Inhibition of Bruton's Tyrosine Kinase Suppresses Cancer Stemness and Promotes Carboplatin-induced Cytotoxicity Against Bladder Cancer Cells

Materials and Methods

Bioinformatics analysis of BTK expression. In the beginning, we evaluated the BTK expression pattern in human bladder cancer and normal tissues using the Gene Expression database of Normal and Tumor tissues (GENT2). The data were analyzed from the microarray data of published urothelial carcinoma transcriptome GSE32894, GSE31684 and GSE13507. The hazard ratio of overall survival was investigated.

Cell lines, reagents and chemicals. T24 human BLCA and HT 1197 human BLCA cell lines were purchased from Bioresources Collection and Research Center (Hsin Chu, Taiwan, ROC). The BTK inhibitor ibrutinib was purchased from Sigma-Aldrich, Merck KGaA (Darmstadt, Germany). Carboplatin was purchased from Accela ChemBio. Inc. (San Diego, CA, USA). alamarBlue™ Cell Viability reagent, L-glutamate, fetal bovine serum, TrypLE Express Enzyme solution, McCoy's 5a basal medium (for the culture of T24 cells) and minimum essential medium (Eagle) with Earle's balanced salt solution (for the culture of HT 1197 cells) were purchased from Gibco, Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Anti-IgG1-allophycocyanin (APC) and anti-CD133-APC human monoclonal antibodies were obtained from BD Biosciences (San Jose, CA, USA).

Human BLCA cell culture. The T24 and HT 1197 human BLCA cell lines were cultured in complete media (90% basal medium containing 1.5 g/l sodium bicarbonate and 10% fetal bovine serum) in a humidified cell culture incubator at 37°C and 5% CO2. The cells were detached using TrypLE Express Enzyme solution for cell passage.

Cell surface marker recognition for analyzing the stemness of BLCA cells. The parental BLCA cells were harvested and incubated with anti-IgG1-APC and anti-CD133-APC human monoclonal antibodies. Following 30 min of incubation at room temperature in the dark, the cells were centrifuged at 300 × g for 10 min at 25°C. The supernatant was then aspirated and the cell pellet was resuspended and gently mixed with 1 ml phosphate-buffered saline. Finally, the cells were analyzed and sorted using a flow cytometer (FACSAria™ II cell sorter; BD Biosciences, San Jose, CA, USA). The harvested CD133⁺-side population (SP) cells were maintained in serum-free medium with basal medium-containing basic fibroblast growth factor (10 ng/ml), leukemia inhibitory factor (10 ng/ml) and epidermal growth factor (20 ng/ml), and incubated in a low-adherent Petri dish in a humidified cell culture incubator at 37°C and 5% CO₂ for 7 days and the medium refreshed every 3 days.

Western blotting. SP and parental BLCA cells were harvested and lysed in cold RIPA lysis buffer containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich; Merck KGaA). Total protein was quantified using a Bradford protein assay and the protein lysate was separated via gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis. The separated proteins were subsequently electrotransferred onto a polyvinylidene fluoride membrane (Sigma-Aldrich) and blocked. The membranes were then incubated with primary antibodies to the following: BTK, phosphorylated mammalian target of rapamycin (mTOR), total mTOR and β-actin, respectively. Following incubation with primary antibody, the membranes were incubated with specific horseradish peroxidase-conjugated secondary antibody. All antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Protein bands were quantified using chemiluminescence (EMD Millipore, Billerica, MA, USA) and a ChemiDoc™ Imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal protein loading was confirmed by analyzing the expression levels of β-actin.

Knockdown of BTK expression. BTK expression was knocked down in BLCA cells using short hairpin RNA against BTK (shBTK). A pLKO.1-puromycin-based vector expressing shBTK was purchased from Academia Sinica (Taipei, Taiwan, ROC). The BLCA cells were transfected with 10 μg/ml shBTK for 4 h prior to the replacement of the cell culture medium.

Wound-healing assay. A wound-healing assay was performed to determine the cell migratory ability of transfected and non-transfected cells. Briefly, a 200-μl pipette tip was used to scratch the surface of the cell monolayer. Following 24-hour incubation, the cell migratory ability was quantified using an inverted microscope equipped with a CCD camera (Olympus Corporation, Tokyo, Japan) and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Cytotoxicity assay of the BTK inhibitor, ibrutinib, in combination with carboplatin in BLCA cells. BLCA cells were treated independently with carboplatin (0-100 μM) or ibrutinib (0-20 μM), or in combination, for 24 and 48 h, and the cell viability was subsequently determined. Cell viability assay was performed using alamarBlue reagent. Briefly, after washing the cells with phosphate-buffered saline, the cell culture medium was replaced with basal medium-containing alamarBlue reagent and incubated in a cell culture incubator for another 3 hours. The fluorescence intensity was measured using an excitation wavelength of 544 nm and an emission wavelength of 590 nm on an ELISA reader (SpectraMax M2^e; Molecular Devices, LLC, Sunnyvale, CA, USA). The cell viability (%) was calculated using the following equation: [(Fluorescence_treated−Fluorescence_control)/Fluorescencecontrol] ×100.

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Figure 1.

Forest plot of studies included in the meta-analysis of the expression of Bruton's tyrosine kinase (BTK) and the risk of bladder cancer mortality. Data were adapted from GENT2 platform (http://gent2.appex.kr/gent2/). CI: Confidence intervaI; HR: hazard ratio; TE: Ln(HR); seTE: standard error of HR.

Statistical analysis. All data were analyzed using SPSS 20.0 software (IBM Corp., Armonk, NY, USA) and are presented as the mean±SEM of three or more independent experiments. Statistical differences between two groups were analyzed using a one-way ANOVA, followed by a post hoc test. A value of p<0.05 was considered to indicate a statistically significant difference.