Original Article - Basic and Translational Research Investig Clin Urol 2016;57:63-72. http://dx.doi.org/10.4111/icu.2016.57.1.63 pISSN 2466-0493 • eISSN 2466-054X Upregulated expression of BCL2, MCM7, and CCNE1 indicate cisplatin-resistance in the set of two human bladder cancer cell lines: T24 cisplatin sensitive and T24R2 cisplatin resistant bladder cancer cell lines Sung Han Kim1, Jin-Nyoung Ho2,3, Hyunjin Jin2,3, Sang Chul Lee3, Sang Eun Lee3, Sung-Kyu Hong3, Jeong Woo Lee4, Eun-Sik Lee4, Seok-Soo Byun3 1Department of Urology, National Cancer Center, Goyang, 2Biomedical Research Institute and 3Department of Urology, Seoul National University Bundang Hospital, Seongnam, 4Department of Urology, Seoul National University Hospital, Seoul, Korea Purpose: The mechanism of resistance to cisplatin during treatment of bladder cancer (BC) has been a subject of intense investiga- tion in clinical research. This study aims to identify candidate genes associated with resistance to cisplatin, in order to understand the resistance mechanism of BC cells to the drug, by combining the use of microarray profiling, quantitative reverse transcription- polymerase chain reaction (RT-PCR), and Western blot analyses. Materials and Methods: The cisplatin sensitive human BC cell line (T24) and the cisplatin resistant BC cell line, T24R2, were used for microarray analysis to determine the differential expression of genes that are significant in cisplatin resistance. Candidate upregulated genes belonging to three well-known cancer-related KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways (p53 tumor suppressor, apoptosis, and cell cycle) were selected from the microarray data. These candidate genes, differentially expressed in T24 and T24R2, were then confirmed by quantitative RT-PCR and western blot. A fold change ≥2 with a p-value <0.05 was considered significant. Results: A total of 18 significantly upregulated genes were detected in the three selected cancer-related pathways in both micro- array and RT-PCR analyses. These genes were PRKAR2A, PRKAR2B, CYCS, BCL2, BIRC3, DFFB, CASP6, CDK6, CCNE1, STEAP3, MCM7, ORC2, ORC5, ANAPC1, and ANAPC7, CDC7, CDC27, and SKP1. Western blot analyses also confirmed the upregulation of BCL2, MCM7, and CCNE1 at the protein level, indicating their crucial association with cisplatin resistance. Conclusions: The BCL2, MCM7, and CCNE1 genes might play distinctive roles in cisplatin resistance in BC. Keywords: Cell line; Cisplatin; Drug resistance; Microarray analysis; Urinary bladder neoplasms 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. Received: 15 October, 2015 • Accepted: 16 November, 2015 Corresponding Author: Seok-Soo Byun Department of Urology, Seoul National University Bundang Hospital, 82 Gumi-ro 173beon-gil, Bundang-gu, Seongnam 13620, Korea TEL: +82-31-787-7349, FAX: +82-31-787-4057, E-mail: [email protected] ⓒ The Korean Urological Association, 2016 www.icurology.org 63 Kim et al INTRODUCTION cisplatin f rom 0.039 to 40.0 µg/mL [6,10]. T24R2 cells were resistant to treatment with cisplatin (Pfizer, Seoul, Korea) Cisplatin-based combination chemotherapy protocols at concentration up to 2 µg/mL [11] and proven significantly such as methotrexate, vinblastine, doxorubicin and cisplatin, more resistant to cisplatin treatment than other cell or gemcitabine and cisplatin have been a mainstay for lines in our previous studies [6,9-12]. To measure cell advanced (or metastatic) bladder cancer (BC) treatment for chemosensitivity, exponentially growing cells were incubated a number of years and have prolonged the survival of these with different concentrations of cisplatin (0.1–100 µg/mL) patients by up to 19 months [1-4]. However, about 30% of for four days, as previously described [6,9-11]. All assays were patients fail to respond to the initial chemotherapy and in performed in triplicate. Preliminary experiments confirmed most cases, the cancer recurs within one year, and patients that cisplatin at these concentrations had no direct cytotoxic usually die after 2–3 years of chemotherapy due to the effect during first 24 hours of treatment. The half maximal emergence of cisplatin resistant (CR) cancer [1,2]. This wide- inhibitory concentrations (IC50) f or T24R2 and T24 cell lines spread occurrence of CR has resulted in many studies that were determined treated with 20 µg /mL and 1.25 µg/mL of have investigated the molecular mechanisms underlying cisplatin, respectively (not shown in data) [6,10]. cisplatin resistance in BC with the aim of developing an effective therapeutic regimen for BC patients with CR. 2. Microarray sample preparation and analysis of In the last few decades, an increased understanding gene expression of genetic and molecular biological properties of both Total RNA was extracted from T24 and T24R2 cell lines anticancer agents and anticancer resistance has led to the using the mirVanaTM isolation kit (mirVana microRNA clinical development of mechanism-based therapeutics and Isolation Kit, Ambion [Life Technologies], Austin, TX, USA) targeted strategies for the clinical treatment of advanced BC in accordance with the manufacturer’s instructions. RNA [3,4]. Numerous experiments have also tried to understand samples that had high RNA integrity numbers (RIN>9.0; and overcome CR in BC cells in the laboratory [5,6]. Recently, RIN, developed by Agilent Technologies, Santa Clara, CA, there has been a rapid progress in the understanding of USA) and had A260/A280 absorbance ratios ranging from 1.8 CR using microarray analysis that permits a simultaneous to 2.1 were used for cDNA synthesis. The amplification cycles and quick expression profiling of tens of thousands of of RNA to cDNA and cDNA to biotin-labeled aRNA were genes, leading to the identification of genes that might performed using the GeneChip IVT Express kit (Affymetrix, be significant in CR [7,8]. Several previously published Santa Clara, CA, USA). The generation of biotin-labeled experiments from our laboratory have been performed to aRNA by in vitro transcription, hybridization to the array try to define the CR mechanism and CR-related genes and and washing and scanning were performed according to the molecular pathways using microarray methods with BC cell manufacturer’s instructions. lines including CR and reported multiple important findings o f CR [5-11]. 3. Microarray data normalization and analysis In this study, we performed microarray profiling, GenPlex software ver. 3.0 (ISTECH Inc., Goyang, Korea) quantitative real time polymerase chain reaction (qRT- was used for analyzing the CEL file data. The MAS5 PCR), and western blot analyses on a CR and a cisplatin algorithm was used for obtaining the gene expression sensitive BC cell line, to investigate potential candidate summary. GeneChip Human Genome HG-U133 Plus 2.0 genes that might be significant in CR. Knowing which genes array data was analyzed using the Microarray Suite, are differentially expressed between the CR and sensitive MicroDB, and the Data Mining Tool software (Affymetrix) cell lines will help us gain a better understanding of the containing 54,120 probe sets of 38,573 gene clusters in the resistance mechanism of cisplatin. UniGene database. After Global scaling normalization, the normalized data was then log transformed to base MATERIALS AND METHODS 2. The gene expression levels in T24R and T24R2 were normalized and analyzed by (Affymetrix). Gene clusters 1. Cell lines and culture conditions were analyzed using GeneCluster 1.0 (MIT, Cambridge, MA, Three experimental paired sets of two BC cell lines USA). Patterns of altered gene expression between the cell used: a cisplatin-sensitive control cell line T24 (ATCC, lines were explored using the self-organizing map cluster Manassas, VA, USA) and a CR cell line T24R2 generated analysis tool [13]. The genes showing altered expression were by the treatment of T24 cells with serial dilutions of then categorized based on their biological function using 64 www.icurology.org http://dx.doi.org/10.4111/icu.2016.57.1.63 Cisplatin-resistance in human bladder cancer Species Direction Gene Sequence Product (bp) Human hGAPDH 5'-forward-3' TGC ACC ACC AAC TGC TTAG 177 5'-reverse-3' AGA GGC AGG GATGAT GTT C Human MCM7 5'-forward-3' TCG GAT TGT GAA GAT GAA CA 132 5'-reverse-3' TATATT TCT GGG GCG ATTGA Human ORC5 5'-forward-3' TGA ACC CGT GGT TAA AGG AG 156 5'-reverse-3' CCC GGA TCT GTG TCA TCT TT Human CDK6 5'-forward-3' AGA GAC AGG AGT GGC CTT GA 185 5'-reverse-3' TGA AAG CAA GCA AAC AGG TG Human CCNE1 5'-forward-3' AGC GGT AAG AAG CAG AGC AG 189 5'-reverse-3' TTT GAT GCC ATC CAC AGA AA Human ANAPC7 5'-forward-3' GGC ACA GAT GTT GGA TCC TT 211 5'-reverse-3' AAT GGC CTT GGC TCC TAAAT Human CDC7 5'-forward-3' TCA GCA GTC CAC CAC AAA AG 169 5'-reverse-3' AGG GCT CTC ATG TGA AATGG Human CDC27 5'-forward-3' TGC TGA CGT GTT TCT TGT CC 134 5'-reverse-3' TTG CAC TGC CTT TCA TTC TG Human ANAPC1 5'-forward-3' TTG GAA TTG CTC TTC CCA TC 107 5'-reverse-3' GGA AAG ATC CTG ACG TCC AA Human ORC2 5'-forward-3' TCA ATG CTC CTC TCA TGT GG 144 5'-reverse-3' TAA GTG GCA GGG ATC CAG AC Human SKP1 5'-forward-3' CAC TGG AGG CTG CTG ACA TA 126 5'-reverse-3' TAG GAG GGA AGC TGG AAA CA Human CYCS 5'-forward-3' CAA AAA CAA GGG CCA GATGT 136 5'-reverse-3' GCT ACC ACA CTG GAC AGC AA Human STEAP3 5'-forward-3' AGA TCC TGG TGG ATG TGA GC 118 5'-reverse-3' ACA TTG AAG GCC TTG ACC AC Human PRKAR2B 5'-forward-3' GAG GGC ACT TGG CAA TTAAA 131 5'-reverse-3' CCA AGG CCA GCA CAT AAC TT Human BCI2 5'-forward-3' ATC GCC CTG TGG ATG ACT GAG 129 5'-reverse-3' CAG CCA GGA GAA ATC AAA CAG AGG Human BIRC3 5'-forward-3' ATGAAT GGA CAG CCC TGA AG 125 5'-reverse-3' GAA TGC TGC ACA GAG ACC AA Human DFFB 5'-forward-3' AAA CCA CCC ACA AGC TCA AC 148 5'-reverse-3' TTA AAA TGA TGC CCA CGT CA Human CASP6 5'-forward-3' GAG TTC GAG ACC AGC CTG AC 110 Fig.