|Year : 2013 | Volume
| Issue : 4 | Page : 668-671
Apoptosis-related molecular differences for response to tyrosin kinase inhibitors in drug-sensitive and drug-resistant human bladder cancer cells
Jixia Li1, Bo Lv2, Xiangyong Li1, Zhiwei He3, Keyuan Zhou3
1 Institute of Biochemistry and Molecular Biology, Guangdong Medical College, Dongguan, China
2 Department of Emergency and Critical Care Medicine, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong, China
3 Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Guangdong Medical College, Dongguan, China
|Date of Web Publication||11-Feb-2014|
Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Guangdong Medical College, Dongguan-523808, Guangdong
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
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 2 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.
Keywords: Bladder cancer, gefitinib, lapatinib, tyrosine kinase inhibitor
|How to cite this article:|
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
|How to cite this URL:|
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 [serial online] 2013 [cited 2020 Jul 13];9:668-71. Available from: http://www.cancerjournal.net/text.asp?2013/9/4/668/126478
| > Introduction|| |
Bladder cancer is a common malignant disease in the USA and is the 4 th most common cancer in men and the 10 th in women.  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. , 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.  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.  The EGFR-associated 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. , 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 likely to respond to TKIs. Recent data showed that tumor response is not associated with a higher proportion of EGFR-positive tumor cells or more intensive EGFR staining in lung cancer.  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 (drug-sensitive) 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% CO 2 incubator in MEM containing 10% FBS and penicillin/streptomycin. Cells were seeded (5 × 10 3 cells/well) in 96-well plates with 10% FBS/MEM and incubated at 37°C in a 5% CO 2 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 1 h at 37°C in a 5% CO 2 incubator. Absorbance was measured at 490 and 690 nm.
Cells were seeded (4 × 10 5 cells/well) in 60-mm dishes with 10% FBS/MEM and incubated overnight at 37°C in a 5% CO 2 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 min 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 one-way 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.
|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|
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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 do 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 UM-UC-5 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|
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|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|
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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.
|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|
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| > Discussion|| |
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 8 activates effector caspases by catalytic cleavage.  Inhibitors of apoptosis proteins (IAPs) family consists of an evolutionarily conserved group of apoptosis inhibitors. , 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. , In agreement with previous study, our data showed EGFR inhibitors reverses TRAIL resistance by inhibition IAP family proteins (survivin). 
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,  and stabilized by activation of Chk1.  It has been proposed that claspin behaves as a tumor suppressor since downregulation promotes apoptosis following genotoxic stress.  Conversely, claspin seems to behave as an oncogene in other instances since overexpression promotes cellular proliferation.  Upregulated claspin has been suggested to be a sensitive marker of abnormally proliferating cells.  In this study, we found EGFR inhibitors induced apoptosis by suppressing Claspin protein.
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.
| > 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|| |
|1.||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. |
|2.||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. |
|3.||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. |
|4.||Shrader M, Pino MS, Brown G, Black P, Adam L, Bar-Eli M, et al. Molecular correlates of gefitinib responsiveness in human bladder cancer cells. Mol Cancer Ther 2007;6:277-85. |
|5.||Johnson LN. Protein kinase inhibitors: Contributions from structure to clinical compounds. Q Rev Biophys 2009;42:1-40. |
|6.||McHugh LA, Kriajevska M, Mellon JK, Griffiths TR. Combined treatment of bladder cancer cell lines with lapatinib and varying chemotherapy regimens-evidence of schedule-dependent synergy. Urology 2007;69:390-4. |
|7.||Shrader M, Pino MS, Lashinger L, Bar-Eli M, Adam L, Dinney CP, et al. Gefitinib reverses TRAIL resistance in human bladder cancer cell lines via inhibition of AKT-mediated X-linked inhibitor of apoptosis protein expression. Cancer Res 2007;67:1430-5. |
|8.||Dancey JE, Freidlin B. Targeting epidermal growth factor receptor-are we missing the mark? Lancet 2003;362:62-4. |
|9.||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. |
|10.||Deveraux QL, Reed JC. IAP family proteins-suppressors of apoptosis. Genes Dev 1999;13:239-52. |
|11.||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. |
|12.||Deveraux QL, Takahashi R, Salvesen GS, Reed JC. X-linked IAP is a direct inhibitor of cell-death proteases. Nature 1997;388:300-4. |
|13.||Kasof GM, Gomes BC. Livin, a novel inhibitor of apoptosis protein family member. J Biol Chem 2001;276:3238-46. |
|14.||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. |
|15.||Chini CC, Wood J, Chen J. Chk1 is required to maintain claspin stability. Oncogene 2006;25:4165-71. |
|16.||Chini CC, Chen J. Human claspin is required for replication checkpoint control. J Biol Chem 2003;278:30057-62. |
|17.||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. |
|18.||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. |
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