Expression of endothelins and their receptors in glioblastoma cell lines

Abstract

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.

The perivascular microenvironment in primary central nervous system lymphomas: the role of chemokines and the endothelin B receptor

“…ET family expression has been reported in many tumors in recent years and the ET family has been described as a family of diverse tumor growth factors [19][20][21][22]. In particular, expression of ET-1 and ETBR is upregulated in various tumors and in some reactive cells, including in brain tumor and reactive glia cells [23][24][25]. Additionally, Buckanovich et al [26] have suggested that the ETBR is involved in the immune escape mechanism of human ovarian cancer tissue.…”

Endothelin B Receptor Expression in Human Astrocytoma: Association With Clinicopathological Variables and Survival Outcomes

“…Since multivariate Cox's proportional hazards model showed that both high expression of the ETB receptor and high Ki-67 LI were independent factors for shorter DFS and OS in astrocytoma patients, proliferation is unlikely the sole mechanism underlying the ET-1/ETB pathway; the ET-1/ETB pathway-mediated survival/anti-apoptotic effect of ET-1 may be another important mechanism, as suggested by other studies (Paolillo et al, 2010;Stiles et al, 1997). The molecular mechanisms underlying the cellular responses to ETB receptor activation in human astrocytomas have been well studied in cell lines (Paolillo et al, 2006(Paolillo et al, , 2010.…”

“…Egidy et al (2000) reported that while the ETA receptor was highly expressed in glioblastoma vessels and in some scattered glioblastoma areas, the ETB receptor was mainly found in tumor cells. Paolillo et al (2006) showed that human glioblastoma cell lines could release ET-1 and express functional ETB receptors, while the ETA receptor appeared to be absent or barely detectable. They also demonstrated that selective ETB receptor antagonists, but not ETA receptor antagonists, blocked proliferation and induced apoptosis in several astrocytoma cell lines (Paolillo et al, 2010), suggesting that the ETB receptor could be a therapeutic target for astrocytomas, particularly glioblastomas.…”

Gene Expression Analysis of anEGFRIndirectly Related Pathway IdentifiedPTENandMMP9as Reliable Diagnostic Markers for Human Glial Tumor Specimens

“…Human glioma cell line U138-MG, derived from a glioblastoma multiforme patient and widely employed, was purchased from ATCC (Rockville, Md, USA); PRT-HU2 cells, previously described [14], were analogously derived from a glioblastoma multiforme patient. U138-MG and PRT-HU2 cells were cultivated in D-MEM medium supplemented with 10% FBS, 100 units/mL penicillin, 0.1 mg/mL streptomycin, and 1% L-glutamine (Invitrogen, Paisley, UK).…”

“…Total RNA was extracted as previously described [14] and accurately quantified using spectrophotometric and fluorimetric (Quant-it RNA Assay, Invitrogen) approaches. The gene-specific primers were designed using the “Primer3 input” software (http://frodo.wi.mit.edu/primer3/), and their specificity was verified using the Primer-BLAST software (http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome/).…”