Novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines, induce cell cycle arrest and apoptosis in prostate cancer cells

Abstract

Abstract. Advanced hormone-refractory prostate cancer is associated with poor prognosis and limited treatment options. Members of the pyrrolo-1,5-benzoxazepine (PBOX) family of compounds exhibit anti-cancer properties in cancer cell lines (including multi-drug resistant cells), ex vivo patient samples and in vivo mouse tumour models with minimal toxicity to normal cells. Recently, they have also been found to possess anti-angiogenic properties in vitro. However, both the apoptotic pathways and the overall extent of the apoptotic response induced by PBOX compounds tend to be cell-type specific. Since the effect of the PBOX compounds on prostate cancer has not yet been elucidated, the purpose of this study was to investigate if PBOX compounds induce antiproliferative effects on hormone-refractory prostate cancer cells. We examined the effect of two representative PBOX compounds, PBOX-6 and PBOX-15, on the androgen-independent human prostate adenocarcinoma cell line, PC3. PBOX-6 and -15 displayed anti-proliferative effects on PC3 cells, mediated initially through a sustained G 2 /M arrest. G 2 /M arrest, illustrated as DNA tetraploidy, was accompanied by microtubule depolymerisation and phosphorylation of antiapoptotic proteins Bcl-2 and Bcl-x L and the mitotic spindle checkpoint protein BubR1. Phosphorylation of BubR1 is indicative of an active mitotic checkpoint and results in maintenance of cell cycle arrest. G 2 /M arrest was followed by apoptosis illustrated by DNA hypoploidy and PARP cleavage and was accompanied by degradation of BubR1, Bcl-2 and Bcl-x L . Furthermore, sequential treatment with the CDK1-inhibitor, flavopiridol, synergistically enhanced PBOXinduced apoptosis. In summary, this in vitro study indicates that PBOX compounds may be useful alone or in combination with other agents in the treatment of hormone-refractory prostate cancer. IntroductionProstate cancer is the leading cause of cancer and the second leading cause of cancer-related deaths among men in Europe and the USA (1). Early diagnosis following widespread PSA screening has resulted in most patients presenting with localized tumours. These patients generally exhibit good survival rates following radiotherapy or surgery. However, a number of patients still progress to or are diagnosed with locally advanced or metastatic tumours. While androgendeprivation therapy can slow the development of advanced metastatic prostate cancer, most tumours will still progress within a few years to an androgen-independent state (2). At this stage, treatment options become limited. The current treatment of choice for patients with advanced hormonerefractory prostate cancer is docetaxel-based chemotherapy. While these treatments significantly improve the overall survival of patients (3,4), the prognosis still remains poor for patients presenting with advanced stage prostate cancer. Therefore, the development of improved treatments and combinations are required.Our research group have established that some members of the pyrrolo-1,5-benzoxazepine (PBOX) famil...

Evolving therapeutic concepts in prostate cancer based on genome-wide analyses (Review)

“…Numerous studies have explored CDK inhibitors to target tumor cells with inactivated pRB and E2F-induced cyclin/CDK hyperactivity. The pan-CDK (CDK1, CDK2, CDK4/6) inhibitor flavoperidol was found to enhance apoptosis in androgenindependent PC-3 prostate cancer cells (67). An increase in apoptosis and a decrease in angiogenesis was detected when combined with docetaxel in a mouse model of prostate cancer (68,69).…”

Pre-clinical evaluation of a novel class of anti-cancer agents, the Pyrrolo-1, 5-benzoxazepines.

“…Given that the tubulin-targeting pro-apoptotic PBOXs destabilise the microtubules, which in turn compromises kinetochore attachment, the SAC is critical in delaying progression to anaphase. Activation of the spindle assembly checkpoint by means of phosphorylation and activation of BUBR1 was also observed in cancer cells following PBOX-6 and -15 exposure 15, 18. PBOX-15- induced mitotic block was compromised following BUBR1 siRNA, conclusively demonstrating the critical importance of the SAC initiated by BUBR1 in maintaining PBOX-15-induced mitotic arrest 15.…”

“…PBOX-15- induced mitotic block was compromised following BUBR1 siRNA, conclusively demonstrating the critical importance of the SAC initiated by BUBR1 in maintaining PBOX-15-induced mitotic arrest 15. Reduction of BUBR1 protein levels associated with a decline in PBOX-15-induced mitotic block and the onset of apoptosis 15, 18, 21. Furthermore, increased polyploidy was observed in BUBR1-depleted cells exposed to PBOX-15 thus confirming the importance of SAC in preventing genomic instability.…”

Involvement of AMP-activated protein kinase in mediating pyrrolo-1,5-benzoxazepine–induced apoptosis in neuroblastoma cells

“…Additionally, these novel microtubule targeting agents have been shown to induce apoptosis in a numerous in vivo murine cancer models including chronic myeloid leukaemia (CML) [12] and breast cancer [5]. Unlike many chemotherapeutic agents currently used to treat cancers including neuroblastoma, the PBOX compounds are not substrates for many MDR drug efflux pumps [7,8] indicating that they may possess advantages over standard chemotherapy which is currently in clinical use.…”

“…The pyrrolo-1,5-benzoxazepine (PBOX) compounds are a series of novel microtubule-targeting agents which possess the ability to potently induce cell cycle arrest and apoptosis in a number of cancer cell lines, derived from both solid tumours and haematological malignancies [4][5][6][7][8][9][10]. We previously demonstrated that one member of this family of compounds, PBOX-15, induces apoptosis both in ex vivo B-cell lymphocytic leukaemia cells harbouring poor prognostic indicators and fludarabine resistance-associated p53 deletions [11], and in primary CML patient samples including those resistant to Imatinib, the current frontline treatment for CML [12].…”