Dual targeting of tumour cells and host endothelial cells by novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines

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 tends 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 anti-proliferative 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 anti-apoptotic proteins Bcl-2 and Bclx 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 PBOX-induced 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.3

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

“…These values are within a similar range (1-6 and 0.2-0.4 μM for PBOX-6 and -15, respectively) to those observed in other cell lines such as CML K562 cells, promyelocytic leukaemia HL60 cells, ovarian carcinoma A2780 cells and a range of mammary carcinoma cell lines (10,11,14,16). This reduction in PC3 cell proliferation was mediated through a significant G 2 /M arrest followed by treatment for 24 h in either fresh media (as a control) or media containing flavopiridol (0.1-0.5 μM).…”

“…Interestingly, these compounds exert only minimal toxicity to normal peripheral blood mononuclear cells (PBMCs) (8) or bone marrow cells (13). In addition, we have also found that PBOXs displayed antiangiogenic properties in vitro (10). An added advantage of PBOX compounds over many clinically used anti-cancer agents such as docetaxel is that they do not appear to be sub- ONCOLOGY REPORTS 24: 1499-1507 Novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines, induce cell cycle arrest and apoptosis in prostate cancer cells strates of the ABC-transporters, P-glycoprotein and BCRP, since they are capable of inducing apoptosis in multidrugresistant cancer cells expressing these transporters with similar efficacy as in transporter-negative drug-sensitive cells (14).…”

“…Our research group have established that some members of the pyrrolo-1,5-benzoxazepine (PBOX) family of compounds are capable of inducing apoptosis in cancerous cell lines derived from both haematological malignancies [promyelocytic leukaemia HL60 cells, Jurkat T-lymphoma cells, Hut-78 lymphoma cells, T cell leukaemia CEM cells and chronic myeloid leukaemia (CML) K562 cells] and solid tumours (breast carcinoma MCF-7 cells and ovarian A2780 cells) (5)(6)(7)(8)(9)(10). These results are supported by studies revealing impaired growth of tumours in a mouse 4T1 breast carcinoma tumour model (11), and a CML mouse model (12) and apoptosis in ex vivo CML and chronic lymphocytic leukaemia (CLL) patient samples (12,13).…”

Novel cis-restricted β-lactam combretastatin A-4 analogues display anti-vascular and anti-metastatic properties in vitro

“…As we previously reported that exposure of normal breast epithelial cells to CA-432 induced only a minimal amount of cytotoxicity with an IC 50 value of greater than 10 mM (19), it is interesting that the compounds had such a potent effect on non-cancerous endothelial cells. This phenomenon has been reported with CA-4P (38) and numerous other microtubuletargeting agents (22,39) and is postulated to be attributable to a variety of mechanisms including enhanced uptake mechanisms in endothelial cells (40) or differences in endothelial cell tubulin composition, its post-translational modifications or its microtubule-associated proteins (41,42).…”

“…HUVECs (60,000 cells/chamber) were cultured on BD Falcon™ 4-chamber glass slides (BD Biosciences, Bedford, MA, USA) for 24 h. Following treatment for 16 h, the cells were fixed in 100% methanol at -20˚C and the microtubular network was detected by indirect immunofluorescence as previously described (22). Briefly, the slides were sequentially incubated in a blocking solution [5% (w/v) BSA/0.1% (v/v) Triton X-100/PBS], monoclonal anti-α-tubulin antibodies (Merck Biosciences, Nottingham, UK), fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse antibodies (DakoCytomation, Glostrup, Denmark) and finally 0.2 µg/ml propidium iodide (to stain DNA).…”