C1q-tumour necrosis factor-related protein 8 (CTRP8) is a novel interaction partner of relaxin receptor RXFP1 in human brain cancer cells.

We report a novel ligand-receptor system composed of the leucine-rich G-protein-coupled relaxin receptor, RXFP1, and the C1q-tumour necrosis factor-related protein 8 (CTRP8) in human primary brain cancer, a tumour entity devoid of the classical RXFP1 ligands, RLN1-3. In structural homology studies and computational docking experiments we delineated the N-terminal region of the globular C1q region of CTRP8 and the leucine-rich repeat units 7 and 8 of RXFP1 to mediate this new ligand-receptor interaction. CTRP8 secreted from HEK293T cells, recombinant human (rh) CTRP8, and short synthetic peptides derived from the C1q globular domain of human CTRP8 caused the activation of RXFP1 as determined by elevated intracellular cAMP levels and the induction of a marked pro-migratory phenotype in established glioblastoma (GB) cell lines and primary cells from GB patients. Employing a small competitor peptide, we were able to disrupt the CTRP8-RXFP1-induced increased GB motility. The CTRP8-RXFP1-mediated migration in GB cells involves the activation of PI3K and specific protein kinase C pathways and the increased production/secretion of the potent lysosomal protease cathepsin B (cathB), a known prognostic marker of GB. Specific inhibition of CTRP8-induced cathB activity effectively blocked the ability of primary GB to invade laminin matrices. Finally, co-immunoprecipitation studies revealed the direct interaction of human CTRP8 with RXFP1. Our results support a therapeutic approach in GB aimed at targeting multiple steps of the CTRP8-RXFP1 signalling pathway by a combined inhibitor and peptide-based strategy to block GB dissemination within the brain.

Relaxin enhances the collagenolytic activity and in vitro invasiveness by upregulating matrix metalloproteinases in human thyroid carcinoma cells.

In this study, we identified differential expression of immunoreactive matrix metalloproteinase 2 (MMP2)/gelatinase A, membrane-anchored MT1-MMP/MMP14, and human relaxin-2 (RLN2) in human benign and malignant thyroid tissues. MMP2 and MT1-MMP were detected in the majority of thyroid cancer tissues and colocalized with RLN2-positive cells. MMP2 was mostly absent in goiter tissues and, similar to RLN2, may serve as a marker for thyroid cancer. MMP2 and MT1-MMP were identified as novel RLN2 targets. RLN2 caused a significant downregulation of tissue inhibitor of MMP (TIMP) 3 protein levels but did not change the expression levels of MMP13, and TIMP1, TIMP2, and TIMP4 in human thyroid carcinoma cells. RLN2 failed to affect the expression of MMP1, 3, 8, and 9 in the thyroid carcinoma cells investigated. Stable RLN2 transfectants secreted enhanced levels of bioactive MMP2 which contributed to the increased collagenolytic activity and in vitro invasiveness into collagen matrix by human thyroid cancer cells. Three-dimensional reconstitution of confocal fluorescent microscopy images revealed larger-sized invadopodia, with intense MT1-MMP accumulation at the leading migrating edge in RLN2 transfectants when compared with enhanced green fluorescent protein clones. In RLN2 transfectants actin stress fibers contributed to pseudopodia formation. In conclusion, enhanced tumor cell invasion by RLN2 involves the formation of MT1-MMP-enriched invadopodia that lead to increased collagenolytic cell invasion by human thyroid cancer cells.

Simple sequential injection analysis system for rapid determination of microalbuminuria.

A simple, specific and sensitive sequential injection analysis (SIA) system based on non-immunoassay fluorescent detection has been developed for the determination of urinary albumin. The specific binding of the dye Albumin Blue 580 (AB 580) to albumin in urine generated high emission fluorescent signals. The excitation and emission wavelengths were set at 590 and 610 nm, respectively. The analytical range was obtained from 1 to 100 mg L(-1), with a detection limit of 0.3 mg L(-1) (S/N=3). The SIA system gave high precision with relative standard deviations (R.S.D.s) of 0.9% and 1.4% when evaluated with 15 and 100 mg L(-1) albumin (n=15), respectively. The method exhibited good reproducibility, as assessed by performing four analytical curves on different days, and intra-run CVs (2.3-3.3%) and inter-run CVs (3.8%) were obtained. Rapid operation was achieved with a sample throughput of 37 h(-1). This method was successfully applied to the determination of urinary albumin, and the method was highly correlated with the immunoturbidimetric method (r(2)=0.965; n=72).