FTY720, a synthetic sphingoid base analog, was examined as a new sphingosine kinase inhibitor, which converts endogenous sphingosine into its phosphate form. With 20 micrometer of FTY720, sphingosine accumulated in the LLC-PK1 cells in a time- and dose-dependent manner. The FTY720 treated cells showed a high concentration of fragmented DNA, a high caspase-3 like activity and TUNEL staining cells. It was also found that the sphingosine and sphinganine level increased in a time- and dose-dependent manner within 12 h after the FTY720 treatment. The sphingosine kinase activity was reduced by FTY720 as much as other sphingosine kinase inhibitors, N, N-dimethylsphingosine (DMS), dl-threo-dihydrosphingosine (DHS). The fragmented DNA content as a result of the 20 micrometer of FTY720 treatment and by 5 micrometer of the exogenously added BSA-sphingosine complex indicated typical apoptosis. Under similar conditions, the accumulated sphingosine concentration in all the cells was almost identical even though the sphingosine distribution inside the cells was somewhat different. These results indicate that the FTY720 induced apoptosis is associated with the inhibition of the sphingosine kinase activity and is strongly associated with the successive accumulation of sphingosine.
Animals ; Apoptosis/*physiology ; Caspases/biosynthesis ; Cell Line ; DNA Fragmentation ; Endothelial Cells/drug effects ; Enzyme Inhibitors/*pharmacology ; Kidney/cytology ; Phosphotransferases (Alcohol Group Acceptor)/*antagonists & inhibitors/physiology ; Propylene Glycols/*pharmacology ; Research Support, Non-U.S. Gov't ; Sphingosine/pharmacology/*physiology ; Swine ; Up-Regulation

Sphingolipids as an important regulator play a critical role in the cell biological functions. Among them, ceramide (Cer) and sphingosine (Sph) induce apoptosis and inhibit cell proliferation; on the contrary sphingosine 1-phosphate (S1P) promotes cell survival and proliferation. The balance between ceramide/sphingosine and S1P forms a so-called "sphingolipid-rheostat", which decides the cell fate. Sphingosine kinases, which catalyze the phosphorylation of sphingosine to S1P, are critical regulators of this balance. Here, we review the role of sphingosine kinase 1 (SphK1) in regulating fundamental biological processes and tumorigenesis and the potential of SphK1 as a new target for cancer therapeutics.
Amino Alcohols ; pharmacology ; Animals ; Apoptosis ; drug effects ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Ceramides ; metabolism ; Enzyme Activation ; Enzyme Inhibitors ; pharmacology ; Humans ; Lysophospholipids ; metabolism ; Neoplasms ; metabolism ; pathology ; Neovascularization, Pathologic ; Phosphorylation ; Phosphotransferases (Alcohol Group Acceptor) ; antagonists & inhibitors ; metabolism ; Sphingosine ; analogs & derivatives ; metabolism ; Thiazoles ; pharmacology

Sphingosine kinase 1 (SphK1) plays critical roles in cell biological functions. Here we investigated the effects of SphK1 inhibitor SKI II on hepatoma HepG2 cell cycle progression and invasion. Cell survival was determined by SRB assay, cell cycle progression was assayed by flow cytometry, the ability of cell invasion was measured by Matrigel-Transwell assay and protein expression was detected by Western blotting. The results showed that SKI II markedly inhibited HepG2 cell survival in a dose-dependent manner, induced G1 phase arrest in HepG2 cell and inhibited cell invasion. SKI II markedly decreased the expressions of G1-phase-related proteins CDK2, CDK4 and Cdc2 and the levels of cell invasion-associated proteins MMP2 and MMP9. The results showed that SKI II inhibited cell cycle progression and cell invasion, implying SphK1 as a potential target for hepatoma treatment.
CDC2 Protein Kinase ; Cell Movement ; drug effects ; Cell Survival ; drug effects ; Cyclin-Dependent Kinase 2 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Cyclin-Dependent Kinases ; metabolism ; G1 Phase ; drug effects ; Hep G2 Cells ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Phosphotransferases (Alcohol Group Acceptor) ; antagonists & inhibitors ; Thiazoles ; pharmacology

OBJECTIVETo study the effect of the sphingosine kinase 1 (SphK1) inhibitor N,N-dimethylsphingosine (DMS) in combination with chemotherapeutic drugs (DDP, 5-Fu, MMC) on the proliferation of gastric cancer cells (SGC7901) in vitro, and to evaluate whether SphK1 inhibitors could be used as synergetic agents in chemotherapy.

METHODSSGC7901 cells were incubated in vitro with DMS (1 micromol/L) and 5-Fu, DDP, MMC at different concentrations in combination or separately for 24 h. The effects on the growth and survival of SGC7901 cells were determined by MTT assay. The inhibition rates were assessed by response surface analysis and the interactive relationships between the combined drugs were evaluated on the basis of positive/negative values of the cross product coefficients in the response surface equation.

RESULTSThe growth inhibition rate of the gastric cancer cells by treatment with DMS (1 micromol/L) was (10.23 +/- 0.74)%. The growth inhibition rates of the gastric cancer cells treated with 5-Fu (1, 5 and 25 microg/ml) for 24 h were (9.95 +/- 3.24)%, (21.04 +/- 2.19)%, and (45.49 +/- 3.60)%, respectively. The growth inhibition rates of the gastric cancer cells treated with DDP (0.5, 2.5 and 12.5 microg/ml) for 24 h were (9.38 +/- 0.79)%, (19.61 +/- 0.90)%, and (29.83 +/- 0.54)%, respectively. The growth inhibition rates of the gastric cancer cells treated with MMC (0.1, 0.5 and 2.5 microg/ml) for 24 h were (15.35 +/- 0.77)%, (24.72 +/- 0.83)%, and (30.68 +/- 0.28)%, respectively. There were significant differences among the inhibition rates caused by different concentrations of the drugs (P < 0.05). When 1 micromol/L DMS was used in combination with 5-Fu (1, 5, and 25 microg/ml) for 24 h, the growth inhibition rates of the cancer cells were (16.76 +/- 0.41)%, (27.28 +/- 0.29)% and (52.56 +/- 3.60)%, respectively. When 1 micromol/L DMS was used in combination with DDP (0.5, 2.5, and 12.5 microg/ml) for 24 h, the growth inhibition rates of the cancer cells were (15.35 +/- 0.86)%, (25.57 +/- 0.27)%, (36.37 +/- 0.51)%, respectively. When 1 micromol/L DMS was used in combination with MMC (0.1, 0.5, and 2.5 microg/ml) for 24 h, the growth inhibition rates of the cancer cells were (21.02 +/- 0.28)%, (32.10 +/- 0.27)%, (36.36 +/- 0.28)%, respectively. There were also significant differences among the growth inhibition rates caused by different concentrations of the drugs alone and in combination groups (P < 0.05).

CONCLUSIONSDMS can suppress the proliferation of SGC7901 cells in vitro, and there are evident synergetic effects when it is used in combination with chemotherapeutic drugs. The results of this study indicate that SphK1 inhibitors may become novel and promising chemotherapeutic sensitizers.

Antibiotics, Antineoplastic ; pharmacology ; Antimetabolites, Antineoplastic ; pharmacology ; Antineoplastic Agents ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Drug Synergism ; Enzyme Inhibitors ; pharmacology ; Fluorouracil ; pharmacology ; Humans ; Mitomycin ; pharmacology ; Phosphotransferases (Alcohol Group Acceptor) ; antagonists & inhibitors ; Sphingosine ; analogs & derivatives ; pharmacology ; Stomach Neoplasms ; pathology

Our previous studies indicate that phosphatidylinositol 4-kinase IIα can promote the growth of multi-malignant tumors via HER-2/PI3K and MAPK pathways. However, the molecular mechanisms of this pathway and its potential for clinical application remain unknown. In this study, we found that PI4KIIα could be an ideal combinatorial target for EGFR treatment via regulating EGFR degradation. Results showed that PI4KIIα knockdown reduced EGFR protein level, and the expression of PI4KIIα shows a strong correlation with EGFR in human breast cancer tissues (r = 0.77, P < 0.01). PI4KIIα knockdown greatly prolonged the effects and decreased the effective dosage of AG-1478, a specific inhibitor of EGFR. In addition, it significantly enhanced AG1478-induced inhibition of tumor cell survival and strengthened the effect of the EGFR-targeting anti-cancer drug Iressa in xenograft tumor models. Mechanistically, we found that PI4KIIα suppression increased EGFR ligand-independent degradation. Quantitative proteomic analysis by stable isotope labeling with amino acids in cell culture (SILAC) and LC-MS/MS suggested that HSP90 mediated the effect of PI4KIIα on EGFR. Furthermore, we found that combined inhibition of PI4KIIα and EGFR suppressed both PI3K/AKT and MAPK/ERK pathways, and resulted in downregulation of multiple oncogenes like PRDX2, FASN, MTA2, ultimately leading to suppression of tumor growth. Therefore, we conclude that combined inhibition of PI4KIIα and EGFR exerts a multiple anti-tumor effect. Dual inhibition of EGFR at protein and activity level via combinatorial blocking of PI4KIIα presents a novel strategy to combat EGFR-dependent tumors.
Animals ; Antineoplastic Agents ; pharmacology ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Survival ; drug effects ; ErbB Receptors ; antagonists & inhibitors ; metabolism ; Female ; HSP90 Heat-Shock Proteins ; metabolism ; Humans ; MCF-7 Cells ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Minor Histocompatibility Antigens ; Mitogen-Activated Protein Kinases ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphotransferases (Alcohol Group Acceptor) ; antagonists & inhibitors ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Quinazolines ; pharmacology ; Transplantation, Heterologous ; Tyrphostins ; pharmacology