Numerous studies have demonstrated the participation of sphingolipids in signal transduction and the regulation of cell growth and apoptosis. [1][2][3] We are continuing attempts to elucidate the biological function of glycosphingolipids (GSLs) found in various invertebrates that do not have sialic acids. 4) With the aim of discovering new anticancer drugs, we have synthesized ten glycolipids found in invertebrates not having sialic acids. Malignant melanoma is very difficult to cure because of its rapid growth, metastasis, and resistance to chemotherapy.5) Many therapeutic approaches to suppress melanoma growth and metastasis have been attempted.
5)Therefore, drugs inhibiting the proliferation of melanoma cells would be of great value. Hess et al. reported that focal adhesion kinase (FAK) increased invasion and migration in aggressive human melanoma cells. 6) They found FAK to be phosphorylated and activated in these cells. 6) Downstream of FAK signaling molecules are phosphatidyliositol 3-kinase (PI3-kinase)-protein kinase B (PKB/Akt) and extracellular signal-regulated kinase (Erk). In previous reports, Erk and Akt pathways were activated in B16 melanoma cells. [7][8][9][10] Further, Davies et al. detected B-raf somatic missense mutations in 60-70% of melanoma cell lines and that the Erk and Akt signaling pathways are constitutively activated.
11)Both these pathways are known to be critically important for cell growth. Furthermore, dominant negative FAK mutants suppressed the metastatic growth of B16F10 cells by downregulating cell proliferation.12) From these reports, FAK and the downstream signal molecules, Erk and Akt, might be targets in the treatment of melanoma. In this paper, we report that glycosphingolipid 7 suppressed cell proliferation and that this effect was associated with suppression of the activation of FAK, Erk, and Akt in melanoma B16F10 cells.
MATERIALS AND METHODS
Cells and ReagentsA mouse melanoma cell line, B16F10 was obtained from Prof. Yoshikazu Sugimoto (Kyoritsu University of Pharmacy) and maintained in Dulbecco's modified Eagle's medium (Nissui Seiyaku, Tokyo) supplemented with 5% heat-inactivated fetal calf serum (FCS, Nippon Bio-Supply Center, Tokyo).Glycolipids 1-10 were synthesized as described previously [13][14][15][16] and dissolved in DMSO to give a stock solution of 50 mM. The stock solution was stored for up to 1 month at 4°C. On the day of experiments, dilutions were made by diluting with incubation medium to 0.5 mM (final DMSO concentration, 1%). Further dilutions were made in medium containing 1% DMSO solvent. Antibodies (Abs) against actin, cyclin D1, 2, and 3, CDK2, and CDK4 were purchased from Santa Cruz Biotechnology (CA, U.S.A.). Antibodies (Abs) against phosphotyrosine (PY) and FAK were purchased from Transduction Lab (KY, U.S.A.), and Abs against Y397-phospho-FAK, pRb and Ser788-, Ser795-, Ser807/811-phosphopRb were from Cell Signaling (MA, U.S.A.). Enhanced chemiluminescence reagents were obtained from GE Healthcare Bio-Sciences (Piscataway, NJ, U.S.A.).Cell Grow...
