P2X7 receptors are a potential novel target for anti-glioma therapies

Journal of Inflammation P2X7 receptors are a potential novel target for anti-glioma therapies Mastura Monif 0 Terence J O'Brien 2 Kate J Drummond 1 Christopher A Reid 3 Simon V Liubinas 1 David A Williams 0 0 Department of Physiology, The University of Melbourne , Melbourne, Victoria 3010 , Australia 1 Department of Surgery, Royal Melbourne Hospital, The University of Melbourne , Melbourne, Victoria 3010 , Australia 2 Department of Medicine, Royal Melbourne Hospital, The University of Melbourne , Melbourne, Victoria 3010 , Australia 3 Howard Florey Institute, The University of Melbourne , Melbourne, Victoria 3010 , Australia Background: Human gliomas pose significant morbidity and mortality to those afflicted by them, and currently there are no curative treatment modalities available for these highly invasive tumours. Methods: With the approval from the human ethics committee, patients diagnosed with brain tumour (glioma) were recruited for this study. At the time of surgical resection, freshly resected tumour as well as 'peri-tumour' tissue were taken directly from theatre to the laboratory and were successfully cultured. Confocal fluorescence microscopy techniques and immunohistochemistry were used for characterization of human glioma cultures. Dye uptake experiments and confocal microscopy were utilized for P2X7 receptor (P2X7R) pore activity. Results: We reveal human glioma cultures to contain microglia in close association with glioma (tumour) cells. Both glioma cells and microglia were found to express the purinergic, ATP sensing, P2X7R. P2X7R protein expression was increased in microglia derived from tumour when compared to 'peri-tumour' tissue. The pore capacity of P2X7R in tumour-associated microglia was functional, as evidenced by dye uptake experiments. Importantly, inhibition of P2X7R with the synthetic antagonist, brilliant blue G (BBG) resulted in a significant decrease in the number of glioma cells in culture. Conclusions: P2X7R was found to be over-expressed in grade IV human gliomas and its pore capacity was functional. Antagonism of P2X7R with BBG resulted in a decrease in tumour cell number. This identifies P2X7R as a promising therapeutic target to combat human glioma proliferation. Microglia; P2X7; Glioma; Cancer; P2X7 pore - Background Gliomas are the most common type of intrinsic brain tumour and a major cause of morbidity and mortality for those afflicted by these highly invasive tumours. The majority of adult gliomas are high-grade astrocytomas, comprising grade 3 anaplastic astrocytoma (AA) and grade 4 glioblastoma multiforme (GBM) [1]. In addition to tumour cells, gliomas contain microglia, which are known to contribute to the tumour mass [2]. Microglia are the immunocompetent cells of the central nervous system. Under normal conditions microglia assume a quiescent/ resting (ramified) phenotype, but in the setting of brain injury or neoplasia microglia become activated [2]. Activated microglia are capable of releasing various immunomodulatory molecules that could alter the course of tumourogenesis [3]. The mechanisms controlling the transition from ramified to activated microglia are not fully understood. We have recently shown that the purinergic receptor, P2X7R, is involved in this transition [4]. The P2X7R is an ATP sensing receptor expressed in cells of haemopoeitic and immunological origin such as monocytes, macrophages, mast cells and microglia [5]. Increased P2X7R expression in microglia in the brain has been reported in the setting of Alzheimers disease [6,7], multiple sclerosis [8], brain ischemia [9] and spinal cord injury [10]. Pharmacological blockade of P2X7R has been shown to be neuroprotective in an animal model of Alzheimers disease [6], enhance recovery in animal models of spinal cord injury [10], and reduce neuroinflammation in an experimental model of autoimmune encephalomyelitis [11]. P2X7R over-expression is also reported in a number of cancers, including those of the breast [12], prostate [12], thyroid [13] pancreas [14], melanoma [15,16], chronic lymphocytic leukemia [17], human neuroblastoma [18], the rat C6 glioma model, and more recently human glioblastoma [19]. However, the role that P2X7R plays in the biology of brain neoplasms is unknown. The P2X7R has dual ionic conductance states. Transient stimulation with agonist (most commonly ATP) opens a P2X7R channel permeable to small cations, whereas sustained agonist stimulation leads to a pore state permeable to moieties of up to 900 Da [20,21]. The P2X7R pore activity has been most commonly associated with consequent cell death, apoptosis or cytolysis [20-23]. Recently, we showed that in transfected rat primary hippocampal neuron glia mixed cultures over-expression of P2X7R was sufficient to induce microglial activation and proliferation [4]. The trophic effects observed were dependant on P2X7R pore activity (not channel), and there was no evidence of P2X7R-induced cell death. Whether P2X7R has a similar action in the setting of human brain tumours is not known. Previous studies have raised questions about the fundamental biological role of P2X7R in the setting of cancer and cell trophism. To explain the over-expression of a purported cytolytic receptor in settings of cell trophism, some have suggested that the receptor must therefore be non-functional to allow trophism rather than cell death [24]. It has also been argued that in the setting of cancer/cell trophism P2X7R is fully functional (intact channel and pore conductance) and it indeed serves a homeostatic anti-tumour function designed to have pro-apoptotic effects to deal with the growing tumour burden [12]. In contrast some studies have shown no evidence of P2X7R-mediated apoptosis, and attribute the trophism/tumour growth to P2X7R function itself [18]. In this study, we reveal increased P2X7R protein expression in microglia cultured from human brain tumour versus peri-tumour (region of macroscopically normal brain surrounding the frank tumour). Pore activity was evident in microglia, indicative of a normally functioning receptor. These observations are also highly supportive of a trophic rather than cell killing role for P2X7R pore. Importantly, total inhibition of P2X7R activity (channel and pore conductances) with brilliant blue G (BBG) reduced the number of tumour cells in culture. The results from this study identify P2X7R as a potential anti-tumour therapeutic target. Methods Human tumour and peri-tumour cultures Protocols for obtaining and handling human brain tissue were reviewed and approved by the Human Research Ethics Committee of the Royal Melbourne Hospital, Victoria, Australia. Written informed consent to study brain tumour/peri-tumour tissue excised during tumour surgery was obtained from patients prior to the operation. Tumour tissue was obtained during routine tumour resection/debulking, and where safe peri-tumour was obtained during the same operation. The peri-tumour tissue c (...truncated)