2.1 Cell Culture: MDA-MB-231, MCF7, T47D and HEK-293 cells were purchased from the National Centre of Cell Culture (NCCS), Pune, Maharashtra, India. MDA-MB-231 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM high glucose with L-glutamine; Lonza, Houston, Texas, US), MCF7 and HEK-293 in Eagle's Minimum Essential Medium (EMEM; Lonza supplemented with non-essential amino acids (NEAA) from MP biomedicals, Santa Ana, CA, USA), and T47D in Roswell Park Memorial Institute-1640 (RPMI; Lonza, Houston, Texas, US) media. The media were supplemented with heat-inactivated 10% fetal bovine serum (Gibco,Thermo Fisher Scientific, Massachusetts, US), 100 IU mg/mL penicillin/streptomycin (Gibco,Thermo Fisher Scientific, Massachusetts, US) and cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. ST09 was dissolved in dimethyl sulfoxide (DMSO, MP Biomedicals, Santa Ana, California, USA) such that all treatments had equal concentrations of DMSO between 0.1–0.2%. Cisplatin (MP biomedicals, Santa Ana, California, USA) was dissolved in water.
2.2 RNA preparation and HiSeq2500 Sequencing: Drug Treatment, RNA isolation, and Library preparation: As described(11)(8), for the RNA-seq experiment, 0.75 x 10^6 cells were seeded in each well of a 6-well plate. MDA-MB-231 cells were then treated with 50nM ST09, MCF7 and T47D with 100nM of ST09. The mRNA and miRNA libraries were prepared as described(11)(8).
2.3 Differential expression analysis:
2.3.1 mRNA-seq, miRNA-seq: Data analysis was carried out as described(11)(8), beginning with filtering raw reads output from the Illumina Hiseq2500 platform.
2.3.2 Enrichment and Pathway analysis for mRNA-seq data: As described(11)(8), clusterprofileR(12) was used for pathway analysis and the significant pathways were considered based on P-value.
2.4 Integrated analysis of mRNA-miRNA: miRtarvis+(13,14) and miRmapper(15) tools were used for studying the interaction between mRNA-miRNA. Every miRNA target was verified further using TargetScan(16) and miRDB(17).
2.5 Target validation and Transfection of miR-mimic 197a-5p,siRNA GPX3: The validation of GPX3 as a target of miR-197-5p was performed using TargetScan(18) and miRDB(17) (Supplementary Figure 2). miR-mimic for miR-17a-5p was purchased from Applied Biological Materials (ABM) Inc.(Vancouver, Canada), siRNA for GPX3 was purchased from Santa Cruz Biotechnology(Dallas, Texas, United States) and anti-miR for miR-197-5p was purchased from Thermo Fisher Scientific, Massachusetts, US. 100µM stocks were prepared for all with nuclease free water. The working concentration for miR-mimic was optimised to 75nM and 50pM for siRNA.Whereas, 1 and 2.5nM of anti-miR was used in combination with 20,40nM of ST09. Briefly, 0.75x10^6 MDA-MB-231 cells/ml were seeded in 6 well plates and treated with ST09 for 24h. Transfection was done using Oligofectamine and Opti-MEM. 48h after transfection, cells were collected for LDH and western blot assays for mimic, siRNA experiments. MTT assay was performed for anti-miR experiment.
2.6 Real time PCR: Real time PCR was done for samples using iTaq™ Universal SYBR® Green Supermix(Biorad,Berkeley, California). Β-actin primers were used as housekeeping genes to normalise mRNA and U3 snRNA for miRNA. All the primers used in the study are tabulated in Supplementary Table 1. The PCR conditions used for amplification are 95℃ 5 min initial denaturation, 40 cycles, 95 C for 20 s, 60 C for 20 s, 72 C for 20 s) followed by a melt curve stage. Comparative Ct(cycle number at which fluorescence crosses threshold level) analysis was used for relative quantification of gene expression(19).
2.7 GPX activity assay: This assay was performed using the Glutathione Peroxidase Activity Colorimetric Assay Kit (Biovision, Cambridge, UK). MDA-MB-231 cells were seeded at a density of 1 x 105 cells/well in a 6-well plate. The following day, the cells were treated with 50 nM ST09 drug for 12 h. ST09 treated and untreated cells were transfected with 75 nM miR-197a-5p mimic (Applied Biological Materials Inc., Vancouver, Canada) using oligofectamine (Thermo Fisher Scientific, Massachusetts, US). Cells were harvested post 12 h, and the assay was performed according to the manufacturer's protocol. The GPX activity was measured for 2 h. A bar graph was plotted for each condition where GPX activity was normalised with its respective protein concentration.
2.8 Plasmid construct, transfection, and luciferase assay: The 3’UTR of GPX3(Supplementary Figure 2) was cloned into the pmirGlo vector downstream of the firefly luciferase gene and denoted pmirGlo-GPX3-3’UTR. Luciferase assay was performed using the dual-luciferase reporter assay system (Promega, Wisconsin, US). Briefly, HEK-293 cells were seeded at a density of 10,000 cells/well in a 96-well plate. Following day, transfection with 1 µg pmirGlo-GPX3-3'UTR alone or in combination with miR-197a-5p mimic (75 nM) was performed in Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific, Massachusetts, US) using Lipofectamine 2000 (Thermo Fisher Scientific, Massachusetts, US). The firefly luciferase activity was measured post 48 h of transfection. The bar graph was plotted with firefly luminescence normalised with the renilla luminescence.
2.9 Phalloidin Staining: Cells were grown on gelatin-coated coverslips and then treated with 20,40nM of ST09 for 48h under culture conditions. Cells were then fixed by adding 4% paraformaldehyde(PFA, MP biomedicals, Santa Ana, California, USA), rinsed three times with phosphate-buffered saline (PBS) and then permeabilized with 0.5% triton X-100-PBS for 15 min at room temperature. 3% BSA(MP biomedicals, Santa Ana, California, USA) was used for blocking. 1 µg/mL of phalloidin-FITC (P5282, Sigma Aldrich, Missouri, US) for 30 min at room temperature was used to label F-actin. Coverslips were then mounted using mounting media(DABCO-glycerol+DAPI 1 μg/mL, Sigma Aldrich, Missouri, US). Samples were then analysed by fluorescence microscope from Nikon under the 40X objective.
2.10 Wound healing Assay: MDA-MB-231 cells (0.75×106 cells/well) were seeded into 24-well plates and scraped with the end of 20 µl pipette tips. The plates were washed with PBS to remove detached cells and then incubated with the complete growth medium containing 40nM and 60nM of ST09 for 24 h. Cell migration was observed under a phase-contrast microscope at 4× magnification after 0 and 24 h post-induction of injury. The ratio of cell migration was calculated as the percentage of the remaining cell-free area compared with the area of the initial scratched area using Image J software version 1.40 (National Institutes of Health, Bethesda, MD, USA). The changes in the open wound area represent the motility of cells across the scratch wound.
2.11 Immunoblotting: MDA-MB-231 cells were incubated with different concentrations of ST09 (0,20,40 nM) for 48h, and western blotting was performed as described(4,5,11). Briefly, the membrane was probed with appropriate primary antibodies NF-KB(Biolegend,San Diego, California, US), Cyclin B1(Cell Signaling Technology,Massachusetts, US), GPX3(Cloud Clone Corp, Houston, Texas, US), followed by probing with the HRP-conjugated secondary anti-rabbit(Cell Signaling Technology,Massachusetts, US).
2.12 Drug combination study by MTT assay: MTT assay was performed as described earlier(4,5). Cells were with various concentrations of Cisplatin (2,2.5 µM) and ST09 (10,20 nM)(Supplementary Table 2). The combination study of Cisplatin with ST09 was carried out as described(20,21).
2.13 Breast Cancer Mice tumor model: The study for the breast cancer tumor model was performed as described earlier(5). The Institutional animal ethics committee (Reg. No. 1994/GO/ReBi/S/17/CPCSEA) approved the study, and all experiments were performed following institutional and national guidelines and regulations of the CPCSEA. 1 x 10^6 Ehrlich ascites breast adenocarcinoma (EAC) cells were injected to induce solid tumors in the left thigh region of Swiss albino mice. After animals had developed tumor of size ~ 300 mm3 the animals were segregated in 4 groups: control(n=5) and ST09 treated(n=5), Cisplatin treated(n=5), Cisplatin+ST09 treated(n=5). The treated group animals were then subjected to 10 doses of 10mg/kg of body weight(bd wt) of ST09, 1mg/kg bd wt of Cisplatin, and ST09(10mg/kg body weight)+Cisplatin(1 mg/kg bd wt) intraperitoneally (i.p) every alternate day. The experiment was repeated twice with five animals and changes in the tumor size and body weight were observed for 20 days from the day of treatment. Tumor volume was calculated as described (8).
2.14 Drug toxicity and side effect assessment on ST09, Cisplatin treatment: As described earlier(8), EAC tumor-induced mice in the treatment group were treated with ST09 for 20 days and then were evaluated for drug toxicity.
2.15 Breast carcinoma xenograft: Nude mice (n = 14) were implanted with MDA-MB-231(5 10^6) cells. The mice were given 14 oral doses of either the vehicle control (control, n = 7) or ST09 (20 mg/kg; treated, n = 7) daily. Changes in the tumor size were observed for 24 days from the day of treatment. Tumor volume was calculated using the formula V = 0.5x a x b2, where V is tumor volume, a, and b are major and minor tumor diameters.
2.16 ST09 bioavailability studies:
2.16.1 Chromatographic conditions: As described (6), the separation and retention of analytes were performed on a Shiseido cap cell pack C18 column (S-5, 4.6 mm × 250 mm). The mobile phase for ST09 was composed of 2% Acetic acid in HPLC grade water (A) and HPLC grade Acetonitrile (B) (40:60, v/v). With 1 mL/min flow rate and 312 nm detection wavelength, 20μL of samples were injected in columns maintained at room temperature. The mobile phase was degassed and filtered through a 0.45 μm membrane filter.
2.16.2 Sample preparation: As described(6),Swiss albino mice were divided into two groups (n = 2) and ST09 was administered intraperitoneally to both groups. The mice in both groups were administered a single dose of ST09 at 20 mg/Kg b.wt and blood collected at different time points (5, 10 and 15 min and 1, 3, 6, 12 and 24 h). As described(6), plasma samples were collected by centrifuging at 1500 rpm for 15 min. Briefly, the analyte was extracted from plasma using protein precipitation procedure. An equal volume of acetonitrile was added to the plasma, vortexed for 2 min, centrifuged at 14,000 rpm 5 min. The upper organic layer was collected and used for the analysis.
2.16.3 HPLC analysis: As described(6), to evaluate linearity, a mice plasma calibration curve was prepared with a gradient of ST09 (97.7% purity; 100, 200, 300, 400 and 500 μM) in the mobile phase. Selectivity was assessed by comparing the chromatograms of blank plasma and plasma spiked with known concentrations of ST09. LOQ and LOD for ST09 were calculated using the linear regression method.
2.17 Breast tumor sample processing: Breast tumor samples and their matched normal samples were collected in RNAlater solution(Thermo Fisher Scientific, Massachusetts, US) from BGS Global Hospitals, Bangalore, India, with informed consent from the donors. The experimental protocols of this study were approved by the medical ethics committee of the hospital (BGS Global Hospitals IEC/Approval/2018-05/06/01A/May 2018), and all methods were carried out in accordance with relevant guidelines and regulations. The tissues were homogenised in Trizol Reagent (Ambion), and RNA extraction was done per the manufacturer’s protocol.
2.18 Statistical analysis: The Graph pad prism 5 tools were used for statistical analysis. To compare control versus treatment groups, Student’s t-test, two-way ANOVA, and Tukey’s test were performed. P values less than 0.05 were considered significant, and the bar graphs are represented as Mean +SEM.