Curcumin inhibits GPVI-mediated platelet activation by interfering with the kinase activity of Syk and the subsequent activation of PLCgamma2.

Turmeric (Curcuma longa), a herbal remedy and culinary spice, has been used in traditional Indian culture for millennia. An active ingredient found in turmeric is curcumin (diferuloylmethane). In the current study, we investigated the antiplatelet properties of this naturally occurring compound. Curcumin inhibited human platelet aggregation and dense granule secretion induced by GPVI agonist convulxin in a concentration-dependent manner. At 50 microM, it effectively inhibited the maximal extent of aggregation and dense granule secretion to as much as 75%. It also dramatically inhibited the activation-dependent tyrosine phosphorylation of Y753 and Y759 on PLCgamma2, but did not affect the phosphorylation of Y145 residue on the cytosolic adaptor protein SLP-76. Interestingly, curcumin had no significant effect on the phosphorylation of Y525/Y526 present on the activation loop of Syk (spleen tyrosine kinase), but had a significant inhibitory effect on in vitro Syk kinase activity. Moreover, the inhibitory action of curcumin is not due to an inhibition of thromboxane generation because all our studies were performed using aspirin-treated platelets. We conclude that curcumin inhibits platelet activation induced by GPVI agonists through interfering with the kinase activity of Syk and the subsequent activation of PLCgamma2.

Anticancer effects of fenretinide in human medulloblastoma.

N-(4-hydroxyphenyl) retinamide (4-HPR, fenretinide) a synthetic retinoid is in clinical trials for the treatment of several malignancies. However, its biological effects and therapeutic value in childhood brain tumor medulloblastoma (MB) has not been investigated. In this study, we report for the first time that fenretinide (2.5-10 microM) induces apoptotic cell death in human MB cells. We observed significant inhibition of cell survival in four MB cell lines (D425MED, D458MED, D283MED and D341MED) as determined by MTT assays. These results were further supported by inhibition of anchorage-independent colony formation in soft agar. Fenretinide-induced decrease in cell viability was in part due to activation of caspase-3 dependent cell death, which was further supported by the cleavage of poly(ADP-ribose) polymerase-1 (PARP-1), a caspase-3 substrate. Cell death was partially prevented by the antioxidant, l-ascorbic acid suggesting that free radical intermediates might be involved in fenretinide effects. These results suggest that pharmacologically achievable concentrations of fenretinide are effective in killing MB cells and thus show its therapeutic potential to treat human MB.

A novel kinase, AATYK induces and promotes neuronal differentiation in a human neuroblastoma (SH-SY5Y) cell line.

Apoptosis Associated Tyrosine Kinase (AATYK), a novel protein recently isolated from differentiating 32D mouse myeloid cells, contains a putative tyrosine kinase domain and several binding motifs for src homology 2 (SH-2) and src homology 3 (SH-3) domain containing proteins. We observed that AATYK is expressed in different regions of the brain. Although it might play a role in normal nervous system development by modulating apoptosis, little is known regarding its function in the brain or its intracellular localization and kinase activity. Recognizing its homology with Insulin like growth factor-I (IGF-I) receptor (IGF-IR) and the critical role of IGF-I in neuronal survival, we hypothesized that AATYK plays an important role in neuronal differentiation/apoptosis. To test this hypothesis, we transfected the human adrenergic neuroblastoma (NB):SH-SY5Y cells with AATYK cDNA under a tetracycline-repressible promoter and established stable cell lines that readily express AATYK on removal of tetracycline. AATYK immunoprecipitated from these cell lysates is an active kinase. Indirect immunofluorescent staining of the clones revealed AATYK to be localized in the cytoplasm. By itself, AATYK overexpression for short duration (2-3 days) did not induce differentiation in the stable SH-SY5Y clones. On the other hand, overexpression for longer periods (7-8 days) per se, significantly (P<0.05-0.001) increased the percent of differentiated cells as well as the neurite length. AATYK-induced differentiation was in the same range as the differentiation induced by agents like all-trans retinoic acid (RA), 12-O-Tetradecanoyl phorbol 13-acetate (TPA) and IGF-I. In addition, AATYK significantly promoted the neuronal differentiation induced by these agents. Our results demonstrate for the first time that AATYK is an active, non-receptor, cytosolic kinase which induces neuronal differentiation and also promotes differentiation induced by other agents in the SH-SY5Y cells.

Role of MAP kinase pathways in primitive neuroectodermal tumors.

Mitogen-activated protein kinase and Phosphatidylinositol-3 kinase/Akt-mediated signaling pathways play a major role in controlling cell proliferation, differentiation and cell death. Phosphorylation and dephosphorylation of their specific Thr/Tyr residues is critical in determining their activity. We determined the expression pattern and activity of MAP kinases and Akt in Primitive Neuroectodermal Tumors (PNETs). The kinase activity of extracellular signal-regulated kinase (ERK) was higher in both primary tumors and cell lines, as evident from the increased phosphorylation of ERK1 and ERK2. We did not observe the activation of C-jun N-terminal kinase (JNK) or p38 MAPK The expression of Raf-1, a kinase acting upstream of ERK, was significantly increased in primary tumors compared to normal brain. The PI-3 kinase-activated phosphorylation of Akt was also higher in primary tumors. These results suggest that activation of the Raf-1/ERK module of the MAP kinase pathway play an important role in PNETs.