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Institute of Biochemistry and Molecular Biology I, Heinrich-Heine-University Düsseldorf, D-40001 Düsseldorf, Germany
2To whom correspondence should be addressed. E-mail: wilhelm.stahl{at}uni-duesseldorf.de.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • astaxanthin • canthaxanthin • carotenoids • connexin • gap junction • phosphorylation
Epidemiologic studies reveal an association between nutritional habits and the prevention of several types of cancer. However, there is a lack of knowledge on the nature of the chemopreventive food constituents and their mechanism of action (1). Preventive properties have been assigned to antioxidant micronutrients, including the carotenoid group. Scavenging of reactive oxygen species that are deleterious to DNA and other cellular macromolecules has been discussed as a mechanism of chemoprevention. However, there is increasing evidence that carotenoids and other antioxidants exhibit biological properties beyond their antioxidant potential, such as regulatory effects on intra- and intercellular signaling and gene expression (2).
Cell culture studies provide evidence that carotenoids affect cell proliferation by interference with the progression of the cell cycle, modulation of the insulin-like growth factor system, or by effects on gap junctional intercellular communication (GJIC) (3,4). GJIC is mediated by microdomains of the plasma membrane, which contain an array of channels providing a direct link between the cytosol of neighboring cells (5). Each channel is composed of 2 connexons (hemi-channels), which are connected in the intermembrane gap, permitting small molecules up to 
1000 Da to shuttle from one cell to another. Multiple pathways for the regulation of GJIC are known, including effects on the rate of transcription of connexin genes as well as stabilization of connexin mRNA. Connexins may also be modified post-translationally, and phosphorylation is a common modification of these proteins (6).
All of the major carotenoids present in the human organism, which include 
- and ß-carotene, lutein, zeaxanthin, ß-cryptoxanthin, and lycopene, stimulate GJIC (7). Stimulation of intercellular communication is associated with the inhibition of the growth of preneoplastic foci in chemically transformed cells (8,9). GJIC is disturbed in most tumor cells, and restoring cell-to-cell communication may be a strategy in cancer prevention.
Astaxanthin and the structurally closely related canthaxanthin (Fig. 1) are carotenoids used as colorants in animal feed for poultry and for farmed salmon and trout. They are efficient antioxidants but are usually not found in considerable amounts in human serum. In animal studies, cancer-preventive effects were demonstrated for both carotenoids (10). Canthaxanthin stimulates GJIC in various cell systems (7), whereas little is known about the effects of astaxanthin. In the present study, we compared the effects of canthaxanthin and astaxanthin on GJIC in primary human skin fibroblasts.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cell culture.
Primary human fibroblasts were obtained from biopsy material of fetal foreskin (kind gift from Dr. P. Brenneisen, Institute of Biochemistry and Molecular Biology I, University of Düsseldorf). Because the basal communication in this cell system is 7–10 communicating cells, it provides a suitable tool with which to measure stimulatory and inhibitory effects on GJIC. Cells were grown in DMEM (Sigma) supplemented with 10% (v:v) fetal calf serum (FCS, Greiner bio-one), 2 mmol/L L-glutamine (as glutamax from Invitrogen) and penicillin/streptomycin (PAA Laboratories) in 35-mm plastic dishes. Incubation was at 37°C in a humidified atmosphere containing 5% CO2. At confluence of 90%, 3% FCS was added to primary human fibroblasts; after another 24 h of incubation, the cells were exposed to carotenoids and RA. Carotenoid stock solutions were prepared in THF and diluted 1:1000 in DMEM (3% FCS) to obtain a final concentration in the incubation medium within the range of 0.001–10 µmol/L. Incubation medium of solvent controls contained 0.1% (v:v) THF; incubation time was between 0 and 72 h. The study was approved by the ethics committee of the University of Düsseldorf, Germany.
Gap junctional communication assay. GJIC was determined by the dye-transfer assay as described earlier (11). For microinjection of Lucifer Yellow CH (10% in 0.33 mol/L LiCl) a micromanipulator/microinjector system (FemtoJet and InjectMan) was used; 10 cells were injected/dish, and means were calculated to evaluate communication. Each experiment was repeated 4 or 6 times.
Statistical analysis.
Means ± SEM were calculated and the data are presented as a percentage of control. All data were analyzed by SAS 8.2 using a repeated-measures ANOVA to test the hypothesis that the independent variables (treatment, day, incubation time, time point) have no effect; tests for contrasts to compare the different levels of the independent variables were carried out. An -level of 5% was used for analysis.
Immunohistochemistry. For immunohistochemistry, cells were grown in complete medium until they reached 90% confluence. After treatment with carotenoids, cells were washed with PBS, fixed with methanol, and blocked with normal goat serum. Cells were incubated with a polyclonal anti-connexin Cx43 antibody (Sigma); diluted 1:1500 in PBS with 1% (v:v) normal goat serum overnight at 4°C. Cells were washed with PBS, and incubated with an Alexa 546-coupled goat anti-rabbit IgG (H+L) secondary antibody (Molecular Probes; diluted 1:800 in PBS) for 45 min at 37°C. Images were taken with a fluorescence microscope coupled to a CCD camera (12).
Western blot analysis. Medium was removed, cells were washed twice with PBS and lysed with 0.5% SDS. Lysates were stored at –80°C. Cell lysates were sonicated, and protein levels were determined with a protein detection assay (BioRad). Sample blue buffer (30% sucrose, 10% SDS, 0.1% bromophenol blue, 0.2% dithiothreitol) was added, samples were heated for 5 min at 95°C and loaded onto gels [SDS-PAGE, 10% (v:v) acrylamide (Roth)]. SDS-PAGE–separated proteins were blotted onto a nitrocellulose membrane (Amersham Biosciences) using a semidry blotter (VWR) and a 3-buffer system (anode-1-buffer: 300 mmol/ L Tris, 10% (v:v) methanol, pH 10.4; anode-2-buffer: 25 mmol/ L Tris, 10% (v:v) methanol, pH 10.4; cathode-buffer: 25 mmol/ L Tris, 10% (v:v) methanol, 40 mmol/L L-glycine, pH 9.4). Protein transfer was checked with Ponceau S staining (Sigma) and after washing with PBS, the membranes were blocked overnight in tris-buffered saline Tween (TBST) containing 5% milk powder (Roth). For immunodetection of Cx43, a polyclonal rabbit anti-Cx43 antibody [Sigma; 1 h, 1:1000 in TBST (1% milk powder)] was used as primary antibody and a horseradish peroxide (HRP)-conjugated goat anti-rabbit as secondary antibody [PerbioScience; 1 h, 1:10,000 in TBST (1% milk powder)].
Immunodetection of the loading control ß-tubulin was performed with a monoclonal mouse anti-ß-tubulin antibody [Santa Cruz Biotechnology; 1 h, 1:500 in TBST (1% milk powder)] as primary antibody and a HRP-conjugated goat anti-mouse secondary antibody [Perbio Science; 1 h, 1:5000 in TBST (1% milk powder)]. After incubation with the antibody, membranes were washed for 1.5 h in TBST. TBST was changed at least 5 times. The membranes were developed with SuperSignal West Femto (Perbio Science) and exposed to X-ray films (Kodak Biomax Film, Sigma) for an adequate amount of time. Western blot analysis was done at least 3 times and figures show representative blots.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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80% at this level. Inhibition was even more pronounced (95%) at 10 µmol/L astaxanthin compared with control (P < 0.0001) after both incubation times. After 72 h of treatment, the inhibitory effect of 10 µmol/L astaxanthin was stronger (P = 0.0214) than after incubation with 1 µmol/L. Compared with control, no cellular responses were found at 0.01 and 0.001 µmol/L. Incubation time influenced GJIC at 0.1 µmol/L (P = 0.0033, Fig. 2B): after 72 h treatment GJIC was decreased by 30% compared with the control level (P = 0.0451). During incubation, cells were fully viable as determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromid (MTT) assay (data not shown).
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70% of control. Similarly, the stimulatory effect of canthaxanthin was reversed when the compound was removed from the incubation mixture (Fig. 3).
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60% of the control level (Fig. 4). Compared with control, GJIC was also lower at 24 h of incubation (P < 0.0001) and reached only 2% of the control level.
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Connexin location. Changes in connexin trafficking, expression, and phosphorylation are the biochemical mechanisms responsible for modulation of intercellular communication via gap junctions. Using immunofluorescence, we investigated the intracellular location of connexin43 protein, a major channel protein in primary human skin fibroblasts. No changes in connexin43 location were detected upon incubation with astaxanthin. Most of the connexin protein was located in the plasma membrane (data not shown). A slight increase in the amount of connexin43 protein was detected when cells were exposed to canthaxanthin and RA; again, most of the protein was located in the plasma membrane.
Connexin phosphorylation pattern. The effects of astaxanthin and canthaxanthin on connexin43 expression and phosphorylation were investigated by Western blot analysis using ß-tubulin as the loading control (Fig. 5). Compared with control, the total amount of connexin43 protein was increased after incubation with canthaxanthin at 24 (Fig. 5A) and 72 h (Fig. 5B). Analysis of connexin43 using SDS-PAGE resolved 3 bands, reflecting different phosphorylation states of the protein, assigned as P0 (unphosphorylated protein) and the higher phosphorylation states P1 and P2. No changes in the phosphorylation pattern were detected when the cells were exposed to canthaxanthin (Fig. 5A and B); the pattern was similar to that in the solvent or in the positive control. However, a significant modification of the phosphorylation pattern was revealed when cells were incubated with astaxanthin. At 1 and 10 µmol/L, the intensity of the P2 band decreased after 24 (Fig. 5C) and 72 h (Fig. 5D) of incubation; within this concentration range, a significant inhibition of GJIC by astaxanthin was measured (Fig. 2B). The increase of GJIC after removal of astaxanthin (Fig. 3) was accompanied by the reappearance of the P2 band (Fig. 6A and B). Based on the present findings, we conclude that astaxanthin affects the phosphorylation pattern of connexin43 with a substantial effect on channel function.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-carotene, lycopene, lutein, and ß-cryptoxanthin stimulate GJIC and enhance connexin43 expression in cell culture. The effect is apparently independent of the provitamin A activity of carotenoids and is not correlated with their antioxidant properties. The molecular mechanism for upregulated expression is not yet known. It is still under debate whether the parent compounds or biologically active metabolites are responsible for activation and whether RA receptor–dependent pathways play a role in the regulation of GJIC. The phosphorylation pattern of connexin43 was not changed when the cells were exposed to canthaxanthin, indicating that such post-translational modifications are not involved. In contrast to canthaxanthin, astaxanthin was a strong suppressor of GJIC, inducing changes in the phosphorylation state of the connexin43 protein. Correlated with impaired GJIC, the P2 band (highest phosphorylation state) disappeared. Thus, hypophosphorylation of connexin43 is potentially responsible for the loss of GJIC. It should be noted that increases in connexin protein levels, as observed to a minor extent with astaxanthin, do not necessarily lead to an increased GJIC. Phosphorylation/dephosphorylation of functional connexin proteins in the membrane may influence channel gating and regulate channel function (6).
Withdrawing astaxanthin from the culture medium leads to a recovery of GJIC (Fig. 3) and the reappearance of the P2 band (Fig. 6). Connexin43 contains several possible phosphorylation sites (21 serine and 6 tyrosine residues), and phosphorylation was implicated in the regulation of cellular communication through a number of mechanisms. A loss of the P2 form of connexin43 was observed when rat liver epithelial cells were exposed to dicumarol, an inhibitor of GJIC (15); similar effects were described for heptanol and oleamide (16,17), in agreement with the data provided in the present work. However, hyperphosphorylation of connexin proteins was noted in several cell lines after incubation with tumor-promoting phorbol esters, accompanied by a loss of GJIC (12,18,19). It appears that a complex regulatory network is responsible for the fine-tuning of intercellular communication via gap junctions, making use of different phosphorylation sites at the connexin proteins.
Stimulatory effects of astaxanthin and astaxanthin tetrasodium diphosphate were determined in C3H/10T1/2 cells (20,21). After 7 d of exposure to the carotenoids, GJIC was increased 3-fold at levels from 0.001 to 0.1 µmol/L, determined in a dye transfer assay, monitored after scrape loading. No stimulation was found with 1 µmol/L of astaxanthin. Induction of GJIC for both compounds was correlated with their inhibitory effects on the formation of methylcholanthrene-induced preneoplastic foci. In the case of astaxanthin tetrasodium diphosphate, connexin43 protein was upregulated 4-fold at 0.1 and 1 µmol/L; only minor effects were measured with the parent astaxanthin. The discrepancy between the 2 studies related to the functional assay (dye transfer) may be due to different experimental conditions and cell types (22).
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FOOTNOTES |
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3 Abbreviations used: Cx, connexin; FCS, fetal calf serum; GJIC, gap junctional intercellular communication; HRP, horseradish peroxidase; RA, retinoic acid; TBST, tris-buffered saline Tween; THF, tetrahydrofuran.
Manuscript received 2 May 2005. Initial review completed 10 June 2005. Revision accepted 15 August 2005.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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