Gonadotropin-releasing hormone (GnRH) receptors are expressed in prostate cancer, specifically in the most aggressive stage of the tumor (castration-resistant prostate cancer, CRPC) for which the standard treatment, docetaxel-based chemotherapy, can only improve the median survival time by few months. of GnRH agonists, experiments were performed in p53-null PC3 cells. We found that GnRH agonists fail to increase Bax expression and do not potentiate the cytotoxic activity of docetaxel. These results may provide a rationale for novel combination treatment strategies, especially for docetaxel-resistant CRPC patients expressing a functional p53 protein. Introduction Prostate cancer is the most commonly diagnosed cancer for men and the second leading cause of cancer-related deaths among men in Western Countries [1]. Most prostate cancers are dependent on the presence of androgens for growth and survival, and androgen ablation therapy, aimed to block androgen secretion/activity, represents the most effective initial treatment [2], [3]. This therapy includes surgical or chemical castration, achieved by: administration of gonadotropin-releasing hormone (GnRH) analogs; blocking of the binding of androgens to their receptor by antiandrogens; inhibition of steroidogenic enzymes. Unfortunately, despite an excellent initial response, in approximately 2 to 3 3 years, most order KRN 633 prostate cancers will progress to castration-resistant prostate cancer (CRPC) stage with increased proliferation and Rabbit Polyclonal to RPC5 malignancy [4], [5]. For CRPC patients, taxane-based chemotherapy represents the treatment of choice [6], [7]. order KRN 633 Docetaxel acts by binding to tubulin to promote polymerization and prevents microtubule depolymerization in the absence of guanosine triphosphate. It has also been shown to induce tumor cell death by affecting the expression/activity of multiple cancer-specific targets, including downregulation of the antiapoptotic protein Bcl-2 and upregulation of the proapoptotic order KRN 633 protein Bax [8], [9]. However, despite the initial demonstration of a better survival with docetaxel-based chemotherapy, the improvement was found to be only a progression-free survival of few months [6], [10]. Thus, treatment of patients order KRN 633 with CRPC that progresses after docetaxel-based chemotherapy remains a significant clinical challenge. The identification of novel strategies aimed at overcoming docetaxel resistance will likely improve the therapeutic options for these patients. GnRH was first identified as the hypothalamic key regulator of the reproductive functions. By binding to specific receptors (GnRH-R) on pituitary gonadotropes GnRH activates the pituitary-gonadal axis. GnRH agonists, when given continuously and at high doses, desensitize pituitary GnRH-R, thus suppressing gonadal steroid secretion; on the basis of their activity, these compounds represent the most widely and successfully utilized medical treatment for androgen-responsive prostate cancer [2], [11]. It is now well established that GnRH receptors are expressed in prostate cancer cells, specifically in CRPC cells and tissues [12]C[15]. These receptors (as well as GnRH receptors in breast and gynecological cancer cells and tissues) have been first characterized in terms of binding affinity. However, contrasting results have been reported: one class of low-affinity binding sites [12], [13], [16]C[18]; two types of receptors (one with high affinity and one with low affinity) [19]C[21]; one single class of high affinity GnRH binding sites [22]C[25]. In particular, we reported the presence of low affinity GnRH receptors in prostate cancer cells [12], [13]. The reason for this discrepancy is still a matter of debate; however, it might be due to the different experimental conditions adopted (different cancer cell lines and ligands, evaluation of the binding affinity in cancer cells/tissues expressing the binding sites and for 10 minutes at 4C). Caspase-3-like activity was assessed by following the proteolytic cleavage of the colorimetric substrate Ac-DEVD-pNA. Samples were read at 405 nm in a spectrophotometer using a 100 L quarz cuvette. The pan-caspase inhibitor z-VAD-fmk was used to confirm assay specificity. Colony Formation Assay For the development of docetaxel-resistant cells (DU145-R), DU145 cells seeded in 10-cm dishes were serially treated with docetaxel (10 nmol/L, once a week) until they developed the ability to grow and divide in the presence of the drug (8 weeks). To investigate whether GnRH agonists might resensitize DU145-R cells to the activity of docetaxel, a standard clonogenic assay was performed. DU145-R cells were seeded at 1,000 cells/well in six-well plates and allowed to attach for 24 hours. Cells were then treated with GnRH-A (10?6 mol/L) for 24 hours followed by docetaxel (10 nmol/L) for 72 hours. At the end of the treatment, cells were rinsed and.