Polymethoxyflavones (PMFs) exhibit a broad spectrum of biological properties, including anticancer, antiatherogenic, and neuroprotective effects. The aim of this study is to investigate the neurotrophic effects of 5-demethylnobiletin, a hydroxylated PMF found in citrus plants, and gardenin A, a synthetic PMF analogue, on neurite outgrowth and neuronal differentiation in PC12 cells. The results of this study showed that 5-demethylnobiletin and gardenin A (10-20 ?M) potently induce neurite outgrowth in PC12 cells, accompanied by the expression of neuronal differentiation and synapse formation marker proteins, growth-associated protein-43 (GAP-43), and synaptophysin. We observed that the addition of K252a, a TrKA antagonist, significantly inhibited NGF-induced neurite outgrowth in PC12 cells, but 5-demethylnobiletin- or gardenin A-induced neurite outgrowth was not affected. Treatment with 5-demethylnobiletin and gardenin A markedly induced the phosphorylation of both cyclic AMP response element-binding protein (CREB) and CRE-mediated transcription, which was suppressed through the administration of the inhibitor 2-naphthol AS-E phosphate (KG-501) or using CREB siRNA. Furthermore, our results showed that MAPK/ERK kinase 1/2 (MEK1/2), protein kinase A (PKA), and protein kinase C (PKC) inhibitors blocked the CRE transcription activity and neurite outgrowth induced through 5-demethylnobiletin or gardenin A. Consistently, increased ERK phosphorylation and PKA and PKC activities were observed in PC12 cells treated with 5-demethylnobiletin or gardenin A. These results reveal for the first time that 5-demethylnobiletin and gardenin A promote neuritogenesis through the activation of MAPK/ERK-, PKC-, and PKA-dependent, but not TrkA-dependent, CREB signaling pathways in PC12 cells.
5-Hydroxy-3,6,7,8,3,4-hexamethoxyflavone (5-OH-HxMF), a hydroxylated polymethoxyflavone, is found exclusively in the Citrus genus, particularly in the peels of sweet orange. In this research, we report the first investigation of the neurotrophic effects and mechanism of 5-OH-HxMF in PC12 pheochromocytoma cells. We found that 5-OH-HxMF can effectively induce PC12 neurite outgrowth accompanied with the expression of neuronal differentiation marker protein growth-associated protein-43(GAP-43). 5-OH-HxMF caused the enhancement of cyclic AMP response element binding protein (CREB) phosphorylation, c-fos gene expression and CRE-mediated transcription, which was inhibited by 2-naphthol AS-E phosphate (KG-501), a specific antagonist for the CREB-CBP complex formation. Moreover, 5-OH-HxMF-induced both CRE transcription activity and neurite outgrowth were inhibited by adenylate cyclase and protein kinase A (PKA) inhibitor, but not MEK1/2, protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K) or calcium/calmodulin-dependent protein kinase (CaMK) inhibitor. Consistently, 5-OH-HxMF treatment increased the intracellular cAMP level and downstream component, PKA activity. We also found that addition of K252a, a TrKA antagonist, significantly inhibited NGF- but not 5-OH-HxMF-induced neurite outgrowth. These results reveal for the first time that 5-OH-HxMF is an effective neurotrophic agent and its effect is mainly through a cAMP/PKA-dependent, but TrKA-independent, signaling pathway coupling with CRE-mediated gene transcription. A PKC-dependent and CREB-independent pathway was also involved in its neurotrophic action.
?-Catulin is an oncoprotein that helps sustain proliferation by preventing cellular senescence. Here, we report that ?-catulin also drives malignant invasion and metastasis. ?-Catulin was upregulated in highly invasive non-small cell lung cancer (NSCLC) cell lines, where its ectopic expression or short-hairpin RNA-mediated attenuation enhanced or limited invasion or metastasis, respectively. ?-Catulin interacted with integrin-linked kinase (ILK), a serine/threonine protein kinase implicated in cancer cell proliferation, antiapoptosis, invasion, and angiogenesis. Attenuation of ILK or ?-catulin reciprocally blocked cell migration and invasion induced by the other protein. Mechanistic investigations revealed that ?-catulin activated Akt-NF-?B signaling downstream of ILK, which in turn led to increased expression of fibronectin and integrin ?v?3. Pharmacologic or antibody-mediated blockade of NF-?B or ?v?3 was sufficient to inhibit ?-catulin-induced cell migration and invasion. Clinically, high levels of expression of ?-catulin and ILK were associated with poor overall survival in patients with NSCLC. Taken together, our study shows that ?-catulin plays a critical role in cancer metastasis by activating the ILK-mediated Akt-NF-?B-?v?3 signaling axis.
Luteolin (3,4,5,7-tetrahydroxyflavone), a food-derived flavonoid, has been reported to exert neurotrophic properties that are associated with its capacity to promote neuronal survival and neurite outgrowth. In this study, we report for the first time that luteolin induces the persistent expression of microRNA-132 (miR-132) in PC12 cells. The correlation between miR-132 knockdown and a decrease in luteolin-mediated neurite outgrowth may indicate a mechanistic link by which miR-132 functions as a mediator for neuritogenesis. Furthermore, we find that luteolin led to the phosphorylation and activation of cAMP response element binding protein (CREB), which is associated with the up-regulation of miR-132 and neurite outgrowth. Moreover, luteolin-induced CREB activation, miR-132 expression and neurite outgrowth were inhibited by adenylate cyclase, protein kinase A (PKA) and MAPK/ERK kinase 1/2 (MEK1/2) inhibitors but not by protein kinase C (PKC) or calcium/calmodulin-dependent protein kinase II (CaMK II) inhibitors. Consistently, we find that luteolin treatment increases ERK phosphorylation and PKA activity in PC12 cells. These results show that luteolin induces the up-regulation of miR-132, which serves as an important regulator for neurotrophic actions, mainly acting through the activation of cAMP/PKA- and ERK-dependent CREB signaling pathways in PC12 cells.
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