Treatment with K6PC-5, a selective stimulator of SPHK1, ameliorates intestinal homeostasis in an animal model of Huntington's disease.

Emerging evidence indicates that Huntington's disease (HD) may be described as multi-organ pathology. In this context, we and others have contributed to demonstrate that the disease is characterized by an impairment of the homeostasis of gastro-intestinal (GI) tract. Sphingolipids represent a class of molecules involved in the regulation and maintenance of different tissues and organs including GI system. In this study, we investigated whether the alteration of Sphingosine-1-phosphate (S1P) metabolism, previously described in human HD brains and animal models, is also detectable peripherally in R6/2 HD mice. Our findings indicate, for the first time, that sphingolipid metabolism is perturbed early in the disease in the intestinal tract of HD mice and, its modulation by K6PC-5, a selective activator of S1P synthesis, preserved intestinal integrity and homeostasis. These results further support the evidence that modulation of sphingolipid pathways may represent a potential therapeutic option in HD and suggest that it has also the potential to counteract the peripheral disturbances which may usually complicate the management of the disease and affect patient's quality of life.

Neonatal therapy with PF543, a sphingosine kinase 1 inhibitor, ameliorates hyperoxia-induced airway remodeling in a murine model of bronchopulmonary dysplasia.

Hyperoxia (HO)-induced lung injury contributes to bronchopulmonary dysplasia (BPD) in preterm newborns. Intractable wheezing seen in BPD survivors is associated with airway remodeling (AWRM). Sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) signaling promotes HO-mediated neonatal BPD; however, its role in the sequela of AWRM is not known. We noted an increased concentration of S1P in tracheal aspirates of neonatal infants with severe BPD, and earlier, demonstrated that Sphk1-/- mice showed protection against HO-induced BPD. The role of SPHK1/S1P in promoting AWRM following exposure of neonates to HO was investigated in a murine model. Therapy using PF543, the specific SPHK1 inhibitor, during neonatal HO reduced alveolar simplification followed by reduced AWRM in adult mice. This was associated with reduced airway hyperreactivity to intravenous methacholine. Neonatal HO exposure was associated with increased expression of SPHK1 in lung tissue of adult mice, which was reduced with PF543 therapy in the neonatal stage. This was accompanied by amelioration of HO-induced reduction of E-cadherin in airway epithelium. This may be suggestive of arrested partial epithelial mesenchymal transition (EMT) induced by HO. In vitro studies using human primary airway epithelial cells (HAEpCs) showed that SPHK1 inhibition or deletion restored HO-induced reduction in E-cadherin and reduced formation of mitochondrial reactive oxygen species (mtROS). Blocking mtROS with MitoTempo attenuated HO-induced partial EMT of HAEpCs. These results collectively support a therapeutic role for PF543 in preventing HO-induced BPD in neonates and the long-term sequela of AWRM, thus conferring a long-term protection resulting in improved lung development and function.

Vaticanol C, a phytoalexin, induces apoptosis of leukemia and cancer cells by modulating expression of multiple sphingolipid metabolic enzymes.

Resveratrol (RSV) has recently attracted keen interest because of its pleiotropic effects. It exerts a wide range of health-promoting effects. In addition to health-promoting effects, RSV possesses anti-carcinogenic activity. However, a non-physiological concentration is needed to achieve an anti-cancer effect, and its in vivo bioavailability is low. Therefore, the clinical application of phytochemicals requires alternative candidates that induce the desired effects at a lower concentration and with increased bioavailability. We previously reported a low IC50 of vaticanol C (VTC), an RSV tetramer, among 12 RSV derivatives (Ito T. et al, 2003). However, the precise mechanism involved remains to be determined. Here, we screened an in-house chemical library bearing RSV building blocks ranging from dimers to octamers for cytotoxic effects in several leukemia and cancer cell lines and their anti-cancer drug-resistant sublines. Among the compounds, VTC exhibited the highest cytotoxicity, which was partially inhibited by a caspase 3 inhibitor, Z-VAD-FMK. VTC decreased the expression of sphingosine kinase 1, sphingosine kinase 2 and glucosylceramide synthase by transcriptional or post-transcriptional mechanisms, and increased cellular ceramides/dihydroceramides and decreased sphingosine 1-phosphate (S1P). VTC-induced sphingolipid rheostat modulation (the ratio of ceramide/S1P) is thought to be involved in cellular apoptosis. Indeed, exogenous S1P addition modulated VTC cytotoxicity significantly. A combination of SPHK1, SPHK2, and GCS chemical inhibitors induced sphingolipid rheostat modulation, cell growth suppression, and cytotoxicity similar to that of VTC. These results suggest the involvement of sphingolipid metabolism in VTC-induced cytotoxicity, and indicate VTC is a promising prototype for translational research.

Identification of the Antidepressant Vilazodone as an Inhibitor of Inositol Polyphosphate Multikinase by Structure-Based Drug Repositioning.

Inositol polyphosphate multikinase (IPMK) is required for the biosynthesis of inositol phosphates (IPs) through the phosphorylation of multiple IP metabolites such as IP3 and IP4. The biological significance of IPMK's catalytic actions to regulate cellular signaling events such as growth and metabolism has been studied extensively. However, pharmacological reagents that inhibit IPMK have not yet been identified. We employed a structure-based virtual screening of publicly available U.S. Food and Drug Administration-approved drugs and chemicals that identified the antidepressant, vilazodone, as an IPMK inhibitor. Docking simulations and pharmacophore analyses showed that vilazodone has a higher affinity for the ATP-binding catalytic region of IPMK than ATP and we validated that vilazodone inhibits IPMK's IP kinase activities in vitro . The incubation of vilazodone with NIH3T3-L1 fibroblasts reduced cellular levels of IP5 and other highly phosphorylated IPs without influencing IP4 levels. We further found decreased Akt phosphorylation in vilazodone-treated HCT116 cancer cells. These data clearly indicate selective cellular actions of vilazodone against IPMK-dependent catalytic steps in IP metabolism and Akt activation. Collectively, our data demonstrate vilazodone as a method to inhibit cellular IPMK, providing a valuable pharmacological agent to study and target the biological and pathological processes governed by IPMK.

Galangin Suppresses Renal Inflammation via the Inhibition of NF-κB, PI3K/AKT and NLRP3 in Uric Acid Treated NRK-52E Tubular Epithelial Cells.

Renal inflammation can result in renal injury. Uric acid (UA) is the final product of purine metabolism in humans and because of the lack of urate oxidase, UA may accumulate in tissues, including kidney, causing inflammation. Galangin was isolated from a traditional Chinese medicine plant and possesses several beneficial effects, working as an anti-oxidant, anti-mutagenic, anti-tumor, anti-inflammatory, anti-microbial, and anti-viral agent. Therefore, this study aimed at investigating the molecular mechanism of galangin in the attenuation of UA induced renal inflammation in normal rat kidney epithelial cells NRK-52E. Our findings suggested that galangin treatment efficiently protected NRK-52E cells against UA induced renal inflammation by decreasing tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-18, prostaglandin E2 (PGE2), and nitric oxide (NO) release, and it inhibited nitric oxide synthase (iNOS), prostaglandin endoperoxide synthase 2 (PTGS2), TNF-α, IL-1ß, and IL-18 mRNA expression. In addition, galangin was not exerting any cytotoxicity at the concentrations that were effective against inflammation as assessed by CCK8 assay. Moreover, western blotting showed that galangin treatment effectively inhibited nuclear factor-kappa B (NF-κB), phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) and nucleotide-binding domain- (NOD-) like receptor protein 3 (NLRP3) signaling pathway activation. Taken together, these findings suggested that galangin plays a pivotal role in renal inflammation by suppressing inflammatory responses, which might be closely associated with the inhibition of NLRP3 inflammasome, NF-κB and PI3K/AKT signaling pathway activation.

NADP+ is an endogenous PARP inhibitor in DNA damage response and tumor suppression.

ADP-ribosylation is a unique posttranslational modification catalyzed by poly(ADP-ribose) polymerases (PARPs) using NAD+ as ADP-ribose donor. PARPs play an indispensable role in DNA damage repair and small molecule PARP inhibitors have emerged as potent anticancer drugs. However, to date, PARP inhibitor treatment has been restricted to patients with BRCA1/2 mutation-associated breast and ovarian cancer. One of the major challenges to extend the therapeutic potential of PARP inhibitors to other cancer types is the absence of predictive biomarkers. Here, we show that ovarian cancer cells with higher level of NADP+, an NAD+ derivative, are more sensitive to PARP inhibitors. We demonstrate that NADP+ acts as a negative regulator and suppresses ADP-ribosylation both in vitro and in vivo. NADP+ impairs ADP-ribosylation-dependent DNA damage repair and sensitizes tumor cell to chemically synthesized PARP inhibitors. Taken together, our study identifies NADP+ as an endogenous PARP inhibitor that may have implications in cancer treatment.

Prolonged treatment with mevalonolactone induces oxidative stress response with reactive oxygen species production, mitochondrial depolarization and inflammation in human glioblastoma U-87 MG cells.

Mevalonate pathway impairment has been observed in diverse diseases, including Mevalonate Kinase Deficiency (MKD). MKD is a hereditary auto-inflammatory disorder, due to mutations at mevalonate kinase gene (MVK), encoding mevalonate kinase (MK) enzyme. To date, the most accredited MKD pathogenic hypothesis suggests that the typical MKD phenotypes might be due to a decreased isoprenoid production rather than to the excess and accumulation of mevalonic acid, as initially supported. Nevertheless, recent studies provide clear evidences that accumulating metabolites might be involved in MKD pathophysiology by exerting a toxic effect. Our work aims at describing the effects of accumulating mevalonolactone, mostly produced by a dehydration reaction due to mevalonic acid accumulation, using an in vitro cellular model mimicking the glial component of the central nervous system (human glioblastoma U-87 MG cells). In order to mimic its progressive increase, occurring during the disease, U-87 MG cells have been treated repeatedly with growing doses of mevalonolactone, followed by the assessment of oxidative stress response (evaluated by measuring SOD2 and HemeOX expression levels), ROS production, mitochondrial damage and inflammatory response (evaluated by measuring IL1B expression levels). Our results suggest that protracted treatments with mevalonolactone induce oxidative stress with augmented ROS production and mitochondrial damage accompanied by membrane depolarization. Furthermore, an increment in IL1B expression has been observed, thus correlating the accumulation of the metabolite with the development of a neuroinflammatory response. Our experimental work suggests to reconsider the presence of a possible synergy between the two major MKD pathogenic hypotheses in attempt of unravelling the different pathogenic pathways responsible for the disease.

Structural modeling identifies Plasmodium vivax 4-diphosphocytidyl-2C-methyl-d-erythritol kinase (IspE) as a plausible new antimalarial drug target.

Malaria parasites utilize Methylerythritol phosphate (MEP) pathway for synthesis of isoprenoid precursors which are essential for maturation and survival of parasites during erythrocytic and gametocytic stages. The absence of MEP pathway in the human host establishes MEP pathway enzymes as a repertoire of essential drug targets. The fourth enzyme, 4-diphosphocytidyl-2C-methyl-d-erythritol kinase (IspE) has been proved essential in pathogenic bacteria, however; it has not yet been studied in any Plasmodium species. This study was undertaken to investigate genetic polymorphism and concomitant structural implications of the Plasmodium vivax IspE (PvIspE) by employing sequencing, modeling and bioinformatics approach. We report that PvIspE gene displayed six non-synonymous mutations which were restricted to non-conserved regions within the gene from seven topographically distinct malaria-endemic regions of India. Phylogenetic studies reflected that PvIspE occupies unique status within Plasmodia genus and reflects that Plasmodium vivax IspE gene has a distant and non-conserved relation with human ortholog Mevalonate Kinase (MAVK). Structural modeling analysis revealed that all PvIspE Indian isolates have critically conserved canonical galacto-homoserine-mevalonate-phosphomevalonate kinase (GHMP) domain within the active site lying in a deep cleft sandwiched between ATP and CDPME-binding domains. The active core region was highly conserved among all clinical isolates, may be due to >60% ß-pleated rigid architecture. The mapped structural analysis revealed the critically conserved active site of PvIspE, both sequence, and spacially among all Indian isolates; showing no significant changes in the active site. Our study strengthens the candidature of Plasmodium vivax IspE enzyme as a future target for novel antimalarials.

Characterization and validation of Entamoeba histolytica pantothenate kinase as a novel anti-amebic drug target.

The Coenzyme A (CoA), as a cofactor involved in >100 metabolic reactions, is essential to the basic biochemistry of life. Here, we investigated the CoA biosynthetic pathway of Entamoeba histolytica (E. histolytica), an enteric protozoan parasite responsible for human amebiasis. We identified four key enzymes involved in the CoA pathway: pantothenate kinase (PanK, EC 2.7.1.33), bifunctional phosphopantothenate-cysteine ligase/decarboxylase (PPCS-PPCDC), phosphopantetheine adenylyltransferase (PPAT) and dephospho-CoA kinase (DPCK). Cytosolic enzyme PanK, was selected for further biochemical, genetic, and phylogenetic characterization. Since E. histolytica PanK (EhPanK) is physiologically important and sufficiently divergent from its human orthologs, this enzyme represents an attractive target for the development of novel anti-amebic chemotherapies. Epigenetic gene silencing of PanK resulted in a significant reduction of PanK activity, intracellular CoA concentrations, and growth retardation in vitro, reinforcing the importance of this gene in E. histolytica. Furthermore, we screened the Kitasato Natural Products Library for inhibitors of recombinant EhPanK, and identified 14 such compounds. One compound demonstrated moderate inhibition of PanK activity and cell growth at a low concentration, as well as differential toxicity towards E. histolytica and human cells.

A multitarget approach to drug discovery inhibiting Mycobacterium tuberculosis PyrG and PanK.

Mycobacterium tuberculosis, the etiological agent of the infectious disease tuberculosis, kills approximately 1.5 million people annually, while the spread of multidrug-resistant strains is of great global concern. Thus, continuous efforts to identify new antitubercular drugs as well as novel targets are crucial. Recently, two prodrugs activated by the monooxygenase EthA, 7947882 and 7904688, which target the CTP synthetase PyrG, were identified and characterized. In this work, microbiological, biochemical, and in silico methodologies were used to demonstrate that both prodrugs possess a second target, the pantothenate kinase PanK. This enzyme is involved in coenzyme A biosynthesis, an essential pathway for M. tuberculosis growth. Moreover, compound 11426026, the active metabolite of 7947882, was demonstrated to directly inhibit PanK, as well. In an independent screen of a compound library against PyrG, two additional inhibitors were also found to be active against PanK. In conclusion, these direct PyrG and PanK inhibitors can be considered as leads for multitarget antitubercular drugs and these two enzymes could be employed as a "double-tool" in order to find additional hit compounds.

GDF11 Attenuates Development of Type 2 Diabetes via Improvement of Islet ß-Cell Function and Survival.

Growth differentiation factor 11 (GDF11) has been implicated in the regulation of islet development and a variety of aging conditions, but little is known about the physiological functions of GDF11 in adult pancreatic islets. Here, we showed that systematic replenishment of GDF11 not only preserved insulin secretion but also improved the survival and morphology of ß-cells and improved glucose metabolism in both nongenetic and genetic mouse models of type 2 diabetes (T2D). Conversely, anti-GDF11 monoclonal antibody treatment caused ß-cell failure and lethal T2D. In vitro treatment of isolated murine islets and MIN6 cells with recombinant GDF11 attenuated glucotoxicity-induced ß-cell dysfunction and apoptosis. Mechanistically, the GDF11-mediated protective effects could be attributed to the activation of transforming growth factor-ß/Smad2 and phosphatidylinositol-4,5-bisphosphate 3-kinase-AKT-FoxO1 signaling. These findings suggest that GDF11 repletion may improve ß-cell function and mass and thus may lead to a new therapeutic approach for T2D.

Phosphate Solubilization and Gene Expression of Phosphate-Solubilizing Bacterium Burkholderia multivorans WS-FJ9 under Different Levels of Soluble Phosphate.

Phosphate-solubilizing bacteria (PSB) have the ability to dissolve insoluble phosphate and enhance soil fertility. However, the growth and mineral phosphate solubilization of PSB could be affected by exogenous soluble phosphate and the mechanism has not been fully understood. In the present study, the growth and mineral phosphate-solubilizing characteristics of PSB strain Burkholderia multivorans WS-FJ9 were investigated at six levels of exogenous soluble phosphate (0, 0.5, 1, 5, 10, and 20 mM). The WS-FJ9 strain showed better growth at high levels of soluble phosphate. The phosphate-solubilizing activity of WS-FJ9 was reduced as the soluble phosphate concentration increased, as well as the production of pyruvic acid. Transcriptome profiling of WS-FJ9 at three levels of exogenous soluble phosphate (0, 5, and 20 mM) identified 446 differentially expressed genes, among which 44 genes were continuously up-regulated when soluble phosphate concentration was increased and 81 genes were continuously down-regulated. Some genes related to cell growth were continuously up-regulated, which would account for the better growth of WS-FJ9 at high levels of soluble phosphate. Genes involved in glucose metabolism, including glycerate kinase, 2-oxoglutarate dehydrogenase, and sugar ABC-type transporter, were continuously down-regulated, which indicates that metabolic channeling of glucose towards the phosphorylative pathway was negatively regulated by soluble phosphate. These findings represent an important first step in understanding the molecular mechanisms of soluble phosphate effects on the growth and mineral phosphate solubilization of PSB.

Uncovering oxysterol-binding protein (OSBP) as a target of the anti-enteroviral compound TTP-8307.

The genus Enterovirus (e.g. poliovirus, coxsackievirus, rhinovirus) of the Picornaviridae family of positive-strand RNA viruses includes many important pathogens linked to a range of acute and chronic diseases for which no approved antiviral therapy is available. Targeting a step in the life cycle that is highly conserved provides an attractive strategy for developing broad-range inhibitors of enterovirus infection. A step that is currently explored as a target for the development of antivirals is the formation of replication organelles, which support replication of the viral genome. To build replication organelles, enteroviruses rewire cellular machinery and hijack lipid homeostasis pathways. For example, enteroviruses exploit the PI4KIIIß-PI4P-OSBP pathway to direct cholesterol to replication organelles. Here, we uncover that TTP-8307, a known enterovirus replication inhibitor, acts through the PI4KIIIß-PI4P-OSBP pathway by directly inhibiting OSBP activity. However, despite a shared mechanism of TTP-8307 with established OSBP inhibitors (itraconazole and OSW-1), we identify a number of notable differences between these compounds. The antiviral activity of TTP-8307 extends to other viruses that require OSBP, namely the picornavirus encephalomyocarditis virus and the flavivirus hepatitis C virus.

Anti-cancer effect of pristimerin by inhibition of HIF-1α involves the SPHK-1 pathway in hypoxic prostate cancer cells.

BACKGROUND: Hypoxia is a typical character of locally advanced solid tumours. The transcription factor hypoxia-inducible factor 1α (HIF-1α) is the main regulator under the hypoxic environment. HIF-1α regulates various genes to enhance tumour progression, angiogenesis, and metastasis. Sphingosine kinase 1 (SPHK-1) is a modulator of HIF-1α. METHODS: To investigate the molecular mechanisms of pristimerin in association with SPHK-1 pathways in hypoxic PC-3 cancer cells. Vascular endothelial growth factor (VEGF) production, cell cycles, and SPHK-1 activity were measured, and western blotting, an MTT assay, and an RNA interference assay were performed. RESULTS: Pristimerin inhibited HIF-1α accumulation in a concentration- and-time-dependent manner in hypoxic PC-3 cells. Pristimerin suppressed the expression of HIF-1α by inhibiting SPHK-1. Moreover, inhibiting SPHK-1 with a sphingosine kinase inhibitor enhanced the suppression of HIF-1α, phosphorylation AKT, and glycogen synthase kinase-3ß (GSK-3ß) by pristimerin under hypoxia. Furthermore, a reactive oxygen species (ROS) scavenger enhanced the inhibition of HIF-1α and SPHK-1 by pristimerin. CONCLUSION: Taken together, these findings suggest that pristimerin can exert an anti-cancer activity by inhibiting HIF-1α through the SPHK-1 pathway.

Glucocorticoids limit acute lung inflammation in concert with inflammatory stimuli by induction of SphK1.

Acute lung injury (ALI) is a severe inflammatory disease for which no specific treatment exists. As glucocorticoids have potent immunosuppressive effects, their application in ALI is currently being tested in clinical trials. However, the benefits of this type of regimen remain unclear. Here we identify a mechanism of glucocorticoid action that challenges the long-standing dogma of cytokine repression by the glucocorticoid receptor. Contrarily, synergistic gene induction of sphingosine kinase 1 (SphK1) by glucocorticoids and pro-inflammatory stimuli via the glucocorticoid receptor in macrophages increases circulating sphingosine 1-phosphate levels, which proves essential for the inhibition of inflammation. Chemical or genetic inhibition of SphK1 abrogates the therapeutic effects of glucocorticoids. Inflammatory p38 MAPK- and mitogen- and stress-activated protein kinase 1 (MSK1)-dependent pathways cooperate with glucocorticoids to upregulate SphK1 expression. Our findings support a critical role for SphK1 induction in the suppression of lung inflammation by glucocorticoids, and therefore provide rationales for effective anti-inflammatory therapies.

Modification of sphingolipid metabolism by tamoxifen and N-desmethyltamoxifen in acute myelogenous leukemia--Impact on enzyme activity and response to cytotoxics.

The triphenylethylene antiestrogen, tamoxifen, can be an effective inhibitor of sphingolipid metabolism. This off-target activity makes tamoxifen an interesting ancillary for boosting the apoptosis-inducing properties of ceramide, a sphingolipid with valuable tumor censoring activity. Here we show for the first time that tamoxifen and metabolite, N-desmethyltamoxifen (DMT), block ceramide glycosylation and inhibit ceramide hydrolysis (by acid ceramidase, AC) in human acute myelogenous leukemia (AML) cell lines and in AML cells derived from patients. Tamoxifen (1-10 µM) inhibition of AC in AML cells was accompanied by decreases in AC protein expression. Tamoxifen also depressed expression and activity of sphingosine kinase 1 (SphK1), the enzyme-catalyzing production of mitogenic sphingosine 1-phosphate (S1-P). Results from mass spectroscopy showed that tamoxifen and DMT (i) increased the levels of endogenous C16:0 and C24:1 ceramide molecular species, (ii) nearly totally halted production of respective glucosylceramide (GC) molecular species, (iii) drastically reduced levels of sphingosine (to 9% of control), and (iv) reduced levels of S1-P by 85%, in vincristine-resistant HL-60/VCR cells. The co-administration of tamoxifen with either N-(4-hydroxyphenyl)retinamide (4-HPR), a ceramide-generating retinoid, or a cell-deliverable form of ceramide, C6-ceramide, resulted in marked decreases in HL-60/VCR cell viability that far exceeded single agent potency. Combination treatments resulted in synergistic apoptotic cell death as gauged by increased Annexin V binding and DNA fragmentation and activation of caspase-3. These results show the versatility of adjuvant triphenylethylene with ceramide-centric therapies for magnifying therapeutic potential in AML. Such drug regimens could serve as effective strategies, even in the multidrug-resistant setting.

Vectorization efforts to increase Gram-negative intracellular drug concentration: a case study on HldE-K inhibitors.

In this paper, we present different strategies to vectorize HldE kinase inhibitors with the goal to improve their gram-negative intracellular concentration. Syntheses and biological effects of siderophoric, aminoglycosidic, amphoteric, and polycationic vectors are discussed. While siderophoric and amphoteric vectorization efforts proved to be disappointing in this series, aminoglycosidic and polycationic vectors were able for the first time to achieve synergistic effects of our inhibitors with erythromycin. Although these effects proved to be nonspecific, this study provides information about the required stereoelectronic arrangement of the polycationic amines and their basicity requirements to fulfill outer membrane destabilization resulting in better erythromycin synergies.

Sphingosine kinase isoforms as a therapeutic target in endocrine therapy resistant luminal and basal-A breast cancer.

Sphingosine kinase signaling has become of increasing interest as a cancer target in recent years. Two sphingosine kinase inhibitors, sphingosine kinase inhibitor (SKI)-II and ABC294640, are promising as potential breast cancer therapies. However, evidence for their therapeutic properties in specific breast cancer subtypes is currently lacking. In this study, we characterize these drugs in luminal, endocrine-resistant (MDA-MB-361) and basal-A, triple-negative (MDA-MB-468) breast cancer cells and compare them with previously published data in other breast cancer cell models. Both SKI-II and ABC294640 demonstrated greater efficacy in basal-A compared with luminal breast cancer. ABC294640, in particular, induced apoptosis and blocked proliferation both in vitro and in vivo in this triple-negative breast cancer system. Furthermore, Sphk expression promotes survival and endocrine therapy resistance in previously sensitive breast cancer cells. Taken together, these results characterize sphingosine kinase inhibitors across breast cancer cell systems and demonstrate their therapeutic potential as anti-cancer agents.

Isoflurane activates intestinal sphingosine kinase to protect against renal ischemia-reperfusion-induced liver and intestine injury.

BACKGROUND: Renal ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury and often leads to multiorgan dysfunction and systemic inflammation. Volatile anesthetics have potent antiinflammatory effects. We aimed to determine whether the representative volatile anesthetic isoflurane protects against acute kidney injury-induced liver and intestinal injury and to determine the mechanisms involved in this protection. METHODS: Mice were anesthetized with pentobarbital and subjected to 30 min of left renal ischemia after right nephrectomy, followed by exposure to 4 h of equianesthetic doses of pentobarbital or isoflurane. Five hours after renal IRI, plasma creatinine and alanine aminotransferase concentrations were measured. Liver and intestine tissues were analyzed for proinflammatory messenger RNA (mRNA) concentrations, histologic features, sphingosine kinase-1 (SK1) immunoblotting, SK1 activity, and sphingosine-1-phosphate concentrations. RESULTS: Renal IRI with pentobarbital led to severe renal, hepatic, and intestinal injury with focused periportal hepatocyte vacuolization; small-intestinal apoptosis; and proinflammatory mRNA up-regulation. Isoflurane protected against renal IRI and reduced hepatic and intestinal injury via induction of small-intestinal crypt SK1 mRNA, protein and enzyme activity, and increased sphingosine-1-phosphate. We confirmed the importance of SK1 because mice treated with a selective SK inhibitor or mice deficient in the SK1 enzyme were not protected against hepatic and intestinal dysfunction with isoflurane. CONCLUSIONS: Isoflurane protects against multiorgan injury after renal IRI via induction of the SK1/sphingosine-1-phosphate pathway. Our findings may help to unravel the cellular signaling pathways of volatile anesthetic-mediated hepatic and intestinal protection and may lead to new therapeutic applications of volatile anesthetics during the perioperative period.

Isoflurane protects human kidney proximal tubule cells against necrosis via sphingosine kinase and sphingosine-1-phosphate generation.

BACKGROUND/AIMS: We previously showed that the inhalational anesthetic isoflurane protects against renal ischemia reperfusion injury in part via sphingosine kinase (SK)-mediated synthesis of sphingosine-1-phosphate (S1P). In this study, we tested the hypothesis that isoflurane directly targets renal proximal tubule cells via SK activation, S1P synthesis and activation of S1P receptors to initiate cytoprotective signaling. METHODS AND RESULTS: Isoflurane-mediated phosphorylation of extracellular signal-regulated kinase (ERK) and Akt and induction of HSP70 in human kidney proximal tubule (HK-2) cells were inhibited by dimethylsphingosine (DMS), an SK inhibitor, and VPC23019, an S1P(1/3) receptor selective antagonist, in HK-2 cells. A selective S1P(1) receptor agonist, SEW2781, mimicked isoflurane-induced phosphorylation of ERK and Akt and induction of HSP70. Moreover, isoflurane-mediated protection against H(2)O(2)-induced necrosis of HK-2 cells was significantly attenuated by an S1P(1/3) receptor antagonist, VPC23019, and by SK inhibitors DMS or 4-[[4- (4-chlorophenyl)-2-thiazolyl]amino]phenol. Finally, overexpression of the SK1 enzyme in HK-2 cells protected against H(2)O(2)-induced necrosis. CONCLUSIONS: Collectively, our study demonstrates that S1P released via isoflurane-mediated SK1 stimulation produces direct anti-necrotic effects probably via S1P(1) receptor-mediated cytoprotective signaling (ERK/Akt phosphorylation and HSP70 induction) in HK-2 cells. Our findings may help to unravel the cellular signaling pathways of volatile anesthetic-mediated renal protection and lead to new therapeutic applications of volatile anesthetics during the perioperative period.