ABSTRACT
Human glioblastoma cells are strikingly refractory to ATP-stimulated, P2X7 receptor (P2X7R)-mediated cytotoxicity. To elucidate the mechanistic basis of this feature, we investigated P2X7R-dependent responses in wild type and P2X7R-transfected U138 cells. Mouse GL261 glioma cells were used as an additional control. Here, we report that wild type U138 glioma cells expressed the P2X7R to very low level. Contrary to human U138 cells, mouse GL261 cells showed strong P2X7R expression and P2X7R-dependent responses. Transfection of wild type P2RX7 into U138 cells fully restored P2X7R-dependent responses. P2RX7 transfection conferred a negligible in vitro growth advantage to U138 cells, while strongly accelerated in vivo growth. In silico analysis showed that the P2RX7 gene is seldom mutated in specimens from glioblastoma multiforme (GBM) patients. These observations suggest that the P2X7R might be an important receptor promoting GBM growth.
ABSTRACT
Malignant pleural mesothelioma (MPM) is an aggressive tumor refractory to anti-blastic therapy. MPM cells show several genetic and biochemical defects, e.g. overexpression of oncogenes, downregulation of onco-suppressor genes, dysregulation of microRNA, or alteration of intracellular Ca2+ homeostasis and of apoptosis. No information is as yet available on purinergic signalling in this tumor. Signalling via the P2X7 (P2RX7 or P2X7R) purinergic receptor is attracting increasing attention as a pathway involved in cancer cell death or proliferation. In this report we show that the P2X7R is expressed by three MPM cell lines established from MPM patients but not by mesothelial cells from healthy subjects (healthy mesothelial cells, HMCs). MPM cell proliferation was inhibited by in vitro incubation in the presence of selective P2X7R antagonists, as well as by stimulation with the P2X7R agonist BzATP. Systemic administration of the selective P2X7R blocker AZ10606120 inhibited in vivo growth of MPM tumors whether implanted subcutaneously (s.c.) or intraperitoneally (i.p.). Our findings suggest that the P2X7R might be a novel target for the therapy of mesothelioma.
Subject(s)
Lung Neoplasms/metabolism , Mesothelioma/metabolism , Pleural Neoplasms/metabolism , Purinergic P2X Receptor Antagonists/pharmacology , Receptors, Purinergic P2X7/metabolism , Adult , Animals , Apoptosis , Biopsy , Cell Line, Tumor , Cell Proliferation , Cytoplasm/metabolism , Humans , Male , Mesothelioma, Malignant , Mice , Mice, Nude , MicroRNAs/metabolism , Signal TransductionABSTRACT
Lymphocyte growth and differentiation are modulated by extracellular nucleotides and P2 receptors. We previously showed that the P2X7 receptor (P2X7R or P2RX7) is overexpressed in circulating lymphocytes from chronic lymphocytic leukemia (CLL) patients. In the present study we investigated the P2X7R/NLRP3 inflammasome axis in lymphocytes from a cohort of 23 CLL patients. P2X7R, ASC and NLRP3 were investigated by Western blot, PCR and transfection techniques. P2X7R was overexpressed and correlated with chromosome 12 trisomy in CLL patients. ASC mRNA and protein were also overexpressed. On the contrary, NLRP3 was dramatically down-modulated in CLL lymphocytes relative to lymphocytes from healthy donors. To further investigate the correlation between P2X7R, NLRP3 and cell growth, NLRP3 was silenced in THP-1 cells, a leukemic cell line that natively expresses both NLRP3 and P2X7R. NLRP3 silencing enhanced P2X7R expression and promoted growth. On the contrary, NLRP3 overexpression caused accelerated apoptosis. The P2X7R was also up-modulated in hematopoietic cells from NLRP3-KO mice. In conclusion, we show that NLRP3 down-modulation stimulates P2X7R expression and promotes growth, while NLRP3 overexpression inhibits cell proliferation and stimulates apoptosis. These findings suggest that NLRP3 is a negative regulator of growth and point to a role of the P2X7R/NLRP3 axis in CLL.
Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Receptors, Purinergic P2X7/metabolism , Aged , Aged, 80 and over , Animals , Apoptosis , CARD Signaling Adaptor Proteins/metabolism , Cell Proliferation , Female , HEK293 Cells , Humans , Inflammasomes/immunology , Inflammasomes/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , NLR Family, Pyrin Domain-Containing 3 Protein/antagonists & inhibitors , NLR Family, Pyrin Domain-Containing 3 Protein/deficiency , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , RNA Interference , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , RNA, Small Interfering/genetics , THP-1 CellsABSTRACT
Extracellular ATP, released upon microbial infection, cell damage, or inflammation, acts as an alert signal toward immune cells by activating P2 receptors. The nucleotide causes microvesicle (MV) shedding from immune and nonimmune cells. Here, we show that IL-18 associates with MVs shed by human ex vivo macrophages upon P2X receptor stimulation. MV shedding was potently induced by ATP and by the P2X7 agonist 3'-benzoylbenzoyl adenosine 5'-triphosphate, while it was greatly reduced by P2X irreversible inhibitor-oxidized ATP and by the specific P2X7 inhibitors KN-62, A-740003, and A-438079. Peculiarly, the P2X7 subtype was highly present in the MVs, while on the contrary the P2X3 and P2X4 subtypes were almost absent. The Ca(2+) ionophore A23187 mimicked the effect of 3'-benzoylbenzoyl adenosine 5'-triphosphate suggesting that an intracellular Ca(2+) increase was sufficient to evoke MV shedding. Caspase inhibitors Ac-YVAD-CMK or Z-YVAD-CMK did not block the cleavage of MV-associated pro-IL-18. Pro-IL-18 formation in macrophages did not require pretreatment of cells with LPS, as the procytokine was already present in unprimed macrophages and did not decrease by incubating cells with the LPS-binding antibiotic polymyxin B nor with the TLR-4 intracellular inhibitor CLI-095. These data reveal a nucleotide-based mechanism responsible for the shedding of MV to which IL-18 is associated.
