The SW872 human adipocyte cell line was obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). Dulbecco’s modified Eagle’s medium with F12 (DMEM/F12), fetal bovine serum (FBS) and phosphate-buffered saline (PBS) were purchased from Gibco Life Technologies (Grand Island, NY, USA). Curcumin (99%) and 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The ApoStrand™ ELISA Apoptosis Detection kits were purchased from Chemicon International (Temecula, CA, USA). Rabbit polyclonal antibodies against PARP (1:1,000; cat. no. sc-25780), Bax (1:500; cat. no. sc-493), Bcl-2 (1:500; cat. no. sc-492), caspase-3 (1:800; cat. no. sc-7148), β-actin (1:800; cat. no. sc-1616-R) and cytochrome c (1:500; cat. no. sc-7159) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

The SW872 cells were grown in DMEM/F12 (3:1) containing 10% FBS, penicillin (100 U/ml) and streptomycin (100 U/ml) (Gibco Life Technologies) at 37°C in 5% CO₂. SW872 cells that formed a confluent monolayer were induced to differentiate using DMEM/F12 containing 1% bovine serum albumin (BSA; Sigma-Aldrich) and 0.6 mol/l oleic acid for three days, until >90% of the cells had reached maturity.

The cell viability of mature adipocytes was evaluated using an MTT assay in 96-well plates. The mature SW872 adipocytes were treated with 10, 20, 40, 60 or 80 μmol/l of curcumin, and were incubated for 24, 48 and 72 h (every 12-wells was a group). The contents of each well was added into 20 μl of the MTT dye (50 μg/ml) and incubated for 4 h at 37°C. The medium was then discarded. Dimethyl sulfoxide (DMSO; 150 μl) was added into each well, and the absorbance was measured at 620 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

SW872 cells were cultured in 6-well plates and grown to maturity, as described above. The cells were then treated with or without 40 μmol/l curcumin for 24 h. Cells were washed with PBS and then stained with 0.1 μg/ml DAPI for 30 min at 20°C. Images were acquired using an Olympus IX-70 inverted fluorescent microscope (magnification, x400; Olympus Corporation, Tokyo, Japan) and the percentage of cells that contained condensed chromatin and/or fragmented nuclei was determined.

Tests were performed in 96-well plates. The SW872 cells were seeded (5,000 cells/well), grown to confluence, induced to differentiate and grown to maturity. Curcumin (0, 10, 20 or 40 μmol/l) in 0.01% DMSO carrier was added for 24 h with the wash buffer. Following washing, cells were incubated with 100 μl peroxide substrate for 1 h, and absorbance was read using an ELISA plate reader (Spectra Max M2; Molecular Devices, Sunnyvale, CA, USA) at 405 nm. The reaction was stopped by the addition of 100 μl l% sodium dodecyl sulfate.

Mature SW872 adipocytes were incubated with 0, 10, 20 or 40 μmol/l of curcumin for 24 h, or with 40 μmol/l curcumin for 24, 48 or 72 h. Cells were washed twice with cold PBS and then treated with ice-cold lysates containing 1 μg/ml aprotinin, 1 μg/ml leupeptin and 1 mmol/l PMSF (20). The lysates were collected following centrifugation at 15,000 × g for 15 min at 4°C. Once samples had been stratified, protein content was isolated by collecting the middle transparent liquid. Samples were prepared with 2-mercaptoethanol and denatured by heating at 100°C for 6 min. Protein samples were resolved by SDS-PAGE (Beijing Saichi Biological Technology Co., Ltd., Beijing, China), transferred to the nitrocellulose membrane (Bio-Rad Laboratories, Inc.) and immunoblotted with primary antibodies against caspase-3, PARP, Bax, Bcl-2 and cytochrome c. Subsequently, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit polyclonal immunoglobulin G secondary antibodies (1:10,000; cat. no. sc-2004; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Images were acquired and quantified using a ChampGel-3200 Digital Imaging system (Cell Biosciences, Inc., Santa Clara, CA, USA).

Differences among groups were assessed using one-way analysis of variance (ANOVA). Data are expressed as the mean ± standard deviation. Statistical analyses were performed using the SPSS 13.0 statistical program (version 13.01 S; Beijing Stats Data Mining Co., Ltd., Beijing, China). P<0.05 was considered to indicated a statistically significant difference.

The effect of curcumin on cell viability was determined in the mature adipocytes. The results are shown in Fig. 1. A reduction in cell viability was observed in mature adipocytes treated with 40, 60 and 80 μmol/l of curcumin, for 24, 48 and 72 h. Furthermore, curcumin treatment reduced cell viability in a time- and dose-dependent manner.

In order to assess whether the reduction in cell number following treatment with curcumin was due to increased apoptosis, an ssDNA ELISA kit was used to determine cell apoptosis. As shown in Fig. 2A, exposure of adipocytes to curcumin resulted in a dose-dependent increase in the level of apoptosis. In order to determine the direct effect of curcumin on nuclear morphology, DAPI staining was used to visualize nuclear shrinkage and fragmentation (20). When the mature SW872 adipocytes were treated with curcumin (40 μmol/l) for 48 h, the cells exhibited morphological features characteristic of apoptotic cells, such as bright nuclear condensation, DNA fragmentation and perinuclear apoptotic bodies, as demonstrated by DAPI staining (Fig. 2B).

In order to investigate the effect of curcumin on the Bcl-2 family in SW872 cells, western blotting was used to detect the expression of the Bax (proapoptotic) and Bcl-2 (antiapoptotic) proteins, and thus evaluate the changes in the Bax/Bcl-2 ratio (Fig. 3A). The SW872 cells were treated with 20 or 40 μmol/l of curcumin for 24 h, and with 40 μmol/l of curcumin for 24, 48 or 72 h, and a time- and dose-dependent increase in Bax expression and a decrease in Bcl-2 expression was observed (Fig. 3B). The Bax/Bcl-2 ratio was significantly increased, by 146 and 220% following treatment with 20 and 40 μmol/l of curcumin, respectively, compared with the control cells (P<0.05).

Following an increase in the Bax/Bcl-2 ratio in the mitochondrial membrane, mitochondria release cytochrome c into the cytosol, leading to the subsequent activation of caspase-3 for apoptosis (21). The effects of curcumin on the protein expression of cytochrome c in SW872 adipocytes are shown in Fig. 4. The expression of cytochrome c in the cytoplasm significantly increased, of 121 and 129%, when SW872 cells were treated with 20 and 40 μmol/l of curcumin, respectively for 24 h compared with that in the control cells (P<0.05; Fig. 4A). Furthermore, the expression of cytochrome c also showed a significant increase, of 129, 139% and 151%, in cells treated with 40 μmol/l of curcumin for 24, 48 and 72 h, respectively (all P<0.05; Fig. 4B).

The release of Cytochrome c from mitochondria into the cytosol is an important event in the activation of caspase-3 (13). Treatment of adipocytes with 40 and 20 μmol/l of curcumin for 24 h (Fig. 5A), and with 40 μmol/l for 24, 48 and 72h (Fig. 5B) significantly stimulated caspase-3 expression in a time- and dose-dependent manner (P<0.05), with a maximal increase of 17 kDa. However, the activation of caspase-3 results in the cleavage of a number of proteins, the most important of which is PARP. PARP is thought to have a multifunctional role in apoptosis, DNA repair and recombination, as well as in the maintenance of chromosomal stability (22). Cleavage of this protein leads to its inactivation and thus prevents the futile DNA repair cycle. Treatment of cells with 20 or 40 μmol/l of curcumin for 24 h (Fig. 5C), or with 40 μmol/l of curcumin for 24, 48 and 72 h (Fig. 5D) significantly induced PARP cleavage, compared with the control cells, with a maximal cleavage of 85kD (P<0.05).