Korean J Physiol Pharmacol 2018; 22(1): 43-51
Published online January 1, 2018 https://doi.org/10.4196/kjpp.2018.22.1.43
Copyright © Korean J Physiol Pharmacol.
Ye-Na Ha1,#, Hye Youn Sung1,#, San-Duk Yang2, Yun Ju Chae1, Woong Ju3,*, and Jung-Hyuck Ahn1,*
1Department of Biochemistry, School of Medicine, Ewha Womans University, Seoul 07985, 2Department of Biomedical Sciences, Seoul National University, College of Medicine, Seoul 03080, 3Department of Obstetrics and Gynecology, School of Medicine, Ewha Womans University Seoul 07985, Korea
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Although cisplatin is one of the most effective antitumor drugs for ovarian cancer, the emergence of chemoresistance to cisplatin in over 80% of initially responsive patients is a major barrier to successful therapy. The precise mechanisms underlying the development of cisplatin resistance are not fully understood, but alteration of DNA methylation associated with aberrant gene silencing may play a role. To identify epigenetically regulated genes directly associated with ovarian cancer cisplatin resistance, we compared the expression and methylation profiles of cisplatin-sensitive and -resistant human ovarian cancer cell lines. We identified α-
Keywords: Cisplatin resistance, DNA methylation, Ovarian cancer, α-N-acetylgalactosaminidase
Ovarian cancer is the most lethal in gynecological cancer worldwide, accounting for 200,000 new cases and 125,000 deaths every year . The high mortality of ovarian cancer is associated with the fact that >70% of patients are diagnosed at an advanced stage due to lack of acceptable screening tools for early detection and absence of specific symptoms at early stages . The current standard therapy for advanced ovarian cancer includes tumor-debulking surgery, followed by taxanes combined with platinum (cisplatin/carboplatin)-based therapy. However, over 80% of patients who initially respond to standard chemotherapy eventually develop recurrence with fully chemoresistant disease . Although cisplatin is one of the most effective chemotherapeutic agents for ovarian cancer, the emergence of chemoresistance is a major challenge to successful therapy. Cisplatin is a small-molecule platinum compound, and its anticancer activity is mediated by the formation of intra- and interstrand cisplatin-DNA adducts that activate apoptotic cell death . The mechanisms of cisplatin resistance are not fully understood, but this resistance can result from multifactorial changes at the molecular and cellular levels, including reduced cisplatin accumulation by active efflux or impaired influx, increased detoxification, increased DNA repair, deregulated expression of transporters, and altered expression and activation of genes involved in apoptosis . Recent studies suggest that anticancer drug resistance, including cisplatin resistance, can be mediated by aberrant DNA methylation. In particular, aberrant promoter hypermethylation and subsequent gene silencing can affect sensitivity to cisplatin by inactivating genes that are critical for response to the drug .
To identify epigenetically regulated genes directly associated with ovarian cancer cisplatin resistance, we measured the response of 11 human ovarian cancer cell lines to cisplatin and classified them into three groups based on cytotoxicity: sensitive, intermediate, and resistant. We compared expression and methylation profiles of cisplatin-sensitive and -resistant human ovarian cancer cell lines and identified α-
The human ovarian cancer cell lines studied were SK-OV-3, ES-2, PA-1, Caov-3, TOV-21G, TOV-112D, OV-90, OVCAR-3, and MDAH 2774, which were purchased from the American Type Culture Collection (Manassas, VA, USA), as well as A2780 and A2780cis, which were purchased from the European Collection of Cell Cultures (London, UK). All cell lines were initially cultured using the media and supplements recommended by the suppliers. Table S1 summarizes the components of culture media for individual cell lines (Table S1). All 11 cell lines were grown as monolayers and attached cells were fully disaggregated by trypsinization between passages. The cell lines were maintained in a 95% humidified and 5% CO2 atmosphere at 37℃.
The cisplatin cytotoxicity of the 11 human ovarian cell lines (SK-OV-3, ES-2, PA-1, Caov-3, TOV-21G, TOV-112D, OV-90, OVCAR-3, MDAH 2774, A2780, and A2780cis) was determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) assays. Briefly, 2×104 cells were seeded onto 96-well plates and incubated at 37℃ overnight. The medium was exchanged with fresh medium containing different cisplatin concentrations (0–100 µM; Sigma-Aldrich; Merck KGaA). After incubation for 48 h, 20 µl of the 2.5 mg/ml MTT solution was added to each well and the plates were further incubated for 2 h at 37℃. One hundred microliters of dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA) was added to solubilize the MTT formazan product by oscillating for 10 min at 37℃. Absorbance at 540 nm was measured using a microplate reader (Molecular Devices LLC, Sunnyvale, CA, USA). Dose-response curves were plotted as the percentage of the control, which was obtained from the sample with no drug exposure. Half-maximal inhibitory concentration (IC50) was calculated as the concentration of cisplatin that reduces cell growth by 50% under the experimental conditions using a nonlinear regression analysis with GraphPad Prism5 software (GraphPad Software, Inc., La Jolla, CA, USA). The 11 human ovarian cell lines were classified into three groups: sensitive, intermediate, and resistant.
Total RNA was extracted from eight human ovarian cancer cell lines (SK-OV-3, PA-1, Caov-3, TOV-21G, TOV-112D, OV-90, and OVCAR-3) using the RNeasy mini kit (Qiagen, Inc., Valencia, CA, USA) and amplified and labeled according to the Affymetrix GeneChip Whole Transcript Sense Target Labeling protocol (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The resulting labeled cDNA was hybridized to Affymetrix Human Gene 1.0 ST Arrays (Thermo Fisher Scientific, Inc.). The scanned raw expression values were background-corrected, normalized, and summarized using the Robust Multiarray Averaging approach in the Bioconductor “affy” package (Bioconductor, http://www.bioconductor.org/). The resulting log2-transformed data were used for further analyses. To identify differentially expressed genes, we applied moderated t-statistics based on an empirical Bayesian approach . Significantly up-regulated and down-regulated genes were defined as those with ≥1.5-fold difference in expression level between cisplatin-resistant and -sensitive groups after correction for multiple testing (Benjamini-Hochberg false-discovery rate [BH FDR]-adjusted p<0.01) .
Genomic DNA was extracted from eight human ovarian cell lines (SK-OV-3, PA-1, Caov-3, TOV-21G, TOV-112D, OV-90, and OVCAR-3) using the QIAmp mini kit (Qiagen, Inc.), according to the manufacturer's instructions. For genome-wide screening of DNA methylation, the Illumina HumanMethylation450 BeadChip (Illumina, Inc., San Diego, CA, USA) was used, which targets 450,000 specific CpG sites. DNA methylation values were described by β-values, which were determined by subtracting the background obtained from negative controls on the array and calculating the ratio of the methylated signal intensity to the sum of the methylated and unmethylated signals. β-values range from 0 (completely unmethylated) to 1 (fully methylated) on a continuous scale for each CpG site. To identify differentially methylated CpG sites, we calculated the difference in mean β-value (Δβ; mean β-value in resistant group – mean β-value in sensitive group). If the absolute difference in mean β-values (|Δβ|) was >0.3, the sites were defined as differentially methylated CpG sites.
One microgram of total RNA was converted to cDNA using Superscript II reverse transcriptase and oligo-(dT)12-18 primers (both from Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. RT-qPCR was performed in a 20-µl reaction mixture containing 1 µl cDNA, 10 µl SYBR Premix EX Taq (Takara Bio, Inc., Otsu, Japan), 0.4 µl 50× Rox reference dye (Takara Bio, Inc.), and 200 nM primers for each gene. The primer sequences were:
To demethylate methylated CpG sites, the eight human ovarian cell lines were treated with 10 µM 5-aza-dc (Sigma-Aldrich; Merck KGaA) for three days at 37℃. Each day, the medium was exchanged with fresh medium supplemented with 10 µM of 5-aza-dc.
To establish a transient expression system, cisplatin-sensitive TOV-112D and cisplatin-resistant SK-OV-3 cells were transfected with pCMV6-AC-NAGA (OriGene Technologies, Inc., Rockville, MD, USA) or pEGFP-N3 (Clontech, Mountain View, CA, USA) DNA constructs using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Briefly, the cells were plated at a density of 6×105 cells/well in 6-well plates containing antibiotic-free complete growth medium and allowed to grow overnight. Two micrograms of each plasmid DNA and 5 µl of Lipofectamine 2000 were diluted separately in Opti-MEM medium (Gibco; Thermo Fisher Scientific, Inc.) to a total volume of 250 µl. The diluted plasmid DNAs and Lipofectamine 2000 were mixed and incubated at room temperature for 20 min to generate the transfection mixtures. The transfection mixtures were added to each well of the 6-well plates, and incubated at 37℃ for 24 h in a 5% CO2 incubator.
Pre-designed small interfering RNA (siRNA) against
The sensitivity of the transfected cells to cisplatin was determined using an MTT assay, described in the “Cisplatin cytotoxicity assay” section.
Proteins (30 µg) were resolved using denaturing 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in 5% skim milk in Tris-buffered saline with 0.1% Tween 20 (TBST) and subsequently incubated overnight at 4℃ with the following primary antibodies: rabbit anticaspase-3 polyclonal antibody (1:1,000, Cell signalling technology, Danvers, MA, USA and mouse anti-α-tublin monoclonal antibody (1:10,000, Sigma-Aldrich; Merck KGaA). After washing, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase for 1 h at room temperature. Chemiluminescence was detected using West Save Star (AbFrontier, Seoul, Korea) according to the manufacturer's protocol. Bands were visualized using an Image Quant LAS mini 4000 (General Electric life sciences, Chicago, IL, USA) and quantified using Image J software (http://rsb.info.nih.gov/ij/index.html).
Data are expressed as mean±standard deviation (SD) of ≥3 independent experiments. Statistical analyses were carried out using GraphPad Prism5 software (GraphPad Software, Inc.). An unpaired
We determined the cytotoxicity of cisplatin in 11 human ovarian cancer cell lines (PA-1, TOV-21G, TOV-112D, Caov-3, A2780, A2780cis, MDAH2774, ES-2, OVCAR-3, OV-90, and SK-OV-3) using an MTT assay. Table 1 illustrates the cytotoxicity of cisplatin in the 11 human ovarian cancer cell lines in order of increasing cytotoxic response: PA-1, TOV-21G, TOV-112D, Caov-3, A2780, MDAH2774, A2780cis, ES-2, OVCAR-3, OV-90, and SK-OV-3. Based on the IC50 values for cisplatin, we classified these cell lines into three groups: sensitive (PA-1, TOV-21G, TOV-112D, Caov-3, and A2780), intermediate (MDAH2774, A2780cis, and ES-2), and resistant (OVCAR-3, OV-90, and SK-OV-3).
We performed mRNA expression microarray analysis on five cisplatin-sensitive cell lines and three cisplatin-sensitive cell lines. We identified differentially expressed genes by performing moderated t-statistics based on an empirical Bayesian approach . Differentially expressed genes were defined as genes whose levels were up-regulated or down-regulated by at least 1.5-fold between cisplatin-sensitive and -resistant groups after correction for multiple testing (BH FDR-adjusted p<0.01) .
To investigate the mechanism by which
The Illumina HumanMethylation450 BeadChip contained 11 CpG sites within the promoter region of the
Next, we assessed whether
We investigated whether
We further explored how
The cleavage status of caspase 3, an indicator of apoptosis was determined in the NAGA-overexpressed SK-OV-3 cells after treatment with cisplatin (30 µM for 10 h). Cisplatin-induced caspase 3 cleavage was increased by the overexpression of
To determine whether the mitochondrial pathway was involved in cisplatin-induced apoptosis in SK-OV-3 cells, we examined changes in mitochondrial membrane potential. As shown in Fig. S1, the mitochondrial membrane potential did not significantly change in SK-OV-3 cells after 8 h of cisplatin treatment regardless of the level of
Taken together, our data suggest that NAGA is involved in the regulation of cisplatin sensitivity by promoting apoptosis in human ovarian cancer cells.
Previous studies have been reported that aberrant changes in DNA methylation play an important role in the development of cisplatin resistance in human cancer cells, including ovarian cancer. In particular aberrant hypermethylation of CpG sites within promoter regions can affect the sensitivity of cancer to chemotherapy by inducing silencing of genes responsible for drug response .
By comparing expression and methylation profiling of cisplatin-sensitive and -resistant human ovarian cancer cell lines, we identified a candidate gene,
Cisplatin exerts its cytotoxic effects mainly through apoptosis and alteration or defiance against apoptotic signaling process could confer cisplatin resistance. There are two major types of cell death pathways, namely, the extrinsic pathway and the intrinsic pathway. The extrinsic pathway is initiated when ligands bind to the death receptors such as tumor necrosis factor-α (TNFα) receptor 1, CD95 (Fas) and TNF-related apoptosis inducing ligand (TRAIL) followed by receptor clustering and recruitment of adaptor molecules into a death-inducing signaling complex (DISC), which then activates an initiator caspase, procaspase 8. Activated caspase 8 then propagates the apoptotic signal via the activation of the executioner caspase 3. The intrinsic pathway also known as mitochondrial pathway is initiated by a variety of receptor-independent stimuli such as DNA damage which trigger mitochondrial membrane permeabilization and subsequently result in the release of proapoptotic mitochondrial proteins and cytochrome c into cytosol. The initiator caspase procaspase 9 is activated through the dimerization with apoptosis promoting activating factor-1 (APAF-1) and formation of an active apoptosome complex containing cytochrome c, APAF-1 and caspase 9. Bcl-2 family proteins regulate DNA damage-induced apoptosis by preventing formation of mitochondrial pores and inhibiting the release of mitochondrial cytochrome c. . It has been reported that p53 play a critical role in cisplatin-induced cell death via two mechanisms, transcription-dependent and -independent pathway. In the transcription dependent pathway, p53 upregulates the expression of pro-apoptotic proteins such as PUMA, Bax and BID which are involved in the regulation of the intrinsic pathway and activates death receptors such as CD95, DR5 receptors which mediate the extrinsic pathway. In addition, p53 can also suppress anti-apoptotic proteins such as Survivin. In the transcription-independent pathway, p53 localizes to the mitochondria following cytotoxic insults where it interacts with Bcl-2 and Bcl-XL and inhibits their anti-apoptotic function at the outer mitochondrial membrane. In addition to mitochondrial p53, cytosolic p53 induces the activation of Bax, subsequently resulting in mitochondrial membrane permeabilization and cytochrome c release .
The caspase 3 assay and mitochondrial permeability detection assay showed overexpression of
In the present study, we provide novel evidence that
Components and supplementation of culture media for the human ovarian cancer cell lines in this studykjpp-22-43-s001.pdf
Cisplatin-induced apoptosis in p53-null SK-OV-3 cells proceeds through mitochondria-independent pathway.kjpp-22-43-s002.pdf
A CpG site is hypermethylated within the
The DNA methylation status of CpG sites within the
Eight ovarian cancer cell lines were treated with 5-aza-2′-deoxycytidine and
Cisplatin-resistant SK-OV-3 cells and cisplatin-sensitive TOV-112D cells were transiently transfected with
Transient depletion of
Cisplatin-resistant SK-OV-3 cells and cisplatin-sensitive TOV-112D cells were transiently transfected with siNC and siNAGA. After 24 h of transfection, knockdown of
Regulation of cisplatin-induced apoptosis by ectopic expression of
Cisplatin-resistant SK-OV-3 cells were transiently transfected with
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