the general population, subgroups at risk owing to lifestyle or other environmental factors, individuals with precancerous lesions and cancer survivors at risk for secondary tumors (1). Among the leading candidates for cancer chemoprevention are dietary carotenoids, pigments in plants that play a crucial role in protection from oxidative damage (2). There is abundant epidemiological and laboratory evidence that carotenoids possess potent cancer chemopreventive properties, independent of their antioxidant activity or their potential for conversion to retinoids (3,4). Unfortunately, three major clinical trials of high-dose supplemental ß-carotene, the carotenoid most frequently identified as protective against lung cancer, failed to demonstrate protection. In contrast, in two of these studies conducted in high-risk smokers and/or asbestos-exposed workers, lung cancer incidence actually increased (57). The third study in largely non-smoking US physicians did not demonstrate protection or risk (8). In studies conducted in ferrets, one of the few laboratory models which absorb ß-carotene to a comparable level as do humans, high-dose ß-carotene was found to induce lung pathology. Molecular changes in the lung were consistent with a ß-carotene induced deficiency of retinoic acid as a consequence of enhanced catabolism of this important regulator of cell differentiation (9). These data suggest that the use of carotenoids without potential for conversion to vitamin A may provide protection and avoid this toxicity. Recent studies in the ferret model using lycopene, a non-provitamin A carotenoid, showed protection against tobacco-induced pathology, without toxicity (10).

Astaxanthin (AST), another non-provitamin A carotenoid, is found predominantly as a dietary source in shrimp, lobster and salmon, and as such is not a major circulating carotenoid in humans, as are lycopene and ß-carotene. In experimental animal studies AST has been shown to be capable of inhibiting chemically-induced oral and bladder carcinogenesis (11,12), and to be an effective immuno-stimulating agent (1315). As with other carotenoids, AST is a powerful lipid-phase antioxidant, and has been reported to suppress production of inflammatory cytokines (16). Based on this evidence, AST has significant cancer chemopreventive potential.

Delivery of highly lipophilic carotenoids, such as AST, to biological systems has met with formidable challenges. The most commonly employed method of delivery is in ‘beadlet’ form, a micro-disbursed solution of carotenoids in vegetable oil in a water-soluble matrix. Unfortunately, only ß-carotene, canthaxanthin (CTX) and lycopene have been so formulated, and studies using beadlets in most laboratory animals have been confounded by poor absorption. Delivery of AST and other carotenoids in cell culture was made possible by using the solvent tetrahydrofuran (THF), although this solvent is unsuitable for animal and clinical use (17). The need for a water-soluble and/or water-dispersible delivery system for carotenoids has led to the development of a highly bioavailable, water-dispersible, disodium salt disuccinate ester of astaxanthin (dAST). This compound formed a pseudosolution in water at concentrations of up to 8 mg/ml (~10 mM), and bioavailability in vitro was enhanced by the addition of ethanol as a co-solvent to maintain the compound in monomeric form (18). Oral administration to mice resulted in rapid absorption, cleavage to free AST and accumulation in target tissue (19). dAST was shown to be an effective scavenger of free radicals in the aqueous phase using an in vitro human polymorphonuclear leukocyte assay with complete inhibition of the induced superoxide anion at millimolar concentrations. This compound has also been shown to significantly protect against cardiac ischemia–reperfusion injury, generally considered to result from oxidative stress, at doses up to 75 mg/kg in a rat model of experimental infarction (20) and at 50 mg/kg in canines (21). In the 10T1/2 cell model of in vitro carcinogenesis, the compound was biologically active when delivered in an aqueous ethanolic formulation, causing upregulated expression of connexin 43 (Cx43) protein and increased gap junctional communication (GJIC) (22). Unfortunately its chemopreventive potential in this system could not be evaluated since dAST was found to cause morphological changes in the cell monolayer which obscured detection of neoplastically transformed foci, perhaps as a consequence of the recently discovered activity of succinate as a signal transducing molecule (23). For these and other reasons, AST was reformulated by synthesis of the tetrasodium diphosphate AST (pAST) derivative. This compound exhibits ~3-fold enhanced aqueous dispersibility (25.21 mg/ml) over the dAST derivative.

In the present study, we examine the ability of pAST to inhibit chemically-induced neoplastic transformation in the 10T1/2 model system. Since previous studies have revealed that the chemopreventive activity of carotenoids is directly correlated with their ability to upregulate Cx43 expression and consequently to enhance GJIC (24), we have also examined Cx43 expression and GJIC in these treated cultures. Cx43 is now widely regarded as a tumor suppressor gene, and most human and animal tumors are deficient in GJIC [reviewed in (25)]. GJIC involves the transfer of small molecules and ions <1 kDa in size through aqueous channels (connexons) that span the plasma membrane of adjoining cells; it is through these channels that growth inhibitory signals are hypothesized to pass. This concept of growth control via junctional communication, first proposed by Loewenstein (26), has received strong support recently with the demonstration that connexin 32 (Cx32) knock-out mice are more susceptible to physically- and chemically-induced liver carcinogenesis (27,28), whereas heterologous deletion of Cx43 increases susceptibility to lung carcinogenesis (29). We report that pAST is more potent than the parent carotenoid AST in its ability to upregulate Cx43 protein expression, induce functional GJIC and completely inhibit carcinogen-induced neoplastic transformation at 10–6 M. In addition, the derivatized compound was cleaved by cellular phosphatases as evidenced by the cellular uptake of non-esterifed AST. In view of these findings, further cell culture and animal studies utilizing this derivative are warranted.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
The racemic tetrasodium diphosphate derivative of AST (>97% purity by HPLC) was synthesized from commercial AST and its structure verified (see below for synthetic methodology). The chemical structures of the three stereoisomers, (3R,3'R)-, (3S,3'S)- and (3R,3'S; meso)-tetrasodium diphosphate astaxanthin are shown in Figure 1. The racemic pAST used in this study comprised the statistical mixture of the above stereoisomers in a 1:1:2 ratio. Non-esterified, all-E synthetic AST utilized for biological tests (>96% purity by HPLC; conditions described below) was obtained from Sigma (St Louis, MO). CTX and the synthetic retinoid 5,6,7,8-tetrahydro-(5,5,8,8-tetramethyl-2-napthyl) propenyl benzoic acid (TTNPB) were gifts from Hoffman-LaRoche (Nutley, NJ). TTNPB, CTX and AST concentrations were confirmed by comparing their UV absorption and their published extinction coefficients. Carotenoids were stored under nitrogen at –70°C, and care was taken to ensure minimal exposure to direct sunlight or UV light.



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Fig. 1. Structure of the of the tetrasodium diphosphate astaxanthin derivative. The racemic mixture of stereoisomers evaluated in the current study, referred to as pAST in the text, contains (3S,3'S)-pAST, (3R,3'R)-pAST and (3R,3'S; meso)-tetrasodium disphosphate astaxanthin in a 1:1:2 ratio.

 
Synthesis of pAST
Unless otherwise noted, all reagents and solvents were purchased from commercial suppliers and used as received without further purification. Proton and carbon nuclear magnetic resonance (NMR) spectra were obtained on a Bruker AMX 500 spectrometer at 500 MHz for proton NMR (1H NMR) and 202 MHz for phosphorous NMR (31P NMR). Chemical shifts are given in p.p.m. ({delta}) and coupling constants, J, are reported in Hertz (Hz). Tetramethylsilane (TMS) was used as an internal standard for proton spectra. High performance liquid chromatography (HPLC) analysis for in-process control was performed on a Varian Prostar Series 210 liquid chromatograph with a PDA detector using methods A and B. (i) Method A: Waters Symmetry C18, 3.5 µm, 4.6 x 150 mm column; 25°C; mobile phase: [A = water (pH 4.5, 20 mM ammonium acetate), B = acetonitrile], 95% A/5% B (start); hold for 5 min; linear gradient to 100% B over 25 min; hold for 8 min; linear gradient to 5% B over 2 min; flow rate: 1.0 ml/min; detector wavelength: 474 nm (AST 30.5 min). (ii) Method B: Agilent Zorbax SB CN 5 µm, 4.6 x 150 mm column; 25°C; mobile phase: [A = water (pH 6.8, 20 mM ammonium acetate), B = acetonitrile], 80% A/20% B (start); hold for 5 min; linear gradient to 100% B over 20 min; hold for 10 min; linear gradient to 20% B over 5 min; flow rate: 1.0 ml/min; detector wavelength: 474 nm (AST, 30.5 min).

The intermediate rac-3,3'-dihydroxy-ß,ß-carotene-4,4'-dione diphosphoric acid bis-(2-cyano-ethyl) ester was synthesized as follows: a 100 ml round bottom flask wrapped with aluminum foil was equipped with a stir bar under nitrogen at room temperature. Racemic AST (Buckton Scott, India) (0.440 g, 0.74 mmol) was dissolved in methylene chloride (13.2 ml) then reacted with bis (2-cyanoethyl)-N,N-diisopropyl phosphoramidite (0.80 g, 2.95 mmol) and tetrazole (0.21 g, 2.95 mmol). After 14 h, HPLC analysis indicated that the reaction was complete and 7.4 ml of 0.4 M iodine in a solution of pyridine–dichloromethane (DCM)–water (3:1:1) was added dropwise over 15 min. The reaction mixture was diluted with DCM (10 ml) and washed with aqueous sodium thiosulfate (1 M, 2 x 10 ml) and brine (10 ml). The solution was concentrated to dryness to afford 590 mg of dried red solid (83% yield). 1H NMR (CDCl3) {delta} 6.42–6.17 (14 H, m), 5.12–5.06 (2 H, m), 4.59–4.31 (m, 8 H), 2.93–2.83 (8 H, m), 2.07–1.98 (16 H, m), 1.89 (6 H, s), 1.35 (6 H, s), 1.24 (6 H, s); 31P NMR (CDCl3) {delta} –2.62; Anal. calculated for [C52H66O10P2]: 969.05, ESI MS m/z 969.29 [C52H66O10P2]+; HPLC (Method B) 94.0% area under the curve (AUC), tR = 26.0 min.

pAST was synthesized from rac-3,3'-dihydroxy-ß,ß-carotene-4,4'-dione diphosphoric acid bis-(2-cyano-ethyl) (500 mg, 0.52 mmol) by addition of 10 ml of 50% dimethylamine in water in a 250 ml flask under nitrogen (N2) with a stir bar and heated to 40°C. The reaction was complete as determined by HPLC after 4 days and the reaction mixture was concentrated to dryness. The red solid was redissolved in 50 ml of water and then eluted through a sodium ion exchange resin (50 g, Amberlite IR-120 Na+). The solution was concentrated using acetonitrile to form an azeotrope with water. The red solid was then redissolved in a minimum amount of water (~20 ml) and precipitated with the addition of ethanol (20 ml). The precipitate was filtered through a 2 µm filter and dried under vacuum to afford 66 mg. This was again redissolved in 2 ml of water and lyophilized to afford 42 mg (28% yield) of pAST. [1H NMR (CD3OD) {delta} 6.81–6.35 (14 H, m), 4.87–4.83 (2 H, m), 2.07–1.98 (16 H, m), 1.90 (6 H, s), 1.30 (6 H, s), 1.14 (6 H, s); 31P NMR (CDCl3) {delta} 3.28; Anal. calculated for [C40H54O10P2]: 756.80, ESI MS m/z 755.2 [C40H53O10P2]; HPLC (Method B) 97.7% AUC, tR = 14.05 min].

Determination of aqueous solubility/dispersibility
pAST was added to 1 ml of water, the mixture stirred for 2 h and centrifuged for 5 min. The solution was diluted in water and analyzed by UV/vis spectroscopy at 480 nm and absorbance was compared with a standard curve compiled from four standards of known concentration. The solubility was calculated to be 25.21 mg/ml with an extinction coefficient of 93.65 AU ml/cm mg.

Cell lines and culture conditions
Mouse embryonic fibroblast 10T1/2 cells were cultured in Eagle's basal medium with Earle's salts supplemented with 4% fetal calf serum (Atlanta Biologicals, Norcross, GA), 25 µg/ml gentamicin sulfate (Sigma, St Louis, MO) and passaged by trypsin/EDTA (Gibco Invitrogen, Carlsbad, CA). Cells were incubated at 37°C in 5% CO2 as described previously (30), and confluent cells were treated on day 7 after seeding, unless otherwise indicated. dAST was prepared in a formulation of 20% EtOH and water to minimize supramolecular aggregation, then passed through a 0.2 µm sterile filter (22). The final concentration of EtOH in culture medium was 0.2%. CTX was dissolved in THF and added to media as described (17). TTNPB was dissolved in acetone (Sigma, St Louis, MO) and cultures received a final acetone concentration of 0.1%.

Analysis of Cx43 expression
Expression of Cx43 protein in 10T1/2 cells was assessed by western blotting essentially as described (30). In brief, 10T1/2 cells were treated with AST, pAST or retinoids at confluence on day 7 after seeding in 100 mm dishes (Fisher Scientific, Pittsburgh, PA). The cells were harvested after 4 days and total protein content was measured using the Protein Assay Reagent kit (Pierce Chemical, Rockford, IL) according to the manufacturer's instructions. Cell lysates containing 40 µg of protein were analyzed by western blotting using the NuPage Western blotting kit (Invitrogen, Carlsbad, CA). Cx43 was detected using a rabbit polyclonal antibody (Zymed, San Francisco, CA) raised against a synthetic polypeptide corresponding to the C-terminal domain common to mouse, human and rat Cx43. As an endogenous control for equal protein loading, a rabbit polyclonal GAPDH antibody was also used (Zymed). Cx43 and GAPDH immunoreactive bands were visualized by chemiluminescence using an anti-rabbit HRP-conjugated secondary antibody (Pierce Chemical). Images were obtained by exposure to X-ray film as previously described (30) and scanned for digital analysis on the Fluoro-S Imager (Bio-Rad, Richmond, CA).

Immunofluorescence
Expression and assembly of Cx43 into plaques was assessed by immunofluorescence staining essentially as described (31). In brief, confluent cultures of 10T1/2 cells were grown on Permanox plastic 4-chamber slides (Nalge Nunc International, Naperville, IL) and treated for 4 days. Cells were fixed with –20°C methanol overnight, washed in buffer, blocked in 1% bovine serum albumin (Sigma) in PBS, incubated with the rabbit anti-Cx43 antibody and finally detected with Alexa568 conjugated anti-rabbit secondary antibody (Molecular Probes, Eugene, OR). Images were acquired with a Zeiss Axioplan microscope and a Roper Scientific cooled CCD camera.

Gap junctional communication assay
Junctional permeability was assayed by the scrape-loading dye transfer assay (32). In brief, confluent cultures of 10T1/2 cells grown in 60 mm dishes were treated with test compounds for 7 days, and then rinsed with Ca++-free phosphate-buffered saline (PBS). 1.5 ml of 0.2% Lucifer Yellow CH (Sigma) in PBS was added and linear cuts were made on the monolayer using a surgical scalpel. The cultures were incubated for 2 min at 37°C and rinsed thoroughly with PBS. The cultures were then fixed with 2 ml of 5% formaldehyde in PBS. Fluorescent images were digitally quantitated by intensity thresholding using the SigmaScan software program (Jandel Scientific, San Rafael, CA) and the distance of dye transfer away from the cut measured. Approximately 10 measurements were made in each of 2 replicate cultures/concentration.

Inhibition of MCA-induced neoplastic transformation in 10T1/2 cells
Cells were initiated with methylcholanthrene (MCA) 5 µg/ml (Sigma) in acetone for 24 h. Potential inhibitors were added 7 days after the removal of carcinogen and were renewed with weekly medium change for another 4 weeks after which cultures were fixed and stained as previously described. A total of 24 dishes were used per treatment group. Type II and III foci were then quantitated as described (33).

Selective cytotoxicity
Determination of plating efficiencies and growth rates were as previously described (34). In brief, normal 10T1/2 cells and MCA-transformed 10T1/2 cells were plated onto 100 mm plates at a density of 104/dish and treated 24 h later with pAST, AST or EtOH-only as controls. Duplicate cultures were trypsinized and counted after 1, 2, 6 and 8 days by Coulter counter (Coulter Electronics, Hialeah, FL).

Cellular uptake
Cellular levels of AST in pAST- and AST-treated cells were determined in confluent 10T1/2 cells treated with pAST or AST at 10–6 M or cells treated with media-alone as an untreated control. Two sets of cells were harvested by trypsinization, pelleted and snap-frozen in liquid nitrogen at days 1, 4 and 7 after treatment. Cells were shipped frozen on dry ice to Trondheim, Norway for HPLC analysis. AST in the plasma and cell pellet samples was extracted as described with slight modifications (19). Methanol (1.0 ml), water (1.0 ml) and chloroform (3.0 ml) were added to each weighed sample, then mixed with an Ultraturax® mixer for 20 s. Next, water (1.0 ml) and chloroform (3.0 ml) were added to each sample, mixed as above and centrifuged (1700x g, 10 min). An aliquot (2 ml) of the chloroform phase was evaporated with N2 and the residue dissolved in n-hexane:acetone (86:14; 75 µl). Total AST, including all-E-, 9Z- and 13Z-AST, was quantified by HPLC using a phosphoric acid-modified silica gel column, with all-E-AST as an external standard. The flow was 1 ml/min and 470 nm detection wavelength. The employed extinction coefficients (E1cm,1%) at 472 nm in hexane containing 4% chloroform were 2100 for all-E-AST, and 1350 and 1750 for 13Z- and 9Z-AST, respectively.

Statistical analysis
Transformation data were analyzed by one-tailed, two-sample t-tests that incorporated unequal variances. The scrape-loading data were analyzed by paired t-tests.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The following experiments were designed to compare and contrast the properties of pAST, delivered in an aqueous/ethanol solution, with AST itself, delivered as a solution in THF, in terms of their ability to inhibit neoplastic transformation in 10T1/2 cells and upregulate expression of Cx43.

Inhibition of MCA-induced neoplastic transformation
Compounds were added in all cases 7 days after the removal of the carcinogen MCA so as not to potentially interfere with the production of carcinogen-initiated cells. Results are presented as the mean number of foci per dish (Table I). In control cultures treated with acetone only, a spontaneous background rate of about 1 focus/6 dishes was observed (mean 0.17 foci/dish); this was increased to 1.67 foci/dish in cultures exposed to MCA (P > 0.0002). pAST at 10–6 M completely inhibited neoplastic transformation, and at 10–7 M and 10–8 M pAST significantly inhibited transformation (P < 0.04). AST at all tested concentrations inhibited transformation to ~40% of control (0.98 foci/dish) without any clear dose–response curve. Transformation was completely inhibited in cells treated with either pAST or AST at 10–5 M; however, these cultures failed to form complete monolayers, potentially indicating cumulative toxicity at this concentration and invalidating the assay results.


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Table I. Inhibitory effects of carotenoids on MCA-induced transformation

 
pAST induces Cx43 expression
Racemic pAST induced Cx43 expression in comparison with solvent-treated controls at concentrations of 10–6 and 10–7 M, and was equipotent to CTX at these concentrations (~5- and 2-fold induction). CTX, included as a positive carotenoid control, was strongly active at 10–5 M as previously reported (~7-fold induction) (35). AST was essentially inactive across these concentrations. Surprisingly, induction was not observed at 10–5 M with either compound, again suggesting potential toxicity at this increased concentration. As expected, Cx43 expression was induced about 13-fold by the synthetic retinoid TTNPB at 10–8 M included as positive retinoid control (Figure 2).



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Fig. 2. pAST induces Cx43 expression in 10T1/2 cells. Confluent cultures of 10T1/2 cells were treated for 4 days, harvested for western blotting then probed with antibodies to Cx43 and GAPDH as described. (A) Western blot; lane 1: TTNPB 10–8 M in acetone (final acetone concentration in assay 0.1%) as positive control. Lanes 2–4: CTX at 10–5 M, 10–6 M and 10–7 M in THF; lanes 5–7: AST 10–5 M, 10–6 M and 10–7 M in THF; lanes 8–10: racemic pAST 10–5 M, 10–6 M and 10–7 M in 20% EtOH/water; lane 11: media alone. Equal protein loading was confirmed by detection with GAPDH. (B) Quantitation of (A): relative induction levels of Cx43 expression plotted versus untreated control. The figure is representative of two separate experiments.

 
These studies demonstrate that the novel pAST compound delivered in an aqueous ethanol formulation is superior to AST itself delivered in THF in suppressing transformation and inducing Cx43.

pAST-induced Cx43 is assembled into junctional plaques
In untreated confluent cultures of 10T1/2 cells, we have previously reported low levels of junctional communication, of Cx43 expression and infrequent, small Cx43 immunoreactive plaques. Treatment with racemic pAST at 10–6 M increased the frequency of Cx43 plaques in regions of cell/cell apposition over untreated controls. In contrast, few such plaques were seen in cultures treated with AST at 10–6 M; a frequency that appeared less than in untreated cultures. At the lowest concentration of AST (10–8 M) tested, gap junction assembly was comparable to untreated cultures. Cells treated with TTNPB at 10–8 M as expected exhibited the most extensive levels of immunoreactive plaques, whereas CTX at 10–5 M produced plaques equivalent to pAST at 10–6 M. This location of Cx43 immunoreactive junctional plaques is consistent with the formation of functional gap junctions. Representative photomicrographs are shown in Figure 3.



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Fig. 3. pAST induces immunoreactive plaques in regions of cell/cell contact. Cells were treated for 4 days as in Figure 2, then fixed and immunostained with Cx43 antibody. (A) Medium control; (B) pAST 10–6 M; (C) AST 10–6 M; (D) CTX 10–5 M and (E) TTNPB, 10–8 M. Arrows indicate plaque location.

 
Induction of GJIC
To assess the functional capacity of these junctional plaques for direct intercellular GJIC, scrape-loading dye transfer assays were performed. Racemic pAST at 10–9–10–6 M increased the extent of junctional communication ~4-fold over that seen in untreated controls. Dye-transfer in cultures treated with AST at 10–6 M was below that observed in controls, whereas 10–7 M dye transfer was enhanced to levels seen in pAST-treated cultures. Thereafter, communication decreased in a dose-responsive manner (Figure 4). The positive controls CTX 10–5 M and TTNPB 10–8 M increased dye transfer by 4-fold and 6-fold, respectively, over controls. Neither THF nor EtOH at concentrations used to deliver AST and pAST, respectively, influenced dye-transfer (data not shown).



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Fig. 4. Comparative induction of gap junctional communication by pAST (filled cirlces) and AST (filled squares). Confluent cultures of 10T1/2 cells were treated with the indicated concentrations for 7 days. Communication was assessed by scrape-loading assays as described. In these cultures a dye-transfer value of 60 is equal to transfer of dye 1 mm from the region of scrape-load. All values for pAST except at 10–10 M were highly significantly different from controls (P < 0.001). For AST, at 10–7 and 10–8 M values were increased from controls (P < 0.001), whereas at 10–6 M values were significantly decreased from controls (P < 0.05).

 
Cytotoxicity assay
When added to sparsely seeded cultures of 10T11/2 cells or to MCA-transformed 10T1/2 cells, and maintained for the 8-day duration of the assay, no decreases on colony formation were observed for either AST or pAST over the concentration range 10–5–10–7 M (data not shown). To determine if these compounds were inducing more subtle changes in growth rates, cells were seeded, treated 24 h later, and growth curves measured over the next 7 days. At concentrations of pAST and AST from 10–6 to 10–7 M, these compounds caused no statistically significant dose-dependent decreases in growth rate as compared with cells treated with media alone (data not shown). This demonstrates that pAST and AST are not cytotoxic and do not have differential cytotoxicity with respect to normal and transformed cells. However, as noted above in the transformations assays, when exposed for 4 weeks to AST or pAST at 10–5 M with weekly retreatments, both compounds inhibited the development of a complete monolayer of cells, an observation suggesting potential cumulative cytotoxicity at this concentration.

Cellular uptake studies
Studies were performed to assess the cellular uptake of AST and to assess the ability of phosphatases to cleave pAST to free AST. Free AST levels in pAST-treated cells on days 1, 4 and 7 were 5.4, 7.0 and 3.6 µg/g wet weight of cells, whereas levels in AST-treated cells were 117, 557 and 204 µg/g, respectively. On day 4, when most of the Cx43 and functional studies were performed, AST-treated cells exhibited ~100-fold higher levels of free AST as compared with pAST-treated cells. By day 7, when cells would be retreated in the transformation assays, AST levels indicated a tendency to decrease in both groups while at the same time maintaining the wide differential in uptake. Thus a proportion of pAST in these culture systems is cleaved, presumably by cellular or serum phosphatases, to yield free AST which associated with the cell monolayer.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present paper, we demonstrate that a novel racemic tetrasodium diphosphate derivative of astaxanthin (pAST) in aqueous formulation demonstrated 100% efficacy in preventing carcinogen-induced neoplastic transformation at a concentration of 10–6 M and was significantly more active than the parent AST compound at this concentration (Table I). Although we did not compare this activity directly with that of CTX, previously the most potent carotenoid in this respect, in all previous studies using identical protocols, CTX only achieved complete inhibition of transformation at a 10-fold higher concentration; at 10–6 M ~40% inhibition was obtained (34,36). pAST also upregulated Cx43 expression with equivalent or greater potency than that observed previously for other carotenoids delivered in an organic vehicle (30,35) and was also more active than AST (Figure 2). pAST-treated cells were found to assemble Cx43 into immunoreactive plaques in the regions of cell/cell contact, consistent with formation of gap junctions (Figure 3). This was confirmed by functional studies utilizing a scrape-loading dye transfer technique, which demonstrated that pAST-treated cells were more extensively coupled than solvent-alone control treated cells at all concentrations tested except the lowest of 10–10 M. AST at 10–6 M reduced the low background level of dye transfer seen in control cells; only at lower concentrations of 10–7 and 10–8 M did AST increase dye-coupling (Figure 4). However, at no concentration did AST exert greater effects on dye-coupling or suppression of transformation than pAST. This is somewhat surprising in view of the ~20 to 100-fold greater cellular concentrations of free AST in AST-treated cells than in cells treated with pAST. Technical problems precluded quantitation by HPLC of pAST uptake into cells. One explanation for these data may be that the AST utilized for cellular studies, which differed in source from the AST used to synthesize pAST, contained small amounts of an inhibitor of junctional communication not present in pAST; thus, at 10–6 M AST, dye-transfer was inhibited whereas at lower concentrations the effects of AST became dominant. In this context, partially oxidized ß-carotene, in contrast to unmodified ß-carotene, has been reported to inhibit junctional communication in cultures of adenocarcinoma cells (37). Alternatively, AST itself may inhibit GJIC at this concentration without being overtly cytotoxic.

The differential effects of AST and pAST are again evident in the studies of Cx43 induction (Figure 2). Surprisingly, Cx43 levels detected by western blotting were not increased by AST over the entire dose range studied (10–6–10–8 M), in spite of the ability of AST to increase functional junctional communication at these concentrations (Figure 4) and partially suppress neoplastic transformation (Table II). In contrast, pAST increased total Cx43 levels and was approximately equipotent to CTX in this respect. One prediction of the greater potency of pAST delivered in aqueous solvent, over AST delivered in THF, would be that pAST-treated cells accumulated high concentrations of biologically active material, assumed to be pAST. Unfortunately, as stated above, technical difficulties in chromatographic evaluation precluded determination of intact pAST in cells; however, measurements of AST demonstrated that pAST was cleaved to yield free AST and was detectable by HPLC in cell pellets within 1 day of treatment. This cleavage is the expected result of the action of cellular phosphatases or phosphatases present in serum-supplemented culture medium. The activity of pAST at concentrations as low as 10–8 M in the transformation assays (Table I) and 10–9 M in the GJIC assays (Figure 4) suggests that pAST possesses intrinsic activity. The data obtained with pAST clearly demonstrated that this water-dispersible molecule has the ability to inhibit neoplastic transformation and enhance junctional communication to an extent greater than the most potent previously tested carotenoid, CTX. These results are summarized in Table II.


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Table II. Summary of the actions in 10T1/2 cells of carotenoids and the retinoid TTNPB

 
Regardless of mechanism, it appears that the actions of pAST and AST on neoplastic transformation reported here are complex and do not appear solely as a result of their effects on GJIC. Although it is possible that GJIC and Cx43 expression, measured after 7 and 4 days treatment, respectively, may be qualitatively different from that occurring after the 28-day treatment used in the transformation assays, previous studies had shown that although GJIC progressively increased with treatment duration, relative potencies were the same when measured after 7 or 28 days (30). The results obtained with AST illustrate this lack of correlation most clearly: it had little effect on Cx43 levels; although over a narrow concentration range it increased GJIC, over the entire concentration range it decreased transformation. Correlations between GJIC, Cx43 expression and inhibition of transformation are more apparent in the case of pAST, yet are not as clear as in previous studies. For example, at 10–6 M pAST completely suppressed transformation and increased the expression of Cx43 to levels comparable with those previously seen after treatment with CTX at this concentration. However, as noted above, complete inhibition of transformation by CTX required a 10-fold higher concentration which further enhanced Cx43 expression (34). Moreover, although enhanced expression of Cx43 by pAST was clearly dose-dependent, being maximal at 10–6 M, its effects on GJIC revealed by scrape-loading assays were seen equally at all but the lowest concentration tested. It may be that part of the success of pAST as an in vitro chemopreventive agent may be a consequence of its ability to act as an antioxidant/free radical scavenger in both the aqueous and lipid phases of the cell (39) and to enhance GJIC by virtue of Cx43 upregulation. The ability of AST to increase GJIC and to suppress transformation without apparently increasing Cx43 could also reflect its activity as an antioxidant. It is known, for example that other antioxidants can counteract the ability of tumor promoters to decrease Cx43-mediated GJIC (40). Other mechanisms must be considered; in studies in cell culture and in mice, AST itself has been shown to inhibit the production of inflammatory cytokines such as TNF-{alpha}, prostaglandins and NO. This was reported to result from inhibition of activation of the nuclear transcription factor NF-{kappa}B, probably as a consequence of scavenging by AST of intracellular reactive oxygen species known to activate this inflammation pathway (16). Certainly the possible involvement of the NF-{kappa}B pathway in the response of 10T1/2 cells to AST cannot be ignored; however, the role of NO in 10T1/2 transformation is unclear, having been reported to either enhance (41,42) or inhibit (43) this process.

The inhibition of transformation caused by pAST in contrast to AST may largely result from its ability to upregulate GJIC as a consequence of upregulated Cx43 expression. Our earlier studies of retinoid (44,45) and carotenoid (30) suppression of transformation and enhancement of growth control in 10T1/2 cells strongly support the concept of growth control via GJIC. Even in established tumor cells, reestablishment of GJIC can have profound effects on cell behavior. Restoration has been achieved by pharmacological and molecular means. In the 10T1/2 system, pharmacological agents that increase cellular levels of cAMP were found to restore GJIC between quiescent non-transformed cells and neoplastically transformed cells leading to cell-cycle arrest of the transformed cells in direct proportion to the extent of GJIC (46). The use of connexin-containing plasmids to force expression in cancer cell lines of diverse origins resulted in decreased tumorigenicity of these cells as xenografts in nude mice and reduced expression of indices of neoplasia in culture [reviewed in (47)]. Conversely, inhibition of GJIC would be expected to enhance carcinogenesis. As previously mentioned, recent studies of connexin knock-out mice have confirmed this linkage. Mice deficient in the liver-expressed Cx32 were found to be more susceptible to liver carcinogens (27); similarly, increased liver tumor incidence was found in mice expressing a dominant negative Cx32 construct (48). Interestingly, knock-out mice were resistant to tumor promotion by phenobarbital, previously shown to inhibit Cx32-mediated GJIC in the liver (49). Although the homozygous Cx43 knock-out is lethal, hemizygous deletion of Cx43 resulted in mice more prone to lung carcinogenesis (50). Moreover, a wide variety of tumor promoters, agents that enhance the process of carcinogenesis, although they are not intrinsically carcinogenic have been shown to inhibit GJIC both in vivo and in cell culture.

It remains to be determined if carotenoids can increase GJIC and Cx43 expression in vivo in experimental animals or clinically. However topical retinoic acid will dramatically increase Cx43 expression in human skin (51) and carotenoids are active in organotypic culture of human keratinocytes (52,53). These data suggest that if sufficient concentrations can be reached, perhaps by the use of these highly bioavailable derivatives of AST, modulation of Cx43 expression, at least in keratinocytes, will be possible. It may be that the protective effects of ß-carotene against cancer progression in the uterine cervix (54) and the oral mucosa (55) may be, in part, a consequence of this modulation. As previously mentioned, in both these anatomic sites Cx43 is downregulated early in the process of carcinogenesis, even in dysplastic tissue (56). The issue of carotenoid-induced lung toxicity after high dose administration, as occurred with ß-carotene in smokers (6,7) and in a ferret model (9), may be avoided by the use of non-provitamin A carotenoids, as has been demonstrated for lycopene (10). ß-carotene also has the potential to act as a pro-oxidant under high oxygen partial pressure and to be toxic (57)—toxicity which may be avoided by the use of carotenoids, such as AST, which lack this activity (58). The availability of water-dispersible bioavailable carotenoid derivatives, such as described here, will facilitate their use in in vitro and in vivo model systems and aid in the full characterization of their effects in mammalian cells.


    Notes
 
6 Present address: Catalyst Biosciences, Inc., 290 Utah Avenue, South San Francisco, CA 94080, USA Back


    Acknowledgments
 
We wish to thank Dr Leo Cheung, Biostatistics Core, CRCH for performing statistical analysis of the transformation data.

Conflict of Interest Statement: None declared.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 11, 2005; revised April 21, 2005; accepted May 3, 2005.