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Department of Food Science and Biotechnology, National Chung-Hsing University, Taichung, Taiwan, Republic of China and * Department of Food & Beverage Management, National Kaohsiung Hospitality College, Taiwan, Republic of China
2To whom correspondence should be addressed. E-mail: mlhuhu{at}dragon.nchu.edu.tw.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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870%). In contrast to the effects on migration and invasion, lycopene enhanced nm23-H1 expression at both the protein and mRNA levels; the effects were also bell shaped, and at 5 µmol/L, lycopene enhanced nm23-H1 protein and mRNA expressions by 220 ± 33 and 153 ± 22% (P < 0.01), respectively. These bell-shaped effects of lycopene may be related to autoxidation of lycopene at elevated concentrations (
10 µmol/L). Significant correlations existed between nm23-H1 protein expression and migration (r2 = 0.78, P < 0.001) and between nm23-H1 protein expression and invasion (r2 = 0.84, P < 0.001) in lycopene-treated SK-Hep-1 cells. We conclude that lycopene has significant antimigration and anti-invasion activity, and that this effect is associated with its induction of nm23-H1 expression.
KEY WORDS: • lycopene • migration • invasion • nm23-H1 • hepatocellular carcinoma cell
Tumor metastasis, the process by which tumor cells leave a primary tumor to colonize other sites of the body, is a major cause of death for cancer patients. Metastasizing cells must first disseminate from the primary tumor, invade the surrounding tissue, intravasate and extravasate the circulatory system, arrest, initiate angiogenesis, and colonize distant sites, while evading the immune system (1,2). The nm23 gene was first identified as a gene whose expression was reduced in highly metastatic rodent tumors relative to poorly metastatic tumor cells (3). It is located on chromosome 17q 21 and codes for an 18.5-kDa protein containing 166 amino acids with nucleoside diphosphate kinase and protein-histidine kinase activities, as well as serine auto phosphorylation activity (4,5). The transfection of nm23 cDNA into various cancer cell lines results in the suppression of metastatic potential of motility, invasion, or colonization (6–12), indicating that nm23 is a potential metastasis suppressor gene that could function on the invasion and migration steps of the metastatic pathway. Eight human nm23 genes have been characterized to date, of which the H1 gene is most closely correlated with the metastatic phenotype in human breast carcinoma, colorectal carcinoma, ovarian carcinoma, and hepatocarcinoma (13–19).
Carotenoids including ß-carotene and lycopene possess several common biological functions such as photoprotection, antioxidant effects, and immunomodulatory and anticancer activity (20,21). Elevated intakes of lycopene have been associated with lowered risk of several types of cancer (22–24). Studies suggested that the anticancer effects of carotenoids such as lycopene are related to their effectiveness as antioxidants, singlet oxygen quenchers, and free radical scavengers (25–30). However, the mechanisms by which lycopene decreases the risk of cancer are not well understood.
Kozuki et al. (31) suggested that the antioxidative property of lycopene may partly explain its anti-invasive action. By contrast, Collins (32) indicated that the antioxidant property of carotenoids is not related to their anticancer ability. Recently, several studies showed that carotenoids affect the transcription of various genes, such as connexin 43 (32–34). In the present study, we hypothesized that lycopene may exert its antimetastatic effects through upregulation of an antimetastatic gene, nm23-H1. Because liver cancer is the most endemic cancer in Taiwan and in much of the world, we employed a highly invasive hepatocarcinoma, SK-Hep-1 cells, to examine the effects of lycopene on cell migration and invasion and the possible mechanisms underlying such actions.
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MATERIALS AND METHODS |
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Cell culture and lycopene incorporation.
SK-Hep-1 cells were grown in DMEM medium containing 10% (v:v) FBS, 0.37% (wt:v) NaHCO3, penicillin (100 kU/L), streptomycin (100 kU/L) in a humidified incubator under 5% CO2 and 95% air at 37°C. The cells were harvested at 90% confluence (106 cells/dish). The survival rate of cells was always >95% by Trypan-blue assay (35). A stock THF-lycopene solution (20 mmol/L) was freshly prepared before each experiment, and the concentration of the stock solution was always 19 mmol/L, as determined using an extinction coefficient of 1.85 x 105 (mol/L)–1 cm–1 at 472 nm after 1:104 dilution in THF. The purity of commercial lycopene was 97%, which compares well with the 98% purity claimed by the supplier (Wako). THF-lycopene was added to the culture medium at a final concentration of 1, 2.5, 5, 10, or 20 µmol/L. The final concentration of THF in the culture medium was 0.1% (v:v, 1.2 µmol/L), which did not affect the assays described below. SK-Hep-1 cells (106 cells/dish) were incubated with THF-lycopene at 37°C in the dark for 2 h, as described in other cell lines (36,37). The cells were than washed 3 times in PBS (pH7.4).
MTT assay. The effect of lycopene on cell viability was estimated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenol tetrazolium bromide (MTT) assay, as described previously (38). Cells were cultured in 24-well plates at 1 x 104 cells/well in DMEM for 24 h, and each well was washed and then filled with 1 mL of DMEM containing various concentrations of lycopene and incubated for 2 h at 37°C, washed twice in PBS, and then incubated in DMEM for 24 h to observe cytotoxicity, if any (the longest incubation time for various assays employed in this study was 24 h). Each well was then incubated with MTT for 1 h, after which the liquid was removed, and dimethyl sulfoxide was added to dissolve the solid residue. The optical density at 570 nm of each well was then determined using a microplate reader (FLUOstar OPTIMA, BMG Labtechnologies).
Cell migration assay. Tumor cell migration was assayed in transwell chambers (Costar) according in the methods reported by Repesh (39) with some modifications. Briefly, transwell chambers (Costar) with 6.5-mm polycarbonate filters of 8-µm pore size were used. After preincubation with lycopene or ß-carotene for up to 24 h, Sk-Hep-1 cells (5 x 108/L) were finally suspended in DMEM (100 µL, serum free), placed in the upper transwell chamber, and then incubated for 5 h at 37°C. Then, the cells on the upper surface of the filter were completely wiped away with a cotton swab. The cells on the lower surface of the filter were fixed in methanol, stained with Giemsa, and counted under a microscope. For each replicate, the tumor cells in 10 randomly selected fields were determined, and the counts were averaged.
Cell invasion assay. The procedure reported by Repesh (39) for the cell invasion assay was similar to that for cell migration. The invasion of tumor cells was assessed in transwell chambers with a 6.5-mm-diameter polyvinyl/pyrrolidone–free polycarbonate filter of 8-µm pore size. Each filter was coated with 100 µL of a 1:20 diluted matrigel in cold DMEM to form a thin continuous film on the top of the filter. The number of cells was adjusted to 5 x 108/L and a 100-µL aliquot containing 5 x 104 cells was added to each of triplicate wells in DMEM containing 10% FBS. After incubation for 24 h, cells were stained and counted as described above, and the number of cells invading the lower side of the filter was measured as invasive activity.
Western blotting. Expression levels of endogenous nm23-H1 protein were determined by immunoblotting. Briefly, the medium was removed and cells were lysed with 20% SDS containing 1 mmol/L phenylmethylsulfonyl fluoride. The lysate was sonicated for 30 s on ice, followed by centrifugation at 12,000 x g for 30 min at 4°C. An amount of protein (40 µg) from the supernatant was resolved by SDS-PAGE and transferred onto a nitrocellulose membrane. After blocking with TBS buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.4) containing 5% nonfat milk, the membrane was incubated with anti-nm23-H1 monoclonal antibody (BD Biosciences) followed by horseradish peroxidase-conjugated anti-mouse IgG, and then visualized using an ECL chemiluminescent detection kit (Amersham).
RT-PCR (RNA isolation and sequencing). Total cellular RNA was isolated from cell culture (RNAzol-kit), reverse-transcribed into cDNA (MMLV-Reverse Transcriptase, Gibco/BRL) using oligo (dT)15 as primers, and then coamplified with 4 primer bases on nm23-H1 and ß-actin (internal control) sequences. The primers for amplifying nm23-H1 cDNA were 5'-CTGCGAACCACGTGGGT-3', located in the 5'-untranslated region, and 5'-TCGGGGATGGTAACACTGTA-3', located in the 3' untranslated region. The primers for amplifying ß-actin cDNA were 5'-GTGGGGCGCCCCAGGCACCA-3' and 5'-CTCCTTAATGTCACGCACGATTTC-3'. PCR amplification was performed with a thermal cycler, as follows: denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 90 s, followed by a final incubation at 72°C for 7 min. The optimal number of cycles was 29. The sizes of the amplification products of nm23-H1 and ß-actin were 702 and 541 bp, respectively. The PCR products were subjected to 1% agarose gel electrophoresis and stained with ethidium bromide. The relative nm23-H1 levels were quantitated by Matrox Inspector 2.1 software.
Statistical analysis. Values are expressed as means ± SD and analyzed using 1-way ANOVA followed by Duncan’s multiple range test for comparisons of group means. Differences were considered significant at P < 0.05.
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RESULTS |
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120 and 870%, respectively, compared with ß-carotene).
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-tocopherol.
Because the antimigration and anti-invasion effects of 10 µmol/L lycopene were lower than those of 5 µmol/L lycopene, we suspected that autoxidation of lycopene might have occurred at elevated concentrations of lycopene (10 µmol/L). Indeed, we found that a combination of 10 µmol/L lycopene with 10 µmol/L -tocopherol during the 2-h incubation with SK-Hep-1 cells reduced the oxidation of lycopene from 35 to 12% (data not shown) and that the combined incubation significantly increased the antimigration and anti-invasion ability and the expression of nm23-H1 induced by 10 µmol/L lycopene, whereas -tocopherol itself at 10 µmol/L had no effect (Table 1).
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DISCUSSION |
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870%). These findings indicate that lycopene is a potent antimetastatic nutrient. Our results agree with those of Kozuki et al. (31), who showed that lycopene inhibits the invasion of rat ascites hepatoma AH109A cells in a dose-dependent manner up to 5 µmol/L. Rooprai et al. (41) also showed that lycopene is able to reduce the motility of brain tumor cells. Although Okajima et al. (29) found no significant effects of lycopene on the invasion of rat urinary bladder transitional cell carcinomas, it should be noted that these authors used tomato juice rather than pure lycopene. The decrease in the metastasis-associated phenotypes, such as cell migration and cell invasion, induced by lycopene treatment may be mediated in part by its upregulation of nm23-H1 expression because these effects are highly correlated. Moreover, the expression of nm23-H1 protein was similar to that of nm23-H1 mRNA, indicating that induction of nm23-H1 protein in response to lycopene may rely entirely on upregulation at the transcriptional level.
An interesting observation of the present study is that the effects of lycopene on cell migration, invasion, and the expression of nm23-H1 (at both the protein and mRNA levels) were all bell-shaped; i.e., the effects were all lower at 10 µmol/L lycopene than at 5 µmol/L. In agreement with the present finding, carotenoids including ß-carotene and lycopene were shown to have lowered effectiveness as antioxidants and anticarcinogens in vitro at concentrations > 10 µmol/L (42,43). A possible explanation for the bell-shaped effects is that the antioxidant activity of ß-carotene may shift into a prooxidant activity, depending on carotenoid concentration inside the cells and on the oxygen tension of the biological environment as well as on cell redox status (44). Here, our results suggest that lycopene at concentrations 10 µmol/L may undergo more rapid autoxidation and lead to decreased effects because concomitant incubation of SK-Hep-1 cells with -tocopherol and lycopene (both at 10 µmol/L) protected against lycopene autoxidation and increased the anti-invasion, antimigration and nm23-H1-enhancing effects of lycopene. These results appear to support the notion that the antimetastatic effects are also related to the antioxidant properties of lycopene (29–31,45). However, this issue is unsettled because ß-carotene, a carotenoid similar to lycopene, inhibits only cell migration, not cell invasion.
The concentrations of lycopene (1–5 µmol/L) used in the present study are relatively high compared with plasma lycopene concentrations in humans (50–900 nmol/L) (22,23,46). However, plasma levels of lycopene can increase markedly with lycopene supplementation. For instance, Edwards et al. (47) reported an increase in plasma lycopene from 428 to 960 nmol/L over 3 wk using 18.4 mg/d lycopene from 240 g (1 cup/d) of tomato juice. In addition, pure lycopene was reported to be 3 times as bioavailable to humans as lycopene from steamed and pureed tomatoes (48). Thus, it appears that the concentrations of lycopene used in our study are not exceedingly high and may be reached in vivo by lycopene supplementation.
In summary, we demonstrated that lycopene has significant antimigration and anti-invasion activities against SK-Hep-1 cells, and that this effect is associated with its induction of nm23-H1 expression. Further studies are warranted to verify the in vivo relevance of these findings.
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FOOTNOTES |
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3 Abbreviations used: FBS, fetal bovine serum; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenol tetrazolium bromide; THF, tetrahydrofuran.
Manuscript received 12 March 2005. Initial review completed 29 April 2005. Revision accepted 27 May 2005.
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