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Cucurbitacin B and cucurbitacin I suppress adipocyte differentiation through inhibition of STAT3 signaling
Food and Chemical Toxicology 64 (2014) 217–224
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Cucurbitacin B and cucurbitacin I suppress adipocyte differentiation through inhibition of STAT3 signaling Cho-Rong Seo a,1, Dong Kwon Yang b,1, No-Joon Song a, Ui Jeong Yun c, A-Ryeong Gwon c, Dong-Gyu Jo c, Jae Youl Cho d, Keejung Yoon d, Jee-Yin Ahn e, Chu Won Nho f, Woo Jin Park b, Seung Yul Yang g, Kye Won Park a,⇑ a
Department of Food Science and Biotechnology, College of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea College of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea d Department of Genetic Engineering, College of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea e Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea f Functional Food Center, Korea Institute of Science and Technology, Gangneung Institute, Gangwon-do 210-340, Republic of Korea g Department of Horticulture, Sunchon National University, 413 Jungangno, Suncheon, Jeonnam 540-742, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 26 October 2013 Accepted 27 November 2013 Available online 3 December 2013 Keywords: Cucurbitacin B Adipocyte differentiation STAT3 PPARc Cucurbitacin I
a b s t r a c t Cucurbitacin B, a member of the cucurbitaceae family, can act as a STAT3 signaling inhibitor to regulate the growth of hepatocellular carcinoma. STAT3 signaling has been shown to inhibit adipocyte differentiation through C/EBPa and PPARc. Based on these studies, we hypothesized that cucurbitacin B would prevent PPARc mediated adipocyte differentiation through STAT3 signaling. To test this hypothesis, mesenchymal C3H10T1/2 and 3T3-L1 preadipocyte cells were treated with a sub-cytotoxic concentration of cucurbitacin B. Cucurbitacin B treatment inhibits lipid accumulation and expression of adipocyte markers including PPARc and its target genes in a dose-dependent manner. Cucurbitacin B treatment impairs STAT3 signaling as manifested by reduced phosphorylation of STAT3 and suppression of STAT3 target gene expression in preadipocytes. The anti-adipogenic effects of cucurbitacin B are significantly blunted in cells with STAT3 silenced by introducing small interfering RNA. Finally, our data show that cucurbitacin I, another cucurbitacin family member, also inhibits adipocyte differentiation by suppressing STAT3 signaling. Together, our data suggest the possibility of utilizing cucurbitacins as a new strategy to treat metabolic diseases and implicate STAT3 as a new target for the development of functional foods and drugs. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The global epidemic of obesity is characterized by accumulation of excess fat in the body (Kopelman, 2000). Excess fat in obese individuals can be generated from excess calories by lipogenesis and stored in the body’s metabolic tissues resulting in metabolic diseases (Kahn and Flier, 2000; Spiegelman and Flier, 2001). Bioactive compounds isolated from edible foods and herbal extracts are extensively studied as alternatives and tools for identification of new molecular targets for anti-obesity (Hasani-Ranjbar et al., 2009). Resveratrol found in grapes, red wine, and nuts increases lipolysis, lowers body fat, and mimics calorie restriction (Baur et al., 2006; Howitz et al., 2003). Recent investigation on resveratrol also highlighted Sirtuin 1 (Sirt1) (Picard et al., 2004) and ⇑ Corresponding author. Tel.: +82 31 290 7804; fax: +82 31 290 7882. 1
E-mail address: [email protected] (K.W. Park). These authors contributed equally.
0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.11.040
phosphodiesterase 4 (PDE4) (Park et al., 2012) as potential molecular targets for metabolic diseases. Sirt1 senses the nutritional status of metabolic tissues and acts as a metabolic master regulator (Feige et al., 2008). Competitive inhibition of cAMP-degrading phosphodiesterase by resveratrol activates AMP-activated protein kinase, resulting in protection against metabolic diseases associated with aging (Park et al., 2012). Thus, studies on signaling pathways targeted by bioactive molecules can illuminate new players in metabolism and alternative future strategies for obesity-related metabolic diseases. Signal transducer and activator of transcription 3 (STAT3) signaling has been shown to play roles in cancer progression, inflammation, stem cell self-renewal, and differentiation (Debnath et al., 2012). STAT3 is phosphorylated and activated by cytokines and growth factors binding to the epidermal growth factor, fibroblast growth factor receptors, hepatocyte growth factor receptors, platelet growth factor receptor, and vascular endothelial growth factor receptor (Grivennikov and Karin, 2010). In contrast, the
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transforming growth factor-b receptor pathway inhibits STAT3 signaling at least in part by suppressing STAT3 phosphorylation (Starsichova et al., 2010). Once STAT3 is phosphorylated, it induces the transcription of genes involved in cell cycle progression, angiogenesis, and apoptosis. Knockdown of STAT3 using STAT3 small interfering RNA (siRNA) resulted in induction of Fas and Fas-Associated protein with Death Domain (FADD) expression, indicating that STAT3 also represses the expression of certain genes (Clarkson et al., 2006). Interestingly, a recent study showed that STAT3 mediates adipogenesis through activation of CCAAT/enhancer-binding protein a (C/EBPa) and peroxisome proliferator-activated receptor c (PPARc) expression (Zhang et al., 2011). Therefore, modulation of STAT3 levels can be a useful strategy against metabolic diseases in addition to various STAT3-associated diseases (Debnath et al., 2012). STAT3 signaling plays roles in adipocyte differentiation through inhibition of critical adipogenic transcription factors, C/EBPa and PPARc (Zhang et al., 2011). Cucurbitacin family members found in pumpkins, cucumbers, and melons have been shown to act as STAT3 inhibitors in certain cancer cells (Chambliss and Jones, 1966; Chan et al., 2010; Sun et al., 2010; van Kester et al., 2008). However, the role of cucurbitacin family members in adipocyte differentiation has not been investigated (Chen et al., 2005). Based on these observations, we hypothesized that Cucurbitacin B would impair adipogenesis through suppression of STAT3 signaling. To test this, the commonly used mesenchymal C3H10T1/2 and 3T3L1 preadipocyte cells were differentiated into adipocytes and treated with sub-cytotoxic concentrations of Cucurbitacin B. In this study, we demonstrate a new role of cucurbitacin B and cucurbitacin I in adipogenesis and further determine the underlying mechanism of how these cucurbitacins exert inhibitory effects in adipocyte differentiation.
2. Materials and methods 2.1. Cell culture Cucurbitacins and the STAT3 inhibitor WP-1066 were purchased from Sigma Aldrich (St. Louis, MO) and dissolved in dimethyl sulphoxide (DMSO) (Sigma, St. Louis, MO). Mouse 3T3-L1 and C3H10T1/2 cell lines were purchased from the American Type Culture Collection (Rockville, MD). C3H10T1/2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone, Logan, UT) supplemented with 10% fetal bovine serum (FBS) (Hyclone) and antibiotics (penicillin and streptomycin, Hyclone), and were incubated at 37 °C under 5% CO2. 3T3-L1 preadipocytes were cultured in DMEM supplemented with 10% fetal calf serum (Hyclone). Mouse embryonic fibroblasts (MEFs) were freshly isolated from E13.5-E14.5 embryos. For adipocyte differentiation, cells were seeded at 5 104/ml in 6-well tissue culture plates, and confluent cells were incubated for 2 days in DMEM supplemented with 10% FBS, 1 lM dexamethasone (Sigma, St. Louis, MO), 0.5 mM isobutyl-1-methylxanthine (Sigma), and 5 lg/ml insulin (Sigma). Cells were refreshed with DMEM containing 10% FBS and 5 lg/ml insulin every 2 days. GW7845 (20 nM, a PPARc ligand, kindly provided by the Tontonoz Lab) was further supplemented for the adipocyte differentiation of C3H10T1/2 cells and MEFs. After differentiation, cells were fixed with 4% paraformaldehyde in PBS at room temperature for 4 h, and stained with 0.5% Oil Red O (Sigma) in a mixture of isopropanol and distilled water at a 3:2 ratio for 45 min. Cells were washed with water, and photographed under a microscope. To quantify the intracellular triglyceride content, stained cells from at least two independent experiments were resolved with isopropanol and measured with a spectrophotometer at 520 nm.
2.2. Cell viability assays Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louise, MO) assay. 3T3-L1 cells were seeded at 1.5 104 cells per well in 96-well plates and incubated in culture medium until 70–80% confluence. After the cells reached confluence, cells were treated with 100, 200, 300 nM of cucurbitacin B in triplicate. After 24, 48, and 72 h, MTT (5 mg/mL in PBS) was added and cells were incubated at 37 °C for an additional 4 h. The formazan crystals were dissolved in 200 lL DMSO and absorbance was measured at 520 nm using a microplate reader.
2.3. Expression analysis Total RNA was isolated from 3T3-L1 cells using TRIzol reagent (Invitrogen, Carlsbad, CA). First-strand cDNA (cDNA) was synthesized from 0.5 lg of total RNA using the AMV Reverse Transcription System kit (Promega, Madison, WI) with random primers. After cDNA synthesis, the final 25 ll volume of the amplification mixture containing Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), primers, and cDNA was subjected to 40 amplification cycles of polymerase chain reaction (PCR) using a Thermal Cycler Dice (Takara, Shiga, Japan). Expression was normalized to 36B4. All real-time PCRs were performed at least twice. 4cycle threshold (CT) was used to calculate the differences between the target CT value and the control (36B4) for each sample: 4CT = CT(target)-CT(control). The relative expression level was calculated using 2DCT. The oligonucleotide primer (Integrated DNA Technologies, San Diego, CA) sequences used for PCR were as follows: peroxisome proliferator-activated receptor c (PPARc), PPARc F, 50 -CCATTCTGGCCCACCAAC-30 and PPARc R, 50 -AATGCGAGTGGTCTTCCATCA-30 ; adipocyte binding protein 2 (aP2), aP2 F, 50 -CACCGCAGACGACAGGAAG-30 and aP2 R, 50 - GCACCTGCACCAGGGC-30 ; cluster of differentiation 36 (CD36), CD36 F, 50 -GGCCAAGCTAT TGCGACAT-30 and CD36 R, 50 -CAGATCCGAACACAGCGTAGA-30 ; CCAAT-enhancerbinding proteins (C/EBPa), C/EBPa F, 50 -GCGGGCAAAGCCAAGAA-30 and C/EBPa R, 50 -GCGTTCCCGCCGTACC-30 ; adiponectin F, 50 -CCGGAACCCCTG GCAG-30 and adiponectin R, 50 -CTGAACGCTGAGCGATACACA-30 ; acidic ribosomal phosphoprotein P0 (36B4), 36B4 F, 50 -AGATGCAGCAGATCCGCAT-30 and 36B4 R, 50 -GTTCTTGCCCATCAGCACC-30 . Western blotting was performed as previously described (Song et al., 2013). Cells were harvested in 200 ll of sample buffer and heated at 100 °C for 10 min. The proteins were separated by 10% SDS–PAGE and electrophoretically transferred to nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ). Membranes were blocked with blocking buffer (5% skim milk in TBS, 0.1% Tween 20) for 30 min at room temperature and treated with STAT3, phosphorylated STAT3 (PSTAT3) (sc-7196; Santa Cruz Biotechnology, Santa Cruz, CA), and b-actin antibodies (A5316; Sigma), followed by incubation with horseradish peroxidase-conjugated secondary antibody (Zymed Laboratories, San Francisco, CA). Antibody binding was detected on X-ray film using Enhanced Chemiluminescence Western Blotting Detection Reagent (Amersham Biosciences). 2.4. STAT3 inhibition studies Scramble control, signal transducer and activator of transcription 3 (STAT3)specific oligos were synthesized by Genolution Pharmaceuticals, Inc. (Seoul, Korea). Two independent small interfering RNAs (siRNAs) were used to silence STAT3 expression. The sense sequences of STAT3-specific siRNA are as follows: Stat3 #1: 50 -GAGU UGAAUUAUCAGCUUAUU-30 ; Stat3 #2: 50 -CAUCAAUCCUGUGGUAUA AUU-30 . The sense sequence of control nonspecific scramble RNA is 50 -CC UCGUGCCGUUCCAUCAGG UAGUU-30 . Cells plated at a density of 1 105 cells per well in a 6-well plate were transfected with 50 nmol of scrambled RNA, STAT3-specific siRNA using RNAiMAX (Invitrogen), as previously described (Song et al., 2013; Zhang et al., 2011). Cells were treated with siRNA for 8 h, and the medium was exchanged. After 48 h, cells were processed using differentiation protocols. Transfection was carried out in duplicated wells and repeated three times. For WP-1066 (STAT3 inhibitor, Calbiochem, Gibbstown, NJ) treatment experiments, C3H10T1/2 cells were treated with 10 lM in the presence of Cu B or Cu I and then allowed to differentiate to adipocytes for 5 days. 2.5. Statistical analysis Data are presented as the means ± SEM. Differences in gene expression and lipid accumulation were analyzed using a two-tailed unpaired Student’s t-test or using analysis of variance followed by Student–Newman–Keuls tests. Statistical significance was defined as P < 0.05. All computations were performed using statistical analysis software (PASW Statistics 17).
3. Results 3.1. Cucurbitacin B inhibits lipid accumulation during adipocyte differentiation Since cucurbitacin family members including cucurbitacin B have previously been shown to have cytotoxic effects in certain cancer cells (Lee et al., 2010; Thoennissen et al., 2009), the potential cytotoxic effect of cucurbitacin B in preadipocytes was evaluated (Fig. 1). Treatment of 3T3-L1 cells with 100–300 nM cucurbitacin B for 24 h did not significantly affect cell viability (Fig. 1B). Similarly, cucurbitacin B treatment for 48 and 72 h did not decrease cell numbers (Fig. 1C and D). Thus, we used cucurbitacin B at 100–300 nM to assess its effects in adipocyte differentiation.
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Fig. 1. Cell viability of 3T3-L1 cells with cucurbitacin B (Cu B) treatment at the indicated concentrations. (A) Chemical structure of cucurbitacin B (25-acetoxy-2b,16a,20btrihydroxy-9b-methyl-19nor-10a-lanosta-5,23-diene-3,11,22-trione). (B-D) Cell viability in 3T3-L1 cells was measured upon DMSO (ctrl) or cucurbitacin B (Cu B) treatment (100, 200, and 300 nM) for 24 h (B), 48 h (C), and 72 h (D). Values are expressed as the means ± SEM from three independent experiments.
To test our hypothesis of cucurbitacin B as an anti-adipogenic compound, preadipocyte 3T3-L1 cells were treated with cucurbitacin B and differentiated into adipocytes under adipogenic conditions for 5 days. As expected, 20 lM resveratrol, a well known anti-adipogenic compound, significantly suppressed lipid accumulation as assessed by Oil Red O staining. Remarkably, 200–300 nM cucurbitacin B treatment inhibited lipid accumulation more potently than 20 lM resveratrol treatment (Fig. 2A and C). To further confirm the anti-adipogenic effects, mesenchymal C3H10T/12 cells were differentiated into adipocytes. Consistently, cucurbitacin B also suppressed lipid accumulation in C3H10T1/2 cells (Fig. 2B), further indicating the anti-lipogenic effects of cucurbitacin B during adipocyte differentiation. 3.2. Cucurbitacin B suppresses the expression of adipocyte markers Adipocyte differentiation is dictated by the master regulator PPARc followed by induction of target genes involved in triglyceride synthesis and preadipocyte differentiation into adipocytes. 3T3-L1 cells were differentiated into adipocytes in the presence of 100–300 nM cucurbitacin B and the expression of adipocyte markers were measured by real-time PCR. Similar to the effects on lipid accumulation, the mRNA expression of PPARc, aP2, C/ EBPa, adiponectin, and CD36 was significantly suppressed in a dose-dependent manner after 5 days of adipocyte differentiation (Fig. 2D). The effects of cucurbitacin B on lipid accumulation and expression of adipocyte markers indicate that cucurbitacin B is a natural anti-adipogenic compound.
expression of C/EBPd and Cyclin D1 and suppress the expression of KLF5 (Clarkson et al., 2006; Song et al., 2013). Consistently, cucurbitacin B treatment of 3T3-L1 cells reduced the expression of C/EBPd and Cyclin D1 but induced the expression of KLF5 (Fig. 3A). Similar regulatory responses for the expression of STAT3 target genes by cucurbitacin B was also observed in C3H10T1/2 cells (Fig. 3B). Resveratrol treatment, however, did not affect the expression of these genes, providing support that cucurbitacin B has specific effects on STAT3 in preadipocytes (Fig. 3B). Since activation of STAT3 signaling by various cascades induces STAT3 phosphorylation, the levels of STAT3 phosphorylation can be considered a surrogate marker of STAT3 activation (Lee et al., 2010). To directly investigate the effects of cucurbitacin B on STAT3 activity, STAT3 phosphorylation upon cucurbitacin B treatment was examined. Preadipocyte 3T3-L1 cells were treated with cucurbitacin B and probed against a STAT3 phospho-specific antibody to measure the levels of phosphorylated STAT3. The phosphorylated STAT3 was normalized to the total amount of STAT3 protein. Consistent with inhibitory effects on STAT3, cucurbitacin B treatment for 12 h reduced the levels of phosphorylated STAT3 (Fig. 3C). Cucurbitacin B suppressed the phosphorylation levels to 50% compared to DMSO treatments in a quantification analysis (Fig. 3D). Therefore, the STAT3 inhibition by cucurbitacin B can suppress the levels of phosphorylated STAT3 followed by inhibition on C/ EBPa and PPARc. Thus, analysis of target gene expression and STAT3 phosphorylation indicate that cucurbitacin B has regulatory effects on STAT3 signaling in preadipocytes.
3.3. Cucurbitacin B inhibits STAT3 signaling in preadipocytes
3.4. STAT3 signaling may be the key to the anti-adipogenic effects of cucurbitacin B
The inhibitory effects of cucurbitacin B on adipogenesis prompted us to investigate the molecular mechanism of cucurbitacin B action in preadipocytes. Previous studies showed that cucurbitacin B can suppress the cell cycle in cancer cells through impairment of STAT3 signaling (Thoennissen et al., 2009). Therefore, we hypothesized that STAT3 signaling may mediate the inhibitory effects of cucurbitacin B during adipocyte differentiation. To investigate this possibility, preadipocyte 3T3-L1 cells were treated with cucurbitacin B and the effects on STAT3 signaling were assessed. Activation of STAT3 signaling has been shown to induce
To demonstrate that STAT3 signaling is required for the antiadipogenic actions of cucurbitacin B, the anti-adipogenic effects of cucurbitacin B treatment were assessed in cells with STAT3 expression abrogated by the introduction of small interfering RNAs (siRNA). We employed two independent previously verified siRNAs that effectively silence STAT3 mRNA expression (Song et al., 2013; Zhang et al., 2011). 3T3-L1 cells were transfected with control scrambled siRNA or STAT3-targeted siRNAs and cells were differentiated into adipocytes. As shown previously, STAT3 expression was successfully reduced in the cells transfected with the two
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Fig. 2. Cucurbitacin B inhibits adipocyte differentiation of 3T3-L1 and C3H10T1/2 cells. (A) 3T3-L1 cells were treated with cucurbitacin B (Cu B) for 5 days during adipocyte differentiation. Oil red O staining was performed to assess lipid accumulation. An anti-adipogenic compound, resveratrol (20 lM, RSV) and DMSO (ctrl) were used as controls. (B) C3H10T1/2 cells were treated with various concentrations of Cu B (100, 200, and 300 nM) and induced to differentiate, followed by Oil Red O staining. (C) Lipid accumulation in 3T3-L1 cells was quantified by measuring the extracted Oil red O dye at 520 nM. (D) Cu B suppresses the expression of adipocyte markers. 3T3-L1 cells were treated with the indicated concentrations of Cu B for 5 days during adipocyte differentiation and mRNA expression of adipogenic genes encoding PPARc, aP2, C/EBPa, adiponectin, and CD36 was measured by real-time PCR. Data shown represent the mean ± SEM and are representative of three independent experiments. Mean with different letters at each samples are significantly different (P < .05) by Student–Newman–Keuls test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
independent siRNAs compared to control cells transfected with scrambled siRNA (Fig. 4A). Lipid accumulation was also reduced in STAT3 siRNA-transfected cells. As expected, cucurbitacin B treatment of control scrambled siRNA (sc)-transfected cells suppressed lipid accumulation. However, the anti-lipogenic effects of cucurbitacin B in STAT3 siRNA-transfected cells were significantly compromised compared to the effects in scrambled siRNA (sc)-transfected control cells (Fig. 4B and C). The requirement for STAT3 signaling was further supported by the expression levels of adipocyte markers. The expression of adipocyte markers such as PPARc and C/ EBPa were reduced by cucurbitacin B in control scrambled siRNA-transfected cells, whereas the regulatory action of cucurbitacin B on the expression of these adipocyte markers was markedly blunted in STAT3 siRNA-transfected cells (Fig. 4D). These data demonstrate that STAT3, at least in part, mediates the anti-adipogenic actions of cucurbitacin B during adipocyte differentiation. 3.5. Cucurbitacin I also inhibits adipocyte differentiation Cucurbitacin is a class of triterpene hydrocarbon compounds mainly found in plants with more than 40 different variants (Chambliss and Jones, 1966). To test whether other cucurbitacins also inhibit adipocyte differentiation, another commercially available cucurbitacin family member, cucurbitacin I was selected and tested for its effects on adipocyte differentiation.
Similar to cucurbitacin B, cucurbitacin I treatment inhibited lipid accumulation in 3T3-L1 (Fig. 5A and C) and C3H10T1/2 cells (Fig. 5B). Expression of PPARc, C/EBPa, and CD36 was also suppressed by cucurbitacin I treatment during adipocyte differentiation (Fig. 5D). These data suggest that cucurbitacin I also suppress lipid accumulation. 3.6. Cucurbitacin I suppresses adipogenesis via STAT3 signaling The similar anti-adipogenic effects of cucurbitacin B and cucurbitacin I suggest common molecular actions on adipocyte differentiation of preadipocytes. The expression of STAT3 target genes were measured to show the effects of cucurbitacin I on STAT3 signaling. Cucurbitacin I treatment reversed the expression of STAT3 target genes such as Cyclin D1 and KLF5 similar to that by cucurbitacin B (Fig. 6A), suggesting the possibility of cucurbitacin I as a STAT3 inhibitor. To show STAT3 requirement for the effects of cucurbitacin I, 3T3-L1 cells were transfected with siRNAs targeted to STAT3 and differentiated into adipocytes. Cucurbitacin I treatment suppressed lipid accumulation in scrambled control siRNA-transfected cells, whereas anti-lipogenic effects of cucurbitacin I were significantly reduced in STAT3 targeted siRNA-transfected cells (Fig. 6B). These data show that the anti-adipogenic effects of cucurbitacin I can also be mediated by STAT3 signaling and further raise the
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Fig. 3. Cucurbitacin B inhibits STAT3 activity in preadipocytes. (A) Expression of STAT3 target genes are regulated by Cu B in 3T3-L1 cells. 3T3-L1 cells were treated with DMSO (ctrl), 200 nM, 300 nM cucurbitacin B (Cu B) for 12 h and the expression of STAT3 target genes was assessed. C/EBPd and Cyclin D1 (CycD1), known STAT3-induced genes, were suppressed by Cu B treatment. Expression of KLF5, a gene known to be downregulated by STAT3, was induced by Cu B. (B) STAT3 target genes are selectively regulated by Cu B in C3H10T1/2 cells. C3H10T1/2 cells were treated with Cu B (200 nM) or resveratrol (RSV, 20 lM) for 12 h and the expression of STAT3 target genes was measured. STAT3 target genes were regulated by Cu B but not by resveratrol treatment. (C-D) Cu B suppresses STAT3 phosphorylation (P-STAT3) levels in 3T3-L1 cells. 3T3-L1 cells were treated for 12 h with 100 nM or 300 nM of Cu B and protein expression was measured by immunoblot analysis (C) followed by the measurement of relative intensities of P-STAT3/STAT3 (D). Immunoblotting was performed using anti-STAT3 and anti-phopho-Tyr STAT3 antibodies and were quantified by NIH Image J software. Numbers indicate fold changes relative to controls. The b-Actin antibody was probed to verify equal protein loading. Data shown represent the mean ± SEM and are representative of three independent experiments. Statistical significance was determined relative to the control by the Student’s t-test (P < 0.05; P < 0.005; P < 0.0005).
Fig. 4. STAT3 mediates the anti-adipogenic effects of Cucurbitacin B . (A) Transient transfection of 3T3-L1 cells with two independent STAT3-targeted siRNAs (si#1 or si#2) effectively reduced STAT3 expression. (B–C) Silencing the expression of STAT3 blunted the inhibitory effects of cucurbitacin B (Cu B) on adipocyte differentiation in 3T3-L1 cells. Control scrambled (sc) and STAT3 targeted siRNA-transfected cells (si#1 and si#2) were treated with DMSO or Cu B (100 nM) and then differentiated for 5 days. Differentiated cells were stained with Oil Red O (B) followed by quantification (C). (D) Knockdown of STAT3 attenuated the effects of Cu B on expression of adipocyte markers. Expression of PPARc and C/EBPa in control scrambled and STAT3-specific siRNA-transfected cells treated with DMSO (ctrl) or Cu B (100 nM) for 5 days was measured by realtime PCR. Representative data from three experiments are shown. Values are means ± SD. Statistically significant differences in gene expression upon CuB treatment between the control and STAT3 siRNA-transfected cells were determined using Student’s t-test (NS, not significant; P < 0.05; P < 0.005; P < 0.0005). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
possibility that other cucurbitacin family members are also antiadipogenic compounds. We also examined whether STAT3 acts as a mediator of the anti-adipogenic actions of cucurbitacins in the presence of STAT3
pharmacological inhibitor. The degree of inhibition of lipid accumulation in response to cucurbitacin B and I treatment was significantly reduced in STAT3-inhibitor (WP-1066) treated cells compared to DMSO-treated controls, further corroborating that
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Fig. 5. Cucurbitacin I also inhibits adipocyte differentiation. (A) 3T3-L1 cells were treated with the indicated concentrations of cucurbitacin I (Cu I) for 5 days during adipocyte differentiation. Oil red O staining was performed to visualize lipid accumulation. Resveratrol (20 lM, RSV) and DMSO (ctrl) were used as controls. (B) C3H10T1/2 cells were treated with various concentrations of Cu I (300, 400, and 500 nM) and induced to differentiate, followed by Oil Red O staining. (C) Lipid accumulation in 3T3-L1 cells was quantified by measuring the extracted Oil red O dye at 520 nM. Data shown represent the mean ± SEM from three independent experiments. Mean with different letters at each samples are significantly different (P < .05) by Student–Newman–Keuls test. (D) Cu I suppresses the expression of adipocyte markers. 3T3-L1 cells were treated with the indicated concentrations of Cu I for 5 days during adipocyte differentiation and mRNA expression of adipogenic genes encoding PPARc, C/EBPa, and CD36 was measured by real time PCR. Representative data from three experiments are shown. Statistically significant differences in gene expression was determined relative to the control (DMSO) by the Student’s t-test (P < 0.05; P < 0.005; P < 0.0005). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the inhibitory action of both cucurbitacin B and I in adipocyte differentiation is mediated through inhibition of STAT3 (Fig. 6C). To further verify the similarity of the anti-lipogenic effects of cucurbitacin B and I, primary mouse embryonic fibroblasts were freshly isolated from mouse embryos and induced to differentiate into adipocytes in the presence of cucurbitacin B or cucurbitacin I (Fig. 6D). Both cucurbitacin B and I similarly suppressed expression of PPARc, aP2, C/EBPa, and CD36 although cucurbitacin B seemed to have more potent effects. These data raise the possibility that the similar biological actions and underlying molecular mechanisms of other cucurbitacin family members in adipocyte differentiation.
4. Discussion STAT3 signaling is required for adipocyte differentiation (Zhang et al., 2011). Thus, we investigated the possibility that cucurbitacin B, a STAT3 inhibitor in certain cancer cells, would also inhibit adipocyte differentiation of preadipocytes. In this study, we show for the first time the anti-adipogenic effects of cucurbitacin B in multipotent C3H10T1/2, preadipocyte 3T3-L1, and freshly isolated primary cells. We also present evidence that cucurbitacin B acts on STAT3 signaling. Furthermore, our data show that STAT3 signaling is at least in part essential for the anti-adipogenic actions of cucurbitacin B. More than 40 members of the cucurbitacin family have been isolated from the cucurbitaceae family and others including cucumber, pumpkins, gourds, some mushrooms, and marine mollusks (Alghasham, 2013). Cucurbitacins have an impact on various
biological activities, showing anti-tumor, anti-inflammatory, and anti-infectious effects. Most cucurbitacins, including A, B, E, I, and Q, have been shown to affect cancer cell growth, whereas cucurbitacin B and E are effective in radical and oxidative damage (Tannin-Spitz et al., 2007). Cucurbitacin R and dihydrocucurbitacin B are also known for their efficacy on the immune system (Rios, 2010). However, until the present study, anti-adipogenic activity of cucurbitacin family members has not been implied. Thus, it should be interesting to investigate in future studies whether other cucurbitacin family members also possess anti-lipogenic activity and act though STAT3 signaling. Our current data suggest the possibility that cucurbitacins at least cucurbitacin B and I, if not all members of the cucurbitacin family, could be beneficial in preventing lipid accumulation and metabolic diseases. These data further raise questions whether cucurbitacin containing herbal products such as cucumber and pumpkins may be effective against obesity and related diseases. Interestingly, extracts from cucurbita moschata have been shown to decrease body weight and fat storage in high fat diet-induced obese mouse models (Choi et al., 2007). Thus, it is possible that cucurbitacins may partly mediate the anti-obese actions of cucurbita moschata. The possible anti-lipogenic effects of cucurbitacin B and I should be studied in animal models and humans before addressing these further questions. In conclusion, we show that cucurbitacin B inhibits adipocyte differentiation of multiple adipogenic cells. Mechanism studies indicate that cucurbitacin B exerts its inhibitory effects on adipogenesis through the inhibition of STAT3 signaling. We further show that another family member, cucurbitacin I, also possesses anti-adipogenic activity. Therefore, our data provide evidence for
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Fig. 6. The anti-adipogenic effect of cucurbitacin I is also mediated through STAT3 signaling. (A) Expression of STAT3 target genes are regulated by cucurbitacin I (Cu I) in 3T3L1 cells. 3T3-L1 cells were treated with DMSO (ctrl) or Cu I (300, 400 nM) for 12 h and the expression of STAT3 target genes assessed. Similar to the effects of Cu B, expression of Cyclin D1 (CycD1) and KLF5 were regulated by treatment with Cu I. (B) Cucurbitacin I (Cu I) acts through STAT3 signaling. Silencing STAT3 compromised the inhibitory effects of Cu I treatment on lipid accumulation in 3T3-L1 cells. Control scrambled (sc) and siRNA-transfected cells (si#2) were treated with DMSO or Cu I (400 nM) for 5 days during adipocyte differentiation. Differentiated cells were stained with Oil Red O followed by quantification. (C) Pharmacological disruption of STAT3 signaling blunts the inhibitory effects of both Cu B and Cu I on lipid accumulation. C3H10T1/2 cells were treated with 10 lM WP-1066 (STAT3 inhibitor) and differentiated in the presence of Cu B (200 nM) or Cu I (400 nM) for 5 days, and quantified by measuring the Oil red O dye extracted in isopropanol at 520 nm. (D) Both Cu B and Cu I inhibit the expression of adipocyte markers in primary mouse embryonic fibroblasts. Primary mouse embryonic fibroblasts were treated with 300 nM of Cu B and Cu I for 10 days and the mRNA expression of adipogenic genes encoding PPARc, aP2, C/EBP, and CD36 was measured by real-time PCR. Representative data from three experiments are shown. Values are means ± SD. Statistical significance was determined by comparison to the control using Student’s t-test (NS, not significant; P < 0.05; P < 0.005; P < 0.0005).
the previously unidentified role of cucurbitacin family members in adipogenesis, and further raise the possibility that cucurbitacins may be a potential strategy for the prevention of metabolic diseases in humans. 5. Conflict of Interest The authors have declared that there is no conflict of interest. Acknowledgments This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. 2011-0014302). This study was also supported by a grant (No. PJ008777) from the Rural development Administration and Jeonnam Agricultural Research & Extension Services, Republic of Korea. References Alghasham, A.A., 2013. Cucurbitacins – a promising target for cancer therapy. Int. J. Health Sci. (Qassim). 7, 77–89. Baur, J.A., Pearson, K.J., Price, N.L., Jamieson, H.A., Lerin, C., Kalra, A., Prabhu, V.V., Allard, J.S., Lopez-Lluch, G., Lewis, K., Pistell, P.J., Poosala, S., Becker, K.G., Boss, O., Gwinn, D., Wang, M., Ramaswamy, S., Fishbein, K.W., Spencer, R.G., Lakatta, E.G., Le Couteur, D., Shaw, R.J., Navas, P., Puigserver, P., Ingram, D.K., de Cabo, R., Sinclair, D.A., 2006. Resveratrol improves health and survival of mice on a highcalorie diet. Nature 444, 337–342. Chambliss, O.L., Jones, C.M., 1966. Cucurbitacins: specific insect attractants in Cucurbitaceae. Science 153, 1392–1393. Chan, K.T., Li, K., Liu, S.L., Chu, K.H., Toh, M., Xie, W.D., 2010. Cucurbitacin B inhibits STAT3 and the Raf/MEK/ERK pathway in leukemia cell line K562. Cancer Lett. 289, 46–52.
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Song, N.J., Yoon, H.J., Kim, K.H., Jung, S.R., Jang, W.S., Seo, C.R., Lee, Y.M., Kweon, D.H., Hong, J.W., Lee, J.S., Park, K.M., Lee, K.R., Park, K.W., 2013. Butein is a novel antiadipogenic compound. J. Lipid Res. 54, 1385–1396. Spiegelman, B.M., Flier, J.S., 2001. Obesity and the regulation of energy balance. Cell 104, 531–543. Starsichova, A., Lincova, E., Pernicova, Z., Kozubik, A., Soucek, K., 2010. TGF-beta1 suppresses IL-6-induced STAT3 activation through regulation of Jak2 expression in prostate epithelial cells. Cell. Signal. 22, 1734–1744. Sun, C., Zhang, M., Shan, X., Zhou, X., Yang, J., Wang, Y., Li-Ling, J., Deng, Y., 2010. Inhibitory effect of cucurbitacin E on pancreatic cancer cells growth via STAT3 signaling. J. Cancer Res. Clin. Oncol. 136, 603–610. Tannin-Spitz, T., Bergman, M., Grossman, S., 2007. Cucurbitacin glucosides: antioxidant and free-radical scavenging activities. Biochem. Biophys. Res. Commun. 364, 181–186.
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Cucurbitacin B and cucurbitacin I suppress adipocyte differentiation through inhibition of STAT3 signaling Cho-Rong Seo a,1, Dong Kwon Yang b,1, No-Joon Song a, Ui Jeong Yun c, A-Ryeong Gwon c, Dong-Gyu Jo c, Jae Youl Cho d, Keejung Yoon d, Jee-Yin Ahn e, Chu Won Nho f, Woo Jin Park b, Seung Yul Yang g, Kye Won Park a,⇑ a
Department of Food Science and Biotechnology, College of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea College of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea d Department of Genetic Engineering, College of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea e Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea f Functional Food Center, Korea Institute of Science and Technology, Gangneung Institute, Gangwon-do 210-340, Republic of Korea g Department of Horticulture, Sunchon National University, 413 Jungangno, Suncheon, Jeonnam 540-742, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 26 October 2013 Accepted 27 November 2013 Available online 3 December 2013 Keywords: Cucurbitacin B Adipocyte differentiation STAT3 PPARc Cucurbitacin I
a b s t r a c t Cucurbitacin B, a member of the cucurbitaceae family, can act as a STAT3 signaling inhibitor to regulate the growth of hepatocellular carcinoma. STAT3 signaling has been shown to inhibit adipocyte differentiation through C/EBPa and PPARc. Based on these studies, we hypothesized that cucurbitacin B would prevent PPARc mediated adipocyte differentiation through STAT3 signaling. To test this hypothesis, mesenchymal C3H10T1/2 and 3T3-L1 preadipocyte cells were treated with a sub-cytotoxic concentration of cucurbitacin B. Cucurbitacin B treatment inhibits lipid accumulation and expression of adipocyte markers including PPARc and its target genes in a dose-dependent manner. Cucurbitacin B treatment impairs STAT3 signaling as manifested by reduced phosphorylation of STAT3 and suppression of STAT3 target gene expression in preadipocytes. The anti-adipogenic effects of cucurbitacin B are significantly blunted in cells with STAT3 silenced by introducing small interfering RNA. Finally, our data show that cucurbitacin I, another cucurbitacin family member, also inhibits adipocyte differentiation by suppressing STAT3 signaling. Together, our data suggest the possibility of utilizing cucurbitacins as a new strategy to treat metabolic diseases and implicate STAT3 as a new target for the development of functional foods and drugs. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The global epidemic of obesity is characterized by accumulation of excess fat in the body (Kopelman, 2000). Excess fat in obese individuals can be generated from excess calories by lipogenesis and stored in the body’s metabolic tissues resulting in metabolic diseases (Kahn and Flier, 2000; Spiegelman and Flier, 2001). Bioactive compounds isolated from edible foods and herbal extracts are extensively studied as alternatives and tools for identification of new molecular targets for anti-obesity (Hasani-Ranjbar et al., 2009). Resveratrol found in grapes, red wine, and nuts increases lipolysis, lowers body fat, and mimics calorie restriction (Baur et al., 2006; Howitz et al., 2003). Recent investigation on resveratrol also highlighted Sirtuin 1 (Sirt1) (Picard et al., 2004) and ⇑ Corresponding author. Tel.: +82 31 290 7804; fax: +82 31 290 7882. 1
E-mail address: [email protected] (K.W. Park). These authors contributed equally.
0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.11.040
phosphodiesterase 4 (PDE4) (Park et al., 2012) as potential molecular targets for metabolic diseases. Sirt1 senses the nutritional status of metabolic tissues and acts as a metabolic master regulator (Feige et al., 2008). Competitive inhibition of cAMP-degrading phosphodiesterase by resveratrol activates AMP-activated protein kinase, resulting in protection against metabolic diseases associated with aging (Park et al., 2012). Thus, studies on signaling pathways targeted by bioactive molecules can illuminate new players in metabolism and alternative future strategies for obesity-related metabolic diseases. Signal transducer and activator of transcription 3 (STAT3) signaling has been shown to play roles in cancer progression, inflammation, stem cell self-renewal, and differentiation (Debnath et al., 2012). STAT3 is phosphorylated and activated by cytokines and growth factors binding to the epidermal growth factor, fibroblast growth factor receptors, hepatocyte growth factor receptors, platelet growth factor receptor, and vascular endothelial growth factor receptor (Grivennikov and Karin, 2010). In contrast, the
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transforming growth factor-b receptor pathway inhibits STAT3 signaling at least in part by suppressing STAT3 phosphorylation (Starsichova et al., 2010). Once STAT3 is phosphorylated, it induces the transcription of genes involved in cell cycle progression, angiogenesis, and apoptosis. Knockdown of STAT3 using STAT3 small interfering RNA (siRNA) resulted in induction of Fas and Fas-Associated protein with Death Domain (FADD) expression, indicating that STAT3 also represses the expression of certain genes (Clarkson et al., 2006). Interestingly, a recent study showed that STAT3 mediates adipogenesis through activation of CCAAT/enhancer-binding protein a (C/EBPa) and peroxisome proliferator-activated receptor c (PPARc) expression (Zhang et al., 2011). Therefore, modulation of STAT3 levels can be a useful strategy against metabolic diseases in addition to various STAT3-associated diseases (Debnath et al., 2012). STAT3 signaling plays roles in adipocyte differentiation through inhibition of critical adipogenic transcription factors, C/EBPa and PPARc (Zhang et al., 2011). Cucurbitacin family members found in pumpkins, cucumbers, and melons have been shown to act as STAT3 inhibitors in certain cancer cells (Chambliss and Jones, 1966; Chan et al., 2010; Sun et al., 2010; van Kester et al., 2008). However, the role of cucurbitacin family members in adipocyte differentiation has not been investigated (Chen et al., 2005). Based on these observations, we hypothesized that Cucurbitacin B would impair adipogenesis through suppression of STAT3 signaling. To test this, the commonly used mesenchymal C3H10T1/2 and 3T3L1 preadipocyte cells were differentiated into adipocytes and treated with sub-cytotoxic concentrations of Cucurbitacin B. In this study, we demonstrate a new role of cucurbitacin B and cucurbitacin I in adipogenesis and further determine the underlying mechanism of how these cucurbitacins exert inhibitory effects in adipocyte differentiation.
2. Materials and methods 2.1. Cell culture Cucurbitacins and the STAT3 inhibitor WP-1066 were purchased from Sigma Aldrich (St. Louis, MO) and dissolved in dimethyl sulphoxide (DMSO) (Sigma, St. Louis, MO). Mouse 3T3-L1 and C3H10T1/2 cell lines were purchased from the American Type Culture Collection (Rockville, MD). C3H10T1/2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone, Logan, UT) supplemented with 10% fetal bovine serum (FBS) (Hyclone) and antibiotics (penicillin and streptomycin, Hyclone), and were incubated at 37 °C under 5% CO2. 3T3-L1 preadipocytes were cultured in DMEM supplemented with 10% fetal calf serum (Hyclone). Mouse embryonic fibroblasts (MEFs) were freshly isolated from E13.5-E14.5 embryos. For adipocyte differentiation, cells were seeded at 5 104/ml in 6-well tissue culture plates, and confluent cells were incubated for 2 days in DMEM supplemented with 10% FBS, 1 lM dexamethasone (Sigma, St. Louis, MO), 0.5 mM isobutyl-1-methylxanthine (Sigma), and 5 lg/ml insulin (Sigma). Cells were refreshed with DMEM containing 10% FBS and 5 lg/ml insulin every 2 days. GW7845 (20 nM, a PPARc ligand, kindly provided by the Tontonoz Lab) was further supplemented for the adipocyte differentiation of C3H10T1/2 cells and MEFs. After differentiation, cells were fixed with 4% paraformaldehyde in PBS at room temperature for 4 h, and stained with 0.5% Oil Red O (Sigma) in a mixture of isopropanol and distilled water at a 3:2 ratio for 45 min. Cells were washed with water, and photographed under a microscope. To quantify the intracellular triglyceride content, stained cells from at least two independent experiments were resolved with isopropanol and measured with a spectrophotometer at 520 nm.
2.2. Cell viability assays Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louise, MO) assay. 3T3-L1 cells were seeded at 1.5 104 cells per well in 96-well plates and incubated in culture medium until 70–80% confluence. After the cells reached confluence, cells were treated with 100, 200, 300 nM of cucurbitacin B in triplicate. After 24, 48, and 72 h, MTT (5 mg/mL in PBS) was added and cells were incubated at 37 °C for an additional 4 h. The formazan crystals were dissolved in 200 lL DMSO and absorbance was measured at 520 nm using a microplate reader.
2.3. Expression analysis Total RNA was isolated from 3T3-L1 cells using TRIzol reagent (Invitrogen, Carlsbad, CA). First-strand cDNA (cDNA) was synthesized from 0.5 lg of total RNA using the AMV Reverse Transcription System kit (Promega, Madison, WI) with random primers. After cDNA synthesis, the final 25 ll volume of the amplification mixture containing Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), primers, and cDNA was subjected to 40 amplification cycles of polymerase chain reaction (PCR) using a Thermal Cycler Dice (Takara, Shiga, Japan). Expression was normalized to 36B4. All real-time PCRs were performed at least twice. 4cycle threshold (CT) was used to calculate the differences between the target CT value and the control (36B4) for each sample: 4CT = CT(target)-CT(control). The relative expression level was calculated using 2DCT. The oligonucleotide primer (Integrated DNA Technologies, San Diego, CA) sequences used for PCR were as follows: peroxisome proliferator-activated receptor c (PPARc), PPARc F, 50 -CCATTCTGGCCCACCAAC-30 and PPARc R, 50 -AATGCGAGTGGTCTTCCATCA-30 ; adipocyte binding protein 2 (aP2), aP2 F, 50 -CACCGCAGACGACAGGAAG-30 and aP2 R, 50 - GCACCTGCACCAGGGC-30 ; cluster of differentiation 36 (CD36), CD36 F, 50 -GGCCAAGCTAT TGCGACAT-30 and CD36 R, 50 -CAGATCCGAACACAGCGTAGA-30 ; CCAAT-enhancerbinding proteins (C/EBPa), C/EBPa F, 50 -GCGGGCAAAGCCAAGAA-30 and C/EBPa R, 50 -GCGTTCCCGCCGTACC-30 ; adiponectin F, 50 -CCGGAACCCCTG GCAG-30 and adiponectin R, 50 -CTGAACGCTGAGCGATACACA-30 ; acidic ribosomal phosphoprotein P0 (36B4), 36B4 F, 50 -AGATGCAGCAGATCCGCAT-30 and 36B4 R, 50 -GTTCTTGCCCATCAGCACC-30 . Western blotting was performed as previously described (Song et al., 2013). Cells were harvested in 200 ll of sample buffer and heated at 100 °C for 10 min. The proteins were separated by 10% SDS–PAGE and electrophoretically transferred to nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ). Membranes were blocked with blocking buffer (5% skim milk in TBS, 0.1% Tween 20) for 30 min at room temperature and treated with STAT3, phosphorylated STAT3 (PSTAT3) (sc-7196; Santa Cruz Biotechnology, Santa Cruz, CA), and b-actin antibodies (A5316; Sigma), followed by incubation with horseradish peroxidase-conjugated secondary antibody (Zymed Laboratories, San Francisco, CA). Antibody binding was detected on X-ray film using Enhanced Chemiluminescence Western Blotting Detection Reagent (Amersham Biosciences). 2.4. STAT3 inhibition studies Scramble control, signal transducer and activator of transcription 3 (STAT3)specific oligos were synthesized by Genolution Pharmaceuticals, Inc. (Seoul, Korea). Two independent small interfering RNAs (siRNAs) were used to silence STAT3 expression. The sense sequences of STAT3-specific siRNA are as follows: Stat3 #1: 50 -GAGU UGAAUUAUCAGCUUAUU-30 ; Stat3 #2: 50 -CAUCAAUCCUGUGGUAUA AUU-30 . The sense sequence of control nonspecific scramble RNA is 50 -CC UCGUGCCGUUCCAUCAGG UAGUU-30 . Cells plated at a density of 1 105 cells per well in a 6-well plate were transfected with 50 nmol of scrambled RNA, STAT3-specific siRNA using RNAiMAX (Invitrogen), as previously described (Song et al., 2013; Zhang et al., 2011). Cells were treated with siRNA for 8 h, and the medium was exchanged. After 48 h, cells were processed using differentiation protocols. Transfection was carried out in duplicated wells and repeated three times. For WP-1066 (STAT3 inhibitor, Calbiochem, Gibbstown, NJ) treatment experiments, C3H10T1/2 cells were treated with 10 lM in the presence of Cu B or Cu I and then allowed to differentiate to adipocytes for 5 days. 2.5. Statistical analysis Data are presented as the means ± SEM. Differences in gene expression and lipid accumulation were analyzed using a two-tailed unpaired Student’s t-test or using analysis of variance followed by Student–Newman–Keuls tests. Statistical significance was defined as P < 0.05. All computations were performed using statistical analysis software (PASW Statistics 17).
3. Results 3.1. Cucurbitacin B inhibits lipid accumulation during adipocyte differentiation Since cucurbitacin family members including cucurbitacin B have previously been shown to have cytotoxic effects in certain cancer cells (Lee et al., 2010; Thoennissen et al., 2009), the potential cytotoxic effect of cucurbitacin B in preadipocytes was evaluated (Fig. 1). Treatment of 3T3-L1 cells with 100–300 nM cucurbitacin B for 24 h did not significantly affect cell viability (Fig. 1B). Similarly, cucurbitacin B treatment for 48 and 72 h did not decrease cell numbers (Fig. 1C and D). Thus, we used cucurbitacin B at 100–300 nM to assess its effects in adipocyte differentiation.
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Fig. 1. Cell viability of 3T3-L1 cells with cucurbitacin B (Cu B) treatment at the indicated concentrations. (A) Chemical structure of cucurbitacin B (25-acetoxy-2b,16a,20btrihydroxy-9b-methyl-19nor-10a-lanosta-5,23-diene-3,11,22-trione). (B-D) Cell viability in 3T3-L1 cells was measured upon DMSO (ctrl) or cucurbitacin B (Cu B) treatment (100, 200, and 300 nM) for 24 h (B), 48 h (C), and 72 h (D). Values are expressed as the means ± SEM from three independent experiments.
To test our hypothesis of cucurbitacin B as an anti-adipogenic compound, preadipocyte 3T3-L1 cells were treated with cucurbitacin B and differentiated into adipocytes under adipogenic conditions for 5 days. As expected, 20 lM resveratrol, a well known anti-adipogenic compound, significantly suppressed lipid accumulation as assessed by Oil Red O staining. Remarkably, 200–300 nM cucurbitacin B treatment inhibited lipid accumulation more potently than 20 lM resveratrol treatment (Fig. 2A and C). To further confirm the anti-adipogenic effects, mesenchymal C3H10T/12 cells were differentiated into adipocytes. Consistently, cucurbitacin B also suppressed lipid accumulation in C3H10T1/2 cells (Fig. 2B), further indicating the anti-lipogenic effects of cucurbitacin B during adipocyte differentiation. 3.2. Cucurbitacin B suppresses the expression of adipocyte markers Adipocyte differentiation is dictated by the master regulator PPARc followed by induction of target genes involved in triglyceride synthesis and preadipocyte differentiation into adipocytes. 3T3-L1 cells were differentiated into adipocytes in the presence of 100–300 nM cucurbitacin B and the expression of adipocyte markers were measured by real-time PCR. Similar to the effects on lipid accumulation, the mRNA expression of PPARc, aP2, C/ EBPa, adiponectin, and CD36 was significantly suppressed in a dose-dependent manner after 5 days of adipocyte differentiation (Fig. 2D). The effects of cucurbitacin B on lipid accumulation and expression of adipocyte markers indicate that cucurbitacin B is a natural anti-adipogenic compound.
expression of C/EBPd and Cyclin D1 and suppress the expression of KLF5 (Clarkson et al., 2006; Song et al., 2013). Consistently, cucurbitacin B treatment of 3T3-L1 cells reduced the expression of C/EBPd and Cyclin D1 but induced the expression of KLF5 (Fig. 3A). Similar regulatory responses for the expression of STAT3 target genes by cucurbitacin B was also observed in C3H10T1/2 cells (Fig. 3B). Resveratrol treatment, however, did not affect the expression of these genes, providing support that cucurbitacin B has specific effects on STAT3 in preadipocytes (Fig. 3B). Since activation of STAT3 signaling by various cascades induces STAT3 phosphorylation, the levels of STAT3 phosphorylation can be considered a surrogate marker of STAT3 activation (Lee et al., 2010). To directly investigate the effects of cucurbitacin B on STAT3 activity, STAT3 phosphorylation upon cucurbitacin B treatment was examined. Preadipocyte 3T3-L1 cells were treated with cucurbitacin B and probed against a STAT3 phospho-specific antibody to measure the levels of phosphorylated STAT3. The phosphorylated STAT3 was normalized to the total amount of STAT3 protein. Consistent with inhibitory effects on STAT3, cucurbitacin B treatment for 12 h reduced the levels of phosphorylated STAT3 (Fig. 3C). Cucurbitacin B suppressed the phosphorylation levels to 50% compared to DMSO treatments in a quantification analysis (Fig. 3D). Therefore, the STAT3 inhibition by cucurbitacin B can suppress the levels of phosphorylated STAT3 followed by inhibition on C/ EBPa and PPARc. Thus, analysis of target gene expression and STAT3 phosphorylation indicate that cucurbitacin B has regulatory effects on STAT3 signaling in preadipocytes.
3.3. Cucurbitacin B inhibits STAT3 signaling in preadipocytes
3.4. STAT3 signaling may be the key to the anti-adipogenic effects of cucurbitacin B
The inhibitory effects of cucurbitacin B on adipogenesis prompted us to investigate the molecular mechanism of cucurbitacin B action in preadipocytes. Previous studies showed that cucurbitacin B can suppress the cell cycle in cancer cells through impairment of STAT3 signaling (Thoennissen et al., 2009). Therefore, we hypothesized that STAT3 signaling may mediate the inhibitory effects of cucurbitacin B during adipocyte differentiation. To investigate this possibility, preadipocyte 3T3-L1 cells were treated with cucurbitacin B and the effects on STAT3 signaling were assessed. Activation of STAT3 signaling has been shown to induce
To demonstrate that STAT3 signaling is required for the antiadipogenic actions of cucurbitacin B, the anti-adipogenic effects of cucurbitacin B treatment were assessed in cells with STAT3 expression abrogated by the introduction of small interfering RNAs (siRNA). We employed two independent previously verified siRNAs that effectively silence STAT3 mRNA expression (Song et al., 2013; Zhang et al., 2011). 3T3-L1 cells were transfected with control scrambled siRNA or STAT3-targeted siRNAs and cells were differentiated into adipocytes. As shown previously, STAT3 expression was successfully reduced in the cells transfected with the two
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Fig. 2. Cucurbitacin B inhibits adipocyte differentiation of 3T3-L1 and C3H10T1/2 cells. (A) 3T3-L1 cells were treated with cucurbitacin B (Cu B) for 5 days during adipocyte differentiation. Oil red O staining was performed to assess lipid accumulation. An anti-adipogenic compound, resveratrol (20 lM, RSV) and DMSO (ctrl) were used as controls. (B) C3H10T1/2 cells were treated with various concentrations of Cu B (100, 200, and 300 nM) and induced to differentiate, followed by Oil Red O staining. (C) Lipid accumulation in 3T3-L1 cells was quantified by measuring the extracted Oil red O dye at 520 nM. (D) Cu B suppresses the expression of adipocyte markers. 3T3-L1 cells were treated with the indicated concentrations of Cu B for 5 days during adipocyte differentiation and mRNA expression of adipogenic genes encoding PPARc, aP2, C/EBPa, adiponectin, and CD36 was measured by real-time PCR. Data shown represent the mean ± SEM and are representative of three independent experiments. Mean with different letters at each samples are significantly different (P < .05) by Student–Newman–Keuls test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
independent siRNAs compared to control cells transfected with scrambled siRNA (Fig. 4A). Lipid accumulation was also reduced in STAT3 siRNA-transfected cells. As expected, cucurbitacin B treatment of control scrambled siRNA (sc)-transfected cells suppressed lipid accumulation. However, the anti-lipogenic effects of cucurbitacin B in STAT3 siRNA-transfected cells were significantly compromised compared to the effects in scrambled siRNA (sc)-transfected control cells (Fig. 4B and C). The requirement for STAT3 signaling was further supported by the expression levels of adipocyte markers. The expression of adipocyte markers such as PPARc and C/ EBPa were reduced by cucurbitacin B in control scrambled siRNA-transfected cells, whereas the regulatory action of cucurbitacin B on the expression of these adipocyte markers was markedly blunted in STAT3 siRNA-transfected cells (Fig. 4D). These data demonstrate that STAT3, at least in part, mediates the anti-adipogenic actions of cucurbitacin B during adipocyte differentiation. 3.5. Cucurbitacin I also inhibits adipocyte differentiation Cucurbitacin is a class of triterpene hydrocarbon compounds mainly found in plants with more than 40 different variants (Chambliss and Jones, 1966). To test whether other cucurbitacins also inhibit adipocyte differentiation, another commercially available cucurbitacin family member, cucurbitacin I was selected and tested for its effects on adipocyte differentiation.
Similar to cucurbitacin B, cucurbitacin I treatment inhibited lipid accumulation in 3T3-L1 (Fig. 5A and C) and C3H10T1/2 cells (Fig. 5B). Expression of PPARc, C/EBPa, and CD36 was also suppressed by cucurbitacin I treatment during adipocyte differentiation (Fig. 5D). These data suggest that cucurbitacin I also suppress lipid accumulation. 3.6. Cucurbitacin I suppresses adipogenesis via STAT3 signaling The similar anti-adipogenic effects of cucurbitacin B and cucurbitacin I suggest common molecular actions on adipocyte differentiation of preadipocytes. The expression of STAT3 target genes were measured to show the effects of cucurbitacin I on STAT3 signaling. Cucurbitacin I treatment reversed the expression of STAT3 target genes such as Cyclin D1 and KLF5 similar to that by cucurbitacin B (Fig. 6A), suggesting the possibility of cucurbitacin I as a STAT3 inhibitor. To show STAT3 requirement for the effects of cucurbitacin I, 3T3-L1 cells were transfected with siRNAs targeted to STAT3 and differentiated into adipocytes. Cucurbitacin I treatment suppressed lipid accumulation in scrambled control siRNA-transfected cells, whereas anti-lipogenic effects of cucurbitacin I were significantly reduced in STAT3 targeted siRNA-transfected cells (Fig. 6B). These data show that the anti-adipogenic effects of cucurbitacin I can also be mediated by STAT3 signaling and further raise the
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Fig. 3. Cucurbitacin B inhibits STAT3 activity in preadipocytes. (A) Expression of STAT3 target genes are regulated by Cu B in 3T3-L1 cells. 3T3-L1 cells were treated with DMSO (ctrl), 200 nM, 300 nM cucurbitacin B (Cu B) for 12 h and the expression of STAT3 target genes was assessed. C/EBPd and Cyclin D1 (CycD1), known STAT3-induced genes, were suppressed by Cu B treatment. Expression of KLF5, a gene known to be downregulated by STAT3, was induced by Cu B. (B) STAT3 target genes are selectively regulated by Cu B in C3H10T1/2 cells. C3H10T1/2 cells were treated with Cu B (200 nM) or resveratrol (RSV, 20 lM) for 12 h and the expression of STAT3 target genes was measured. STAT3 target genes were regulated by Cu B but not by resveratrol treatment. (C-D) Cu B suppresses STAT3 phosphorylation (P-STAT3) levels in 3T3-L1 cells. 3T3-L1 cells were treated for 12 h with 100 nM or 300 nM of Cu B and protein expression was measured by immunoblot analysis (C) followed by the measurement of relative intensities of P-STAT3/STAT3 (D). Immunoblotting was performed using anti-STAT3 and anti-phopho-Tyr STAT3 antibodies and were quantified by NIH Image J software. Numbers indicate fold changes relative to controls. The b-Actin antibody was probed to verify equal protein loading. Data shown represent the mean ± SEM and are representative of three independent experiments. Statistical significance was determined relative to the control by the Student’s t-test (P < 0.05; P < 0.005; P < 0.0005).
Fig. 4. STAT3 mediates the anti-adipogenic effects of Cucurbitacin B . (A) Transient transfection of 3T3-L1 cells with two independent STAT3-targeted siRNAs (si#1 or si#2) effectively reduced STAT3 expression. (B–C) Silencing the expression of STAT3 blunted the inhibitory effects of cucurbitacin B (Cu B) on adipocyte differentiation in 3T3-L1 cells. Control scrambled (sc) and STAT3 targeted siRNA-transfected cells (si#1 and si#2) were treated with DMSO or Cu B (100 nM) and then differentiated for 5 days. Differentiated cells were stained with Oil Red O (B) followed by quantification (C). (D) Knockdown of STAT3 attenuated the effects of Cu B on expression of adipocyte markers. Expression of PPARc and C/EBPa in control scrambled and STAT3-specific siRNA-transfected cells treated with DMSO (ctrl) or Cu B (100 nM) for 5 days was measured by realtime PCR. Representative data from three experiments are shown. Values are means ± SD. Statistically significant differences in gene expression upon CuB treatment between the control and STAT3 siRNA-transfected cells were determined using Student’s t-test (NS, not significant; P < 0.05; P < 0.005; P < 0.0005). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
possibility that other cucurbitacin family members are also antiadipogenic compounds. We also examined whether STAT3 acts as a mediator of the anti-adipogenic actions of cucurbitacins in the presence of STAT3
pharmacological inhibitor. The degree of inhibition of lipid accumulation in response to cucurbitacin B and I treatment was significantly reduced in STAT3-inhibitor (WP-1066) treated cells compared to DMSO-treated controls, further corroborating that
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Fig. 5. Cucurbitacin I also inhibits adipocyte differentiation. (A) 3T3-L1 cells were treated with the indicated concentrations of cucurbitacin I (Cu I) for 5 days during adipocyte differentiation. Oil red O staining was performed to visualize lipid accumulation. Resveratrol (20 lM, RSV) and DMSO (ctrl) were used as controls. (B) C3H10T1/2 cells were treated with various concentrations of Cu I (300, 400, and 500 nM) and induced to differentiate, followed by Oil Red O staining. (C) Lipid accumulation in 3T3-L1 cells was quantified by measuring the extracted Oil red O dye at 520 nM. Data shown represent the mean ± SEM from three independent experiments. Mean with different letters at each samples are significantly different (P < .05) by Student–Newman–Keuls test. (D) Cu I suppresses the expression of adipocyte markers. 3T3-L1 cells were treated with the indicated concentrations of Cu I for 5 days during adipocyte differentiation and mRNA expression of adipogenic genes encoding PPARc, C/EBPa, and CD36 was measured by real time PCR. Representative data from three experiments are shown. Statistically significant differences in gene expression was determined relative to the control (DMSO) by the Student’s t-test (P < 0.05; P < 0.005; P < 0.0005). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the inhibitory action of both cucurbitacin B and I in adipocyte differentiation is mediated through inhibition of STAT3 (Fig. 6C). To further verify the similarity of the anti-lipogenic effects of cucurbitacin B and I, primary mouse embryonic fibroblasts were freshly isolated from mouse embryos and induced to differentiate into adipocytes in the presence of cucurbitacin B or cucurbitacin I (Fig. 6D). Both cucurbitacin B and I similarly suppressed expression of PPARc, aP2, C/EBPa, and CD36 although cucurbitacin B seemed to have more potent effects. These data raise the possibility that the similar biological actions and underlying molecular mechanisms of other cucurbitacin family members in adipocyte differentiation.
4. Discussion STAT3 signaling is required for adipocyte differentiation (Zhang et al., 2011). Thus, we investigated the possibility that cucurbitacin B, a STAT3 inhibitor in certain cancer cells, would also inhibit adipocyte differentiation of preadipocytes. In this study, we show for the first time the anti-adipogenic effects of cucurbitacin B in multipotent C3H10T1/2, preadipocyte 3T3-L1, and freshly isolated primary cells. We also present evidence that cucurbitacin B acts on STAT3 signaling. Furthermore, our data show that STAT3 signaling is at least in part essential for the anti-adipogenic actions of cucurbitacin B. More than 40 members of the cucurbitacin family have been isolated from the cucurbitaceae family and others including cucumber, pumpkins, gourds, some mushrooms, and marine mollusks (Alghasham, 2013). Cucurbitacins have an impact on various
biological activities, showing anti-tumor, anti-inflammatory, and anti-infectious effects. Most cucurbitacins, including A, B, E, I, and Q, have been shown to affect cancer cell growth, whereas cucurbitacin B and E are effective in radical and oxidative damage (Tannin-Spitz et al., 2007). Cucurbitacin R and dihydrocucurbitacin B are also known for their efficacy on the immune system (Rios, 2010). However, until the present study, anti-adipogenic activity of cucurbitacin family members has not been implied. Thus, it should be interesting to investigate in future studies whether other cucurbitacin family members also possess anti-lipogenic activity and act though STAT3 signaling. Our current data suggest the possibility that cucurbitacins at least cucurbitacin B and I, if not all members of the cucurbitacin family, could be beneficial in preventing lipid accumulation and metabolic diseases. These data further raise questions whether cucurbitacin containing herbal products such as cucumber and pumpkins may be effective against obesity and related diseases. Interestingly, extracts from cucurbita moschata have been shown to decrease body weight and fat storage in high fat diet-induced obese mouse models (Choi et al., 2007). Thus, it is possible that cucurbitacins may partly mediate the anti-obese actions of cucurbita moschata. The possible anti-lipogenic effects of cucurbitacin B and I should be studied in animal models and humans before addressing these further questions. In conclusion, we show that cucurbitacin B inhibits adipocyte differentiation of multiple adipogenic cells. Mechanism studies indicate that cucurbitacin B exerts its inhibitory effects on adipogenesis through the inhibition of STAT3 signaling. We further show that another family member, cucurbitacin I, also possesses anti-adipogenic activity. Therefore, our data provide evidence for
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Fig. 6. The anti-adipogenic effect of cucurbitacin I is also mediated through STAT3 signaling. (A) Expression of STAT3 target genes are regulated by cucurbitacin I (Cu I) in 3T3L1 cells. 3T3-L1 cells were treated with DMSO (ctrl) or Cu I (300, 400 nM) for 12 h and the expression of STAT3 target genes assessed. Similar to the effects of Cu B, expression of Cyclin D1 (CycD1) and KLF5 were regulated by treatment with Cu I. (B) Cucurbitacin I (Cu I) acts through STAT3 signaling. Silencing STAT3 compromised the inhibitory effects of Cu I treatment on lipid accumulation in 3T3-L1 cells. Control scrambled (sc) and siRNA-transfected cells (si#2) were treated with DMSO or Cu I (400 nM) for 5 days during adipocyte differentiation. Differentiated cells were stained with Oil Red O followed by quantification. (C) Pharmacological disruption of STAT3 signaling blunts the inhibitory effects of both Cu B and Cu I on lipid accumulation. C3H10T1/2 cells were treated with 10 lM WP-1066 (STAT3 inhibitor) and differentiated in the presence of Cu B (200 nM) or Cu I (400 nM) for 5 days, and quantified by measuring the Oil red O dye extracted in isopropanol at 520 nm. (D) Both Cu B and Cu I inhibit the expression of adipocyte markers in primary mouse embryonic fibroblasts. Primary mouse embryonic fibroblasts were treated with 300 nM of Cu B and Cu I for 10 days and the mRNA expression of adipogenic genes encoding PPARc, aP2, C/EBP, and CD36 was measured by real-time PCR. Representative data from three experiments are shown. Values are means ± SD. Statistical significance was determined by comparison to the control using Student’s t-test (NS, not significant; P < 0.05; P < 0.005; P < 0.0005).
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