2.1 Designing IL17A antagonists
We have generated many derivatives of RA to inhibit angiogenesis via disruption of IL17A. The computational program NOVA was used to design the derivatives. However, 4 compounds were selected according to the ease of synthesis. These 4 compounds were salts and base derivateives of rosmarinic acid. Sodium rosmarinate (NaR) and silver rosmarinate (AgR) were two salts and diamine caffeate/rosmarinate (FLVM) and imidazole caffeate/rosmarinate (FLVZ) were two bases. All of these compounds were suitable to bind the active pockets of IL17A protein. Additionally, they showed binding to VEGF and p-gp proteins. Despite the lower percentage of homology (13.3%) and similarity (19.7%) (Fig. 1), the active pockets of these two proteins (IL17A and VEGF) could predicted to be bound by NaR, AgR, FLVM and FLVZ thereby inhibiting angiogenesis or providing anti-angiogenic efficiency.
2.2 Synthesis and Characterization
2.2.1 Chemicals and Reagent
Solvents and chemicals were purchased from Fluka™, Aldrich™, and JT Baker™, USA. Melting points were measured using the Stuart Scientific SMP1 melting point apparatus. UV–Vis spectra were recorded in dimethyl sulfoxide (DMSO) solution with a Perkin Elmer Lambda 25 UV-Vis spectrophotometer. Infrared (IR) spectra were recorded using the Perkin Elmer System 2000 spectrophotometer and the KBr disc method in the range of 4000 to 400 lb pressure at room temperature (RT). 1H, 13C, and nuclear magnetic resonance (NMR) spectra were recorded on a Bruker™ 500 MHz-NMR spectrophotometer relative to N3 and Me in DMSO. Elemental analysis was conducted using a Perkin Elmer™ 2400 Series-11 CHN analyzer. Molar conductivity measurements were carried out using a Jenway™ 4510 conductivity meter and DMSO solvent.
The melting points was measured by the Stuart Scientific SMP1 melting point apparatus. UV–Vis spectra were recorded in DMSO solution with a Perkin Elmer Lambda 25 UV-Vis spectrophotometer. Infrared (IR) spectra were recorded by the Perkin Elmer System 2000 spectrophotometer by using the KBr disc method in the range 4000–400 at room temperature. 1H, 13C NMR spectra were recorded on a Bruker 500 MHz-NMR spectrophotometer relative to SiMe4 and Me4Sn in DMSO solvent. Elemental analysis was conducted by the Perkin Elmer 2400 Series-11 CHN analyzer. Molar conductivity measurements were carried out with a Jenway 4510 conductivity meter using a DMSO solvent. A Capillary apparatus was used to determine the melting points of the compounds.
2.2.2 Synthesis of sodium rosmarinate (NaR)
Rosmarinic acid (0.5 g, 1.37 mM) was dissolved in 20 mL 1,4-dioxane (dry). Sodium hydride (NaH) was heated to dry (at 80 oC) for 30 minutes and 6 weight equivalents (0.2 g, 8.32 mM) were added to the Rosemarinic acid solution. The mixture was stirred at room temperature for 30 minutes under an inert atmosphere. The reaction mixture was filtered twice and was washed with fresh 1,4-dioxane (2 × 3 mL). The filtrate was evaporated using rotary evaporator (70 mbar, 60 oC) to obtain a thick yellowish fluid. Diethyl ether (20 mL) was added to the round bottom flask which contained the compound and this was stirred for 5 minutes. Beige colored fluffy precipitates appeared, these were filtered and dried under vacuum. Yield: 0.46 g (72%).
2.2.2(a) Characterization of rosmarinic acid and sodium rosmarinate (NaR)
Rosmarinic acid. FT-IR (KBr, ? cm-1) 1727, 1709, 1645, 1616, 1520, 1463, 1356, 1284, 1459, 1231, 1199, 1152, 1111, 1074, 979, 817. 1H NMR (500 MHz, DMSO-d6, d ppm): 9.68, 9.21, 8.83, 8.77, 7.50 (d, J = 15 Hz), 7.08, 7.03 (dd, J = 8 Hz), 6.80 (d, J = 8 Hz), 6.70 (s, 1H), 6.85 (d, J = 8Hz, 1H), 6.54 (dd, J = 8 Hz, 1H), 6.28 (d, J = 16 Hz, 1H), 5.06 (q, 1H), 2.90-3.02 (m, 2H). 13C NMR (125.1 MHz, DMSO-d6, d ppm): 170.8, 165.9, 148.5, 145.9, 145.5, 144.8, 143.9, 127.2, 125.3, 121.6, 120.0, 116.6, 115.7, 115.3, 114.8, 113.2, 72.7, 36.0.
Sodium rosmarinate (NaR). Beige colored fluffy precipitates appeared, were filtered and were dried under vacuum. Yield: 0.46 g (72%). FT-IR (KBr disc): 2857, 2921, 2956 (C-Haliph stretch), 1693 (C=O), 1630 (C=Cnon-arom), 1602 (C=Carom), 1524, 1446, 1361, 1265 (CH & CH2 bendings, broad), 1182, 1163, 1116, 1073, 1036, 977. 1H NMR (500 MHz, DMSO-d6, d ppm): 9.68, 9.21, 8.83, 8.77, 7.50 (d, J = 15 Hz), 7.08, 7.03 (dd, J = 8 Hz), 6.80 (d, J = 8 Hz), 6.70 (s, 1H), 6.85 (d, J = 8Hz, 1H), 6.54 (dd, J = 8 Hz, 1H), 6.28 (d, J = 16 Hz, 1H), 5.6 (q, 1H), 2.90-3.02 (m, 2H). X-ray was used to confirm the presence of the sodium ion with rosmarinic acid (Fig. 9).
2.2.2 Synthesis of silver rosmarinate (AgR)
Rosmarinic acid (0.5 g, 1.37 mM) and silver acetate (1.36g, 8.22 mM) were mixed in methanol (100 mL) in a round bottom flask. The mixture was stirred at room temperature for 2 days. The reaction mixture was filtered thrice using a pad of celites (Diatomaceous earth Or SiO2). A Black colored solution was evaporated slowly at room temperature to obtain a black crystalline shiny material.
2.2.2(a) Characterization of silver rosmarinate (AgR)
AgR was recrystalized using diethyl ether and methanol solvent system. Yield: 0.52 g (84%). FT-IR (KBr, ? cm-1) 1696, 1628, 1602, 1520, 1441, 1363, 1256, 1160, 1117, 1074, 977, 853, 813. 1H NMR (500 MHz, DMSO-d6, d ppm): 7.45 (d, J = 15 Hz), 7.06, 7.00 (d, J = 8 Hz), 6.78 (s, 1H), 6.69 (s, 1H), 6.64 (d, J = 8 Hz, 1H), 6.53 (d, J = 8 Hz, 1H), 6.25 (d, J = 16 Hz, 1H), 5.00 (q, 1H), 2.86-3.07 (m, 2H). 13C NMR (125.1 MHz, DMSO-d6, d ppm): 171.5, 166.0, 148.5, 145.6, 145.3, 144.8, 143.8, 128.1, 125.3, 121.3, 119.9, 116.6, 115.8, 115.3, 114.8, 113.2, 73.8, 36.4. X-ray was used to confirm the presence of the silver ion with rosmarinic acid (Fig. 9).
2.2.3 Synthesis of FLVM
Flavomin was synthesized by the reaction between caffeic acid and 1,3-diamine (1:1) at 70 °C which was stirred for roughly 48 h using hotplate magnetic stirrer. The resultant product was isolated and filtered using 1,4 dioxane. The filtrate was evaporated and a black yellow product was obtained. The product was sticky and only dissolved in the DMSO. The product was characterized by NMR and FT-IR.
2.2.3(a) Characterization of FLVM
The synthesis of the designed compound was carried out by reacting caffeic acid with 1,3-diamine at 70 °C. 1H NMR (500 MHz, DMSO-d6, d, ppm); 1.58 (s, H), 1.84 (qnt, 2H), 2.67 (t, 2H), 3.21 (t, 2H), 6.41 (d, J = 7.5 Hz, 1H), 6.62 (d, J = 3 Hz, 1H), 6.81 (d, J = 3.5 Hz, 1H), 7.12 (s, 1H), 7.36 (d, J = 7.5 Hz, 1H), 8.46 (br. s, 1H), 9.28 (br. s, 2H). 13C NMR (125.1 MHz, DMSO-d6, d, ppm); 33.6, 39.4, 41.6, 115.2, 117.4, 120.8, 123.8, 128.9, 141.4, 145.2, 146.1, 171.9. FT-IR (KBr, ? cm-1); 3415 (OH stretch), 3012, 3067 (C-Harom), 2999, 2915 (C-Haliph stretch), 1639 (C=Caliph), 1564 (C=Carom), 1434, 1312 (CH2 bending).
2.2.4 Synthesis of FLVZ
To prepare the imidazole rosmarinate, 2.0 g (29.3 mM) imidazole was mixed with 1.64 g (29.3 mM) of potassium hydroxide powder in a round bottom flask using DMSO. The solution was stirred and refluxed for 3-4 h at 50 °C. The obtained cloudy material (bromo imidazole) was filtered, evaporated and stored for next steps. To obtain the imidazole rosmarinate, sodium caffeate was prepared using 2g (12 mM) caffeic acid and 0.02 g (12 mM) sodium hydride. The reactants were dissolved in the 1,4 dioxane solution and stirred at room temperature for 3 – 4 h. The resultant solution was filtered twice, evaporated and isolated by adding diethyl ether. The filtrate was collected (Na – caffeate). Bromo imidazole and sodium caffeate (1:1) were mixed together in a dual open neck round bottom flask and the flask was setup to pass the nitrogen gas through H2SO4. The mixer solution was stirred at 70 oC for 4 h. The viscous liquid with a tea color appeared and was characterized by NMR and FT-IR.
2.2.4(a) Characterization of FLVZ
FLVZ: 13C NMR (125.1 MHz, DMSO-d6, d, ppm); 30.55, 30.61, 30.71, 30.83, 48.04, 66.28, 119.16, 119.84, 122.38, 134.89. FT-IR (KBr, ? cm-1); 3389 (OH stretch), 3008 (C- Harom), 1832 (C=Caliph), 1564 (C=Carom), 1427, 1288 (CH2 bending).
2.2.5 Characterization of sodium and silver rosmarinate: X-ray spectroscopy
Silver and sodium also were detected using the Tecnai T20 TEM engaged with the INCA energy dispersive x-ray spectrometer at 120 kV. Electron probes at 120kV voltage and 14° angle were used for this spectroscopy and counting time was 100 s.
2.3 Prediction of physicochemical properties
ACD ILAB ver 12.01 and PKCSM (Douglas et al., 2015) was used to determine the ADME and physicochemical properties of the new RA derivatives (NaR, AgR, FLVM, and FLVZ). The scores for oral and CNS activity are shown in supplementary Table S1 and they were set as the optimum value for the new analogues.
The logP (Octanol/Water), logD7.4 (Octanol/Water) for aqueous solubility and intrinsic aqueous solubility (logS), solubility at pH 7.4 (logS7.4), absorption, BBB penetration, P-gp transport, hERG inhibition, plasma protein binding and toxicity of the compounds were predicted before being tested experimentally. The computational program PKCSM version 1.0 was used to predict these properties. The prediction of efflux ratios for BBB was calculated using previously described algorithmic models (Khan et al., 2016).
2.4 Prediction of binding interaction with IL17A, VEGF and P-gp
The IL17A, VEGF and p-gp binding affinity and efficiency of the compounds were predicted using the LeadIT program windows version 2.1.9. The “flexible docking calculation” was used to predict the inhibitory potential of FLVM and FLVZ towards VEGF and IL17A. The binding activity and anti-angiogenic efficiency of the ligand with VEGF and IL17A were assessed using LeadIT ver 2.1.9 (http://www.biosolveit.de/LeadIT/). 3D crystal structures of VEGF and IL17A proteins were downloaded from the RCSB database (PDB code: 3QTK and 4HR9 respectively). The active sites of VEGF and IL17A were predicted using DoGSiteScorer (Volkamer et al., 2012). For VEGF, 19 pockets were detected and the most active pocket P0 was selected based on its high score of volume [ų], surface [Ų], lipo surface [Ų], depth [Å], and simple score (2280.96, 2431.33, 1459.52, 27.59, and 0.64, respectively) relative to other pockets. Similarly, for IL17A, these scores were 1038.40, 1096.81, 713.19, 20.98, and 0.67, respectively. Chains B, C, E, and F of VEGF and A and B of IL17A were selected for the docking study. The ligand-protein interaction pose with lowest binding affinity was selected as the optimum effect of the compounds. The binding efficiency of the inhibitors was measured using hydrogen and desolvation (Hyde) energies. Molecular docking of the 3D crystal structure of P-gp (PDB code: 3G60) with the new derived compounds was performed using LeadIT. All molecular docking was performed three times. Molecular docking of P-glycoprotein (PDB code: 3G60) was performed with the new derived compounds.
2.5 Quantitative structure activity relationship (QSAR) and structure activity relationship (SAR)
QSAR of the compounds was determined using 37 training set compounds and 4 test set compounds using BuildQSAR ver 2.0. The QSAR was assessed by MLR analysis. To identify the mechanism by which these compounds (NaR, AgR, FLVM and FLVZ) act in cells, SAR was determined from their structure and chemical substituents. SAR was obtained from the molecular docking interactions between the substituents and the target molecule.
2.6 In vitro physicochemical properties
2.6.1 Thermal and chemical stability
The compounds were treated at 60 °C in an oven for 48 h within a glass chamber and incubated with LiOH (Khan et al., 2016a). The purity of the resultant compound was then determined by HPLC. To test chemical stability, the compound was mixed with aqueous HCL (pH 2.0) and Krebs-Heneseleit bicarbonate buffer (pH 7.4) at 37 °C for 2 h and again the purity of the resultant compound was detected by HPLC.
2.6.2 Stability in Human Serum
The compounds were mixed with the human serum (Sigma), (preheated at 37°C) with the resulting concentration of 0.5 mM (Khan et al., 2016a). The mixer solution (1000 µL) was then collected in a 2 ml tube. This serum solution was deprotonated using methanol (1 ml) and vortexed to mix properly. The solution was then centrifuged for 10 min at 10,000 rpm and filtered through a 0.45 µm PTFE filters (Biofil). The purity of the filtered compound was then determined by X-ray spectroscopy and RP-HPLC on a Agilent 1200 series coupled to a photodiode array detector (Agilent, CA, USA). The compound was quantified by the area of the peak, UV profile and retention time.
2.6.3 Ionization Constants (pKa) and Lipophilicity (logD 7.4)
The ionization constants of the compounds were determined by potentiometric titration with the GlpKa apparatus (Sirius Analytical Instruments Ltd, Forest Row, East Sussex, UK). The ionization constants (pKas) were measured by at least four separate titrations for each compound: different aqueous solutions (ionic strength adjusted to 0.15M with KCl) of the compounds (20 mL, about 1 mM) were initially acidified to pH 1.8 with 0.5 N HCl; the solutions were then titrated with standardized 0.5 N KOH to pH 12.2. All titrations were performed under an N2 atmosphere and at controlled temperature (25.0 ±0.1°C).
The partition coefficient for the neutral (un-ionized) form of the compounds (log P) and the distribution coefficient at physiological pH (log D7.4) between n-octanol and water were obtained using the shake-flask technique at room temperature. In the shake-flask experiments HCl 0.1N and phosphate 50 mM buffers (pH 1.0 and 7.4 respectively) were used as aqueous phases. The ionic strength was adjusted to 0.15 M with KCl. The organic (n-octanol) and aqueous phases were mutually saturated by shaking for 4 h. The compounds were solubilized in the buffered aqueous phase at a concentration of about 0.1 mM and an appropriate amount of n-octanol was added. The two phases were shaken for about 20 min, by which time the partitioning equilibrium of solutes is reached, and then centrifuged (10000 rpm, 10 min). The concentration of the solutes was measured in the aqueous phase by UV spectrophotometer (UV-2501PC, Shimadzu). Each log P or log D value is an average of at least six measurement. All the experiments were performed avoiding exposure to light. The concentrations of FLVM and FLVZ were determined by our validated HPLC method.
2.7 Antioxidant properties
2.7.1 DPPH radical scavenging assay
The free radical quenching activity of the compounds and ascorbic acid (standard reference) were determined by the DPPH (Khan et al., 2013). The compounds were mixed with methanol (methanol:water as 1:1) and DPPH (100 µL, 200 µmol L-1) and added in the mixer solution at room temperature for 30 min. The UV absorbance of the compounds were recorded at 517 nm and the EC50 was calculated from the linear regression of the plotted percentage inhibition (I%) of below equation 2.3,
I%=(1- AsampleAblank )×100
……………………….(2.3)
where, Ablank is the absorbance of the control reaction (containing all reagents except the test material).
2.7.2 FRAP assay
The antioxidant activity of the compounds was determined by the FRAP (Ferric reducing antioxidant power) (Benzie and Strain, 1996). FRAP working solution (300 mmol L-1 acetate buffer, pH 3.6, 10 mmol L-1 TPTZ in 40 mmol L-1 HCl and 20 mmol L-1 FeCl3 in a ratio of 10:1:1) and compounds were mixed and kept for 8 min. The absorbance of the compounds was recorded at 600 nm using a Tecan microplate reader and the antioxidant activity was calculated as nmol Fe2+ equivalent amount of compound (µg) from the standard curve of ferrous sulfate (FeSO4.7H2O) (reference standard).
2.8 Cell based screening
2.8.1 Cell culture
MDCK, MDCK-MDR1 lines were collected from NIH, USA. Other lines of R28 were purchased from Kerafast, USA, and CCD 18co, HCT 116 and human endothelial cell line EA.hy926 were bought from the American Type Culture Collection (ATCC, Manassas, VA, USA) and human glioblastoma cell line U87 MG and DBTRG MG (multiple patient-derived cultures) were collected from the school of medicine, USM, Kelantan and cultured in Dulbecco’s modified eagle medium (DMEM) (ATCC, USA) supplemented with 10% heat inactivated fetal bovine serum (HIFBS) (GIBCO), 100 units/mL penicillin–streptomycin (GIBCO), and 2 mM glutamine (GIBCO) in a humidified incubator with 5% CO2 at 37 ?C. HCT 116 cells were cultured in RPMI media with different contents of above supplemental (growth factors, serum etc). Rosmarinic acid (RA) and Avastin were purchased from Sigma (Germany). MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), trypsin, fibrinogen, aprotinin, phosphate buffered saline (PBS), and thrombin also were purchased from Sigma. Matrigel matrix (10 mg/mL) was obtained from BD Bioscience (USA). About 10 mg of the compounds (96% purity) was dissolved in sterile, cell-cultured tested DMSO (0.1%) (Sigma, USA) to prepare a 10 mg/mL stock solution, which was stored in a moisture controlled environment at RT. Other chemicals used were of analytical grade.
The human cell lines were cultured in DMEM medium (ATCC, USA) supplemented with 10% heat inactivated fetal bovine serum (HIFBS) (GIBCO, USA), 100 units/mL penicillin–streptomycin (GIBCO), and 2 mM glutamine (GIBCO) in a humidified incubator with 5% CO2 at 37 ?C. Rosmarinic acid (RA) and Avastin were purchased from Sigma (Germany). MTT, (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), trypsin, fibrinogen, aprotinin, phosphate buffered saline (PBS), and thrombin were purchased from Sigma, Germany. Matrigel matrix (10 mg/ml) was obtained from BD Bioscience, USA. Roughly 10mg of 96% pure RA powder was dissolved in sterile, cell-cultured tested dimethyl sulfoxide (DMSO) (0.1%) (Sigma, USA) to prepare 10 mg/ml stock solutions which were stored in moisture controlled environment at room temperature. Other chemicals used were in analytical grade.
2.8.2 Cell viability assay
The human umbilical vein endothelial cell line EA.hy926 was used to screen the effects of compounds. MTT assay was used to perform the proliferation assays (Khan et al., 2016a). The human gliobalstoma multiforme type IV cell line U87 MG was used to measure the anti-proliferative activity of the compounds. The effects of the compounds on IL17A production (1.56, 3.12, 6.25, 12.5, 25, 50 ng/ml) and on the proliferation of cancer and normal cell was also investigated. The study was conducted on 70% confluent cells seeded in the 96 wells plate. Cells were treated with six concentrations (6.25, 12.5, 25, 50, 100, and 200 µg/mL) of the compounds including Avastin (standard drug) in triplicates for 72 h at 37 °C and 5% CO2. The MTT salt (20 µl of 5 mg/ml stock solution) was added after 72 h and again incubated for 4 h. The media of each well was discarded after 4 hours and DMSO (0.1%, 100 µl) was added in each well. The absorbance of the samples was recorded at 490 — 570 nm and the cell viability activity IC50 was calculated by following equation 2.4,
Inhibition=1-absorbanceoftreatedabsorbanceofuntreated×100
………………………….(2.4)
Proliferation=absorbanceoftreatedabsorbanceofuntreated×100-100
…………………(2.5)
The % inhibition was plotted against the concentration tested using Microsoft Excel, and the IC50 was calculated using the regression analysis.
2.8.3 ROS activity
ROS reagent (fluorescent dye i.e. 2’,7’-dichloro fluorescein diacetate (DCFH-DA) was used to determine the ROS expression in U87 MG cells. The expression and/or production level of the ROS was analysed on U87 MG cells according to previously described studies and details were described (Mohan et al., 2012). U87 MG cells were seeded in a 48 well plate for 2 days. At 80% confluence cells were treated with FLVM and FLVZ for 24 hour. Cells were washed with PBS and 100 µl ROS reagents (fluorescent dye i.e. 2’,7’-dichloro fluorescein diacetate (DCFH-DA) was added in each well. Cells were incubated at 37°C for 30 min. Cell lysis buffer was added to the cells after the incubation period and centrifuged at 10,000 rpm for 10 min. Supernatant was collected and diluted with PBS and measured in a Tecan fluorescence microplate reader at 485 to 520 nm. Standard curves using a ROS standard were used to calculate the ROS concentrations in each experimental sample.
2.8.4 Ex vivo BBB permeability assay
BBB permeability of compounds was determined using MDCK-MDR1 cells (passage number 2, obtained from NIH, USA), which were seeded onto 0.33 cm2 polycarbonate filters at a density of 60,000 cells/cm2 and maintained in culture in 24-well plates according to a previously described protocol with slight modifications (Wang et al., 2005; Taub et al., 2005). Cells were grown for 3–4 d to become a confluent monolayer that expressed the P-gp after seeding, and their integrity was determined by measurement of the transepithelial electrical resistance (TEER, ohm square centimeter) with a volt-ohm meter (Millicell-ERS, Millipore Corporation, Billerica, MA, USA). The background TEER, that is to say the resistance of the filter alone was subtracted, and only the cell monolayers with TEER > 1,000 Ocm2 were used (pre and post study). Cells were cultured with HBSS (Hank’s balanced salt solution containing millimolar Hepes buffer, pH ~7.4) at 37 °C, which was incubated for 30 min with warmed HBSS. At first, HBSS was added to the receiver compartments and the compounds were independently added to the donor compartments at a concentration of 10 mg/mL DMSO (0.1%). Samples were collected from both the receiver and donor compartments after 90 min of incubation. Drug permeation across the monolayer was measured in both apical to basolateral (A–B) and basolateral to apical (B–A) directions. The apparent permeability, Papp (in cm s–1) was calculated as following equation 1:
Papp= dQdt ×1 (A×Co)
…………(S1)
where dQdt
is the transport rate of the compound (moles per second), A
is the area of the cell monolayer (centimeter square), and Co
is the initial donor concentration (moles per liter).
2.9 Anti-angiogenic activity
2.9.1 Migration Assay
70% confluent EA.hy926 and U87 MG cells were used for the migration assay. A scratch wound was created in the middle of the well using a 0.1 ml pipette tip. After scratching the cell monolayer, detached cells were washed gently by PBS. The cells were treated with 4 concentrations (i.e. 2IC50, IC50, 1/2 IC50 and 1/4 IC50) of NaR, AgR, FLVM and FLVZ based on the IC50 for 24 h. Avastin was used as standard reference. The percentage of inhibition of migration was calculated by measuring the gap (in Image J soft) of the scratch at 0h, 12h and 24 h on the photomicrograph. The percentage of inhibition was calculated relative to zero time using the equation below (3.1) and the results are displayed as average ± SD, (n = 6).
%inhibition (migration)=1-the width at the th hourthe width at zero time×100
……………………..(3.1)
2.9.2 Capillary-like tube formation assay
The 3D collagen (Matrigel, BD Bioscience, USA) with growth factors was used to form the tubes of the human umbilical vein endothelial cells EA.hy926. Matrigel (100 µl) was seeded in 48 well plates and incubated for 30 min at 37 ?C. NaR, AgR, FLVM and FLVZ treated EA.hy926 cells (2IC50, IC50, 1/2 IC50 and 1/4 IC50 doses) were seeded into this matrigel. The network formation of the tubes was then monitored under a microscope after 16 h. Because of the effect of the new compounds, EA.hy926 cannot form the tubes and capillary network related to control. The branch points, number of tubes, covered area by cells were calculated from the web-based Image Analysis Win-Tube module of Wimasis software (Wimasis GmbH).
2.9.3 Rat aorta ring assay
Male Sprague Dawley Albino rats (180-220 mg, 6-8 weeks old) were collected from the USM animal house to perform rat aortic ring assays. Experiments were conducted according to animal ethics guideline approved by Animal Ethics Committee, Universiti Sains Malaysia(USM), Reference Number: USM/Animal Ethics Approval/2015/(658).
Aortic ring were used as angiogenic assay (Nicosia et al., 1990). Thoracic aorta were collected from male Sprague Dawley rates (12 – 14 weeks old). The cleaned aorta (removing fat tissue) was cut into small pieces and put in the membrane coated (M199 media supplemented with 3 mg/ml fibrinogen, 1 mg/ml aprotinin and 200 µl L-glutamin) 48 well plate. After seeding, the plate was incubated for 1 hour at 37 °C. Thrombin (10µl), (50 NIH U/ml in 1% bovine serum albumin in 0.15 M NaCl) was added into each well and reincubated at 37 °C for 1 hour to solidify. After forming the gel, the rings were treated with the NaR, AgR, FLVM FLVZ and Avastin at six concentrations (6.25, 12.5, 25, 50, 100 and 200 µg/ml) prepared in M199 media supplemented with 20% HIFBS, 0.1% 6-aminocaproic acid, 1% L-glutamine, 1% amphotericin B, and 0.6% gentamicin. The old media was discarded and rings were treated again at day 3. The length of the vessels outgrowth from the rings (primary tissue explants) was measured using Image-J software. The formation of vessel was affected by the new derivatives and percentage inhibition (IC50) was calculated using a non-linear regression analysis as below equation:
% Inhibition (vessel)=1-AoA×100
Where, Ao = distance of blood vessel growth in treated rings in µm and A = distance of blood vessel growth in the control in µm.
2.9.4 CAM Assay
Chicken embryos at 5 – 7 days old were used in this assay following previously described methods with slight modification (Dohle et al., 2009). Eggs were wiped with sterile rough tissues to remove the feathers and dirt. The eggs were positioned vertically (towards long axis) and marked on the the wide upper side with a pencil since embryos are located in this area. The egg was then cracked by hitting this area (marked side). The shell was then delicately removed to make sure the egg (yolk and egg white) remains undamaged. The eggs were incubated at 37.5 °C and 60% humidity in the presence of sufficient O2 supply. The new derivatives were applied on egg blood vessels at two concentrations (IC50 and 1/2 IC50). The eggs were monitored for 7 days and the inhibition of blood flow and affected vessels were observed by analysis of photomicrograph and the marked region.
2.10 IL17A and VEGF ELISA assay
The effect of NaR, AgR, FLVM and FLVZ against the expression of IL17A and VEGF (DuoSet ELISA Kit R&D Systems, USA) was assessed by sandwich ELISA assay in the U87 MG cells. 70% confluent U87 MG cells were treated for 48 h with NaR, AgR, FLVM and FLVZ. The cells were then lysed using 1 ml lysis buffer. The lysates were centrifuged and supernatant was collected and placed into the antibody-coated 96 well plate. The rest of the procedure was followed according to the instruction manual. The level of expression of the protein was calculated in comparison to a standard curve of IL17A and VEGF.
2.11 Apoptotic activity
2.11.1 Determination of nuclear condensation by Hoechst 33258 stain: 20,000 cells were seeded per well in 48 wells plate. After 48 hr , when 70-80% confluence was reached, U87 MG glioma cells were treated with the compounds. Cells were fixed by adding 4%, paraformaldehyde in each well and incubated for 20 min. After incubation, the cells were washed with PBS and 100 µl of hoechst 33258 stain (1 µg/ml in PBS) was added. After 30 min of incubation, cells were observed under an inverted microscope to determine cell morphology. Cells with bright condensed nuclei and shrunken cytoplasms indicative of apoptosis were observed. These apoptotic cells were counted in four fields per well. The percentage of apoptotic cells were determined by following equation:
% Apoptotic index= number of apoptotic cellstotal number of live cells ×100
2.11.3 Determination of mitochondrial potential: U87 MG cells were used to study the mitochondrial inhibitory potential of the compounds. Cells were treated as described in a hoechst staining assay. The only difference is that rhodamine 123 (5µg/ml PBS) was added prior to the hoechst. Definition and calculation of mitochondrial potential condition are similar to nuclear condensation as unhealthy mitochondria appear bright and shiny with irregular shapes.
2.12 Effect of RA derivatives on apoptotic targets of Caspase 3/ 7, 8, 9
The assay was carried out according to manufacturer’s instructions (Promega, USA). The cells were seeded in 96 well plates at 20 x 104 cells/well in 200 µl medium and incubated for 24 hr to allow the attachment of cells. The treated cells were incubated for approximately 18 hr. Then, 100 µl of the caspase reagent containing cell lysis buffer and specific luminogenic substrate for caspase 3/7, 8 and 9 was added to each well. After incubating the plates for 30 min, the luminescence intensity was measured using microplate reader (Tecan, Switzerland). The effect of the compounds on caspase activity is represented by fold change upon treatment with NaR, AgR, FLVM and FLVZ. The fold changes in the caspase activity were measured by the following formula:
Fold change= Ls- LbLc- Lb
where: Ls=
Luminescence reading of wells treated with tested extract
Lb=
Luminescence reading of blank
Lc=
Luminescence reading of wells treated with vehicle
The results were stated as mean of fold changes in caspase activity in comparison to control ± SD (n=3).
2.13 In vivo detection of compounds in mice blood and brain
BALB/c male mice weighing 20-28 g were used for this experiment. Mice were treated with 50 mg/kg of NaR and AgR by oral gavaging of 500 µL of compound dissolved in 0.1% DMSO. After 2-4 hours, animals were euthanized and sacrificed to collect the blood and brain. Blood was collected by syringe from a heart punch into a test tube. The brain was removed immediately and rinsed with cold distilled water and homogenized at 4?C in an equal volume of water in respect to brain weight. After homogenization with a the Potter-Elvehjem homogenizer, an equal volume of acetonitrile containing 0.1% acetic acid was added to blood samples and brain tissue homogenates to precipitate proteins. The samples were sonicated, vortexed, and then centrifuged for 10min at 2150g. The clear supernatant was filtered by 0.45 µm PTFE filters and analyzed by x-ray energy spectroscopy and HPLC.
2.14 Luciferase reporter gene assay (10 cancer pathways)
Luciferase gene reporter assay was conducted following Vikram et al. 2012. In brief, U87 MG cells were transfected using Trans Fact liposome transfection reagent (Promega, USA). Cells were incubated overnight and treated with the compounds Na+ and Ag+ salts. After 24 h, the luciferase reporter system was used to detect luciferase activity in a luminometer. Afterwards, firefly/renilla activity was determined in a luminometer. The fold change was calculated from the luminescence reading as following:
Fold Change= TfireflyTrenillaCfireflyCrenilla
Where, T = treated and C = control
2.15 HPLC
The compounds were detected by HPLC. An HPLC analytical method was developed on Agilent 1200 series coupled to a photodiode array detector (Agilent, CA, USA). Chromatographic separation was achieved at room temperature (25?C) with a C4 reversed-phase column (Thermo Scientific™, USA 250×
4.6 mm, 5-µM particle size) using a gradient solvent system that consisted of a mobile phase of acetonitrile and 0.05% formic acid, 50:50 (v/v), pH 5, running at an isocratic mode at flow rate 1.0 mL/min. The injected volume per sample was 100 µl and the total run time was 15 min. The absorbance was set at 200 – 450 nm and the UV measurements for quantification were performed at 220 nm. Quantification was carried out using HPLC retention time and UV spectrum of the analyte. The method was specific and sensitive with a lower limit of quantification of 1 ng/mL and the ChemStation LC3D software was used to ensure quantification of compounds. The variation of intraday and interday was minimal < 10%.
2.16 Statistical analysis
Microsoft excel 2013 and GraphPad prism V6.02 were used for statistical analysis. All data were evaluated with one-way ANOVA multiple comparisons test. A p value <0.05 was considered statistically significant. Data are reported as mean ± SEM.