Human ovarian cancer cell lines A2780 (cisplatin sensitive human epithelial ovarian cancer cell line) and A2780cis (the resistant variant), human breast cancer cell lines MCF7 and MDA-MB-231, and human glioblastoma cancer cell lines U-87 MG and T98G were purchased from European Collection of Authenticated Cell Cultures (England). A2780 and A2780cis cell lines were grown in complete RPMI-1640 medium containing 10 % FBS, 2 mM glutamine, 0.1 mg/ml each of penicillin and streptomycin. To maintain A2780cis resistance to cisplatin, 1 μM of cisplatin (Sigma-Aldrich, St. Louis, USA) was added to the media every 3 passages. MCF7 and MDA-MB-231 cell lines were grown in EMEM containing 10 % FBS, 2 mM glutamine, 1 % non-essential amino acids (NEAA), 0.1 mg/ml each of penicillin and streptomycin. U-87 MG and T98G cell lines were cultured in EMEM, 2 mM Glutamine, 10 % FBS and 1 % Sodium Pyruvate (NaP). All cell cultures were kept in 5 % (v/v) CO2 humidified atmosphere at 37 °C (Binder, Germany). Morphological phenotypes of cell lines were assessed when the cell density was up to 70 % confluence using Eclipse TS100 inverted light microscope (Nikon, Japan).
Cell Viability Assay
Cells were plated into 96-well-plates (1×104 cells/well) for MTT assay and allowed to attach O/N. Different concentrations of cisplatin were used to treat cells for 24 hrs. MTT solution (Roche, Germany) was added to each well and incubated for 4 hrs at 37 °C. Then absorbance values were measured at 550 nm using Multiskan Ascent absorbance plate reader (Thermo Labsystems, Germany). Cell viability determined as following:
Cell viability (%) = (average OD value of experimental group/average OD value of control group) *100 %
Gene expression analysis by qRT-PCR
Total RNA was extracted from A2780, A2780cis, MCF7, MDA-MB-231, U-87 MG, T98G cell lines and from A2780 treated with 5-azacytidine (5-aza) using the RNeasy Mini kit (Qiagen, Germany). First-strand cDNA synthesis was carried out from 3 μg total RNAs were reverse transcriptase to cDNAs using M-MLV reverse transcriptase (Invitrogen, USA) for 2 hrs at 37 °C. To calculate the relative expression of EMT regulating genes; (CDH1, EPCAM, SNAIL1, TWIST2), and ABC transporters (ATP-binding cassette transporters) genes; (MDR1- multidrug resistance protein 1 gene, MRP1- MDR1-related protein 1, MRP2- MDR-related protein 2), quantitative real-time PCR was performed using Maxima™ SYBR™ Green/ROX 2x qPCR Master Mix (Thermo Scientific, USA) for 40 amplification cycles using StepOne™ Real-Time PCR System (Applied Biosystems, USA). Relative transcript fold changes were calculated using the ΔΔCt method with GAPDH as a reference gene. All reactions were run in triplicate. Primers sequences are detailed in Table 1.
In order to select the candidate genes for the methylation study, A2780 cells were cultured and treated with 0.1 mM 5-Azacytidine (Sigma-Aldrich, USA). Culture medium was removed every 24 hrs and replaced by a fresh medium containing 0.1 mM 5-aza. Treated and mock treated cells were collected after 7 treatment days and total RNAs were extracted as described above.
Genomic DNAs from A2780 and A2780cis cells were extracted using the QIAamp DNA Mini kit (Qiagen, Germany) according to the manufacturer's instructions. Isolated DNAs were quantified using NanoVue Plus (GE Healthcare Life Sciences, Germany).
Quantitative PCR-based methylation analysis (Methylscreen assay) was performed to analyze DNA methylation of genes that have differential expression between A2780 and the resistant variant cells and that expression increased after 5-aza treatment. Methylscreen assay is based on combined restriction digestion of DNA with methylation sensitive and methylation dependent restriction enzymes, MSRE and MDRE respectively 76. Genomic DNA of A2780 and A2780cis cells were divided into four parts and treated with different digestions: (1) Rs: two methylation-sensitive enzymes MSRE (HhaI + AciI) or (HpaII + AciI) depending on the frequency of their restriction sites within the studied fragments, which are cutting only unmethylated DNA, (2) Rd: one methylation-dependent restriction enzyme McrBC (MDRE), which is cutting only methylated DNA or (3) Rsd: both MSRE and MDRE enzymes (double digest, DD), and (4) R0: neither MSRE nor MDRE (mock control). Each 50 ml reaction contained 1 mg of gDNA, 1x CutSmart Buffer, 100 μg/mL BSA, 1 mM guanosine-5'-triphosphate, 3 % glycerol and 10 U of each enzyme used in restriction reaction, 50 % glycerol was used instead of enzymes in mock reaction in order to keep restriction digest cocktail homogeneity. Digestions were incubated at 37 °C for 6 hrs followed by inactivation of the enzymes at 65 °C for 20 min. The enzymes, CutSmart Buffer, BSA, and guanosine-5'-triphosphate were purchased from New England Biolabs, USA. Restricted samples were analyzed by qPCR with locus-specific PCR primers and SYBR Green dye. An in-silico analysis was performed using EMBOSS Cpgplot sequence analysis tool (https://www.ebi.ac.uk/Tools/seqstats/emboss_cpgplot/) from European Bioinformatics Institute (EMBL-EBI) to identify the CpG sites associated with the proximal promoter and transcription start site (TSS) for four genes. Sets of locus-specific PCR primers were designed to amplify gDNA at proximal CpG located within 1000 bp (±) of the transcription start site for each gene. Primers sequences, genomic loci, numbers of CpGs nucleotides and number of restriction sites contained in amplified amplicons are listed in Table 2. The PCR amplification was performed in 20 ml volume with 10 ml Maxima™ SYBR™ Green/ROX 2x qPCR Master Mix (Thermo Scientific, USA), 300 nM of each primer and 2 ml (40 ng) of digested template DNA using the qPCR System. The PCR conditions were as follows: 95 °C for 10 min, and 45 cycles of 95 °C for 1 min and temperature for optimized annealing for 1 min. Amplification for each sample was performed in triplicate in a 48-well plate. All primer pairs were tested to identify the annealing temperature for optimal efficiency and melting curve analysis was conducted after the reaction to verify the amplification of the desired products.
Calculations of DNA Methylation Occupancy
The Ct values from R0, Rs, Rd and Rsd, reactions were used to calculate the initial amount of DNA in each digest before PCR as following:
CMs = 2-Ct(Rs); CRd = 2-Ct(Rd); CRsd = 2-Ct(Rsd); CR0= 2-Ct(R0).
The DNA methylation (%) was calculated as following:
hypermethylated DNA fraction (HM) = Rs/(R0-Rsd) x 100; unmethylated DNA fraction (UM) = Rd/(R0-Rsd) x 100; intermediately methylated DNA fraction (IM) = 1-HM-UM. If or ΔCt(Rd-R0) or ΔCt(Rs-R0) < 1.0, The DNA methylation (%) was calculate as following: HM = 1-UM, UM = 1-HM 76,77.
GraphPad Prism Version 7.0 (GraphPad Software, La Jolla, CA, USA) was used to generate graphical figures and to perform statistical analysis. Data are expressed as the mean ±SD. Statistical significance was defined as *= P ≤ 0.05, **= P ≤ 0.01, ***= P ≤ 0.001.