Chemicals and Reagents
Antibodies used for this study and their sources are as follows: IDO 1 #13268-I-AP, IDO 2 #25053-1-AP, TDO 2 #15880-I-AP, BIN 1 #15880-I-AP, JAK 2 #17670-1-AP, and IκB-α #10268-1-AP, were purchased from Proteintech, San Ying Biotechnology, Wuhan, China. JAK 1 (3332S), and p-JAK 2 (3776S), from Cell Signaling Technology. STAT 1 (ET1606-39), and STAT 3 (ET1607-38) from HangZhou HuaAn Biotechnology Co., Ltd. p-JAK 1 #11149-1, Tyr 1022, p-STAT 1 #11044, Tyr 701, p-STAT 3 #11045, Tyr 705, and NFκB-p65 #21014, from Signalway antibody, Baltimore Ave. USA. IDO 1 primers were obtained from Bioengineering (Shanghai) Co., Ltd. Interferon-γ was obtained from GenScript Piscataway, NJ USA. Nylon wool fiber for T-cell purification (#18369-10) from Polysciences, Inc. Warrinton PA. USA. Every other chemicals and solvents were purchased from Sigma-Aldrich. Reagents used in this study were of the highest available analytic grade (purity ≥ 98%).
Authentication of Plant Material
LML and LMS, collected from trees around the eastern part of Nigeria, in July 2017, authenticated by the Department of Botany herbarium team, the University of Ibadan, where a voucher specimen exists in the herbarium (UIH-22812). Subsequently, leaves (LML) and stem (LMS) were separated, air-dried in a room protected from light.
Preparation of plant Extract
Dried leaves and stem, were crushed separately in an electric grinder. From the powder of LML and LMS: 10 g was extracted with 100 mL of 75% aqueous ethanol by maceration for 48 hours at 25 °C. The extract was filtered, and dried by low pressure, and stored at 4°C, protected from light.
HPLC Analysis of Extract
According to the method described by Vijay et al.  with slight modification as described in brief; 10 mg of powdered plant extracts of LML and LMS were dissolved in 10 mL of methanol to get a concentration of 1mg/mL, subsequently, the solution was filtered using a 0.45μm syringe filter (Millipore) for sterilization. 1 mg of each standard (epicatechin, quercetin, and rutin) was dissolved individually in 1mL of methanol and sterile filtered through 0.45 μm syringe filter (Millipore), before HPLC analysis. The prepared samples of extracts and standards were used for HPLC. The SSI 1500 HPLC series equipped with a DAD detector connected to the system processor was used for analysis. The system explored Empower software with standard certification for analysis of the results; maximum pressure of 2500 psi and a minimum of 1500 psi was maintained. The HPLC of solvents (60:40 ratio of methanol and water) was run at 300 nm wavelength using reverse phase C-18 column (5 µm, 250 mm X 4.6 mm). During the run, a flow rate of 1mL/min was maintained using a binary mode of gradient system. To, identity the compounds, standards of epicatechin, quercetin, and rutin were used. The peaks were identified by comparing the retention time (RT) of the standard with that of different peaks obtained in HPLC analysis of extracts confirmed by UV-spectra online.
Enzyme assay for IDO1 activity
MDA-MB 231 and MCF-7 cells were seeded in a 96-well plate (1 × 105 cells/well) and treated with LML and LMS doses of 1µg/mL, 5µg/mL, 10µg/mL, 15µg/mL, 20µg/mL and 30µg/mL for 2 h before addition of INF-γ (50 ng/mL) for another 24 h to determine the IC50. Dimethylsulfoxide (DMSO, 0.5%) and epacadostat (25 nM) were used as negative and positive controls respectively. IDO1 activity was estimated by measuring the concentration of L-kynurenine in cell culture media according to the method described by Travers et al. . In Brief; 100μL of culture medium was incubated with IDO buffer and then mixed with 25μL of 30 % trichloroacetic acid, incubated for 30 minutes at 50 °C and thereafter centrifuged at 10,000 rpm for 5 min. 100 µL of the supernatant was transferred into a new 96 well plate, and an equal volume of freshly prepared 2% w/v p-dimethylaminobenzaldehyde in acetic acid was added. The optical density was measured at 480 nm using Thermo Scientific VarioskanFlash Multimode Reader. The kynurenine concentration was determined from L-kynurenine standard curve. Each assay was performed in triplicate and data presented as mean ± standard error of mean.
Cell viability assay
MDA-MB 231 and MCF-7 cells were seeded in a 96-well plate (1 × 105 cells/well) and treated with LML and LMS doses of 1, 5, 10, 15, 20 and 30 µg/mL for 2 h. INF-γ (50 ng/mL) was added for another 24 h at 37 °C in a humidified incubator with 5% CO2, to determine the cell viability. Dimethylsulfoxide (DMSO, 0.5%) and epacadostat (25 nM) were used as negative and positive controls respectively. The cell viability was determined by replacing the initial culture medium with 100 µL of fresh culture medium containing 10% of the Cell Counting Kit-8 (CCK-8, Beyotime) in each well of the microplate. After 1 hour of incubation at 37 °C in a humidified incubator with 5% CO2, the absorbance was read at 450 nm on a Thermo Scientific VarioskanFlash Multimode Reader. Cell viability was calculated as a percentage of control (DMSO, 0.5%).
Cells were harvested in RIPA buffer supplemented with a protease inhibitor cocktail (Sigma, Shanghai, China) at the end of the treatment period. The concentration of protein was measured by using a BCA protein assay kit (Bestbio, Shanghai, China). Western blotting was conducted according to standard procedures, as described in our earlier study, Wonganan et al. . In brief; well-mixed aliquots of total cell lysates with loading buffer were boiled for 3-5 min, and separated on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel. After electrophoresis, gels were cut according to molecular weight with the aid of Blue Plus® II Protein Marker (TransGen Biotech Co., LTD Cat. No. DM111-01) and proteins were transferred to nitrocellulose membranes and 5% bovine serum albumin or 5% defatted milk powder were used to block for 1 h at room temperature and then incubated overnight at 4 °C with each of the following antibodies: anti-IDO1, anti-IDO2, anti-TDO2, anti-BIN1, anti-phospho-STAT1, anti- STAT1, anti-phospho-STAT2, anti-STAT2, anti-phospho-STAT3, anti-STAT3, anti-NFκB-p65, anti-IκB-α and GAPDH across all gel for each protein. Afterward, the membranes were incubated with an appropriate peroxidase-conjugated secondary antibody 511203 (Zen Bioscience, Chengdu). The protein bands were determined by the detection of enhanced chemiluminescence solution (Amersham Biosciences, Piscataway, NJ, USA).
Evaluation of IDO1 activity/expression in pCMV3-IDO1-transfected HEK293A cells
HEK293A cells were seeded in a 6-well plate, overnight for proper attachment. pCMV3-IDO1 plasmids (0.94 μg/μL) were transfected into cells by using lipofectamine® 2000 according to the manufacturer’s instructions. After 6 h of incubation, the transfection complex was replaced with fresh medium, and the transfected cells were used for subsequent experiments 12 h later. Transfected cells were treated with different concentrations of LML and LMS (5, 10 and 20 µM) and epacadostat (25 nM) served as a positive control. After 2 h of incubation, the transfected and treated cells were lysed with RIPA lysis buffer, and IDO 1 activity was estimated while the same amount of protein samples were separated on SDS-PAGE to determine the IDO1 expression by Western blot analysis.
Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR)
Total cellular RNA isolated from treated MDA-MB 231 and MCF-7 cells using TRIzol reagent (Invitrogen) by following the manufacturer’s protocol was reverse-transcribed using SuperScript III Reverse Transcriptase (Invitrogen) with oligo dT18 primer. Equal amounts of cDNA were subjected to real-time quantitative PCR with the fluorescent dye SYBR Green I by following the manufacturer’s protocol (TransGen Biotech, Beijing, China). The primer pairs used in the assay for IFN-γ induced IDO 1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as follows: IDO 1, (forward); 5′-AA C AGC GCC TTT AGC AAA GTG TCC CGT TCT TG-3′ (reverse); 5′-AGC GCC TTG CAC GTC TAG TTC TGG GAT GC-3′ and GAPDH, (forward); 5′-TGC ACC ACC AAC TGC TTA GC-3′, (reverse); 5′-GGC ATG GAC TGT GGT CAT GAG-3′. The samples were run in triplicate and the relative expression levels of IDO 1 were determined by normalizing the expression of each target gene to that of IFN-γ using the 2-ΔΔCt method.
Co-culture and T cell Proliferation Assay
Primary T cells were isolated from mouse spleen by following the method described by Flaherty and Reynolds , with little modifications as follows in brief: Spleens from freshly sacrificed mouse were collected into a 60 mm dish containing 3 ml of PBS+. Afterward, a square of sterile nylon mesh was placed over the tissue using sterilized forceps. The thumb side of a syringe plunger (3-10 mL) was used to gently smash the tissue against the mesh. This was followed by pipetting the suspension up and down a few times to break up the remaining soluble clumps and a new square piece of nylon mesh was placed over the opening of a 15 mL conical tube and the suspension filtered through to remove the remaining debris of splenic tissues. Cells were centrifuged at 475 x g for 5 min at 4oC, then the supernatant discarded and re-suspend the cell pellet in 1 mL of ice-cold 1X cell lysing solution per spleen for 1 min to lyse erythrocytes. Then 10 ml of PBS+ was added on top of the lysing solution, and the tube inverted twice, and centrifuged at 475 x g for 5 min at 4oC. After centrifugation, the supernatant (splenocytes) was aspirated and re-suspended in another 10 mL of PBS+. Primary T cells from the splenocytes were purified by using nylon wool mesh (Polysciences, Inc. USA).
MDA MB-231 cells were incubated with T-Cells isolated from mice as described above. Cancer cells were incubated with T-cells at a ratio of 1:10 or 1:5 (cancer cells: T-Cells) for 48 h at 37 °C in a humidified incubator with 5% CO2. At the end of co-culture experiments, conditioned medium (Dulbecco's Modified Eagle Medium (DMEM) plus 10% Fetal Bovine Serum (FBS) was discarded by pipetting, and cell viability was estimated using CCK 8 counting kit as described above.
Statistical analysis was performed with GraphPad Prism 5.0 software (GraphPad, La Jolla, CA, USA). Experiments were repeated at least three times and representative results presented. The data were compared by the Student-T test for each concentration of LML and LMS and one-way ANOVA across the groups followed by Dunnett’s post-hoc test. Differences were considered statistically significant for values at p < 0.05.