Cell line and reagents

The A2780 human ovarian cancer cell line was obtained from the cell bank of Pasture Institute of Iran (NCBI). Pyrogallol was procured from Sigma-Aldrich Chemical Co. (St. Louis, MO) and was dissolved in distilled water at 100mM as a stock solution. As well as, 2,2-diphenyl-1- pikrilhydrazil (DPPH), dimethyl sulfoxide (DMSO), 3- [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT), were obtained from Sigma. All solution obtained in molecular grade. The study was conducted in accordance with the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies [38].

Cell cytotoxicity

The A2780 human ovarian cancer cell line was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium complemented with 10% Fetal Bovine Serum (FBS) (Gibco, USA) and 1% penicillin/streptomycin (Sigma, USA). Cells were sustained at 37 °C under 5% of CO2 and 95% humidity. After every 4 days, old media was changed with fresh media, to stimulate growth until the cells reach a confluence of 75–80%. The MTT assay was performed to investigate the cytotoxicity and viability percentage of A2780 cells in the treatment with cisplatin and pyrogallol individually and in combination with each other. The cells were cultured in 96-well plates at the number of 1 × 104 cells/well and were incubated for 24 h at 37 °C until the cells were attached to the cell culture plate. Then, the medium of the cells was replaced with the drug treatment medium. Cells were treated with cisplatin (at 0-6μg/ml) and pyrogallol (at 0-20μg/ml) separately for 48 h. To conduct the combination treatment, concentrations of 0.5, 1, 3 and 6 of pyrogallol were combined with concentrations of 0–6 μg/ml of cisplatin. After 48 h of cell treatment, to investigate cell viability, 20 μl of MTT solution (5 mg/ml) was added to each well of the plate and the cells were incubated for 3 h at 37 °C in the dark. Then, 100μl of DMSO was added to the wells to dissolve the formed formazan crystals, and optical absorbance was measured by plate reader at 490-nm wavelength. The percentage of cell viability in the treatment with the studied drugs was calculated using the formula below:

$$ \% }\,} = \frac}\,}\,}\,} - }\,}\,}}}}\,}\,}\,} - }\,}\,}}} \times 00 $$

Synergistic effect of pyrogallol and cisplatin

Compusyn software was used to study the synergistic or antagonistic effect of cisplatin and pyrogallol. Basically, one of the important factors in pharmacology and combination drug therapy is an index called Combination Index (CI). This index is a measure of the effect of the combination of drugs and shows that the combination drug has a synergistic or antagonistic effect. The software uses the following formula to calculate the index:

$$ } = \left( } \right)}/\left( }} \right)} + \, \left( } \right)}/\left( }} \right)} $$

where, (D)A and (D)B represent the doses of drugs A and B, respectively, which must be combined to obtain effective Fa. As well, (DX)A and (DX)B indicate the doses of drugs A and B, respectively, that are used individually to obtain effective Fa. CI < 1 represents the synergistic effect of medicinal compounds, CI = 1 additive effect and CI > 1 the effect of the combination drug as antagonism [6].

Colony formation

Clonogenic assay or colony formation assay is an in vitro cell viability assay based on the ability of a single cell to grow and form a cell colony. A cell colony is defined as at least 50 cells. This assay essentially tests each cell in the population for its ability to undergo unlimited division [4].

This method can be used to determine the effect of drugs and agents with cytotoxic effects. In the present study, to perform the colony formation test, first, 500 cells were cultured in each well of a 12-well plate After 24 h, the treatment with the IC10s of studied drugs including pyrogallol, cisplatin and pyrogallol/cisplatin combination was done. After 48 h, the drug medium was replaced with fresh medium and the cells were incubated at 37 °C for 14 days. Then, to stain the colonies, the supernatant of the cells was removed and 1 ml of the mixture of ethanol and acetic acid was added to each well. After the cells were fixed, crystal violet stain was added to the wells and then the wells were incubated for 1 h at room temperature. In the next step, washing was done with distilled water and after the plate dried, the colonies were counted. The ratio of the number of colonies to the number of cultured cells was calculated.

Migration assay

In the scratch test method, the attached cells on the bottom of the cell culture plate are scratched and a cell-free area is created. The migration of cells from around the scratched area toward the center is analyzed by imaging. In this study, cells were cultured at a density of 95% in 12-well plates to examine the effect of drug treatment on the inhibition of cell migration. A vertical scratch was made in the middle of the well by the tip and the isolated cells were washed with PBS and the medium containing pyrogallol, cisplatin and the combination of the two agents at a concentration of IC10 was added to each well. Imaging of the cells at the scratch area was done at 0, 24, 48 and 72-h intervals. The extent of the scratch area at different intervals was calculated using ImagJ software.

Flow cytometry

The amount of cell apoptosis was determined by annexin V-fluorescein isothiocyanate (FITC) staining and PI labeling according the protocol of kit’s manufacturer. Annexin V can be used to identify the externalization of phosphatidylserine throughout the progression of apoptosis, and therefore can identify cells in the early stages of apoptosis. For the investigation of apoptosis rate under the treatment of studied drugs, 1 × 106 cells/well were cultured. After 24 h, the cells were treated with the IC30 of pyrogallol and cisplatin alone and their combination. After 48 h of cell treatment, the cells were separated by trypsin and then the cell mass was separated by centrifugation. The collected cells were washed twice with cold PBS and then, 90μl of annexin binding buffer (10mM HEPES/NaOH, pH 7.4, 140mM NaCl, 2.5mM CaCl2) was added to the cells and the cells were completely suspended in the buffer. Then 5μl of annexin V-FITC and PI were added to the cells and the cells were incubated for 20 min in the dark, and then they were examined using a flow cytometry device. Viable cells are negative for both annexin V-FITC and PI stain. Early apoptotic cells are positive for annexin V-FITC and negative for PI, and late apoptotic cells are strongly positive for both staining. Necrotic cells are displayed as PI positive and annexin V-FITC negative.

Investigation of the antioxidant capacity of pyrogallol

DPPH is a stable chromogenic radical with dark purple color and its inhibition test is done based on electron donation of antioxidants to neutralize DPPH radical. This reaction is accompanied by the color change of DPPH, and the color change serves as an indicator of antioxidant effectiveness. The result obtained for a compound is generally compared with a conventional antioxidant compound such as ascorbic acid. To perform the test, a 100µM alcoholic solution of DPPH was prepared and to investigate the antioxidant activity, it was mixed with an equal amount of pyrogallol (at 0-20μg/ml). The samples were incubated for 30 min in the dark at room temperature and then absorbance was read by a spectrophotometer at 517 nm wavelength. Prepared DPPH substance is used as a blank. Next, the scavenging rate of free radicals in DPPH solution is calculated with the following formula:

$$ }\,}\left( \% \right) = \, \left( }\,} - }\,}} \right)/ \, \left( }\,}} \right) $$

Measurement of cellular superoxide dismutase activity

Superoxide dismutase (SOD) catalyzes the conversion of superoxide anion into hydrogen peroxide and molecular oxygen. In this assay, SOD activity is measured using tetrazolium salt, which produces a water-soluble formazan dye after reduction with superoxide anion. The rate of formation of formazan is limited by the presence of SOD in the environment and it can be measured photometrically, in such a way that an increase in the activity of SOD causes a decrease in the amount of color and lower absorption. In this study, in order to check the level of SOD activity, cells were cultured at the rate of 1 × 106 cells/well in a 6-well plate. After 24 h, the cells were treated with concentrations of 0, 1, 3, 6, 10, 12 and 20μg/ml of pyrogallol and incubated for 48 h at 37 °C in a CO2 incubator. Then, the cells were separated without using trypsin and with the help of scraping and were lysed by repeated freezing and thawing in cold PBS. After centrifugation, the cell supernatant was collected and reagents were added for SOD assay according to the kit protocol. After 20 min of incubation at 37 °C, absorbance reading was done by a plate reader at 450-nm. The amount of SOD inhibitory activity was calculated by the following formula:

$$ }\, } \left( \% \right) = \frac}\,}1 - }\,}3} \right) - \left( }\,} - }\,}2} \right) }}}1 - }3} \right)}} \times 100 $$

qPCR analysis

qPCR was used to determine the effect of the studied compounds on the apoptotic pathway (CASP-3, -8, -9, BCL2) as well as the expression of mir-15a and two of the target genes involved in cell proliferation pathway and metastasis (CDC25A, KRAS). First, 1 × 106 cells/well were cultured in a 6-well plate. Then the cells are treated with IC30 concentration of pyrogallol, cisplatin and pyrogallol-cisplatin combination. After 48 h, RNA was extracted using RNX-Plus solution. The cDNA synthesis of miR-15a was completed by specific miRNA primers in the form of stem-loop and MMLV enzyme. Oligo-dt and random-hexamer primers were used for total cell cDNA synthesis. The real-time PCR was completed by specific primers for miR-15a, miRNA target genes as well as genes of the apoptotic pathway using the SYBR Green method. The U6 gene was used as miRNA internal control and GAPDH as internal control for target genes. Amplification was conducted with an initial denaturation for 5 min at 94 °C, followed by 35 cycles of amplification (94 °C for 15s, annealing temperature for 10s, and 72 °C for 10s) using Mic qPCR cycler (Bio-Molecular Systems). All amplification reactions were performed in triplicate. Relative gene expression was analyzed using the 2-ΔΔCt method.

. Statistical analysis

Prism software was used to investigate the results of data analysis related to migration test, colony formation, antioxidant activity, flow cytometry and also to calculate the expression level in the treated and control samples and compare them. To make comparisons two group, t-test was used and ANOVA test were used for comparison of more than two groups. P-value < 0.05 was considered as a significance level.