The Proton-Sensing G-Protein Coupled Receptor GPR4 Promotes Angiogenesis in Head and Neck Cancer.
The Proton-Sensing G-Protein Coupled Receptor GPR4 Promotes Angiogenesis in Head and Neck Cancer.
复制标题
DOI：
10.1371/journal.pone.0152789
复制
发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Huang Z
中科院分区：
文献类型：
--
作者：
Jing Z;Xu H;Chen X;Zhong Q;Huang J;Zhang Y;Guo W;Yang Z;Ding S;Chen P;Huang Z
头颈部鳞状细胞癌(SCCHN)是一种侵袭性疾病，存活率低，是全球第六大常见癌症。胃食道反流是SCCHN患者的常见事件。GPR4是一种质子敏感型G蛋白偶联受体，可被酸中毒激活。本研究的目的是探讨GPR4在SCCHN酸暴露和肿瘤血管生成中的作用。在本研究中，我们证实了在SCCHN细胞中过表达GPR4可以在pH为5.9时增加IL6、IL8和VEGFA的表达和分泌。这种作用可被p38抑制剂SB203580所抑制。Western印迹分析表明，在pH值为5.9的GPR4感染细胞中，p38的磷酸化水平升高，SB203580可抑制这一作用。在试管形成实验中，HMEC-1细胞与对照组和GPR4感染的SCCHN细胞的条件培养液(CM，pH 5.9，6.5，7.4)孵育。在pH值为5.9时，GPR4感染细胞与CM共同孵育的HMEC-1细胞较对照细胞管长明显增加，表明GPR4在酸性pH条件下具有促血管生成作用。IL-6、IL-8和VEGFA中和抗体可抑制HMEC-1细胞的管状形成。在体内，采用鸡绒毛膜尿囊膜(CAM)模型观察GPR4对血管生成的影响。将对照组和GPR4感染的SCCHN细胞接种于细胞表面，每24 h加入5μL DMEM/F12(pH值分别为5.9、6.5、7.4)，4d后取上层细胞，用Image-Pro Plus6.0软件计算血管面积与细胞间黏附分子面积的比值。在pH值为5.9时，GPR4感染细胞比对照细胞募集到更多的血管。综上所述，我们认为GPR4通过在酸性细胞外pH条件下诱导p38介导的IL6、IL8和VEGFA的分泌来诱导SCCHN的血管生成。
Squamous cell carcinoma of the head and neck (SCCHN) is an aggressive disease with poor survival and is the sixth most common cancer worldwide. Gastroesophageal reflux is a common event in SCCHN patients. GPR4 is a proton-sensing G-protein coupled receptor, which can be activated by acidosis. The objective of this study was to explore the role of GPR4 in acid exposure and tumor angiogenesis in SCCHN. In this study, we confirmed that overexpressing GPR4 in SCCHN cells could increase the expression and secretion of IL6, IL8 and VEGFA at pH 5.9. This effect could be inhibited by SB203580 (a p38 inhibitor). Western blot analysis indicated that phosphorylation of p38 increased in GPR4 infected cells at pH 5.9, which could be inhibited by SB203580. In tube formation assay, HMEC-1 cells were incubated with conditioned medium (CM, pH 5.9, 6.5, 7.4) derived from control and GPR4 infected SCCHN cells. Tube length was significantly increased in HMEC-1 cells incubated with CM from GPR4 infected cells compared with control cells at pH5.9, which indicated the pro-angiogenic effect of GPR4 in acidic pH. The neutralizing antibodies of IL6, IL8 and VEGFA could inhibit tube formation of HMEC-1 cells. In vivo, the effect of GPR4 on angiogenesis was investigated with the chick chorioallantoic membrane (CAM) model. Control and GPR4 infected SCCHN cells were seeded onto the upper CAM surface (n = 5 in each group) and 5 μL DMEM/F12 (pH 5.9, 6.5, 7.4) was added to the surface of the cell every 24 h. Four days later, the upper CAM were harvested and the ratio of the vascular area to the CAM area was quantified using Image-Pro Plus 6.0 software. GPR4 infected cells could recruit more vascular than control cells at pH5.9. In conclusion, we suggested that GPR4 induces angiogenesis via GPR4-induced p38-mediated IL6, IL8 and VEGFA secretion at acidic extracellular pH in SCCHN.