Galanin Receptor subtypes 2 as therapeutic targets in Head and Neck Squamous cell Carcinoma

目的：甘丙氨酸及其受体GALR1和GALR2被认为是肿瘤抑制因子，是头颈部鳞状细胞癌（HNSCC）的治疗靶点。先前，我们证明了GALR1和GALR2信号通路的功能。在GALR1和GALR2沉默的HNSCC细胞中，我们发现重新表达的GALR1通过细胞外调节蛋白激酶1/2（ERK1/2）介导的对细胞周期蛋白依赖性激酶抑制剂（CKI）和cyclinD1的作用抑制肿瘤细胞增殖。另一方面，在galr2转染的HNSCC细胞中，甘丙肽抑制增殖并诱导凋亡。甘丙肽刺激也介导cyclin D1表达降低，CKI表达增加，p27^<Kip1>和p57^<Kip2>。这些作用与GALR1相似，但GALR2也诱导caspase-3依赖性细胞凋亡，Annexin-V染色和DNA片段分析证实了这一点。因此，我们了解GALR1和GALR2作为肿瘤抑制因子的功能，但GALR2的更多信号通路尚不清楚。在本研究中，我们研究了GALR2在p53突变而GALR1不表达的HNSCC细胞中的信号通路。实验设计：稳定转染剪接位点突变导致46-bp p53脱框缺失的HNSCC细胞系，表达GALR2，并通过免疫印迹检测phospho-ERK1/2的表达。结果：galr2转染细胞经甘丙肽处理后，细胞形态发生改变，细胞数量明显减少，这在模拟转染细胞中没有观察到。在转染galr2的细胞中，甘丙氨酸诱导细胞外调节蛋白激酶1/2（ERK1/2）的激活并抑制细胞增殖，而不是模拟。断掉的细胞。丙氨酸刺激也介导了细胞周期蛋白D1表达的降低。用erk1 /2特异性抑制剂U0126预处理可阻止cyclin D1表达的抑制。结论：本研究表明，重表达的GALR2也通过与GALR1几乎相同的途径抑制肿瘤细胞的增殖。然而，erk1 /2特异性抑制剂不能阻止GALR2介导的细胞凋亡。本研究提示GALR2通过不同的信号通路诱导细胞凋亡或细胞周期阻滞，但需要进一步的研究来详细了解GALR2信号通路。少

Purpose : Galanin and its receptors, GALR1 and GALR2, are known as tumor suppressor and focused as therapeutic targets in head and neck squamous cell carcinoma(HNSCC). Previously, we demonstrated that the function of signaling pathways of GALR1 and GALR2.In HNSCC cells with silenced GALR1 and GALR2, we showed that reexpressed GALR1 suppresseed tumor cell proliferation via the extracellular-regulated protein kinase-1/2(ERK1/2)-mediated effects on the cyclin-dependent kinase inhibitors(CKI) and cyclinD1.On the other hands, in the GALR2-transfected HNSCC cells, galanin suppressed proliferation and induced apoptosis. Galanin stimulation also mediated decreased expression of cyclin D1 and increased expression of the CKI, p27^<Kip1> and p57^<Kip2>. These effects were similar to GALR1, but GALR2 also induced caspase-3-dependent apoptosis, which was confirmed by Annexin-V staining and DNA fragmentation analysis. Thus, we understood the function of GALR1 and GALR2 as tumor suppressor, but the s … More ignaling pathway of GALR2 is still unclear. In this study, we investigated the signaling pathway of GALR2 in HNSCC cells that have mutant p53 and do not express GALR1.Experimental Design : The HNSCC cell line with a splice site mutation causing a 46-bp p53 off-frame deletion, was stably transfected to express GALR2 and examined the expression of phospho-ERK1/2 by immunoblotting. Results : Galanin treatment of the GALR2-transfected cells caused morphologic changes and a marked decrease in cell number that were not observed in the mock transfected cells. In the GALR2-transfected cells, galanin induced activation of the extracellular-regulated protein kinase-1/2(ERK1/2) and suppresses cell proliferation, not the mock. transected cells. Galanin stimulation also mediated decreased expression of cyclin D1.Pretreatment with the ERK1/2-specific inhibitor U0126 prevented the suppression of cyclin D1 expression. Conclusion : This study shows that reexpressed GALR2 also suppresses tumor cell proliferation via the almost same pathway for GALR1.However, the ERK1/2-specific inhibitor could not prevent the GALR2 mediated apoptosis. This study suggest that GALR2 uses the different signaling pathways to induce apoptosis or cell cycle arrest, but further investigations are need to understand the GALR2 signaling pathway in detail. Less