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A natural piper-amide-like compound NED-135 exhibits a potent inhibitory effect on the invasive breast cancer cells
Chemico-Biological Interactions 237 (2015) 58–65
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A natural piper-amide-like compound NED-135 exhibits a potent inhibitory effect on the invasive breast cancer cells Eun-Sook Kim a,1, Hyunkyung Cho b,1, Chaemin Lim b, Joo-Youn Lee b, Da-In Lee a, Sanghee Kim b,⇑, Aree Moon a,⇑ a b
College of Pharmacy, Duksung Women’s University, Samyang-ro, Dobong-gu, Seoul 132-714, Republic of Korea College of Pharmacy, Seoul National University, Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
a r t i c l e
i n f o
Article history: Received 5 February 2015 Received in revised form 17 April 2015 Accepted 8 May 2015 Available online 14 May 2015 Keywords: NED-135 MMP-9 Cell invasion
a b s t r a c t Invasiveness and metastasis are the primary factors indicating poor prognosis in breast cancer patients. To identify a novel lead compound for the development of therapeutics for the treatment of breast cancer through inhibiting invasion, we screened the natural piper amide-like compounds library that we previously constructed. Among the compounds tested, (E)-3-(3,4-dimethoxyphenyl)-N-(4-hydroxyphenethyl)acrylamide (NED-135) showed potent inhibitory effects on matrix metalloproteinase (MMP)-9 and invasiveness of MCF10A human breast epithelial cells treated with an inflammatory lipid, sphingosine-1-phosphate (S1P). The invasive phenotypes of MDA-MB-231 and Hs578T triple-negative breast cancer cells were significantly inhibited by NED-135. NED-135 efficiently inhibited the S1P-induced MMP-9 expression at the transcriptional level with a comparable degree to FTY720, a known antagonist of S1P. We further showed that NED-135 significantly inhibited activation of S1P-induced signaling molecules, Akt, ERKs, and p38 MAPK. Computational similarity analysis led us to postulate that NED-135 and FTY720 may exert anti-invasive effects on breast cells possibly via different mechanisms. Due to its novel structural and functional features, we suggest that NED-135 can be used as a novel lead compound against breast cancer in an inflammatory microenvironment and highly invasive triple-negative breast cancer. Ó 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Breast cancer is one of the most frequently diagnosed types of cancers among women [1]. Invasiveness and metastasis are the primary causes of mortality in breast cancer patients. Inflammation has been linked with poor prognosis in breast cancer [2], and chronic inflammation is involved in breast cancer recurrence [3]. Many studies have demonstrated a close association between an inflammatory microenvironment and breast cancer progression [4–7]. Sphingosine-1-phosphate (S1P) is considered an inflammatory lipid since its level rises locally in inflamed tissues [8,9] and in chronic inflammatory diseases such as rheumatoid arthritis [10]. We and others have suggested a crucial role of S1P in breast cancer aggressiveness [11–13]. Among breast cancers, triple-negative breast cancers that lack the estrogen receptor, progesterone receptor, and HER2 have been associated with poor ⇑ Corresponding authors. Tel.: +82 2 880 2487; fax: +82 2 888 0649 (S. Kim). Tel.: +82 2 901 8394; fax: +82 2 901 8386 (A. Moon). E-mail addresses: [email protected] (S. Kim), [email protected] (A. Moon). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.cbi.2015.05.006 0009-2797/Ó 2015 Elsevier Ireland Ltd. All rights reserved.
prognosis and tumor aggressiveness [14]. Much attention has been focused on triple-negative breast cancer since there are no drugs proven effective against this type of cancer [3]. Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play a role in degradation of extracellular matrix (ECM) proteins, especially the major collagen constituents of the basement membrane. MMPs are deeply involved in tumor invasion and migration potential and have been implicated as biomarkers for various kinds of cancers. Breast cell invasion is associated with enhanced expression of MMP-2 and MMP-9 [15– 18]. Expression of MMP-9 is induced under various physiological conditions such as wound healing, inflammation, tumor invasion and metastasis [19,20]. Elevated expression of MMP-9 is associated with poor prognosis in several cancers including breast cancer [21– 23]. Recently, we revealed a relationship between MMP-9 and S1P by showing that S1P induces the invasive phenotype of human breast epithelial cells through MMP-9 up-regulation [12,13]. Piper amides, the most common constituents of the genus Piper [24], have diverse and versatile biological activities including anti-inflammatory and anti-tumor activities [25]. We have previously constructed a library of piper amide-like compounds
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from the bifunctional b-phosphono-N-hydroxysuccinimidyl ester without chromatographic purification [26]. The 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) mediated coupling reaction and an Horner–Wadsworth–Emmons type reaction provided the desired a,b-unsaturated amide. In an attempt to discover new lead compounds for the treatment of breast cancer through inhibiting MMP-9 expression and invasion/migration, we screened our piper amide-like compound library [26]. We evaluated the inhibitory activities of randomly selected library members against S1P-induced MMP-9 expression and investigated the effects of these compounds on breast cell invasion. Here, we identified (E)-3-(3,4-dimethoxyphenyl)N-(4-hydroxyphenethyl)acrylamide-135 (NED-135) as a novel lead compound with anti-invasive activity against breast cells.
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2.3. Cell lines MCF10A, MDA-MB-231 and Hs578T cell lines were cultured as previously described [27,28]. 2.4. Gelatin zymogram assay Cells were cultured in serum-free DMEM/F12 medium for 48 h. Gelatinolytic activity of the conditioned medium was determined using the gelatin zymogram assay as previously described [27]. 2.5. In vitro invasion assay
2. Materials and methods
In vitro invasion assays were performed using 24-well Transwell units with polycarbonate filters (Corning Costar, Cambridge, MA), as previously described [29].
2.1. Reagents
2.6. Immunoblot analysis
The chemicals used in the synthesis of NED-135 were purchased from Sigma–Aldrich Chemical Co. In addition, FTY720 was obtained from Selleck Chemicals (Huston, TX, USA). S1P was purchased from Sigma–Aldrich (St Louis, MO, USA).
Immunoblot analysis was performed, as previously described [17]. Anti-p38 MAPK, anti-phospho-p38 MAPK, anti-ERK1/2, anti-phospho-ERK1/2, anti-Akt, anti-phospho-Akt, and anti-b-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-MMP-2 antibody was purchased from R&D Systems (Minneapolis, MN, USA). Anti-MMP-9, anti-S1P3, anti-Gaq, and anti-PLC-b4 antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
2.2. Chemistry The selected library member, NED-135, was resynthesized and purified by chromatography. Reaction was monitored by TLC analysis using silica gel 60 F-254 thin-layer chromatography plates. Flash column chromatography was performed on silica gel (230– 400 mesh). Melting points were measured using a Buchi B-540 melting point apparatus without correction. 1H NMR (600 MHz) spectra and 13C NMR (150 MHz) spectra were recorded in d units relative to the non-deuterated solvent as the internal reference. IR spectra were measured on a Fourier Transform Infrared spectrometer. High-resolution mass spectra (HRMS) were recorded using fast atom bombardment (FAB). (E)-3-(3,4-dimethoxyphenyl)-N-(4-hydroxyphenethyl)acrylami de (NED-135): To a solution of 3,4-dimethoxycinnamicacid (1.04 g, 5 mmol) in CH2Cl2 (15 mL) was added N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (1.44 g, 7.50 mmol) and 1-hydroxybenzotriazole hydrate (743 mg, 5.50 mmol) at 0 °C. After stirring for 15 min, tyramine (755 mg, 5.50 mmol) and N,N-diisopropylethylamine (0.18 mL, 1 mmol) in CH2Cl2 (10 mL) were added. The reaction mixture was warmed to room temperature and stirred for 20 h. The mixture was then extracted twice with CH2Cl2. The combined organic layers were washed with water and brine, dried with MgSO4, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (hexane/EtOAc, 3:1) to afford the product as a white solid (1.40 g, 86%). The NMR and HPLC analysis of the synthesized compound revealed that it was very pure and contained no notable impurities. The confirmation of chemical structure of NED-135 was based on its spectroscopic data; m.p.: 161–162 °C. IR (CHCl3): tmax/cm1 3271 (NH), 3015 (OH), 1654 (CO), 1595 (C@C), 1260 (C–O–C) and 1140 (CH). 1H NMR (600 MHz, CDCl3), d (ppm): 2.79 (t, 2H, J = 6.9 Hz, CH2), 3.60 (q, 2H, J = 6.6 Hz, CH2), 3.87 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 5.55 (br s, 1H, OH), 5.57 (br s, 1H, NH), 6.17 (d, 1H, J = 15.5 Hz, CH), 6.79 (td, 2H, J = 2.5, 9.0 Hz, Ar), 6.82 (d, 1H, J = 8.2 Hz, Ar), 6.98 (d, 1H, J = 1.8 Hz, Ar), 7.03–7.06 (m, 3H, Ar), 7.54 (d, 1H, J = 15.6 Hz, CH). 13C NMR (150 MHz, CDCl3), d (ppm): 34.75 (C13), 40.97 (C12), 55.88 (C21), 55.95 (C22), 109.68 (C3), 111.08 (C6), 115.59 (C16, C18), 118.30 (C8), 122.02 (C4), 127.72 (C5), 129.88 (C15, C19), 130.59 (C14), 141.13 (C7), 149.13 (C2), 150.63 (C1), 154.59 (C17), 166.27 (C9). FAB-MS (m/z) calcd. C19H22NO4 328.1549 ([M+H]+), found 328.1557.
2.7. Luciferase reporter assay Luciferase and beta-galactosidase activities were assayed using a luciferase assay kit (Promega, Madison, WI, USA) and a Galacto-Light Kit (Tropix Inc., Bedford, MA, USA), respectively, and measured with a luminometer (Tuner Designs, Sunnyvale, CA, USA) as previously described [18]. For MMP-9 promoter assays, full-length human MMP-9 promoter-luciferase constructs [30] were kindly provided by Etty N. Benveniste (Department of Cell Biology, University of Alabama, Birmingham, AL, USA). 2.8. RT-PCR RNA was extracted from cells using Trizol (Invitrogen, Carlsbad, CA, USA) and reverse transcribed with Superscript-III reverse transcriptase (Invitrogen). RT-PCR was performed using primers for MMP-9. The thermocycler conditions used were as previously described [13]. Equal volumes of each PCR product were analyzed by means of 2% agarose gel electrophoresis. 2.9. b-Arrestin translocation assay The b-arrestin translocation assay was done using the b-arrestin PathHunter™ assay kit (DiscoveRx, Fremont, CA, USA) according to manufacturer’s protocol [31]. Data were collected in duplicate from a single point profile with 10 lM NED-135. 2.10. Computational similarity method FCFP_4 (functional class extended-connectivity fingerprint of maximum diameter 4) were computed in Pipeline Pilot v9.1 [32] for the FTY720 and NED-135. 2D Similarity was calculated using Molecular Similarity component in Pipeline Pilot, which provide the Tanimoto coefficient. For estimating 3D similarity, conformers for each active NED compounds were generated using OMEGA v2.5 with default parameters [33]. Atom typing, energy calculations and geometry optimization in OMEGA were performed using the MMFF (Merck Molecular Force Field). Numerous common
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pharmacophore models with different combinations of variants were developed using the PHASE v3.9 module of the Schrödinger molecular modeling software [34] with conformers. The best model provided superior alignment with five active compounds and contained three features. The matched conformer of NED-135 was used to calculate shape similarity with OpenEye ROCS v3.2, which is a shape-based molecule superposition method [35]. The best hit selection conformer was set to 100 and other parameters were set to the software’s default values. Two molecules were aligned by means of a solid-body optimization process that maximizes a Gaussian-based volume overlap parameterized to reproduce hard-sphere volume. From the obtained shape similarities, we computed the electrostatic similarity by OpenEye EON v2.2 [36]. The parameters were at default settings. The electrostatic potential maps between two pre-aligned small molecules were compared and the Electrostatic Tanimoto score was calculated using EON. The electrostatic maps were visualized using OpenEye VIDA v4.2 [37].
A
3. Results and discussion 3.1. Screening a piper amide-like compound library for inhibition of MMP-9
B
To identify novel lead compounds that exert inhibitory effects on S1P-induced MMP-9 expression in breast cells, 23 compounds were randomly selected from a 250 member piper amide-like compound library. Inhibitory effects on S1P-induced MMP-9 expression were assessed in MCF10A human breast epithelial cells using a gelatin zymogram assay. Among the 23 compounds tested, five compounds, NED-032, NED-085, NED-097, NED-118, and NED-135 (Fig. 1A) significantly suppressed MMP-9 expression triggered by S1P in MCF10A cells (Fig. 1B). We conducted a MTT assay to evaluate the toxicity of NED compounds in MCF10A cells. NED compounds did not affect the cell viability at 10 lM (data not shown).
3.2. Effect of NED-135 on invasive phenotypes of S1P-treated MCF10A and triple-negative breast cancer cells We next performed an in vitro invasion assay to investigate the effects of the five identified compounds on breast cell invasion. Treatment of MCF10A cells with NED-097 and NED-135 significantly reduced S1P-induced invasive phenotype by 22.5% and 43.7%, respectively (Fig. 2A). FTY720, a known antagonist of S1P receptor 1 [38,39], was used as a positive control. In our previous study, FTY720 exhibited potent inhibition of S1P-induced MMP-9 expression and invasion in MCF10A cells [12]. FTY720 inhibited the invasive phenotype by 53.8%. We selected NED-135 for further studies because it exhibited the most potent inhibitory activity among the tested compounds. We examined the effect of NED-135 on the invasive capacities of highly invasive triple-negative breast cancer cell lines, MDA-MB-231 and Hs578T. NED-135 caused a significant decrease in the invasive ability of these cell lines (Fig. 2B and C). FTY720 inhibited invasion in Hs578T cells with comparable efficiency as NED-135, while invasion of MDA-MB-231 cell was not affected by FTY720 treatment. These data demonstrate that NED-135 inhibits the invasive phenotype not only in MCF10A cells treated with S1P, but also in the originally invasive breast cancer cells, MDA-MB-231 and Hs578T. The results suggest a potential application of NED-135 for the development of anti-invasive agents for triple-negative breast cancers where no effective therapeutic interventions are currently available.
Fig. 1. Screening the piper amide-like compound library for inhibition of MMP-9. (A) Chemical structures of NED-032, NED-085, NED-097, NED-118 and NED-135. (B) MCF10A cells were treated with 10 lM S1P and 10 lM NED compounds for 48 h. Conditioned media from cell were subjected to gelatin zymogram assay. Intensities of 92 kDa MMP-9 bands were quantitated. MMP-9 expression (%) was calculated with the MMP-9 band intensity from cells treated with S1P alone as 100%. Values are means + s.e.m. of triplicate samples. *,**Statistically different from control (S1P alone) at p < 0.05 and p < 0.01, respectively.
E-cadherin functions as an invasion suppressor by protecting epithelial integrity and is downregulated in most cancer cells, whereas vimentin contributes to invasion and metastasis of cancer cells [40,41]. We next detected the expressions of E-cadherin and vimentin in MDA-MB-231 cells treated with NED-135. As shown in Fig. 2D, NED-135 increased the level of E-cadherin and inhibited that of vimentin, suggest that E-cadherin and vimentin may be involved in the NED-135-mediated inhibitory effect on invasion.
3.3. NED-135 inhibits MMP-9 expression at the transcriptional level Treatment of MCF10A cells with NED-135 significantly inhibited S1P-induced MMP-9 expression at the protein level with a similar efficiency as FTY720 as evidence by the gelatin zymogram assay (Fig. 3A) and immunoblot analysis (Fig. 3B). We next investigated whether NED-135 inhibits S1P-induced MMP-9 up-regulation at the transcriptional level. RT-PCR analysis (Fig. 3C) and the luciferase
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A
B
C
D
Fig. 2. Effects of NED compounds on the invasive phenotype of breast cells. (A) In vitro invasion assay was conducted on MCF10A cells treated with 10 lM S1P and 10 lM FTY720 or 10 lM NED compounds for 24 h. Invaded cells were stained with 0.5% crystal violet and then observed by microscope (magnification, 200). Invasion ability (%) was calculated with the invasiveness of cells treated with S1P alone as 100%. *,**Statistically different from S1P-treated cells at p < 0.05 and p < 0.01, respectively. (B and C) An in vitro invasion assay was conducted on MDA-MB-231 and Hs578T cells. *,**Statistically different from control at p < 0.05 and p < 0.01, respectively. (D) MDA-MB-231 cells were treated with NED-135 for 24 h. Immunoblot analysis was performed to detect the levels of E-cadherin and vimentin. *,**Statistically different from control at p < 0.05 and p < 0.01, respectively.
reporter assay (Fig. 3D) showed that mRNA level and the promoter activity of MMP-9 were significantly inhibited by NED-135. These results demonstrate that NED-135 inhibits S1P-induced MMP-9 expression at the transcriptional level.
phosphorylation of Akt, ERKs and p38 MAPK at comparable degree as FTY720 (Fig. 4B–D). These data imply that NED-135 may exert its effects via inhibition of the S1P-triggered signaling pathways responsible for breast cell invasion. 3.5. Effect of NED-135 on S1P receptors
3.4. Effect of NED-135 on S1P-induced activation of signaling molecules We previously demonstrated that S1P induces expressions of S1P3, Gaq, and PLC-b4 and leads to activations of Akt, ERKs, and p38 MAPK, which play crucial roles in MMP-9 up-regulation and breast cell invasion [12]. We next tested the effect of NED-135 on S1P-induced activation of these signaling molecules in MCF10A cells. NED-135 inhibited the S1P-induced expressions of S1P3, Gaq, and PLC-b4 (Fig. 4A). It inhibited the S1P-induced
To address the molecular mechanisms of NED-135 and to determine whether the effect of NED-135 is associated with the S1P receptors, we performed a b-arrestin translocation assay [31] in agonist and antagonist formats against all S1P receptor subtypes. Unlike FTY720, which is a prototype S1P receptor modulator [39], NED-135 exhibited no discernible agonistic or antagonistic activity against any of the S1P receptor subtypes (Fig. 5). The result suggests that the anti-invasive ability of NED-135 in breast cells may not be due to the modulation of S1P receptors.
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A
Gelatin zymogram assay S1P FTY720 NED-135
-
+ -
+ + -
C RT-PCR
+ +
S1P FTY720 NED-135
-
+ -
+ + -
+ +
B
MMP-9 β -actin
∗
6 5 4 3 2 1 0
∗∗
D
Immunoblot analysis S1P FTY720 NED-135 MMP-9 MMP-2
-
+ -
+ + -
+ +
Luciferase assay MMP-9 promoter activity
Relative MMP-2/MMP-9
MMP-9 MMP-2
1.2
∗
∗
1.0 0.8 0.6 0.4 0.2
0 S1P FTY720 NED-135
-
+ -
+ + -
+ +
Fig. 3. Effect of NED-135 on S1P-induced MMP-9 up-regulation. (A and B) Gelatin zymogram assay and immunoblot analysis were conducted on conditioned media from cells treated with 10 lM S1P and 2 lM FTY720 or 2 lM NED-135 for 48 h. (C and D) RT-PCR and luciferase assay were conducted on cells treated with 10 lM S1P and 2 lM FTY720 or 2 lM NED-135 for 24 h. *,**Statistically different from S1P-treated cells at p < 0.05 and p < 0.01, respectively.
A
B
C
D
Fig. 4. Effect of NED-135 on S1P-induced activation of signaling molecules. Cells were treated with 10 lM S1P and 2 lM FTY720 or 2 lM NED-135 for 24 h (A), 30 min (B) or 5 min (C and D). Immunoblot analysis was performed to detect the levels of S1P3, Gaq, and PLC-b4, phosphorylated Akt (pAkt), Akt, phosphorylated ERKs (pERKs), ERKs, and phosphorylated p38 (pp38) MAPK, and p38 MAPK. *Statistically different from S1P-treated cells at p < 0.05.
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Fig. 5. Activity of NED-135 with the selected b-arrestin translocation assay. NED-135 was tested in agonist and antagonist mode with the S1P receptor assays at 10 lM. For agonist assay, data were normalized to the maximal and minimal responses observed in the presence of S1P and vehicle. For antagonist assay, data were normalized to the maximal and minimal responses observed in the presence of EC80 concentration of S1P (S1P1/EDG1: 495 nM, S1P2/EDG5: 160 nM, S1P3/EDG3: 130 nM, S1P4/EDG6: 660 nM, S1P5/EDG8: 240 nM). Data represent the average of duplicates. Table 1 3D-similarity analysis of NED-135 and FTY720.
Electorstatic Tanimoto_pba Electrostatic Tanimoto_coulb Electrostatic Tanimoto_comboc Shape Tanimotod
3.6. Molecular mechanisms of NED-135
NED-135/NED-135
NED-135/FTY720
1 1 2 1
0.096 0.029 0.448 0.544
a Value of electrostatic Tanimoto using full Poisson–Boltzmann (PB) electrostatics. b Value of electrostatic Tanimoto using only the coulmobic part of PB electrostatics. c Sum of electrostatic Tanimoto_pb and shape Tanimoto accounted both shape and electrostatic mismatch. d Shape Tanimoto between the given molecule and the query. The values were calculated using the tool EON and ROCS from OpenEye [36,37]. The higher values indicate more similarity.
From the above results, we supposed that FTY720 and NED-135 exert their effects via different targets and mechanisms. They are structurally very distinct and may therefore function in dissimilar ways [42]. This notion was supported by computational similarity analysis. Similarity was evaluated on the basis of 2D and 3D molecular aspects of NED-135 and FTY720. We calculated the Tanimoto coefficient [43,44], a widely used similarity coefficient, from FCFP 4 fingerprints. Higher values indicate greater similarity than lower ones. The calculated coefficient of the two compounds was 0.2, reflecting a very low similarity between the two compounds (data not shown).
Fig. 6. 3D shape and electrostatic similarity analysis. (A) The electrostatic potential maps of FTY720 and NED-135. (B) The overlaid potential map of NED-135 on FTY720. Ligands are depicted as ball-and-stick structures. The carbon atoms of FTY720 are colored blue, and the carbon atoms of NED-135 are colored gray. In the electrostatic potential map, positive charges are colored blue and negative charges are colored red. Electron density surface of FTY720 in Fig. 6B is represented as a mesh.
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We also performed 3D-Similarity analysis. We built a pharmacophore model with the active NED compounds and employed it to select the biologically-active conformers. Based on the selected conformers, the 3D shapes and electrostatic similarities of FTY720 and NED-135 were computed. The value of the electrostatic Tanimoto combo was only 0.448 (Table 1), which indicates that NED-135 possesses many distinct features from FTY720. The electrostatic potential maps of FTY720 and NED-135 were depicted in Fig. 6A. The overlaid potential map revealed distinct differences in the electrostatic surfaces of the two molecules (Fig. 6B). Overall, our computational results suggest that FTY720 and NED-135 may be structurally dissimilar and therefore, bind to different targets. 4. Conclusions In summary, piper amide-like compounds were evaluated with the ultimate goal of identifying a novel lead compound for the development of therapeutics for the treatment of breast cancer by inhibiting MMP-9 expression and the invasive phenotype. Among the compounds that exhibited suppression of MMP-9 expression triggered by S1P, NED-135 displayed a remarkable percentage of reduction of S1P-induced invasion of MCF10A cells. It inhibited the invasive phenotypes of MDA-MB-231 and Hs578T triple-negative breast cancer cells. NED-135 markedly inhibited the S1P-induced MMP-9 up-regulation at the transcriptional level, with a comparable degree to FTY720. Unlike FTY720, NED-135 exhibited no activity against S1P receptors, suggesting that its anti-invasive ability may not be due to the results of the modulation of S1P receptors. Since computational similarity analysis indicated a very low similarity between NED-135 and FTY720, it is reasonable to postulate that they may exert similar effects on breast cell invasion via different modes of action. Here, we identified a novel compound, NED-135, that shows a potent inhibitory effect on the invasive phenotype in breast cells stimulated by an inflammatory lipid S1P as well as against invasive triple-negative breast cells. We expect that NED-135 may be a novel chemical tool to identify a new mechanism of breast cancer cell invasion that can serve as a target for drug discovery. In addition, owing to the comparable potency to FTY720, NED-135 can be used as a lead structure for the development of therapeutics for the treatment of breast cancer with novel modes. Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version.
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Guo, Oroxylin A suppresses invasion through down-regulating the expression of matrix metalloproteinase-2/9 in MDA-MB-435 human breast cancer cells, Eur. J. Pharmacol. 603 (2009) 22–28. [17] M.S. Kim, E.J. Lee, H.R. Kim, A. Moon, P38 kinase is a key signaling molecule for H-Ras-induced cell motility and invasive phenotype in human breast epithelial cells, Cancer Res. 63 (2003) 5454–5461. [18] H. Song, S.H. Ki, S.G. Kim, A. Moon, Activating transcription factor 2 mediates matrix metalloproteinase-2 transcriptional activation induced by p38 in breast epithelial cells, Cancer Res. 66 (2006) 10487–10496. [19] J.F. Woessner Jr, Matrix metalloproteinases and their inhibitors in connective tissue remodelling, FASEB J. 5 (1991) 2145–2154. [20] I.S. Shin, K.S. Ahn, N.R. Shin, C.M. Jeon, O.K. Won, Y.W. Chin, K. Lee, S.R. Oh, Homoegonol attenuates the asthmatic responses induced by ovalbumin challenge, Arch. Pharm. Res. 37 (2014), http://dx.doi.org/10.1007/s12272013-0327-8. [21] J.M. Pellikainen, K.M. Ropponen, V.V. Kataja, J.K. Kellokoski, M.J. Eskelinen, V.M. Kosma, Expression of matrix metalloproteinase (MMP)-2 and MMP-9 in breast cancer with a special reference to activator protein-2, HER2, and prognosis, Clin. Cancer Res. 10 (2004) 7621–7628. [22] J. Chen, L.J. Chen, H.C. Zhou, R.B. Yang, Y. Lu, Y.L. Xia, W. Wu, L.W. Hu, Prognostic value of matrix metalloproteinase-9 in gastric cancer: a metaanalysis, Hepatogastroenterology 61 (2014) 518–524. [23] B. Yang, F. Tang, B. Zhang, Y. Zhao, J. Feng, Z. Rao, Matrix metalloproteinase-9 overexpression is closely related to poor prognosis in patients with colon cancer, World J. Surg. Oncol. 12 (2014) 24–30. [24] V.S. Parmer, S.C. Jains, K.S. Bisht, R. Jain, P. Taneja, A. Jha, O.D. Tyagi, A.K. Prasad, J. Wengel, C.E. Olsen, P.M. Boll, Phytochemistry of the genus Piper, Phytochemistry 46 (1997) 597–673. [25] R.M.P. Gutierrez, A.M.N. Gonzalez, C. Hoyo-Vadillo, Alkaloids from piper: a review of its phytochemistry and pharmacology, Mini-Rev. Med. Chem. 13 (2013) 163–193. [26] S. Kim, C. Lim, S. Lee, S. Lee, H. Cho, J.Y. Lee, D.S. Shim, H.D. Park, S. Kim, Column chromatography-free solution-phase synthesis of a natural piper-amide-like compound library, ACS Comb. Sci. 15 (2013) 208–215. [27] A. Moon, M.S. Kim, T.G. Kim, S.H. Kim, H.E. Kim, Y.Q. Chen, H.R. Kim, H-ras, but not N-ras, induces an invasive phenotype in human breast epithelial cells: a role for MMP-2 in the H-ras-induced invasive phenotype, Int. J. Cancer 85 (2000) 176–181. [28] M.S. Koh, A. Moon, Activation of H-Ras and Rac1 correlates with epidermal growth factor-induced invasion in Hs578T and MDA-MB-231 breast carcinoma cells, Biochem. Biophys. Res. Commun. 406 (2011) 25–29. [29] C.L. Hyder, G. Lazaro, J.W. Pylvänäinen, M.W.G. Roberts, S.M. Qvarnström, J.E. 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[38] S.W. Paugh, S.G. Payne, S.E. Barbour, S. Milstien, S. Spiegel, The immunosuppressant FTY720 is phosphorylated by sphingosine kinase type 2, FEBS Lett. 554 (2003) 189–193. [39] H. Rosen, P.J. Gonzalez-Cabrera, M.G. Sanna, S. Brown, Sphingosine 1phosphate receptor signalling, Annu. Rev. Biochem. 78 (2009) 743–768. [40] Q.S. Zhu, K. Rosenblatt, K.L. Huang, G. Lahat, R. Brobey, S. Bolshakov, T. Nguyen, Z. Ding, R. Belousov, K. Bill, X. Luo, A. Lazar, A. Dicker, G.B. Mills, M.C. Hung, D. Lev, Vimentin is a novel AKT1 target mediating motility and invasion, Oncogene 30 (2011) 457–470. [41] N. Pec´ina-Slaus, Tumor suppressor gene E-cadherin and its role in normal and malignant cells, Cancer Cell Int. 3 (2003) 17. [42] M.A. Johnson, G.M. Maggiora, Concepts and Applications of Molecular Similarity, John Wiley & Sons, New York, 1990. [43] P. Willett, J.M. Barnard, G.M. Downs, Chemical similarity searching, J. Chem. Inf. Comput. Sci. 38 (1998) 983–996. [44] T.T. Tanimoto, IBM Internal Report, Armonk IBM Corp, New York, 1957.
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Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
A natural piper-amide-like compound NED-135 exhibits a potent inhibitory effect on the invasive breast cancer cells Eun-Sook Kim a,1, Hyunkyung Cho b,1, Chaemin Lim b, Joo-Youn Lee b, Da-In Lee a, Sanghee Kim b,⇑, Aree Moon a,⇑ a b
College of Pharmacy, Duksung Women’s University, Samyang-ro, Dobong-gu, Seoul 132-714, Republic of Korea College of Pharmacy, Seoul National University, Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
a r t i c l e
i n f o
Article history: Received 5 February 2015 Received in revised form 17 April 2015 Accepted 8 May 2015 Available online 14 May 2015 Keywords: NED-135 MMP-9 Cell invasion
a b s t r a c t Invasiveness and metastasis are the primary factors indicating poor prognosis in breast cancer patients. To identify a novel lead compound for the development of therapeutics for the treatment of breast cancer through inhibiting invasion, we screened the natural piper amide-like compounds library that we previously constructed. Among the compounds tested, (E)-3-(3,4-dimethoxyphenyl)-N-(4-hydroxyphenethyl)acrylamide (NED-135) showed potent inhibitory effects on matrix metalloproteinase (MMP)-9 and invasiveness of MCF10A human breast epithelial cells treated with an inflammatory lipid, sphingosine-1-phosphate (S1P). The invasive phenotypes of MDA-MB-231 and Hs578T triple-negative breast cancer cells were significantly inhibited by NED-135. NED-135 efficiently inhibited the S1P-induced MMP-9 expression at the transcriptional level with a comparable degree to FTY720, a known antagonist of S1P. We further showed that NED-135 significantly inhibited activation of S1P-induced signaling molecules, Akt, ERKs, and p38 MAPK. Computational similarity analysis led us to postulate that NED-135 and FTY720 may exert anti-invasive effects on breast cells possibly via different mechanisms. Due to its novel structural and functional features, we suggest that NED-135 can be used as a novel lead compound against breast cancer in an inflammatory microenvironment and highly invasive triple-negative breast cancer. Ó 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Breast cancer is one of the most frequently diagnosed types of cancers among women [1]. Invasiveness and metastasis are the primary causes of mortality in breast cancer patients. Inflammation has been linked with poor prognosis in breast cancer [2], and chronic inflammation is involved in breast cancer recurrence [3]. Many studies have demonstrated a close association between an inflammatory microenvironment and breast cancer progression [4–7]. Sphingosine-1-phosphate (S1P) is considered an inflammatory lipid since its level rises locally in inflamed tissues [8,9] and in chronic inflammatory diseases such as rheumatoid arthritis [10]. We and others have suggested a crucial role of S1P in breast cancer aggressiveness [11–13]. Among breast cancers, triple-negative breast cancers that lack the estrogen receptor, progesterone receptor, and HER2 have been associated with poor ⇑ Corresponding authors. Tel.: +82 2 880 2487; fax: +82 2 888 0649 (S. Kim). Tel.: +82 2 901 8394; fax: +82 2 901 8386 (A. Moon). E-mail addresses: [email protected] (S. Kim), [email protected] (A. Moon). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.cbi.2015.05.006 0009-2797/Ó 2015 Elsevier Ireland Ltd. All rights reserved.
prognosis and tumor aggressiveness [14]. Much attention has been focused on triple-negative breast cancer since there are no drugs proven effective against this type of cancer [3]. Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play a role in degradation of extracellular matrix (ECM) proteins, especially the major collagen constituents of the basement membrane. MMPs are deeply involved in tumor invasion and migration potential and have been implicated as biomarkers for various kinds of cancers. Breast cell invasion is associated with enhanced expression of MMP-2 and MMP-9 [15– 18]. Expression of MMP-9 is induced under various physiological conditions such as wound healing, inflammation, tumor invasion and metastasis [19,20]. Elevated expression of MMP-9 is associated with poor prognosis in several cancers including breast cancer [21– 23]. Recently, we revealed a relationship between MMP-9 and S1P by showing that S1P induces the invasive phenotype of human breast epithelial cells through MMP-9 up-regulation [12,13]. Piper amides, the most common constituents of the genus Piper [24], have diverse and versatile biological activities including anti-inflammatory and anti-tumor activities [25]. We have previously constructed a library of piper amide-like compounds
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from the bifunctional b-phosphono-N-hydroxysuccinimidyl ester without chromatographic purification [26]. The 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) mediated coupling reaction and an Horner–Wadsworth–Emmons type reaction provided the desired a,b-unsaturated amide. In an attempt to discover new lead compounds for the treatment of breast cancer through inhibiting MMP-9 expression and invasion/migration, we screened our piper amide-like compound library [26]. We evaluated the inhibitory activities of randomly selected library members against S1P-induced MMP-9 expression and investigated the effects of these compounds on breast cell invasion. Here, we identified (E)-3-(3,4-dimethoxyphenyl)N-(4-hydroxyphenethyl)acrylamide-135 (NED-135) as a novel lead compound with anti-invasive activity against breast cells.
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2.3. Cell lines MCF10A, MDA-MB-231 and Hs578T cell lines were cultured as previously described [27,28]. 2.4. Gelatin zymogram assay Cells were cultured in serum-free DMEM/F12 medium for 48 h. Gelatinolytic activity of the conditioned medium was determined using the gelatin zymogram assay as previously described [27]. 2.5. In vitro invasion assay
2. Materials and methods
In vitro invasion assays were performed using 24-well Transwell units with polycarbonate filters (Corning Costar, Cambridge, MA), as previously described [29].
2.1. Reagents
2.6. Immunoblot analysis
The chemicals used in the synthesis of NED-135 were purchased from Sigma–Aldrich Chemical Co. In addition, FTY720 was obtained from Selleck Chemicals (Huston, TX, USA). S1P was purchased from Sigma–Aldrich (St Louis, MO, USA).
Immunoblot analysis was performed, as previously described [17]. Anti-p38 MAPK, anti-phospho-p38 MAPK, anti-ERK1/2, anti-phospho-ERK1/2, anti-Akt, anti-phospho-Akt, and anti-b-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-MMP-2 antibody was purchased from R&D Systems (Minneapolis, MN, USA). Anti-MMP-9, anti-S1P3, anti-Gaq, and anti-PLC-b4 antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
2.2. Chemistry The selected library member, NED-135, was resynthesized and purified by chromatography. Reaction was monitored by TLC analysis using silica gel 60 F-254 thin-layer chromatography plates. Flash column chromatography was performed on silica gel (230– 400 mesh). Melting points were measured using a Buchi B-540 melting point apparatus without correction. 1H NMR (600 MHz) spectra and 13C NMR (150 MHz) spectra were recorded in d units relative to the non-deuterated solvent as the internal reference. IR spectra were measured on a Fourier Transform Infrared spectrometer. High-resolution mass spectra (HRMS) were recorded using fast atom bombardment (FAB). (E)-3-(3,4-dimethoxyphenyl)-N-(4-hydroxyphenethyl)acrylami de (NED-135): To a solution of 3,4-dimethoxycinnamicacid (1.04 g, 5 mmol) in CH2Cl2 (15 mL) was added N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (1.44 g, 7.50 mmol) and 1-hydroxybenzotriazole hydrate (743 mg, 5.50 mmol) at 0 °C. After stirring for 15 min, tyramine (755 mg, 5.50 mmol) and N,N-diisopropylethylamine (0.18 mL, 1 mmol) in CH2Cl2 (10 mL) were added. The reaction mixture was warmed to room temperature and stirred for 20 h. The mixture was then extracted twice with CH2Cl2. The combined organic layers were washed with water and brine, dried with MgSO4, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (hexane/EtOAc, 3:1) to afford the product as a white solid (1.40 g, 86%). The NMR and HPLC analysis of the synthesized compound revealed that it was very pure and contained no notable impurities. The confirmation of chemical structure of NED-135 was based on its spectroscopic data; m.p.: 161–162 °C. IR (CHCl3): tmax/cm1 3271 (NH), 3015 (OH), 1654 (CO), 1595 (C@C), 1260 (C–O–C) and 1140 (CH). 1H NMR (600 MHz, CDCl3), d (ppm): 2.79 (t, 2H, J = 6.9 Hz, CH2), 3.60 (q, 2H, J = 6.6 Hz, CH2), 3.87 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 5.55 (br s, 1H, OH), 5.57 (br s, 1H, NH), 6.17 (d, 1H, J = 15.5 Hz, CH), 6.79 (td, 2H, J = 2.5, 9.0 Hz, Ar), 6.82 (d, 1H, J = 8.2 Hz, Ar), 6.98 (d, 1H, J = 1.8 Hz, Ar), 7.03–7.06 (m, 3H, Ar), 7.54 (d, 1H, J = 15.6 Hz, CH). 13C NMR (150 MHz, CDCl3), d (ppm): 34.75 (C13), 40.97 (C12), 55.88 (C21), 55.95 (C22), 109.68 (C3), 111.08 (C6), 115.59 (C16, C18), 118.30 (C8), 122.02 (C4), 127.72 (C5), 129.88 (C15, C19), 130.59 (C14), 141.13 (C7), 149.13 (C2), 150.63 (C1), 154.59 (C17), 166.27 (C9). FAB-MS (m/z) calcd. C19H22NO4 328.1549 ([M+H]+), found 328.1557.
2.7. Luciferase reporter assay Luciferase and beta-galactosidase activities were assayed using a luciferase assay kit (Promega, Madison, WI, USA) and a Galacto-Light Kit (Tropix Inc., Bedford, MA, USA), respectively, and measured with a luminometer (Tuner Designs, Sunnyvale, CA, USA) as previously described [18]. For MMP-9 promoter assays, full-length human MMP-9 promoter-luciferase constructs [30] were kindly provided by Etty N. Benveniste (Department of Cell Biology, University of Alabama, Birmingham, AL, USA). 2.8. RT-PCR RNA was extracted from cells using Trizol (Invitrogen, Carlsbad, CA, USA) and reverse transcribed with Superscript-III reverse transcriptase (Invitrogen). RT-PCR was performed using primers for MMP-9. The thermocycler conditions used were as previously described [13]. Equal volumes of each PCR product were analyzed by means of 2% agarose gel electrophoresis. 2.9. b-Arrestin translocation assay The b-arrestin translocation assay was done using the b-arrestin PathHunter™ assay kit (DiscoveRx, Fremont, CA, USA) according to manufacturer’s protocol [31]. Data were collected in duplicate from a single point profile with 10 lM NED-135. 2.10. Computational similarity method FCFP_4 (functional class extended-connectivity fingerprint of maximum diameter 4) were computed in Pipeline Pilot v9.1 [32] for the FTY720 and NED-135. 2D Similarity was calculated using Molecular Similarity component in Pipeline Pilot, which provide the Tanimoto coefficient. For estimating 3D similarity, conformers for each active NED compounds were generated using OMEGA v2.5 with default parameters [33]. Atom typing, energy calculations and geometry optimization in OMEGA were performed using the MMFF (Merck Molecular Force Field). Numerous common
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pharmacophore models with different combinations of variants were developed using the PHASE v3.9 module of the Schrödinger molecular modeling software [34] with conformers. The best model provided superior alignment with five active compounds and contained three features. The matched conformer of NED-135 was used to calculate shape similarity with OpenEye ROCS v3.2, which is a shape-based molecule superposition method [35]. The best hit selection conformer was set to 100 and other parameters were set to the software’s default values. Two molecules were aligned by means of a solid-body optimization process that maximizes a Gaussian-based volume overlap parameterized to reproduce hard-sphere volume. From the obtained shape similarities, we computed the electrostatic similarity by OpenEye EON v2.2 [36]. The parameters were at default settings. The electrostatic potential maps between two pre-aligned small molecules were compared and the Electrostatic Tanimoto score was calculated using EON. The electrostatic maps were visualized using OpenEye VIDA v4.2 [37].
A
3. Results and discussion 3.1. Screening a piper amide-like compound library for inhibition of MMP-9
B
To identify novel lead compounds that exert inhibitory effects on S1P-induced MMP-9 expression in breast cells, 23 compounds were randomly selected from a 250 member piper amide-like compound library. Inhibitory effects on S1P-induced MMP-9 expression were assessed in MCF10A human breast epithelial cells using a gelatin zymogram assay. Among the 23 compounds tested, five compounds, NED-032, NED-085, NED-097, NED-118, and NED-135 (Fig. 1A) significantly suppressed MMP-9 expression triggered by S1P in MCF10A cells (Fig. 1B). We conducted a MTT assay to evaluate the toxicity of NED compounds in MCF10A cells. NED compounds did not affect the cell viability at 10 lM (data not shown).
3.2. Effect of NED-135 on invasive phenotypes of S1P-treated MCF10A and triple-negative breast cancer cells We next performed an in vitro invasion assay to investigate the effects of the five identified compounds on breast cell invasion. Treatment of MCF10A cells with NED-097 and NED-135 significantly reduced S1P-induced invasive phenotype by 22.5% and 43.7%, respectively (Fig. 2A). FTY720, a known antagonist of S1P receptor 1 [38,39], was used as a positive control. In our previous study, FTY720 exhibited potent inhibition of S1P-induced MMP-9 expression and invasion in MCF10A cells [12]. FTY720 inhibited the invasive phenotype by 53.8%. We selected NED-135 for further studies because it exhibited the most potent inhibitory activity among the tested compounds. We examined the effect of NED-135 on the invasive capacities of highly invasive triple-negative breast cancer cell lines, MDA-MB-231 and Hs578T. NED-135 caused a significant decrease in the invasive ability of these cell lines (Fig. 2B and C). FTY720 inhibited invasion in Hs578T cells with comparable efficiency as NED-135, while invasion of MDA-MB-231 cell was not affected by FTY720 treatment. These data demonstrate that NED-135 inhibits the invasive phenotype not only in MCF10A cells treated with S1P, but also in the originally invasive breast cancer cells, MDA-MB-231 and Hs578T. The results suggest a potential application of NED-135 for the development of anti-invasive agents for triple-negative breast cancers where no effective therapeutic interventions are currently available.
Fig. 1. Screening the piper amide-like compound library for inhibition of MMP-9. (A) Chemical structures of NED-032, NED-085, NED-097, NED-118 and NED-135. (B) MCF10A cells were treated with 10 lM S1P and 10 lM NED compounds for 48 h. Conditioned media from cell were subjected to gelatin zymogram assay. Intensities of 92 kDa MMP-9 bands were quantitated. MMP-9 expression (%) was calculated with the MMP-9 band intensity from cells treated with S1P alone as 100%. Values are means + s.e.m. of triplicate samples. *,**Statistically different from control (S1P alone) at p < 0.05 and p < 0.01, respectively.
E-cadherin functions as an invasion suppressor by protecting epithelial integrity and is downregulated in most cancer cells, whereas vimentin contributes to invasion and metastasis of cancer cells [40,41]. We next detected the expressions of E-cadherin and vimentin in MDA-MB-231 cells treated with NED-135. As shown in Fig. 2D, NED-135 increased the level of E-cadherin and inhibited that of vimentin, suggest that E-cadherin and vimentin may be involved in the NED-135-mediated inhibitory effect on invasion.
3.3. NED-135 inhibits MMP-9 expression at the transcriptional level Treatment of MCF10A cells with NED-135 significantly inhibited S1P-induced MMP-9 expression at the protein level with a similar efficiency as FTY720 as evidence by the gelatin zymogram assay (Fig. 3A) and immunoblot analysis (Fig. 3B). We next investigated whether NED-135 inhibits S1P-induced MMP-9 up-regulation at the transcriptional level. RT-PCR analysis (Fig. 3C) and the luciferase
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A
B
C
D
Fig. 2. Effects of NED compounds on the invasive phenotype of breast cells. (A) In vitro invasion assay was conducted on MCF10A cells treated with 10 lM S1P and 10 lM FTY720 or 10 lM NED compounds for 24 h. Invaded cells were stained with 0.5% crystal violet and then observed by microscope (magnification, 200). Invasion ability (%) was calculated with the invasiveness of cells treated with S1P alone as 100%. *,**Statistically different from S1P-treated cells at p < 0.05 and p < 0.01, respectively. (B and C) An in vitro invasion assay was conducted on MDA-MB-231 and Hs578T cells. *,**Statistically different from control at p < 0.05 and p < 0.01, respectively. (D) MDA-MB-231 cells were treated with NED-135 for 24 h. Immunoblot analysis was performed to detect the levels of E-cadherin and vimentin. *,**Statistically different from control at p < 0.05 and p < 0.01, respectively.
reporter assay (Fig. 3D) showed that mRNA level and the promoter activity of MMP-9 were significantly inhibited by NED-135. These results demonstrate that NED-135 inhibits S1P-induced MMP-9 expression at the transcriptional level.
phosphorylation of Akt, ERKs and p38 MAPK at comparable degree as FTY720 (Fig. 4B–D). These data imply that NED-135 may exert its effects via inhibition of the S1P-triggered signaling pathways responsible for breast cell invasion. 3.5. Effect of NED-135 on S1P receptors
3.4. Effect of NED-135 on S1P-induced activation of signaling molecules We previously demonstrated that S1P induces expressions of S1P3, Gaq, and PLC-b4 and leads to activations of Akt, ERKs, and p38 MAPK, which play crucial roles in MMP-9 up-regulation and breast cell invasion [12]. We next tested the effect of NED-135 on S1P-induced activation of these signaling molecules in MCF10A cells. NED-135 inhibited the S1P-induced expressions of S1P3, Gaq, and PLC-b4 (Fig. 4A). It inhibited the S1P-induced
To address the molecular mechanisms of NED-135 and to determine whether the effect of NED-135 is associated with the S1P receptors, we performed a b-arrestin translocation assay [31] in agonist and antagonist formats against all S1P receptor subtypes. Unlike FTY720, which is a prototype S1P receptor modulator [39], NED-135 exhibited no discernible agonistic or antagonistic activity against any of the S1P receptor subtypes (Fig. 5). The result suggests that the anti-invasive ability of NED-135 in breast cells may not be due to the modulation of S1P receptors.
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A
Gelatin zymogram assay S1P FTY720 NED-135
-
+ -
+ + -
C RT-PCR
+ +
S1P FTY720 NED-135
-
+ -
+ + -
+ +
B
MMP-9 β -actin
∗
6 5 4 3 2 1 0
∗∗
D
Immunoblot analysis S1P FTY720 NED-135 MMP-9 MMP-2
-
+ -
+ + -
+ +
Luciferase assay MMP-9 promoter activity
Relative MMP-2/MMP-9
MMP-9 MMP-2
1.2
∗
∗
1.0 0.8 0.6 0.4 0.2
0 S1P FTY720 NED-135
-
+ -
+ + -
+ +
Fig. 3. Effect of NED-135 on S1P-induced MMP-9 up-regulation. (A and B) Gelatin zymogram assay and immunoblot analysis were conducted on conditioned media from cells treated with 10 lM S1P and 2 lM FTY720 or 2 lM NED-135 for 48 h. (C and D) RT-PCR and luciferase assay were conducted on cells treated with 10 lM S1P and 2 lM FTY720 or 2 lM NED-135 for 24 h. *,**Statistically different from S1P-treated cells at p < 0.05 and p < 0.01, respectively.
A
B
C
D
Fig. 4. Effect of NED-135 on S1P-induced activation of signaling molecules. Cells were treated with 10 lM S1P and 2 lM FTY720 or 2 lM NED-135 for 24 h (A), 30 min (B) or 5 min (C and D). Immunoblot analysis was performed to detect the levels of S1P3, Gaq, and PLC-b4, phosphorylated Akt (pAkt), Akt, phosphorylated ERKs (pERKs), ERKs, and phosphorylated p38 (pp38) MAPK, and p38 MAPK. *Statistically different from S1P-treated cells at p < 0.05.
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Fig. 5. Activity of NED-135 with the selected b-arrestin translocation assay. NED-135 was tested in agonist and antagonist mode with the S1P receptor assays at 10 lM. For agonist assay, data were normalized to the maximal and minimal responses observed in the presence of S1P and vehicle. For antagonist assay, data were normalized to the maximal and minimal responses observed in the presence of EC80 concentration of S1P (S1P1/EDG1: 495 nM, S1P2/EDG5: 160 nM, S1P3/EDG3: 130 nM, S1P4/EDG6: 660 nM, S1P5/EDG8: 240 nM). Data represent the average of duplicates. Table 1 3D-similarity analysis of NED-135 and FTY720.
Electorstatic Tanimoto_pba Electrostatic Tanimoto_coulb Electrostatic Tanimoto_comboc Shape Tanimotod
3.6. Molecular mechanisms of NED-135
NED-135/NED-135
NED-135/FTY720
1 1 2 1
0.096 0.029 0.448 0.544
a Value of electrostatic Tanimoto using full Poisson–Boltzmann (PB) electrostatics. b Value of electrostatic Tanimoto using only the coulmobic part of PB electrostatics. c Sum of electrostatic Tanimoto_pb and shape Tanimoto accounted both shape and electrostatic mismatch. d Shape Tanimoto between the given molecule and the query. The values were calculated using the tool EON and ROCS from OpenEye [36,37]. The higher values indicate more similarity.
From the above results, we supposed that FTY720 and NED-135 exert their effects via different targets and mechanisms. They are structurally very distinct and may therefore function in dissimilar ways [42]. This notion was supported by computational similarity analysis. Similarity was evaluated on the basis of 2D and 3D molecular aspects of NED-135 and FTY720. We calculated the Tanimoto coefficient [43,44], a widely used similarity coefficient, from FCFP 4 fingerprints. Higher values indicate greater similarity than lower ones. The calculated coefficient of the two compounds was 0.2, reflecting a very low similarity between the two compounds (data not shown).
Fig. 6. 3D shape and electrostatic similarity analysis. (A) The electrostatic potential maps of FTY720 and NED-135. (B) The overlaid potential map of NED-135 on FTY720. Ligands are depicted as ball-and-stick structures. The carbon atoms of FTY720 are colored blue, and the carbon atoms of NED-135 are colored gray. In the electrostatic potential map, positive charges are colored blue and negative charges are colored red. Electron density surface of FTY720 in Fig. 6B is represented as a mesh.
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We also performed 3D-Similarity analysis. We built a pharmacophore model with the active NED compounds and employed it to select the biologically-active conformers. Based on the selected conformers, the 3D shapes and electrostatic similarities of FTY720 and NED-135 were computed. The value of the electrostatic Tanimoto combo was only 0.448 (Table 1), which indicates that NED-135 possesses many distinct features from FTY720. The electrostatic potential maps of FTY720 and NED-135 were depicted in Fig. 6A. The overlaid potential map revealed distinct differences in the electrostatic surfaces of the two molecules (Fig. 6B). Overall, our computational results suggest that FTY720 and NED-135 may be structurally dissimilar and therefore, bind to different targets. 4. Conclusions In summary, piper amide-like compounds were evaluated with the ultimate goal of identifying a novel lead compound for the development of therapeutics for the treatment of breast cancer by inhibiting MMP-9 expression and the invasive phenotype. Among the compounds that exhibited suppression of MMP-9 expression triggered by S1P, NED-135 displayed a remarkable percentage of reduction of S1P-induced invasion of MCF10A cells. It inhibited the invasive phenotypes of MDA-MB-231 and Hs578T triple-negative breast cancer cells. NED-135 markedly inhibited the S1P-induced MMP-9 up-regulation at the transcriptional level, with a comparable degree to FTY720. Unlike FTY720, NED-135 exhibited no activity against S1P receptors, suggesting that its anti-invasive ability may not be due to the results of the modulation of S1P receptors. Since computational similarity analysis indicated a very low similarity between NED-135 and FTY720, it is reasonable to postulate that they may exert similar effects on breast cell invasion via different modes of action. Here, we identified a novel compound, NED-135, that shows a potent inhibitory effect on the invasive phenotype in breast cells stimulated by an inflammatory lipid S1P as well as against invasive triple-negative breast cells. We expect that NED-135 may be a novel chemical tool to identify a new mechanism of breast cancer cell invasion that can serve as a target for drug discovery. In addition, owing to the comparable potency to FTY720, NED-135 can be used as a lead structure for the development of therapeutics for the treatment of breast cancer with novel modes. Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version.
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