apoptosis related molecular differences for response to tyrosin kinase inhibitors in drug sensitive and drug resistant human bladder cancer cells

Original Article Apoptosis-related molecular differences for response to tyrosin kinase inhibitors in drug-sensitive and drug-resistant human bladder cancer cells ABSTRACT Context: The epidermal growth factor receptor (EGFR) family is reportedly overexpressed in bladder cancer, and tyrosine kinaseinhibitors (TKIs) have been suggested as treatment Gefitinib is a selective inhibitor of the EGFR and lapatinib is a dual inhibitor of both the EGFR and HER2 (human EGFR type receptor) Both compounds compete with the binding of adenosine triphosphate (ATP) to the tyrosine kinase domain of the respective receptors to inhibit receptor autophosphorylation causing suppression of signal transduction Unfortunately, resistance to these inhibitors is a major clinical problem Aims: To compare the apoptosis signaling pathway(s) induced by gefitinib and lapatinib, in UM-UC-5 (drug-sensitive) and UM-UC-14 (drug-resistant) bladder cancer cells and to identify molecular differences that might be useful predictors of their efficacy Materials and Methods: Cell proliferation, cell cycle and apoptosis assay were used to detect the effect of TKIs on UM-UC-5 and UM-UC-14 cells Molecular differences for response to TKIs were examined by protein array Results: TKIs strongly inhibited cell proliferation and induced cell cycle G1 arrest and apoptosis in UM-UC-5 cells Most notable apoptosis molecular differences included decreased claspin, trail, and survivin by TKIs in the sensitive cells In contrast, TKIs had no effect on resistant cells Conclusions: Claspin, trail, and survivin might be used to determine the sensitivity of bladder cancers to TKIs KEY WORDS: Bladder cancer, gefitinib, lapatinib, tyrosine kinase inhibitor INTRODUCTION Jixia Li, Bo Lv1, Xiangyong Li, Zhiwei He2, Keyuan Zhou2 Institute of Biochemistry and Molecular Biology, Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Guangdong Medical College, Dongguan, Department of Emergency and Critical Care Medicine, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong, China Bladder cancer is a common malignant disease in the USA and is the 4th most common cancer in men and the 10th in women.[1] Genetic factors, including oncogenes, such as the epidermal growth factor receptor (EGFR) and tumor suppressor genes, are one of the risk factors for the development of bladder cancer Bladder cancer highly expresses EGFR and/ or HER2.[2,3] Transgene-driven overexpression of the EGFR within the bladder enhances tumor progression in mice, providing direct support for its importance in the biology of this disease.[4] EGFR is a cell-surface receptor, belonging to the EGFR family of receptor tyrosine kinases The EGFR family comprises four members, EGFR (HER1, ErbB1), ErbB2 (HER2), ErbB3 (HER3), and ErbB4 (HER4) The EGFR family members have four ectodomains, a single transmembrane domain and a cytoplasmic tail containing the active tyrosine kinase domain Following the kinase domain, a C-terminal tail contains autophosphorylation sites that recruit signaling molecules.[5] The EGFRassociated signaling pathway plays an important role in the development and progression of cancers It has become one of the most important targets for anticancer drug discovery, and a large number of different small molecule and antibody-based EGFR antagonists have been tested in clinical trials Three small molecule EGFR tyrosine kinase inhibitors (TKIs) are in clinical use and include gefitinib (ZD1839, Iressa), erlotinib (Tarceva) and lapatinib (GW 572016, Tykerb) All are based on a 4-anilinoquinazoline scaffold and target the ATP site to inhibit receptor autophosphorylation causing suppression of signal transduction Gefitinib and erlotinib, selective EGFR inhibitors, target the active form of the kinase and have been approved for nonsmall-cell lung cancer Lapatinib, a dual inhibitor of EGFR and HER2, preferentially targets the inactive conformation and has been approved for HER2 positive breast cancer [6,7] Although TKIs display a survival advantage in clinical trials, only a minority of patients seem to respond to this approach Resistance to TKIs has become a major clinical problem Research is needed to identify and validate predictive factors that can be used to select patients with disease 668 Journal of Cancer Research and Therapeutics - October-December 2013 - Volume - Issue For correspondence: Dr Keyuan Zhou, Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Guangdong Medical College, Dongguan-523808, Guangdong, China E-mail: kyz@ gdmc edu.cn Access this article online Website: www.cancerjournal.net DOI: 10.4103/0973-1482.126478 PMID: 24518715 Quick Response Code: Li, et al.: Molecular differences for response to tyrosine kinase inhibitors in drug-sensitive and resistant human bladder cancer cells likely to respond to TKIs Recent data showed that tumor response is not associated with a higher proportion of EGFRpositive tumor cells or more intensive EGFR staining in lung cancer.[8] Therefore, the identification of other predictive markers is extremely important In this study, we compared the apoptosis signaling pathway(s) induced by TKIs (gefitinib and lapatinib), in UM-UC-5 (drugsensitive) and UM-UC-14 (drug-resistant) bladder cancer cell lines and identified molecular differences as predictors of their efficacy Here, we report that claspin, trail, and survivin substantially suppressed by TKIs in UM-UC-5 cells MATERIALS AND METHODS Eagle’s minimum essential medium (MEM) was purchased from Invitrogen (Carlsbad, CA) Fetal bovine serum (FBS) was purchased from Gemini Bio-products (Calabasa, CA) and the antibiotics (penicillin and streptomycin) were from Invitrogen (Carlsbad, CA) The human apoptosis array kit was purchased from R&D (Minneapolis, MN) The protein assay kit was from Bio-Rad (Hercules, CA) The CellTiter96 Aqueous One Solution Cell Proliferation Assay Kit was from Promega (Madison, WI) The bladder cancer cell lines (UM-UC-5 and UM-UC-14, Department of Urology, University of Texas M D Anderson Cancer Center) were cultured in monolayers at 37°C in a 5% CO2 incubator in MEM containing 10% FBS and penicillin/ streptomycin Cells were seeded (5 × 103 cells/well) in 96-well plates with 10% FBS/MEM and incubated at 37°C in a 5% CO2 incubator overnight Cells were then fed with fresh medium and treated with gefitinib (2.5 μM) or lapatinib (2.5 μM) After culturing for various times, 20 μl of Cell Titer 96 Aqueous One Solution were added to each well, and the cells were then incubated for h at 37°C in a 5% CO2 incubator Absorbance was measured at 490 and 690 nm Cells were seeded (4 × 105 cells/well) in 60-mm dishes with 10% FBS/MEM and incubated overnight at 37°C in a 5% CO2 incubator Cells were then starved in serum-free medium for 24 h followed by treatment for 24 h with gefitinib (2.5 μM) or lapatinib (2.5 μM) in 10% FBS/MEM The cells were trypsinized and then washed twice with cold PBS, and fixed with ice-cold 70% ethanol at -20°C overnight Cells were then washed twice with PBS, incubated with 20 mg/mL RNase A and 200 mg/mL propidium iodide in PBS at room temperature for 30 in the dark and subjected to flow cytometry using the FACSCalibur flow cytometer Data were analyzed using ModFit LT (Verity Software House, Inc., Topsham, ME) Annexin V and propidium iodide staining was used to visualize apoptotic cells in a similar procedure as described above but the cells were not prefixed with 70% ethanol Cells were stained using the Annexin V-FITC Apoptosis Detection Kit (MBL International Corporation, Watertown, MA) and propidium iodide according to the manufacturer's instructions Cells were analyzed by two-color flow cytometer The emission fluorescence of the Annexin V conjugate was detected and recorded through a 530/30 bandpass filter in the FL1 detector Propidium iodide was detected in the FL2 detector through a 585/42 bandpass filter Apoptotic cells were only those that stained positive for Annexin V and negative for propidium iodide, located in the bottom right quadrant Each cell line was cultured to 90% confluence and then starved 24 h in serum-free media They were treated, respectively, with 2.5 μM gefitinib or lapatinib in culture medium containing 10% FBS for 24 h and then harvested Cell samples were disrupted and then proteins were extracted The protein concentration was determined using a dye-binding protein assay kit (Bio-Rad) as described in the manufacturer’s manual Following the instructions provided with the protein arrays, cell lysates were subjected to Proteome Profiler™ Array analysis As necessary, data are expressed as means ± standard error (S.E.) and significant differences were determined using oneway ANOVA A probability value of p < 0.05 was used as the criterion for statistical significance RESULTS We first examined the effects of TKIs on UM-UC-5 and UM-UC-14 cell proliferation Cells were treated with 2.5 μM of the agents (gefitinib and lapatinib) dissolved in DMSO (vehicle) for 72 h Gefitinib and lapatinib caused a decrease to 44% and 33% (P < 0.05) of control, respectively [Figure 1] in UM-UC-5 cells; while in UM-UC-14 cells, both drugs had little effect on cell proliferation We next assessed the effects of TKIs on cell cycle progression and apoptosis in both cells After treatment with TKIs, cells were stained with propidium iodide and analyzed by flow cytometry Cell cycle distribution analysis showed that the TKIs treatments for 24 h result in an increased accumulation of cells in G1-phase [Figure 2] in UM-UC-5 cells but Figure 1: TKIs inhibit cell proliferation in UM-UC-5 cells Cells were treated with gefitinib (2.5 μM) or lapatinib (2.5 μM) in 10% FBS/MEM for various times At the end of each treatment time, cell growth was measured by MTS assay Data are shown as means ±S.E The asterisks (*) indicate a significant difference (P < 0.05) between groups treated with drugs and the group treated with DMSO Journal of Cancer Research and Therapeutics - October-December 2013 - Volume - Issue 669 Li, et al.: Molecular differences for response to tyrosine kinase inhibitors in drug-sensitive and resistant human bladder cancer cells Figure 2: TKIs induce significant G1 arrest in UM-UC-5 cells Cells were starved in serum-free medium for 24 h and then treated with gefitinib (2.5 μM) or lapatinib (2.5 μM) for 24 h Cell cycle analysis was performed using flow cytometry Data are shown as means ± S.E The asterisks (*) indicate a significant difference (P < 0.05) between groups treated with drugs and the group treated with DMSO Figure 3: TKIs induce apoptosis in UM-UC-5 cells (a) Cells were starved in serum-free medium for 24 h and then treated with gefitinib (2.5 μM) or lapatinib (2.5 μM) for 24 h Apoptosis was analyzed by flow cytometry Data are shown as means ± S.E The asterisk (*) indicates a significant difference (P < 0.05) between groups treated with TKIs and the group treated with DMSO DISCUSSION Figure 4: UM-UC-5 cells are sensitive to TKIs and show decreased expression of TRAIL, claspin, and survivin (a) Results of apoptosis protein array analysis using UM-UC-5 and UM-UC-14 cells treated or not treated with gefitinib (b) Results of apoptosis protein array analysis using UM-UC-5 and UM-UC-14 cells treated or not treated with lapatinib For A and B, both cell lines were treated with 2.5 mM gefitinib or lapatinib for 24 h, and the cell lysates were hybridized to the apoptosis array In the array, each signal is spotted in duplicate Hybridization signals at the corners serve as controls Apoptosis can be activated through two pathways: The extrinsic pathway (mediated by death receptors) or the intrinsic pathway (mediated by mitochondria) The death receptor pathway is activated in response to the engagement of ligands such as TNF-α with their receptors (TRAIL-R1 and TRAIL-R2) This in turn induces the recruitment of adapter proteins (FADD) to form the death-inducing signal complex (DISC), which activates caspase-8 In turn, caspase activates effector caspases by catalytic cleavage.[9] Inhibitors of apoptosis proteins (IAPs) family consists of an evolutionarily conserved group of apoptosis inhibitors.[10,11] Human members of the family include c-IAP-1, c-IAP-2, XIAP, surivivin, livin, and NAIP In general, the IAP proteins function through direct interactions to inhibit the activity of several caspases, including caspase-3, caspase-7, and caspase-9.[12,13] In agreement with previous study, our data showed EGFR inhibitors reverses TRAIL resistance by inhibition IAP family proteins (survivin).[7] not affect on UM-UC-14 cells After gefitinib or lapatinib treatment for 24 h, no differences in numbers of apoptotic cells were observed in UM-UC-14 cells However, treatment with TKIs resulted in significant apoptosis in UM-UC-5 cells [Figure 3] These results suggested that TKIs inhibited cell proliferation by inducing G1 arrest and apoptosis in UMUC-5 cells Claspin is a mediator of Chk1 signal transduction at the replication checkpoint and in response to DNA damage Its expression is negatively regulated by both proteosome- and caspase-mediated degradation,[14] and stabilized by activation of Chk1.[15] It has been proposed that claspin behaves as a tumor suppressor since downregulation promotes apoptosis following genotoxic stress.[16] Conversely, claspin seems to behave as an oncogene in other instances since overexpression promotes cellular proliferation.[17] Upregulated claspin has been suggested to be a sensitive marker of abnormally proliferating cells.[18] In this study, we found EGFR inhibitors induced apoptosis by suppressing Claspin protein To assess the direct effects of TKIs on apoptosis signaling pathway, we used protein array to measure the level of apoptosis-related proteins Results showed in sensitive cells both drugs strongly downregulated antiapoptotic proteins such as claspin, survivin, and death receptors (TRAIL R1, TRAIL R2), but in resistant cells, TKIs had no effect [Figure 4] These suggest claspin, TRAIL, and Survivin might be used to determine the sensitivity of bladder cancers to TKIs In sensitive cell, both drugs significantly downregulated TRAIL R1, TRAIL R2, survivin, and claspin TKIs were the apoptosis inducer mainly by death receptor pathways In resistant cell, both drugs had little effect on these proteins The reason is that these proteins are downstream molecular of Akt and MAP kinases signaling pathway Upstream signaling unchanged after inhibitors treated resistant cell; thus, downstream proteins had no change 670 Journal of Cancer Research and Therapeutics - October-December 2013 - Volume - Issue Li, et al.: Molecular differences for response to tyrosine kinase inhibitors in drug-sensitive and resistant human bladder cancer cells CONCLUSION In summary, we first compared the signaling pathway(s) induced by gefitinib and lapatinib in human bladder cancer cell and confirmed that TKIs were effective in suppressing EGFR/ErbB2 and inducing apoptosis by mainly inhibiting death receptor (TRAIL), claspin and survivin proteins expression in sensitive cells, whereas TKIs had no effect in resistant cells These molecular differences could be predictors of their efficacy in human bladder cancer cells REFERENCES 12 van Rhijn BW, Burger M, Lotan Y, Solsona E, Stief CG, Sylvester RJ, et al Recurrence and progression of disease in non-muscle-invasive bladder cancer: From epidemiology to treatment strategy Eur Urol 2009;56:430-42 McHugh LA, Sayan AE, Mejlvang J, Griffiths TR, Sun Y, Manson MM, et al Lapatinib, a dual inhibitor of ErbB-1/-2 receptors, enhances effects of combination chemotherapy in bladder cancer cells Int J Oncol 2009;34:1155-63 Wang X, Zhang S, MacLennan GT, Eble JN, Lopez-Beltran A, Yang XJ, et al Epidermal growth factor receptor protein expression and gene amplification in small cell carcinoma of the urinary bladder Clin Cancer Res 2007;13:953-7 Shrader M, Pino 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Lancet 2003;362:62-4 Martinez-Ruiz G, Maldonado V, Ceballos-Cancino G, Grajeda JP, Melendez-Zajgla J Role of Smac/DIABLO in cancer progression J Exp Clin Cancer Res 2008;27:48 Deveraux QL, Reed JC IAP family proteins-suppressors of apoptosis Genes Dev 1999;13:239-52 Deveraux QL, Roy N, Stennicke HR, Van Arsdale T, Zhou Q, Srinivasula SM, et al IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases EMBO J 1998;17:2215-23 Deveraux QL, Takahashi R, Salvesen GS, Reed JC X-linked IAP is a direct inhibitor of cell-death proteases Nature 1997;388:300-4 Kasof GM, Gomes BC Livin, a novel inhibitor of apoptosis protein family member J Biol Chem 2001;276:3238-46 Semple JI, Smits VA, Fernaud JR, Mamely I, Freire R Cleavage and degradation of Claspin during apoptosis by caspases and the proteasome Cell Death Differ 2007;14:1433-42 Chini CC, Wood J, Chen J Chk1 is required to maintain claspin stability Oncogene 2006;25:4165-71 Chini CC, Chen J Human claspin is required for replication checkpoint control J Biol Chem 2003;278:30057-62 Lin SY, Li K, Stewart GS, Elledge SJ Human Claspin works with BRCA1 to both positively and negatively regulate cell proliferation Proc Natl Acad Sci U S A 2004;101:6484-9 Tsimaratou K, Kletsas D, Kastrinakis NG, Tsantoulis PK, Evangelou K, Sideridou M, et al Evaluation of claspin as a proliferation marker in human cancer and normal tissues J Pathol 2007;211:331-9 Cite this article as: Li J, Lv B, Li X, He Z, Zhou K Apoptosis-related molecular differences for response to tyrosin kinase inhibitors in drug-sensitive and drug-resistant human bladder cancer cells J Can Res Ther 2013;9:668-71 Source of Support: Grants from National Natural Science Foundation of China (81201710), Guangdong Natural Science Foundation (S2012010008259) and Guangdong Medical College for the research grant (no XG-1101 and GX0306), Conflict of Interest: None declared Journal of Cancer Research and Therapeutics - October-December 2013 - Volume - Issue 671 Copyright of Journal of Cancer Research & Therapeutics is the property of Medknow Publications & Media Pvt Ltd and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission However, users may print, download, or email articles for individual use