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Sensitivity Profiles of Human Prostate Cancer Cell Lines to an 80 Kinase Inhibitor Panel

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Sensitivity Profiles of Human Prostate Cancer Cell Lines to an 80 Kinase Inhibitor Panel ANTICANCER RESEARCH 36: 633-642 (2016) Sensitivity Profiles of Human Prostate Cancer Cell Lines to an 80 Kinase Inhibitor Panel AMY J. BURKE1, HUSNAIN ALI1, ENDA O’CONNELL2, FRANCIS J. SULLIVAN1,3 and SHARON A. GLYNN1,4,5 1Prostate Cancer Institute, National University of Ireland Galway, Galway, Ireland; 2Screening Core, National Centre for Biomedical Engineering Science, National University of Ireland Galway, Galway, Ireland; 3HRB Clinical Research Facilities Galway, National University of Ireland Galway, Galway, Ireland; 4Discipline of Pathology, Lambe Institute for Translational Research, School of Medicine, National University of Ireland Galway, Galway, Ireland; 5Apoptosis Research Centre, National University of Ireland Galway, Galway, Ireland Abstract: Background: Taxanes and anti-androgen diagnosed with prostate cancer and 27,540 men will die of therapies are routinely used for the treatment of metastatic cancer of the prostate during 2015 (http://seer.cancer.gov). prostate cancer, however the majority of patients eventually Several choices exist for the treatment of early prostate develop resistance. Materials and Methods: Eighty kinase cancer, including androgen deprivation therapy, radical inhibitors were screened regarding their ability to inhibit cell prostatectomy, external-beam radiation and prostate viability in CWR22, 22Rv1, PC-3 and DU145 prostate brachytherapy, with similar outcomes (1). In patients who cancer cells using automated toxicity assays. Four kinase develop metastatic disease, androgen deprivation therapy and inhibitors were selected for further investigation. Results: No taxanes remain the main therapeutic strategies. While these significant difference in sensitivity patterns was found treatment approaches extend patient survival, they are not between the androgen receptor wild-type CWR22 and its curative and eventually patients develop refractory disease androgen receptor mutant variant 22Rv1, indicating that and progress. The taxane family, that includes paclitaxel, androgen receptor mutation did not impact on kinase docetaxel and the newly-approved cabazitaxel are natural or inhibitor sensitivity in this model. Metastatic PC-3 and semi-synthetic plant derivatives widely used in the treatment DU145 prostate cancer cell lines were less sensitive to of castrate-resistant metastatic prostate cancer. Their kinase inhibitors than the non-metastatic CWR22 and 22Rv1. predominant mechanism of action is through mitotic arrest All four cell lines responded to GSK-3 inhibitor BIO, and (2, 3), by inhibiting microtubule dynamics (4). Since 2010 MEK inhibitor PD198306. DU145 cells were resistant to new drugs have been approved for use in patients with p75NTR/TrkA and CHK4 inhibitors, RO-082750 and metastatic castration-resistant prostate cancer, including Ryuvidine. Conclusion: Kinase inhibition may be an androgen receptor inhibitors (abiraterone acetate and appropriate strategy for the treatment of prostate cancer. enzalutamide), drugs targeting bone metastasis and the microenviroment (alpharadin), immunotherapeutics Prostate cancer is the second most common cancer diagnosed (Sipuleucel-T) and a new previously-mentioned taxane in men globally (1.1 million cases per year), accounting for (cabazitaxel) (5). While these drugs extend life they are not 15% of all cancer cases in men worldwide (http:// curative and therefore there is a need to explore new globocan.iarc.fr) in 2012. In the United States, the National potential therapeutic targets for prostate cancer. Kinase Cancer Institute (NCI) estimates that 220,800 men will be inhibitors represent potential agents for the development of a more personalized approach to treating prostate cancer, however clinical trials on inhibitors such as imatinib (PDGF and c-Kit inhibitor) or lapatinib (EGFR and HER2 inhibitor) Correspondence to: Sharon A. Glynn, Discipline of Pathology, failed to show benefit in phase II clinical trials, indicating Lambe Institute for Translational Research, School of Medicine, that other kinases may be more appropriate targets (6). National University of Ireland Galway, Galway, Ireland. Tel: +353 Herein, we present preliminary data on the efficacy of 80 91494242, Fax: +353 91494242, e-mail: [email protected] kinase inhibitors in vitro against a panel of prostate cancer Key Words: Prostate cancer, kinase inhibitors, androgen receptor, cell lines. The prostate cancer cell lines tested for cell therapy, resistance. viability after kinase inhibitor exposure, represent the 0250-7005/2016 $2.00+.40 633 ANTICANCER RESEARCH 36: 633-642 (2016) transition from non-metastatic androgen-dependent (CWR22 dilution in DMSO, and further diluted 1 in 10 in appropriate media - non metastatic, androgen receptor wildtype), to non- and added to cell culture plates. This was left in culture for 72 h at metastatic androgen-independent (22Rv1 - non metastatic, 37˚C in a 5% CO2 incubator. After 72 h Alamar Blue solution was added to the drug-treated plates and incubated for 8 h at 37˚C in a androgen receptor) and finally androgen receptor-negative 5% CO2 incubator before being read at 530 nm/ 620 nM using a metastatic prostate cancer (PC-3 and DU145 metastatic, Victor X5 Multilabel plate reader. A percentage viability curve was androgen receptor negative). This reflects the clinical path calculated based on these values. Error was presented at +/– the where androgen-sensitive localised prostate cancer percentage coefficient variant (%CV). All cytotoxicity assays were progresses to castrate-resistant disease, and later to castrate- conducted in triplicate. resistant metastatic disease. Western blot analysis. Cells were seeded in 10 cm3 dishes at a cell Materials and Methods density of 1×106 per dish and allowed to reach 80% confluence. Cells were rinsed twice with cold PBS and lysed directly on the dish with cold RIPA buffer (#89900, Fisher Scientific, Dublin, Ireland) Chemicals. The Tocriscreen kinase inhibitor toolbox (Cat. 3514) supplemented with protease inhibitors (#78410, Fisher Scientific, containing 80 kinase inhibitors was purchased from Bio-Techne Dublin, Ireland), scraped, and spun at 14,000 × g for 15 min at 4˚C. (Abingdon, United Kingdom). Unless otherwise stated, all Supernatant was collected and stored at –20˚C for western blot chemicals were obtained from Sigma-Aldrich, Dublin, Ireland. analysis of protein expression. Extracted proteins were quantified using a BCA kit. The expression of p75NTR, TrkA, GSK-3α/β, Cell lines. DU145, PC-3, 22Rv1 and CWR22 were obtained from CDK4 and MEK1/2 were detected using primary anti-p75NTR rabbit the American Type Culture Collection (ATCC) (Manassas, VA, polyclonal antibody #07-476 (Merck Millipore, Cork, Ireland), and USA) and cultured according to recommendations. In brief, CWR22 anti-TrkA rabbit monoclonal antibody #2508, anti-GSK-3α/β XP and 22Rv1 were cultured in RPMI 1640 medium with L-glutamine rabbit monoclonal antibody #5676, anti-MEK1/2 rabbit polyclonal (Sigma #R8758), and supplemented with 10% fetal bovine serum antibody #9122 and anti-CDK4 mouse monoclonal antibody #2906 (FBS) (Sigma #F7524). DU145 was cultured in Minimum Essential (Cell Signalling Technology, Danvers, MA, USA). The anti-p75NTR Medium (1×) with Earles (Gibco #22561-021, Biosciences, Dublin, antibody was diluted 1:1,000, and TrkA antibody 1:1,000, anti-GSK- Ireland) supplemented with 10% FBS. PC-3 was cultured in F12 3α/β antibody 1:1,000, anti-MEK1/2 antibody 1:1,000 and anti- Nutrient Mixture (HAM) medium, with L-glutamine (Gibco CDK4 antibody 1:2,000 in 5% skimmed milk reconstituted in 1x #21765-029) supplemented with 10% FBS. With the exception of tris-buffered saline (TBS) (pH8) 0.1% Tween. These dilutions were RPMI 1640 and FBS (Sigma), all media and supplements were added to the transfer membrane, and shaken overnight at 4˚C, Gibco (Biosciences, Dublin, Ireland). All media contained 1% of following a 1 h RT blocking in 5% skimmed milk in TBS. Mouse (100X) antibiotic-antimycotic (Life Technologies). monoclonal anti-β-actin antibody (Fisher Scientific, Dublin, Ireland) (#10624754) was used to confirm equal protein loading. Secondary Alamar Blue Assay optimisation and Z-Factor calculation. The antibodies used were IRDye 800CW goat anti-rabbit IgG (LI-COR seeding density for the four prostate cancer cell lines and Alamar Biosciences, Cambridge, UK) (#926-32211) and IRDye 680LT goat Blue incubation time were optimised to ensure a Z factor of 0.5-1. anti-mouse IgG (LI-COR Biosciences) (#926-68020) and detection The Z factor is a statistical method used to determine the robustness was imaged on the LI-COR ODYSSEY CLx imaging system. of the assay. A Z-factor of 0.5-1 demonstrates a highly robust assay, while a Z-factor of 0-0.5 indicates a below-par robustness (7). Statistical analysis. Data analysis was performed using the CWR22, 22Rv1, PC3 and DU145 were seeded at a range of densities GraphPad Prism Version 5. All statistical tests were two-sided, and in 96 well plates and treated with 10 μM paclitaxel or 1% DMSO an association was considered statistically significant when p-values control, followed by an Alamar Blue assay at 72 h post-treatment. were less than 0.05. For the kinase panel screen a two-way ANOVA Treated cells were incubated with Alamar Blue (93.33 μM) for 2, 4, with Bonferroni’s multiple comparisons test was used to determine 6 and 8 h. The Z factor for each treatment was calculated based on whether there were significant differences in the sensitivity of values obtained from the entire plate, and values from
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