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Original Research Article

DTT 2022; 1(1): 1-11

Published online July 31, 2022 https://doi.org/10.58502/DTT.22.003

Copyright © The Pharmaceutical Society of Korea.

Selective Anticancer Effects by Oxyclozanide in Triple-Negative Breast Cancer Expressing S100A9

Hyun Hwan Hwang1, Ki Ho Chang2, Yi Young Choi2, Ii-Du Jung2, In-Cheol Kang3, Youngjin Choi4, Mi Jin O1, Chang-Keun Cho1 , Pureum Kang1 , Hye-Jung Park1 , Choon-Gon Jang1 , Seok-Yong Lee1

1School of Pharmacy, Sungkyungkwan University, Suwon, Korea
2Department of RND Center, Ahngook Pharm, Seoul, Korea
3Department of Biological Science, Hoseo University, Asan, Korea
4Department of Food Science & Technology, Hoseo University, Asan, Korea

Correspondence to:Seok-Yong Lee, sylee@skku.edu

Received: April 7, 2022; Revised: May 3, 2022; Accepted: May 25, 2022

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ((http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

S100A9 binds to the receptor for advanced glycation end products (RAGE), forming a complex that activates various signaling pathways in cancer cells. Accordingly, inhibitors of this interaction are potential therapeutic targets. We identified and characterized oxyclozanide, a small molecule that has selective anti-cancer effects in vitro and in vivo through the inhibition of interactions between S100A9 and RAGE in triple-negative breast cancer (TNBC) cells expressing S100A9. Based on a ProteinChip array, oxyclozanide strongly inhibited the interaction between S100A9, RAGE, and toll-like receptor 4. In addition, oxyclozanide inhibited the proliferation of TNBC cells in an S100A9-specific manner. This inhibition was confirmed by Western blotting analysis, which indicated that oxyclozanide decreased pERK expression, but increased cleaved PARP, specifically in TNBC cells expressing S100A9. In human umbilical vein endothelial cells, oxyclozanide inhibited processes important for angiogenesis, e.g., basic fibroblast growth factor-induced endothelial cell tube formation, proliferation, and migration. Furthermore, with respect to the angiogenic switch, oxyclozanide induced thrombospondin- 1 (TSP-1) expression in S100A9-expressing TNBC cells. In a xenograft animal model, oxyclozanide significantly delayed tumor growth, but also suppressed the phosphorylation of ERK and induced TSP-1 in S100A9-positive tumors. The results of this study suggest that oxyclozanide is a potential drug candidate targeting S100A9-positive TNBC.

Keywordsoxyclozanide, S100A9, RAGE, TLR4, triple-negative breast cancer

The S100A9 protein belongs to the S100 family, consists of 114 amino acids (including an EF-hand calcium-binding domain), and has a molecular weight of 13.2 kDa (Schäfer and Heizmann 1996). Each S100A9 monomer has a calcium-binding site with high-affinity at the C-terminus and a low-affinity calcium binding site at the N-terminus (Schäfer and Heizmann 1996). S100A9 is most commonly found in the homodimer or heterodimer form together with S100A8 in cells (Itou et al. 2001; Korndörfer et al. 2007). It is localized in the cytoplasm and nuclei of various types of cells and is involved in the regulation of many cellular processes, such as cell cycle progression and differentiation, via interactions with target proteins (Cheng et al. 2008). According to clinical studies, S100A9 expression is upregulated in patients with various types of cancer, inflammatory diseases, neurodegenerative disorders, and autoimmune diseases (Vogl et al. 2012; Huang et al. 2022; Mitrović Ajtić et al. 2022). In terms of cellular signaling cascades, S100A9 can activate a wide range of processes at both intra- and extracellular sites by forming a complex with the receptor for advanced glycation end products (RAGE) and toll-like receptor 4 (TLR4) (Gebhardt et al. 2008; Turovskaya et al. 2008; Ichikawa et al. 2011). S100A9 binds to RAGE and activates RAGE-mediated signaling cascades such as the MAPK, PI3K/AKT, and NF-kB signaling pathways, thereby inducing increased expression of the genes involved in cell survival and proliferation (Ghavami et al. 2008; Turovskaya et al. 2008; Wu et al. 2013; Xu et al. 2013; Zhong et al. 2020; Rigiracciolo et al. 2022).

S100 family proteins are involved in angiogenesis (Chen et al. 2014; Nguyen et al. 2021) through the mediation of angiogenic stimulators and inhibitors, e.g., vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), angiopoietin, thrombospondin-1 (TSP-1), Matrix metalloproteinases (MMPs), and Transforming growth factors beta (TGF-β) (Tonini et al. 2003). Accordingly, S100A9 may affect tumor metastasis by influencing the process of angiogenesis (Zhang et al. 2022). Increased S100A9 levels have been detected in numerous human cancers, including those of the breast, cervix, lung, skin, stomach, and colon (Arai et al. 2001, 2004; El-Rifai et al. 2002; Cross et al. 2005; Celis et al. 2006; Chao et al. 2006; Kim et al. 2009; Zhu et al. 2009). The overexpression of S100A8 and S100A9 is regarded as a marker of poor prognosis in invasive ductal breast cancer (Arai et al. 2008) and colorectal cancer (Liu et al. 2022). Moreover, an association between S100A9 and basal subtypes as well as the poor prognostic value of S100A9 has been demonstrated in tumor samples from early breast cancer (Gonçalves et al. 2008). Most tumors expressing ‘basal’ markers are triple-negative breast cancer (TNBC) (Badve et al. 2011). Therefore, elucidating the mechanisms of S100A9 signaling in TNBC may lead to the identification of new therapeutic targets. Based on previous studies of S100A9 (Björk et al. 2009; Källberg et al. 2012), we performed virtual screening for new compounds that bind to S100A9 and inhibit its interaction with RAGE and TLR4 (RAGE/TLR4). The molecule identified in this analysis, oxyclozanide (Fig. 1), has already been identified by Björk et al. (2013). In this study, the effects of oxyclozanide on TNBC cells expressing S100A9 were examined and oxyclozanide was characterized.

Figure 1.Chemical structure of oxyclozanide.

Reagents

Oxyclozanide (Sigma-Aldrich, Darmstadt, Germany), human S100A9 (Sino Biological, Beijing, China), recombinant human RAGE Fc chimera protein (R&D Systems, Minneapolis, MN, USA), recombinant human TLR4/MD-2 complex protein (R&D Systems), and Cy5 fluorescent dye (GE Healthcare Life Sciences, Little Chalfont, UK) were purchased. A protein microarray panel was derived from ProteoChipTM (Proteogen Inc., Seoul, Korea). Anti-pERK, anti-PARP, and anti-β-actin antibodies were obtained from Cell Signaling (Beverly, MA, USA). Anti-TSP-1 antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Cell lines

TNBC cell lines (MDA-MB-468 and MDA-MB-231) and human umbilical vein endothelial cells (HUVECs) were purchased from ATCC (Manassas, VA, USA). Cancer cells were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. HUVECs were cultured in M199 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cell cultures were then maintained at 37℃ in a humidified atmosphere with 5% CO2.

Molecular docking simulation

Ligand-flexible molecular docking simulations were conducted using the Glide module in the Maestro 9.9 package (Schrodinger Inc., Cambridge, MA, USA). The three-dimensional coordinates of S100A9 were obtained from the structure of the S100A9/CHAPS complex (PDB ID 1IRJ) in the RCSB Protein Data Bank. The CHAPS structure was manually deleted to construct a model of S100A9. The protein structure was prepared using the Protein Preparation Wizard tool in the Maestro 9.9 package. Using this tool, all hydrogen atoms were added to S100A9, protonation states were optimized, and structural water molecules near the heavy atoms were preserved. The modeled protein structure was further relaxed using the energy-minimization method. A molecular grid was generated for the CHAPS-binding site of S100A9 using the Receptor Grid Generation tool in Glide. The final grid size was optimized to obtain a rectangular box of 16 × 20 × 20 Å. A molecular docking simulation was performed using the Glide module in Maestro 9.9 under Extra Precision (XP) mode.

ProteinChip-based S100A9-RAGE/TLR4 interaction assay

1) ProteinChip-based S100A9 inhibitor screening

A solution of 100 μg/mL RAGE/TRL4-MD-2 (in a dilution buffer consisting of 30% glycerol in phosphate-buffered saline [PBS], pH 7.4) was spotted on the ProteinChip. The RAGE/TRL4 array chip was incubated overnight in a humidity chamber at 4℃ and then rinsed two times with PBST (0.5% Tween-20 in PBS) for 10 min. After extensive rinsing, the RAGE/TRL4 array was subsequently spotted with a mixture of Cy5-labeled S100A9 protein and oxyclozanide in a humidity chamber at 30℃ for 1 h. Cy5-labeled S100A9 and oxyclozanide were diluted to 6 μg/mL and 100 μM with 1 mM CaCl2, 10 μM ZnCl2, and 10% glycerol in HEPES-buffered saline (HBS), respectively. After rinsing with PBST and distilled water, the chip was dried using N2 gas. The fluorescence intensity of the mixture was measured at a specific control spot and competitive inhibition was evaluated.

2) Detection and data analysis

The assay chip was scanned using a GenePix 4100A Scanner (Molecular Devices, Sunnyvale, CA, USA) and images were saved in the TIFF file format. The scanned images were processed using GenePix Pro 6.0 (Molecular Devices). Using Microsoft Origin 6.1 and Excel, the final experimental data were analyzed (Microsoft, Redmond, WA, USA and OriginLab, Northampton, MA, USA).

Surface plasmon resonance (SPR)

SPR analysis was performed following the previously described methods (Björk et al. 2009, 2013) using the Reichert SPR7500DC System (Reichert Technologies, Depew, NY, USA). In a direct binding assay, oxyclozanide was injected over human S100A9 immobilized on a CMDH chip at a density of ~3 kRU. Oxyclozanide was consecutively diluted in 100% DMSO and then diluted 50-fold in 10 mM HEPES, 0.15 M NaCl, pH 7.4, containing 0.005% Surfactant P20 (HBS-P buffer). Before injection, S100A9 was incubated with oxyclozanide for at least 1 h in assay buffer (HBS-P containing 1 mM Ca2+, 20 mM Zn2+, and 1% v/v DMSO). The scrubber was used to evaluate binding data.

TNBC cell proliferation assay

Inhibition of cell proliferation was assayed using Vybrant® MTT Cell Proliferation Assay Kits [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] according to the manufacturer’s protocol. MDA-MB-231 and MDA-MB-468 cells were plated at 8,000 cells/well on 96-well culture plates. Cells were treated with oxyclozanide (3, 10, and 30 μM) and incubated for 72 h. Then, 10 μL of 12 mM MTT solution was added to each well, and cells were incubated for 4 h at 37℃. After cells were labeled with MTT, all but 25 μL of the medium was removed from the wells. Then, 50 μL of DMSO was added to each well and mixed with a pipette, followed by incubation at 37℃ for 10 min. Each sample was mixed again and absorbance was read at 540 nm using an ELISA reader.

Western blotting

Total proteins were extracted using RIPA lysis buffer (50 mM Tris-HCl [pH 8.0], 1% NP-40, 0.5% sodium deoxycholate, 150 mM NaCl, 1 mM PMSF) and samples were resolved by SDS-PAGE on an 8% or 10% gel. Western blotting was performed using either anti-pERK (1:1000; Cell Signaling), anti-PARP (1:1000; Cell Signaling), or anti-TSP-1 (1:200; Santa Cruz) antibodies. The membranes were stripped and reprobed using anti-β-actin (1:1000; Cell Signaling) for protein loading.

Angiogenesis assay

1) HUVEC tube formation assay

HUVEC tube formation was assayed as described previously, with some modifications (Brooks et al. 1994). First, 24-well plates were coated with Matrigel (250 μL) and incubated for 30 min at 37℃ to allow the Matrigel solution to form a gel. Then, medium including oxyclozanide and bFGF (25 ng/mL) with 100 μL of HUVECs (4 × 104 cells) was transferred to the 24-well plates. For the control, 0.5% DMSO in the fresh medium was added and the 24-well plates were incubated for 18 h at 37℃. After the incubation process, the capillary tubes were fixed and stained using Diff-Quick solution (Becton Dickinson, San Diego, CA, USA). Observations were performed under a phase-contrast microscope.

2) HUVEC proliferation assay

HUVEC proliferation was examined according to the MTT assay protocol. HUVECs (1 × 104 cells per well) were plated on 96-well tissue culture plates coated with gelatin and allowed to adhere overnight. Cells were treated with oxyclozanide and incubated for 72 h. Then, 50 μL of a 1 mg/mL MTT solution was added to each well, and the cells were incubated for 2 h at 37℃. After the supernatants were discarded, formazan crystals were dissolved in 100 μL of DMSO. Absorbance was measured at 570 nm using an ELISA reader. Control cells were incubated in the absence of bFGF, whereas bFGF-control cells were incubated in the presence of bFGF alone.

3) Wound-healing HUVEC migration assay

Six-well culture dish was added with HUVECs (2 × 105 cells). A wound area was generated with a sterilized 200 μL micropipette tip in confluent cultures of HUVECs on the dish. After rinsing cellular debris with PBS, the cells were treated with different concentrations of oxyclozanide in the presence or absence of bFGF. The width of the wound area was photographed after 4 h and 8 h using inverted microscopy (Olympus, Japan).

Tumor xenograft study

To establish a nude mouse xenograft model, BALB/c nude mice (Japan SLC Inc., Shizuoka, Japan) aged 5-6 weeks and weighing about 20 ± 2 g were used. Animal care and all experimental procedures were approved by Institution Animal Care and Use Committee of Ahngook Pharm (Seoul, Korea). The animals were fed a standard rodent chow diet, provided tap water ad libitum, and maintained under a 12 h dark/light cycle at 22℃. MDA-MB-231 and MDA-MB-468 (8 × 106 cells/0.2 mL) in PBS were injected into the mammary fat pad of each mouse. When the tumor size reached a volume of approximately 60-80 mm3, the mice were randomly divided into 5 groups (n = 7 mice per group), i.e., vehicle mice, oxyclozanide-treated mice (62.5, 125, or 250 mg/kg), and cisplatin-treated mice. Next, 1% carboxymethyl cellulose was administered to the vehicle mice every day, 62.5, 125, or 250 mg/kg oxyclozanide was administered to oxyclozanide-treated mice every day, and 5 mg/kg of cisplatin was injected into the cisplatin-treated mice every week. Tumor dimensions were measured using digital calipers and volume was calculated using the following formula: Tumor volume = length × width2 × 0.5.

Docked pose of oxyclozanide upon S100A9 and SPR analysis

S100A9 has four α-helices, and the spatial arrangement of these helices is maintained by a hydrophobic cluster in the interior of the S100A9 monomer. This hydrophobic cluster is partially exposed to the solvent at the hinge region and the inter-helix area between H3 and H4, and the cluster forms a hydrophobic patch on the S100A9 surface (Itou et al. 2002). Based on the molecular docking simulation, oxyclozanide is bound to the hydrophobic patch formed at the hinge region between helices H3 and H4 (Fig. 2A). The oxygen atoms in the hydroxyl and carbonyl group of oxyclozanide formed stable interactions with the hydrophobic residues Leu49, Phe48, and Glu52 in the hinge region of S100A9 (Fig. 2B). We used SPR to analyze the binding of oxyclozanide to immobilized S100A9 with Ca2+ and Zn2+. Oxyclozanide showed the ability to bind to immobilize human S100A9 in a dose-dependent manner (Fig. 2C). The calculated dissociation constant (KD) of oxyclozanide and S100A9 was 60 μM, with Kon = 194 M−1 s−1 and Koff = 0.01249 s−1, respectively.

Figure 2.Docked pose of oxyclozanide on S100A9 and an SPR analysis. (A) Docked structure of S100A9 and oxyclozanide (gray) in the hinge region. (B) Binding mode of S100A9 and oxyclozanide showing various residue interactions. (C) Dose-dependent binding of oxyclozanide to S100A9 using a surface plasmon resonance (SPR) analysis. The oxyclozanide concentrations (from top to bottom) are 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 μM, respectively.

Inhibitory effects of oxyclozanide on S100A9-RAGE/TLR4 interactions on the ProteinChip

We used the ProteinChip to identify the inhibitory effects of oxyclozanide on the interaction between S100A9 and RAGE/TLR4. The RAGE and TLR4 array was spotted with a mixture of Cy5-labeled S100A9 and oxyclozanide (0.4, 2, 10, 50, or 250 μM) in a humidity chamber at 30℃ for 1 h. Competitive inhibition was determined by measuring the relative fluorescence intensity of the mixture versus the intensity of the control spot. Oxyclozanide inhibited, in a dose-dependent manner, the interactions between S100A9 and both RAGE (Fig. 3A) and TLR4/MD2 (Fig. 3B). The half-maximal inhibitory concentrations of oxyclozanide for S100A9 binding to RAGE and TLR4 were 6.6 ± 2.6 μM and 4.5 ± 1.1 μM respectively.

Figure 3.Oxyclozanide inhibited the interaction between S100A9 and the receptor for RAGE/TLR4 on the ProteinChip. The RAGE and TLR4 array were subsequently spotted with a mixture of Cy5-labeled S100A9 protein and oxyclozanide in a humidity chamber at 30℃ for 1 h. Competitive inhibition was determined by measuring the relative fluorescence intensity of the mixture versus the control spot.

Inhibitory effects of oxyclozanide on cell growth in TNBC cells

To evaluate the inhibitory effect of oxyclozanide on the growth of TNBC cells dependent on S100A9 expression, two cell lines, MDA-MB-468 (expressing S100A9) and MDA-MB-231 (not expressing S100A9), were used (Fig. 4A). Both cell lines were treated with different concentrations (0, 3, 10, or 30 μM) for 72 h. Oxyclozanide had higher cytotoxicity in MDA-MB-468 cells than in MDA-MB-231 cells, with IC50 values of 3.02 ± 0.08 μM and 8.68 ± 0.05 μM, respectively (Fig. 4B). We found that the IC50 value for oxyclozanide in MDA-MB-468 cells was more than three-fold lower than that of MDA-MB-231 cells, suggesting that oxyclozanide has strong inhibition selectivity in MDA-MB-468 cells, in which growth is specifically driven by the S100A9 pathway.

Figure 4.Oxyclozanide inhibits the proliferation of TNBC cells in an S100A9 expression-dependent manner. (A) Western blot detection of S100A9 in MDA-MB-231 and MDA-MB-468 cells. (B) Viability of TNBC cell lines with or without S100A9 expression. The cells were treated with three concentrations (3, 10, and 30 μM) of oxyclozanide or with 0.1% dimethyl sulfoxide (DMSO) as a control for 72 h. After treatment with oxyclozanide, the cells were subjected to an MTT assay. Data represent the means ± SD from duplicate wells.

Effects of oxyclozanide on signaling molecules in TNBC cells expressing S100A9

The effect of oxyclozanide on ERK phosphorylation was investigated by treating TNBC cells with 30 μM oxyclozanide for 4 h and 8 h. As shown in Fig. 5A, oxyclozanide inhibited the phosphorylation of ERK in the MDA-MB-468 cell line expressing S100A9, but oxyclozanide did not affect on ERK phosphorylation in MDA-MB-231 cells that did not express S100A9. Therefore, the suppression of ERK phosphorylation by oxyclozanide was correlated with its inhibitory effects on proliferation in TNBC cells expressing S100A9. To estimate the effect of oxyclozanide on apoptosis, we assessed the expression of apoptosis-related proteins. The two TNBC cell lines were incubated with 30 μM oxyclozanide for 8 h and 24 h. Following protein extraction, Western blotting was performed to examine the expression of cleaved poly (ADP-ribose) polymerase (PARP). Oxyclozanide increased the cleavage of PARP in the MDA-MB-468 cell line, but had only a minor effect in the MDA-MB-231 cell line (Fig. 5B). The term angiogenic switch describes a characteristic of cells involved in the continuous formation of tumors in which cancer cells must down-regulate natural angiogenesis inhibitors such as TSP-1 (thrombospondin-1), while equally up-regulating angiogenesis stimulators such as VEGF (Bergers and Benjamin, 2003). TSP-1 protein levels were slightly induced by oxyclozanide in MDA-MB-468 cells, but not in MDA-MB-231 cells. VEGF and HIF-1α levels were not affected by oxyclozanide treatment (data not shown). These data suggest that oxyclozanide inhibits TSP-1, an anti-angiogenic factor, in TNBC cells expressing S100A9 (Fig. 5B).

Figure 5.Oxyclozanide has effects on multiple signaling molecules in TNBC cells expressing S100A9. (A) MDA-MB 231 and MDA-MB-468 cells were treated with 30 μM oxyclozanide (OXC) for 4 h and 8 h, and phosphorylated ERK (p-ERK) was assayed by Western blotting. (B) The two TNBC cell types were treated with 30 μM oxyclozanide for 8 h and 24 h, and full-length PARP, cleaved PARP, and TSP-1 were assayed by Western blotting. β-Actin was used as a loading control.

Inhibitory effects of oxyclozanide on bFGF-induced endothelial cell tube formation, proliferation, and migration

To examine the anti-angiogenic effect of oxyclozanide, in vitro bFGF-induced HUVEC proliferation, tube formation, and migration assays were performed. The new capillary formation is required for the initial steps of angiogenesis, which involves various processes, including endothelial cell activation, proliferation, and migration. We evaluated the effects of oxyclozanide on bFGF-induced tube formation in HUVEC. bFGF stimulated the formation of capillary-like structures by HUVEC, and this action was significantly suppressed by the addition of oxyclozanide (Fig. 6B). To evaluate tube formation by endothelial cells in a quantitative manner, tube length was measured using an imaging analyzer. Oxyclozanide suppressed tube length following bFGF-induced tube formation. This effect was statistically significant for oxyclozanide concentrations of 5 to 50 μM (Fig. 6A). For specific evaluation of vascular endothelial cell proliferation, a key initial step in angiogenesis, we examined whether oxyclozanide inhibited bFGF-induced HUVEC proliferation using an MTT assay. Oxyclozanide potently suppressed bFGF-induced HUVEC proliferation in a dose-dependent manner (Fig. 7, IC50 = 4.8 ± 0.9 μM). For further investigation of the anti-angiogenic effects, we examined vascular endothelial cell migration, an essential step in angiogenesis. Oxyclozanide perturbed bFGF-induced HUVEC migration compared with DMSO-treated control group in a dose and time-dependent fashion, especially oxyclozanide significantly suppressed bFGF-induced HUVEC migration at a concentration of 50 μM after 4 h and 8 h of incubation (Fig. 8).

Figure 6.Oxyclozanide inhibited bFGF-induced endothelial cell tube formation. (A) Tube formation was evaluated by measurements of tube length after treatment with three concentrations (0.5, 5, and 50 μM) of oxyclozanide (OXC). Data represent means ± S.D. *p < 0.05, **p < 0.01 compared to the control (DMSO 0.5%). (B) Representative images of the effects of oxyclozanide on bFGF-induced tube formation in HUVECs (40×). (+) bFGF: bFGF alone, DMSO: bFGF plus DMSO 0.5%, 2-Me: bFGF plus 2-methoxy estradiol 3 μM, oxyclozanide: bFGF plus 0.5, 5, and 50 μM oxyclozanide.

Figure 7.Oxyclozanide inhibited bFGF-induced endothelial cell proliferation. HUVECs were incubated with three concentrations (0.5, 5, and 50 μM) of oxyclozanide (OXC) in the presence of bFGF (25 ng/mL) for 72 h at 37℃ in 5% CO2 with humidity. Cell proliferation was estimated using an MTT assay. Data represent the means ± SD from duplicate wells.

Figure 8.Oxyclozanide inhibited bFGF-induced endothelial cell migration. (A) Migration of HUVECs was performed using cell scratch wound healing assay. Images from the same area were captured after 4 h and 8 h wound infliction and compared migration in the controls (DMSO 0.5%) for each concentration. Data represent the means ± SD. *p < 0.05 as compared to the control (DMSO 0.5%). (B) Image of HUVEC migration obtained at 4 h and 8 h after treatment with bFGF alone and bFGF plus 0.5% DMSO, 3 μM 2-methoxy estradiol, and oxyclozanide (OXC) (40×).

Inhibitory effect of oxyclozanide in an S100A9-expressing xenograft model

Based on the in vitro findings, we expanded our analysis to an in vivo xenograft nice mouse model. TNBC cells expressing S100A9 (MDA-MB-468) or not expressing S100A9 (MDA-MB-231) were subcutaneously injected into the flanks of animals and tumors were allowed to reach solidity before drug treatment. Oxyclozanide-treated mice did not show signs of visible toxicity for doses of 62.5, 125, or 250 mg/kg administered daily. In vehicle-treated animals, tumor volume increased rapidly (Fig. 9). Tumors not expressing S100A9 treated with oxyclozanide were similar to vehicle tumors (Fig. 9A). However, tumors expressing S100A9 had significantly smaller volumes after oxyclozanide treatment, especially for 250 mg/kg (Fig. 9B). To further confirm these findings, Western blotting was performed to determine the effects of oxyclozanide on key indicators of tumor growth. Eight hours after a single administration of 250 mg/kg oxyclozanide, we evaluated the expression levels of pERK, cleaved PARP, and TSP-1 in isolated tumor tissues. As shown in Fig. 9C, pERK levels were significantly diminished, cleavage of PARP expression increased slightly, and TSP-1 protein levels were induced in oxyclozanide-treated tumors. However, VEGF and HIF-1α levels were not affected by oxyclozanide treatment (data not shown). Collectively, our results suggest that oxyclozanide has potent anti-tumor efficacy via the inhibition of S100A9 with RAGE in vivo.

Figure 9.Oxyclozanide suppressed tumor growth in an S100A9-positive xenograft model. (A) Mice with xenograft tumors established from MDA-MB-231 cell lines were given 125 and 250 mg/kg oxyclozanide orally every day and tumor volume was recorded twice a week for 22 days. (B) Mice with S100A9-positive tumors (MDA-MB-468 cell lines) were given 62.5, 125, and 250 mg/kg oxyclozanide orally every day, and tumor volume was recorded twice a week for 28 days. Data represent the means ± SD (n = 7). *p < 0.05 as compared to the vehicle group. (C) Western blotting was performed to determine the effects of oxyclozanide on key indicators of tumor growth. Expression levels of pERK, cleaved PARP, and TSP-1 were measured from MDA-MB-468 xenografts 8 h post-treatment.

S100A9 plays an important role in cancer processes via structural interactions with RAGE or TLR4-MD2. It binds to and activates various signaling pathways, including MAPK and NF-kB, in cancer cells. S100A9 expression differs among cancer types, including breast cancer, colon cancer, hepatocellular carcinoma, gastric cancer, and non-small cell lung cancer. Owing to its potential value as a prognostic indicator, S100A9 is regarded as a relevant therapeutic target and may lead to cancer drug discovery. A recent study demonstrated that quinoline-2-carboxamides (tasquinimod) bind to S100A9 and inhibit the interactions of S100A9 with RAGE and TLR4-MD2 (Björk et al. 2009). More recently, tasquinimod was tested in patients with metastatic prostate cancer in a phase III trial and found to have anti-angiogenic properties (Williamson et al. 2013). Although S100A9-targeted compounds have been developed, the optimal therapeutic strategy has not been determined.

Here, we characterized oxyclozanide, a highly selective small-molecule inhibitor targeting the interaction between S100A9 and RAGE. We found that oxyclozanide had a prominent inhibitory effect on the S100A9/RAGE complex and its signaling pathway in S100A9-expressing TNBC cells, as evidenced by its inhibitory effects on growth in vitro and in vivo. Oxyclozanide binding to S100A9 was detected in an SPR analysis, and inhibition of the interaction between S100A9 and RAGE/TLR4 was observed using a ProteinChip-based protein-protein interaction assay system, a new screening technology based on competition among protein-protein interactions. Up-regulated in numerous cancer types and associated with tumor cell growth and survival, S100A9 binds to RAGE and triggers RAGE-mediated cell signaling involving MAPK, leading to the up-regulation of genes involved in cell survival and proliferation. With respect to selectivity, oxyclozanide treatment resulted in the dose- and S100A9-dependent inhibition of proliferation in TNBC cells. As a functional correlate of the inhibition of cell growth, multiple cell signaling pathways were analyzed to determine the effects of oxyclozanide on survival, apoptosis, and angiogenesis in TNBC cells. Oxyclozanide inhibited the expression of pERK and induced cleaved PARP exclusively in TNBC cells expressing S100A9, resulting in the regulation of proliferation and survival in cancer cells. Tumor progression is controlled by the balance between pro-angiogenic and anti-angiogenic factors. For a tumor to grow continuously, cancer cells must down-regulate angiogenesis inhibitors, such as TSP-1 (Sargiannidou et al. 2001). TSP-1 expression was induced by oxyclozanide in TNBC cells expressing S100A9. Taken together, these data demonstrate that oxyclozanide exhibits potent anti-TNBC effects in an S100A9-specific manner. In the tumor microenvironment, angiogenesis is the process by which new capillary blood vessels are generated, which is important for tumor growth. HUVEC is an experimental model for endothelial cells. Oxyclozanide strongly suppressed bFGF-induced HUVEC tube formation, proliferation, and migration. Thus, these data support the role for oxyclozanide as a putative inhibitor of endothelial cell functions in vitro.

A xenograft model was used to evaluate the in vivo anti-cancer efficacy of S100A9. TNBC cells expressing or not expressing S100A9 were subcutaneously injected into nude mice and the drug was administered when the size of the tumors was palpable. The growth rate was slower for S100A9-negative xenografts treated with oxyclozanide than for vehicle tumors. However, the growth of S100A9-positive tumors was largely delayed by oxyclozanide treatment, especially at 250 mg/kg. Key indicators of tumor growth, i.e., pERK, cleaved PARP, and TSP-1, were evaluated in S100A9-positive tumors. As expected, pERK levels were significantly diminished, cleavage PARP expression was slightly increased, and TSP-1 protein levels were higher in oxyclozanide treated tumors.

Our findings show that oxyclozanide has potent anti-cancer activity in cells and preclinical TNBC models in which S100A9 is expressed. These data strongly support the applicability of oxyclozanide for the treatment of patients with TNBC when S100A9 is overexpressed.

KH Chang, YY Choi and ID Jung are employees of Ahngook Pharm., Seoul, Republic of Korea. The other authors have no other conflicts of interest with regard to the content of this article.

This work was supported by grants from the Korean Healthcare Technology R&D Project through the Korean Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (Grant HI13C2148).

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Article

Original Research Article

DTT 2022; 1(1): 1-11

Published online July 31, 2022 https://doi.org/10.58502/DTT.22.003

Copyright © The Pharmaceutical Society of Korea.

Selective Anticancer Effects by Oxyclozanide in Triple-Negative Breast Cancer Expressing S100A9

Hyun Hwan Hwang1, Ki Ho Chang2, Yi Young Choi2, Ii-Du Jung2, In-Cheol Kang3, Youngjin Choi4, Mi Jin O1, Chang-Keun Cho1 , Pureum Kang1 , Hye-Jung Park1 , Choon-Gon Jang1 , Seok-Yong Lee1

1School of Pharmacy, Sungkyungkwan University, Suwon, Korea
2Department of RND Center, Ahngook Pharm, Seoul, Korea
3Department of Biological Science, Hoseo University, Asan, Korea
4Department of Food Science & Technology, Hoseo University, Asan, Korea

Correspondence to:Seok-Yong Lee, sylee@skku.edu

Received: April 7, 2022; Revised: May 3, 2022; Accepted: May 25, 2022

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ((http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

S100A9 binds to the receptor for advanced glycation end products (RAGE), forming a complex that activates various signaling pathways in cancer cells. Accordingly, inhibitors of this interaction are potential therapeutic targets. We identified and characterized oxyclozanide, a small molecule that has selective anti-cancer effects in vitro and in vivo through the inhibition of interactions between S100A9 and RAGE in triple-negative breast cancer (TNBC) cells expressing S100A9. Based on a ProteinChip array, oxyclozanide strongly inhibited the interaction between S100A9, RAGE, and toll-like receptor 4. In addition, oxyclozanide inhibited the proliferation of TNBC cells in an S100A9-specific manner. This inhibition was confirmed by Western blotting analysis, which indicated that oxyclozanide decreased pERK expression, but increased cleaved PARP, specifically in TNBC cells expressing S100A9. In human umbilical vein endothelial cells, oxyclozanide inhibited processes important for angiogenesis, e.g., basic fibroblast growth factor-induced endothelial cell tube formation, proliferation, and migration. Furthermore, with respect to the angiogenic switch, oxyclozanide induced thrombospondin- 1 (TSP-1) expression in S100A9-expressing TNBC cells. In a xenograft animal model, oxyclozanide significantly delayed tumor growth, but also suppressed the phosphorylation of ERK and induced TSP-1 in S100A9-positive tumors. The results of this study suggest that oxyclozanide is a potential drug candidate targeting S100A9-positive TNBC.

Keywords: oxyclozanide, S100A9, RAGE, TLR4, triple-negative breast cancer

Introduction

The S100A9 protein belongs to the S100 family, consists of 114 amino acids (including an EF-hand calcium-binding domain), and has a molecular weight of 13.2 kDa (Schäfer and Heizmann 1996). Each S100A9 monomer has a calcium-binding site with high-affinity at the C-terminus and a low-affinity calcium binding site at the N-terminus (Schäfer and Heizmann 1996). S100A9 is most commonly found in the homodimer or heterodimer form together with S100A8 in cells (Itou et al. 2001; Korndörfer et al. 2007). It is localized in the cytoplasm and nuclei of various types of cells and is involved in the regulation of many cellular processes, such as cell cycle progression and differentiation, via interactions with target proteins (Cheng et al. 2008). According to clinical studies, S100A9 expression is upregulated in patients with various types of cancer, inflammatory diseases, neurodegenerative disorders, and autoimmune diseases (Vogl et al. 2012; Huang et al. 2022; Mitrović Ajtić et al. 2022). In terms of cellular signaling cascades, S100A9 can activate a wide range of processes at both intra- and extracellular sites by forming a complex with the receptor for advanced glycation end products (RAGE) and toll-like receptor 4 (TLR4) (Gebhardt et al. 2008; Turovskaya et al. 2008; Ichikawa et al. 2011). S100A9 binds to RAGE and activates RAGE-mediated signaling cascades such as the MAPK, PI3K/AKT, and NF-kB signaling pathways, thereby inducing increased expression of the genes involved in cell survival and proliferation (Ghavami et al. 2008; Turovskaya et al. 2008; Wu et al. 2013; Xu et al. 2013; Zhong et al. 2020; Rigiracciolo et al. 2022).

S100 family proteins are involved in angiogenesis (Chen et al. 2014; Nguyen et al. 2021) through the mediation of angiogenic stimulators and inhibitors, e.g., vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), angiopoietin, thrombospondin-1 (TSP-1), Matrix metalloproteinases (MMPs), and Transforming growth factors beta (TGF-β) (Tonini et al. 2003). Accordingly, S100A9 may affect tumor metastasis by influencing the process of angiogenesis (Zhang et al. 2022). Increased S100A9 levels have been detected in numerous human cancers, including those of the breast, cervix, lung, skin, stomach, and colon (Arai et al. 2001, 2004; El-Rifai et al. 2002; Cross et al. 2005; Celis et al. 2006; Chao et al. 2006; Kim et al. 2009; Zhu et al. 2009). The overexpression of S100A8 and S100A9 is regarded as a marker of poor prognosis in invasive ductal breast cancer (Arai et al. 2008) and colorectal cancer (Liu et al. 2022). Moreover, an association between S100A9 and basal subtypes as well as the poor prognostic value of S100A9 has been demonstrated in tumor samples from early breast cancer (Gonçalves et al. 2008). Most tumors expressing ‘basal’ markers are triple-negative breast cancer (TNBC) (Badve et al. 2011). Therefore, elucidating the mechanisms of S100A9 signaling in TNBC may lead to the identification of new therapeutic targets. Based on previous studies of S100A9 (Björk et al. 2009; Källberg et al. 2012), we performed virtual screening for new compounds that bind to S100A9 and inhibit its interaction with RAGE and TLR4 (RAGE/TLR4). The molecule identified in this analysis, oxyclozanide (Fig. 1), has already been identified by Björk et al. (2013). In this study, the effects of oxyclozanide on TNBC cells expressing S100A9 were examined and oxyclozanide was characterized.

Figure 1. Chemical structure of oxyclozanide.

Materials and Methods

Reagents

Oxyclozanide (Sigma-Aldrich, Darmstadt, Germany), human S100A9 (Sino Biological, Beijing, China), recombinant human RAGE Fc chimera protein (R&D Systems, Minneapolis, MN, USA), recombinant human TLR4/MD-2 complex protein (R&D Systems), and Cy5 fluorescent dye (GE Healthcare Life Sciences, Little Chalfont, UK) were purchased. A protein microarray panel was derived from ProteoChipTM (Proteogen Inc., Seoul, Korea). Anti-pERK, anti-PARP, and anti-β-actin antibodies were obtained from Cell Signaling (Beverly, MA, USA). Anti-TSP-1 antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Cell lines

TNBC cell lines (MDA-MB-468 and MDA-MB-231) and human umbilical vein endothelial cells (HUVECs) were purchased from ATCC (Manassas, VA, USA). Cancer cells were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. HUVECs were cultured in M199 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cell cultures were then maintained at 37℃ in a humidified atmosphere with 5% CO2.

Molecular docking simulation

Ligand-flexible molecular docking simulations were conducted using the Glide module in the Maestro 9.9 package (Schrodinger Inc., Cambridge, MA, USA). The three-dimensional coordinates of S100A9 were obtained from the structure of the S100A9/CHAPS complex (PDB ID 1IRJ) in the RCSB Protein Data Bank. The CHAPS structure was manually deleted to construct a model of S100A9. The protein structure was prepared using the Protein Preparation Wizard tool in the Maestro 9.9 package. Using this tool, all hydrogen atoms were added to S100A9, protonation states were optimized, and structural water molecules near the heavy atoms were preserved. The modeled protein structure was further relaxed using the energy-minimization method. A molecular grid was generated for the CHAPS-binding site of S100A9 using the Receptor Grid Generation tool in Glide. The final grid size was optimized to obtain a rectangular box of 16 × 20 × 20 Å. A molecular docking simulation was performed using the Glide module in Maestro 9.9 under Extra Precision (XP) mode.

ProteinChip-based S100A9-RAGE/TLR4 interaction assay

1) ProteinChip-based S100A9 inhibitor screening

A solution of 100 μg/mL RAGE/TRL4-MD-2 (in a dilution buffer consisting of 30% glycerol in phosphate-buffered saline [PBS], pH 7.4) was spotted on the ProteinChip. The RAGE/TRL4 array chip was incubated overnight in a humidity chamber at 4℃ and then rinsed two times with PBST (0.5% Tween-20 in PBS) for 10 min. After extensive rinsing, the RAGE/TRL4 array was subsequently spotted with a mixture of Cy5-labeled S100A9 protein and oxyclozanide in a humidity chamber at 30℃ for 1 h. Cy5-labeled S100A9 and oxyclozanide were diluted to 6 μg/mL and 100 μM with 1 mM CaCl2, 10 μM ZnCl2, and 10% glycerol in HEPES-buffered saline (HBS), respectively. After rinsing with PBST and distilled water, the chip was dried using N2 gas. The fluorescence intensity of the mixture was measured at a specific control spot and competitive inhibition was evaluated.

2) Detection and data analysis

The assay chip was scanned using a GenePix 4100A Scanner (Molecular Devices, Sunnyvale, CA, USA) and images were saved in the TIFF file format. The scanned images were processed using GenePix Pro 6.0 (Molecular Devices). Using Microsoft Origin 6.1 and Excel, the final experimental data were analyzed (Microsoft, Redmond, WA, USA and OriginLab, Northampton, MA, USA).

Surface plasmon resonance (SPR)

SPR analysis was performed following the previously described methods (Björk et al. 2009, 2013) using the Reichert SPR7500DC System (Reichert Technologies, Depew, NY, USA). In a direct binding assay, oxyclozanide was injected over human S100A9 immobilized on a CMDH chip at a density of ~3 kRU. Oxyclozanide was consecutively diluted in 100% DMSO and then diluted 50-fold in 10 mM HEPES, 0.15 M NaCl, pH 7.4, containing 0.005% Surfactant P20 (HBS-P buffer). Before injection, S100A9 was incubated with oxyclozanide for at least 1 h in assay buffer (HBS-P containing 1 mM Ca2+, 20 mM Zn2+, and 1% v/v DMSO). The scrubber was used to evaluate binding data.

TNBC cell proliferation assay

Inhibition of cell proliferation was assayed using Vybrant® MTT Cell Proliferation Assay Kits [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] according to the manufacturer’s protocol. MDA-MB-231 and MDA-MB-468 cells were plated at 8,000 cells/well on 96-well culture plates. Cells were treated with oxyclozanide (3, 10, and 30 μM) and incubated for 72 h. Then, 10 μL of 12 mM MTT solution was added to each well, and cells were incubated for 4 h at 37℃. After cells were labeled with MTT, all but 25 μL of the medium was removed from the wells. Then, 50 μL of DMSO was added to each well and mixed with a pipette, followed by incubation at 37℃ for 10 min. Each sample was mixed again and absorbance was read at 540 nm using an ELISA reader.

Western blotting

Total proteins were extracted using RIPA lysis buffer (50 mM Tris-HCl [pH 8.0], 1% NP-40, 0.5% sodium deoxycholate, 150 mM NaCl, 1 mM PMSF) and samples were resolved by SDS-PAGE on an 8% or 10% gel. Western blotting was performed using either anti-pERK (1:1000; Cell Signaling), anti-PARP (1:1000; Cell Signaling), or anti-TSP-1 (1:200; Santa Cruz) antibodies. The membranes were stripped and reprobed using anti-β-actin (1:1000; Cell Signaling) for protein loading.

Angiogenesis assay

1) HUVEC tube formation assay

HUVEC tube formation was assayed as described previously, with some modifications (Brooks et al. 1994). First, 24-well plates were coated with Matrigel (250 μL) and incubated for 30 min at 37℃ to allow the Matrigel solution to form a gel. Then, medium including oxyclozanide and bFGF (25 ng/mL) with 100 μL of HUVECs (4 × 104 cells) was transferred to the 24-well plates. For the control, 0.5% DMSO in the fresh medium was added and the 24-well plates were incubated for 18 h at 37℃. After the incubation process, the capillary tubes were fixed and stained using Diff-Quick solution (Becton Dickinson, San Diego, CA, USA). Observations were performed under a phase-contrast microscope.

2) HUVEC proliferation assay

HUVEC proliferation was examined according to the MTT assay protocol. HUVECs (1 × 104 cells per well) were plated on 96-well tissue culture plates coated with gelatin and allowed to adhere overnight. Cells were treated with oxyclozanide and incubated for 72 h. Then, 50 μL of a 1 mg/mL MTT solution was added to each well, and the cells were incubated for 2 h at 37℃. After the supernatants were discarded, formazan crystals were dissolved in 100 μL of DMSO. Absorbance was measured at 570 nm using an ELISA reader. Control cells were incubated in the absence of bFGF, whereas bFGF-control cells were incubated in the presence of bFGF alone.

3) Wound-healing HUVEC migration assay

Six-well culture dish was added with HUVECs (2 × 105 cells). A wound area was generated with a sterilized 200 μL micropipette tip in confluent cultures of HUVECs on the dish. After rinsing cellular debris with PBS, the cells were treated with different concentrations of oxyclozanide in the presence or absence of bFGF. The width of the wound area was photographed after 4 h and 8 h using inverted microscopy (Olympus, Japan).

Tumor xenograft study

To establish a nude mouse xenograft model, BALB/c nude mice (Japan SLC Inc., Shizuoka, Japan) aged 5-6 weeks and weighing about 20 ± 2 g were used. Animal care and all experimental procedures were approved by Institution Animal Care and Use Committee of Ahngook Pharm (Seoul, Korea). The animals were fed a standard rodent chow diet, provided tap water ad libitum, and maintained under a 12 h dark/light cycle at 22℃. MDA-MB-231 and MDA-MB-468 (8 × 106 cells/0.2 mL) in PBS were injected into the mammary fat pad of each mouse. When the tumor size reached a volume of approximately 60-80 mm3, the mice were randomly divided into 5 groups (n = 7 mice per group), i.e., vehicle mice, oxyclozanide-treated mice (62.5, 125, or 250 mg/kg), and cisplatin-treated mice. Next, 1% carboxymethyl cellulose was administered to the vehicle mice every day, 62.5, 125, or 250 mg/kg oxyclozanide was administered to oxyclozanide-treated mice every day, and 5 mg/kg of cisplatin was injected into the cisplatin-treated mice every week. Tumor dimensions were measured using digital calipers and volume was calculated using the following formula: Tumor volume = length × width2 × 0.5.

Results

Docked pose of oxyclozanide upon S100A9 and SPR analysis

S100A9 has four α-helices, and the spatial arrangement of these helices is maintained by a hydrophobic cluster in the interior of the S100A9 monomer. This hydrophobic cluster is partially exposed to the solvent at the hinge region and the inter-helix area between H3 and H4, and the cluster forms a hydrophobic patch on the S100A9 surface (Itou et al. 2002). Based on the molecular docking simulation, oxyclozanide is bound to the hydrophobic patch formed at the hinge region between helices H3 and H4 (Fig. 2A). The oxygen atoms in the hydroxyl and carbonyl group of oxyclozanide formed stable interactions with the hydrophobic residues Leu49, Phe48, and Glu52 in the hinge region of S100A9 (Fig. 2B). We used SPR to analyze the binding of oxyclozanide to immobilized S100A9 with Ca2+ and Zn2+. Oxyclozanide showed the ability to bind to immobilize human S100A9 in a dose-dependent manner (Fig. 2C). The calculated dissociation constant (KD) of oxyclozanide and S100A9 was 60 μM, with Kon = 194 M−1 s−1 and Koff = 0.01249 s−1, respectively.

Figure 2. Docked pose of oxyclozanide on S100A9 and an SPR analysis. (A) Docked structure of S100A9 and oxyclozanide (gray) in the hinge region. (B) Binding mode of S100A9 and oxyclozanide showing various residue interactions. (C) Dose-dependent binding of oxyclozanide to S100A9 using a surface plasmon resonance (SPR) analysis. The oxyclozanide concentrations (from top to bottom) are 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 μM, respectively.

Inhibitory effects of oxyclozanide on S100A9-RAGE/TLR4 interactions on the ProteinChip

We used the ProteinChip to identify the inhibitory effects of oxyclozanide on the interaction between S100A9 and RAGE/TLR4. The RAGE and TLR4 array was spotted with a mixture of Cy5-labeled S100A9 and oxyclozanide (0.4, 2, 10, 50, or 250 μM) in a humidity chamber at 30℃ for 1 h. Competitive inhibition was determined by measuring the relative fluorescence intensity of the mixture versus the intensity of the control spot. Oxyclozanide inhibited, in a dose-dependent manner, the interactions between S100A9 and both RAGE (Fig. 3A) and TLR4/MD2 (Fig. 3B). The half-maximal inhibitory concentrations of oxyclozanide for S100A9 binding to RAGE and TLR4 were 6.6 ± 2.6 μM and 4.5 ± 1.1 μM respectively.

Figure 3. Oxyclozanide inhibited the interaction between S100A9 and the receptor for RAGE/TLR4 on the ProteinChip. The RAGE and TLR4 array were subsequently spotted with a mixture of Cy5-labeled S100A9 protein and oxyclozanide in a humidity chamber at 30℃ for 1 h. Competitive inhibition was determined by measuring the relative fluorescence intensity of the mixture versus the control spot.

Inhibitory effects of oxyclozanide on cell growth in TNBC cells

To evaluate the inhibitory effect of oxyclozanide on the growth of TNBC cells dependent on S100A9 expression, two cell lines, MDA-MB-468 (expressing S100A9) and MDA-MB-231 (not expressing S100A9), were used (Fig. 4A). Both cell lines were treated with different concentrations (0, 3, 10, or 30 μM) for 72 h. Oxyclozanide had higher cytotoxicity in MDA-MB-468 cells than in MDA-MB-231 cells, with IC50 values of 3.02 ± 0.08 μM and 8.68 ± 0.05 μM, respectively (Fig. 4B). We found that the IC50 value for oxyclozanide in MDA-MB-468 cells was more than three-fold lower than that of MDA-MB-231 cells, suggesting that oxyclozanide has strong inhibition selectivity in MDA-MB-468 cells, in which growth is specifically driven by the S100A9 pathway.

Figure 4. Oxyclozanide inhibits the proliferation of TNBC cells in an S100A9 expression-dependent manner. (A) Western blot detection of S100A9 in MDA-MB-231 and MDA-MB-468 cells. (B) Viability of TNBC cell lines with or without S100A9 expression. The cells were treated with three concentrations (3, 10, and 30 μM) of oxyclozanide or with 0.1% dimethyl sulfoxide (DMSO) as a control for 72 h. After treatment with oxyclozanide, the cells were subjected to an MTT assay. Data represent the means ± SD from duplicate wells.

Effects of oxyclozanide on signaling molecules in TNBC cells expressing S100A9

The effect of oxyclozanide on ERK phosphorylation was investigated by treating TNBC cells with 30 μM oxyclozanide for 4 h and 8 h. As shown in Fig. 5A, oxyclozanide inhibited the phosphorylation of ERK in the MDA-MB-468 cell line expressing S100A9, but oxyclozanide did not affect on ERK phosphorylation in MDA-MB-231 cells that did not express S100A9. Therefore, the suppression of ERK phosphorylation by oxyclozanide was correlated with its inhibitory effects on proliferation in TNBC cells expressing S100A9. To estimate the effect of oxyclozanide on apoptosis, we assessed the expression of apoptosis-related proteins. The two TNBC cell lines were incubated with 30 μM oxyclozanide for 8 h and 24 h. Following protein extraction, Western blotting was performed to examine the expression of cleaved poly (ADP-ribose) polymerase (PARP). Oxyclozanide increased the cleavage of PARP in the MDA-MB-468 cell line, but had only a minor effect in the MDA-MB-231 cell line (Fig. 5B). The term angiogenic switch describes a characteristic of cells involved in the continuous formation of tumors in which cancer cells must down-regulate natural angiogenesis inhibitors such as TSP-1 (thrombospondin-1), while equally up-regulating angiogenesis stimulators such as VEGF (Bergers and Benjamin, 2003). TSP-1 protein levels were slightly induced by oxyclozanide in MDA-MB-468 cells, but not in MDA-MB-231 cells. VEGF and HIF-1α levels were not affected by oxyclozanide treatment (data not shown). These data suggest that oxyclozanide inhibits TSP-1, an anti-angiogenic factor, in TNBC cells expressing S100A9 (Fig. 5B).

Figure 5. Oxyclozanide has effects on multiple signaling molecules in TNBC cells expressing S100A9. (A) MDA-MB 231 and MDA-MB-468 cells were treated with 30 μM oxyclozanide (OXC) for 4 h and 8 h, and phosphorylated ERK (p-ERK) was assayed by Western blotting. (B) The two TNBC cell types were treated with 30 μM oxyclozanide for 8 h and 24 h, and full-length PARP, cleaved PARP, and TSP-1 were assayed by Western blotting. β-Actin was used as a loading control.

Inhibitory effects of oxyclozanide on bFGF-induced endothelial cell tube formation, proliferation, and migration

To examine the anti-angiogenic effect of oxyclozanide, in vitro bFGF-induced HUVEC proliferation, tube formation, and migration assays were performed. The new capillary formation is required for the initial steps of angiogenesis, which involves various processes, including endothelial cell activation, proliferation, and migration. We evaluated the effects of oxyclozanide on bFGF-induced tube formation in HUVEC. bFGF stimulated the formation of capillary-like structures by HUVEC, and this action was significantly suppressed by the addition of oxyclozanide (Fig. 6B). To evaluate tube formation by endothelial cells in a quantitative manner, tube length was measured using an imaging analyzer. Oxyclozanide suppressed tube length following bFGF-induced tube formation. This effect was statistically significant for oxyclozanide concentrations of 5 to 50 μM (Fig. 6A). For specific evaluation of vascular endothelial cell proliferation, a key initial step in angiogenesis, we examined whether oxyclozanide inhibited bFGF-induced HUVEC proliferation using an MTT assay. Oxyclozanide potently suppressed bFGF-induced HUVEC proliferation in a dose-dependent manner (Fig. 7, IC50 = 4.8 ± 0.9 μM). For further investigation of the anti-angiogenic effects, we examined vascular endothelial cell migration, an essential step in angiogenesis. Oxyclozanide perturbed bFGF-induced HUVEC migration compared with DMSO-treated control group in a dose and time-dependent fashion, especially oxyclozanide significantly suppressed bFGF-induced HUVEC migration at a concentration of 50 μM after 4 h and 8 h of incubation (Fig. 8).

Figure 6. Oxyclozanide inhibited bFGF-induced endothelial cell tube formation. (A) Tube formation was evaluated by measurements of tube length after treatment with three concentrations (0.5, 5, and 50 μM) of oxyclozanide (OXC). Data represent means ± S.D. *p < 0.05, **p < 0.01 compared to the control (DMSO 0.5%). (B) Representative images of the effects of oxyclozanide on bFGF-induced tube formation in HUVECs (40×). (+) bFGF: bFGF alone, DMSO: bFGF plus DMSO 0.5%, 2-Me: bFGF plus 2-methoxy estradiol 3 μM, oxyclozanide: bFGF plus 0.5, 5, and 50 μM oxyclozanide.

Figure 7. Oxyclozanide inhibited bFGF-induced endothelial cell proliferation. HUVECs were incubated with three concentrations (0.5, 5, and 50 μM) of oxyclozanide (OXC) in the presence of bFGF (25 ng/mL) for 72 h at 37℃ in 5% CO2 with humidity. Cell proliferation was estimated using an MTT assay. Data represent the means ± SD from duplicate wells.

Figure 8. Oxyclozanide inhibited bFGF-induced endothelial cell migration. (A) Migration of HUVECs was performed using cell scratch wound healing assay. Images from the same area were captured after 4 h and 8 h wound infliction and compared migration in the controls (DMSO 0.5%) for each concentration. Data represent the means ± SD. *p < 0.05 as compared to the control (DMSO 0.5%). (B) Image of HUVEC migration obtained at 4 h and 8 h after treatment with bFGF alone and bFGF plus 0.5% DMSO, 3 μM 2-methoxy estradiol, and oxyclozanide (OXC) (40×).

Inhibitory effect of oxyclozanide in an S100A9-expressing xenograft model

Based on the in vitro findings, we expanded our analysis to an in vivo xenograft nice mouse model. TNBC cells expressing S100A9 (MDA-MB-468) or not expressing S100A9 (MDA-MB-231) were subcutaneously injected into the flanks of animals and tumors were allowed to reach solidity before drug treatment. Oxyclozanide-treated mice did not show signs of visible toxicity for doses of 62.5, 125, or 250 mg/kg administered daily. In vehicle-treated animals, tumor volume increased rapidly (Fig. 9). Tumors not expressing S100A9 treated with oxyclozanide were similar to vehicle tumors (Fig. 9A). However, tumors expressing S100A9 had significantly smaller volumes after oxyclozanide treatment, especially for 250 mg/kg (Fig. 9B). To further confirm these findings, Western blotting was performed to determine the effects of oxyclozanide on key indicators of tumor growth. Eight hours after a single administration of 250 mg/kg oxyclozanide, we evaluated the expression levels of pERK, cleaved PARP, and TSP-1 in isolated tumor tissues. As shown in Fig. 9C, pERK levels were significantly diminished, cleavage of PARP expression increased slightly, and TSP-1 protein levels were induced in oxyclozanide-treated tumors. However, VEGF and HIF-1α levels were not affected by oxyclozanide treatment (data not shown). Collectively, our results suggest that oxyclozanide has potent anti-tumor efficacy via the inhibition of S100A9 with RAGE in vivo.

Figure 9. Oxyclozanide suppressed tumor growth in an S100A9-positive xenograft model. (A) Mice with xenograft tumors established from MDA-MB-231 cell lines were given 125 and 250 mg/kg oxyclozanide orally every day and tumor volume was recorded twice a week for 22 days. (B) Mice with S100A9-positive tumors (MDA-MB-468 cell lines) were given 62.5, 125, and 250 mg/kg oxyclozanide orally every day, and tumor volume was recorded twice a week for 28 days. Data represent the means ± SD (n = 7). *p < 0.05 as compared to the vehicle group. (C) Western blotting was performed to determine the effects of oxyclozanide on key indicators of tumor growth. Expression levels of pERK, cleaved PARP, and TSP-1 were measured from MDA-MB-468 xenografts 8 h post-treatment.

Discussion

S100A9 plays an important role in cancer processes via structural interactions with RAGE or TLR4-MD2. It binds to and activates various signaling pathways, including MAPK and NF-kB, in cancer cells. S100A9 expression differs among cancer types, including breast cancer, colon cancer, hepatocellular carcinoma, gastric cancer, and non-small cell lung cancer. Owing to its potential value as a prognostic indicator, S100A9 is regarded as a relevant therapeutic target and may lead to cancer drug discovery. A recent study demonstrated that quinoline-2-carboxamides (tasquinimod) bind to S100A9 and inhibit the interactions of S100A9 with RAGE and TLR4-MD2 (Björk et al. 2009). More recently, tasquinimod was tested in patients with metastatic prostate cancer in a phase III trial and found to have anti-angiogenic properties (Williamson et al. 2013). Although S100A9-targeted compounds have been developed, the optimal therapeutic strategy has not been determined.

Here, we characterized oxyclozanide, a highly selective small-molecule inhibitor targeting the interaction between S100A9 and RAGE. We found that oxyclozanide had a prominent inhibitory effect on the S100A9/RAGE complex and its signaling pathway in S100A9-expressing TNBC cells, as evidenced by its inhibitory effects on growth in vitro and in vivo. Oxyclozanide binding to S100A9 was detected in an SPR analysis, and inhibition of the interaction between S100A9 and RAGE/TLR4 was observed using a ProteinChip-based protein-protein interaction assay system, a new screening technology based on competition among protein-protein interactions. Up-regulated in numerous cancer types and associated with tumor cell growth and survival, S100A9 binds to RAGE and triggers RAGE-mediated cell signaling involving MAPK, leading to the up-regulation of genes involved in cell survival and proliferation. With respect to selectivity, oxyclozanide treatment resulted in the dose- and S100A9-dependent inhibition of proliferation in TNBC cells. As a functional correlate of the inhibition of cell growth, multiple cell signaling pathways were analyzed to determine the effects of oxyclozanide on survival, apoptosis, and angiogenesis in TNBC cells. Oxyclozanide inhibited the expression of pERK and induced cleaved PARP exclusively in TNBC cells expressing S100A9, resulting in the regulation of proliferation and survival in cancer cells. Tumor progression is controlled by the balance between pro-angiogenic and anti-angiogenic factors. For a tumor to grow continuously, cancer cells must down-regulate angiogenesis inhibitors, such as TSP-1 (Sargiannidou et al. 2001). TSP-1 expression was induced by oxyclozanide in TNBC cells expressing S100A9. Taken together, these data demonstrate that oxyclozanide exhibits potent anti-TNBC effects in an S100A9-specific manner. In the tumor microenvironment, angiogenesis is the process by which new capillary blood vessels are generated, which is important for tumor growth. HUVEC is an experimental model for endothelial cells. Oxyclozanide strongly suppressed bFGF-induced HUVEC tube formation, proliferation, and migration. Thus, these data support the role for oxyclozanide as a putative inhibitor of endothelial cell functions in vitro.

A xenograft model was used to evaluate the in vivo anti-cancer efficacy of S100A9. TNBC cells expressing or not expressing S100A9 were subcutaneously injected into nude mice and the drug was administered when the size of the tumors was palpable. The growth rate was slower for S100A9-negative xenografts treated with oxyclozanide than for vehicle tumors. However, the growth of S100A9-positive tumors was largely delayed by oxyclozanide treatment, especially at 250 mg/kg. Key indicators of tumor growth, i.e., pERK, cleaved PARP, and TSP-1, were evaluated in S100A9-positive tumors. As expected, pERK levels were significantly diminished, cleavage PARP expression was slightly increased, and TSP-1 protein levels were higher in oxyclozanide treated tumors.

Our findings show that oxyclozanide has potent anti-cancer activity in cells and preclinical TNBC models in which S100A9 is expressed. These data strongly support the applicability of oxyclozanide for the treatment of patients with TNBC when S100A9 is overexpressed.

Conflict of interest

KH Chang, YY Choi and ID Jung are employees of Ahngook Pharm., Seoul, Republic of Korea. The other authors have no other conflicts of interest with regard to the content of this article.

Acknowledgements

This work was supported by grants from the Korean Healthcare Technology R&D Project through the Korean Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (Grant HI13C2148).

Fig 1.

Figure 1.Chemical structure of oxyclozanide.
Drug Targets and Therapeutics 2022; 1: 1-11https://doi.org/10.58502/DTT.22.003

Fig 2.

Figure 2.Docked pose of oxyclozanide on S100A9 and an SPR analysis. (A) Docked structure of S100A9 and oxyclozanide (gray) in the hinge region. (B) Binding mode of S100A9 and oxyclozanide showing various residue interactions. (C) Dose-dependent binding of oxyclozanide to S100A9 using a surface plasmon resonance (SPR) analysis. The oxyclozanide concentrations (from top to bottom) are 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 μM, respectively.
Drug Targets and Therapeutics 2022; 1: 1-11https://doi.org/10.58502/DTT.22.003

Fig 3.

Figure 3.Oxyclozanide inhibited the interaction between S100A9 and the receptor for RAGE/TLR4 on the ProteinChip. The RAGE and TLR4 array were subsequently spotted with a mixture of Cy5-labeled S100A9 protein and oxyclozanide in a humidity chamber at 30℃ for 1 h. Competitive inhibition was determined by measuring the relative fluorescence intensity of the mixture versus the control spot.
Drug Targets and Therapeutics 2022; 1: 1-11https://doi.org/10.58502/DTT.22.003

Fig 4.

Figure 4.Oxyclozanide inhibits the proliferation of TNBC cells in an S100A9 expression-dependent manner. (A) Western blot detection of S100A9 in MDA-MB-231 and MDA-MB-468 cells. (B) Viability of TNBC cell lines with or without S100A9 expression. The cells were treated with three concentrations (3, 10, and 30 μM) of oxyclozanide or with 0.1% dimethyl sulfoxide (DMSO) as a control for 72 h. After treatment with oxyclozanide, the cells were subjected to an MTT assay. Data represent the means ± SD from duplicate wells.
Drug Targets and Therapeutics 2022; 1: 1-11https://doi.org/10.58502/DTT.22.003

Fig 5.

Figure 5.Oxyclozanide has effects on multiple signaling molecules in TNBC cells expressing S100A9. (A) MDA-MB 231 and MDA-MB-468 cells were treated with 30 μM oxyclozanide (OXC) for 4 h and 8 h, and phosphorylated ERK (p-ERK) was assayed by Western blotting. (B) The two TNBC cell types were treated with 30 μM oxyclozanide for 8 h and 24 h, and full-length PARP, cleaved PARP, and TSP-1 were assayed by Western blotting. β-Actin was used as a loading control.
Drug Targets and Therapeutics 2022; 1: 1-11https://doi.org/10.58502/DTT.22.003

Fig 6.

Figure 6.Oxyclozanide inhibited bFGF-induced endothelial cell tube formation. (A) Tube formation was evaluated by measurements of tube length after treatment with three concentrations (0.5, 5, and 50 μM) of oxyclozanide (OXC). Data represent means ± S.D. *p < 0.05, **p < 0.01 compared to the control (DMSO 0.5%). (B) Representative images of the effects of oxyclozanide on bFGF-induced tube formation in HUVECs (40×). (+) bFGF: bFGF alone, DMSO: bFGF plus DMSO 0.5%, 2-Me: bFGF plus 2-methoxy estradiol 3 μM, oxyclozanide: bFGF plus 0.5, 5, and 50 μM oxyclozanide.
Drug Targets and Therapeutics 2022; 1: 1-11https://doi.org/10.58502/DTT.22.003

Fig 7.

Figure 7.Oxyclozanide inhibited bFGF-induced endothelial cell proliferation. HUVECs were incubated with three concentrations (0.5, 5, and 50 μM) of oxyclozanide (OXC) in the presence of bFGF (25 ng/mL) for 72 h at 37℃ in 5% CO2 with humidity. Cell proliferation was estimated using an MTT assay. Data represent the means ± SD from duplicate wells.
Drug Targets and Therapeutics 2022; 1: 1-11https://doi.org/10.58502/DTT.22.003

Fig 8.

Figure 8.Oxyclozanide inhibited bFGF-induced endothelial cell migration. (A) Migration of HUVECs was performed using cell scratch wound healing assay. Images from the same area were captured after 4 h and 8 h wound infliction and compared migration in the controls (DMSO 0.5%) for each concentration. Data represent the means ± SD. *p < 0.05 as compared to the control (DMSO 0.5%). (B) Image of HUVEC migration obtained at 4 h and 8 h after treatment with bFGF alone and bFGF plus 0.5% DMSO, 3 μM 2-methoxy estradiol, and oxyclozanide (OXC) (40×).
Drug Targets and Therapeutics 2022; 1: 1-11https://doi.org/10.58502/DTT.22.003

Fig 9.

Figure 9.Oxyclozanide suppressed tumor growth in an S100A9-positive xenograft model. (A) Mice with xenograft tumors established from MDA-MB-231 cell lines were given 125 and 250 mg/kg oxyclozanide orally every day and tumor volume was recorded twice a week for 22 days. (B) Mice with S100A9-positive tumors (MDA-MB-468 cell lines) were given 62.5, 125, and 250 mg/kg oxyclozanide orally every day, and tumor volume was recorded twice a week for 28 days. Data represent the means ± SD (n = 7). *p < 0.05 as compared to the vehicle group. (C) Western blotting was performed to determine the effects of oxyclozanide on key indicators of tumor growth. Expression levels of pERK, cleaved PARP, and TSP-1 were measured from MDA-MB-468 xenografts 8 h post-treatment.
Drug Targets and Therapeutics 2022; 1: 1-11https://doi.org/10.58502/DTT.22.003

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