Toxicology in Vitro 20 (2006) 1098–1105 www.elsevier.com/locate/toxinvit 0887-2333/$ - see front matter  2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2006.01.020 Inhibitory eVect of anethole on T-lymphocyte proliferation and interleukin-2 production through down-regulation of the NF-AT and AP-1 Sung Su Yea a,b,c,¤ , Haeng-Soon Jeong a , Chun Yeon Choi a,b , Kyung-Ran Park a,b , Sangtaek Oh c , Jae-Gook Shin c , Chul-Ho Yun d a Department of Biochemistry, College of Medicine, Inje University, 633-165 Gaegum-dong, Busanjin-gu, Busan 614-735, Republic of Korea b Biohealth Product Research Center, Inje University, Busan 614-735, Republic of Korea c Pharmacogenomics Research Center, Inje University, Busan 614-735, Republic of Korea d School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea Received 23 November 2005; accepted 26 January 2006 Available online 13 March 2006 Abstract Anethole is a naturally occurring alkenylbenzene found in a variety of foods and essential oils. In the present study, we investi- gated the eVect of anethole on T-cell function and the regulatory mechanism of its eVect. Direct addition of anethole to B6C3F1 mouse splenocyte cultures produced a concentration-dependent inhibition of the lymphoproliferative response to concanavalin A stimulation. Anethole inhibited phorbol 12-myristate 13-acetate (PMA) plus ionomycin (Io)-induced interleukin-2 (IL-2) mRNA expression and protein secretion in EL4 mouse T-cells as determined by quantitative/competitive RT-PCR and ELISA, respectively. To further characterize the mechanism for the transcriptional regulation of IL-2, an electrophoretic mobility shift assay was per- formed to evaluate the binding activity of the nuclear factor of activated T-cells (NF-AT), activator protein-1 (AP-1), nuclear factor- B (NF-B), and octamer binding protein (Oct) in PMA/Io-stimulated EL4 cells. Anethole decreased the NF-AT and AP-1 binding activity, but no signiWcant eVect was observed on NF-B or Oct binding activity. These results suggest that anethole suppress T-cell proliferation and IL-2 production and that the inhibition is mediated, at least in part, through the down-regulation of NF-AT and AP-1.  2006 Elsevier Ltd. All rights reserved. Keywords: Anethole; Interleukin-2 (IL-2); Nuclear factor of activated T-cells (NF-AT); Activator protein-1 (AP-1) Abbreviations: PMA, phorbol 12-myristate 13-acetate; Io, ionomycin; IL-2, interleukin-2; RT-PCR, reverse transcription-polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; NF-AT, nuclear factor of activated T-cells; AP-1, activator protein-1; NF-B, nuclear factor-B; Oct, octamer binding protein; GRAS, generally recognized as safe; PHA, phytohemagglutinin; ConA, concanavalin A; DMSO, dimethylsulfoxide; FBS, fetal bovine serum; LPS, lipopolysaccharide; DTT, dithiothreitol; PMSF, phenyl-methylsulfonyl Xuoride. * Corresponding author. Address: Department of Biochemistry, College of Medicine, Inje University, 633-165 Gaegum-dong, Busanjin-gu, Busan 614- 735, Republic of Korea. Tel.: +82 51 890 6892; fax: +82 51 894 5801. E-mail address: ssyea@inje.ac.kr (S.S. Yea).