Synthesis NSAID Triazolothiadiazine derivatives
Synthesis of 4-amino-3-substituted-1,2,4-triazole-5-thiones (Compounds 1-8), was carried out by melting some aralkyl carboxylic acid derivatives with thiocarbohydrazide according to the procedure described previously [25,29]. Synthesis of 3,6-disubstituted-7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines (1a-8c) was carried out by reacting relevant 4-amino-3-substituted-1,2,4-triazole-5-thiones (1-8) with appropriate phenacyl halides in anhydrous ethanol under reflux, 3,6-disubstituted-7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines (1a-8c) [25,29]. Physical properties and spectral data characterizing the structure of the synthesized compounds were reported previously [24,25].
Drugs and Chemicals
JNK inhibitor (cat. # 420119) was provided from Calbiochem, Sorafenib (cat. # S7397), and DAPT (cat. # S2215) was provided from Selleck chemicals, Taxol was provided from (Bristol Myers Squibb). Camptothecin was purchased from Sigma (cat. # C9911).
Nine hepatocellular carcinoma cell lines, and one colon carcinoma cell line were obtained from the following sources: Huh7 (JCRB0403), HepG2 (ATCC HB-8065), Hep3B (ATCC HB-8064), PLC (ATCC CRL-8024), SK-Hep1 (ATCC HTB52) Mahlavu , FOCUS , SNU182 (ATCC CRL-2235), SNU387 (ATCC CRL-2237), SNU475 (ATCC CRL-2236) and, HCT116 (ATCC CCL-247). Cells were grown in DMEM, (Dulbecco's Modified Eagle Medium) or RPMI (Roswell Park Memorial Institute)-1640 growth medium supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acids, 1% L-glutamine, 1% penicillin and streptomycin (GIBCO, Invitrogen) at 37°C under 5% CO2. All cell lines used in this study are STR (short tandem repeat) authenticated. Cells are regularly tested for mycoplasma contamination using mycoplasma detection kit (MycoAlert™, Lonza). The passaging of the cells did not exceed 8-10 passages (2 times a week) throughout the experiments.
Sulforhodamine B (SRB) assay
Cells were inoculated (1000-5000cell) for 24h and treated with the increasing concentrations of compounds (40-0.01µM) for 72h. Cells were washed, and plates were stained with SRB (Sigma Aldrich) as described previously . The absorbance values were obtained at 515nm using a plate reader (ELx800, BioTek).
Real-time cell growth analysis (RT-CES)
Cells were plated on to E-96 plates cells (1000-5000 cells/well). Cells were treated with the compounds, and cell index (CI) values were recorded (RT-CES, xCELLigence, ACEA Biosciences) and analyzed as described previously . Cell growth curves were generated using the time-zero normalized CI values.
Cell cycle analysis by flow cytometer
HCC cells were inoculated in 100mm culture dishes. After 24h, cells were treated with compound 7b and its DMSO control. 24h later, cell pellets were collected and re-suspended in 1 ml, ice-cold 1xPBS, and fixed by adding 2.5mL, 70% ice-cold ethanol. Next, the cell pellets were re-suspended in Propidium iodide (PI) solution (50µg/mL PI (Sigma Aldrich), 0.1µg/mL RNaseA (Fermentas), 0.05% Triton-X-100, 1xPBS) and incubated for at 37 oC for 40 minutes at dark. Cell cycle analysis was performed using FACSCalibur (BD Biosciences) and CellQuest Software (Becton Dickinson).
Detection of ER-stress
Total RNA was isolated from cells via the Nucleospin RNA II kit (Macherey-Nagel) according to the manufacturer’s protocol. First strand cDNA synthesis was performed using RevertAid First Strand cDNA synthesis kit (Thermo Scientific). Semi-quantitative reverse transcriptase PCR (RT-PCR) assay was performed using XBP1 specific primers. The primer sequences; GAPDH forward primer: GGCTGAGAACGGGAAGCTTGTCAT, GAPDH reverse primer: CAGCCTTCTCCATGGTGGTGAAGA, XBP1 forward primer: TTACGAGAGAAAACTCATGGCC, XBP1 reverse primer: GGGTCCAAGTTGTCCAGAATGC.
Reactive Oxygen Species (ROS) detection
Huh7 and Mahlavu cells were seeded into 10 cm culture dish (100,000-300,000 cells/dish). Next day, cells were treated with compound 7b, DMSO or Selenium (Se) deficient serum-free media for 8, 12 or 24h. Flow cytometric analysis of cells for ROS induction at 12h was done with the Oxidative Stress kit (MCH100111, Merck Millipore) using MUSE™ Cell Analyzer. In parallel, cells were also visualized under a fluorescence microscope (Nikon Eclipse Ti-E), which were incubated with ROS assay solution (10 mM HEPES buffer, 10mM glucose, 1 µM DCFH-DA (Dichloro-dihydro-fluorescein diacetate) in 1 x PBS). Se-deficient serum-free medium was used as the positive control .
Western Blot analysis
HCC cells were treated with increasing concentrations of compound 7b (+: IC50, ++: 2 x IC50) or with DMSO control. After 24h, cells were harvested by scraping, washed with PBS, lysed with RIPA lysis buffer on ice and centrifuged at 13000 rpm for 20 min. The supernatants were collected, and the protein concentration was measured using Bradford assay. For SDS-PAGE, 20-50 µg of protein was prepared, and samples were run using the Novex® NuPAGE® Bis-Tris Electrophoresis system according to the manufacturer’s protocol. Transfer of proteins to nitrocellulose membrane was done via XCell IITM Blot Module. The blots were incubated with primary antibodies against PARP-1 (Santa Cruz Biotechnology Cat# sc-8007, 1:1,000 dilution), SAPK/JNK (Cell Signaling Technology Cat# 9252, 1:300 dilution), Phospho-SAPK/JNK (Thr183/Tyr185) (Cell Signaling Technology Cat# 9251, 1:200 dilution), phospho-p38 (Thr180/Tyr182) (Cell Signaling Technology Cat# 9211S, 1:500 dilution), phospho-c-Jun (Santa Cruz Biotechnology Cat# sc-822, 1:200 dilution), phospho-ASK1 (Thr845) (Cell Signaling Technology Cat# 3765, 1:500 dilution), phospho-ASK1 (Ser966) (Genscript, Cat# A00340, 1:500 dilution), phospho-ASK1 (Ser83) (Abcam Cat# ab47304, 1:500 dilution), phospho-MKK7 (Ser271/Thr275) (Cell Signaling Technology Cat# 4171, 1:500 dilution), phospho-MKK4 (S257 + T261) (Abcam Cat# ab4760, 1:300 dilution), calnexin (Sigma-Aldrich Cat# C4731, 1:5000 dilution), and actin (Santa Cruz Biotechnology Cat# sc-1616, 1:5000 dilution) in 0.1% TBST at 4°C overnight, followed by secondary antibody incubations with HRP-conjugated goat anti-mouse IgG (Sigma-Aldrich Cat# A0168, 1:5000 dilution), rabbit anti-goat IgG (Sigma-Aldrich Cat# A8919, 1:5000 dilution), or goat anti-rabbit IgG (Sigma-Aldrich Cat# A6154, 1:5000 dilution) for 1h at room temperature. Proteins were visualized by using the enhanced chemiluminescence (ECL) system.
In vivo mouse xenograft experiments
All animals received human care, and study protocols comply with the institution's guidelines. Animal ethics committee of Bilkent University approved the study protocol. In addition, all studies were reported in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. Mahlavu cells prepared in DMEM (10,000,000 cells/mouse) were injected subcutaneously (SC) to the flank of 8-16 weeks old male nude mice as described previously . Drug treatment was initiated once the tumor volume reached 150mm3. The subjects received compound 7b (100 mg/kg) in simple syrup (16 g glucose in 9 g ddH2O) by oral gavage, and the control group mice received 100µl simple syrup only twice a week for 21 days. Nude mice were not treated for the following 3 weeks and imaged with Magnetic Resonance Imaging (acquired with 3-TESLA Siemens MAGNETOM Trio, UMRAM Center, Bilkent University) following intraperitoneal (ip) injection of anesthesia regimen consisting of 10 mg/kg xylaxin and 90 mg/kg ketamine.
Detection of liver cancer stem cell (LCSC) enrichment by flow cytometry
Huh7 and Mahlavu cells were seeded onto 100mm culture dishes. The next day cells were treated with compound 7b, sorafenib, DAPT, or DMSO control at their IC50 concentrations and with different combinations of sorafenib and compound 7b. Fluorescence labeling of LCSCs was done using primary antibodies against CD133 (Miltenyi Biotec Cat# 130-090-664), and anti-biotin-PE (Miltenyi Biotec Cat# 130-090-756), EpCAM (Miltenyi Biotec Cat# 130-080-301), or CD90 (Miltenyi Biotec Cat# 130-095-403) for flow cytometry analysis as described previously . Mouse-IgG-FITC (Miltenyi Biotec Cat# 130-092-213), and mouse-IgG-biotin antibodies (Miltenyi Biotec Cat# 130-093-018) were used as isotype controls. DAPT (Notch pathway inhibitor) was used as a positive control for cancer stem cell inhibition. Results for each treatment group were compared to that of DMSO control. Changes in the positivity of CD133+/EpCAM+ cells or CD90+ cells were indicative of enrichment or reduction of the LCSC population. BD Accuri C6 and Novoctye flow cytometer (ACEA Biosciences) were used for flow cytometric analysis.
Sphere formation assay
Sphere formation was triggered as described previously  in ultra-low attachment 96-well plates. Images of spheres and measurements of sphere size and number were assessed using light microscopy (Zeiss) after 6-12 days of incubation.
Cell migration assay
To test the effect of compound 7b on the migration capacity of Huh7 and Mahlavu cells, RT-CES DP system was used. The lower chamber of the 16 well CIM-plate was filled with 160 µl of 10% FBS containing complete DMEM, and the upper chamber was placed on top of the lower chamber. After 1 hour at 37°C, cells that were prepared in the presence of different concentrations of compound 7b (2 µM for Huh7 and 8 µM for Mahlavu), Taxol (20ng/ml) or DMSO inside serum-free DMEM, to be seeded into the upper chamber (30000-50000 cell/well). CIM-plates were placed into the system after 30 min of incubation at room temperature (RT), and CI values were obtained every 15 min for 24 hours. Time-zero normalized CI values were used to generate time-dependent migration curves for each experimental group.
Data were obtained from three independent experiments, and all experiments were carried out with n≥3 biological replicates. Statistical analysis for in vitro data was done using a Student's t-test. All in vivo experimental data were analyzed using ANOVA, n=5-6 mice/group (Graphpad Prism version 7.0, or Microsoft Excel). * p<0.05, ** p<0.01, *** p<0.001.