This study was approved by Kerman University of Medical Science Ethical & Research Committee (ethical code: IR.KMU.REC.1398.397). The volunteers were informed about the objective and procedure of the study and those who were willing to participate, donate blood sample and sign an informed consent were recruited. All methods were performed in accordance with relevant guidelines and applicable regulations.
Acute Lymphoblastic Leukemia cell line (Nalm-6, NCBI C212) was obtained from the Pasteur Institute collection, Tehran, Iran. Nalm-6 cells were cultured in RPMI-1640 with 2mM L-glutamine (Gibco™ A1049101) containing 10% fetal bovine serum (FBS) (Gibco™ A3160402), 1% antibiotic (Penicillin–Streptomycin Solution 100X, Biowest, L0022) in a humidified atmosphere of 5% CO2 incubator at 37 °C. ZME was prepared according to methanolic extract protocol that Saedi Dezaki et al. applicated in their study . The stock of DOX (EBEWE Pharma, Austria) was also diluted into considered concentration for treatment.
Trypan Blue assay
To evaluate the apoptotic effects of ZME and DOX on cell viability, Nalm-6 cells (250×105 cells/mL) were seeded in 12-well plate and incubated in the presence of the various concentrations of ZME (20, 40, 80, 100, 200 µg/mL) and DOX (5, 10, 20, 40, 80,100 nM) individually for 24, 48, and 72 h. After that, the cell suspension was centrifuged and the cell pellet was suspended in a serum-free complete medium. Next, one part of 0.4 % trypan blue (Gibco™ 15250061) and one part of cell suspension was mixed and then allowed mixture to incubate 2 minutes at room temperature. The total number of unstained (viable) and stained (non-viable) cells was manually counted by Neubauer chamber and light microscope (ECLIPSE E100, Nikon). Finally, the percentage of viable cells was calculated as “Viability (%) = viable cells / viable cells + death cells ×100” .
Determination of Combination Index and Dose Reduction Index
To estimate the interaction between ZME and DOX, the combination index (CI) was calculated using CompuSyn Software (ComboSyn, Inc., Paramus, NJ, USA) according to the classic isobologram equation: “CI = (D)1/(Dx)1 + (D)2/(Dx)2”, where (Dx)1 and (Dx)2 represent the individual dose of ZME and DOX required to inhibit a given level of viability index, and (D)1 and (D)2 are the doses of ZME and DOX necessary to produce the same effect in combination, respectively. Since different CI values (<1, =1, >1 indicate synergism, additive effect, and antagonism, respectively) can be observed at different levels of growth inhibition (fraction affected, FA), CI versus FA plots were applied to present the data using MS Excel. The dose which may be decreased in a combination for a given level of effect as compared to the concentration of individual drug alone defined as dose reduction index (DRI) and calculated as follow: (DRI)1 = (Dx)1/(D)1 and (DRI)2 = (Dx)2/(D)2 [23, 24].
In vitro screening of the cytotoxicity effect of ZME and DOX towards cancer cell lines was measured using MTT colorimetric assay. The metabolization of thiazolyl blue tetrazolium bromide into formazan crystals by Nalm-6 alive cells was assessed by this test . Hence, 1×104 Nalm-6 cells were seeded in 96-well plates with various concentrations of ZME (20, 40, 80, 100, 200 µg/mL) and DOX (5, 10, 20, 40, 80,100 nM) individually for 24, 48, and 72 h. Afterward, the plate was centrifuged at 700×g for 10 min and the supernatant was removed. The cells were incubated with 100 µL MTT solution (0.5 mg/mL; (M5655, Sigma) at 37 °C. After 4 h, the formazan crystals were solubilized by the addition of 150 μL dimethylsulfoxide (DMSO) (Merck, CAS 67-68-5) at each well and optical absorbance was evaluated at 570 nm with an enzyme-linked immunosorbent assay reader. The percentage of metabolic activity of treated cells was calculated relative to untreated cells which were set as negative control. In addition, MTT test was performed for combination dose of ZME/DOX (100 µg/mL ZME +10 nM DOX, 100 µg/mL ZME +20 nM DOX, 200 µg/mL ZME +10 nM DOX, 200 µg/mL ZME +20 nM DOX). In addition to untreated cells, NALM-6 were treated with the highest concentrations of DMSO (were used in our study) as a negative control owing to use it for dissolving ZME (0.01% and 0.1%).
The flowcytometry technique was used to assess the effect of ZME and DOX on the induction of early and late apoptosis using annexin V-propidium iodide (PI) staining . Consequently, 4×105 NALM-6 cells were seeded into six-well cell culture plates. Then, after 48 h, the cells were collected and they were washed with PBS. Flowcytometry was performed using Annexin-V Apoptosis Detection Kit (Mab Tag, AnxF100PI) and the results were analyzed using the FlowJo.7.6.1 software.
RNA isolation and preparation of cDNA
YTzol Pure RNA (Yekta Tajhiz Azma, YT9066) was used to isolate total RNA from untreated (control) and treated cells with 100 µg/mL ZME, 10 nM DOX and ZME/DOX combination (100 µg/mL ZME and 10 nM DOX). Quantity of RNA samples was assessed by NanoDrop (NanoDrop ND-1000; Thermo Scientific, Wilmington, DE) at A260/A280 ratio. The quality and purity of extracted RNA were illustrated by agarose gel electrophoresis. Reverse transcription (RT) reaction was carried out according to the manufacturer instructions using the RevertAid First Strand cDNA Synthesis kit (Thermo Scientific Fermentas, K1622).
Quantitative Real-time PCR
Changes in mRNA expression of desired genes were surveyed by real-time PCR. Quantitative real-time PCR was performed by 10 μL containing Real Q Plus 2x Master Mix Green (Amplicon, Denmark, A325402), 1.5 μL of the cDNA product, 1 μL of forward and reverse primers (10 pmol of each other), and 7.5 μL of nuclease-free water. Thermal cycling conditions included an initial activation step at 95 °C for 15 min followed by 40 cycles, a denaturation step at 95 °C for 15s and a combined annealing/elongation step at 60 °C for 60s. The reaction took place in the RotorGene® Q Real-time PCR System (Qiagen, USA). A melting curve analysis was performed to verify the specificity of the products. The fold change was measured relative to the control and calculated after adjusting for the B-actin reference gene using Ct (2−ΔΔCT) method. Nucleotide sequences of the primers used for real-time RT-PCR listed in Table. 1.
ZME Effect on Normal Cells
Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor using density gradient centrifugation using Ficoll- Hypaque density gradient (Lymphodex, Germany). Isolated cells were washed two times by PBS. Thus, the pellet was resuspended in 1 mL complete media (containing RPMI-1640 with 2mM L-glutamine, 10% FBS and 1% antibiotic) and cultured in T75 flasks with 8 mL complete media at the same condition used for Nalm-6 cells. These cells were treated with 100 and 200 µg/mL ZME and incubated at 37 °C. Then, metabolic activity and apoptotic treated cells were assessed using MTT and flowcytometry after 48h, respectively.
The crystal structure of P53 (PDB ID: 1TUP), BCL2 (PDB ID: 2XA0) was obtained from protein data bank (https://www.rcsb.org). In addition, the DNA chain (1TUP) and BH3 (2XA0) peptide were eliminated by using the software MOE 2019.102 (Molecular Operating Environment). Thymol (CID:6989), carvacrol (CID:10364) structures have also been acquired from PubChem compound database. CASTp (Computed Atlas of Surface Topography of proteins) server was used to predict the P53 and BCL2 pocket and cavities of active sites for ligands binding. CASTp identifies not only the cavities but also pockets and pocket mouth opening. Here, AutoDock Tools 4.2 has been used for molecular docking. The grid box dimension has been adjusted to 126x126x126 Å surrounding the active site of the protein in order to ensure the free rotation of the ligands in the inner side of the grid. The docking run numbers have been estimated to be 100. The resulting poses have been chosen according to the corresponding binding energy. By using protein-ligand interaction profiler server, the interactions of ligand and residues of protein were investigated. Ultimately, the 3D shapes of the final interaction were drawn by using the software Chimera 1.12.
Experimental data are expressed by mean ± standard deviation (SD) to compare the mean values among experimental groups using SPSS version 18.0 (SPSS, Inc., Chicago, IL, USA). All tests were done in duplicate or triplicate. Statistical analysis of MTT and Trypan blue data was calculated by Two-way ANOVA test and One-way ANOVA analysis was used to evaluate the data of flowcytometry and Quantitative real-time PCR. Statistically different values were defined significant at *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001.