u-0126 and verbenachalcone

Past research has shown that natural products of plant and marine origins and their congeners enhance the actions of neuritogenic factors of the central nervous system (CNS) such as nerve growth factor (NGF). However, the role of fluorine substitutions in their structure-activity relationship (SAR) has not been explored. We have synthesized a trifluoromethyl analog of verbenachalcone (VC), a pharmacologically active natural compound previously shown to potentiate NGF activity. This analog, designated C278, enhances neurite outgrowth and proliferation of NeuroScreen-1™ (NS-1) cells, a subclone of PC12 pheochromocytoma cells. C278 increases the percentage of neurite bearing cells in the presence of suboptimal doses of NGF in comparison with controls treated with NGF alone. In addition, C278 stimulates cell growth in reduced serum and serum-free cell culture conditions based on our observation of increases in cell number and metabolic assessment with MTT reduction and resazurin assays. The addition of C278 partially restored inhibition of NGF-induced neurite outgrowth by the mitogen-activated protein kinase kinase (MEK) inhibitors PD98059 and U0126. Short-term sequential exposure of cells to U0126, C278, and NGF enhanced phosphorylation of extracellular signal-regulated kinase (ERK) in comparison with cells treated with only the MEK inhibitor and NGF. C278 also attenuated cell growth arrest caused by exposure to PD98059, U0126 and the phosphatidylinositol-3 kinase (PI3K) inhibitor, LY294002 but did not alter phosphorylation of Akt, a classic downstream target of PI3K during cell survival. These data suggest that C278 promotes NGF-dependent neurite outgrowth in NS-1 cells through a MEK signaling pathway by a mechanism that alters short-term activation of ERK. In contrast, C278 promotes PI3K-mediated survival independently of Akt phosphorylation.

Topics: Animals; Butadienes; Cell Line; Cell Proliferation; Chalcone; Chalcones; Chlorofluorocarbons, Methane; Culture Media, Serum-Free; Drug Evaluation, Preclinical; Flavonoids; Models, Biological; Neural Stem Cells; Neurites; Nitriles; PC12 Cells; Protein Kinase Inhibitors; Rats

This study uses NeuroScreen-1 (NS-1) cells, a derivative of pheochromocytoma (PC12) cells, to examine neurite outgrowth induced by a novel synthetic verbenachalcone derivative, DSRB20-022 (C22). We treated NS-1 cells with varying concentrations of C22 in the presence of 2ng/mL nerve growth factor (NGF). A dose-dependent effect of C22 was observed at concentrations of 2microM and above, resulting in significant enhancement of NGF-dependent neurite outgrowth in NS-1 cells. C22 did not exhibit neuritogenic activity in the absence of NGF, but promoted a concentration-dependent increase in neurite-bearing cells without inducing cytotoxicity. Cell viability assays showed that C22 and the parent compound verbenachalcone (VC) are neuroprotective and enhanced survival of NS-1, PC12, and the murine neuro-2A (N2a) cell lines under conditions of serum deprivation. The results show that augmentation of NGF-induced neurite outgrowth by C22 in NS-1 was dependent on MAP kinase. Furthermore, the neuroprotective function of C22 and VC was accompanied by suppression of caspase-3/7 activation. However, C22 and VC exerted their antagonistic effects on caspase-3/7 activation through potentially different mechanisms of action.

Topics: Animals; Butadienes; Caspase 3; Caspase 7; Cell Line; Cell Line, Tumor; Cell Survival; Chalcones; Coumarins; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Mice; Nerve Growth Factor; Neurites; Neuroprotective Agents; Nitriles; Oligopeptides; Rats