Inhibitory effects of oxyresveratrol and cyanomaclurin on adipogenesis of 3T3-L1 cells

journal of functional foods 15 (2015) 207–216

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Inhibitory effects of oxyresveratrol and cyanomaclurin on adipogenesis of 3T3-L1 cells Hui-Yuan Tan, Iris M.Y. Tse, Edmund T.S. Li, Mingfu Wang * School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, China

A R T I C L E

I N F O

A B S T R A C T

Article history:

Many phenolics are known to possess anti-obesity properties through mechanisms that inhibit

Received 23 September 2014

adipogenesis, stimulate lipolysis or induce apoptosis. This study aimed to examine the anti-

Received in revised form 19 January

adipogenic effects of two phenolics, oxyresveratrol and cyanomaclurin in 3T3-L1 cells. At

2015

doses that they did not induce cytotoxicity (based on the lactate dehydrogenase assay), the

Accepted 5 March 2015

exposure of oxyresveratrol (0–100 µM) or cyanomaclurin (0–600 µM) in the first three days

Available online

of differentiation led to a dose-dependent decrease of triacylglycerol accumulation on day 9 post-confluent. Results of cell proliferation and cell cycle study demonstrated that both

Keywords:

compounds inhibited differentiation through inducing cell cycle arrest by retaining

Oxyresveratrol

the preadipocytes in the G1 phase during the first two days post-confluent. Their anti-

Cyanomaclurin

adipogenic properties were also attributed to the down-regulation of protein expression of

Adipogenesis

major transcriptional factors in adipogenesis, including peroxisome proliferator-activated

3T3-L1 cells

receptor γ (PPAR γ) and CCAAT/Enhancer-binding proteins α (C/EBP α), and the induction of

Transcriptional factors

cell cycle arrest through regulating cyclin D1, cyclin-dependent kinase 4 (CDK4) and cyclin-

Cell cycle arrest

dependent kinase inhibitor 1B (P27/kip1) expression during the mitotic clonal expansion period. © 2015 Elsevier Ltd. All rights reserved.

1.

Introduction

Obesity, a disorder of excess body fat accumulation, is a risk factor known to contribute to a variety of comorbidities including diabetes, lipidaemia and hypertension (Bray & Bouchard, 2003; Kushner & Bessesen, 2007). The high risk and prevalence of obesity has led to the exploration of strategies for its prevention and treatment. Previous anti-obesity strategies mainly focused on the suppression of adipocyte hypertrophy, which is one of the three major events (together with adipocyte hyperplasia and angiogenesis) involved in the development of obesity (Avram, Avram, & James, 2007). However, extensive studies in anti-obesity have illustrated that adipocyte

hyperplasia is also a target for obesity treatment. Adipocyte hyperplasia, also referred to as “adipogenesis”, is a process of preadipocyte proliferation and differentiation by which the proliferative fibroblast-like preadipocytes develop into mature round, lipid-filled adipocytes. Treatment with effective phenolic compounds to inhibit adipogenesis is considered a promising anti-obesity approach. Oxyresveratrol (Fig. 1A), which frequently occurs in mulberry (Morus alba L.), is a natural phenol with high solubility in aqueous solution and low toxicity (Li, Cheng, Cho, He, & Wang, 2007; Lorenz, Roychowdhury, Engelmann, Wolf, & Horn, 2003). Oxyresveratrol is an analogue of resveratrol with an additional hydroxyl group on its aromatic ring (Galindo et al., 2011; Lorenz et al., 2003). It possesses similar biological activities as

* Corresponding author. School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, China. Tel.: +852 22990338; fax: +852 22990348. E-mail address: [email protected] (M. Wang). Chemical compounds: Oxyresveratrol (PubChem CID: 5281717); Cyanomaclurin (PubChem CID: 44257130). http://dx.doi.org/10.1016/j.jff.2015.03.026 1756-4646/© 2015 Elsevier Ltd. All rights reserved.

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Fig. 1 – Structures of oxyresveratrol (A) and cyanomaclurin (B).

resveratrol and has been demonstrated to show antiinflammatory (Chung et al., 2003), antioxidative (Lorenz et al., 2003), anti-browning (Li et al., 2007), and neuroprotective (Andrabi et al., 2004; Chao, Yu, Ho, Wang, & Chang, 2008) properties. It has also been considered as a potent free radical scavenger and tyrosinase inhibitor (Lorenz et al., 2003; Shin et al., 1998). Cyanomaclurin (Fig. 1B) is a flavanoid first separated from the Indian dyestuff Jackwood (Artocarpus integrifolia) (Perkin, 1905). It is composed of benzene rings and dihydropyran heterocycles with hydroxyl groups and its basic structure is similar to catechin. Limited research in exploring the bioactivities of cyanomaclurin has been carried out, while our previous studies indicated that it was an effective trapping agent of acrolein (ACR) and 4-hydroxy-trans-2-nonenal (HNE) by working as sacrificial nucleophile, where ACR and HNE were both cytotoxic lipid-derived α,β-unsaturated aldehydes (Zhu et al., 2009). It has been well documented that resveratrol inhibits lipogenesis and differentiation of 3T3-L1 adipocytes through suppressing the expression of specific adipogenic transcription factors and enzymes (Chen, Li, Li, Shan, & Zhu, 2011; Rayalam, Yang, Ambati, Della Fera, & Baile, 2008), while catechin can suppress adipocyte differentiation and stimulate lipolysis by modulating the expression of key transcription factors and hormone sensitive lipase (Furuyashiki et al., 2004; Lee, Kim, Kim, & Kim, 2009). Taking into consideration the structure similarity with these known anti-obesity phenolics, we hypothesize that oxyresveratrol and cyanomaclurin might also inhibit adipogenesis. In this study, we examined the antiadipogenic properties of oxyresveratrol and cyanomaclurin and further uncovered the underlying mechanisms using the 3T3-L1 cell model. This research helps to identify novel agents with anti-obesity properties.

2.

Materials and methods

2.1.

Materials

Oxyresveratrol (CAS registry no. 29700-22-9) was prepared by Dr. Zongping Zheng (State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China) from M. alba. Its

structure was confirmed by spectral analysis. Dulbecco’s Modified Eagle’s Medium (DMEM), foetal bovine serum (FBS), penicillin-streptomycin, and insulin were purchased from GIBCO (Grand Island, NY, USA). NaHCO3, sodium pyruvate, indomethacin (Indo), dexamethasone (DEX), dimethyl sulphoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Oil Red O were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The CytoTox 96 nonradioactive cytotoxicity assay kit was purchased from Promega Co. (Madison, WI, USA). The Triglycerides LiquiColor Test (Mono) kit was purchased from Stanbio Laboratory (Boerne, TX, USA). Bio-Rad Protein Assay Dye Reagent Concentrate was purchased from Bio-Rad Laboratories (Hercules, CA, USA). Protease inhibitor cocktail, phosphatase inhibitor cocktail B, phosphatase inhibitor cocktail C, mouse monoclonal anti-PPARγ, rabbit polyclonal anti-C/EBPα, mouse monoclonal anti-β-actin, rabbit polyclonal anti-CDK2, rabbit polyclonal anti-CDK4, mouse monoclonal anti-cyclin D1, rabbit polyclonal anti-cyclin A, goat antirabbit IgG-HRP and goat anti-mouse IgG-HRP were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal anti-p27 KIP 1 was obtained from Abcam (Cambridge, MA, USA). Polyvinylidene diflouride membrane was obtained from GE Healthcare (Little Chalfont, UK). SuperSignal West Pico Chemiluminescent Substrate was obtained from Thermo Scientific (Rockford, IL, USA).

2.2.

Preparation of cyanomaclurin

The wood of A. heterophyllus (2 kg) purchased from Wenchang County, Hainan Province, P. R. China was ground using a minigrinder and transferred into a glass flask. 95% ethanol (10L) was added and the extraction was performed by sonication. The extraction was repeated three times and the extracts were combined and concentrated on a rotor evaporator at 50 °C under vacuum to remove ethanol and water. The dried extract was subjected to Amberlite XAD16 column chromatography by successive elution with different proportions of water/ethanol mixtures (H2O-95% EtOH, v/v, 1:0, 4:1, 3:2, 2:3, 1:4, 1:19) to yield 6 fractions (Fractions 1–6). Fraction 5 (39.59 g) was applied to column chromatography on silica gel and eluted with dichloromethane/methanol (10:1, v/v) to obtain 11 fractions (Sub-fractions 1–11). Sub-fractions 4–9 (17.71 g) were pooled together and first purified with a Sephedex LH-20 column eluted with methanol/water (3:2, v/v) and then subjected to a silica gel column (eluted with dichloromethane/methanol (20:1, v/v)) to get cyanomaclurin. The structure of cyanomaclurin was confirmed by HPLC, TLC and NMR analysis.

2.3.

Cell culture

3T3-L1 preadipocytes (American Type Culture Collection, Rockville, MD, USA) were cultured in complete medium containing DMEM supplemented with 10% (v/v) FBS, 4.5 mg/mL glucose, 1.5 mg/mL NaHCO3, 4 mM glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin-streptomycin at 37 °C in 95% air-5% CO2 atmosphere. The fresh complete medium was renewed every 2 days and the cells were sub-cultured when they reach a confluence of 80%.

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2.4.

Adipocyte differentiation of 3T3-L1 cells

3T3-L1 preadipocytes were maintained post-confluence for 2 days. To induce differentiation, post-confluent 3T3-L1

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preadipocytes (day 0) were stimulated for 3 days (from day 0 to day 3) with induction medium containing DMEM with 10% (v/v) FBS supplemented with 125 µM of Indo, 1 µM of DEX and 5 µg/mL of insulin (IDI). After 3 days, and every 2 days

Fig. 2 – Oxyresveratrol and cyanomaclurin inhibit lipid accumulation in 3T3-L1 cells. Cytotoxicity of (A) oxyresveratrol and (B) cyanomaclurin on 3T3-L1 cells. Postconfluent 3T3-L1 cells were induced differentiation with IDI plus the phenolics of fixed concentrations or vehicle for 72 hours. The cytotoxicities of phenolics (tested by LDH assay and expressed as the percent LDH release) are presented as means ± S.D. (n = 3 per group). TG assays of (C) oxyresveratrol and (D) cyanomaclurin on 3T3-L1 cells. Oil Red O Staining assays of (E) oxyresveratrol and (F) cyanomaclurin on 3T3-L1 cells. Postconfluent 3T3-L1 cells were induced differentiation with IDI plus the phenolics of fixed concentrations or vehicle for 3 days. On Day 3, the differentiation medium was changed into transition medium and changed every two days until Day 9. On Day 9 after induction of differentiation, cells were lysed for TG and protein assays or stained with Oil Red O. TG levels are presented as means ± S.D. (n = 6 per group). Within each experiment, bars topped by different letters are significantly different (p ≤ 0.05; post hoc Duncan’s multiple range test).

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thereafter, cells were switched to fresh maintenance medium containing DMEM with 10% (v/v) FBS supplemented with 5 µg/ mL of insulin. After 9 days, cells were harvested for further experiments.

2.5. Preparation of oxyresveratrol and cyanomaclurin stock solutions Oxyresveratrol (100 mM) and 1000 mM of cyanomaclurin stock solutions were prepared by dissolving the individual phenolic compound in dimethyl sulphoxide (DMSO), respectively, and stored at −80 °C. For each experiment, cells exposed to oxyresveratrol or cyanomaclurin were premixed with culture medium. The final concentration of DMSO in culture medium is no more than 0.1%. Unless otherwise noted, “vehicle” refers to 0.1% DMSO in culture medium.

2.6.

Cytotoxicity assay

Preconfluent 3T3-L1 preadipocytes were seeded in 96-well plates at a density of 4000 cells/100 µL/well. Vehicle or phenolics were added to culture medium with or without cells at fixed concentrations. At the end of experiment, LDH assay was conducted by analysing the LDH release into the culture medium with a commercial kit (CytoTox 96 nonradioactive cytotoxicity assay kit). The total LDH was determined after the cells were thoroughly lysed with lysis solution. The percentage of LDH release was then calculated to determine cytotoxicity. % LDH release = (LDH in culture medium/LDH in culture medium + LDH in cells) × 100.

2.7.

Cell proliferation assay

Preconfluent 3T3-L1 preadipocytes were seeded in 96-well plates at a density of 4000 cells/100 µL/well. Phenolics, in fixed doses ranging, were added to the culture medium. At the end of experiment, MTT assay was performed as described by Hsu et al. with some adjustment (Hsu, Huang, & Yen, 2006). The culture medium was removed and replaced by 100 µL of MTT solution (0.5 mg/mL) dissolved in serum-free DMEM, and then the cells were incubated for 3 hours at 37 °C in an incubator. All unreacted dye was removed, and then the insoluble formazan crystals were dissolved in 100 µL/well acidified isopropanol (0.04M HCl). The plates with covers were swirled in the dark for 20 min at room temperature prior to measurement of absorbance at 595 nm by a microplate reader (Bio-Rad, IMark). The percentage (%) of control was expressed as the percentage of viable cells compared to vehicle as a 100% reference.

2.8.

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Oil Red O staining

Cells in wells of 6-well plates were washed with phosphatebuffered saline (PBS) twice, fixed in 1 mL/well 4% (v/v) formaldehyde for 30 minutes at 4 °C. At the end of fixation, cells were washed with PBS and with RO water twice, respectively, and then air dried for 2 min. 1 mL/well 0.36% (w/v) working Oil Red O solution was added to stain fat droplets for 1 hour at room temperature. After two washes with RO water, the fat droplets in the cells were observed under an inverted light microscope and pictured as described by Giri et al. (2006) with some adjustment.

2.9.

Triacylglycerol (TG) content assay

The cell extracts were collected across a treatment period of 9 days with intervals. After two washes with PBS, the cell extracts were collected by scraping the cell cultures with a cell scraper into 0.3 mL/well (for 6-well plates) lysing buffer containing 50 mM Tris, 1 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM β-mercaptoethanol at pH 7.5. The harvested cells, after passing through a pipette several times, were then sonicated with a microtip for 30 seconds at 40 W (Branson Sonifier 250, VWR Scientific). After centrifugation at 15,000 g for 5 min at 4 °C, the supernatants were assayed for triglyceride content according to the manufacturer’s protocol [Triglycerides LiquiColor Test (Mono) kit] and for the amount of total protein by Bradford method against BSA standards of 0, 0.05, 0.1, 0.2, 0.4, and 0.8 mg/mL. Results were expressed as total TG per cellular protein.

2.10. Cell cycle analysis by Fluorescence-activated cell sorting (FACS) In wells of 6-well plates, postconfluent 3T3-L1 preadipocytes were differentiated according to the IDI protocol with or without the test phenolic compounds for 24 hours. Then, cells were harvested and fixed with 70% ethanol at −40 °C overnight. After washing with PBS, cells were stained with Vindelov’s reagent for 30 minutes (Vindeløv, 1977). Fluorescence-activated cell sorting (FACS) analysis was performed on Becton–Dickinson FACS AriaIII instrument (Becton–Dickinson, San Jose, CA, USA) and data were analysed with Winlist and Modfit software.

2.11.

Western blotting analysis

Two-day postconfluent (designated day 0) growth-arrested 3T3L1 preadipocytes were induced to differentiate with or without

Fig. 3 – Oxyresveratrol and cyanomaclurin inhibit the expression of PPARγ and C/EBPα in 3T3-L1 cells. Postconfluent 3T3-L1 cells were induced differentiation with IDI plus 100 µM oxyresveratrol, 600 µM cyanomaclurin or vehicle for 3 days. At the indicated time points, 3T3-L1 cells were harvested for protein extraction and then subjected to western blotting analysis. Treated with (A, B, C) oxyresveratrol or (D, E, F) cyanomaclurin, the relative expressions of PPARγ and C/EBPα in 3T3-L1 cells were quantified densitometrically using the software ImageJ 1.47v, and data are expressed as ~fold changes in protein levels relative to those of controls. The control value was set at 1. Equal sample loading was verified by staining the blots with Coomassie Brilliant Blue R-250. OXY = oxyresveratrol; CYM = cyanomaclurin. Each value is mean ± S.D. obtained from three independent experiments. Within each experiment, bars topped by different letters are significantly different (p ≤ 0.05; post hoc Duncan’s multiple range test).

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the phenolics for 3 days. At different time points after the induction of differentiation, cells were harvested and lysed in ice-cold lysis buffer [25 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA disodium salt, 1 mM dithiothreitol, 1% Triton X-100] with added 4% protease inhibitor cocktail, 1% phosphatase inhibitor cocktail B and 2% phosphatase inhibitor cocktail C for protein collection. The protein concentration was measured using the Bradford Reagent (Bio-Rad Protein Assay Dye Reagent Concentrate). Thirty micrograms of protein of each sample were applied for electrophoresis on 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to polyvinylidene diflouride membrane. Membranes were blocked with 5% nonfat dried milk powder in PBST or TBST buffer (0.1% v/v Tween-20 in PBS or TBS) overnight at 4 °C. The membranes were then incubated for 1–2 h with specific antibodies at room temperature. Equal sample loading was verified either by anti-β-actin or by staining the blots with Coomassie Brilliant Blue R-250. Membranes were developed using SuperSignal West Pico Chemiluminescent Substrate. The bands were quantified densitometrically using the software ImageJ 1.47v.

2.12.

Statistical analysis

The data were expressed as means ± standard deviation. Student’s t-test was used to assess the differences between the means and one-way ANOVA with Duncan corrections was used to determine significance for multiple comparisons. For all analyses, the accepted level of significance was p ≤ 0.05. The calculations were performed using SPSS statistical package version 11.0 for Windows.

3.

Results

3.1. Oxyresveratrol and cyanomaclurin inhibit lipid accumulation in 3T3-L1 cells To evaluate the cytotoxicity of oxyresveratrol and cyanomaclurin, LDH assay was conducted by measuring the LDH content released into culture supernatant at 72 hours after the induction of differentiation. According to the results (Fig. 2A and B), dose limits of 100 µM for oxyresveratrol and 600 µM for cyanomaclurin were set in the later studies as below these

Fig. 4 – Oxyresveratrol and cyanomaclurin suppress postconfluent mitotic clonal expansion of 3T3-L1 cells. Postconfluent 3T3-L1 cells were induced differentiation with IDI plus the phenolics or vehicle for 72 hours. Effects of (A) oxyresveratrol and (C) cyanomaclurin on 3T3-L1 cell proliferation were tested by MTT assay and the degrees of cell proliferation (expressed as the percentage of control) are presented as means ± S.D. (n = 6 per group). Cytotoxicities of (B) oxyresveratrol and (D) cyanomaclurin were tested by LDH assay and the cytotoxicities of phenolics (expressed as the percent LDH release) are presented as means ± S.D. (n = 3 per group). Within each experiment, bars topped by different letters are significantly different (p ≤ 0.05; post hoc Duncan’s multiple range test).

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Fig. 5 – Cell cycle assay of oxyresveratrol and cyanomaclurin on 3T3-L1 cells. Postconfluent 3T3-L1 preadipocytes were differentiated with IDI plus (A) oxyresveratrol, (B) cyanomaclurin or vehicle for 24 hours. The harvested cells were stained with Vindelov’s reagent and analysed by FACS.

concentrations they didn’t induce any significant cytotoxicity in 3T3-L1 cells. The TG assay (Fig. 2C and D) showed both oxyresveratrol and cyanomaclurin inhibited intracellular triacylglycerol accumulation of the cells in a dose dependent manner on day 9 after the differentiation induction. The TG content in 3T3-L1 cells was reduced to 68% of control cells with the treatment of 100 µM of oxyresveratrol, and to 41% of control cells with the treatment of 600 µM of cyanomaclurin. The Oil Red O staining (Fig. 2E and F) further confirmed that oxyresveratrol and cyanomaclurin significantly suppressed lipid accumulation on day 9.

3.2. Oxyresveratrol and cyanomaclurin inhibit the expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer-binding protein α (C/EBPα) in 3T3-L1 cells Both oxyresveratrol and cyanomaclurin inhibited the protein expression of PPARγ and C/EBPα in a time dependent manner (Fig. 3). Oxyresveratrol exerted stronger suppression effect on PPARγ expression (Fig. 3A and C) during the late stage of differentiation, and down-regulated C/EBPα expression (Fig. 3B and C) at a relatively early stage (on day 4), while cyanomaclurin reduced PPARγ expression (Fig. 3D and F) at an early stage and exerted a long-lasting inhibitory effect against C/EBPα expression (Fig. 3E and F).

3.3. Oxyresveratrol and cyanomaclurin suppress postconfluent mitotic clonal expansion of 3T3-L1 cells The effects of oxyresveratrol and cyanomaclurin on 3T3-L1 cell proliferation during the postconfluent mitotic clonal expansion period were also determined. As shown in Fig. 4, both of oxyresveratrol and cyanomaclurin inhibited postconfluent mitotic clonal expansion of 3T3-L1 cells in a dose-dependent manner without inducing significant cytotoxicity. At 72 hours after the induction of differentiation, 100 µM of oxyresveratrol led up to around 45% inhibition of cell proliferation as compared

to the vehicle, while 600 µM of cyanomaclurin induced 57% inhibition of cell proliferation. Since oxyresveratrol and cyanomaclurin were identified to affect the mitotic clonal expansion of postconfluent 3T3-L1 cells, further studies were conducted to explore their effects on cell cycle changes.

3.4. Oxyresveratrol and cyanomaclurin induce cell cycle arrest in 3T3-L1 cells Fluorescence-activated cell sorting (FACS) was carried out. The results (Fig. 5, Tables 1 and 2) indicated that oxyresveratrol and cyanomaclurin induce cell cycle arrest dose-dependently during the mitotic clonal proliferation stage of 3T3-L1 cells.

3.5. Effects of oxyresveratrol and cyanomaclurin on several cell cycle regulatory molecules Cyclins and cyclin-dependent kinases (CDKs) are two major groups of regulatory molecules which work collaboratively to determine a cell’s progression (Nabel, 2002). Our data showed that oxyresveratrol and cyanomaclurin significantly repressed the expression of cyclin A and cyclin-dependent kinase 2 (CDK 2) during the S and G2 phases (Fig. 6). Furthermore, expressions of two regulatory molecules, cyclin D1 and cyclin-dependent

Table 1 – Cell cycle assay of oxyresveratrol on 3T3-L1 cells#. Treatment

G0/G1 (%)

S (%)

G2/M (%)

No differentiation Vehicle OXY 40 µM OXY 80 µM OXY 100 µM

83.88 ± 4.55b,c 69.74 ± 5.17a 69.36 ± 1.87a 78.64 ± 2.19b 86.00 ± 2.91c

5.95 ± 2.88a 15.21 ± 2.01b 16.46 ± 3.66b 10.00 ± 0.93a 7.20 ± 2.24a

10.16 ± 4.35a,b 15.04 ± 4.09b 14.18 ± 3.09b 11.36 ± 1.63a,b 6.80 ± 4.05a

#

Results are presented as means ± S.D. (n = 3 per group). Within each cell cycle stage, different letters are significantly different (p ≤ 0.05; post hoc Duncan’s multiple range test).

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Table 2 – Cell cycle assay of cyanomaclurin on 3T3-L1 cells#. Treatment

G0/G1 (%)

S (%)

G2/M (%)

No differentiation Vehicle CYM 200 µM CYM 400 µM CYM 600 µM

83.88 ± 4.55b 69.74 ± 5.17a 74.26 ± 2.50a 82.30 ± 5.09b 86.77 ± 2.75b

5.95 ± 2.88a 15.21 ± 2.01b,c 18.98 ± 2.26c 11.19 ± 0.49b 4.90 ± 3.59a

10.16 ± 4.35a 15.04 ± 4.09a 6.75 ± 4.65a 6.51 ± 4.64a 8.32 ± 4.50a

#

Results are presented as means ± S.D. (n = 3 per group). Within each cell cycle stage, different letters are significantly different (p ≤ 0.05; post hoc Duncan’s multiple range test).

kinase 4 (CDK 4), activated in the G1 stage, were down-regulated by oxyresveratrol or cyanomaclurin treatment (Fig. 6). Moreover, the expression of cyclin-dependent kinase inhibitor 1B (p27/kip1) was up-regulated in oxyresveratrol or cyanomaclurin treated cells.

4.

Discussion

In recent decades, extensive studies have demonstrated many beneficial properties such as antioxidant, anti-mutagenic,

Fig. 6 – Effects of oxyresveratrol and cyanomaclurin on several cell cycle regulatory molecules. Postconfluent 3T3L1 cells were induced differentiation with IDI plus (A) 100 µM oxyresveratrol, (B) 600 µM cyanomaclurin or vehicle. At the indicated time points, cells were harvested, lysed for protein collection and then subjected to western blotting analysis.

anti-thrombotic, anti-inflammatory and anti-obesity activities for phenolic compounds (Balasundram, Sundram, & Samman, 2006; Cho et al., 2010; Fernández, Sáenz, & García, 1998; Thériault, Caillet, Kermasha, & Lacroix, 2006). An increasing number of phenolic compounds, including chlorogenic acid, catechin and resveratrol, have been observed to fight obesity either in in vitro or in vivo studies (Chen et al., 2011; Cho et al., 2010; Rains, Agarwal, & Maki, 2011). Compared with drug therapies, these natural phenolics are considered to be associated with fewer side effects as they are commonly distributed in foodstuffs and natural herbs. Various mechanisms are suggested to associate with the potential anti-obesity properties of these phenolics including the induction of apoptosis in preadipocytes or adipocytes, the blocking of adipocytes differentiation, or the inhibition of intracellular triglyceride and GPDH accumulation in adipocyte (Williams et al., 2013; Yun, 2010). In our study, two natural phenolics, oxyresveratrol and cyanomaclurin have been discovered to inhibit the adipogenesis process of 3T3-L1 cells. Both oxyresveratrol and cyanomaclurin suppressed lipid accumulation in 3T3-L1 cells dose dependently when administered at the onset of differentiation (Day 0–3). The conversion of preadipocytes to mature adipocytes requires a variety of regulatory proteins to adjust the expression of adipocyte-specific genes in various time point of differentiation. PPARγ and C/EBPα have been well recognized to function cooperatively in activating adipocyte genes and consequently leading to adipocyte differentiation (Gregoire, Smas, & Sul, 1998). PPARγ is regarded as the “master regulator” of adipogenesis and also required at the differentiated state (Rosen & MacDougald, 2006). C/EBPα functions to transactivate the promoters of various adipocyte genes, such as GLUT4, SCD1, 422/aP2, PEPCK, leptin, and the insulin receptor (Darlington, Ross, & MacDougald, 1998). Exposing 3T3-L1 cells to oxyresveratrol or cyanomaclurin was found to restrain the expression of PPARγ and C/EBPα significantly. The inhibitory effects of cyanomaclurin in PPARγ and C/EBPα expression were greater than that of oxyresveratrol, which might be one mechanism leading to cyanomaclurin’s strong suppression effect against lipid accumulation. To identify other pathways that might contribute to the studied phenolics’ anti-adipogenic properties, their effects in mediation of the postconfluent mitotic clonal expansion of 3T3L1 cells were investigated. After reaching confluence, growth arrested preadipocytes will undergo a clonal expansion phase when they receive an appropriate combination of mitogenic and adipogenic signals and then withdraw from the cell cycle before adipocyte conversion. It was found that oxyresveratrol and cyanomaclurin exerted significant dose-dependent antiproliferative effects at the mitotic clonal expansion stage of adipogenesis process. The anti-proliferative effects were further confirmed by FACS analysis on cell cycle changes during the mitotic clonal expansion stage of 3T3-L1 cells. With IDI induction, postconfluent 3T3-L1 preadipocytes undergo clonal amplification and go through the interphase, mitotic phase and cytokinesis of cell cycle periods. The treatment of cells with oxyresveratrol or cyanomaclurin interrupted the cell cycle progression. They dose-dependently restrained postconfluent 3T3-L1 preadipocytes in the G0/G1 stage of cell cycle, which led to cell cycle arrest and the inhibition of cell proliferation. 100 µM of oxyresveratrol and 600 µM of cyanomaclurin were

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found to induce a similar DNA histogram type as the normal growth-arrested preadipocytes. With the onset of clonal amplification, 3T3-L1 preadipocytes pass through the G1-S checkpoint and enter the S phase between 12 and 16 hours (Tang, Otto, & Lane, 2003). During this time period, expression of cyclin A was activated and remained constant to 24 hours in phenolics-free cells (Fig. 6). Similar to cyclin A, CDK 2 expression was up-regulated with the entry of S phase and reached a maximum at 24 hours (Fig. 6). In oxyresveratrol or cyanomaclurin treated cells, expression of cyclin A and CDK 2 were both suppressed, indicating a block of G1 to S phase transition, which supports the results of FACS analysis. The above studies manifest that oxyresveratrol and cyanomaclurin suppressed adipogenesis of 3T3-L1 cells through inducing cell cycle arrest at the G0/G1 phase during the postconfluent mitotic clonal expansion period. In further studies, the expression of cyclin D1 and CDK 4 were found to be restrained by oxyresveratrol or cyanomaclurin treatment (Fig. 6). As the first cyclins to be expressed when the growth-arrested cells reenter the cell cycle, the D-type cyclins interact combinatorially with CDK 4 and CDK 6, and the cyclin D-CDK 4/6 complexes are essential in mediating the G1 progression and the G1 to S phase transition (Johnson & Walker, 1999; Nabel, 2002). The most important function of the cyclin D-CDK 4/6 complexes is to phosphorylate the retinoblastoma tumor suppressor protein (Rb). The Rb protein binds to and inhibits the E2F family members which regulate the expression of many genes required for the entry of S phase. Phosphorylation of Rb by D-type cyclin kinases leads to the release of E2F family members and subsequently the expression of the E2F-regulated genes for cell cycle progression (Johnson & Walker, 1999; Nabel, 2002). By down-regulating the expression of cyclin D1 and CDK 4, oxyresveratrol or cyanomaclurin might block the cells from entering the S phase through reducing the Rb protein phosphorylation thus inhibiting E2F’s transcriptional activation capacity. Moreover, the expression of p27/kip1 was found to be up-regulated in oxyresveratrol or cyanomaclurin treated cells (Fig. 6). p27/kip1 belongs to the CIP/KIP family which is a group of cyclin-CDK inhibitors that act as the key components of the molecular network regulating cell cycle progression. In proliferating cells, cyclin D-dependent kinases bind and sequester CIP/KIP proteins to reduce the levels of unbound CIP/KIP proteins, which relieve the cyclin E-CDK 2 complex from CIP/KIP protein inhibition and lead to an irreversible cell cycle transition from G1 to S phase (Nabel, 2002). In our study, oxyresveratrol or cyanomaclurin treatment up-regulated the expression of p27/kip1, which may constrain cyclin E-CDK 2 complex and result in G1-phase arrest. However, more researches are needed to verify the influence of oxyresveratrol or cyanomaclurin on Rb-E2F pathway and p27/kip1 induced inhibitory effects.

5.

Conclusion

In this study, two natural phenolics, oxyresveratrol and cyanomaclurin, were identified to suppress adipogenesis of 3T3-L1 cells. The anti-adipogenic properties of these phenolics were attributed to down-regulation of PPARγ and C/EBPα

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expression, and the induction of cell cycle arrest through regulating cyclin D1, CDK 4 and P27/kip1 expression during the mitotic clonal expansion period.

Acknowledgement We would like to thank The Hong Kong Jockey Club Charities Trust (HKJCCT) for funding the project of ″R&D Laboratory for Testing of Chinese Medicines″.

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