Suppression of STAT3 and HIF-1 Alpha Mediates Anti-Angiogenic Activity of Betulinic Acid in Hypoxic PC-3 Prostate Cancer Cells

Abstract

Background

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that regulates various cellular processes such as cell survival, angiogenesis and proliferation. In the present study, we examined that betulinic acid (BA), a triterpene from the bark of white birch, had the inhibitory effects on hypoxia-mediated activation of STAT3 in androgen independent human prostate cancer PC-3 cells.

Methodology/Principal Findings

BA inhibited the protein expression and the transcriptional activities of hypoxia-inducible factor-1α (HIF-1α) under hypoxic condition. Consistently, BA blocked hypoxia-induced phosphorylation, DNA binding activity and nuclear accumulation of STAT3. In addition, BA significantly reduced cellular and secreted levels of vascular endothelial growth factor (VEGF), a critical angiogenic factor and a target gene of STAT3 induced under hypoxia. Furthermore, BA prevented in vitro capillary tube formation in human umbilical vein endothelial cells (HUVECs) maintained in conditioned medium of hypoxic PC-3 cells, implying anti-angiogenic activity of BA under hypoxic condition. Of note, chromatin immunoprecipitation (ChiP) assay revealed that BA inhibited binding of HIF-1α and STAT3 to VEGF promoter. Furthermore, silencing STAT3 using siRNA transfection effectively enhanced the reduced VEGF production induced by BA treatment under hypoxia.

Conclusions/Significance

Taken together, our results suggest that BA has anti-angiogenic activity by disturbing the binding of HIF-1α and STAT3 to the VEGF promoter in hypoxic PC-3 cells.

About the Authors:

Jimin Shin

Contributed equally to this work with: Jimin Shin, Hyo-Jeong Lee, Soo-Jin Jeong

Affiliation: College of Oriental Medicine, Kyung Hee University, Seoul, South Korea

Hyo-Jeong Lee

Contributed equally to this work with: Jimin Shin, Hyo-Jeong Lee, Soo-Jin Jeong

Affiliation: College of Oriental Medicine, Kyung Hee University, Seoul, South Korea

Deok-Beom Jung

Affiliation: College of Oriental Medicine, Kyung Hee University, Seoul, South Korea

Ji Hoon Jung

Affiliation: College of Oriental Medicine, Kyung Hee University, Seoul, South Korea

Hyo-Jung Lee

Affiliation: College of Oriental Medicine, Kyung Hee University, Seoul, South Korea

Eun-Ok Lee

Affiliation: College of Oriental Medicine, Kyung Hee University, Seoul, South Korea

Seok Geun Lee

Affiliation: College of Oriental Medicine, Kyung Hee University, Seoul, South Korea

Beom Sang Shim

Affiliation: College of Oriental Medicine, Kyung Hee University, Seoul, South Korea

Seung Hoon Choi

Affiliation: College of Oriental Medicine, Kyung Hee University, Seoul, South Korea

Seong Gyu Ko

Affiliation: College of Oriental Medicine, Kyung Hee University, Seoul, South Korea

Kwang Seok Ahn

Affiliation: College of Oriental Medicine, Kyung Hee University, Seoul, South Korea

Soo-Jin Jeong

Contributed equally to this work with: Jimin Shin, Hyo-Jeong Lee, Soo-Jin Jeong

Affiliation: College of Oriental Medicine, Kyung Hee University, Seoul, South Korea

Sung-Hoon Kim

* E-mail: [email protected]

Affiliation: College of Oriental Medicine, Kyung Hee University, Seoul, South Korea

Introduction

Signal transducer and activator of transcription 3 (STAT3) is one of STAT protein family and constitutively active in a wide range of human cancer cells [1]. Activated STAT3 proteins by cytokines and growth factors form homo- or heterodimers, and then translocate from the cytoplasm to the nucleus, where they are binding to the promoter of various gene products involved in anti-apoptosis (bcl-2, bcl-xL and survivin), proliferation (cyclin D1), and angiogenesis (vascular endothelial growth factor (VEGF)) [2]. Interestingly, recent studies reported that STAT3 is activated in response to hypoxia, a common feature of various solid tumors [3], [4]. Activated STAT3 mediates the up-regulation of hypoxia inducible factor alpha (HIF-1α), a major regulator to adapt under hypoxic conditions by increasing its stability and transcriptional activity [5]. Thus, recently STAT3 and HIF-1α are attractive target molecules by natural compounds and herbal extracts in cancer research.

Betulinic acid (BA), initially reported as a human melanoma-specific inhibitor, is a triterpenoid mainly derived from the bark of the white birch (Betula pubescens) [6]. Recent evidences suggest the anti-cancer effects of BA [7], [8], anti-inflammatory [9] and anti-viral [10] activities via various signaling pathways such as epidermal growth factor receptor (EGFR) [11], hedgehog [12], signal transducer and activator of transcription 3 (STAT3) [13] and nuclear factor-kappa B (NF-κB) [14]. Nonetheless, there is no evidence that BA mediates anti-cancer activity through inhibiting STAT3 signaling in solid tumors.

Thus, in the present study, we investigated the roles of STAT3 and HIF-1 α in BA induced anti-angiogenic activity in hypoxic PC-3 prostate cancer cells by MTT assay, Western blotting, immunocytochemistry, ELISA and EMSA.

Results

Cytotoxic effect of betulinic acid (BA) against PC-3 cells

Cytotoxic effect of BA (Fig. 1) was evaluated by MTT assay. PC-3 cells were treated with various concentrations of BA (0, 12.5, 25, 50 or 100 µM) for 24 h. Cell viability was reduced to 78.49±5.67 and 62.64±1.26% at concentrations of 12.5 and 25 µM, respectively, and sustained to∼60% at over 25 µM (Fig. 2A).

[Figure omitted. See PDF.]

Figure 1. Chemical structure of betulinic acid (BA).

Molecular weight  =  456.

https://doi.org/10.1371/journal.pone.0021492.g001

[Figure omitted. See PDF.]

Figure 2. Effect of betulinic acid (BA) on hypoxia-induced HIF-1α activation in PC-3 cells.

(A) PC-3 cells were treated with various concentrations of BA (0, 12.5, 25, 50 or 100 µM) for 24 h. Cell viability was analyzed by MTT assay. (B) Cells were exposed to normoxia or hypoxia for 0.5, 2, 4, 6, 8, 12 or 24 h. Cell lysates were prepared and subjected to Western blotting to determine the expression of HIF-1α. (C) Cells were treated with or without BA (5 or 10 µM) under normoxic or hypoxic condition for 4 h. Cell lysates were prepared and subjected to Western blotting to determine the expression of HIF-1α. (D) Nuclear extract was prepared from the cells treated with BA (0, 10, 20 or 40 µM) under normoxia or hypoxia for 4 h. HIF-1α transcription activity was measured by using TransAM HIF-1 transcription factor assay kit. Data represent means ± S.D. ##, p<0.01 vs normoxia control, and *, p<0.05 and ** < 0.01 vs hypoxia control.

https://doi.org/10.1371/journal.pone.0021492.g002

Effect of betulinic acid (BA) on hypoxia-induced HIF-1α activation in PC-3 cells

Hypoxia is a hallmark of solid tumors [15] and HIF-1α is a transcription factor that responses to hypoxia [16]. To examine whether BA can affect HIF-1α induced by hypoxia, we first determined the best time point of hypoxia-induced HIF-1α expression in PC-3 cells. Cells were exposed to normoxia or hypoxia for 0.5, 2, 4, 6, 8, 12 or 24 h. HIF-1α expression was dramatically induced under hypoxic condition for 4 h (Fig. 2B). Then, cells were treated with or without BA under hypoxia for 4 h. BA decreased hypoxia-induced HIF-1α expression in a dose-dependent manner compared with hypoxia control (Fig. 2C). In addition, hypoxia significantly activated HIF-1α transcription while BA treatment inhibited the hypoxia-mediated transcriptional activation of HIF-1α in a dose-dependent manner (Fig. 2D). These results suggest that BA has the ability to inhibit the expression as well as transcription of HIF-1α in hypoxic PC-3 cells.

Effect of betulinic acid (BA) on hypoxia-induced STAT3 activation in PC-3 cells

Recent studies reported that a transcription factor STAT3 is involved in the transcriptional regulation of HIF-1α [17]. In our study, hypoxia enhanced phospho-STAT3 level while normoxia did not affect it. BA treatment inhibited hypoxia-mediated STAT3 phosphorylation in a dose-dependent manner (Fig. 3A). Also, EMSA revealed that BA prevented the STAT3/DNA binding activity under hypoxia in a dose-dependent manner (Fig. 3B). Furthermore, imunocytochemical (ICC) staining with anti-HIF-1α antibody showed a significant nuclear expression of HIF-1α under hypoxic condition. In contrast, BA treatment attenuated HIF-1α expression in the nucleus in hypoxic PC-3 cells (Fig. 3C), suggesting its inhibitory effect on the nuclear translocation of HIF-1α.

[Figure omitted. See PDF.]

Figure 3. Effect of betulinic acid (BA) on hypoxia-induced STAT3 activation in PC-3 cells.

PC-3 cells were treated with or without BA (5 or 10 µM) under normoxic or hypoxic condition for 4 h. (A) Cell lysates were prepared and subjected to Western blotting for phospho-STAT3 and STAT3. (B) Nuclear extracts were prepared and applied to EMSA to analyze the STAT3-DNA binding activity. (C) Cells were treated with or without BA (10 µM) under hypoxia. Immunocytochemistry was performed for STAT3. DAB (brown) and hematoxylin-eosin was used as a substrate and a counterstaining, respectively.

https://doi.org/10.1371/journal.pone.0021492.g003

Effects of betulinic acid (BA) on hypoxia-induced angiogenesis

Hypoxia is one of angiogenesis inducers through HIF-1α activation [18]. Thus, the inhibitory effect of BA was evaluated on hypoxia-mediated angiogenesis. VEGF, a critical angiogenesis factor [19], was evaluated at the secreted cellular and protein levels by ELISA and Western blotting, respectively. BA significantly reduced VEGF production in a dose-dependent manner by ELISA (Fig. 4A). Consistently, BA attenuated VEGF protein expression in a dose-dependent manner by Western blotting (Fig. 4B).

[Figure omitted. See PDF.]

Figure 4. Effect of betulinic acid (BA) on hypoxia-induced angiogenesis.

(A and B) PC-3 cells were treated with 0, 5 or 10 µM BA for 24 h. (A) VEGF levels in the culture supernatants were measured by using a Quantikine VEGF ELISA kit. (B) Cell lysates were prepared and subjected to Western blotting to determine VEGF expression. Graphs represent relative band intensities of VEGF/β-actin. Data represent means ± S.D. ##, p<0.01 vs normoxia control, and *, p<0.05 and ** <0.01 vs hypoxia control. (C) HUVECs were treated with VEGF (20 ng/ml) as positive control or the culture supernatant from PC-3 cells treated with or without BA (10 µM) under normoxia or hypoxia. Tube formation assay was performed using growth factor reduced Matrigel. Cells were fixed with Diff-Quick solution, photographed randomly under an Axiovert S 100 light microscope at ×100 magnification and counted.

https://doi.org/10.1371/journal.pone.0021492.g004

Additionally, HUVEC tube formation assay, which is known as a typical angiogenesis in vitro model, was performed to confirm anti-angiogenic effect of BA on hypoxia-mediated angiogenesis. VEGF was used as a positive control of angiogenesis induction. HUVECs mixed with the supernatants from PC-3 cells were cultured in the absence or presence of BA under hypoxia. As shown in Fig. 4C, hypoxia-induced tube formation was prevented by BA treatment in PC-3 cells while clear tube formation was exhibited in untreated control under hypoxia, suggesting that BA inhibits hypoxia-mediated angiogenesis.

Effects of betulinic acid (BA) on the binding of STAT3 and HIF1 α to VEGF promoter in hypoxic PC-3 cells

Recent studies revealed that STAT3 activation is directly link to the transcriptional regulation of VEGF by binding to the VEGF promoter [20], [21]. In light of this event, we conducted chromatin immunoprecipitation (ChiP) assay. As shown in Fig. 5A, the binding activity of STAT3 and HIF-1α to the VEGF promoter was detected under hypoxia (lanes 5-8) compared with normoxia (lanes 1-4). Notably, BA treatment suppressed the binding of STAT3 and HIF-1α to VEGF promoter in hypoxic condition (lanes 9-12).

[Figure omitted. See PDF.]

Figure 5. Effect of betulinic acid (BA) on STAT3 binding on the VEGF promoter in hypoxic PC-3 cells.

(A) PC-3 cells were treated with or without BA (10 µM) under normoxia or hypoxia for 4 h. The immunoprecipitated DNA with rabbit normal IgG, HIF-1α or STAT3 antibody was amplified by PCR analysis for VEGF promoter. (B) Cells were transiently transfected with siRNA for scramble or STAT3 for 24 h and treated with or without BA (10 µM) for 18 h under hypoxia. VEGF levels in the culture supernatants were measured by using a Quantikine VEGF ELISA kit. Data represent means ± S.D. #, p<0.05 vs control, and *, p<0.05 vs control siRNA. Cell lysates were subjected to Western blotting for phospho-STAT3, STAT3 and HIF-1α.

https://doi.org/10.1371/journal.pone.0021492.g005

In order to confirm the critical role of STAT3 in anti-angiogenic regulation of BA in hypoxic PC-3 cells, STAT3 siRNA transfection was carried out in PC-3 cells. Treatment with either BA or STAT3 siRNA reduced the production of VEGF by 39.6% and 45.9% respectively, compared with untreated control. Furthermore, BA treatment significantly reduced VEGF production by 63.25% in STAT3 siRNA-transfected PC-3 cells (Fig. 5B). Western blotting revealed that siRNA for STAT3, but not control, efficiently blocked STAT3 (Fig. 5B).

Discussion

Prostate cancer classified as an adenocarcinoma is the second most common malignant tumors in American men, with estimates of 192,280 new cases and approximately 27,360 deaths in 2009 [22], [23]. Betulinic acid (BA), a plant-derived pentacyclic lupane-type triterpenoid, can be extracted from various plants such as Sarracenia flava [24], Diospyros spp., Inga punctata [25], Ziziphus spp., and Vauquelinia corymbosa [26]. Several groups reported anti-cancer activity of BA in various cancers including lung, colorectal, breast, prostate and cervical cancer [27], but not normal cells [28]. Also, BA completely inhibited tumour growth without toxicity in athymic mice bearing human melanomas [6]. Moreover, anti-cancer activity of BA was exerted by inducing apoptosis in the cancer cells. For example, BA-induced apoptosis was independent of p53 in neurorectodermal tumor [29] and melanoma cells [30]. In neuroblastoma cells, BA induced apoptosis through loss of the mitochondrial membrane potential, reactive oxygen species (ROS) production and caspase activation [31].

Interestingly, Karna and colleagues recently reported that BA inhibited the expression of HIF-1α and vascular endothelial growth factor (VEGF) in human endometrial cancer cells [32]. However, the regulatory mechanisms whereby BA inhibits angiogenesis are not fully understood. In the present study, we found that BA suppressed hypoxia-mediated protein accumulation, transcriptional activation and nuclear localization of HIF-1α in PC-3 cells. Consistent with the results of Karna's paper, our data also showed that BA significantly inhibited VEGF secretion and protein expression in hypoxic PC-3 cells. Additionally, in vitro tube formation assay further confirmed anti-angiogenenic effect of BA in hypoxic PC-3 cells.

Recently, Niu and colleagues suggested that constitutively activated STAT3 up-regulated VEGF and induced tumor angiogenesis [20]. Also, Wei and colleagues reported that STAT3 activation regulates the expression of VEGF and human pancreatic cancer angiogenesis Furthermore, several papers described the role of STAT3 as a potential modulator of HIF-1α-induced VEGF signaling in cancer cells [4], [33]. In this regard, the effect of BA on STAT3 and HIF-1α activation was examined in hypoxic PC-3 cells in our study. Consistent with the evidences by Pandey and colleagues that BA suppressed STAT3 activation in multiple myeloma cells [13], BA prevented hypoxia-induced tyrosine phosphorylation, DNA binding activity and nuclear translocalization of STAT3, suggesting the inhibitory effect of BA on STAT3 activation.

VEGF promoter contains various transcription factor binding sites including STAT3 [20] as well as HIF-1 [34]. Physical interaction of STAT3 with HIF-1 controls VEGF transcriptional activation by their binding to the VEGF promoter [4]. In our study, hypoxia promoted the binding of STAT3 and HIF-1α to the VEGF promoter in PC-3 cells. In contrast, BA remarkably inhibited the binding of STAT3 and HIF-1α to the VEGF promoter site under hypoxic condition. Additionally, silencing STAT3 using its specific siRNA significantly enhanced BA-mediated inhibition of VEGF production, implying the involvement of STAT3 in anti-angiogenic regulation of BA in hypoxic PC-3 cells. Similar to our study, Gariboldi and colleagues reported that NVP-AEW541, a IGFR1 inhibitor, disrupted IGF/STAT3/HIF1 pathway in human glioblastoma cells [35]. Leeman-Neill and colleagues also reported that Guggulsterone inhibited STAT3 and HIF-1α and suggested a biologic rationale for further clinical investigation BA for human head and neck squamous cell carcinoma (HNSCC) therapy [36].

Collectively, our data demonstrate that BA suppressed expression and transactivation of hypoxia-induced HIF-1α, STAT3, VEGF as well as capillary tube formation in PC-3 cells. It is noteworthy that anti-cancer activity of BA is exerted by inhibiting angiogenesis via inhibition of binding of STAT3 and HIF-1α to the VEGF promoter in PC-3 cells. Thus, our findings suggest that BA can be a potent anti-angiogenic agent by targeting STAT3/HIF-1α/VEGF signaling for prostate cancer therapy.

Materials and Methods

Compounds

Betulinic acid (BA) (Figure 1) was purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in dimethyl sulfoxide (DMSO) as a 10 mM stock solution for experimental use.

Cell culture

Human prostate cancer cell line PC-3 was obtained from American Type Culture Collection (ATCC, Rockville, MD) and maintained in RPMI1640 (Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimyotic solution. Human umbilical vein endothelial cells (HUVECs) were isolated from fresh human umbilical cord vein and maintained in EBM-2 (Lonza, Valais, Switzerland) supplemented with 2% FBS, 0.04% hydrocortisone, 0.1% VEGF, 0.1% IGF-1, 0.4% hFGF-B, 0.1% hEGF, 0.1% ascorbic acid, and 1% heparin.

Hypoxia induction

Cells were incubated in anaerobic incubator at 94% N2, 5% CO2 and 1% O2 (Thermo scientific, Rockford, IL) as previously described [37].

Cytotoxicity assay

To evaluate cytotoxicity of BA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed as previously described [38]. PC-3 cells were plated onto 96-well microplates at a density of 1×104 cells per well and exposed to various concentrations of BA (0, 12.5, 25, 50 or 100 µM) for 24 h. MTT solution (1 mg/ml) (Sigma-Aldrich) was added onto each well and incubated for 2 h at 37°C. Extraction buffer (20% SDS and 50% dimethylformamide) was then added and optical density (OD) was measured using microplate reader (Tecan Austria GmbH, Grödig, Austria) at 570 nm. Cell viability was calculated as a percentage of viable cells in BA-treated group versus untreated control by following equation.

Cell viability (%)  =  [OD (BA) - OD (Blank)] / [OD (Control) - OD (Blank)] ×100

Western blot analysis

Whole-cell extracts were prepared using lysis buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 1% triton X-100, 0.01% SDS, 1 mM EDTA (pH 8.0) and protease inhibitor cocktail tablets (Roche Applied Science, Inndianapolis, IN)]. Nuclear and cytoplasmic extracts were obtained by fractionated by using NE-PER nuclear and cytoplasmic extraction reagents (Thermo scientific, Rockford, IL). Protein samples were separated on 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was blocked in 5% nonfat skim milk, and probed with primary antibodies for HIF-1α (1∶500, Gene Tex, Irvine, CA), STAT3 (1∶1000, Cell Signaling, Danvers, MA), phospho-STAT3 (1∶500, Cell Signaling, Danvers, MA), VEGF (1∶500, Santa Cruz Biotechnologies, Santa Cruz, CA) and β-actin (Sigma, St. Louis, MO) overnight at 4°C. The membranes were exposed to HRP-conjugated secondary antibodies for 2 h at room temperature and protein expression was detected by using enhanced chemiluminescence (ECL) Western blotting detection reagent (GE Health Care Bio-Sciences, Piscataway, NJ).

HIF-1α transcription activity assay

HIF-1α transcriptional activity was analyzed by HIF-1α transcription factor assay using TransAM HIF-1 transcription factor assay kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions. Briefly, nuclear extracts were added onto 96-well microplate coated with oligonucleotides containing hypoxia response element (HRE) (5′-TACGTGCT-3′) from the erythropoietin (EPO) gene. HIF dimers present in nuclear extracts bind with high specificity to this response element and are subsequently detected with an antibody directed against HIF-1α. Addition of a secondary antibody conjugated to horseradish peroxidase (HRP) provides a sensitive colorimetric readout that is easily quantified by spectrophotometry. Values are expressed as optical density (OD) at 450 nm with a reference wavelength of 655 nm.

Immunocytochemistry

PC-3 cells were seeded on 4-chamber slides at a density of 3×104 cells per chamber and treated either with or without BA (10 µM) under hypoxia as previously described [37]. The cells were fixed in 4% formaldehyde solution for 10 min at room temperature and blocked in blocking buffer (10% BSA/Triton X-100 in PBS) containing 6% horse serum for 1 h at room temperature. The slides were incubated with anti-STAT3 (1:100) antibody overnight at 4°C and then probed with anti-mouse or rabbit biotinylated antibodies (Vector Labs, Burlingame, CA) for 1.5 h at room temperature. The expression was detected by using Vector ABC complex/HRP kit (Vector Labs, Burlingame, CA) and color-developed with 3,3′-diaminobenzidine tetrahydrochloride in dark. The specimens were then counterstained with hematoxylin-eosin (Sigma-Aldrich, St. Louis, MO) and analyzed under a microscope (Leica Microsystems Res., Wetzlar, Germany).

Electrophoretic mobility shift assay (EMSA)

The STAT3-DNA binding was analyzed by electrophoretic mobility shift assay (EMSA) using Gelshift Chemiluminescent EMSA kit (Active Motif, Carlsbad, CA) as previously described [39]. Briefly, nuclear extracts were prepared from anethole-treated cells and incubated with STAT3 consensus oligonucleotides (5′-CTT CAT TTC CCG TAA ATC CCT AAA GCT-3′) (Santa Cruz Biotechnologies, Santa Cruz, CA). The DNA-protein complex formed was separated from free oligonucleotides on 5% native polyacrylamide gels. Chemiluminescent detection was performed using ECL reagents according to the vendor's protocols (GE Health Care Bio-Sciences, Piscataway, NJ).

In vitro tube formation assay

In vitro tube formation assay was performed as previously described [40]. Matrigel (BD) was added on 24-well plates and polymerized by incubating for 1 h at 37°C. HUVECs were seeded onto Matrigel coated plates and incubated in EBM-2 supplemented with VEGF (20 ng/ml) or the supernatant from PC-3 cells treated with BA (0 or 10 µM) under normoxia or hypoxia for 24 h. After 8 h incubation, cells were fixed with 4% formaldehyde and randomly chosen fields were photographed under an Axiovert S 100 light microscope (Carl Zeiss, Weimar, Germany) at 100×magnification.

Enzyme-linked immunosorbent assay (ELISA) for VEGF

PC-3 cells were plated onto 60-mm dish at a density of 1×106 cells/plate and incubated in the absence or presence of BA (10 µM) under normoxia or hypoxia for 24 h. VEGF level in the supernatant was measured by using human VEGF ELISA kit according to the manufacturer's protocol (Biosource International Inc., Camarillo, CA).

Chromatin immunoprecipitation (ChiP) assay

PC-3 cells were plated onto 100-mm dishes at a density of 1.5×106 cells/dish, treated with BA for 4 h under normoxic or hypoxic condition and then 1% formaldehyde and 0.125 M glycine. Soluble chromatins were isolated by using EZ-Zyme chromatin prep kit (Millipore, Billerica, MA) and immunoprecipitated with antibodies of normal rabbit IgG (EMD biosciences, Gibbstown, NJ), HIF-1α or STAT3. Histone/DNA crosslinks were reversed by adding 5 M NaCl at 65°C for 4 h, followed by phenol/chloroform extraction and ethanol precipitation. PCR reaction was performed to amplify VEGF promoter using ChiP primers (sense 5′-AGACTCCACAGTGCATACGTG-3′ and antisense 5′-AGTGTGTCCCTCTGACAATG-3′.

siRNA trasnfection

PC-3 cells were transiently transfected with scramble or STAT3 siRNA (SantaCruz biotechnology, SantaCruz, CA) at 50 nM by using INTERFERin siRNA transfection reagent (Polyplus-transfection Inc., New York, NY). After incubation for 24 h, the cells were treated with BA and maintained for 18 h under hypoxia.

Statistical analysis

All data were expressed as means ± S.D. Statistical significance was analyzed by student's t-test.

Author Contributions

Conceived and designed the experiments: JS H-JeL S-JJ S-HK. Performed the experiments (Cytotoxicity assay, Western blotting, HIF-1α transcription, IHC, tube formation assay, ELISA and ChiP assay): JS DBJ. Performed the experiments (EMSA): JHJ. Analyzed the data: JS H-JeL H-JuL E-OL SGL BSS SHC SGK KSA. Wrote the paper: JS S-JJ S-HK.

Citation: Shin J, Lee H-J, Jung D-B, Jung JH, Lee H-J, Lee E-O, et al. (2011) Suppression of STAT3 and HIF-1 Alpha Mediates Anti-Angiogenic Activity of Betulinic Acid in Hypoxic PC-3 Prostate Cancer Cells. PLoS ONE6(6): e21492. https://doi.org/10.1371/journal.pone.0021492

References

1. Bowman T, Garcia R, Turkson J, Jove R (2000) STATs in oncogenesis. Oncogene 19: 2474–2488.T. BowmanR. GarciaJ. TurksonR. Jove2000STATs in oncogenesis.Oncogene1924742488

2. Aggarwal BB, Sethi G, Ahn KS, Sandur SK, Pandey MK, et al. (2006) Targeting signal-transducer-and-activator-of-transcription-3 for prevention and therapy of cancer: modern target but ancient solution. Ann N Y Acad Sci 1091: 151–169.BB AggarwalG. SethiKS AhnSK SandurMK Pandey2006Targeting signal-transducer-and-activator-of-transcription-3 for prevention and therapy of cancer: modern target but ancient solution.Ann N Y Acad Sci1091151169

3. Yee Koh M, Spivak-Kroizman TR, Powis G (2008) HIF-1 regulation: not so easy come, easy go. Trends Biochem Sci 33: 526–534.M. Yee KohTR Spivak-KroizmanG. Powis2008HIF-1 regulation: not so easy come, easy go.Trends Biochem Sci33526534

4. Jung JE, Lee HG, Cho IH, Chung DH, Yoon SH, et al. (2005) STAT3 is a potential modulator of HIF-1-mediated VEGF expression in human renal carcinoma cells. Faseb J 19: 1296–1298.JE JungHG LeeIH ChoDH ChungSH Yoon2005STAT3 is a potential modulator of HIF-1-mediated VEGF expression in human renal carcinoma cells.Faseb J1912961298

5. Jung JE, Kim HS, Lee CS, Shin YJ, Kim YN, et al. (2008) STAT3 inhibits the degradation of HIF-1alpha by pVHL-mediated ubiquitination. Exp Mol Med 40: 479–485.JE JungHS KimCS LeeYJ ShinYN Kim2008STAT3 inhibits the degradation of HIF-1alpha by pVHL-mediated ubiquitination.Exp Mol Med40479485

6. Pisha E, Chai H, Lee IS, Chagwedera TE, Farnsworth NR, et al. (1995) Discovery of betulinic acid as a selective inhibitor of human melanoma that functions by induction of apoptosis. Nat Med 1: 1046–1051.E. PishaH. ChaiIS LeeTE ChagwederaNR Farnsworth1995Discovery of betulinic acid as a selective inhibitor of human melanoma that functions by induction of apoptosis.Nat Med110461051

7. Mullauer FB, Kessler JH, Medema JP (2010) Betulinic acid, a natural compound with potent anticancer effects. Anticancer Drugs 21: 215–227.FB MullauerJH KesslerJP Medema2010Betulinic acid, a natural compound with potent anticancer effects.Anticancer Drugs21215227

8. Fulda S (2009) Betulinic acid: a natural product with anticancer activity. Mol Nutr Food Res 53: 140–146.S. Fulda2009Betulinic acid: a natural product with anticancer activity.Mol Nutr Food Res53140146

9. Mukherjee PK, Saha K, Das J, Pal M, Saha BP (1997) Studies on the anti-inflammatory activity of rhizomes of Nelumbo nucifera. Planta Med 63: 367–369.PK MukherjeeK. SahaJ. DasM. PalBP Saha1997Studies on the anti-inflammatory activity of rhizomes of Nelumbo nucifera.Planta Med63367369

10. De Clercq E (1995) Antiviral therapy for human immunodeficiency virus infections. Clin Microbiol Rev 8: 200–239.E. De Clercq1995Antiviral therapy for human immunodeficiency virus infections.Clin Microbiol Rev8200239

11. Chadalapaka G, Jutooru I, Burghardt R, Safe S (2010) Drugs that target specificity proteins downregulate epidermal growth factor receptor in bladder cancer cells. Mol Cancer Res 8: 739–750.G. ChadalapakaI. JutooruR. BurghardtS. Safe2010Drugs that target specificity proteins downregulate epidermal growth factor receptor in bladder cancer cells.Mol Cancer Res8739750

12. Eichenmuller M, Hemmerlein B, von Schweinitz D, Kappler R (2010) Betulinic acid induces apoptosis and inhibits hedgehog signalling in rhabdomyosarcoma. Br J Cancer 103: 43–51.M. EichenmullerB. HemmerleinD. von SchweinitzR. Kappler2010Betulinic acid induces apoptosis and inhibits hedgehog signalling in rhabdomyosarcoma.Br J Cancer1034351

13. Pandey MK, Sung B, Aggarwal BB (2010) Betulinic acid suppresses STAT3 activation pathway through induction of protein tyrosine phosphatase SHP-1 in human multiple myeloma cells. Int J Cancer 127: 282–292.MK PandeyB. SungBB Aggarwal2010Betulinic acid suppresses STAT3 activation pathway through induction of protein tyrosine phosphatase SHP-1 in human multiple myeloma cells.Int J Cancer127282292

14. Rabi T, Shukla S, Gupta S (2008) Betulinic acid suppresses constitutive and TNFalpha-induced NF-kappaB activation and induces apoptosis in human prostate carcinoma PC-3 cells. Mol Carcinog 47: 964–973.T. RabiS. ShuklaS. Gupta2008Betulinic acid suppresses constitutive and TNFalpha-induced NF-kappaB activation and induces apoptosis in human prostate carcinoma PC-3 cells.Mol Carcinog47964973

15. Zhong H, De Marzo AM, Laughner E, Lim M, Hilton DA, et al. (1999) Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases. Cancer Res 59: 5830–5835.H. ZhongAM De MarzoE. LaughnerM. LimDA Hilton1999Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases.Cancer Res5958305835

16. Smith TG, Robbins PA, Ratcliffe PJ (2008) The human side of hypoxia-inducible factor. Br J Haematol 141: 325–334.TG SmithPA RobbinsPJ Ratcliffe2008The human side of hypoxia-inducible factor.Br J Haematol141325334

17. Niu G, Briggs J, Deng J, Ma Y, Lee H, et al. (2008) Signal transducer and activator of transcription 3 is required for hypoxia-inducible factor-1alpha RNA expression in both tumor cells and tumor-associated myeloid cells. Mol Cancer Res 6: 1099–1105.G. NiuJ. BriggsJ. DengY. MaH. Lee2008Signal transducer and activator of transcription 3 is required for hypoxia-inducible factor-1alpha RNA expression in both tumor cells and tumor-associated myeloid cells.Mol Cancer Res610991105

18. Zhang J, Li L, Kim SH, Hagerman AE, Lu J (2009) Anti-cancer, anti-diabetic and other pharmacologic and biological activities of penta-galloyl-glucose. Pharm Res 26: 2066–2080.J. ZhangL. LiSH KimAE HagermanJ. Lu2009Anti-cancer, anti-diabetic and other pharmacologic and biological activities of penta-galloyl-glucose.Pharm Res2620662080

19. Ellis LM, Hicklin DJ (2008) VEGF-targeted therapy: mechanisms of anti-tumour activity. Nat Rev Cancer 8: 579–591.LM EllisDJ Hicklin2008VEGF-targeted therapy: mechanisms of anti-tumour activity.Nat Rev Cancer8579591

20. Niu G, Wright KL, Huang M, Song L, Haura E, et al. (2002) Constitutive Stat3 activity up-regulates VEGF expression and tumor angiogenesis. Oncogene 21: 2000–2008.G. NiuKL WrightM. HuangL. SongE. Haura2002Constitutive Stat3 activity up-regulates VEGF expression and tumor angiogenesis.Oncogene2120002008

21. Wei D, Le X, Zheng L, Wang L, Frey JA, et al. (2003) Stat3 activation regulates the expression of vascular endothelial growth factor and human pancreatic cancer angiogenesis and metastasis. Oncogene 22: 319–329.D. WeiX. LeL. ZhengL. WangJA Frey2003Stat3 activation regulates the expression of vascular endothelial growth factor and human pancreatic cancer angiogenesis and metastasis.Oncogene22319329

22. Jemal A, Siegel R, Ward E, Hao Y, Xu J, et al. (2009) Cancer statistics, 2009. CA Cancer J Clin 59: 225–249.A. JemalR. SiegelE. WardY. HaoJ. Xu2009Cancer statistics, 2009.CA Cancer J Clin59225249

23. Pulukuri SM, Gondi CS, Lakka SS, Jutla A, Estes N, et al. (2005) RNA interference-directed knockdown of urokinase plasminogen activator and urokinase plasminogen activator receptor inhibits prostate cancer cell invasion, survival, and tumorigenicity in vivo. J Biol Chem 280: 36529–36540.SM PulukuriCS GondiSS LakkaA. JutlaN. Estes2005RNA interference-directed knockdown of urokinase plasminogen activator and urokinase plasminogen activator receptor inhibits prostate cancer cell invasion, survival, and tumorigenicity in vivo.J Biol Chem2803652936540

24. Miles DH, Kokpol U, Zalkow LH, Steindel SJ, Nabors JB (1974) Tumor inhibitors. I. Preliminary investigation of antitumor activity of sarracenia flava. J Pharm Sci 63: 613–615.DH MilesU. KokpolLH ZalkowSJ SteindelJB Nabors1974Tumor inhibitors. I. Preliminary investigation of antitumor activity of sarracenia flava.J Pharm Sci63613615

25. Kingston DG, Munjal RC (1976) Plant anticancer agents. VIII. Constituents of Inga punctata. Lloydia 41: 499–500.DG KingstonRC Munjal1976Plant anticancer agents. VIII. Constituents of Inga punctata.Lloydia41499500

26. Trumbull ER, Bianchi E, Eckert DJ, Wiedhopf RM, Cole JR (1976) Tumor inhibitory agents from Vauquelinia corymbosa (Rosaceae). J Pharm Sci 65: 1407–1408.ER TrumbullE. BianchiDJ EckertRM WiedhopfJR Cole1976Tumor inhibitory agents from Vauquelinia corymbosa (Rosaceae).J Pharm Sci6514071408

27. Kessler JH, Mullauer FB, de Roo GM, Medema JP (2007) Broad in vitro efficacy of plant-derived betulinic acid against cell lines derived from the most prevalent human cancer types. Cancer Lett 251: 132–145.JH KesslerFB MullauerGM de RooJP Medema2007Broad in vitro efficacy of plant-derived betulinic acid against cell lines derived from the most prevalent human cancer types.Cancer Lett251132145

28. Zuco V, Supino R, Righetti SC, Cleris L, Marchesi E, et al. (2002) Selective cytotoxicity of betulinic acid on tumor cell lines, but not on normal cells. Cancer Lett 175: 17–25.V. ZucoR. SupinoSC RighettiL. ClerisE. Marchesi2002Selective cytotoxicity of betulinic acid on tumor cell lines, but not on normal cells.Cancer Lett1751725

29. Rieber M, Strasberg RM (1998) Induction of p53 without increase in p21WAF1 in betulinic acid-mediated cell death is preferential for human metastatic melanoma. DNA Cell Biol 17: 399–406.M. RieberRM Strasberg1998Induction of p53 without increase in p21WAF1 in betulinic acid-mediated cell death is preferential for human metastatic melanoma.DNA Cell Biol17399406

30. Selzer E, Pimentel E, Wacheck V, Schlegel W, Pehamberger H, et al. (2000) Effects of betulinic acid alone and in combination with irradiation in human melanoma cells. J Invest Dermatol 114: 935–940.E. SelzerE. PimentelV. WacheckW. SchlegelH. Pehamberger2000Effects of betulinic acid alone and in combination with irradiation in human melanoma cells.J Invest Dermatol114935940

31. Fulda S, Friesen C, Los M, Scaffidi C, Mier W, et al. (1997) Betulinic acid triggers CD95 (APO-1/Fas)- and p53-independent apoptosis via activation of caspases in neuroectodermal tumors. Cancer Res 57: 4956–4964.S. FuldaC. FriesenM. LosC. ScaffidiW. Mier1997Betulinic acid triggers CD95 (APO-1/Fas)- and p53-independent apoptosis via activation of caspases in neuroectodermal tumors.Cancer Res5749564964

32. Karna E, Szoka L, Palka JA (2010) Betulinic acid inhibits the expression of hypoxia-inducible factor 1alpha and vascular endothelial growth factor in human endometrial adenocarcinoma cells. Mol Cell Biochem 340: 15–20.E. KarnaL. SzokaJA Palka2010Betulinic acid inhibits the expression of hypoxia-inducible factor 1alpha and vascular endothelial growth factor in human endometrial adenocarcinoma cells.Mol Cell Biochem3401520

33. Xu Q, Briggs J, Park S, Niu G, Kortylewski M, et al. (2005) Targeting Stat3 blocks both HIF-1 and VEGF expression induced by multiple oncogenic growth signaling pathways. Oncogene 24: 5552–5560.Q. XuJ. BriggsS. ParkG. NiuM. Kortylewski2005Targeting Stat3 blocks both HIF-1 and VEGF expression induced by multiple oncogenic growth signaling pathways.Oncogene2455525560

34. Forsythe JA, Jiang BH, Iyer NV, Agani F, Leung SW, et al. (1996) Activation of vascular endothelial growth factor gene transcription by Hypoxia-inducible factor 1. Mol Cell Biol 16: 4604–4613.JA ForsytheBH JiangNV IyerF. AganiSW Leung1996Activation of vascular endothelial growth factor gene transcription by Hypoxia-inducible factor 1.Mol Cell Biol1646044613

35. Gariboldi MB, Ravizza R, Monti E (2010) The IGFR1 inhibitor NVP-AEW541 disrupts a pro-survival and pro-angiogenic IGF-STAT3-HIF1 pathway in human glioblastoma cells. Biochem Pharmacol 80: 455–462.MB GariboldiR. RavizzaE. Monti2010The IGFR1 inhibitor NVP-AEW541 disrupts a pro-survival and pro-angiogenic IGF-STAT3-HIF1 pathway in human glioblastoma cells.Biochem Pharmacol80455462

36. Leeman-Neill RJ, Wheeler SE, Singh SV, Thomas SM, Seethala RR, et al. (2009) Guggulsterone enhances head and neck cancer therapies via inhibition of signal transducer and activator of transcription-3. Carcinogenesis 30: 1848–1856.RJ Leeman-NeillSE WheelerSV SinghSM ThomasRR Seethala2009Guggulsterone enhances head and neck cancer therapies via inhibition of signal transducer and activator of transcription-3.Carcinogenesis3018481856

37. Park KY, Lee HJ, Jeong SJ, Kim HS, Kim SH, et al. (2010) 1,2,3,4,6-Penta-O-galloly-beta-D-glucose suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1alpha and signaling in LNCaP prostate cancer cells. Biol Pharm Bull 33: 1835–1840.KY ParkHJ LeeSJ JeongHS KimSH Kim20101,2,3,4,6-Penta-O-galloly-beta-D-glucose suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1alpha and signaling in LNCaP prostate cancer cells.Biol Pharm Bull3318351840

38. Kim SH, Ahn KS, Jeong SJ, Kwon TR, Jung JH, et al. (2011) Janus activated kinase 2/signal transducer and activator of transcription 3 pathway mediates icariside II-induced apoptosis in U266 multiple myeloma cells. Eur J Pharmacol 654: 10–16.SH KimKS AhnSJ JeongTR KwonJH Jung2011Janus activated kinase 2/signal transducer and activator of transcription 3 pathway mediates icariside II-induced apoptosis in U266 multiple myeloma cells.Eur J Pharmacol6541016

39. Choo EJ, Rhee YH, Jeong SJ, Lee HJ, Kim HS, et al. (2011) Anethole exerts antimetatstaic activity via inhibition of matrix metalloproteinase 2/9 and AKT/mitogen-activated kinase/nuclear factor kappa B signaling pathways. Biol Pharm Bull 34: 41–46.EJ ChooYH RheeSJ JeongHJ LeeHS Kim2011Anethole exerts antimetatstaic activity via inhibition of matrix metalloproteinase 2/9 and AKT/mitogen-activated kinase/nuclear factor kappa B signaling pathways.Biol Pharm Bull344146

40. Lee HJ, Kim SA, Lee HJ, Jeong SJ, Han I, et al. (2010) Paeonol oxime inhibits bFGF-induced angiogenesis and reduces VEGF levels in fibrosarcoma cells. PLoS One 5: e12358.HJ LeeSA KimHJ LeeSJ JeongI. Han2010Paeonol oxime inhibits bFGF-induced angiogenesis and reduces VEGF levels in fibrosarcoma cells.PLoS One5e12358