The novel pyrrolo-1,5-benzoxazepine, PBOX-21, potentiates the apoptotic efficacy of STI571 (imatinib mesylate) in human chronic myeloid leukaemia cells☆
Introduction
Chronic myeloid leukaemia (CML) is a haematological stem cell disorder characterised by the presence of a Philadelphia chromosome. This unique and abnormal chromosome, present in ∼95% of CML patients is caused by the reciprocal t9:22 translocation between two genes, Bcr and Abl coding for a constitutively active tyrosine kinase Bcr-Abl [1], [2]. The Bcr-Abl protein has been identified as having a central role in CML pathology and is responsible for the continuous downstream phosphorylation of many signalling pathways routinely activated in normal cells or involved in cell adhesion [3]. Exactly which of these pathways are responsible for the effects of Bcr-Abl is poorly understood however the ras, Janus kinase-signal transducer and activator transcription factor (STAT), mitogen-activated protein kinase and phosphatidyl inositol-3 kinase pathways are all thought to play key roles in rendering leukaemic blast cells resistant to apoptosis.
In 2001 the release of a novel tyrosine kinase inhibitor, known as STI571 (Imatinib mesylate, Gleevec, Glivec®), quickly became the frontline treatment in CML [4]. STI571 binds to the ATP-binding site of Bcr-Abl, inhibiting downstream phosphorylation. Despite the high rates of hematological and cytogenetic [5] responses to STI571 monotherapy, the emergence of resistance to STI571 [6], [7] has led to the evaluation of combination therapies. Many studies have been conducted to date demonstrating the therapeutic efficacy of STI571 in combination with a range of anti-cancer agents including DNA-damaging agents [8], signalling farnesyltransferase inhibitors like SCH66336 [9], the cyclin-dependent kinase inhibitor flavopiridol [10], histone deacetylase inhibitors such as SAHA [11], [12] and mitogen-activated kinase inhibitors PD98059 and U0126 [13], [14] amongst others.
Apoptosis is a cell suicide mechanism invoked in disparate situations to remove redundant, damaged, or infected cells [15], [16]. One of the best characterised mediators of apoptosis is a family of cysteine proteases known as caspases [17]. These proteases are involved in two distinct apoptotic pathways, the extrinsic and intrinsic pathways. The extrinsic pathway is triggered by death receptors binding to their cognate ligands, whilst the intrinsic pathway is initiated in response to situations such as hypoxia or DNA-damage. This pathway is often called the mitochondrial pathway as it involves release of cytochrome c from the mitochondria and apoptosome formation, a complex consisting of cytochrome c, apoptotic protease-activating factor-1, and pro-caspase-9. Both pathways converge at the level of the effector caspases that leads to the typical biochemical and morphological changes of the apoptotic cell. However, caspase inhibition does not always prevent cell death and their targeted disruption does not always impair apoptosis and therefore roles for non-caspase proteases such as serine proteases have recently attracted more interest [18].
Recently we have reported how a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds have potential as anti-cancer agents. These compounds can be divided into two groups with distinct mechanisms of action; those that exert anti-proliferative effects by arresting cancerous cells in the G1 phase of the cell cycle without eliciting cytotoxic effects, e.g., PBOX-21 [19] and those that exhibit pro-apoptotic ability by binding to tubulin and acting as microtubule targeting agents (MTAs), e.g., PBOX-6 (Fig. 1). This second group of MTAs have been shown to cause arrest in the G2/M phase and potently induce apoptosis in a wide range of solid tumours and cell lines derived from the haematopoietic system and also in vivo[20], [21], [22], [23]. More recently we reported that STI571 potentiated the apoptotic efficacy of a representative pro-apoptotic PBOX compound, PBOX-6, and other MTAs such as paclitaxel and nocodazole, by inhibiting endoreplication [21]. The apoptotic-inducing nature of PBOX-6 may be due to the presence of a naphthyl ring which does not form part of the structure of PBOX-21 (Fig. 1).
In the present study, we sought to determine the effects of STI571 which has previously been shown to cause a G1-arrest, with a PBOX compound that acts in the same stage of the cell cycle. Hence, we examined a representative member (PBOX-21) of the first group of PBOX compounds, the non-cytotoxic G1-arresting compounds, and its ability to lower the apoptotic threshold of STI571. Herein we demonstrate that PBOX-21 synergistically enhanced the apoptotic ability of STI571 in Bcr-Abl-positive CML cells including those resistant to STI571 through activation of both caspases and serine proteases. Collectively these findings highlight the potential therapeutic efficacy in combining STI571 with PBOX-21 as a novel anti-leukaemic strategy.
Section snippets
Cell culture
K562 and HL-60 cells were obtained from the European Cell Culture Collection (Salisbury, UK), K562R cells were a generous gift from Dr. Steven Grant, Division of Haematology/Oncology, Medical College of Virginia, Virginia, US [12]. LAMA84 cells were kindly provided by Dr. Jane Apperley and Dr. Junia Melo, Department of Haematology, Imperial College, London, U.K. Cells were cultured in RPMI-1640 (Glutamax) medium supplemented with 10% foetal bovine serum (FBS) (K562, K562R, LAMA84) or 20% FBS
PBOX-21 induces G1-arrest and augments the apoptotic effect of STI571 in LAMA84 CML cells
To determine the effects of STI571 and PBOX-21 on apoptosis and cell cycle arrest in CML cells, LAMA84 cells were exposed to various concentrations of PBOX-21 in the presence or absence of a pharmacologically relevant concentration of STI571 (250 nM) [12], [21]. The DNA content of propidium iodide-labelled cells was determined by flow cytometric analysis after the times indicated. In analysing the effect of the combination treatment over time (Fig. 2A and B), STI571 was combined with 25 μM
Discussion
The tyrosine kinase inhibitor STI571 has rapidly become frontline treatment for Bcr-Abl-positive CML patients since its release on the market in 2001. The emergence of STI571 resistance, intolerance and dose-limiting toxicity has led to the need to develop novel therapeutic strategies for CML patients. One approach to overcome these problems is to combine STI571 with other anti-cancer agents [8], [9], [13], [14], [30], [31]. We have recently shown that STI571 enhances the apoptotic efficacy of
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2010, Biochemical PharmacologyCitation Excerpt :CI values of <1, =1 and >1 indicate synergism, an additive effect and antagonism, respectively [38]. SDS-polyacrylamide gel analysis was performed as previously described [39]. Briefly, whole cell lysates were prepared in Laemmli buffer and run on 12% gels.
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2017, Central Nervous System Agents in Medicinal ChemistryInvolvement of AMP-activated protein kinase in mediating pyrrolo-1,5-benzoxazepine–induced apoptosis in neuroblastoma cells
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We would like to thank Science Foundation Ireland for funding this project and Elisabeth Buchdunger, Novartis for providing the STI571.