BioFactors 33 (2008) 137148 IOS Press

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Withaferin A induces apoptosis and inhibits adipogenesis in 3T3-L1 adipocytes

Hea Jin Parka,1 , Srujana Rayalama , Mary Anne Della-Feraa , Suresh Ambatia, Jeong-Yeh Yanga and Clifton A. Bailea,b,
b Department a Department

of Animal & Dairy Science, University of Georgia, Athens, GA, USA of Foods and Nutrition, University of Georgia, Athens, GA, USA

Received 8 August 2008 Revised 16 September 2008 Accepted 19 September 2008 Abstract. Withaferin A (WA), a highly oxygenated steroidal lactone that is found in the medicinal plant Withania somnifera (also called ashwagandha) has been reported to have anti-tumor, anti-angiogenesis, and pro-apoptotic activity. We investigated the effects of WA on viability, apoptosis and adipogenesis in 3T3-L1 adipocytes. Pre- and post-conuent preadipocytes and mature adipocytes were treated with WA (125 M) up to 24 hrs. Viability and apoptosis were measured by CellTiter-Blue Cell Viability Assay and single strand DNA ELISA Assay, respectively. WA decreased viability and induced apoptosis in all stages of cells. Induction of apoptosis by WA in mature adipocytes was mediated by increased ERK1/2 phosphorylation and altered Bax and Bcl2 protein expression. The effect of WA on adipogenesis was examined by AdipoRedTM Assay after treating with WA (0.11 M) during the differentiation period. WA decreased lipid accumulation in a dose-dependent manner and decreased the expression of peroxisome proliferator-activated receptor , CCAAT/enhancer binding protein and adipocyte fatty acid binding protein. The effects on apoptosis and lipid accumulation were also conrmed with Hoechst staining and Oil Red O staining, respectively. These results show that WA acts on adipocytes to reduce cell viability and adipogenesis and also induce apoptosis. Keywords: Withania somnifera, Bcl2 family, ERK1/2, PPAR , aP2

1. Introduction Obesity is a chronic metabolic disorder characterized by an excess of body fat. While weight loss drugs have become more popular in recent times, such treatments have failed to provide long term maintenance of weight loss in patients suffering from obesity. A decrease in adipose tissue mass may involve loss of lipids through either lipolysis or decreased adipogenesis [20], and an increase in adipose tissue mass may involve increased proliferation and differentiation of preadipocytes [22]. Therefore, natural products that have the ability to inhibit adipogenesis or induce adipocyte apoptosis could be important tools in preventing obesity.
Current address: University of Connecticut, Department of Nutritional Sciences, Storrs, CT, USA Address for correspondence: Clifton A. Baile, 444 Edgar L. Rhodes Center for Animal and Dairy Science, University of Georgia, Athens, GA 30602-2771, USA. Tel.: +1 706 542 2771; Fax: +1 706 542 7925; E-mail: cbaile@uga.edu.
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0951-6433/08/$17.00 2008 IUBMB/IOS Press and the authors. All rights reserved

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Fig. 1. Structure of Withaferin A.

Several natural compounds like genistein [13], ajoene [32], esculetin [30], guggulsterone [31] and xanthohumol [33] were reported to induce apoptosis in 3T3-L1 adipocytes. The 3T3-L1 cell line is an ideal model system to understand adipocyte development. 3T3-L1 preadipocytes can be induced to differentiate into lipid lled mature adipocytes in cell culture [6] when treated with a hormonal cocktail that includes insulin, dexamethasone, and isobutylmethylxanthine (IBMX). During differentiation, the morphology of preadipocytes changes from that of an elongated broblastic-type cell shape to a round shape lled with cytoplasmic lipid vesicles. In addition a complex sequence of changes in gene expression occurs leading to storage of lipid [5]. Peroxisome proliferator-activated receptor (PPAR ), CCAAT/enhancer binding proteins and (C/EBP) and adipocyte fatty acid binding protein (aP2) are considered to play important roles in adipocyte differentiation [5,6]. Withaferin A (WA, Fig. 1), an important prototype of the withanolide class of natural products, is a highly oxygenated steroidal lactone that is found in the medicinal plant Withania somnifera and its related solanaceae species. Withania somnifera (also called ashwagandha) is widely used in Ayurvedic medicine, the traditional medical system of India, to increase longevity and vitality. Further the anti-inammatory, anti-tumor, anti-oxidant and immunomodulatory properties of the herb Withania somnifera have been reported [16,28]. In the current study, we investigated the effect of WA on adipocyte apoptosis and adipogenesis. Although the mechanism of apoptosis induction in tumor cells by WA has been investigated [29], to our knowledge this is the rst study to demonstrate pro-apoptotic and anti-adipogenic effects of WA in adipocytes. We show that WA decreased viability and induced apoptosis in both preadipocytes and mature adipocytes. The apoptosis induction by WA was mediated by increased ERK1/2 phosphorylation and altered Bax and Bcl2 protein expression. Furthermore, WA decreased the expression of adipocyte specic factors leading to decreased adipocyte differentiation and reduced lipid accumulation.

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2. Materials and methods 2.1. Reagents Withaferin A (95.1% HPLC purity) was purchased from Chromadex Inc. (Santa Ana, CA). CellTiter Blue Cell Viability Assay reagent was obtained from Promega (Madison, WI) and ApoStrand ELISA Apoptosis Detection Kit was purchased from BIOMOL (Plymouth Meeting, PA). AdipoRed TM Assay reagent was purchased from Cambrex BioScience (Walkersville, MA). Oil Red O stain and Hoechst stain were obtained from Sigma (St. Louis, MO). Antibodies specic for -actin, Bax, Bcl2, PPAR , C/EBP, and aP2 were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies specic for polyclonal antiphospho-ERK1/2 (Thr202 /Tyr204 ) and total ERK1/2 were from Cell Signaling Technology (Beverly, MA). 2.2. Cell line and cell culture 3T3-L1 mouse embryo broblasts were obtained from American Type Culture Collection (Manassas, VA) and were cultured as described elsewhere [9]. Briey, cells were cultured in DMEM containing 10% bovine calf serum until conuent. Two days after conuence (day 0), the cells were stimulated to differentiate with DMEM containing 10% fetal bovine serum (FBS), 167 nM insulin, 0.5 M isobutylmethylxanthine (IBMX), and 1 M dexamethasone for 2 days. On day 2, differentiating media was replaced with 10% FBS/DMEM medium containing 167 nM insulin and incubated for 2 days, followed by culturing with 10% FBS/DMEM medium for an additional 4 days, at which time >90% of cells were mature adipocytes with accumulated fat droplets. All media contained 1% Penicillin-Streptomycin (10,000 U/ml) and 1% (v/v) 100 mM pyruvate. Cells were maintained at 37 C in a humidied 5% CO2 atmosphere. 2.3. Cell viability assay Tests were performed in 96-well plates. For preconuent preadipocytes, a seeding density of 2500 cells/well was used, and cells were cultured overnight before treatment. Cells were incubated with either dimethyl sulfoxide (DMSO) or increasing concentrations (125 M) of WA for 6, 12 and 24 hrs. For the postconuent preadipocytes, a seeding density of 2500 cells/well was used, and cells were grown to conuency before treatment. For mature adipocytes, cells were grown to maturation as described above. For maturing adipocytes, two day post-conuent preadipocytes were treated with WA (0.11 M) in DMSO throughout the differentiation period. Medium was changed every 2 days. On assay day, the medium was changed and replaced with 100 l fresh 10% FBS/DMEM medium and 20 l Cell Titer-Blue Cell Viability reagents. Cells were then incubated in the dark for 1 hr at 37 C and the uorescent signal was measured at an excitation wavelength of 560 nm and an emission wavelength of 590 nm to determine the resorun concentration, which is proportional to the number of live cells. 2.4. Quantication of lipid content Lipid content was quantied using commercially available AdipoRed TM Assay Reagent according to the manufacturers instructions. AdipoRed TM is a reagent that enables the quantication of intracellular lipid droplets in a high-throughput manner. AdipoRed reagent is a solution of the hydrophilic stain Nile Red. Briey, two day post-conuent preadipocytes were treated with WA (0.11 M) in DMSO through

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out the differentiation period and medium was changed every 2 days. On day 6, culture supernatant was removed and cells were carefully rinsed with 200 l of phosphate buffered saline (PBS). Wells were then lled with 200 l of PBS; 5 l of AdipoRedTM reagent was added and cells were incubated for 10 min at room temperature. Fluorescence was measured with excitation at 485 nm and emission at 572 nm. 2.5. Oil Red O staining Cells were treated with 0.1% DMSO or WA for 0 to 6 days during adipogenesis as described above. On day 6, cells were washed twice with PBS and xed with 10% formalin in PBS (pH 7.4). Cells were then stained with Oil Red O and hematoxylin as described by Suryawan and Hu [27]. After mounting with glycerol gelatin, at least three images for each dish were captured using ImagePro software version 5.1 (MediaCybernetics, Silver Spring, MD). 2.6. Apoptosis assay For measuring the extent of apoptosis, ApoStrand ELISA Apoptosis Detection Kit (Biomol, Plymouth Meeting, PA) was used. Cells were grown in 96 well plates and pre- and post-conuent preadipocytes and mature adipocytes were incubated with either 0.1% DMSO or WA (125 M) for 24 hrs. Cells were then xed and assayed as per the manufacturers instructions. The assay selectively detects single stranded DNA, which occurs in apoptotic cells but not in necrotic cells or cells with DNA breaks in the absence of apoptosis [3]. For Hoechst staining, cells treated with WA were xed with 3.7% formaldehyde in PBS and incubated for 15 min with 5 g/ml Hoechst 33342. After extensive washing, nuclear staining was examined under a uorescence microscope and three images for each dish were captured using ImagePro software version 5.1 (MediaCybernetics, Silver spring, MD). 2.7. Western blot analysis Mature adipocytes were treated with either 0.1% DMSO or 10 M WA for 6, 12 and 24 hr. Likewise maturing preadipocytes were treated with either 0.1% DMSO or WA (0.11 M) for 6 days during differentiation. Whole cell extracts were prepared by washing the cells with PBS and suspending in a lysis buffer (20 mM Tris, pH 7.5, 150 mM sodium chloride, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM -glycerophosphate, 1 mM sodium vanadate (Na 3 VO4 ), 1 g/ml aprotinin, 1 g/ml leupeptin, and 100 g/ml phenylmethylsulfonyl uoride). After 30 min of rocking at 4 C, the mixtures were centrifuged (10,000 x g) for 10 min, and the supernatants were collected as whole cell extracts. The protein concentration was determined by the method of Bradford with bovine serum albumin as the standard. Western blot analysis was performed using the commercial NUPAGE system (Novex/Invitrogen, Carlsbad, CA), in which a lithium dodecyl sulfate (LDS) sample buffer (Tris/glycerol buffer, pH 8.5) was mixed with fresh dithiothreitol and added to samples. Samples were then heated to 70 C for 10 min, separated by 12% acrylamide gels and analyzed by immunoblotting as previously described [14]. Immunoblots were developed using the ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ). All experiments were repeated three times. Representative Western Blots are shown along with the graphs of the quantitative data.

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2.8. Quantitative analysis of Western blot data Measurement of signal intensity on PVDF membranes after Western blotting with various antibodies was performed using a FluorChem TM densitomer with the AlphaEaseFC TM image processing and analysis software (Alpha Innotech Corp., San Leandro, CA). For statistical analysis, all data were expressed as integrated density values (IDV). For Bax, Bcl2, PPAR , C/EBP and aP2, the IDVs were calculated as the density values of the specic protein bands/ -actin density values. For phospho-ERK, the IDVs were calculated as the density values of the specic protein bands/total ERK. Data were expressed as percentage of the control (DMSO) or 0 hr values. 2.9. Statistical analysis ANOVA (GLM procedure, Statistica, version 6.1; StatSoft, Tulsa, OK) was used to determine signicance of treatment effects. Fishers post hoc least signicant difference test was used to determine signicance of differences among means. Statistically signicant differences are dened at the 95% condence index. Data shown are means standard error. 3. Results 3.1. Effect of WA on cell viability and apoptosis in 3T3-L1 preconuent preadipocytes, postconuent preadipocytes and mature adipocytes We rst tested the effect of WA on viability in both preadipocytes (preconuent and postconuent) and mature adipocytes. Cells were treated with WA (125 M) for 6, 12, 24 hrs. As shown in Table 1, WA decreased viability in a dose- and time-dependent manner. In preconuent preadipocytes, WA decreased viability with as little as 1 M after 12 hr and at higher concentrations after 6 hr incubation. In postconuent preadipocytes 1 M WA increased viability by 22.5 1.2% (P < 0.0001), but higher concentrations decreased viability in a dose and time-dependent manner. In mature adipocytes, 25 M WA decreased viability by 53.2 2.7% (P < 0.0001) after only 6 hr of incubation. WA 10 M decreased viability by 30.2 2.3% (P < 0.0001) and 90.1 1.0% (P < 0.0001) at 12 hr and 24 hr in mature adipocytes. To assess whether the reduction in cell number by WA was due to apoptosis, we used an ssDNA ELISA assay (ApoStrand ELISA Apoptosis Detection Kit) as a measure of cellular apoptosis after 24 hr incubation with WA (Table 2). At 10 M and 25 M concentrations, WA increased apoptosis in a dose-dependent manner in all cell stages. The increase in apoptosis was greater in preconuent preadipocytes than in postconuent preadipocytes, and in mature adipocytes 10 M and 25 M WA increased apoptosis by 134.6 16.8% (P < 0.0001) and 216.0 15.9% (P < 0.0001). We selected the 10 M concentration in mature adipocytes for the following study using Western blotting. 3.2. Effect of WA on the expression of Bcl2 and Bax in 3T3-L1 mature adipocytes The effect of WA on apoptosis in mature adipocytes was conrmed with Hoechst staining (Fig. 2A). We next measured Bcl2 and Bax expression by Western blotting after treating with WA 10 M for 6, 12 and 24 hrs in mature adipocytes. WA decreased Bcl2 expression by 45.0 4.5% (P < 0.0001) and 53.6 2.9% (P < 0.0001) at 12 hrs and 24 hrs after treatments, respectively (Fig. 2B). WA also increased Bax expression by 42.6 10.7% (P < 0.05) and 45.4 6.8% (P < 0.05) at 12 hrs and 24 hrs, respectively (Fig. 2C).

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H.J. Park et al. / Withaferin A induces apoptosis and inhibits adipogenesis Table 1 The effect of WA on cell viability (% control) in 3T3-L1 preconuent preadipocytes, postconuent preadipocytes and lipid-lled mature adipocytes Preconuent preadipocytes DMSO WA 1M WA 2.5M WA 5M WA 10M WA 25M DMSO WA 1M WA 2.5M WA 5M WA 10M WA 25M DMSO WA 1M WA 2.5M WA 5M WA 10M WA 25M 6hr 100.00 6.85a 93.69 1.73a 80.39 1.82b 79.82 1.60c 78.26 1.42c 65.91 0.54d 100.00 1.53c 97.32 2.76c 95.33 2.33c 87.03 2.93d 80.68 3.58d,e 55.03 3.39f 100.00 2.39a,b 105.49 2.47a 94.63 2.00b,c 95.02 1.41b,c 90.91 2.25c,d 46.82 2.73h 12hr 100.00 1.88a 84.90 1.23b,c 68.89 1.79d 67.78 1.60d 63.68 1.51d 38.33 0.22e,f 100.00 2.58c 113.59 3.86b 96.53 4.40c 76.68 3.27e 56.04 1.79f 8.77 0.10h 100.00 1.47a,b 98.33 1.75b 95.34 3.35b,c 92.57 1.95c,d 69.77 2.29e 4.68 0.18h,i 24 hr 100.00 2.58a 83.27 3.27b,c 45.62 1.78e 37.67 1.26f 28.74 0.57g 23.97 0.21g 100.00 2.24c 122.50 1.23a 81.89 3.33d,e 55.92 1.72f 17.42 0.49g 8.19 0.05h 100.00 1.55a,b 99.98 1.16a,b 87.57 1.31d 59.15 3.07f 9.87 0.98h 0.85 0.05i

Postconuent preadipocytes

Lipid-lled mature adipocytes

Cells were treated with WA at various concentrations for 6, 12 and 24 hr and cell viability was determined as indicated in Materials and Methods. The experiments were performed in eight replicates for each treatment. Values showed mean SEM. Means not designated by a common superscript are different within the same stage of cells, P < 0.05. Table 2 The effect of WA on apoptosis in 3T3-L1 preconuent preadipocytes, postconuent preadipocytes and lipid-lled mature adipocytes Apoptosis (% Control) DMSO WA 1M WA 2.5M WA 5M WA 10M WA 25M Preconuent preadipocytes 100.00 3.21a 81.38 6.37a 91.52 3.32a 97.50 4.05a 145.81 15.29b 316.18 19.82c Postconuent preadipocytes 100.00 7.35a 119.77 8.61a,b 102.30 4.40a,b 113.25 8.31a,b 127.28 9.09b 197.42 12.98c Lipid-lled mature adipocytes 100.00 5.61a 88.89 6.20a 87.24 4.95a 81.64 2.52a 234.60 16.80b 316.04 15.91c

Cells were treated with WA at various concentrations for 24 hrs and apoptosis was determined as indicated in Materials and Methods. The experiments were performed in eight replicates for each treatment. Values showed mean SEM. Means not designated by a common superscript are different, P < 0.05.

3.3. Effect of WA on ERK1/2 phosphorylation in 3T3-L1 mature adipocytes We determined ERK1/2 phosphorylation after treating with WA 10 M for 6, 12 and 24 hr in mature adipocytes. WA increased ERK1/2 phosphorylation with as little as 6 hr treatment (Fig. 3). Phospho-p44 ERK and phospho-p42 ERK increased by 196.1 8.8% (P < 0.0001) and 228.4 4.2 (P < 0.0001) after 6 hr, and 165.6 27.5% (P < 0.0001) and 219.9 12.8% (P < 0.0001) after 12 hr treatment, respectively.

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Fig. 2. Representative Images of Hoechst staining (A) and the effect of WA on expression of Bcl2 (B) and Bax (C). 3T3-L1 mature adipocytes were treated with WA 10 M for the indicated time periods. All experiments were repeated three times. Vertical bars denote mean SEM. Means which are not denoted by a common superscript are different, P < 0.05.

3.4. Effect of WA on lipid accumulation in 3T3-L1 maturing preadipocytes WA decreased lipid accumulation in a dose-dependent manner in maturing preadipocytes, as shown at Table 3. WA 0.1 M, 0.5 M and 1 M decreased lipid accumulation by 30.7 3.7%, 56.4 3.1% and 89.7 0.5% (P < 0.0001), respectively. During the same periods WA also decreased cell viability. However, when we expressed lipid content on a per cell basis to correct for cell loss, there was still a dramatic decrease in lipid accumulation/cell. WA 0.1 M, 0.5 M and 1 M decreased lipid accumulation/cell by 26.3 4.0% (P < 0.0001), 50.4 3.5% (P < 0.0001) and 87.1 0.6% (P < 0.0001), respectively. 3.5. Effect of WA on the expression of PPAR , C/EBP, and aP2 in 3T3-L1 maturing preadipocytes The effect of WA on lipid accumulation was conrmed with Oil Red O staining (Fig. 4A). To determine whether the decrease in lipid accumulation with WA was related to changes in PPAR , C/EBP and

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H.J. Park et al. / Withaferin A induces apoptosis and inhibits adipogenesis Table 3 The effect of WA on lipid accumulation in 3T3-L1 maturing preadipocytes Lipid Accumulation (% Control) 100.00 3.25a 69.31 3.74b 43.64 3.06c 10.31 0.47d 13.46 1.54d Cell Viability (% Control) 100.00 0.79a 94.04 0.79b 88.04 0.98c 79.83 1.26d 75.81 0.75e Lipid Accumulation/Cell Viability (% Control) 100.00 3.25a 73.70 3.97b 49.56 3.47c 12.91 0.59d 17.76 2.03d

DMSO WA 0.1M WA 0.5M WA 1M Undifferentiated Preadipocytes

Cells were treated with WA at various concentrations during the differentiation period and lipid accumulation and cell viability were determined. Lipid accumulation/cell was calculated. The experiments were performed in eight replicates for each treatment. Values showed mean SEM. Means not designated by a common superscript are different, P < 0.05.

Fig. 3. The effect of WA on phosphorylation of ERK1/2. 3T3-L1 mature adipocytes were treated with 10 M WA for the indicated time periods. All experiments were replicated three times. Vertical bars denote mean SEM. Means which are not denoted by a common superscript are different, P < 0.05.

aP2 expression levels, 3T3-L1 cells were treated with either 0.1% DMSO or WA from 06 days of the differentiation period. On day 6 whole cell lysates were prepared as described previously and subjected to Western blotting. As shown at Fig. 4B4D, WA decreased PPAR expression by 23.5 5.8% (P < 0.05), 40.1 6.3% (P < 0.001), 56.7 3.7% (P < 0.0001) with 0.1 M, 0.5 M and 1 M concentrations, respectively. WA also decreased C/EBP, and aP2 expression in a dose-dependent manner. Expression levels after treatment with WA 1 M were not signicantly different from those in undifferentiated preadipocytes. 4. Discussion The aim of the current study was to determine the effect of withaferin A on the apoptosis and adipogenesis in 3T3-L1 adipocytes. We demonstrated that WA inhibited 3T3-L1 preadipocyte and

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Fig. 4. Representative Images of Oil Red O staining (A) and the effect of WA on expression of aP2 (B), C/EBP (C), and PPAR (D) in maturing 3T3-L1 preadipocytes. All experiments were replicated three times. Vertical bars denote mean SEM. Means which are not denoted by a common superscript are different, P < 0.05.

mature adipocyte proliferation, induced apoptosis and inhibited adipogenesis. Treatment of preconuent preadipocytes with WA inhibited cell viability in a time- and dose-related manner. Since preconuent preadipocytes are actively proliferating and are in the growth phase, decrease in viability can indicate either loss of cells due to cell death or a decrease in cell division, or both. In contrast postconuent preadipocytes are in a resting phase called growth arrest [5] and a decrease in viability at this stage is likely due to cell death alone. An enhanced induction of apoptosis was observed in preconuent preadipocytes treated with WA when compared to postconuent preadipocytes, showing that the dividing cells were more sensitive to the effects of WA than non-dividing postconuent preadipocytes. In mature adipocytes WA caused a decrease in viability followed by an increase in apoptosis. Although mature adipocytes are considered a terminal phase during adipocyte differentiation, a precise stage beyond which adipocytes can be considered terminally differentiated is not clearly dened and mature adipocytes lled with lipid are reported to still be capable of cell division [20]. Hence it is possible that induction of cell death in mature adipocytes by WA might involve both inhibition of cell division and induction of apoptosis. WA-induced apoptosis in cancer cells was associated with activation of caspase 3, inhibition of NFB activity and down-regulation of Bcl2 expression [26]. WA-induced suppression of Bcl2 expression

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in mature 3T3-L1 adipocytes in the current study is in agreement with this previous report. Moreover, WA was reported to inhibit protein kinase C resulting in enhanced reactive oxygen species (ROS) generation, which in turn contributed to apoptosis induction in tumor cells [24]. Malik et al. also reported that withaferin A-induced apoptosis in human leukemia cells was characterized by early ROS generation and mitochondrial membrane potential loss, which preceded release of cytochrome c, translocation of Bax to mitochondria and apoptosis inducing factor to cell nuclei [15]. Interestingly, in our study, WA did not stimulate ROS generation in mature 3T3-L1 adipocytes (data not shown). However, WA increased ERK1/2 phosphorylation after 6 hrs of treatment with WA in the present study. Kaileh et al. [12] showed an increase of ERK phosphorylation after treating murine brosarcoma L929sA cells with Withania somnifera. Although ERK1/2 activation is reported to stimulate cell proliferation [7], other studies indicated that ERK1/2 activation plays an important role in apoptosis induction [1,17]. Members of the mitogen activated protein kinase family and Bcl2 family regulate mitochondrial-dependent apoptosis and ERK signaling pathway functions upstream of Bcl2 downregulation [4,11]. Therefore, it is possible that induction of apoptosis by WA is mediated by altering the expression of Bcl2 family members, which in turn is dependent on ERK1/2 activation [1]. Apoptosis can be initiated via two alternative signal pathways: the extrinsic pathway (the death receptor-dependent pathway), which acts through death receptors on cell surfaces, and the intrinsic pathway (the mitochondria-dependent pathway), which acts through the mitochondria. The mitochondrial death pathway is controlled by members of the Bcl2 family, including the Bcl2 and Bax. Bcl2 can suppress cell death induced by a variety of stress applications. Although the mechanism of Bcl2 antiapoptotic function is not clear, it is postulated that Bcl2 may help maintain mitochondrial integrity and block the activation of caspase cascade that leads to apoptosis. Bcl2 family can be also involved with an extrinsic pathway. TNF, a major extrinsic mediator of apoptosis, binds to TNF-R1 and then this binding has been shown to lead to caspase activation via the TNF receptor-associated death domain (TRADD) and Fas-associated death domain protein (FADD). Following TNF-R1 and Fas activation in mammalian cells a balance between pro- and anti-apoptotic Bcl2 families seems to be established. The role of withaferin A as a Fas ligand could be another key to determine whether the death receptor-dependent pathway is involved in withaferin A induced apoptosis. Increased adipocyte tissue mass is also due to differentiation of preadipocytes and enhanced lipogenesis [22]. In the present study, WA caused an inhibition of lipid accumulation during the adipogenesis phase. WA decreased cell viability as well as lipid content, but calculation of lipid content/viable cell still showed a dramatic decrease in lipid accumulation. Therefore, WA decreased lipid accumulation mainly by decreasing adipogenesis without having a substantial effect on cell viability. Following induction 3T3-L1 adipocytes show a rapid increase in the expression of C/EBP , followed by expression of C/EBP and PPAR [23]. During the terminal stages of differentiation, the mRNA levels for enzymes involved in triacylglycerol metabolism like glycerol-3-phosphate dehydrogenase, fatty acid synthase, and glyceraldehyde-3-phosphate dehydrogenase, increase to a great extent [19,25]. Several reports indicated that a decrease in adipogenesis induced by natural products was associated with a decrease in the expression of PPAR and C/EBP [8,10,18]. PPAR and C/EBP are further involved in the sequential expression of adipocyte-specic protein aP2 [6]. In the current study WA decreased the expression of PPAR , C/EBP and aP2, thereby suppressing the differentiation of preadipocytes to mature adipocytes. The total number of adipocytes was once believed to be stable throughout life; but it is becoming evident that fat cells have a nite life span and can be eliminated by apoptosis [2,20,21]. The adipocyte life cycle comprises various stages, including preadipocytes (growth phase), postconuent preadipocytes (growth

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arrest), maturing preadipocytes (clonal expansion) and mature adipocytes (terminal differentiation). Adipogenesis is intricately related to adipocyte differentiation and constitutes a major role in the adipocyte life cycle. We have shown in the present study that WA induces apoptosis at various stages of the adipocyte life cycle and inhibits adipogenesis. Furthermore, the dose range of WA used in the current study is comparable to that used in other reports [15,26]. Therefore, we conclude that WA may have benecial effects in the treatment of obesity for the reduction of body fat. Acknowledgments This work was supported in part by grants from AptoTec, Inc., the Georgia Research Alliance and by the Georgia Research Alliance Eminent Scholar endowment held by CAB. References
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