Colorimetric heparinase assay for alternative anti-metastatic activity.

Heparanase has been previously associated with the metastatic potential, inflammation, and angiogenesis of tumor cells. Heparanase activity has been detected by means of UV absorption, radiolabeled substrates, electrophoretic migration, and heparan sulfate affinity assays. However, those methods have proven to be somewhat problematic with regards to application to actual biological samples, the accessibility of the immobilized substrates, experimental sensitivity, and the separation of degraded products. Rather than focusing on heparanase activity, then, we have developed a rapid, alternative colorimetric heparinase assay, on the basis of the recent finding that sulfated disaccharides generated from heparin by bacterial heparinase exhibit biological properties comparable to those from heparan sulfate by mammalian heparanase. In this study, the concentrations of porcine heparin and bacterial heparinase I were determined using a Sigma Diagnostics Kit. Morus alba was selected as a candidate through this assay system, and an inhibitor, resveratrol, was purified from its methanol extract. Its anti-metastatic effects on the pulmonary metastasis of murine B16 melanoma cells were also evaluated. Our findings suggest that this assay may prove useful as a diagnostic tool for heparinase inhibition, as an alternative anti-metastatic target.

Inhibition of phospholipase C gamma 1 by the prenylated flavonoids from Sophora flavescens.

The effect on the phospholipase C gamma 1 activity of eleven prenylated flavonoids from Sophora flavescens was investigated. These flavonoids exhibited relatively strong inhibitory activity with IC50 values ranged from 7.5 x 10(-6) M to 35 x 10(-5) M with the exception of kushenol H (4) (IC50 value; > 5.3 x 10(-4) M). The presence of C3-OH resulted in a significant diminution of activity and the configuration of C3-OH is likely to be another factor influencing the activity. In addition, hydration of the C-4"'-C-5"' double bond of the lavandulyl side chain caused complete loss of activity. These data suggest that the presence and configuration of C3-OH are related to the inhibitory activity and the lavandulyl side chain is also important for high inhibitory activity against PLC gamma 1.

Inhibition of phospholipase C gamma 1 activity by amentoflavone isolated from Selaginella tamariscina.

Amentoflavone was isolated as an inhibitor of phospholipase C gamma 1 (PLC gamma 1) and phosphoinositides (PI)-turnover in PLC gamma 1 overexpressing NIH3T3 fibroblasts (NIH3T3 gamma 1) from Selaginella tamariscina (Selaginellaceae) together with other related biflavonoids, isocryptomerin and cryptomerin B. Only amentoflavone inhibited the PLC gamma 1 activity with an IC50 of 29 microM and the formation of total inositol phosphates (IPt) in PDGF-stimulated NIH3T3 gamma 1 with an IC50 of 9.2 microM but did not show inhibitor activity against protein kinase C. Isocryptomerin and cryptomerin B did not show inhibitor activity against PLC gamma 1 at the concentration of 150 microM, and did not inhibit IPt production in PDGF-induced NIH3T3 gamma 1 at the concentration of 180 microM.