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Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel;
*
Department of Oral Pathology and Medicine, School of Dental Medicine, Tel-Aviv University, Tel-Aviv, Israel; and
Institute of Pathology, Sheba Medical Center, Tel-Hashomer, Israel
1To whom correspondence should be addressed. E-mail: bschwart{at}agri.huji.ac.il
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • lycopene • oral cancer cells • gap-junctional communication • connexin 43
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Although this neoplasia is treatable with surgery or radiotherapy in its early stages, most patients are diagnosed only at advanced stages of the disease. At these late stages, therapy outcomes have not dramatically improved in recent years. Reduced incidence of this disease may be attainable through preventive measures (2
,3
). Preventive strategies are designed to suppress, reverse or prevent the formation of premalignant lesions and their subsequent development through the multistep process of initiation, promotion and progression into squamous cell carcinoma (4
,5
).
Epidemiologic studies suggest that a diet rich in lycopene is related to decreased risk of certain diseases, particularly cancers of the digestive tract, prostate and pancreas, as well as cardiovascular disease (6
). Most of the reports concerning the anticarcinogenic activity of carotenoids emphasize their ability to be converted into vitamin A, which has been associated with differentiation and cancer regression (7
,8
). All-trans retinoic acid has been shown to be effective as a therapeutic agent that induces differentiation of promyelocytic leukemia cells (9
), and 13-cis retinoic acid has shown great promise in inhibiting second primaries in head and neck cancer (10
). However, the use of retinoic acid is limited in chemoprevention because of its high toxicity. Carotenoids may provide a nontoxic alternative treatment, but with far less potent activity. ß-Carotene has shown activity against oral leukoplakia, a premalignant lesion in the oral cavity (11
).
Experimental data have demonstrated lycopene’s notable anticarcinogenic effect in vitro (8
,12
). The carotenoid lycopene differs from ß-carotene mainly in the latter’s ability to be converted into vitamin A. The reported biological activities of lycopene include potent antioxidant activity involving singlet-oxygen quenching, peroxyl-radical scavenging and in addition, induction of cell-cell communication and growth control (13
,14
).
The present study was conducted to investigate whether the inhibitory effect exerted by lycopene and ß-carotene on oral cancer cell proliferation is associated with upregulation of connexin 43 mRNA and protein expression, concomitant with enhanced gap-junctional communication.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Synthetic ß-carotene was generously provided by Hoffman-La Roche (Basel, Switzerland). Natural lycopene extracted from tomatoes was prepared by Makhteshim (Beer Sheva, Israel). Tetrahydrofuran (THF)2 was purchased from Sigma Chemical (St. Louis, MO). Rabbit polyclonal anti-connexin 43 was purchased from Zymed Laboratories (San Francisco, CA).
Carotenoid solutions.
Carotenoids were dissolved in THF to a final concentration of 2 mmol/L and stored at -70°C. Immediately before the experiment, aliquots from these stock solutions were added to the cell culture medium under a N2 environment, and stirred vigorously. The final concentration of the carotenoids in the medium was measured by spectrophotometry after extraction in 2-propanol and n-hexane-dichloromethane (12
). The exact carotenoid concentration, which varied within narrow limits in each medium preparation, was recorded. The final THF concentration in each experiment was 0.5%, a concentration that does not affect cell growth compared with control media. All procedures, including incubation with the carotenoids, were performed under dim light.
Cells.
KB-1 human oral tumor cells (kindly supplied by Prof. Ruth Arnon, Weizmann Institute, Rehovot, Israel) were cultured on RPMI medium (Biological Industries, Bet Haemek, Israel) containing penicillin, streptomycin, fungyzone, 5% fetal calf serum (FCS) and 1% L-glutamine.
Assessment of cell growth.
Cells were seeded onto six-well plates (5 x 105 cells/well) in a medium containing 5% FCS. After 1 d, the medium was replaced with medium containing the solubilized carotenoid or THF alone. At different times, cells were harvested by trypsinization and vital cells, excluding trypan blue, were counted in a Bright Line Neubauer counting chamber (Sigma-Aldrich Chemie, Steinheim, Germany).
Protein isolation.
Cell cultures were grown and treated in six-well dishes for different periods of time. The cells were scraped and lysed in 250 µL of lysis buffer (20 mmol/L Tris, pH 7.8, 1% Nonidet P-40, 100 mmol/L NaCl, 50 mmol/L NaF, 10% glycerol, 1 mmol/L sodium orthovanadate). Expression of solubilized connexin 43 was assessed in the supernatant (15
). Protein concentration was determined by the Lowry method (16
) using bovine serum albumin as the standard protein.
Western blot determination of connexin 43.
Protein (30 µg) from the lysates’ supernatants was electrophoresed on a 12% SDS-polyacrylamide gel. Proteins were electroblotted onto a nylon-transfer membrane (Amersham, Buckinghamshire, UK) and subsequently incubated with a polyclonal antibody against the C-terminal region of connexin 43 (rabbit anti-connexin 43, Zymed Laboratories). The bound antibody was visualized using a chemiluminescent detection system (17
).
Immunocytochemistry.
Cells were grown and treated with the different carotenoid solutions for 3 d on chamber slides. Fixation and abrogation of endogenous peroxidase activity were performed in a solution of methanol/hydrogen peroxide/H2O (95:3:2). The fixed cells were incubated with polyclonal connexin 43 antibody (rabbit anti-connexin 43). Detection was performed with a Histostain kit (Histostain-SP, Zymed Laboratories). Counterstaining was performed with hematoxylin (18
).
Connexin 43 reverse transcription-polymerase chain reaction (RT-PCR).
cDNA was synthesized from 1 µg of total RNA [extracted with Tri Reagent (MRC, Cincinnati, OH) according to manufacturer protocol] from control, ß-carotene or lycopene-treated cells, using RT-PCR beads (Amersham, Piscataway, NJ) essentially according to manufacturer’s instructions. RT-PCR was performed using the primers of the connexin 43 gene 5'-ACA TCA GGT CGA CTG TTT CCT-3', bases 579–600; and 5'-ACG ACT GCT GGC TCT GCT T-3', bases 1110–1091. The PCR products were analyzed on a 1% agarose gel in Tris-acetate/EDTA buffer, and molecular weight was confirmed with the 100-bp DNA Ladder (Promega, Madison, WI). Gels were stained with ethidium bromide and photographed (19
,20
).
Transmission electron microscopy (EM).
Cells grown in flasks and incubated with different ß-carotene and lycopene concentrations (3 d) were fixed in 1% glutaraldehyde in 0.1 mol/L PBS, pH 7.4, for 1 h at room temperature. Fixed cells were then removed with a rubber policeman, washed in PBS and postfixed in 1% OsO4 for 1 h. Samples were dehydrated in graded ethanol solutions and embedded in Epon. Ultrathin sections were stained on grids with uranyl acetate and lead citrate and examined with a Jeol 100 CX or 1200 EX transmission electron microscope (Jeol, Tokyo, Japan) (21
).
Scrape-loading and dye transfer.
Cells were treated with ß-carotene (7 µmol/L) lycopene (7 µmol/L) or solvent (THF) devoid of carotenoids for 3 d. At the end of the treatment procedure, the cells were rinsed with PBS before addition of the fluorescent dye mixture; 2 mL of 0.05% lucifer yellow (purchased from Molecular Probe, Eugene, OR) dissolved in PBS were added to the cells and scrape-loaded at room temperature using a rubber policeman or wooden probe. The dye solution was left on the cells for 2 min then discarded, and the plates were rinsed with PBS to remove detached cells and background fluorescence. Media (2 mL) were replaced and cells were examined under a Nikon (Tokyo, Japan) epifluorescence phase microscope illuminated with an Osram HBO 200 W lamp (22
).
Incorporation of lycopene and ß-carotene into KB-1 cells.
KB-1 cells were incubated with the carotenoids for various periods. On d 1, cells were harvested every 2 h until 12 h of incubation, and then at 24, 36 and 72 h. Harvested cells were exposed to different concentrations of lycopene (3–7 µmol/L) or ß-carotene (3–7 µmol/L). SDS (0.5 mL, 10 mmol/L) and ethanol were added to the harvested cell samples (0.5 mL), and the carotenoids were extracted with hexane and separated by HPLC. The intracellular concentrations of lycopene and ß-carotene in the cancer cells after the different incubation periods were analyzed with a multiwavelength detector. The peak identity of each carotenoid was confirmed by comparison with chromatographic (retention time) and spectral characteristics of authentic standard carotenoids (23
).
Statistical analysis.
All values are expressed as mean ± SEM. Data were analyzed by one- or two-way ANOVA, and then differences among means were analyzed using Tukey-Kramer multiple comparison tests. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The treatments became much more effective after 3 d. However, at all time points, the cell-growth suppression induced by lycopene (7 µmol/L) was significantly more potent than that by ß-carotene (7 µmol/L). The carotenoid solvent (THF) did not affect KB-1 cell growth relative to growth in the control media.
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). ß-Carotene had a similar concentration-dependent inhibitory effect on cell proliferation (Fig. 2
B). However, although the highest concentration used (7 µmol/L) caused 62 ± 8% inhibition, the lowest concentration (3 µmol/L) resulted in a much smaller, although still significant inhibition (15 ± 5%) after 3 d.
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shows a representative gel; the densitometric quantitation of the protein relative to the control is depicted in Figure 3
B. Lycopene significantly (P < 0.05) upregulated connexin 43 expression. ß-Carotene tended to upregulate expression (P = 0.07) at 3 µmol/L but not at 7 µmol/L (P = 0.42). Control and THF-treated cells did not differ; thus, the THF results are not shown. Expression of connexin 43 was also assessed by immunocytochemistry and the results were consistent with the Western blot results. Lycopene induced notable connexin 43 expression, localized to the cell membranes (Fig. 4A
), whereas cells treated with ß-carotene did not show this pattern (Fig. 4
B).
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. Densitometric analysis of this gel and of four others is presented in Figure 5
B. Lycopene treatment (3 and 7 µmol/L) significantly upregulated the expression of connexin 43 mRNA compared with the control untreated cells (212 ± 25 and 325 ± 39% of the control, respectively, P < 0.005). The effect of ß-carotene (7 µmol/L) was smaller but significant (162 ± 19% of the control, P < 0.05). At both concentrations, the effects of lycopene were greater than those of ß-carotene (P < 0.05).
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). After the addition of carotenoids, morphological changes were evident, i.e., EM of thin sections from KB-1 oral cancer cells treated with ß-carotene for 3 d revealed the presence of intermediate junctions and desmosomes (Fig. 6
B) and the cultures contained cells that contacted one another. Interestingly, treatment with lycopene for the same period (3 d) resulted in cell cultures with tight contacts between the cells (desmosomes and intermediate junctions) and several intercellular junction-type structures resembling gap junctions (Fig. 6
C).
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). A weaker effect was seen in cells treated with 3 µmol/L lycopene (Fig. 7
E). No effect was observed following treatment with 3 µmol/L ß-carotene (Fig. 7
C) and a very weak effect was detected with 7 µmol/L ß-carotene (Fig. 7
D); the untreated cells (control) did not show any positive reaction (Fig. 7
A, B).
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). At 72 h, the intracellular lycopene concentration increased. At the lowest concentration of ß-carotene tested (3 µmol/L) and as early as 24 h after treatment, intracellular ß-carotene was detected (Table 1
), an effect that was drastically different from any effect of dose or time for lycopene.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. The mode of action by which both carotenoids affect cell proliferation is unclear. Bertram et al. (24
) demonstrated a significant correlation between the ability of diverse carotenoids to inhibit chemically induced neoplastic transformation (25
) and their ability to stimulate junctional communication in 10T1/2 cells via increased levels of connexin 43 (26
–28
). It has previously been shown that increased levels of connexin 43 are associated with increased gap-junctional communication (29
). Moreover, Zhang et al. (30
) recently demonstrated that overexpression of connexin 43 is associated with suppressed proliferation of human osteosarcoma U2OS cells through inhibition of cell-cycle transition from the G1 to the S phase.
In the present study, the expression of connexin 43 in KB-1 cells was upregulated mainly after treatment with lycopene, and to a much lesser extent, with ß-carotene. These findings were confirmed by Western immunoblotting (Fig. 3)
and immunocytochemistry (Fig. 4)
. EM micrographs of KB-1 cells treated with lycopene (Fig. 6
C) showed many connections between neighboring cells and many points of close communication reminiscent of desmosomes, intermediate junctions and gap-junction communications. In contrast, in micrographs from cells treated with ß-carotene (Fig. 6
B), desmosomes and intermediate junctions were evident but no clear gap-junction-type structures were detected. EM micrographs of KB-1 cells incubated in medium without carotenoids (control) showed no clear communication, no signs of desmosomes, no intermediate junctions and no gap junctions between the cells (Fig. 6
A). In KB-1 cells, lycopene was demonstrated to stimulate cell-to-cell communication, as a function of the concentration. ß-Carotene was far less effective (Fig. 7
A–7
F). Similar data have been reported previously (31
); however, the effect was detectable at lower lycopene concentrations (0.1 µmol/L) than the concentrations used in the present study (3, 5 and 7 µmol/L), which are close to the physiologic levels detectable in basal human serum (
1 µmol/L) (32
). In supplemented people, serum lycopene can be doubled (33
).
We addressed the question of whether differences in proliferation inhibition and modulation of gap-junctional communication induced by both carotenoids are due to differences in the uptake of those carotenoids by the cells. We found that significant differences exist between lycopene and ß-carotene cell uptake. Very low concentrations of intracellular lycopene were detectable only after 48 h of incubation with the highest extracellular carotenoid concentration. In contrast, ß-carotene was detectable in large amounts within 24 h of incubation and continued to increase on subsequent days (Table 1
).
Carotenoid activity that takes place mainly within the extracellular domain appears to be more effective at inhibiting proliferation and regulation of cell-cell communication than that taking place mainly in the intracellular domain. The importance of these differences is being investigated in our laboratory. The present study adds to previous findings suggesting that lycopene is an efficient cancer preventive agent in oral carcinogenesis that deserves further investigation.
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FOOTNOTES |
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Manuscript received 4 May 2002. Initial review completed 12 June 2002. Revision accepted 9 September 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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