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* Graduate School of Pharmaceutical Sciences, Chiba University, Japan and Central Research Laboratories, Wakunaga Pharmaceutical Company, Japan
3 To whom correspondence should be addressed: E-mail: horieto{at}p.chiba-u.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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KEY WORDS: • aged garlic extract • methotrexate • IEC-6 cells • intestinal damage
Methotrexate (MTX)4 is a folate antagonist used not only as an antitumor drug but also as an antirheumatoid drug. The use of MTX in cancer chemotherapy is often limited by various side effects, including vomiting, diarrhea, mucositis, and decrease of nutrient absorption (1). The toxic effects are not considered to be a result of direct action on the gastrointestinal tract tissues but rather to be the consequence of an inhibition in dihydrofolate reductase synthesis (2). This enzyme is required to maintain the intracellular pool of tetrahydrofolate during purine and thymidine synthesis. It affects not only tumor cells but also rapidly dividing host cells such as crypt cells of the gastrointestinal mucosa. It is characterized histologically by villus atrophy or crypt loss (3). MTX treatment decreases the surface area of the small intestine because of the damaged and shortened villi, components of the small intestinal mucosa like proteins and lipids, and the number of crypt cells. Cellular edema and bleb formation occur during treatment (4). The treatment also changes the physical structure of brush-border membranes (5).
The chemical and morphologic changes in the small intestine may be triggered by crypt cell damage (6). To minimize the side effects in patients undergoing chemotherapy, it is important to reduce mucosal damage and stimulate tissue repair (6). Such intestinal damage is reported to be prevented by some kinds of nutrients and growth factors. For example, keratinocyte growth factor (7) and insulin-like growth factor-1 (8) stimulate regrowth of the damaged intestine and protect mice from gastrointestinal injury. We have also demonstrated a protective effect of retinol (5), docosahexaenoic acid (9), and synthetic analogs of prostaglandin E1 (4,10) on the MTX-induced damage of the small intestine.
Garlic derivatives have various biologic properties such as antimicrobial and antithrombotic activities, immune system enhancement, and antitumor potential (11). Aged garlic extract (AGE) and its constituents prevent oxidative injury in endothelial cells (12) and suppress cancer growth (13). AGE also protects the small intestine of rats from MTX-induced damage (14). Thus, it has been suggested that AGE has different effects on tumor cells and normal intestinal cells.
MTX induces apoptosis in the small intestine (15). We found that MTX induced apoptosis of IEC-6 cells, an immortalized epithelial cell line derived from neonatal rat ileum. In the present study, we investigated the effect of AGE on MTX-induced cytotoxicity, using IEC-6 cells, and showed that AGE inhibits the MTX-induced apoptosis in IEC-6 cells.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Cell culture. IEC-6 was obtained from American Type Culture Collection. IEC-6 cells were grown in DMEM (Sigma) containing 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, 5% fetal bovine serum (FBS), 100 units/L bovine insulin (Wako Pure Chemical Industries), and 20 mg/L gentamicin sulfate (Nacalai Tesque). Cells were incubated at 37° in 5% CO2 and 95% air. Culture media were changed every 2 d.
MTT assay. The viability of IEC-6 cells was determined by the 3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, IEC-6 cells were plated on a 96-well multiplate and treated with MTX in the presence or absence of AGE for 72 h. MTT solution (5mg/mL, 1:10) was added to each well. Following 4 h of incubation at 37°C, the produced formazan was dissolved with acid-isopropanol solution (0.04 N HCl/isopropanol). The absorbance at 570 nm (reference at 630 nm) was determined by a microplate reader Multiskan JX (Themo LabSystems).
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RESULTS AND DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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IEC-6 cells were treated with MTX (0.01–100) for 72 h (Fig. 1). The viability of IEC-6 cells decreased with the increase of MTX concentration. The MTX-induced loss of viable IEC-6 cells was prevented by the presence of 0.5% AGE (Fig. 2). We have recently found in chromatin condensation, DNA fragmentation, caspase-3 activation, and cytochrome c release in IEC-6 cells with MTX. These changes were returned to the control levels by the presence of AGE (unpublished results). Thymidine incorporation into IEC-6 cells incubated with MTX for 24, 48, and 72 h was markedly increased (Fig. 3). This finding suggested that the salvage pathway that used the added thymidine contributed to the increase of thymidine incorporation, because MTX inhibited the de novo pathway of thymidylate synthesis. The presence of AGE in IEC-6 cells with MTX suppressed the increase of thymidine incorporation, which suggested that AGE prevented the MTX-induced inhibition of dihydrofolate reductase and/or activated the de novo pathway, resulting in suppression of the requirement of extracellular thymidine.
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FOOTNOTES |
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2 Author disclosure: No relationships to disclose.
4 Abbreviations used: AGE, aged garlic extract; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide; MTX, methotrexate.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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1. Jolly LE, Fletcher HP. The effect of repeated oral dosing of methotrexate on its intestinal absorption in the rat. Toxicol Appl Pharmacol. 1977;39:23–32.[Medline]
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15. Verburg M, Renes IB, Meijer HP, Taminiau JA, Buller HA, Einerhand AW, Dekker J. Selective sparing of goblet cells and paneth cells in the intestine of methotrexate-treated rats. Am J Physiol Gastrointest Liver Physiol. 2000;279:G1037–1047.
16. Santos MF, McCormack SA, Guo Z, Okolicany J, Zheng Y, Johnson LR, Tigyi J. Rho proteins play a critical role in cell migration during the early phase of mucosal restitution. J Clin Invest. 1997;100:216–25.[Medline]
17. Li T, Ito K, Horie T. Transport of fluorescein methotrexate by multidrug resistance-associated protein 3 in IEC-6 cells. Am J Physiol Gastrointest Liver Physiol. 2003;285:G602–610.
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