Sphingosine-1-phosphate (S1P) is the main metabolite produced in the process of phospholipid metabolism, which can promote proliferation, migration, and apoptosis of cells, and maintain the barrier function of vascular endothelium. The latest researches showed that S1P can alleviate acute lung injury (ALI) and the inflammation caused by ALI, while the dosage of S1P is still needed to be considered. Mesenchymal stem cells (MSCs) have been a emerging therapy with potential therapeutic effects on ALI because of their characteristics of self-replication and multi-directional differentiation, and their advantages in hematopoiesis, immune regulation, and tissue repair. S1P can promote differentiation of MSCs and participate in immune regulation, while MSCs can regulate the homeostasis of S1P in the body. The synergistic effect of S1P and MSC provides a new treatment method for ALI. This article reviews the production and biological function of S1P, receptor and signal pathway of S1P, the therapeutic effects of S1P on ALI, and the research advances of S1P combined with MSCs in the treatment of ALI, aiming to provide theoretical references for the development of S1P targeted drugs in the treatment of ALI and the search for new combined treatment schemes for ALI.
Acute Lung Injury ; Animals ; Lung/metabolism* ; Lysophospholipids/pharmacology* ; Mice ; Mice, Inbred C57BL ; Sphingosine/pharmacology*

OBJECTIVETo investigate the effects of FTY720, a new immunosuppressive agent, on apoptosis and autophagy in multiple myeloma(MM) cell line U266 and to clarify its molecular mechanism.

METHODSU266 cells were treated with 0, 2.5, 5.0, 10.0 and 20.0 µmol/L FTY720 for 24 hours, and the cell viability was assayed by CCK-8 method. Then U266 cells were treated with 20.0 µmol/L FTY720 for 0, 2, 6 and 24 hours, the cell viability was tested. The apoptotic rates induced by different doses and time points of FTY720 were tested by flow cytometry separately. The expression of LC3B was detected by Western blot after U266 cells treated with different doses of FTY720 to see autophagy. U266 cells were treated with FTY720 ± Bafilomycin A1, an inhibitor of autophagy, for 24 hours, then the cell viability and apoptotic rates were tested. Meanwhile the expression of survivin, anti-apoptotic factors, were tested by Western blot.

RESULTSThe cell viability and the apoptotic rates were inhibited significantly by FTY720 (P < 0.05) in time-dependent and dose-dependent manner. The expression of LC3B-II increased significantly in a dose-dependent manner, it indicated that the autophagy was induced by FTY720. Bafilomycin A1 could rescue the cell viability and apoptotic rates in U266 cells treated with FTY720, and it could also rescue the expression of survivin decreased by FTY720.

CONCLUSIONSFTY720 can cause apoptosis and autophagy of U266 cells. The autophagy promote the apoptosis, which maybe due to the degradation of anti-apoptotic factors such as survivin or their upstream factors in lysosomes through autophagy.

Apoptosis ; drug effects ; Autophagy ; drug effects ; Cell Line, Tumor ; Fingolimod Hydrochloride ; Humans ; Multiple Myeloma ; pathology ; Propylene Glycols ; pharmacology ; Sphingosine ; analogs & derivatives ; pharmacology

Action Potentials ; drug effects ; physiology ; Animals ; Guinea Pigs ; Heart Ventricles ; drug effects ; Lysophospholipids ; pharmacology ; Myocardial Contraction ; drug effects ; physiology ; Myocardium ; Sphingosine ; analogs & derivatives ; pharmacology

OBJECTIVE: To explore the exact mechanisms of programmed cell death (PCD) induced by Type II anti-CD20 mAb in CD20+ non-Hodgkin lymphoma (NHL) cells, and to provide theoretical basis for anti-tumor ability of new CD20 mAb.
 METHODS: After incubation with Rituximab (a Type I anti-CD20 mAb) and Tositumomab (a Type II anti-CD20 mAb), Raji cells were stained by annexin V & propidium iodide (PI). The ratio of programmed death cells were measured by two channel flow cytometry (FCM). Before the treatment of anti-CD20 mAbs, Raji cells was incubated with a caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Z-VAD-FMK) and a dihydroceramide synthase inhibitor fumonisin B1 (FB1) for 30 minutes to assess their inhibitory effect on PCD. High performance liquid chromatography (HPLC) was utilized to compare the ratio of programmed death cells between the pretreatment group (treated by Rituximab and Tositumomab) and the non-pretreatment group. The anti-CD20 mAbs-treated Raji cells were collected, and the ceramide levels in the Raji cells in the different pretreatment groups were also examined by HPLC, and the inhibitory effect of FB1 on the changes of ceramide levels in the Raji cells was measured. The Raji cells were incubated with different concentration C2-ceramide, C2-Ceramide-induced PCD was also evaluated by annexin V & PI staining after 16 hours. 
 RESULTS: Tositumomab (10 µg/mL) but not Rituximab (10 µg/mL) can induce significant PCD (28.6±4.2)% in Raji cells, with significant difference (t=26.48, P<0.01), which cannot be blocked by Z-VAD-FMK with a concentration range from 10 to 30 µmol/L (F=3.01, P>0.05). The cellular ceramide levels in Raji cells were significantly elevated after the treatment of Tositumomab (t=28.48, P<0.01). C2-ceramide can significantly induce PCD in Raji cells in a dose-dependent manner with a concentration range from 5 to 40 µmol/L (F=2.71, P>0.05). The dihydroceramide synthase inhibitor FB1 can significantly inhibit the elevated cellular ceramide levels (F=20.18, P<0.01) and cell programmed death induced by Tositumomab (F=17.02, P<0.01).
 CONCLUSION Type II but not Type I anti-CD20 mAbs can induce caspase independent PCD in CD20+ NHL cells through the elevation of cellular ceramide levels. The PCD is not associated with classic caspase pathway.
Amino Acid Chloromethyl Ketones ; Apoptosis ; drug effects ; Cell Line, Tumor ; drug effects ; Humans ; Lymphoma, Non-Hodgkin ; Rituximab ; pharmacology ; Sphingosine ; analogs & derivatives ; pharmacology

This study is to investigate the effects of ceramide on GSTA1 expression in Caco-2 cells. After being exposed to ceramide for a fixed time, GSTA1 protein expression was detected by Western blotting analysis; GSTA1 mRNA expression was detected by real time PCR; dual luciferase assay was used to analyze GSTA1 transcriptional activity and GSTA1 activity was determined toward androstanedione (AD) as substrate. The data showed that ceramide can significantly induce the expression of protein and GSTA1 mRNA, and increase transcriptional activity and enzyme activity of GSTA1. The results demonstrated that ceramide may increase resistance to chemotherapeutics in Caco-2 cells by up-regulating the expression of GSTA1.
Caco-2 Cells ; Enzyme Activation ; drug effects ; Glutathione Transferase ; genetics ; metabolism ; Humans ; RNA, Messenger ; metabolism ; Sphingosine ; analogs & derivatives ; pharmacology ; Transcriptional Activation ; drug effects ; Up-Regulation

OBJECTIVETo observe the relationship between murine double minute 2 (mdm2) expression and AngII and ceramide induced human umbilical endothelial cells apoptosis.

METHODHuman umbilical endothelial cells (ECs) were cultured in vitro and treated with angiotensin II alone or in combination with losartan (an inhibitor of AT1), PD123319 (an inhibitor of AT2) and FB1 (an inhibitor of ceramidase) respectively. ECs were also treated with different doses of C2-ceramide. The apoptosis of ECs was detected with Tunel, the mdm2 mRNA and protein expressions were measured with reverse transcription-polymerase chain reaction (RT-PCR) and Western blot.

RESULTSPD123319 and FB1 but not losartan inhibited AngII induced ECs apoptosis and down-regulated the AngII induced increased mdm2 expressions. C2-ceramide also induces ECs apoptosis and down-regulated mdm2 expressions at protein and mRNA levels in a dose-dependent manner.

CONCLUSIONSAngII binding with AT2 induces ECs apoptosis via ceramide. AngII and ceramide induce EC apoptosis by inhibiting mdm2.

Angiotensin II ; pharmacology ; Apoptosis ; Cells, Cultured ; Endothelial Cells ; cytology ; drug effects ; Gene Expression ; Humans ; Imidazoles ; pharmacology ; Proto-Oncogene Proteins c-mdm2 ; metabolism ; Pyridines ; pharmacology ; RNA, Messenger ; metabolism ; Sphingosine ; analogs & derivatives ; pharmacology ; Umbilical Veins ; cytology

Recently, a mitochondrial ceramidase has been identified and cloned, whose mitochondrial localization strongly suggests the existence of an unexpected mitochondrial pathway of ceramide metabolism that may play a key role in mitochondrial functions, especially in the regulation of apoptosis. To explore the biological effect of mitochondrial ceramidase on cells, pcDNA 3.1/His-CDase plasmid, containing mitochondrial ceramidase cDNA sequence, was transducted into K562 cells mediated by liposome, and G418 was used to screen for positive colonies. A stable transfected K562 cell line was established and named as 'K562TC'. The difference between K562 and K562TC cells in chemotheraputic cytotoxicity response and serum-withdrawal resistance and Bcl-2 protein expression were evaluated by MTT assay, annexin V/PI test, flow cytometry or Western blotting, respectively. The results showed that although survival was comparable between K562 and K562TC cells after exposed to adriamycin, etoposide or arsenious acid, K562TC cells with elevated Bcl-2 protein expression level as identified by FCM or Western blotting revealed stronger resistance to apoptosis induced by serum withdrawal than their parental cells. Inhibition of mitochondrial ceramidase expression in K562TC cells by its specific antisense oligodeoxynucleotide was correlated with a decrease in Bcl-2 protein level. N, N-dimethylsphingosine, a sphingosine kinase inhibitor, depleted intracellular sphingosine-1-phosphate production, also abrogated Bcl-2 protein expression in K562TC cells, while Bcl-2 protein level in K562 cells was up-regulated by exogenous sphingosine-1-phosphate. It is concluded that mitochondrial ceramidase overexpression in K562 cells leads to markedly elevated level of Bcl-2 protein and results in more resistance to serum withdrawal. This effect is initiated not by sphingosine, the direct metabolite of mitochondrial ceramidase, but via sphingosine-1-phosphate, its phosphorylated form. This is the first evidence that mitochondrial ceramidase, through its sphingoid metabolite sphingosine-1-phosphate, up-regulates Bcl-2 protein expression in K562 cells.
Amidohydrolases ; physiology ; Apoptosis ; Arsenites ; pharmacology ; Ceramidases ; Doxorubicin ; pharmacology ; Etoposide ; pharmacology ; Humans ; K562 Cells ; Lysophospholipids ; physiology ; Mitochondria ; enzymology ; Oligonucleotides, Antisense ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Sphingosine ; analogs & derivatives ; physiology ; Up-Regulation

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

Syl948 is a synthesized selective S1P1 agonist with novel structure. HTRF-IP1 test indicated that Syl948-P, the active form of Syl948 in vitro, has strong activity against S1P1 (EC50: 83 +/- 16 nmol x L(-1)), but its effect on S1P3 was very weak (EC50: 1 026 +/- 90 nmol x L(-1)). In SD rats, oral administration of Syl948 10 mg x kg(-1) significantly decreased the peripheral blood lymphocytes (PBL), with the maximal PBL inhibition rate of 63%, which was as similar as equal dose of fingolimod (FTY720). Oral administration of Syl948 10 mg x kg(-1) had no effect on heart rate of SD rats, which was better than FTY720. Daily oral administration with Syl948 (2 or 4 mg x kg(-1)) significantly prolonged the survival time of the allografts of skin slice on mice. In summary, the above results demonstrated that Syl948 has great selectivity in vitro and good activity in vivo, which indicated its potential use as an anti-rejection drug in skin transplantation.
Animals ; Fingolimod Hydrochloride ; Graft Survival ; drug effects ; Immunosuppressive Agents ; pharmacology ; Lymphocytes ; drug effects ; Mice ; Propylene Glycols ; pharmacology ; Rats ; Receptors, Lysosphingolipid ; agonists ; Skin Transplantation ; Sphingosine ; analogs & derivatives ; pharmacology ; Transplantation, Homologous

OBJECTIVETo investigate the effect and mechanism of FTY720 on acute graft versus host disease (GVHD) in rat small bowel transplantation (SBTx).

METHODSHeterotopic SBTx was performed using a parent (WF)-into-F1 (WFxACI) rat combination. Recipient rats were divided into experimental group (n=6) and control group (n=6). Rats in the experimental group were administered with FTY720 at 0.5 mg/kg for 14 days. Lymphocyte apoptosis in the liver and the mucosa of intestine and graft was detected by TUNEL and flow cytometry 15 days after transplantation. Recipient survival and lymphocyte apoptosis were compared between the two groups.

RESULTSRecipients in the control group died of GVHD after a mean survival time of (16+/-2.1) days. FTY720-treated recipients had a significantly longer survival (>100 days). After administration of FTY720, the percentage of apoptotic lymphocytes was significantly increased in the graft as compared to that in the control group by flow cytometry. The ratio of apoptotic lymphocyte in the liver and graft was also significantly higher in the experimental group by TUNEL.

CONCLUSIONFTY720 effectively induces the lymphocyte apoptosis, inhibits the lesion of target tissues by GVHD, and prolongs the recipient survival.

Animals ; Apoptosis ; drug effects ; Fingolimod Hydrochloride ; Graft vs Host Disease ; immunology ; prevention & control ; Immunosuppressive Agents ; pharmacology ; Intestine, Small ; transplantation ; Lymphocytes ; cytology ; drug effects ; Male ; Propylene Glycols ; pharmacology ; Rats ; Rats, Inbred WF ; Sphingosine ; analogs & derivatives ; pharmacology ; Transplantation, Heterotopic