Interaction of foods with intestinal transporters has generally been ascribed to small molecules, but recently, edible-plant-derived nanoparticles (NPs) have been suggested to affect intestinal function. Here, we examined the effects of NPs contained in edible fruits on intestinal transporters. Apple-derived NPs (APNPs) were isolated by ultracentrifugation and characterized by measurement of particle size distribution and electron microscopy. Human epithelial colorectal adenocarcinoma (Caco-2) cells internalized fluorescently labeled APNPs, suggesting that fruit-derived NPs would be internalized into intestinal epithelial cells in vivo. We found that the mRNA expression levels of several transporters, including organic-anion-transporting polypeptide (OATP) 2B1, were changed in APNP-treated Caco-2 cells. The protein expression and activity of OATP2B1 were also decreased by APNP exposure, as determined by Western blotting and measurements of [ 3 H]estrone-3-sulfate uptake by Caco-2 cells, respectively. These actions required intact APNPs, because sonication or boiling abrogated the effects. Since the content of apple-derived small molecules in APNPs was negligible, the observed decrease of OATP2B1 expression appears to be mediated by large molecules in the APNPs. We further found that the 3′-untranslated region of the OATP2B1 gene was required for the response to APNPs, suggesting that microRNA in the APNPs might be involved. These results propose a novel mechanism, in which large molecules such as microRNA in food could affect intestinal transporters through food-derived NPs, which also demonstrates that food-derived NPs should be useful for delivery of biologically active large molecules to intestinal tissues.

Gastrointestinal toxicity, such as late-onset diarrhea, is a significant concern in irinotecan hydrochloride (CPT-11)-containing regimens. Prophylaxis of late-onset diarrhea has been reported with use of Japanese traditional (Kampo) medicine containing baicalin and with the antibiotic cefixime, and this has been explained in terms of inhibition of bacterial deconjugation of SN-38-glucuronide since unconjugated SN-38 (active metabolite of CPT-11) is responsible for the gastrointestinal toxicity. It is also prerequisite for SN-38 to be accumulated in intestinal tissues to exert toxicity. Based on the fact that liver-specific organic anion transporting polypeptide (OATP) 1B1, a member of the same family as OATP2B1, is known to be involved in hepatic transport of SN-38, we hypothesized that intestinal transporter OATP2B1 contributes to the accumulation of SN-38 in gastrointestinal tissues, and its inhibition would help prevent associated toxicity. We found that uptake of SN-38 by OATP2B1-expressing Xenopus oocytes was significantly higher than that by control oocytes. OATP2B1-mediated uptake of SN-38 was saturable, pH dependent, and decreased in the presence of baicalin, cefixime, or fruit juices such as apple juice. In vivo gastrointestinal toxicity of SN-38 in mice caused by oral administration for consecutive 5 days was prevented by coingestion of apple juice. Thus, OATP2B1 contributes to the uptake of SN-38 by intestinal tissues, triggering gastrointestinal toxicity. So, in addition to the reported inhibition of bacterial b-glucuronidase by cefixime or baicalin, inhibition of OATP2B1 may also contribute to prevention of gastrointestinal toxicity. Apple juice may be helpful for prophylaxis of late-onset diarrhea observed in CPT-11 therapy without disturbance of the intestinal microflora.

Schematic model of the relationship and locations of putative binding sites of substrates and modulators in OATP2B1. Drug–drug interaction and drug–food interaction on OATP2B1 can be predicted by clarification of multiple binding sites.

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Plant-derived nanoparticles exert cytoprotective effects on intestinal cells by delivering their cargo to intestinal tissues. We previously reported that apple-derived nanoparticles (APNPs) downregulate the mRNA of the human intestinal transporter OATP2B1/SLCO2B1 and that the 3'UTR region is required for the response to APNPs. Here, we investigated the involvement of microRNAs (miRNAs) in APNPs in suppressing OATP2B1 expression to demonstrate that APNP macromolecules directly interact with intestinal tissues. Using in silico analysis, seven apple miRNAs were predicted as candidate miRNAs that interact with the SLCO2B1-3'UTR. The APNP-mediated decrease in luciferase activity of pGL3/SLCO2B1-3'UTR was abrogated by inhibitors of mdm-miR-160a-e, -7121a-c, or -7121d-h. Each miRNA mimic reduced the endogenous expression of SLCO2B1 mRNA in Caco-2 cells. The luciferase activity of the truncated pGL3/SLCO2B1-3'UTR, which contains approximately 200 bp around each miRNA recognition element (MRE), was decreased by the miR-7121d-h mimic but little by the other mimics. APNP also reduced the luciferase activity of truncated pGL3/SLCO2B1-3'UTR containing an MRE for miR-7121d-h. Thus, we demonstrated that mdm-miR-7121d-h contributes to the APNP-mediated downregulation of intestinal OATP2B1. Accordingly, plant macromolecules, such as miRNAs, may directly interact with intestinal tissues via nanoparticles.