Abstract：
Glioblastoma (GB) is a very aggressive human brain tumor. The high growth potential and invasiveness make this tumor surgically and pharmacologically untreatable. Our previous work demonstrated that the activation of the M2 muscarinic acetylcholine receptors (M2 mAChRs) inhibited cell proliferation and survival in GB cell lines and in the cancer stem cells derived from human biopsies. The aim of the present study was to investigate the ability of M2 mAChR to modulate cell migration in two different GB cell lines: U87 and U251. By wound healing assay and single cell migration analysis performed by time-lapse microscopy, we demonstrated the ability of M2 mAChRs to negatively modulate cell migration in U251 but not in the U87 cell line. In order to explain the different effects observed in the two cell lines we have evaluated the possible involvement of the intermediate conductance calcium-activated potassium (IKCa) channel. IKCa channel is present in the GB cells, and it has been demonstrated to modulate cell migration. Using the perforated patch-clamp technique we have found that selective activation of M2 mAChR significantly reduced functional density of the IKCa current in U251 but not in U87 cells. To understand whether the M2 mAChR mediated reduction of ion channel density in the U251 cell line was relevant for the cell migration impairment, we tested the effects of TRAM-34, a selective inhibitor of the IKCa channel, in wound healing assay. We found that it was able to markedly reduce U251 cell migration and significantly decrease the number of invadopodia-like structure formations. These results suggest that only in U251 cells the reduced cell migration M2 mAChR-mediated might involve, at least in part, the IKCa channel.
摘要：
胶质母细胞瘤（GB）是一种非常侵袭性的人类脑肿瘤。高生长潜力和侵袭性使这种肿瘤在手术和药理上无法治疗。我们先前的工作表明，M2毒蕈碱乙酰胆碱受体（M2mAChRs）的激活抑制了GB细胞系和源自人类活检的癌症干细胞中的细胞增殖和存活。本研究的目的是研究M2mAChR在两种不同的GB细胞系U87和U251中调节细胞迁移的能力。通过延时显微镜进行的伤口愈合分析和单细胞迁移分析，我们证明了M2mAChRs负调节U251细胞迁移的能力，而不是在U87细胞系中。为了解释在两种细胞系中观察到的不同作用，我们评估了中间电导钙激活钾（IKCa）通道的可能参与。IKCa通道存在于GB细胞中，它已经被证明可以调节细胞迁移。使用穿孔膜片钳技术，我们发现M2mAChR的选择性激活显着降低了U251中IKCa电流的功能密度，而不是U87细胞中的功能密度。为了了解U251细胞系中M2mAChR介导的离子通道密度降低是否与细胞迁移障碍相关，我们测试了IKCa通道的选择性抑制剂TRAM-34的作用，在伤口愈合试验中。我们发现它能够显着减少U251细胞迁移并显着减少侵入足样结构的形成。这些结果表明，只有在U251细胞中，M2mAChR介导的细胞迁移减少可能涉及，至少在某种程度上,IKCa通道.
