Paolillo Mayra, Barbieri Annalisa, Zanassi Patrizia, Schinelli Sergio
Dipartimento di Farmacologia Sperimentale ed Applicata, Università di Pavia, Viale Taramelli 14, 27100, Pavia, Italy.
J Neurooncol. 2006 Aug;79(1):1-7. doi: 10.1007/s11060-005-9111-z. Epub 2006 Mar 24.
The endothelins (ETs) are a family of three peptides named ET-1, ET-2 and ET-3 that have been initially isolated as potent vasoactive peptides; ETs are synthesized as precursor proteins (preproETs) and are activated by proteolytic cleavage. ETs exert their biological effects through the activation of two receptors subtypes, ETA and ETB. Recent studies have shown that, besides its vascular effects, ET-1 appears to play a major role also in the growth and progression of various types of cancers and ETA or ETB are alternatively indicated as mediators of the ET-1 biological effects. In this study we have investigated the expression and the amounts of preproET-1, preproET-2, ETA and ETB receptors mRNA by classical RT-PCR and quantitative real-time PCR in one human low grade astrocytoma cell line and eight human glioblastoma cell lines. PCR products corresponding to ETB receptor and preproET-1 were detectable in all the cell lines whilst ETA receptor and preproET-2 were only detected in five cell lines. Quantitative real-time PCR experiments showed wide differences in the amounts of mRNAs among the cell lines examined. Range values were 0.23-4860.51 fg/mug total cDNA for preproET-1; 0.13-3330.18 fg/mug total cDNA for preproET-2; 0.63-286.12 fg/mug total cDNA for ETA and 14.40-6720.67 fg/mug total cDNA for ETB. We measured the ET-1 released in the extracellular medium by an ELISA assay and we found an excellent correlation (correlation coefficient r = 0.9526, P = 0.0003) between the amounts of preproET-1 mRNA and released ET-1 peptide. Finally, in the 1321N1 cell line ETB receptors are functionally coupled to intracellular signaling pathways because the stimulation of ETB receptors by ET-1 induces the phosphorylation of the extracellular regulated kinases (ERKs). Although the majority of glioblastoma cell lines in culture express ET isoforms and ET receptors, we conclude that ET-1 and the ETB receptors are likely to mediate the effects of the ET system in glioblastoma cell lines.
内皮素（ETs）是由ET - 1、ET - 2和ET - 3三种肽组成的家族，最初作为强效血管活性肽被分离出来；ETs以前体蛋白（前体ETs）的形式合成，并通过蛋白水解切割被激活。ETs通过激活两种受体亚型ETA和ETB发挥其生物学效应。最近的研究表明，除了其血管效应外，ET - 1似乎在各种类型癌症的生长和进展中也起主要作用，并且ETA或ETB被交替认为是ET - 1生物学效应的介质。在本研究中，我们通过经典逆转录聚合酶链反应（RT - PCR）和定量实时PCR，研究了一种人低级别星形细胞瘤细胞系和八种人胶质母细胞瘤细胞系中前体ET - 1、前体ET - 2、ETA和ETB受体mRNA的表达和含量。在所有细胞系中均可检测到与ETB受体和前体ET - 1相对应的PCR产物，而ETA受体和前体ET - 2仅在五个细胞系中被检测到。定量实时PCR实验显示，在所检测的细胞系中，mRNA含量存在很大差异。前体ET - 1的范围值为0.23 - 4860.51 fg/μg总cDNA；前体ET - 2为0.13 - 3330.18 fg/μg总cDNA；ETA为0.63 - 286.12 fg/μg总cDNA；ETB为14.40 - 6720.67 fg/μg总cDNA。我们通过酶联免疫吸附测定（ELISA）检测了细胞外培养基中释放的ET - 1，发现前体ET - 1 mRNA含量与释放的ET - 1肽之间存在极好的相关性（相关系数r = 0.9526，P = 0.0003）。最后，在1321N1细胞系中，ETB受体在功能上与细胞内信号通路偶联，因为ET - 1对ETB受体的刺激会诱导细胞外调节激酶（ERK）的磷酸化。尽管培养的大多数胶质母细胞瘤细胞系表达ET异构体和ET受体，但我们得出结论，ET - 1和ETB受体可能介导ET系统在胶质母细胞瘤细胞系中的作用。
