Diallyl disulfide (DADS) enhances gap-junctional intercellular communication by both direct and indirect mechanisms in rat liver cells

Carine Huard3, Nathalie Druesne1, Denis Guyonnet4, Muriel Thomas1, Anthony Pagniez1, Anne-Marie Le Bon2, Paule Martel1 and Catherine Chaumontet1,5

1 Laboratoire de Nutrition et Sécurité Alimentaire, INRA, Jouy-en-Josas and 2 Unité de Toxicologie Nutritionnelle, INRA, Dijon, France
Present address: 3 AFSSA, LERPRA, Sophia-Antipolis, France and 4 Danone Vitapole, Palaiseau, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Diallyl disulfide (DADS), a sulfur compound from garlic has been shown to exert many biological effects: induction of carcinogen detoxication, inhibition of tumor cell proliferation, etc. These effects are consistent with its anticarcinogenic properties in animal models and could account for garlic protective effects in humans. Our study demonstrates that DADS can improve gap-junctional intercellular communication (GJIC) in vitro. In rat liver epithelial cells (REL cells), using the dye transfer assay, we observe a time-dependent stimulation of GJIC by DADS at non-cytotoxic concentrations. In addition, incubation of cells with DADS for 1 h prevents the inhibition of GJIC induced by 3,5-di-tertio-butyl-4-hydroxytoluene (BHT). We have studied the direct effects of DADS on the regulation of GJIC, and especially on the expression and localization of the connexin expressed in these cells (Cx43): the enhancement of dye transfer (x1.6) by DADS from 1 to 50 µM is associated with an increase (x1.3–1.8) in the amount of Cx43 protein (western blotting) with no alteration of its localization in the cell–cell contact regions of the plasma membrane (immunofluorescence analysis). We have also explored the possibility that DADS might act indirectly on GJIC. On one hand, DADS does not change the amount of E-cadherin, the adhesion molecule expressed in epithelial cells. On the other hand, it induces rapid inhibition of protein glycosylation. The data suggest that DADS could reduce local constraints imposed by glycoproteins, thus facilitating dye transfer. In conclusion, DADS can be included with other plant microconstituents, which have been demonstrated to improve GJIC. Its effect on REL cells can be explained by its ability to enhance the amount of Cx43 and also to diminish the level of glycosylated proteins.

Abbreviations: BHT, 3,5-di-tertio-butyl-4-hydroxytoluene; Cx, connexin; DADS, diallyl disulfide; DAS, diallyl sulfide; DMSO, dimethyl sulfoxide; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; GJ, gap junction; GJIC, gap-junctional intercellular communication; PBS, phosphate-buffered saline; REL cells, rat liver epithelial cells; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid; TTBS, Tween–Tris-buffered saline


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Diallyl disulfide (DADS) is one of the sulfur compounds obtained from crushed garlic and represents 40–60% of garlic essential oil (1,2). Increasing interest is being shown for DADS due to its numerous biological activities (3,4). In particular, DADS could account for the observed protective effects of garlic on cancer development. Indeed several epidemiological studies have suggested a protective effect of a high intake of raw or cooked garlic on gastric cancer. Cohort studies confirm this inverse association for colorectal cancer (5). These results are supported by extensive in vivo data. In rodents, experimental studies have shown that garlic powder and organosulfurs, especially diallyl sulfide (DAS) and DADS, inhibit chemical-induced carcinogenesis in different organs (mammary gland, colon, oesophagus, lung and liver) (610), when administered during the initiation or the promotion stages.

Various mechanisms of action have been proposed to explain DADS anticarcinogenic effects. As far as the initiation phase is concerned, DADS has been shown to reduce the mutagenicity of N-nitrosopiperidine and benzo[a]pyrene as well as the hepatic DNA breaks induced by aflatoxin B1 or N-nitrosodimethylamine (1113). These effects could be related to the modulation of drug-metabolizing enzymes, which play a key role in xenobiotic activation as well as detoxication (14). DADS enhances the activities, protein and mRNA levels of microsomal P450 1A2 and P450 2B1/2. It increases the activities of different phase II enzymes such as glutathione S-transferase, UDP-glucuronyl transferase and epoxide hydrolase (1517).

Up to now, one explanation for DADS anti-promoting effects has been its ability to retard the growth of established tumor cell lines, which has been demonstrated both in vitro and in vivo (18). In particular, DADS has been found to be considerably more efficient than the water-soluble monosulfide S-allyl cystein in retarding the in vitro growth of human cells from colon, skin or lung tumors (19).

To explore other potential mechanisms of action, we focused our study on gap-junctional intercellular communication (GJIC). Gap junctions (GJ) are transmembrane channels, composed of proteins called connexins (Cx); they permit neighboring cells to communicate directly by sharing small cytoplasmic molecules like ions and second messengers (20). Increasing evidence indicates that GJIC alteration is involved in tumor cell development (21). It is hypothesized that it causes a disruption of the growth control of initiated cells by healthy surrounding cells, allowing the clonal proliferation of initiated cells (22,23). Several articles report that some tumor cells display a reduced communication capacity related to Cx gene alteration (24,25). Transfection of Cx genes in these cells results in GJIC recovery, growth normalization and tumorigenicity suppression (26). It has also been reported that many tumor promoters disrupt GJIC (27,28). While, conversely, a few compounds including dietary chemopreventive constituents have been found to stimulate GJIC or to counteract its inhibition by tumor promoters (2935).

We therefore studied the ability of DADS to modulate GJIC. Rat liver epithelial cells (REL cells) are particularly useful to study the modulatory effect of compounds whose target is GJIC and their mechanisms of action (36), so such a line was used in our study. With this model, we have already observed that retinoic acid and two flavonoids, apigenin and tangeretin, can increase GJIC (29).

The effect of DADS on the amount and localization of Cx43, the main Cx expressed in REL cells, was investigated. In addition, we hypothesized that the modulation of GJIC by DADS could result from a modification of cellular adhesion or glycosylation patterns and we therefore examined E-cadherin expression and protein glycosylation.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
DADS (purity 80%) was obtained from Aldrich Chemical Co. (France) and used without further purification, the remaining 20% being diallyl trisulfide and DAS (17). Lucifer yellow CH, dimethyl sulfoxide (DMSO) and butylated hydroxytoluene (BHT) were obtained from Sigma-Aldrich (St Quentin Fallavier, France). D-[6-3H]Glucosamine hydrochloride (15 Ci/mmol) was from Amersham (Buckinghamshire, UK). Cell culture products, phosphate-buffered saline (PBS), Ham's F10 Glutamax and antibiotics (penicillin, streptomycin and gentamycin) were from Invitrogen (Cergy Pontoise, France), and fetal calf serum (FCS) was purchased from Dutscher (Brumath, France). Monoclonal mouse anti-Cx43 and monoclonal mouse anti-E-cadherin were purchased from Transduction Laboratories (Le Pont de Claix, France). Monoclonal mouse anti-{alpha} tubulin was purchased from Sigma-Aldrich. Peroxidase-conjugated anti-mouse IgGs and fluorescein isothiocyanate (FITC)-anti-mouse IgGs were from Jackson ImmunoResearch Laboratories (West Grove, PA). Materials, chemicals and molecular weight markers for western blot came from Bio-Rad (Marnes la Coquette, France).

Cell culture
REL cells were cloned from a REL cell line, established in our laboratory (37) and cryopreserved in liquid nitrogen. Cells were grown in Ham's F10 medium with Glutamax supplemented with 10% FCS, 0.1% gentamycin and 1% penicillin (50 U/ml)–streptomycin (50 µg/ml). Cells were cultivated in a humidified incubator (5% CO2) at 37°C. For all experiments except cell proliferation curve, cells were seeded at the same density (44 000 cells/cm2): 4 x 105 in 35-mm Petri dishes, 8 x 104 in 24-well microplates, 2 x 104 in 96-well microplates or 3 x 104 on epoxy-treated slides (Merck Eurolab, Strasbourg, France). This density made it possible to obtain a confluent monolayer 48 h later. DADS was dissolved in DMSO and added to pure FCS, then sonicated for 2 min. Ham's-F10 was then added to give a 0.1% DMSO final concentration (this concentration did not affect GJIC, data not shown).

Cytotoxicity assay
Cell density determination
Cells were seeded into each of the 35-mm Petri dishes (three dishes per treatment). Twenty-four hours after seeding, cells were incubated with DMSO (0.1%) or DADS (1, 10, 25, 50 or 100 µM) for 24 h. After trypsinization with 0.05% trypsin and 0.02% EDTA, cells from each dish were counted with a Coulter counter-channelizer (Coultronics, Roissy, France).

Neutral red uptake incorporation (38)
For each treatment, incorporation of neutral red was measured in five wells from a 96-well microplate. Cells were incubated for 24 h with DMSO (0.1%) or DADS (1, 10, 25, 50 or 100 µM), and then they were treated 3 h at 37°C with the neutral red solution (50 µg/ml of culture medium) (Sigma-Aldrich). Cells were washed three times with PBS and fixed with destain solution (1% glacial acetic acid, 50% ethanol, 49% distilled water). The neutral red uptake was then determined by spectrophotometry (Labsystems Multiskan MCC340 type 347, Les Ulis, France) at 540 nm.

Cell proliferation curve
1.5 x 105 cells were seeded into each of the 60-mm Petri dishes. One day after seeding, DMSO (0.1%) or DADS (10, 25, 50 or 100 µM) was added to the culture medium for an additional 1, 3, 6 or 8 days. We counted the cells of three separate 60-mm Petri dishes per treatment, with the Coulter counter.

Dye transfer
Intercellular coupling was determined using the microinjection dye transfer method described by Enomoto et al. (39). Cells were plated in each of the 35-mm Petri dishes. From day 1 (for 24 h incubation) or 2 (for short incubations) cells were incubated with DADS or 0.1% DMSO. In kinetic studies, DADS 10 or 25 µM was added for 1, 4, 8 or 24 h. In dose–response studies, two Petri dishes were incubated for 24 h with DMSO or DADS at 1, 5, 10, 25 or 50 µM and all microinjections were performed at day 2. Single cells were microinjected for 1 s with 5% (w/v) lucifer yellow CH (diluted in 0.33 M lithium chloride) using capillaries driven by a micromanipulator (IMT2-SYF, Tokyo, Japan) coupled to a pressure control unit (Eppendorf model 5242, Hamburg, Germany). All manipulations were performed under a microscope equipped with epifluorescence (Olympus, Rungis, France). The glass capillaries (Clark Electromedical Instruments) were prepared by an automatic horizontal puller (Narishige, Tokyo, Japan). Ten minutes after the last microinjection, cells were fixed with 4% formaldehyde in PBS and the dye transfer was quantified by counting the number of fluorescent cells surrounding the microinjected cell as described previously (40). As only about half of the microinjections were successful, 30–40 microinjections/dish were necessary to allow precise quantification of GJIC competency.

For each treatment, data are the mean ± SD of 10–20 fluorescent areas. Data from independent or pooled experiments were examined by an analysis of variance (ANOVA). Then, differences between control (untreated cells) and treated groups were assessed using the Student–Newman–Keuls test (parametric test; used when possible) or Dunn's method (non-parametric test). To compare results from independent experiments, the modulating factor was calculated, as the ratio of the number of dye-coupled cells in treated dishes on the number of dye-coupled cells in control dishes.

Western blot analysis
Cells were plated in 35-mm Petri dishes. Twenty-four hours after seeding, one Petri dish was incubated with DADS 1, 5, 10, 25 or 50 µM or 0.1% DMSO. Total proteins were recovered the following day. Cells were washed twice with PBS and scraped in 2x sample buffer [2x sample buffer: 250 mM Tris–HCl, 20% glycerol, 4% sodium dodecyl sulfate (SDS), 0.02% bromophenol blue, 200 mM DTT]. After sonication, 2x sample buffer was diluted to 1x with water. Proteins (corresponding to 25 x 103 cells) were loaded on an 8 or 15% SDS–polyacrylamide gel for electrophoresis, and then electro-transferred (60 mA, 4°C for 2 h) to a PVDF-membrane (Hybond ECL; Amersham, Orsay, France). Brain protein extracts (Transduction laboratories) were used as Cx43 positive control and liver protein extracts as negative control. The membrane was blocked for 2 h with 5% skimmed milk powder in Tween–Tris-buffered saline (TTBS) containing 0.1% Tween 20 (Merck Eurolab), 2.5 mM NaCl and 20 mM Tris–Cl. Membranes were hybridized overnight at 4°C with anti-Cx43 (1:250) or anti-E-cadherin (1:2500) antibodies, diluted in TTBS + 5% skimmed milk powder. Membranes were then washed four times with TTBS and incubated with peroxidase-conjugated anti-mouse IgGs (1:12 000). The immunopositive reaction was detected using an ECL Plus kit (Enhanced ChemiLuminescence plus) (Amersham) and revealed using an autoradiography film (Hyperfilm ECL, Amersham). Proteins were quantified by densitometry of the autoradiogram using a Las 1000 camera (Fujifilm, Paris, France) and Aida software (Raytest, Strasbourg, France).

To verify the homogeneity of sample deposition, membranes were stripped and re-probed with a monoclonal anti-{alpha}-tubulin (1:20 000) and then with peroxidase-conjugated anti-mouse IgGs (1:12 000). In this case, the immunopositive reaction was detected using an ECL kit (Enhanced ChemiLuminescence) (Amersham).

Immunolocalization
For immunochemistry, cells were seeded onto epoxy-treated slides. Twenty-four hours after seeding, cells were treated with 0.1% DMSO or DADS (1, 5 10, 25 or 50 µM) for 24 h. They were then briefly washed three times with PBS, permeabilized with 0.25% Triton X-100–4% paraformaldehyde for 2 min and fixed with 4% paraformaldehyde in PBS for 30 min. Cells were pre-hybridized for 1 h in PBS–2% bovine serum albumin then incubated with anti-Cx43 (1:50) or anti-E-cadherin (1:250) monoclonal antibodies, followed by FITC anti-mouse IgGs (1:200). After mounting in a Vecta-Shield (Vector Laboratories, Biovalley, Marne la Vallée, France), samples were observed (x400) using an Olympus fluorescent microscope equipped with a camera (DP50) (Olympus, Rungis, France).

Measurement of glycosylation
To study protein glycosylation, we used the [3H]glucosamine-incorporation method (41). To avoid competition between the glucose from the medium and [3H]glucosamine, 5% FCS in MEM medium without glucose (Invitrogen) was used.

Cells were seeded into 24-well microplates (6 wells/concentration) in Ham's F10 medium. Twenty-four hours later, the medium was replaced with MEM medium without glucose, supplemented with 5% FCS, 0.1% gentamycin and penicillin (50 UI/ml)-streptomycin (50 µg/ml).

Two types of experiment were performed: (i) cells were exposed to DMSO (0.1%) or to DADS (1, 5 10, 25 or 50 µM) for 24 h, and, during the last hour, confluent cells were incubated with 2 µCi/ml [3H]glucosamine added to the medium; (ii) cells were co-incubated for 1 h with DADS or DMSO and with 2 µCi/ml [3H]glucosamine. At the end of labeling, cells were washed in PBS and lysed in 2% SDS. Fifty microliters of total lysate (25% of total protein) were precipitated with 10% TCA (trichloroacetic acid) (Merck-Eurolab). After filtration, [3H]glucosamine incorporated in the TCA-insoluble fraction on the filter was estimated by means of a scintillation counter (Packard 1500 Tri-Carb).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Up to 50 µM DADS has no cytotoxic effect
As the cell density of a monolayer influences the ability of cells to come into contact and to communicate, we first determined the effect of increasing concentrations of DADS on cell density and cell viability. We observed that after a 24 h exposure, DADS up to 50 µM had no effect on cell density (Table I), whereas 100 µM DADS caused a 40% decrease. However, even at 100 µM the neutral red uptake was not significantly decreased, suggesting that DADS had a cytostatic effect rather than a cytotoxic one. To confirm this cytostatic effect we studied the effect of DADS in conditions of exponential cell proliferation (Figure 1). We observed an inverse relationship between the concentration of DADS (from 10 to 100 µM) and cell proliferation. Exposure to 10 µM DADS for 8 days did not cause any alteration of the growth rate. DADS reduced the cell number by half (at 50 µM and totally at 100 µM), without any detachment of cells from the dishes, which confirms the absence of cytotoxicity. These observations prompted us to use DADS in the following experiments at concentrations comprised between 1 and 50 µM for 24 h incubations.


View this table:
[in this window]
[in a new window]
 
Table I. Effect of DADS on cell density and neutral red uptake in REL cells

 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. Effect of various concentrations of DADS on REL cell proliferation. 1.5 x 105 cells were seeded into each 60-mm Petri dish. Cell numbers of treated and control dishes were counted, after trypsinization, at 0, 1, 3, 6 and 8 days after the beginning of the DADS treatment (the day after plating). Data are means ± SEM from one experiment performed in triplicate. This experiment was reproduced three times.

 
DADS enhances GJIC and prevents the inhibition of dye transfer by BHT
To assess GJIC, cell–cell transfer of the fluorescent tracer lucifer yellow was analysed as a function of DADS concentration and time. Subconfluent REL monolayers were incubated with DADS at 10 or 25 µM for 1, 4, 8 or 24 h. A stimulation of GJIC by DADS was observed. As reported in Table II, in the presence of DADS 10 or 25 µM, the enhancement was significant after 8 h of treatment (x1.2 at 10 µM) and 24 h (x1.5 at 25 µM). Figure 2 illustrates the stimulation of GJIC in REL cells treated with 25 µM DADS for 24 h in comparison with control cells.


View this table:
[in this window]
[in a new window]
 
Table II. Kinetic analysis of the stimulation of junctional transfer by DADS

 


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 2. Stimulation of junctional transfer by DADS. Pictures show fluorescent microinjection areas of cells cultured under control conditions or in the presence of DADS 25 µM for 24 h.

 
The data of several independent experiments focused on a 24 h incubation with DADS are summarized in Table III. They indicate that the increase of the dye transfer by DADS was observed from 1 µM, and reached a maximum level at 25 µM with a corresponding modulation factor of 1.6.


View this table:
[in this window]
[in a new window]
 
Table III. Level of communication in confluent cells exposed to DADS for 24 h

 
We also examined the effects of DADS on the inhibition of GJIC induced by a tumor promoter, BHT. As shown previously (31), when cells were treated with BHT at 10 µM, dye transfer was reduced in as little as 10 min (Table IV). In co-incubation experiments, 10 µM DADS caused a half reduction of the GJIC inhibition induced by BHT (20% instead of 40% of the corresponding control).


View this table:
[in this window]
[in a new window]
 
Table IV. Level of communication in confluent cells exposed to DADS alone or in combination with a tumor promoter BHT

 
DADS increases the amount of Cx43 but does not modify its phosphorylation state nor its localization
One possible mechanism by which GJIC might be increased is the up-regulation of Cx expression. We studied the amount and/or localization of Cx43 after a 24 h treatment with or without DADS (1, 5, 10, 25 or 50 µM). In order to measure the effect of DADS on the total amount of Cx43, we used a 15% polyacrylamide, which concentrates the different Cx43 species in a single band (Figure 3A). A marked increase in immunolabeling of Cx43 was observed, with 5, 10, 25 and 50 µM DADS, and the corresponding factors were 1.3, 1.2, 1.4 and 1.8 (Figure 3B).



View larger version (39K):
[in this window]
[in a new window]
 
Fig. 3. Effect of DADS on the amount of Cx43 protein. (A) Western blot analysis was performed with total proteins extracted from rat liver, rat brain, untreated cells and cells treated with DADS 1–50 µM on a 15% polyacrylamide gel. (B) Three independent experiments were performed. Each spot was standardized with {alpha}-tubulin protein. The amount of Cx43 protein was expressed as the percentage of control. Results are mean ± SEM of triplicates. *For these treatments significantly higher (by Student–Neuwman–Keuls, P < 0.05) than control.

 
Using a more resolvent gel (8% polyacrylamide gel), which separates non-phosphorylated Cx43 (43 kDa) and phosphorylated Cx43 (45–47 kDa) (42), we observed that DADS did not change the number of detected bands (Figure 4), but rather increased all bands uniformly. The stimulation factors for different concentrations of DADS were approximately the same as in the experiments above (data not shown). Thus, DADS increased the amount of Cx43 without affecting its phosphorylation state.



View larger version (48K):
[in this window]
[in a new window]
 
Fig. 4. Effect of DADS on the amount and the phosphorylation of Cx43 protein. Western blot analysis was performed with total proteins extracted from rat liver, rat brain, untreated cells and cells treated with DADS 1–50 µM on a 10% polyacrylamide gel. P, phosphorylated forms of Cx43; NP, non-phosphorylated form of Cx43.

 
To assess cellular distribution of Cx43, immunocytochemical staining of Cx43 was performed under the same conditions of seeding and incubation. Under control conditions (without DADS treatment), Cx43 staining appeared as brightly fluorescent spots on plasma membranes, at cell–cell contacts (Figure 5). No modification in Cx43 distribution was detected after cell exposure to 10 and 25 µM DADS.



View larger version (138K):
[in this window]
[in a new window]
 
Fig. 5. Immunofluorescent staining of Cx43 protein in control cells and cells treated with DADS for 24 h. Cells were plated on epoxy-treated slides and Cx43 proteins were detected with Cx43 antibodies followed by an FITC-conjugated second antibody.

 
DADS does not change E-cadherin expression
It is admitted that before the formation of GJs a strong adhesive interaction between neighboring cells is required. E-cadherin, a transmembranous Ca2+-dependent cell–cell adhesion molecule specifically expressed in epithelial cells and involved in epithelial formation and integrity (43), is necessary for Cx assembly in GJ (44).

Thus, we investigated the possible relationship between DADS stimulation of GJIC and E-cadherin expression. Cells were treated or not with 1–50 µM DADS for 24 h. Under control conditions, E-cadherin protein of REL cells was detected by western blot (8% polyacrylamide gel) as a band corresponding to a molecular mass of 120 kDa (Figure 6A). No difference was observed between control and DADS-treated cells (Figure 6B), indicating that DADS GJIC stimulation did not result from an induction of E-cadherin expression.



View larger version (45K):
[in this window]
[in a new window]
 
Fig. 6. Effect of DADS on the amount of E-cadherin protein. (A) Western blot analysis (8% polyacrylamide gel) was performed with total proteins extracted from rat liver, rat brain, untreated cells and cells treated with DADS 1–50 µM. (B) Three independent experiments were performed. Each spot was standardized with {alpha}-tubulin protein. The amount of E-cadherin was expressed as the percentage of control. Results are mean ± SEM of triplicates.

 
DADS inhibits the glycosylation state of cell proteins
Wang and Mehta (41,45) show, in various normal and transformed cells, that the treatment by tunicamycin, a glycosylation inhibitor, induces an enhancement of GJIC. They suggest that oligosaccharide moieties of glycoproteins of the plasma membrane impose local constraints that are unfavorable to GJ assembly.

To explore whether the DADS stimulation of GJIC is the consequence of an inhibition of protein glycosylation, we measured the incorporation of the glycosylation precursor [3H]glucosamine into proteins (TCA-insoluble material) (Figure 7) under two different experimental conditions. When cells were incubated with DADS and [3H]glucosamine for 1 h simultaneously, we observed that DADS inhibited the precursor incorporation in a dose-related manner, without any alteration of the total cellular protein content (data not shown). The inhibition of glucosamine incorporation reached 85% at 50 µM. When cells were treated with DADS for 24 h and with [3H]glucosamine for the last hour, a weak inhibition was observed (40% at 50 µM). The data suggest that the inhibition of glycosylation by DADS is dose-dependent and rapid.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 7. Inhibition of glycosylation by DADS in REL cells. The incorporation of 3H-glucosamine into TCA-insoluble material from 50 µl cell lysate was measured as described in Materials and Methods. The data represent the mean ± SEM from six separate experiments. CPM = counts per min.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Until now, only one study concerning the effects of garlic oil on GJIC has been available (46): using the photobleaching method, it was shown that garlic oil increased GJIC in gastric cells; however, the active compounds and their mechanisms of action were not studied. Our study is the first to demonstrate the ability of DADS to enhance GJIC and to prevent its inhibition by a tumor promoter. This sulfur compound is thus a new type of food-borne GJIC stimulator in addition to retinoids (30,34), carotenoids (32,47) and flavonoids (29,33).

In REL cells incubated with DADS, we observed a significant enhancement of dye transfer from 8 h of treatment. The maximum stimulating factor (1.6 at 24 h) is similar to those obtained with retinoic acid and flavonoids on the same cells (29,30). DADS is active on GJIC at a rather low dose. After a 24 h treatment, its effect is significant from 1 µM and up to a maximum at 25 µM. At these concentrations, DADS has no effect on cell proliferation, but it has a cytostatic effect at higher concentrations (50 and 100 µM). This result obtained from liver cells is consistent with studies focused on proliferation of different tumor cell lines which showed a cytostatic effect of DADS at high concentrations (25 µM to 2 mM) (18). Moreover, we found that DADS, is a more potent stimulator of GJIC than DAS (data not shown), which contains a single sulfur atom. Our results suggest the importance of the number of sulfur atoms, already emphasized by Bose et al. for the induction of detoxication enzymes (48).

We explored the possible mechanisms involved in the enhancement of GJIC by DADS and found that DADS can modify GJIC regulation by both direct and indirect mechanisms. Interestingly, western blotting showed DADS increased the amount of Cx43 without changing the phosphorylation state of the protein. Such effects had also been observed for retinoic acid and flavonoids in previous studies (29,30). The increase of Cx43 detected in DADS-treated cells was not associated with any modification of protein localization. We hypothesize that either DADS does not enhance the density of GJ in the plasma membrane or this enhancement is too diffuse to be detected by immunochemistry. As for retinoic acid, in REL cells, DADS did not increase the amount of Cx43 mRNA (not shown), suggesting that the modification of the amount of Cx43 protein could result from an increase in its half-life.

Indirect mechanisms of GJIC regulation also deserved study. Cell–cell recognition and adhesion mediated by E-cadherin seems to be a prerequisite for GJ assembly in the plasma membrane (43). Some modulators of GJIC are known to have indirect effects mediated by E-cadherin expression (49). In our study, whereas 5–50 µM DADS treatment for 24 h increased the amount of Cx43, it did not modify E-cadherin protein expression in REL cells. Another possible indirect mechanism of GJIC regulation was analyzed: the modulation of protein glycosylation, which affects a lot of membrane proteins (but not Cx43, as they are not glycosylated) (45). We observed that DADS, in the concentration range of 5–50 µM, was capable of decreasing [3H]glucosamine incorporation in proteins under two experimental conditions: when cells were incubated with DADS for 23 h prior to tracer incorporation, and when they were co-incubated for 1 h. DADS was more efficient in the latter case, indicating an early effect that is partly lost after a 23 h treatment.

As proposed previously by Wang and Mehta (41) for the glycosylation inhibitor, tunicamycin, DADS inhibition of protein glycosylation could diminish the ratio of bulky glycoproteins in the membranes of adjacent cells. In turn, it could help cells to come into close contact and hemichannels to interlock and form diffuse GJ. This hypothesis is consistent with the increase in Cx43 amount induced by DADS. The latter could reflect an increase of Cx43 half-life due to better stability of GJ in the membranes.

To complete this new insight into the mechanisms of DADS action and since Cx43 is present in many tissues and organs such as the stomach, the colon, the bladder, the breast, the brain, it would be interesting to further analyze its role in GJIC modulation by studying whether DADS modulates GJIC in other cell types expressing Cx43. In particular, one could verify whether DADS is responsible for the effect of garlic oil on GJIC stimulation in gastric cells (46). Recently, Qin et al. (50) have demonstrated that Cx proteins can mediate tumor growth independently of GJIC. Further studies should therefore examine the relationship between the increase in Cx43 amounts and cell functions other than GJIC.

From a nutritional point of view, only limited data comment on the bioavailability of DADS. It can be estimated that 1–25 µmol DADS corresponds to ~50 mg to 2 g of garlic (51). DADS is formed from its natural precursor allicin when a garlic clove is crushed, but the fate of DADS in the body after consumption of garlic has not been clearly established. DADS was found in the breath of human subjects who had eaten dehydrated granular garlic (52). A recent study indicates that 48–72 h after a single oral administration of DADS, allyl mercaptan, allyl methyl sulfide, allyl methyl sulfoxide and allyl methyl sulfone were detected in the stomach, liver, plasma and urine (53), strongly suggesting that once absorbed, DADS is rapidly and extensively metabolized. It would therefore be interesting to test if metabolites have the same effects as DADS on GJIC, in order to identify the active compounds. For pharmacological applications, their ability to restore GJIC in non-communicating cancer cells could be investigated, as other plant microconstituents exhibit such a property (54).

In other respects, we have shown that, whereas DADS treatment alone for 1 h did not have any rapid effect on dye transfer, DADS pre-treatment for 1 h partly antagonized the BHT-induced inhibition of GJIC. Such an effect has been observed previously with two flavonoids, apigenin and tangeretin, in REL cells (31). BHT at rather low concentration (10 µM) reduced the dye transfer by half in 10 min, probably, as suggested by Guan et al. (55), through a modification of GJ permeability. The mechanisms by which DADS can counteract this inhibition remain to be elucidated. Taken together, the ability of DADS to enhance GJIC alone and its capacity to counteract the inhibition induced by a tumor promoter confers to this molecule interesting properties that complete the range of its potential anticarcinogenic activities.


    Notes
 
5 To whom correspondence should be addressed Email: chaumont{at}jouy.inra.fr Back


    Acknowledgments
 
We are grateful to Dr Pierre-Henri Duée and to Dr Hervé Blottière for helpful discussion. We thank Mrs Brigitte Huet and Mr Frédéric Véran for technical assistance. The contribution of Mr Paul Flanzy to the preparation of the photographs and figures is greatly appreciated.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Lawson,L.D., Wang,Z.J. and Hughes,B.G. (1991) Identification and HPLC quantitation of the sulfides and dialk(en)yl thiosulfinates in commercial garlic products. Planta Med., 57, 363–370.[ISI][Medline]
  2. Yan,X., Wang,Z. and Barlow,P. (1992) Quantitative estimation of garlic content in garlic oil based health products. Food Chem., 45, 135–139.[CrossRef][ISI]
  3. Robert,V., Mouille,B., Mayeur,C., Michaud,M. and Blachier,F. (2001) Effects of the garlic compound diallyl disulfide on the metabolism, adherence and cell cycle of HT-29 colon carcinoma cells: evidence of sensitive and resistant sub-populations. Carcinogenesis, 22, 1155–1161.[Abstract/Free Full Text]
  4. Thomas,M., Zhang,P., Noordine,M.L., Vaugelade,P., Chaumontet,C. and Duee,P.H. (2002) Diallyl disulfide increases rat H-ferritin, L-ferritin and transferrin receptor genes in vitro in hepatic cells and in vivo in liver. J. Nutr., 132, 3638–3641.[Abstract/Free Full Text]
  5. Fleischauer,A.T. and Arab,L. (2001) Garlic and cancer: a critical review of the epidemiologic literature. J. Nutr., 131, 1032S–1040S.[Abstract/Free Full Text]
  6. Schaffer,E.M., Liu,J.Z., Green,J., Dangler,C.A. and Milner,J.A. (1996) Garlic and associated allyl sulfur components inhibit N-methyl-N-nitrosourea induced rat mammary carcinogenesis. Cancer Lett., 102, 199–204.[CrossRef][ISI][Medline]
  7. Reddy,B.S., Rao,C.V., Rivenson,A. and Kelloff,G. (1993) Chemoprevention of colon carcinogenesis by organosulfur compounds. Cancer Res., 53, 3493–3498.[Abstract]
  8. Wargovich,M.J., Imada,O. and Stephens,L.C. (1992) Initiation and post-initiation chemopreventive effects of diallyl sulfide in esophageal carcinogenesis. Cancer Lett., 64, 39–42.[ISI][Medline]
  9. Srivastava,S.K., Hu,X., Xia,H., Zaren,H.A., Chatterjee,M.L., Agarwal,R. and Singh,S.V. (1997) Mechanism of differential efficacy of garlic organosulfides in preventing benzo(a)pyrene-induced cancer in mice. Cancer Lett., 118, 61–67.[CrossRef][ISI][Medline]
  10. Takahashi,S., Hakoi,K., Yada,H., Hirose,M., Ito,N. and Fukushima,S. (1992) Enhancing effects of diallyl sulfide on hepatocarcinogenesis and inhibitory actions of the related diallyl disulfide on colon and renal carcinogenesis in rats. Carcinogenesis, 13, 1513–1518.[Abstract]
  11. Guyonnet,D., Belloir,C., Suschetet,M., Siess,M.H. and LeBon,A.M. (2000) Liver subcellular fractions from rats treated by organosulfur compounds from Allium modulate mutagen activation. Mutat. Res., 466, 17–26.[ISI][Medline]
  12. Le Bon,A.M., Roy,C., Dupont,C. and Suschetet,M. (1997) In vivo antigenotoxic effects of dietary allyl sulfides in the rat. Cancer Lett., 114, 131–134.[CrossRef][ISI][Medline]
  13. Sheen,L.Y., Wu,C.C., Lii,C.K. and Tsai,S.J. (2001) Effect of diallyl sulfide and diallyl disulfide, the active principles of garlic, on the aflatoxin B-1-induced DNA damage in primary rat hepatocytes. Toxicol. Lett., 122, 45–52.[CrossRef][ISI][Medline]
  14. Guyonnet,D., Belloir,C., Suschetet,M., Siess,M.H. and LeBon,A.M. (2001) Antimutagenic activity of organosulfur compounds from Allium is associated with phase II enzyme induction. Mutat. Res., 495, 135–145.[ISI][Medline]
  15. Siess,M.H., Le Bon,A.M., Canivenc-Lavier,M.C. and Suschetet,M. (1997) Modification of hepatic drug-metabolizing enzymes in rats treated with alkyl sulfides. Cancer Lett., 120, 195–201.[CrossRef][ISI][Medline]
  16. Hu,X. and Singh,S.V. (1997) Glutathione S-transferases of female A/J mouse lung and their induction by anticarcinogenic organosulfides from garlic. Arch. Biochem. Biophys., 340, 279–286.[CrossRef][ISI][Medline]
  17. Guyonnet,D., Siess,M.H., Le Bon,A.M. and Suschetet,M. (1999) Modulation of phase II enzymes by organosulfur compounds from allium vegetables in rat tissues. Toxicol. Appl. Pharmacol., 154, 50–58.[CrossRef][ISI][Medline]
  18. Knowles,L.M. and Milner,J.A. (2000) Allyl sulfides modify cell growth. Drug Metabol. Drug. Interact., 17, 81–107.[Medline]
  19. Sundaram,S.G. and Milner,J.A. (1996) Diallyl disulfide suppresses the growth of human colon tumor cell xenografts in athymic nude mice. J. Nutr., 126, 1355–1361.[ISI][Medline]
  20. Evans,W.H. and Martin,P.E. (2002) Gap junctions: structure and function (Review). Mol. Membr. Biol., 19, 121–136.[CrossRef][ISI][Medline]
  21. Trosko,J.E. and Ruch,R.J. (1998) Cell–cell communication in carcinogenesis. Front. Biosci., 15, D208–D236.
  22. Yotti,L.P., Chang,C.C. and Trosko,J.E. (1979) Elimination of metabolic cooperation in Chinese hamster cells by a tumor promoter. Science, 206, 1089–1091.[ISI][Medline]
  23. Mesnil,M. (2002) Connexins and cancer. Biol. Cell, 94, 493–500.[CrossRef][ISI][Medline]
  24. King,T.J., Fukushima,L.H., Donlon,T.A., Hieber,A.D., Shimabukuro,K.A. and Bertram,J.S. (2000) Correlation between growth control, neoplastic potential and endogenous connexin43 expression in HeLa cell lines: implications for tumor progression. Carcinogenesis, 21, 311–315.[Abstract/Free Full Text]
  25. Ruch,R.J., Porter,S., Koffler,L.D., Dwyer-Nield,L.D. and Malkinson,A.M. (2001) Defective gap junctional intercellular communication in lung cancer: loss of an important mediator of tissue homeostasis and phenotypic regulation. Exp. Lung Res., 27, 231–243.[CrossRef][ISI][Medline]
  26. Duflot-Dancer,A., Mesnil,M. and Yamasaki,H. (1997) Dominant-negative abrogation of connexin-mediated cell growth control by mutant connexin genes. Oncogene, 15, 2151–2158.[CrossRef][ISI][Medline]
  27. Krutovskikh,V.A., Mesnil,M., Mazzoleni,G. and Yamasaki,H. (1995) Inhibition of rat liver gap junction intercellular communication by tumor-promoting agents in vivo. Association with aberrant localization of connexin proteins. Lab. Invest., 72, 571–577.[ISI][Medline]
  28. Mally,A. and Chipman,J.K. (2002) Non-genotoxic carcinogens: early effects on gap junctions, cell proliferation and apoptosis in the rat. Toxicology, 180, 233–248.[CrossRef][ISI][Medline]
  29. Chaumontet,C., Bex,V., Gaillard-Sanchez,I., Seillan-Heberden,C., Suschetet,M. and Martel,P. (1994) Apigenin and tangeretin enhance gap junctional intercellular communication in rat liver epithelial cells. Carcinogenesis, 15, 2325–2330.[Abstract]
  30. Bex,V., Mercier,T., Chaumontet,C., Gaillard-Sanchez,I., Flechon,B., Mazet,F., Traub,O. and Martel,P. (1995) Retinoic acid enhances connexin43 expression at the post-transcriptional level in rat liver epithelial cells. Cell Biochem. Funct., 13, 69–77.[ISI][Medline]
  31. Chaumontet,C., Droumaguet,C., Bex,V., Heberden,C., Gaillard-Sanchez,I. and Martel,P. (1997) Flavonoids (apigenin, tangeretin) counteract tumor promoter-induced inhibition of intercellular communication of rat liver epithelial cells. Cancer Lett., 114, 207–210.[CrossRef][ISI][Medline]
  32. Bertram,J.S. (1999) Carotenoids and gene regulation. Nutr. Rev., 57, 182–191.[ISI][Medline]
  33. Nielsen,M., Ruch,R.J. and Vang,O. (2000) Resveratrol reverses tumor-promoter-induced inhibition of gap-junctional intercellular communication. Biochem. Biophys. Res. Commun., 275, 804–809.[CrossRef][ISI][Medline]
  34. Ara,C., Massimi,M. and Devirgiliis Conti,L. (2002) Retinoic acid modulates gap junctional intercellular communication in hepatocytes and hepatoma cells. Cell Mol. Life Sci., 59, 1758–1765.[ISI][Medline]
  35. Trosko,J.E. and Ruch,R.J. (2002) Gap junctions as targets for cancer chemoprevention and chemotherapy. Curr. Drug Targets, 3, 465–482.[ISI][Medline]
  36. Vanrullen,I., Chaumontet,C., Martel,P., Honikman-Leban,E. and Elias,Z. (1998) Inhibition of Gap Junction Intercellular Communication. OECD Test Guidelines Programme, France, pp. II-1–II-26.
  37. Carrera,G., Melgar,J., Alary,J., Lamboeuf,Y. and Martel,P. (1992) Cadmium accumulation and cytotoxicity in rat hepatocytes co-cultured with a liver epithelial cell line. Toxicol. In vitro, 6, 201–206.[CrossRef][ISI]
  38. Borenfreund,E., Babich,H. and Martin-Alguacil,N. (1988) Comparisons of two in vitro cytotoxicity assays—the neutral red (NR) and tetrazolium MTT tests. Toxicol. In vitro, 2, 1–6.[CrossRef][ISI]
  39. Enomoto,T., Martel,N., Kanno,Y. and Yamasaki,H. (1984) Inhibition of cell communication between Balb/c 3T3 cells by tumor promoters and protection by cAMP. J. Cell. Physiol., 121, 323–333.[ISI][Medline]
  40. Chaumontet,C., Mazzoleni,G., Decaens,C., Bex,V., Cassio,D. and Martel,P. (1998) The polarized hepatic human/rat hybrid WIF 12-1 and WIF-B cells communicate efficiently in vitro via connexin 32-constituted gap junctions. Hepatology, 28, 164–172.[ISI][Medline]
  41. Wang,Y. and Mehta,P.P. (1995) Facilitation of gap-junctional communication and gap-junction formation in mammalian cells by inhibition of glycosylation. Eur. J. Cell. Biol., 67, 285–296.[ISI][Medline]
  42. Musil,L.S., Cunningham,B.A., Edelman,G.M. and Goodenough,D.A. (1990) Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines. J. Cell. Biol., 111, 2077–2088.[Abstract]
  43. Fujimoto,K., Nagafuchi,A., Tsukita,S., Kuraoka,A., Ohokuma,A. and Shibata,Y. (1997) Dynamics of connexins, E-cadherin and alpha-catenin on cell membranes during gap junction formation. J. Cell. Sci., 110, 311–322.[Abstract/Free Full Text]
  44. Prowse,D.M., Cadwallader,G.P. and Pitts,J.D. (1997) E-cadherin expression can alter the specificity of gap junction formation. Cell. Biol. Int., 21, 833–843.[CrossRef][ISI][Medline]
  45. Wang,Y., Mehta,P.P. and Rose,B. (1995) Inhibition of glycosylation induces formation of open connexin-43 cell-to-cell channels and phosphorylation and triton X-100 insolubility of connexin-43. J. Biol. Chem., 270, 26581–26585.[Abstract/Free Full Text]
  46. Li,X.G., Xie,J.Y., Li,W.M., Ji,J.F., Cui,J.T., Zhao,M., Sun,M. and Lu,Y.Y. (2000) Effects of garlic oil on tumorigenicity and intercellular communication in human gastric cancer cell line. Sci. China Ser. C Life Sci., 43, 82.[ISI]
  47. Stahl,W. and Sies,H. (1998) The role of carotenoids and retinoids in gap junctional communication. Int. J. Vitam. Nutr. Res., 68, 354–359.[ISI][Medline]
  48. Bose,C., Guo,J., Zimniak,L., Srivastava,S.K., Singh,S.P., Zimniak,P. and Singh,S.V. (2002) Critical role of allyl groups and disulfide chain in induction of Pi class glutathione transferase in mouse tissues in vivo by diallyl disulfide, a naturally occurring chemopreventive agent in garlic. Carcinogenesis, 23, 1661–1665.[Abstract/Free Full Text]
  49. Jansen,L.A., Mesnil,M. and Jongen,W.M. (1996) Inhibition of gap junctional intercellular communication and delocalization of the cell adhesion molecule E-cadherin by tumor promoters. Carcinogenesis, 17, 1527–1531.[Abstract]
  50. Qin,H., Shao,Q., Curtis,H., Galipeau,J., Belliveau,D.J., Wang,T., Alaoui-Jamali,M.A. and Laird,D.W. (2002) Retroviral delivery of connexin genes to human breast tumor cells inhibits in vivo tumor growth by a mechanism that is independent of significant gap junctional intercellular communication. J. Biol. Chem., 277, 29132–29138.[Abstract/Free Full Text]
  51. Kerckhoffs,D.A., Brouns,F., Hornstra,G. and Mensink,R.P. (2002) Effects on the human serum lipoprotein profile of beta-glucan, soy protein and isoflavones, plant sterols and stanols, garlic and tocotrienols. J. Nutr., 132, 2494–2505.[Abstract/Free Full Text]
  52. Rosen,R.T., Hiserodt,R.D., Fukuda,E.K., Ruiz,R.J., Zhou,Z., Lech,J., Rosen,S.L. and Hartman,T.G. (2000) The determination of metabolites of garlic preparations in breath and human plasma. Biofactors, 13, 241–249.[ISI][Medline]
  53. Germain,E., Auger,J., Ginies,C., Siess,M.H. and Teyssier,C. (2002) In vivo metabolism of diallyl disulphide in the rat: identification of two new metabolites. Xenobiotica, 32, 1127–1138.[CrossRef][ISI][Medline]
  54. Trosko,J.E. and Chang,C.C. (2001) Mechanism of up-regulated gap junctional intercellular communication during chemoprevention and chemotherapy of cancer. Mutat. Res., 480, 219–229.[ISI]
  55. Guan,X., Hardenbrook,J., Fernstrom,M.J., Chaudhuri,R., Malkinson,A.M. and Ruch,R.J. (1995) Down-regulation by butylated hydroxytoluene of the number and function of gap junctions in epithelial cell lines derived from mouse lung and rat liver. Carcinogenesis, 16, 2575–2582[Abstract]
Received July 4, 2003; accepted September 12, 2003.