4C). Open in a separate window Figure 4 Glucocorticoid receptor (GR) antagonism downregulates the expression of antiapoptotic Bcl-2 and Bcl-xL proteins. indicating a key role of the GR in docetaxel resistance. The capability of the GR antagonists (RU-486 and cyproterone acetate) to revert docetaxel resistance was investigated and revealed significant resensitization of docetaxel-resistant PCa cells for docetaxel treatment in a dose- and time-dependent manner, in which a complete restoration of docetaxel sensitivity was achieved in both androgen receptor (AR)-negative and AR-positive cell lines. Mechanistically, we demonstrated down-regulation of Bcl-xL and Bcl-2 upon GR antagonism, thereby defining potential treatment targets. In conclusion, we describe the involvement of the GR in the acquisition of docetaxel resistance in human PCa. Therapeutic targeting Prostaglandin E2 of the GR effectively resensitizes docetaxel-resistant PCa cells. These findings warrant further investigation of the clinical utility of the GR antagonists in the management of patients with advanced and docetaxel-resistant PCa. and test. Cell culture and reagents PC3, DU145, and 22Rv1 cells were cultured in RPMI-1640 supplemented with FCS, penicillin/streptomycin, Prostaglandin E2 and glutamine. Docetaxel-resistant Prostaglandin E2 cells (PC3-DR, DU145-DR, and 22Rv1-DR) were generated by increasing exposure to docetaxel and subsequently cultured under the presence of 12.5?nM docetaxel (O’Neill release in the intrinsic apoptotic pathway, in docetaxel-resistant cell lines compared with their chemonaive counterparts (Fig. 4B). Interestingly, GR antagonism resulted in decreased expression of antiapoptotic Bcl-xL and Bcl-2 in both docetaxel-resistant cells (Fig. 4B). This suggests that the sensitizing effects of the GR antagonism may be partially mediated via modulation of the Bcl-2/Bcl-xL axis. To further explore this, a selective antagonist for Bcl-2 and Bcl-xL was investigated: ABT-263. Treatment with ABT-263 already induced cell death in PC3-DR and DU145-DR cell lines (Fig. 4C). On top of this, ABT-263 significantly resensitized both docetaxel-resistant cell lines to docetaxel treatment (Fig. 4C). Since this effect with ABT-263 was not as potent as the effect observed with RU-486, other mechanisms in addition to Bcl-xL/Bcl-2 downregulation are presumably involved in the resensitization upon the GR inhibition. TLN1 This notion is supported by the Prostaglandin E2 observation that the sensitivity to docetaxel is enhanced in both docetaxel-resistant cell lines if treated with both RU-486 and ABT-263 compared to RU-486 or ABT-263 alone (Fig. 4C). Open in a separate window Figure 4 Glucocorticoid receptor (GR) antagonism downregulates the expression of antiapoptotic Bcl-2 and Bcl-xL proteins. (A) Docetaxel-resistant cells undergo apoptosis upon treatment with RU-486 (3?M) and docetaxel (30?nM). ***vitroand in tumor biopsies from enzalutamide-pretreated PCa patients (Arora experiments and wrote the manuscript. M Puhr performed and analyzed the immunohistochemical study with the TMA and established the PC3-DR and DU145-DR cell lines. J T Buijs, G van der Horst, and D M Hemmer contributed to the data acquisition and interpretation. K A Marijt designed and cloned the CRISPR/CAS9 plasmids. M S Hwang, M Masood, and S Grimm carried out the western blot analysis of antiapoptotic proteins. J M Metselaar, G Storm, O C Meijer, and Z Culig provided invaluable intellectual input on the study design and concepts. G van der Pluijm supervised J Kroon, provided intellectual input and helped writing the manuscript. All co-authors improved the manuscript and approved its final version. Acknowledgements The authors thank Hetty Prostaglandin E2 Sips for technical assistance and Sander Kooijman for critical reading of the manuscript. We thank Prof. Dr William Watson (University College Dublin) for providing the 22Rv1 parental and 22Rv1 docetaxel-resistant cell lines. Declaration of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Funding J Kroon is supported by NanoNextNL Drug Delivery programme 03D.01. M Puhr is supported by an Austrian Science Fund (FWF) grant number P25639-B19. J T Buijs is supported by the Netherlands Organisation for Scientific Research (NWO, VENI-grant-916.131.10). G van der Horst is supported by the Dutch Cancer Society (KWF, UL-2011-4030)..