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 (+)-, ()-, 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 ()-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 ()-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, ()-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 ()-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 ()-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. ()-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. T47D-KBluc cells were used in the assay of estrogen-like 30 min) at room temperature. Luciferase assay was performed using a activity of SECO compounds. T47D-KBluc cells were established luciferase reporter gene assay kit (Promega). Luciferase activity was by transfection of a triplet ERE (estrogen-responsive element)- determined by Luminessencer-JNR AB 2100 (ATTO, Tokyo), and promoter-luciferase reporter gene construct in T47D human breast quantified as relative luciferase units (RLU). cancer cells naturally expressing estrogen receptors (ERs) and .13) These cells express lucifarase by stimulation of potent estrogen or well-characterized weaker environmental estrogens. T47D-KBluc Results cells were subcultured in ERDF medium supplemented with 10% of FBS at 37 C in a humidified atmosphere of 5% CO2. The Effects of SECOs on adiponectin production by 3T3- culture medium was replaced with ERDF medium with 10% charcoal- L1 adipocytes treated FBS 7 d before the experiments to eliminate trace estrogens 3T3-L1 adipocytes were stimulated with SECO in FBS. compounds in 10% FBS-ERDF medium for 2 d. As shown in Fig. 1, only ()-SECO accelerated adiponec- Assay of adiponectin production of 3T3-L1 adipocytes. 3T3-L1 tin production by 3T3-L1 adipocytes 1.6-fold. In adipocytes were cultured in 10% FBS-ERDF medium supplemented contrast, (+)- and meso-SECO depressed adiponectin with the SECO compounds at 0.3 mM. Two d later, the amount of adiponectin secreted in culture medium was determined with a mouse production. These facts indicate that differences in the adiponectin ELISA kit. structures of SECO compounds have different influences on adiponectin production. Reverse transcription-polymerase chain reaction (RT-PCR) analy- sis. After 3T3-L1 adipocytes were differentiated, the cells were Effects of SECOs on mRNA expression of adiponectin cultured in 10% FBS-ERDF medium supplemented with each SECO To determine the mode of regulation of the various for 2 d. They were harvested and washed twice with PBS. Total RNA SECOs on adiponectin production, the mRNA levels of was prepared using Sepasol RNA-I super (Nacalai Tesque, Kyoto, adiponectin in 3T3-L1 cells treated with the various Japan) according to the manufacturer’s instruction. One microgram of SECOs was examined. As shown in Fig. 2, ()-SECO total RNA was used as a template for the cDNA synthesis reaction the up-regulated the expression of the adiponectin mRNA performed using MMLV-reverse transcriptase and an oligo-dT primer level. On the other hand, (+)- and meso-SECO down- (Toyobo, Osaka, Japan). The resulting cDNA samples were subjected to 30 cycles of PCR amplification performed under the following regulated the expression level. This result was correlated conditions: denaturation at 98 C for 10 s, annealing at 60 C for 2 s, with the adiponectin production levels of 3T3-L1 cells and DNA synthesis at 74 C for 30 s, according to the manufacturer’s treated with the various SECOs. In both cases, SECO recommendations. The PCR reaction was performed using Takara Taq affected the mRNA expression level, regulating the DNA polymerase (Takara, Kyoto, Japan). Mouse -actin cDNA was adiponectin production of 3T3-L1 cells. Effect of Flaxseed Lignan Secoisolariciresinol on Adipogenesis 37 200 A

** 150

100

Control 50 ** Relative adiponectin production (%)

0 Control (+)–SECO (–)–SECO meso –SECO ** p < 0.01 vs control (+)–SECO (–)–SECO Fig. 1. Effects of SECOs on the Adiponectin Production by 3T3-L1 Adipocytes. 3T3-L1 adipocytes were differentiated and cultured in 10% FBS- ERDF medium supplemented with the various SECO compounds at 0.3 mM, and incubated. The control was supplemented with dimethylsulfoxide (DMSO) instead of SECOs. After 2 d, adiponectin secreted in the culture medium was measured with a mouse adiponectin ELISA kit. The data were indicated as relative adiponectin production against control level, and expressed the mean of the four independent measurements. Statistical significance meso –SECO E2 was analyzed by Student’s t test. B β –actin Adiponectin Control + Control + 120 (+)–SECO 100 (–)–SECO meso –SECO 80 Fig. 2. Effects of SECOs on mRNA Expression of Adiponectin by RT-PCR. 60 *** Total RNA was isolated from 3T3-L1 adipocytes treated with *** 0.3 mM SECOs for 2 d and subjected to reverse transcription-PCR. *** -actin mRNA was analyzed as internal control. 40 Relative TG production (%) Effects of SECOs on triglyceride accumulation in 20 3T3-L1 adipocytes *** Differentiation was induced with 10% FBS-ERDF 0 with 5 mg/ml of insulin, 0.25 mM DEX, 0.5 mM IBMX, Control (+)–SECO (–)–SECOmeso –SECO E2 and 0.15 mM the various SECOs added to the medium at *** p < 0.005 vs control the initiation of differentiation. Two days after induc- tion, the medium was changed to adipogenesis pro- Fig. 3. Effects of SECOs on Triglyceride Accumulation in 3T3-L1 gression medium: 10% FBS-ERDF medium supple- Adipocytes. mented with 5 mg/ml of insulin and 0.15 mM SECOs or A, Observation of the amount of lipid droplets. 3T3-L1 E2, and the medium was changed every 2 d. As shown in preadipocytes were grown to confluence in a 48-well culture plate in 10% FBS-ERDF medium and differentiated as to coexistence Fig. 3A, all of SECOs and E2 significantly suppressed with each SECO compound at 0.15 mM. The control cells were TG accumulation. Especially, ()-SECO obviously supplemented with DMSO. Two weeks after differentiation, 3T3-L1 suppressed TG accumulation, and the oil droplets were preadipocytes were observed using a Nikon DIAPHOT-TMD phase much smaller than those in cells treated with the other contrast microscope (magnification, 40) (Nikon, Tokyo). B, Quantitative analysis of TG in 3T3-L1 adipocytes. 3T3-L1 adipo- SECOs or E2. In addition, no cytotoxicity of these cytes were stained with Oil Red O, and TG level was analyzed by substances was observed at this concentration, and there measuring OD at 540 nm. Data were expressed as the means SD were no differences in the rate of differentiation to ðn ¼ 4Þ. Statistical significance was analyzed by Student’s t test. adipocytes among these substances. Quantitative analysis of TG content in 3T3-L1 adipocytes treated with SECO and E2 was performed ()-SECO. To determine the structure-activity relation- by oil red O assay. As indicated in Fig. 3B, TG ship of SECO in estrogen activity, the estrogen activities accumulation in 3T3-L1 cells stimulated with ()- of the SECO compounds were compared. Estrogen- SECO was obviously lower than in control cells. responsible lusiferase reporter gene assay using T47D- KBluc cells was performed. As indicated in Fig. 4, only Estrogen-like activity of SECOs ()-SECO possessed estrogen activity, but the activity It is well known that SECO has estrogen activity, but was much lower than that of E2. The specific estrogen the activity was evaluated using a mixture of (+)- and activity of ()-SECO was less than 1/1,000 that of E2. 38 S. TOMINAGA et al.

18,000 is stimulated by E2 and estrogen-mimicking com- pounds.19,20) It is concluded from these facts that ()- 0.1 nM E2 SECO has estrogen-like activity and that it stimulates 15,000 the cell growth of MCF-7 cells via the estrogen *** receptor. In fact, ()-SECO alone had estrogen-like 12,000 activity among SECO isomers, as indicated in Fig. 4. ** Our data indicate that E2 suppressed TG accumulation in 3T3-L1 cells. ()-SECO suppressed TG accumula- 9,000 tion more strongly than E2. Matured 3T3-L1 cells meso –SECO express estrogen receptor (ER) and , and ER Control 21) 6,000 (+)–SECO regulates insulin sensitivity. ERs have to do with the Relative luciferase unit regulation of TG accumulation in 3T3-L1 adipocytes. (–)–SECO However, (+)- and meso-SECO also suppressed TG 3,000 production without estrogen activity. This suggests that although beneficial effects on lipid metabolism in 3T3- 0 L1 cells are partly due to the estrogen-like activity of 0.010.1 1 10 100 ()-SECO, there are other pathways besides estrogen- Sample conc. (µM) like activity for depressing TG accumulation by ()- ** p < 0.01 , *** p < 0.005 vs control SECO. In addition, the concentration of ()-SECO in the suppression of TG accumulation was significantly Fig. 4. Estrogen-Like Activity of SECOs. lower than that in the stimulation of adiponectin T47D-KBluc cells were seeded at 104 cells per well in 96-well production. It is concluded that there are different plates and cultured in 10% FBS-ERDF medium with various pathways for adipogenesis in 3T3-L1 adipocytes in- concentrations of SECO compounds and 0.1 nM E2 as positive control. Control cells were supplemented with DMSO. Luciferase duced by ()-SECO. assay was performed using a luciferase reporter gene assay kit. 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