A new screening method was developed that evaluates physiologically relevant chemical selectivity of agonists for insulin-signaling pathways. Phosphorylation (pY939) by an insulin-activated insulin receptor of a target peptide (Y939) derived from an insulin receptor substrate-1 (IRS-1) and its subsequent binding to another downstream target, the SH2 domain of PI-3 kinase (SH2N), were detected by surface plasmon resonance (SPR) spectrometry. This method is based on competitive binding of SH2N to pY939 either in a solution or on the gold surface of the SPR sensor chip. With increasing the concentration of pY939 in solution by the insulin-induced kinase reaction of insulin receptor, SH2N bound to pY939 in solution increases and the one on the sensor chip decreases, thereby causing a decrease in the SPR signal. The amount of thus-detected complex pY939-SH2N was found to depend on added insulin concentrations, confirming that the method utilized part of the sequential transduction mechanism of the insulin-signaling pathways. The kinase activity of insulin receptor-agonist complexes increased in the order of IGF-II < IGF-I < insulin, and neither vanadium ions nor thiazolidine-type medicines for NIDDM, troglitazone and pioglitazone, directly acted on both the kinase reaction of insulin receptor or the binding of pY939 to SH2N. The present approach will thus become a general method for screening agonists for one specific pathway in tyrosine phosphorylation of IRS-1 in insulin signaling, which is regulated by specific protein-protein interaction between a phosphorylated tyrosine in IRS-1 and its corresponding SH2 domain-containing protein such as PI-3 kinase, Grb2-Sos, or SHP2.

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The inhibitory action of prostaglandin D2 (PGD2) and its effect on the cell cycle were examined in cell lines SW480 and LS174T of human colon cancer. The growth of the cell lines were assessed 24 h and 48 h after the addition of 1.0 microgram/ml and 10.0 micrograms/ml PGD2. The growth of SW480 cells was inhibited 48 h, but not 24 h, after the addition of 1.0 microgram/ml, and 24 h and 48 h after the addition of 10.0 micrograms/ml, while that of LS174T was inhibited by both doses after 24 h and 48 h. S-Phase DNA synthesis in the SW480 cells was significantly blocked 24 h after the addition of 10.0 micrograms/ml PGD2. The cell cycle of LS174T cells was arrested at the G0 + G1 phase 24 h after the addition of 1.0 microgram/ml and 10.0 micrograms/ml PGD2. The correlation between hepatic metastasis and PGD2 concentration in human cancer tissue was examined. The mean value of PGD2 concentrations in the primary cancer tissue was significantly lower in the hepatic metastasis group than that in the group without hepatic metastasis. These findings suggest that measuring the PGD2 in cancer tissue may be useful for detecting and predicting the hepatic metastasis from human colorectal cancer.

A fluorescent indicator for tyrosine phosphorylation-based insulin signaling is described. Upon binding of insulin to cell-surface insulin receptor, the receptor phosphorylates tyrosine residues of insulin receptor substrate 1 (IRS-1) in the cell. A fluorescent indicator was designed by using synthetic phosphopeptide pY939 derived from the tyrosine phosphorylation domain of IRS-1 and its target protein SH2N containing an N-terminal SH2 domain of PI 3-kinase. The SH2N protein and pY939 phosphopeptide were labeled with fluorescein (F-SH2N) and tetramethylrhodamine (T-pY939), respectively. Formation of a F-SH2N-T-pY939 complex (termed a fluorescence resonance energy-transfer (FRET) pair) was evaluated from a change in a fluorescence emission spectrum based on FRET between the two fluorophores. The FRET pair was formed to dissociate in competition with the unlabeled pY939 phosphopeptide, resulting in a decrease in a pY939 phosphopeptide-dependent FRET emission at 580 nm and causing an increase in emission at 520 nm. Tyrosine phosphorylation by the partially purified insulin receptor of substrate peptide Y939 was detected with this formed FRET pair, and resulting changes in fluorescence emission spectra were observed for insulin concentration from about 1.0 x 10(-9) to 1.0 x 10(-6) M. These results indicated that the FRET pair served as a competitive fluorescent indicator for tyrosine phosphorylation-based insulin signaling.