SK1 Activity Research Articles

Studies of the silkworm enzyme activity and their correlations with economic variables

The experiment was conducted to analyse the larval performance and economic traits of bivoltine silkworm breeds of silkworm (SK-1, SK-6, SK-22, SK-28, SK-33, CSR4, CSR2, NB4D2, DUN6 and APS4) during spring season. The haemolymph total protein, succinate and gultamate dehydrogenase activities were estimated and their correlation with economic traits were also worked out. The results of the study confirmed that among ten bivoltine silkworm breeds, highest haemolymph SDH activity of 3.47 µmoles/ml/mgprotein/min was recorded in the silkworm breed SK1 and least SDH activity of 1.58 µmoles/ml/mg protein/min was recorded in the breed APS4. The highest peak of succinate dehydrogenase activity of 2.65 µmoles/ml/mg protein/min was observed on 7th day of the 5th instar and lowest peak of succinate dehydrogenase activity of 2.39 µmoles/ml/mg protein/min was observed on 4th day of the 5th instar. GDH activity of 0.46 µmoles/ml/mg protein/min was recorded highest in the silkworm breed SK1 and lowest of 0.15 µmoles/ml/mg protein/min was recorded in silkworm breed APS4. The highest peak of haemolymph GDH of 0.36 µmoles/ml/mg protein/min was recorded on 7th day of 5th instar and lowest peak of 0.26 µmoles/ml/mg protein/min was recorded on 4th day of 5th instar. The correlational studies revealed that haemolymph total protein, SDH and GDH were found to be positively corelated with yield by weight and number (cocoon), weight of mature larvae, shell weight ,cocoon weight, shell ratio percent, silk productivity, rate of pupation, fecundity, raw silk percentage and length of filament. Thus, the study revealed that silkworm breeds like SK1, SK6, SK22 and SK28 as productive breeds and hence may be used for future breeding programmes for evolution of new robust silkworm breeds

Synthesis and biological investigation of protein phosphatase 2A‐activating compounds with dimeric tail as non‐small cell lung cancer cell death agents

AbstractFTY720 exerts an anticancer effect through the activation of protein phosphatase 2A (PP2A), which acts as a tumor suppressor, and inhibits SK1 activity. However, FTY720 taken into the body is phosphorylated by endogenous SK2. The structure of FTY720 is well‐studied as a basis for developing SK inhibitors and PP2A activators. We synthesized analogs of PF‐543 with a dimeric tail structure and reported the efficacy of SK1 inhibition. The compounds with this structure had improved anticancer activity and stability compared with PF‐543. To confirm whether the dimeric tail structure could be applied as a PP2A‐active material, we synthesized four types of compounds with a dimeric tail structure and confirmed the anticancer activity and PP2A activation in A549 non‐small cell lung cancer cells. Compounds 2 and 4 were more cytotoxic to A549 cells than RB005, and induced similar levels of cytotoxic and PP2A activation as FTY720.

Novel Dimer Derivatives of PF-543 as Potential Antitumor Agents for the Treatment of Non-Small Cell Lung Cancer.

Lung cancer can be divided into non-small cell lung cancer (NSCLC) and small cell lung cancer, and the incidence and mortality rate are continuously increasing. In many cases, lung cancer cannot be completely treated with surgery, so chemotherapy is used in parallel; however, the treatment often fails due to drug resistance. Therefore, it is necessary to develop a new therapeutic agent with a new target. The expression of sphingosine kinase promotes cancer cell growth and survival and induces resistance to chemotherapeutic agents. Sphingosine-1-phosphate (S1P), produced by sphingosine kinase (SK), has been shown to regulate cancer cell death and proliferation. PF-543, currently known as an SK inhibitor, has been reported to demonstrate low anticancer activity in several cancers. Therefore, in this study, a derivative of PF-543 capable of increasing anticancer activity was synthesized and its efficacy was evaluated by using an NSCLC cell line and xenograft animal model. Based on the cytotoxic activity of the synthesized compound on lung cancer cells, the piperidine forms (Compounds 2 and 4) were observed to exhibit superior anticancer activity than the pyrrolidine forms of the head group (Compounds 1 and 3). Compounds 2 and 4 showed inhibitory effects on SK1 and SK2 activity, and S1P produced by SK was reduced by both compounds. Compounds 2 and 4 demonstrated an increase in the cytotoxicity in the NSCLC cells through increased apoptosis. As a result of using an SK1 and SK2 siRNA model to determine whether the cytotoxic effects of Compounds 2 and 4 were due to SK1 and SK2 inhibition, it was found that the cytotoxic effect of the derivative was SK1 and SK2 dependent. The metabolic stability of Compounds 2 and 4 was superior compared to PF-543, and the xenograft experiment was performed using Compound 4, which had more excellent MS. Compound 4 demonstrated the inhibition of tumor formation. The results of this experiment suggest that the bulky tail structure of PF-543 derivatives is effective for mediating anticancer activity, and the results are expected to be applied to the treatment of NSCLC.

SK1 Inhibitor RB005 Induces Apoptosis in Colorectal Cancer Cells through SK1 Inhibition Dependent and Independent Pathway.

Colorectal cancer (CRC) is the fourth leading cause of cancer- related death globally, with a high incidence rate in economically fast-growing countries. Sphingosine- 1-phosphate (S1P) is a bioactive lipid mediator that plays critical roles in cancer cell proliferation, migration, and angiogenesis converted by the isoforms of sphingosine kinase (SK1 and SK2). SK1 is highly expressed in colorectal cancer; its inhibitors suppress the formation of S1P and increase ceramide levels having a pro-apoptotic function. RB005 is a selective SK1 inhibitor and a structural analog of PP2A activator FTY720. The purpose of this study is to test whether RB005, an SK1 inhibitor, can be used as an anticancer agent by inhibiting the growth of colon cancer cells. We performed MTT and colony-forming assay using colon cancer cell lines HT29 and HCT116 cells to examine the cell toxicity effect of RB005. To determine whether apoptosis of RB005 in colon cancer cell line is due to SK1 inhibition or other mechanisms due to its structural similarity with FTY720, we conducted LC/MS, siRNA knockdown, and PP2A activity experiments. RB005 notably inhibited CRC cell growth and proliferation compared to PF543 and ABC294640 by inducing the mitochondria-mediated intrinsic apoptotic pathway. Apoptotic cell death is caused by increased mitochondrial permeability Initiated by the activation of pro-apoptotic protein BAX, increased ceramides, and activation of PP2A. Also, RB005 treatment in HT29 cells did not change the expression level of SK1, but strikingly decreased SK1 activity and S1P levels. All these events of cell death and apoptosis were less effective when SK1 was knocked down by siRNA. This result indicates that RB005 shows the in-vitro anti-CRC effect by inhibiting SK1 activity and PP2A activation, increasing proapoptotic ceramide levels following the activation of the intrinsic apoptotic pathway.

Transcriptional Regulation of Sphingosine Kinase 1.

Once thought to be primarily structural in nature, sphingolipids have become increasingly appreciated as second messengers in a wide array of signaling pathways. Sphingosine kinase 1, or SK1, is one of two sphingosine kinases that phosphorylate sphingosine into sphingosine-1-phosphate (S1P). S1P is generally pro-inflammatory, pro-angiogenic, immunomodulatory, and pro-survival; therefore, high SK1 expression and activity have been associated with certain inflammatory diseases and cancer. It is thus important to develop an understanding of the regulation of SK1 expression and activity. In this review, we explore the current literature on SK1 transcriptional regulation, illustrating a complex system of transcription factors, cytokines, and even micro-RNAs (miRNAs) on the post transcriptional level.

Targeting shingolipids inhibits alcohol-induced liver disease

Abstract Chronic alcohol consumption is accompanied by intestinal inflammation. However, little is known about how alterations to the intestinal immune system and sphingolipids contribute to pathogenesis of alcoholic liver disease (ALD). We used WT mice, RORγt-deficient mice and sphingosine kinase-deficient mice in a chronic-binge ethanol feeding model. Targeted lipidomics assessed the sphingolipids in gut and liver samples. Ethanol-fed and control mice were treated by SK1 inhibitor. Gut immune cell populations, the amounts of sphingolipids, and the level of liver injury were examined. We found that alcohol intake induces an increase in Th17 cells in the gut. Increased Th17 cells were due to upregulation of SK1 activity and Rorc activation. The pathogenic role of S1P/S1PR1 signaling in ALD was attributable to the migration and activation of Th17 cells. Importantly, deletion of SK1 markedly attenuated alcohol-induced liver inflammation, steatosis, and damage. In conclusion, our findings suggest that S1P signaling was crucial in the pathogenesis of alcoholic liver disease in a Th17 cell dependent manner.

Isolation and Characterization of Plant Growth-Promoting Endophytic Bacteria Paenibacillus polymyxa SK1 from Lilium lancifolium.

Paenibacillus polymyxa is a plant growth-promoting rhizobacterium that has immense potential to be used as an environmentally friendly replacement of chemical fertilizers and pesticides. In the present study, Paenibacillus polymyxa SK1 was isolated from bulbs of Lilium lancifolium. The isolated endophytic strain showed antifungal activities against important plant pathogens like Botryosphaeria dothidea, Fusarium oxysporum, Botrytis cinerea, and Fusarium fujikuroi. The highest percentage of growth inhibition, i.e., 66.67 ± 2.23%, was observed for SK1 against Botryosphaeria dothidea followed by 61.19 ± 3.12%, 60.71 ± 3.53%, and 55.54 ± 2.89% against Botrytis cinerea, Fusarium fujikuroi, and Fusarium oxysporum, respectively. The metabolite profiling of ethyl acetate fraction was assessed through the UHPLC-LTQ-IT-MS/MS analysis, and putative identification was done with the aid of the GNPS molecular networking workflow. A total of 29 compounds were putatively identified which included dipeptides, tripeptides, cyclopeptides (cyclo-(Leu-Leu), cyclo(Pro-Phe)), 2-heptyl-3-hydroxy 4-quinolone, 6-oxocativic acid, anhydrobrazilic acid, 1-(5-methoxy-1H-indol-3-yl)-2-piperidin-1-ylethane-1,2-dione, octadecenoic acid, pyochelin, 15-hydroxy-5Z,8Z,11Z, 13E-eicosatetraenoic acid, (Z)-7-[(2R,3S)-3-[(2Z,5E)-Undeca-2,5-dienyl]oxiran-2-yl]hept-5-enoic acid, arginylasparagine, cholic acid, sphinganine, elaidic acid, gossypin, L-carnosine, tetrodotoxin, and ursodiol. The high antifungal activity of SK1 might be attributed to the presence of these bioactive compounds. The isolated strain SK1 showed plant growth-promoting traits such as the production of organic acids, ACC deaminase, indole-3-acetic acid (IAA), siderophores, nitrogen fixation, and phosphate solubilization. IAA production was strongly correlated with the application of exogenous tryptophan concentrations in the medium. Furthermore, inoculation of SK1 enhanced plant growth of two Lilium varieties, Tresor and White Heaven, under greenhouse condition. In the light of these findings, the P. polymyxa SK1 may be utilized as a source of plant growth promotion and disease control in sustainable agriculture.

Sphingosine kinase and sphingosine-1-phosphate regulate epithelial cell architecture by the modulation of de novo sphingolipid synthesis.

Sphingolipids regulate several aspects of cell behavior and it has been demonstrated that cells adjust their sphingolipid metabolism in response to metabolic needs. Particularly, sphingosine-1-phosphate (S1P), a final product of sphingolipid metabolism, is a potent bioactive lipid involved in the regulation of various cellular processes, including cell proliferation, cell migration, actin cytoskeletal reorganization and cell adhesion. In previous work in rat renal papillae, we showed that sphingosine kinase (SK) expression and S1P levels are developmentally regulated and control de novo sphingolipid synthesis. The aim of the present study was to evaluate the participation of SK/S1P pathway in the triggering of cell differentiation by external hypertonicity. We found that hypertonicity evoked a sharp decrease in SK expression, thus activating the de novo sphingolipid synthesis pathway. Furthermore, the inhibition of SK activity evoked a relaxation of cell-cell adherens junction (AJ) with accumulation of the AJ complex (E-cadherin/β-catenin/α-catenin) in the Golgi complex, preventing the acquisition of the differentiated cell phenotype. This phenotype alteration was a consequence of a sphingolipid misbalance with an increase in ceramide levels. Moreover, we found that SNAI1 and SNAI2 were located in the cell nucleus with impairment of cell differentiation induced by SK inhibition, a fact that is considered a biochemical marker of epithelial to mesenchymal transition. So, we suggest that the expression and activity of SK1, but not SK2, act as a control system, allowing epithelial cells to synchronize the various branches of sphingolipid metabolism for an adequate cell differentiation program.

Field template-based design and biological evaluation of new sphingosine kinase 1 inhibitors

PurposeSphingosine kinase 1 (SK1) is a protooncogenic enzyme expressed in many human tumours and is associated with chemoresistance and poor prognosis. It is a potent therapy target and its inhibition chemosensitises solid tumours. Despite recent advances in SK1 inhibitors synthesis and validation, their clinical safety and chemosensitising options are not well described. In this study, we have designed, synthesised and tested a new specific SK1 inhibitor with a low toxicity profile.MethodsField template molecular modelling was used for compound design. Lead compounds were tested in cell and mouse cancer models.ResultsField template analysis of three known SK1 inhibitors, SKI-178, 12aa and SK1-I, was performed and compound screening identified six potential new SK1 inhibitors. SK1 activity assays in both cell-free and in vitro settings showed that two compounds were effective SK1 inhibitors. Compound SK-F has potently decreased cancer cell viability in vitro and sensitised mouse breast tumours to docetaxel (DTX) in vivo, without significant whole-body toxicity.ConclusionThrough field template screening, we have identified a new SK1 inhibitor, SK-F, which demonstrated antitumour activity in vitro and in vivo without overt toxicity when combined with DTX.

Design, synthesis and biological evaluation of novel benzimidazole amidines as potent multi-target inhibitors for the treatment of non-small cell lung cancer

A series of novel amidino 2-substituted benzimidazoles linked to 1,4-disubstituted 1,2,3-triazoles were synthesized by implementation of microwave and ultrasound irradiation in click reaction and subsequent condensation of thus obtained 4-(1,2,3-triazol-1-yl)benzaldehyde with o-phenylenediamines. In vitro antiproliferative screening of compounds performed on human cancer cell lines revealed that p-chlorophenyl-substituted 1,2,3-triazolyl N-isopropylamidine 10c and benzyl-substituted 1,2,3-triazolyl imidazoline 11f benzimidazoles had selective and potent cytostatic activities in the low nM range against non-small cell lung cancer cell line A549, which could be attributed to induction of apoptosis and primary necrosis. Additional Western blot analyses showed different mechanisms of cytostatic activity between compounds 10c and 11f that could be associated with the nature of aromatic substituent at 1-(1,2,3-triazolyl) and amidino moiety at C-5 position of benzimidazole ring. Specifically, compound 11f abrogated the activity of several protein kinases including TGM2, CDK9, SK1 and p38 MAPK, whereas compound 10c did not have profound effect on the activities of CDK9 and TGM2, but instead showed moderate downregulation of SK1 activity concomitant with a significant reduction in p38 MAPK. Further in silico structural analysis demonstrated that compound 11f bound slightly better to the ATP binding site of p38 MAPK compared to 10c, which correlated well with observed stronger decrement in the expression level of phospho-p38 MAPK elicited by 11f in comparison with 10c.

Investigation of sphingosine kinase 1 in interferon responses during dengue virus infection.

Dengue virus (DENV) regulates sphingosine kinase (SK)-1 activity and chemical inhibition of SK1 reduces DENV infection. In primary murine embryonic fibroblasts (pMEFs) lacking SK1 however, DENV infection is enhanced and this is associated with induction of normal levels of interferon beta (IFN-β) but reduced levels of IFN-stimulated genes (ISGs). We have further investigated this link between SK1 and type I IFN responses. DENV infection downregulates cell-surface IFN-alpha receptor (IFNAR)1 in both wild-type (WT) and SK1−/− pMEF, but, consistent with poor ISG responses, shows reduced induction of phosphorylated (p)-STAT1 and key IFN regulatory factors (IRF)1 and −7 in SK1−/− pMEF. Direct IFN stimulation induced ISGs (viperin, IFIT1), CXCL10, IRF1 and −7 and p-STAT1. Responses, however, were significantly reduced in SK1−/− pMEF, except for IFN-stimulated CXCL10 and IRF7. Poor IFN responses in SK1−/− pMEF were associated with a small reduction in basal cell-surface IFNAR1 and IRF1 mRNA in uninfected SK1−/− compared with WT pMEF. In contrast, treatment of cells with the SK1 inhibitor, SK1-I or expression of an inhibitory SK1 short hairpin RNA (shRNA), both of which reduce DENV infection, does not alter basal IRF1 mRNA or affect type I IFN stimulation of p-STAT1. Thus, cells genetically lacking SK1 can induce many responses normally following DENV infection, but have adaptive changes in IFNAR1 and IRF1 that compromise DENV-induced type I IFN responses. This suggests a biological link between SK1 and IFN-stimulated pathways. Other approaches to reduce SK1 activity, however, do not influence these important antiviral pathways but reduce infection and may be useful antiviral strategies.

Topical Application of Fingolimod Perturbs Cutaneous Inflammation.

The prevalence of allergies, including rhinitis, eczema, and anaphylaxis, is rising dramatically worldwide. This increase is especially problematic in children who bear the greatest burden of this rising trend. Increasing evidence identifies neutrophils as primary perpetrators of the more severe and difficult to manage forms of inflammation. A newly recognized mechanism by which neutrophils are recruited during the early phase of histamine-induced inflammation involves the sphingosine kinase (SK)/sphingosine-1-phosphate axis. This study examines whether topical application of fingolimod, an established SK/sphingosine-1-phosphate antagonist already in clinical use to treat multiple sclerosis, may be repurposed to treat cutaneous inflammation. Using two mouse models of ear skin inflammation (histamine- and IgE-mediated passive cutaneous anaphylaxis) we topically applied fingolimod prophylactically, as well as after establishment of the inflammatory response, and examined ear swelling, SK activity, vascular permeability, leukocyte recruitment, and production of proinflammatory mediators. The present study reveals that when applied topically, fingolimod attenuates both immediate and late-phase responses to histamine with reduced extravasation of fluid, SK-1 activity, proinflammatory cytokine and chemokine production, and neutrophil influx and prevents ear swelling. Intravital microscopy demonstrates that histamine-induced neutrophil rolling and adhesion to the postcapillary venules in the mouse ears is significantly attenuated even after 24 h. More importantly, these effects are achievable even once inflammation is established. Translation into humans was also accomplished with epicutaneous application of fingolimod resolving histamine-induced and allergen-induced inflammatory reactions in forearm skin. Overall, this study demonstrates, to our knowledge for the first time, that fingolimod may be repurposed to treat cutaneous inflammation.

Role of sphingosine 1-phosphate receptors, sphingosine kinases and sphingosine in cancer and inflammation.

Sphingosine kinase (there are two isoforms, SK1 and SK2) catalyses the formation of sphingosine 1-phosphate (S1P), a bioactive lipid that can be released from cells to activate a family of G protein-coupled receptors, termed S1P1-5. In addition, S1P can bind to intracellular target proteins, such as HDAC1/2, to induce cell responses. There is increasing evidence of a role for S1P receptors (e.g. S1P4) and SK1 in cancer, where high expression of these proteins in ER negative breast cancer patient tumours is linked with poor prognosis. Indeed, evidence will be presented here to demonstrate that S1P4 is functionally linked with SK1 and the oncogene HER2 (ErbB2) to regulate mitogen-activated protein kinase pathways and growth of breast cancer cells. Although much emphasis is placed on SK1 in terms of involvement in oncogenesis, evidence will also be presented for a role of SK2 in both T-cell and B-cell acute lymphoblastic leukemia. In patient T-ALL lymphoblasts and T-ALL cell lines, we have demonstrated that SK2 inhibitors promote T-ALL cell death via autophagy and induce suppression of c-myc and PI3K/AKT pathways. We will also present evidence demonstrating that certain SK inhibitors promote oxidative stress and protein turnover via proteasomal degradative pathways linked with induction of p53-and p21-induced growth arrest. In addition, the SK1 inhibitor, PF-543 exacerbates disease progression in an experimental autoimmune encephalomyelitis mouse model indicating that SK1 functions in an anti-inflammatory manner. Indeed, sphingosine, which accumulates upon inhibition of SK1 activity, and sphingosine-like compounds promote activation of the inflammasome, which is linked with multiple sclerosis, to stimulate formation of the pro-inflammatory mediator, IL-1β. Such compounds could be exploited to produce antagonists that diminish exaggerated inflammation in disease. The therapeutic potential of modifying the SK-S1P receptor pathway in cancer and inflammation will therefore, be reviewed.

Downregulation of sphingosine 1-phosphate (S1P) receptor 1 by dexamethasone inhibits S1P-induced mesangial cell migration.

Sphingosine 1-phosphate (S1P) is generated by sphingosine kinase (SK)-1 and -2 and acts mainly as an extracellular ligand at five specific receptors, denoted S1P1-5. After activation, S1P receptors regulate important processes in the progression of renal diseases, such as mesangial cell migration and survival. Previously, we showed that dexamethasone enhances SK-1 activity and S1P formation, which protected mesangial cells from stress-induced apoptosis. Here we demonstrate that dexamethasone treatment lowered S1P1 mRNA and protein expression levels in rat mesangial cells. This effect was abolished in the presence of the glucocorticoid receptor antagonist RU-486. In addition, in vivo studies showed that dexamethasone downregulated S1P1 expression in glomeruli isolated from mice treated with dexamethasone (10 mg/kg body weight). Functionally, we identified S1P1 as a key player mediating S1P-induced mesangial cell migration. We show that dexamethasone treatment significantly lowered S1P-induced migration of mesangial cells, which was again reversed in the presence of RU-486. In summary, we suggest that dexamethasone inhibits S1P-induced mesangial cell migration via downregulation of S1P1. Overall, these results demonstrate that dexamethasone has functional important effects on sphingolipid metabolism and action in renal mesangial cells.

SKIP Is Underexpressed in AML Leading to Sphingosine Kinase Hypofunction

Background: Sphingosine kinase interacting protein (SKIP) has been shown to be mostly silenced by hypermethylation in AML [1]. SKIP interacts with and regulates the function of sphingosine kinase (SK) enzyme. SK activity results in phosphorylation of sphingosine (SPH) to form sphingosine 1 phosphate (S1P), which promotes cell survival and resistance to apoptosis. On the other hand, S1P precursors ceramide (CER) and SPH mediate antiproliferative and apoptotic responses. SKIP has been reported to negatively regulate SK1 activity in fibroblasts. Therefore, we investigated the consequences of SKIP silencing in primary AML cells. In addition, we studied the effects of SKIP re-expression in leukemic cell lines.Methods: CTS and K562 cells were transfected with SKIP gene using standard techniques. SKIP is normally silenced in both cell lines. Intracellular and extracellular S1P, SPH and CER were measured by UPLC-MS/MS. In addition, intracellular SK activity was determined based on C17 S1P production from C17 SPH substrate. Chemosensitivity to doxorubicin, Imatinib and Ara-C in transfected cells was also studied.Results: In Primary AML cells, intracellular S1P and CER concentrations were reduced compared to G-CSF-mobilized peripheral blood mononuclear cells. In addition, SK activity was found to be downregulated in AML primary cells. When we transfected leukemic cell lines with SKIP gene, S1P and CER showed at least 2 fold increase in intracellular and extracellular basal levels compared to vector alone control (Figure 1). Further studies confirmed a significant increase in intracellular SK activity in SKIP transfected compared to vector alone cells, based on C17 S1P production (8.8 ± 2.6 vs 1.4 ± 0.4 ng/106 cells respectively after 24 hrs, p< 0.05). This increase in S1P and CER was associated with increasing apoptotic signals as evidenced by Annexin V staining and cleaved PARP expression. Moreover, chemosensitivity to Imatinib and AraC was significantly increased in SKIP transfected cell lines. These experiments confirm the regulation of SK1 function by SKIP.Conclusion: These data indicate that SKIP is downregulated in AML leading to reduced SK activity, which ultimately inhibits the apoptosis response. [Display omitted] 1. Saied, M.H., et al., Genome wide analysis of acute myeloid leukemia reveal leukemia specific methylome and subtype specific hypomethylation of repeats. PLoS One, 2012. 7(3): p. e33213.EAG and PS contributed equallyJG and DT contributed equally DisclosuresNo relevant conflicts of interest to declare.

PP-063 TWO CASES WITH PRIMARY IMMUNE DEFICIENCY AND NEOPLASTIC LYMPHOID PROLIFERATION

Sphingosine 1-phosphate (S1P) the product of sphingosine kinase (SK) action plays an important role in various pathological conditions like inflammation and cancer. In this study, we show that in the human breast cancer cell line MCF7, epidermal growth factor (EGF) stimulates SK-1 activity in a biphasic manner with a first peak after 15 min and a second delayed activation occurring after 1 h up to 18 h and thereafter declining again. This delayed activation is accompanied by increased mRNA and protein expression of SK-1, but not SK-2. Mechanistically, the transcriptional upregulation is dependent on the classical mitogen-activated protein kinase, protein kinase C (PKC) and the phosphoinositide 3-kinase, since specific inhibitors of these enzymes all abolish the EGF-induced mRNA upregulation and activity of SK-1. Moreover, dexamethasone also suppressed EGF-induced SK-1 mRNA expression and activity which is reversed by the glucocorticoid receptor antagonist RU486. To see whether EGF-induced upregulation of SK-1 is of relevance for tumor progression, we investigated two hallmarks of carcinogenesis, i.e., cell proliferation and migration. Stimulation of cells with EGF leads to enhanced [3H]thymidine incorporation into DNA and also to stimulated migration in a modified Boyden chamber assay. When cells are depleted of SK-1, but not SK-2, by siRNA transfection or by dexamethasone treatment, EGF-induced proliferation and migration are drastically reduced. In summary, these data show that EGF causes an acute stimulation of SK-1 activity and, moreover, triggers a delayed SK activation which is due to increased gene transcription and de novo synthesis of SK-1, which in turn directs cells towards growth and increased motility. Thus, the sphingosine kinase-1 may represent a novel attractive target for cancer therapy.

Antibacterial compounds from Zanthoxylum rhetsa

A new amide, zanthorhetsamide (1), along with nine known compounds (2-10) was isolated from the roots and stem barks of Zanthoxylum rhetsa. The structure was characterized by spectroscopic methods. In addition, the antibacterial activity of the isolates was evaluated. Dihydrochelerythrine (4) exhibited strong activity against methicillin-resistant Staphylococcus aureus SK1 and moderate activity against Escherichia coli TISTR 780 with MIC values of 8 and 16 μg/mL, respectively.

Abstract 3256: Sphingolipid regulation by sphingosine kinase anchoring protein (SKAP) and its implication in cancer

Abstract Background: Sphingolipids are important in cancer cell signalling. Sphingosine 1 phosphate (S1P) promotes cell survival and resistance to apoptosis, while S1P precursors ceramide (CER) and sphingosine (SPH), mediate antiproliferative and apoptotic responses. S1P is generated from SPH by sphingosine kinase (SK) enzymes (SK1 and SK2), with SK activity and localisation regulated by other proteins, including PKC, PKA and a SK anchoring protein (SKAP) that has been reported to negatively regulate SK1 activity in fibroblasts. S1P localisation is thought to play an important role in its function. Based on our preliminary observation in primary AML cells that SKAP expression resulted in an increase in S1P, we have investigated the effect of SKAP transfection on S1P production and localisation. Methods: K562, (and for some confirmatory experiments MCF-7), cells were transfected with the SKAP gene using standard techniques. SKAP is normally silenced in both cell lines. Transfection was confirmed by RNA expression. Intracellular and extracellular S1P and SPH, and intracellular SK activity (based on the production of C17 S1P from C17 SPH, an unnatural SPH that is a SK substrate) in intact cells were measured by LC-MS/MS. Phorbol 12-myristate 13-acetate (PMA) was used to induce membrane associated SK function, and MK-571 and fumitremorgen C (FTC) were used to block S1P efflux through ABCC1 and ABCG2 efflux pumps, respectively. Chemosensitivity to doxorubicin and imatinib in transfected cells was also studied. Results: K562 cells transfected with the SKAP gene showed a 2.5 fold increase in intracellular and extracellular levels of basal S1P compared to vector alone control. (In MCF-7 cells SKAP transfection resulted in an almost 10-fold increase in S1P). Further studies in K562 cells confirmed a significant increase in intracellular SK activity in SKAP transfected compared to vector alone cells, based on C17 S1P production (8.8 ± 2.6 vs 1.4 ± 0.4 ng/106 cells respectively after 24 hrs, p&amp;lt; 0.05). This increase was also observed, though to a lesser extent, in extracellular C17 S1P (678 ± 50 in SKAP vs 462 ± 47 pg/ml in vector alone, p&amp;lt; 0.05). In a proliferation assay this increase in SK activity was associated with a 25% increase in viable cell number (p&amp;lt;0.01 after 3 days). SK activity could be induced further in SKAP cells by the PKC activator PMA, although to a lesser extent than in vector alone cells, while the addition of MK-571 and FTC resulted in a marked increase in intracellular S1P in SKAP and vector alone cells, with a decrease in extracellular S1P levels. These experiments confirm the membrane localisation of SK1 in SKAP transfected cells. SKAP transfection did not affect sensitivity to imatinib or doxorubicin compared to vector alone. Conclusion: These data suggest that SKAP may act as a positive regulator of SK1 activity in cancer cells, an observation that has implications in carcinogenesis and chemosensitivity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3256. doi:1538-7445.AM2012-3256

Sphingosine kinase 1 protects against renal ischemia–reperfusion injury in mice by sphingosine-1-phosphate1 receptor activation

The roles of sphingosine kinases SK1 and SK2 in ischemia-reperfusion injury have not been fully elucidated since studies have found beneficial effects of SK1 while others showed no role in this injury. To help resolve this, we used SK1 or SK2 knockout mice and confirmed that renal ischemia-reperfusion injury induced SK1, but not SK2, in the kidneys. Furthermore, knockout or pharmacological inhibition of SK1 increased injury after renal ischemia-reperfusion injury. In contrast, lack of SK2 conferred renal protection following injury. In addition, we used lentiviral gene delivery to selectively express enhanced green fluorescent protein (EGFP) or human SK1 coexpressed with EGFP (EGFP-huSK1) in the kidney. Mice with kidney-specific overexpression of EGFP-huSK1 had significantly improved renal function with lower plasma creatinine, renal necrosis, apoptosis, and inflammation. Moreover, EGFP-huSK1 overexpression in cultured human proximal tubule (HK-2) cells protected against peroxide-induced necrosis. Selective overexpression of EGFP-huSK1 led to increased HSP27 mRNA and protein expression in vivo and in vitro. Functional protection as well as induction of HSP27 with EGFP-huSK1 overexpression in vivo was blocked with sphingosine-1-phosphate-1 receptor(1) (S1P(1)) antagonism. Thus, our findings suggest that SK1 is renoprotective by S1P(1) activation and perhaps HSP27 induction. Kidney-specific expression of SK1 through lentiviral delivery may be a viable therapeutic option to attenuate renal ischemia-reperfusion injury.

Sphingosine kinase 1 is critically involved in nitric oxide‐mediated human endothelial cell migration and tube formation

Sphingosine kinases (SKs) convert sphingosine to sphingosine 1-phosphate (S1P), which is a bioactive lipid that regulates a variety of cellular processes including proliferation, differentiation and migration. We used the human endothelial cell line EA.hy926 to investigate the effect of nitric oxide (NO) donors on SK-1 expression, and on cell migration and tube formation. We showed that exposure of EA.hy926 cells to Deta-NO (125-1000 microM) resulted in a time- and concentration-dependent up-regulation of SK-1 mRNA and protein expression, and activity with a first significant effect at 250 microM of Deta-NO. The increased SK-1 mRNA expression resulted from an enhanced SK-1 promoter activity. A similar effect was also seen with various other NO donors. In mechanistic terms, the NO-triggered effect occurred independently of cGMP, but involved the classical mitogen-activated protein kinase cascade because the MEK inhibitor U0126 abolished the NO-induced SK-1 expression. The effect of NO was also markedly reduced by the thiol-reducing agent N-acetylcysteine, suggesting a redox-dependent mechanism. Functionally, Deta-NO triggered an increase in the migration of endothelial cells in an adapted Boyden chamber assay, and also increased endothelial tube formation in a Matrigel assay. These responses were both abolished in cells depleted of SK-1. These data show that NO donors up-regulate specifically SK-1 expression and activity in human endothelial cells, and SK-1 in turn critically contributes to the migratory capability and tube formation of endothelial cells. Thus, SK-1 may be considered an attractive novel target to interfere with pathological processes involving angiogenesis.