Antiangiogenic effects of a novel synthetic curcumin ... - Cancer Letters

Cancer Letters 357 (2015) 557–565

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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Original Articles

Antiangiogenic effects of a novel synthetic curcumin analogue in pancreatic cancer Ganji Purnachandra Nagaraju a, Shijun Zhu a, Jasmine E. Ko a, Nakkana Ashritha a, Ramesh Kandimalla a, James P. Snyder b, Mamoru Shoji a, Bassel F. El-Rayes a,* a b

Department of Hematology and Medical Oncology, Emory University, Atlanta, GA 30322, USA Department of Chemistry, Emory University, Atlanta, GA 30322, USA

A R T I C L E

I N F O

Article history: Received 24 November 2014 Accepted 4 December 2014 Keywords: Pancreatic cancer Angiogenesis Curcumin EF31 UBS109

A B S T R A C T

Hypoxia-inducible factors (HIFs) and NF-κB play essential roles in cancer cell growth and metastasis by promoting angiogenesis. Heat shock protein 90 (Hsp90) serves as a regulator of HIF-1α and NF-κB protein. We hypothesized that curcumin and its analogues EF31 and UBS109 would disrupt angiogenesis in pancreatic cancer (PC) through modulation of HIF-1α and NF-κB. Conditioned medium from MIA PaCa-2 or PANC-1 cells exposed to curcumin and its analogues in vitro significantly impaired angiogenesis in an egg CAM assay and blocked HUVEC tube assembly in comparison to untreated cell medium. In vivo, EF31 and UBS109 blocked the vascularization of subcutaneous matrigel plugs developed by MIA PaCa-2 in mice. Significant inhibition of VEGF, angiopoietin 1, angiopoietin 2, platelet derived growth factor, COX-2, and TGFβ secretion was observed in PC cell lines treated with UBS109, EF31 or curcumin. Treatment with UBS109, EF31 or curcumin inhibited HSP90, NF-κB, and HIF-1α transcription in PC cell lines. UBS109 and EF31 inhibited HSP90 and HIF-1α expression even when elevated due to NF-κB (p65) overexpression. Finally, we demonstrate for the first time that curcumin analogues EF31 and UBS109 induce the downregulation of HIF-1α, Hsp90, COX-2 and VEGF in tumor samples from xenograft models compared to untreated xenografts. Altogether, these results suggest that UBS109 and EF31 are potent curcumin analogues with antiangiogenic activities. © 2014 Elsevier Ireland Ltd. All rights reserved.

Introduction Pancreatic cancer is the fourth leading cause of cancer related death in the USA [1]. The majority of patients with pancreatic cancer present with advanced stage disease and the only treatment option is systemic chemotherapy [2]. Approximately 15% of patients with pancreatic cancer present with resectable disease [3]. The vast majority of this group recurs after resection and at that point will require systemic therapy. Although systemic chemotherapy in pancreatic cancer has improved over the past few years with the introduction of several active chemotherapeutic agents, the median survival of patients with advanced stage disease remains below 1 year [3]. Therefore, development of novel therapeutic options in pancreatic cancer is urgently needed. Angiogenesis is an essential process for tumors to grow and metastasize [4]. Targeting angiogenesis has proven to be an effective strategy in several cancers such as colorectal, gastric and lung cancers [5,6]. Angiogenesis is a complex process that depends on the interaction of several growth factors such as vascular endothelial growth

* Corresponding author. Tel.: +1 404 778 5419; fax: +1 404 778 5520. E-mail address: [email protected] (B.F. El-Rayes). http://dx.doi.org/10.1016/j.canlet.2014.12.007 0304-3835/© 2014 Elsevier Ireland Ltd. All rights reserved.

factor (VEGF), angiopoietin, fibroblast growth factor, platelet derived growth factor and tumor growth factor β (TGFβ) [7]. The expression of these growth factors is regulated by transcriptional factors such as nuclear factor κB (NF-κB) and hypoxia induced factor (HIF1α) [8]. The chaperone protein HSP90 has a central role in the stabilization and activation of these transcription factors [9]. In pancreatic cancer, the presence of hypoxia, elevated cytokines, activation of proto-oncogenes and inhibition of tumor suppressor genes activate NF-κB and HIF-1α leading to synthesis and secretion of angiogenic factors [10]. Therefore, targeting NF-κB and HIF-1α can inhibit angiogenesis, and this represents a rational approach for therapy in pancreatic cancer. Curcumin is a natural polyphenol present in turmeric [11,12]. Preclinical evaluation of curcumin has demonstrated significant anticancer effects against pancreatic cancer cell lines [6]. The mechanism of action of curcumin and its analogues, EF31 and UBS109, is believed to be mediated through inhibition of NF-κB and HSP90 [13]. A phase II clinical trial investigated the activity of curcumin in patients with pancreatic cancer [14]. Two patients demonstrated objective benefit from curcumin supplementation, including one patient with stable disease for over 18 months. The limited benefit of curcumin in clinical trials may be related to its low potency, aqueous instability and poor bioavailability. In order to address these

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issues, our group has developed over 100 curcumin analogues with improved solubility and potency. EF31 and UBS109 are two promising curcumin analogues with preliminary data demonstrating higher potency than curcumin [13]. Since curcumin is known to inhibit NF-κB and HSP90, it has the potential to affect several angiogenic growth factors simultaneously. In this study, we evaluated and compared the antiangiogenic effects of curcumin, EF31 and UBS109 in pancreatic cancer cell lines using in vitro and in vivo models. We also studied the mechanism of antiangiogenic action of UBS109 and EF31. Materials and methods Cells and materials Pancreatic cancer cell lines MIA PaCa-2 and PANC-1 were obtained from American Type Culture Collection (ATCC, Manassas, VA) in 2011. Characterization and authentication were reported by ATCC in the accompanying certificate analysis. All cells were verified mycoplasma-free using Lonza’s MycoAlert mycoplasma detection kit (Cat # LT07-318, USA). Cells are routinely tested once in every two months. Cultures had been validated by ATCC to be mycoplasma-free, expressed only basal epithelial cell markers and unique human DNA profile. They were subsequently cultured in DMEM medium (Cellgro Mediatech, Inc, Manassas, VA) supplemented with 10% fetal bovine serum (Invitrogen Corporation, Carlsbad, CA), 50 units/ml penicillin, and 50 μg/ml streptomycin (Life Technologies, Inc., Frederick, MD). Cells were incubated at 37 °C in humidified 5% CO2. Human umbilical vein endothelial cells (HUVEC) were obtained from ATCC. Specific antibodies against HSP90, HIF-1α, VEGF (sc-57496), COX-2, CD-31, PDGFA, FGF2, Ang-1, Ang-2, TGFβ1, β-actin, and HRP (horseradish peroxidase) conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). pCMV4 vector containing either full-length sequence (NFκB) or empty vector plasmids were purchased from Addgene (Cambridge, MA). HUVEC tube formation assay MIA PaCa-2 and PANC-1 cells (2 × 106/well) were seeded in 6-well plates. MIA PaCa-2 cells were treated with curcumin (10 μM), EF31 (750 nM) and UBS109 (250 nM) for 24 hours. PANC-1 cells were treated with curcumin (20 μM), EF31 (1.25 μM) and UBS109 (250 nM) for 24 hours. The medium was discarded (to remove curcumin, EF31, or UBS109) and fresh conditioned (serum free) media were added without treatments. After 24 hours the media were collected from control and treated cells. The protein concentration in the condition medium was determined (Protein Assay Kit; Bio-Rad, Hercules, CA). Equal amounts of protein in conditioned medium were added to 3 × 102 HUVEC in 24-well matrigel coated plates. The experiment was in triplicate. Cells were allowed to grow for 24 hrs. The formation of capillary-like structures was captured with an Olympus IX51 (Olympus Corporation of Americas, Center Valley, PA) inverted microscope with a ×4 objective magnification. The degree of angiogenesis was quantified based on the average number of tubes formed in 5 fields. Egg chorioallantoic membrane (CAM) assay MIA PaCa-2 and PANC-1 cells (2 × 106/well) were seeded in 75 cm2 flasks. MIA PaCa-2 cells were treated with curcumin (10 μM), EF31 (750 nM) and UBS109 (250 nM) for 24 hours. PANC-1 cells were treated with curcumin (20 μM), EF31 (1.25 μM) and UBS109 (250 nM) for 24 hours. The medium was discarded (to remove curcumin, EF31, or UBS109) and fresh conditioned (serum free) media were added without treatments. After 24 hours the media were collected from control and treated cells. Fertilized chicken eggs were incubated at 70% humidity and 37 °C. The protein concentration in the conditioned medium was determined (Protein Assay Kit; BioRad, Hercules, CA). On day 5, 100 μl of fresh medium (control) and conditioned media obtained from treated and untreated cells (concurrent control) were injected into the eggs. Five eggs were used for each treatment group. The CAMs were photographed in ovo with a DP20 Olympus digital camera (Olympus Corporation of the Americas, Center Valley, PA) at a magnification of ×1.5 on day 17. The grafts were then scored for vascularization using AngioQuant software [15]. VEGF activity assay Cells were treated as previously described. VEGF concentration in the conditioned medium was determined using a commercial Human VEGF Quantikine ELISA kit (R&D Systems Inc., Minneapolis, MN). The medium was discarded (to remove curcumin, EF31, or UBS109) and fresh conditioned (serum free) media were added without treatments. After 24 hours the media were collected from control and treated cells. The conditioned media and assay diluents were added to VEGF antibody precoated ELISA plates and incubated for 2 hr at room temperature. Six plates were used for each treatment group. The reaction mixture was aspirated and washed 3 times with washing buffer. An enzyme-linked polyclonal antibody specific for VEGF (VEGF conjugate) 200 μl was added to each well and incubated for 2 hr at room temper-

ature. After washing 3 times with buffer, 200 μl of substrate solution was added to each well to develop color in proportion to the amount of VEGF bound in the initial step. After incubation for 20 min at room temperature, 50 μl stop solution was added to each well to stop color development. The intensity of color was measured at 450 nm within 30 min. λ correction 570 for background. Transient transfection studies Cells were serum starved for 8 hours and transfected with pCMV4 vector containing either full-length sequence (NF-κB-p65) or empty vector or mock (PBS) using Lipofectamine 2000 transfection reagent per the manufacturer’s instructions (Invitrogen). Serum containing DMEM (complete) medium was added after 12 hours. Cells were harvested at 72 hours and lysed for Western blotting. For combination treatments, MIA PaCa-2 cells were treated with curcumin (10 μM), EF31 (750 nM) and UBS109 (250 nM). PANC-1 cells were treated with curcumin (20 μM), EF31 (1.25 μM) and UBS109 (250 nM). Treatment started 24 hours post-transfection and the cells were incubated for another 48 hours before being harvested for further analysis. Western blotting Cells were harvested at the end of treatment as described above. The cells were lysed in RIPA protein extraction buffer containing protease inhibitors (SigmaAldrich, St. Louis, MO). Protein concentration in the lysis buffer was determined using a commercial Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL). Equal amounts of protein were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membrane. Membranes were incubated with primary antibodies followed by HRP-conjugated secondary antibodies. Bound antibodies were visualized using enhanced chemiluminescence. To confirm equal loading, membranes were verified by re-probing with an antibody specific for the housekeeping protein β-actin. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) Total RNA was extracted using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA). The reverse transcription (RT) step was performed with MultiScribeTM reverse transcriptase (Applied Biosystems, USA). To determine the transcript levels from cDNA, qRT-PCR was carried out with 1 μl of cDNA using primers for HSP90, HIF-1α, VEGF, PDGFA, FGF2, Agn-1, TGFβ1, and actin (primer details given in Table 1). The qRTPCR conditions were as follows: initial denaturation at 95 °C for 3 min, followed by 30 cycles at 95 °C for 1 min, 57 °C for 30 sec (depending on primer sets), 72 °C for 1 min, followed by a final extension at 72 °C for 7 min. Melting curve analysis verified a single product. Relative quantities were calculated and standardized by comparison to actin. Subcellular fractionation Cytoplasmic and nuclear fractions were prepared using a nuclear extraction kit according to the manufacturer’s instructions (Chemicon International, Inc., Temecula, CA). Briefly, harvested cells were lysed in cytoplasmic lysis buffer for 30 min at 4 °C and centrifuged (8000 × g for 20 min). The supernatant was collected as the cytosolic fraction. The nuclear pellet was re-suspended in nuclear extraction buffer for

Table 1 Primers used in qRT-PCR. Primer name

Sequence direction 5′ 3′

HSP90-sense HSP90-antisense HIF-1α-sense HIF-1α-antisense VEGF-sense VEGF-antisense Ang-1-sense Ang-1-antisense FGF2-sense FGF2-antisense PDGFA-sense PDGFA-antisense TGFβ1-sense TGFβ1-antisense Actin-sense Actin-antisense

TTC AGA CAG AGC CAA GGT GC CAATGA CAT CAA CTG GGC AAT CAG AGC AGG AAA AGG AGT CA AGT AGC TGC ATG ATC GTC TG GCT ACT GCC ATC CAA TCG AG CTC TCC TAT GTG CTG GCC TT GAT GGT GGT TTG ATG CTT GT CGG ATT TCT TTG TTG CTT TC’ TTG GCT TGG AAA TGT GTT TT AAA AGC AGT CTG CAG GTT TG ATT ATC GGG AAG AGG ACA CG AGA ACA AAG ACC GCA CAC TG CGT GGA GCT GTA CCA GAA ATA TCC GGT GAC ATC AAA AGA TAA TGG CAC CCA GCA CAA TGA A CTA AGT CAT AGT CCG CCT AGA AGC A

Messenger size 167 bp 231 bp 208 bp 205 bp 153 bp 204 bp 106 bp 185 bp

Heat shock protein 90 (HSP90); hypoxia inducible factor-1α (HIF-1α); vascular endothelial growth factor (VEGF); angiopoietin 1 (Ang-1); fibroblast growth factor 2 (FGF2); platelet-derived growth factor alpha polypeptide (PDGFA); transforming growth factor β1 (TGFβ1).


60 min at 4 °C and the nuclear fraction was collected after centrifugation (16,000 × g for 5 min). Western blot analysis was performed for NF-κB, α-tubulin (cytoplasmic marker and loading control) and lamin B (nuclear marker and served as loading control). Electrophoretic mobility shift assay (EMSA) Nuclear lysates were prepared using the nuclear extraction kit from Chemicon International, Inc. (Temecula, CA) and were then analyzed using the non-radioactive EMSA kit from LI-COR Biosciences (Lincoln, NE) per the manufacturer’s protocol. Briefly, 5 μg of nuclear extract was incubated with 1 μl of NF-κB IRD® 700 Infrared dye labeled double-stranded oligonucleotides (5′-AGTTGAGGGGACTTTCCCAGGC3′ and 3′-TCAACTCCCCTGAAAGGGTCCG-5′), 2 μl of 10× binding buffer, 2.5 mM DTT, 0.25% Tween-20, and 1 μg of poly (dl-dC) in a total volume of 20 μl for 30 min at room temperature in dark. Samples were separated on a 6% polyacrylamide gel in 0.25 × Tris–borate–EDTA running buffer for 60 min at 80 V. The gel was scanned by direct infrared fluorescence detection on the Odyssey® Imaging System (LI-COR Bioscience, Lincoln, NE USA). Immunocytochemistry MIA PaCa-2 and PANC-1 cells were grown in 8-well chamber slides and then cells were treated as previously described. Cells were fixed in 4% formalin followed by permeabilization in Triton-X100. Non-specific binding was blocked in 3% BSA in PBS, followed by incubation with anti-p65-NF-κB (1:200 dilution) for 4 hours at room temperature. The cells were washed with PBS three times for 5 min to remove excess primary antibodies and incubated with Alexa Fluor®-conjugated secondary antibodies (anti-mouse or anti-rabbit, 1:500 dilution). Subsequently, 4′-6-diamidino2-phenylindole (DAPI) was used for nuclear counterstaining and slides were then mounted. The cells were examined by fluorescence microscopy (Zeiss LSM 510 Meta Confocal microscope, Peabody, MA). Immunohistochemical (IHC) analyses Tumor samples were fixed partly with 4% formalin, embedded in paraffin and subsequently processed for H&E and for IHC staining HSP90, CD31, VEGF, HIF1α and COX-2. Tissue sections (5 μm) were de-paraffinized in xylene, rehydrated in graded ethanol solutions, permeabilized in 0.1% Triton X-100, and incubated overnight at 4 °C with primary antibodies. Slides were washed twice in 1× PBS and incubated in HRP-conjugated secondary antibodies for 1 hour at room temperature. The sections with HRP-conjugated antibodies were incubated with DAB (3, 3′-diaminobenzidine) solution for 5–10 min. Hematoxylin was used for nuclear counterstaining. Slides were evaluated using the Hamamatsu NanoZoomer 2.0-HT Slide Scanner (Meyer Instruments Houston, TX) at 20× magnification. Slides were evaluated by an experienced pathologist and scored according to the intensity of staining as 0 (no staining), 1, 2, or 3 (increasingly intense staining). Immunoprecipitation Cells were harvested at the end of treatment as described above. Cell lysates from the treated cells were obtained using RIPA buffer containing protease inhibitors (Sigma, St. Louis, MO), homogenized by Dounce homogenizer, and centrifuged at 13,000 × g to separate the cell debris. The protein concentration of the cell lysates was determined. To demonstrate the interaction between HSP90 and HIF-1α proteins in cells, 500 μg of protein lysate was incubated with 10 μg of anti-HSP90 antibody and antiHSP90 antibody/HSP90 complexes were precipitated by protein A/G PLUS-Agarose (sc-2003; Santa Cruz Biotechnology, Dallas, TX). Protein A/G PLUS-Agarose precipitants were washed three times in RIPA buffer and eluted in SDS gel loading buffer by boiling. The samples were resolved by SDS-PAGE and transferred onto PVDF membranes. Blots were incubated with 10 μg of anti-HIF-1α antibody or non-specific mouse anti-IgG followed by HRP-conjugated secondary antibodies. Bound antibodies were visualized using enhanced chemiluminescence.

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The grafts were then scored for vascularization using AngioQuant software [15]. In addition, IHC, Western blot and qRT-PCR were performed as previously described. Statistical analysis Data obtained from the different treatment groups were analyzed using oneway ANOVA by Neumann–Keuls method of Instat. Data are represented in the form of mean ± SD, which was obtained from five individual repetitions.

Results Antiangiogenic effects of curcumin, EF31 and UBS109 The antiangiogenic effects of curcumin, EF31 and UBS109 in MIA PaCa-2 and PANC-1 cells were assessed using three separate assays: HUVEC tube formation, egg CAM assay and in vivo matrigel assay (Fig. 1A–C). In the tube formation assay (Fig. 1A), medium from pancreatic cells treated with curcumin, EF31 or UBS109 significantly impaired the ability of HUVEC to form tubes compared to the medium from the concurrent control (without treatment, PC cells cultured media) treated pancreatic cells and control (fresh medium). HUVEC formed significantly less tubes in the medium from pancreatic cells treated with curcumin, EF31 or UBS109 as compared to that from concurrent control cells. The antiangiogenic effect observed with EF31 or UBS109 was significantly greater than with curcumin. In the egg CAM assay (Fig. 1B), the culture medium from MIA PaCa-2 and PANC-1 cell lines (concurrent control) significantly increased angiogenesis as compared to the medium from curcumin treated cells. EF31 and UBS109 inhibited angiogenesis as compared to the control medium and the medium from the curcumin treated cell lines. EF31 and UBS109 displayed more potent antiangiogenic effects than curcumin. In the matrigel experiment, significant inhibition of angiogenesis was observed in the animals treated with EF31 or UBS109 as compared to the untreated animals (Fig. 1C). The effects were similar in both cell lines. Curcumin, EF31 and UBS109 inhibit VEGF production After observing the antiangiogenic effects of curcumin, EF31 and UBS109 in both pancreatic cancer cell lines, the underlying molecular mechanism for this inhibition was evaluated. Using an ELISA assay, significant inhibition of VEGF in the culture medium of MIA PaCa-2 and PANC-1 cells treated with curcumin, EF31 or UBS109 in comparison to untreated cells was observed (Fig. 2A). Western blot analysis (Fig. 2B) confirmed the decreased VEGF expression in both cell lines treated with curcumin, EF31 or UBS109 as compared to untreated cell lines. The mechanism underlying this diminished VEGF expression is transcriptional inhibition as demonstrated by the reduction in VEGF mRNA by qRT-PCR in the curcumin, EF31, UBS109 treated cell lines as compared to untreated cells (Fig. 3B).

Matrigel plug angiogenesis assay Female athymic (immunodeficient) nude mice (Charles River Laboratories, Wilmington, MA) were maintained in a pathogen-free environment, and all in vivo procedures were approved by the Emory University Institutional Animal Care and Use Committee. MIA PaCa-2 cells (1.5 × 106/0.1 ml of 20% Matrigel gel in serum free media) were injected subcutaneously into the flank of 5 week old mice. Mice bearing established tumors (100–200 mm3) were randomized into treatment groups of 3 and i.v. dosed weekly via the tail vein with vehicle (0.5% carboxymethyl cellulose sodium (CMC) with 5% DMSO in sterile water), 25 mg/kg EF31 or 25 mg/ kg UBS109 formulated in vehicle for three weeks. None of the animals died from the treatment. Thirty days after injection of the cells, the mice were weighed and sacrificed. The tumor plugs were harvested and weighed. Tumor index was determined using standard formula: (Weight of the tumor/Weight of the mice) × 100. The vascular density in the plug was captured using a DP20 Olympus digital camera (Olympus Corporation of the Americas, Center Valley, PA) at a magnification of 1.5×.

Curcumin, EF31 and UBS109 inhibit multiple angiogenic pathways In order to characterize the mechanism of inhibition of transcription of these angiogenic proteins, evaluation of the effects of curcumin, EF31 and UBS109 on HIF-1α, HSP90 and NF-κB was performed. Western blot analysis showed substantial reductions in HIF1α and HSP90 protein expression in both cell lines treated with curcumin, EF31 or UBS109 (Fig. 2B). Treatment with curcumin, EF31 and UBS109 resulted in inhibition of expression of angiopoietin 1 (Ang-1), Ang-2, fibroblast growth factor 2 (FGF-2), tumor growth factor (TGF-β), platelet derived growth factor A (PDGFA), and cyclooxygenase 2 (COX-2) (Fig. 2B).

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Fig. 1. In vitro and in vivo antiangiogenic activities of curcumin and its analogues EF31 and UBS109. (A) Tube formation assay. Human umbilical vein endothelial cells (HUVEC) were seeded on matrigel in tumor cell conditioned media. Tumor cells were either untreated or treated with curcumin and its analogues EF31 and UBS109. Tube formation was observed under bright field microscope after 24 hours incubation at 37 °C. The conditioned media from cells treated with curcumin and its analogues EF31 and UBS109 decreased tube formation. Each bar represents the mean ± standard deviation; statistically significant differences between a and b, b and c, b and d (ANOVA p < 0.001). (B) Egg CAM was assayed in chick embryos as described in Materials and Methods. Conditioned medium from fresh, untreated and treated (curcumin and its analogues EF31 and UBS109) cells (MIA PaCa-2 and PANC-1) were injected into the CAM of chick embryos through a window in the egg shell. Conditioned medium from untreated cells increased angiogenic measurements (length, size and junctions) as compared to media from curcumin treated and fresh media (control) treated cells. Conditioned media from EF31 and UBS109-treated cells significantly decreased angiogenic measurements as compared to conditioned media from curcumin treated cells. Each bar represents the mean ± standard deviation. Statistically significant differences between a and b, b and c, b and d (ANOVA p < 0.001). (C) Matrigel plug assay. MIA PaCa-2 cells were mixed with matrigel and subcutaneously implanted into mice. After three weeks, skin around the implanted matrigel tumors was photographed under visible light. EF31 and UBS109 treated tumors showed significant decrease in angiogenic measurements compared to concurrent controls and controls (normal mice skin). Each bar represents the mean ± standard deviation. Statistically significant differences between a and b, b and c, b and d (ANOVA p < 0.001).

It has been shown previously that treatment of NF-κB overexpressing MIA PaCa-2 and PANC-1 cells with curcumin, EF31 or UBS109 resulted in inhibition of NF-κB localization in the nucleus and DNA binding [13]. In the present investigation, EMSA (Fig. 4A) revealed significant

inhibition of NF-κB activation in both cell lines treated with curcumin, EF31 or UBS109. Similar effects of curcumin, EF31 and UBS109 on NFκB were observed using nuclear translocation as evaluated by immunoblotting and immunocytochemistry (Fig. 4B and C).


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(Fig. 3B). Inhibition of NF-κB activation by curcumin, EF31 or UBS109 resulted in downregulation of HIF-1α, HSP90, VEGF and COX-2. This experiment demonstrates that the antiangiogenic effects of UBS109 and EF31 are mediated through the NF-κB pathway.

The inhibition of HIF-1α by curcumin, EF31 and UBS109 occurs through HSP90 HSP90 is known to be a chaperone protein for HIF-1α and NFκB. Treatment with curcumin, EF31 or UBS109 decreased the expression of HSP90, HIF-1α, COX-2, VEGF and NF-κB (Fig. 3A and B). To evaluate the effects of treatment with curcumin, EF31 or UBS109 on the interaction of HSP90 and other proteins in cells, an immunoprecipitation (IP) experiment was performed. IP confirmed HIF-1α is a client protein of HSP90 (Fig. 3C). Treatment with curcumin, EF31 or UBS109 resulted in inhibition of the interaction between HSP90 and HIF-1α. These data show that curcumin, EF31 and UBS109 can inhibit HIF-1α through inhibition of its interaction with HSP90.

EF31 and UBS109 display antiangiogenic effects in vivo The effects of EF31 and UBS109 on human tumor growth were measured in vivo, using MIA PaCa-2 xenografts. The mean tumor index significantly (p < 0.001) decreased in the mice that received EF31 or UBS109 compared to vehicle treated controls (Fig. 5A).Tumors resected from treated animals were then assayed for protein levels. HSP90, HIF-1α, VEGF, TGFβ, PDGFA, Ang-1, and FGF-2 protein expressions (Fig. 5B) were measurably lower in tumors treated with EF31 or UBS109 as compared to control. As shown in Fig. 5C, EF31 and UBS109 inhibited HIF-1α, HSP90, COX-2, and VEGF expression, determined by IHC. As shown in Fig. 5D, EF31 and UBS109 inhibited HIF-1α, NF-κB, and VEGF mRNA expression, compared to control xenograft tumors. In addition, the expression of CD31 was decreased after treatment with EF31 and UBS109. These results corroborate our in vitro findings that NF-κB inhibition indeed alters angiogenesis signaling pathways leading to reduced tumor burden. Fig. 2. Curcumin, EF31 and UBS109 decrease levels of VEGF, HSP90, HIF-1α, COX2, Ang, TGF-β, PDGFA and FGF2. (A) Curcumin, EF31 and UBS109 treated cells showed significant decreases in VEGF secretion compared to controls. Cells were treated as indicated in Materials and Methods. VEGF concentration in the conditioned medium was determined using Quantikine ELISA kit. Each value represents the mean ± standard deviation, obtained from determinations made on a minimum of five cultures per experimental condition. * P < 0.001. (B) Curcumin, EF31 and UBS109 treated cells showed significant decreases in HSP90, VEGF, HIF-1α, COX-2, Ang, TGF-β, PDGFA and FGF2 protein expression compared to controls. Curcumin, EF31 and UBS109 treated and untreated cell lysates were analyzed by Western blot using antibodies against the indicated proteins.

The antiangiogenic effects of curcumin, EF31 and UBS109 are mediated via NF-κB activation To further define the contribution of NF-κB inhibition to the antiangiogenic effects of curcumin, EF31 or UBS109, MIA PaCa-2 and PANC-1 cells were transfected with p65NF-κB. MIA PaCa-2 and PANC-1 cells transfected with an empty vector were used as controls. The overproduction of NF-κB resulted in increased expression of HIF-1α and HSP90 as well as downstream molecules COX-2 and VEGF (Fig. 3A). The mechanism of overexpression was related to increased transcription as observed by increased HIF-1α, HSP90, COX-2 and VEGF mRNA levels (Fig. 3B). Transfection with the empty vector did not affect HIF-1α, HSP90, COX-2 and VEGF expression. Treatment of transfected cells with curcumin, EF31 or UBS109 resulted in inhibition of HIF-1α, HSP90, COX-2 and VEGF mRNA expression

Discussion Curcumin has demonstrated modest single agent activity against pancreatic cancer in phase II trials [14]. Challenges in the development of curcumin relate to its limited solubility and potency [12]. Curcumin has been shown in preclinical models to affect pancreatic cancer cell proliferation, metastasis and resistance to genotoxic agents. In this study, we focused on the antiangiogenic effects of curcumin. In addition, we compared the effects of curcumin to those of two novel and more potent analogues, EF31 and UBS109. The antiangiogenic effects of curcumin, EF31 and UBS109 were shown using three different assays and in two different pancreatic cancer cell lines. The three compounds had a significant antiangiogenic effect as compared to untreated controls. UBS109 was the most effective antiangiogenic agent. The concentration of UBS109 used in these experiments was 1.2% and 20% of curcumin and EF31, respectively [13]. The increased potency of UBS109 coupled with its improved solubility makes it a potentially useful antiangiogenic agent for clinical development. Curcumin, EF31 and UBS109 inhibited fibroblast growth factors (FGF), TGF-β and COX-2, key growth factors involved in pancreatic cancer angiogenesis, microenvironment remodeling and tumor progression. FGF1, 2, 7 and 10 are overexpressed in pancreatic cancer [16,17]. FGF2 has been associated with pancreatic cancer proliferation, angiogenesis, stromal hyperplasia and invasion [18–20].

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Fig. 3. UBS109, EF31 and curcumin inhibit NF-κB, HIF-1α, COX-2 and VEGF expression. UBS109, EF31 and curcumin inhibit HIF-1α interaction with HSP90 in MIA PaCa-2 and PANC-1 pancreatic cancer cell lines. (A) Untransfected control, empty vector control (EV) and NF-κB-p65 transfected cells were treated with curcumin, EF31 and UBS109. Protein was extracted and evaluated using Western blot as described in the Materials and Methods section. NF-κB-p65 transfection increased NF-κB, HIF-1α, COX-2, VEGF and HSP90 expression. Treatment with curcumin, EF31 or UBS109 resulted in inhibition of NF-κB, HIF-1α, COX-2, VEGF and HSP90 expression. (B) Untransfected control, empty vector control (EV) and NF-κB-p65 transfected cells were treated with curcumin, EF31 and UBS109. mRNA was extracted and evaluated using qRT-PCR as described in the Materials and Methods section. NF-κB-p65 transfection increased NF-κB, HIF-1α, COX-2, VEGF and HSP90 mRNA. Treatment with curcumin, EF31 or UBS109 resulted in inhibition of NF-κB, HIF-1α, COX-2, VEGF and HSP90 mRNA. * represents (p < 0.001) significant difference. (C) Untransfected control, empty vector control (EV) and NFκB-p65 transfected cells were treated with curcumin, EF31 and UBS109. HSP90 interaction with the client protein HIF-1α was evaluated using immunoprecipitation as described in the Materials and Methods section. Treatment with curcumin, EF31 or UBS109 resulted in inhibition of interaction between HSP90 and HIF-1α in transfected and untransfected cell lines.

Inhibition of FGF signaling has been shown to be a promising therapeutic approach in pancreatic cancer models [21]. Alterations in the TGF-β signaling pathway occur in almost all pancreatic cancers [22]. In preclinical models, TGF-β has been shown to play a central role in tumor invasion, EMT transition and resistance to therapy [23]. Elevated levels of TGF-β in plasma were associated with worse outcomes in patients with pancreatic cancer [24]. Similarly, COX-2 has been shown to be over-activated in approximately 50% of pancreatic cancers [25]. In addition to its effects on angiogenesis, COX-2 exerts a key role in regulating pancreatic stellate cells and tumor matrix deposition [26]. Ang1 and 2 are important in lymphangiogenesis [27] and are suspected to contribute to resistance to VEGF inhibitors. Similarly, PDGFA is known to be overexpressed in pancreatic cancer and to contribute to angiogenesis and extracellular tumor matrix [28]. Anti-angiogenic agents have had limited success in clinical trials in pancreatic cancer [29,30]. These trials have evaluated therapeutic agents that selectively target VEGF or VEGFR. The complexity of

angiogenic pathways and the interplay between different cytokines and growth factors suggest that targeting one pathway may have limited clinical impact. Selective pharmaceutical inhibitors of FGF, VEGF, COX-2, PDGF or Ang signaling pathways are currently in clinical trials across several cancer types. Combining these selective inhibitors is not feasible given their overlapping toxicities and potential pharmacokinetic drug interactions. One advantage of UBS109 and EF31 is their ability to simultaneously modulate multiple pathways. In this study, we have demonstrated the ability of these analogues to downregulate the expression of COX-2, VEGF, FGF2, PDGFA, Ang1, Ang2 and TGF-β in both in vitro and in vivo models. UBS109 and EF31 inhibit the transcription of these growth factors and cytokines. To characterize the mechanism of action of curcumin, EF31 and UBS109, we focused on two transcriptional factors: NF-κB and HIF1α. Overexpression of NF-κB by stable transfection resulted in increased transcription of HSP90 and HIF-1α. These effects are in agreement with previously published literature showing NF-κB is


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Fig. 4. UBS109, EF31 and curcumin inhibit NF-κB activation in MIA PaCa-2 and PANC-1 cell lines. (A) Cells were treated with curcumin, EF31 and UBS109. NF-κB was evaluated by electrophoretic mobility assay (EMSA) as described in the Materials and Methods section. Curcumin, EF31 and UBS109 inhibited NF-κB (p65) activation compared to untreated cell lines. (B) MIA PaCa-2 and PANC-1 cell lines were treated with curcumin, EF31 and UBS109. NF-κB nuclear translocation was evaluated by immunoblotting as described in the Materials and Methods section. Treatment with curcumin, EF31 or UBS109 inhibited nuclear translocation compared to untreated cells. (C) MIA PaCa-2 and PANC-1 cell lines were treated with curcumin, EF31 and UBS109. NF-κB nuclear translocation was evaluated by immunocytochemistry as described in the Materials and Methods section. Treatment with curcumin, EF31 or UBS109 inhibited nuclear translocation compared to untreated cells.

involved in transcriptional control of HIF-1α [31–33] and HSP90 [13]. Treatment of these transfected cells with EF31 or UBS109 resulted in inhibition of the increased transcription of HSP90 and HIF1α. EF31 or UBS109 decreased nuclear NF-κB, as demonstrated by

immunocytochemistry, and decreased DNA binding of NF-κB, as shown by EMSA. Taken together, these experiments confirm that the effects of curcumin, EF31 and UBS109 are at least in part mediated through inhibition of NF-κB activation. Constitutive activation

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Fig. 5. UBS109 and EF31 inhibit angiogenic molecules in MIA PaCa-2 pancreatic cancer cell xenografts. (A) Subcutaneous tumor xenografts were established in nude mice using MIA PaCa-2 cell lines. Animals were treated with vehicle, EF31 or UBS109 administered weekly I.V. as described in the Materials and Methods section. Tumors were removed from animals. Tumor index was determined using standard formula: (Weight of the tumor/Weight of the mice) × 100. Treatment with EF31 and UBS109 significantly decreased tumor index compared to the vehicle treated controls. (B) Tumor protein extraction was performed and proteins were evaluated using Western blot as described in the Materials and Methods section. Treatment with EF31 or UBS109 resulted in downregulation of HSP90, VEGF, HIF-1α, COX-2, Ang1, TGF-β, PDGFA and FGF2. (C) Subcutaneous tumor xenografts were established in nude mice using MIA PaCa-2 cell lines. Animals were treated with vehicle, EF31 or UBS109 administered weekly I.V. Tumors were removed from animals. HSP90, CD31, COX-2, VEGF, and HIF-1α were evaluated using IHC as described in the Materials and Methods section. Treatment with EF31 or UBS109 decreased the expression of HSP90, CD31, COX-2, VEGF, and HIF-1α in MIA PaCa2 tumor xenografts. (D) Subcutaneous tumor xenografts were established in nude mice using MIA PaCa-2 cell lines. Animals were treated with vehicle, EF31 or UBS109 administered weekly I.V. Tumors were removed from animals. mRNA extraction was performed and evaluated using qRT-PCR as described in the Materials and Methods section. Treatment with EF31 or UBS109 decreased the mRNA expression of VEGF, HIF-1α, and NF-κB in MIA PaCa2 tumor xenografts. * represents (p < 0.001) significant difference.

of NF-κB signaling has been associated with invasiveness, angiogenesis and chemoresistance [34]. NF-κB can be activated via both the canonical and alternative pathways. In pancreatic cancer, NFκB activation is mediated by the alternative pathway and this is mainly mediated by NF-κB-inducing kinase (NIK) [35]. Curcumin has been shown to inhibit the NIK/IKKα complex [36]. Therefore, curcumin and its analogues EF31 and UBS109 are a rational choice to target NIK and NF-κB in pancreatic cancer [13]. Curcumin and its analogues have been shown to promote apoptosis and decrease tumor growth in several cancer models [37].

UBS109 and EF31 can potentiate the growth inhibitory effects of cytotoxic chemotherapeutic agents in pancreatic cancer [13]. UBS109 and EF31 have increased antitumor and antiangiogenic effects in PC in vitro and animal models. UBS109 and EF31 inhibit angiogenesis in PC through inhibition of NF-κB, HIF-1α and HSP90, which are important in PC growth and resistance to therapy. In addition, the inhibitory effects of these compounds on key regulators of the tumor microenvironment such as TGFβ suggest a potential role for UBS109 and EF31 in targeting the stromal desmoplasia in PC by the anti-inflammatory effects due to inhibition of NF-κB. The multiple


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