Biosci. Biotechnol. Biochem., 73 (1), 35–39, 2009 The Effect of Secoisolariciresinol on 3T3-L1 Adipocytes and the Relationship between Molecular Structure and Activity y Shiori TOMINAGA,1 Takuya SUGAHARA,1;2; Sogo NISHIMOTO,1;3 Manami YAMAWAKI,1 Yuki NAKASHIMA,1 Taro KISHIDA,1;2 Koichi AKIYAMA,4 Masafumi MARUYAMA,1 and Satoshi YAMAUCHI1;2 1Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan 2South Ehime Fisheries Research Center, Ehime University, 1289-1 Funakoshi, Ainan, Ehime 798-4292, Japan 3Center for Marine Environmental Studies (CMES), Ehime University, 2-5 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan 4Integrated Center for Sciences, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan Received June 12, 2008; Accepted October 6, 2008; Online Publication, January 7, 2009 [doi:10.1271/bbb.80393] As we have reported, flaxseed lignan, (+)-secoisolar- In our previous studies,5) stereoisomeric forms of iciresinol (SECO), (À)-SECO, and meso-SECO were SECO, such as (+)-, (À)-, and meso-SECO, were stereoselectively synthesized and their biological func- synthesized, and several biological activities were tions were evaluated. In the present study, we focused compared to determine the structure-activity relation- on the effects of SECOs on the regulation of 3T3-L1 ships of SECO. First, the immunostimulatory activity of adipocytes, and identified the structure-activity relation- SECO compounds was examined, and then IgM pro- ships. Optically active SECO and meso-SECO were duction stimulatory activity toward human hybridoma tested for their effects on lipid metabolism in 3T3-L1 HB4C5 cells was assessed. The optically active com- adipocytes. (À)-SECO accelerated adiponectin produc- pounds, (+)-SECO and (À)-SECO, stimulated IgM tion of 3T3-L1 adipocytes. On the other hand, (+)- and production of HB4C5 cells. These two compounds meso-SECO suppressed the production of adiponectin. accelerated IgM production by about 2-fold, but meso- In addition, triglyceride (TG) accumulation in 3T3-L1 SECO, a diastereomer of (+)- and (À)-SECO, had adipocytes was significantly suppressed by all three no effect on IgM production. This result means that SECOs tested here, as was 17 -estradiol, when the the molecular configuration of the SECO compounds SECOs were added to the medium during induction of was important in the stimulation of immunoglobulin 3T3-L1 preadipocytes to adipocytes. Especially, (À)- production of HB4C5 cells. Moreover, a stereoselective SECO strongly reduced TG accumulation. It is well- synthesis of the SECO-related compounds was impor- known that SECO has estrogen-like activity. Hence the tant in precisely evaluating their biological functions. estrogen-like activity of each SECO compound was The growth-inhibitory effects of SECOs on cancer assessed. Only (À)-SECO had estrogen-like activity. cell lines were examined. meso-SECO weakly sup- pressed the cell growth of mouse colon adenocarcinoma Key words: adipogenesis; flaxseed lignan; secoisolari- colon-26 cells and human breast cancer MCF-7 cells in a ciresinol dose-dependent manner. On the other hand, (À)-SECO stimulated cell growth at 5 mM and suppressed it at more Secoisolariciresinol (SECO) is one of the most than 100 mM, while (+)-SECO did not affect cell activity abundant dietary lignans. It occurs predominantly in a toward these cancer cell lines. This indicates that the glycosylated form in food products.1) SECO is the major effect of SECO compounds is due not only to types of lignan found in flaxseed (Linum usitatissimum L.), and it substituents, but also to molecular configurations. Diet- is present in a polymer containing secoisolariciresinol ary lignans belong to a group of naturally occurring diglucoside (SDG). SECO, SDG, and the polymers are plant compounds called phytoestrogens, and their action known to have a number of health benefits, including is at least partly attributable to their capability to bind reduction of serum cholesterol levels, delay of the onset to estrogen receptors.6) SECO is also a phytoestrogen, of type II diabetes, and decreased formation of breast, structurally similar to the hormone estrogen, and it prostate, and colon cancers.2) Research on the isolation might exert estrogenic effects in the body.7) On the other and synthesis of SECO compounds is in progress by hand, MCF-7 cells have an estrogen receptor, and organic chemists, but the relationship between molecular their growth is stimulated by 17 -estradiol and by structure and biological activity has not been elucidated. estrogen-mimicking compounds. It is assumed that only On the other hand, meso-SECO is not common, and (À)-SECO associated with estrogen receptors on the there have been only two reports on its isolation.3,4) MCF-7 cells and stimulated cell growth, and that (+)- There is no report on stereoselective synthetic research SECO did not associate or did not have estrogen-like and biological investigation of meso-SECO. activity. y To whom correspondence should be addressed. Tel/Fax: +81-89-946-9863; E-mail: [email protected] Abbreviations: DEX, dexamethasone; DMSO, dimethylsulfoxide; instead E2, 17 -estradiol; ELISA, enzyme-linked immunosorbent assay; ER, estrogen receptor; FBS, fetal bovine serum; IBMX, isobutyl-methylxanthine; SDG, secoisolariciresinol diglucoside; SECO, secoisolariciresinol; TG, triglyceride 36 S. TOMINAGA et al. In this study, the effects of three SECOs on adipo- amplified as an internal control. The PCR primer sequences were as genesis were compared using mouse 3T3-L1 adipocytes. follows: for mouse -actin, sense, 50-TGG AAT CCT GTG GCA TCC 3T3-L1 cells are derived from the 3T3 Swiss albino ATG AAA C-30, and antisense, 50-TAA AAC GCA GCT CAG TAA 0 0 mouse cell line, and are used as an in vitro model of CAG TCC G-3 ; for mouse adiponectin, sense, 5 -AGT GGA TCT GAC GAC ACC-30, and antisense, 50-CTG TCA TTC CAA CAT CTC adipocytes to study insulin pathways, obesity, and 0 0 8) C-3 ; and for mouse PPAR , sense, 5 -GCT GTT ATG GGT GAA cardiovascular diseases. ACT CTG-30, and antisense, 50-ATA AGG TGG AGA TGC AGG TTC-30. The amplified PCR products were subjected to electrophoresis Materials and Methods on a 1.0% agarose gel, and were stained using SYBR Gold (Molecular Probes, Eugene, OR). Reagents. Insulin, dexamethasone (DEX), and isobutyl-methylxan- thine (IBMX) were purchased from Sigma (St. Louis, MO). The lipid Oil red O staining. 3T3-L1 preadipocytes were grown to confluence assay kit for the determination of triglyceride (TG) accumulation in in a 48-well culture plate in ERDF medium supplemented with 10% adipocytes was purchased from Primary Cell (Ishikari, Japan). The FBS. Differentiation was induced with 10% FBS-ERDF with 5 mg/ml luciferase reporter gene assay kit was purchased from Promega of insulin, 0.25 mM DEX, and 0.5 mM IBMX, and the SECOs and E2 (Wisconsin, WI). The mouse adiponectin enzyme-linked immunosorb- were added at 0.15 mM at the initiation of differentiation. At day 2 after ent assay (ELISA) kit was from CircuLex (Ina, Japan). induction, the medium was changed to adipogenesis progression medium: 10% FBS-ERDF medium supplemented with 5 mg/ml of SECO compounds. (+)- and (À)-SECO were synthesized by insulin and 0.15 mM SECOs without DEX or IBMX, and the medium previously reported methods.9) (+)-SECO: colorless crystals, mp, was changed every 2 d. 10) 111–112 C (CHCl3-(iso-Pr)2O), 114–115 C in the literature, Two weeks after inoculation, oil red staining performed using a 20 ½ D ¼þ31 (c 0.3, acetone). NMR data agreed with those in the lipid assay kit (Primary Cell; Ishiari Japan) according to the 10) literature. (À)-SECO: mp, 111–112 C (CHCl3-(iso-Pr)2O). manufacturer’s instructions. Briefly, oil red O dissolved in isopropanol 20 11) 20 ½ D ¼32 (c 0.3, acetone). Lit., ½ D ¼35:5 (c 1.07, was kept overnight at room temperature, filtered, mixed with distilled acetone). The purity of these compounds was checked by silica gel water, kept overnight in the cold, and finally filtered twice before use. TLC (EtOAc/hexane = 1/1), and HPLC analysis by DAICEL chiral The final staining solution was 0.2% oil red O in 60% isopropanol. The column AD-H revealed that the optical purity of each SECO was more cells were washed twice with PBS and fixed for at least 15 min with than 95%ee.9) meso-SECO was also synthesized by a method fixing buffer containing formalin. The cells were washed twice with previously reported.5) PBS, then stained for 15 min in a filtered oil red O solutions. After washing with distilled water, the stained cells were observed under a Cells and cell culture. 3T3-L1 preadipocytes obtained from ATCC microscope. Following microscopic observation, they were lysed with were subcultured in ERDF medium (Kyokuto Pharmaceuticals, Tokyo) lysis buffer and TG accumulation was measured by the absorbance at supplemented with 10% fetal bovine serum (FBS) at 37 Cina 540 nm. humidified atmosphere of 5% CO2. Differentiation of 3T3-L1 preadipocytes into adipocytes was initiated by the addition of Assay of estrogen-like activity. T47D-KBluc cells were seeded at 4 0.25 mM DEX, 0.5 mM IBMX, and 5 mg/ml of insulin to the culture 10 cells per well in 96-well plates and cultured in 10% FBS-ERDF medium of confluent cells for 2 d,12) followed by cultivation of cells medium with various concentrations of SECO compounds and 0.1 nM without DEX or IBMX until differentiation. The media were changed 17 -estradiol (E2).13) After 48 h, the cells were washed twice with every 2 d. PBS, and then lysis buffer (Promega, Madison, WI) was added at 20 ml Estrogen-responsive cell line T47D-KBluc cells were obtained from per well and the culture plate was incubated until cells were lysed (15– ATCC.