SphK1 Activity Research Articles

A sphingolipid rheostat controls apoptosis versus apical cell extrusion as alternative tumour-suppressive mechanisms

Evasion of cell death is a hallmark of cancer, and consequently the induction of cell death is a common strategy in cancer treatment. However, the molecular mechanisms regulating different types of cell death are poorly understood. We have formerly shown that in the epidermis of hypomorphic zebrafish hai1a mutant embryos, pre-neoplastic transformations of keratinocytes caused by unrestrained activity of the type II transmembrane serine protease Matriptase-1 heal spontaneously. This healing is driven by Matriptase-dependent increased sphingosine kinase (SphK) activity and sphingosine-1-phosphate (S1P)-mediated keratinocyte loss via apical cell extrusion. In contrast, amorphic hai1afr26 mutants with even higher Matriptase-1 and SphK activity die within a few days. Here we show that this lethality is not due to epidermal carcinogenesis, but to aberrant tp53-independent apoptosis of keratinocytes caused by increased levels of pro-apoptotic C16 ceramides, sphingolipid counterparts to S1P within the sphingolipid rheostat, which severely compromises the epidermal barrier. Mathematical modelling of sphingolipid rheostat homeostasis, combined with in vivo manipulations of components of the rheostat or the ceramide de novo synthesis pathway, indicate that this unexpected overproduction of ceramides is caused by a negative feedback loop sensing ceramide levels and controlling ceramide replenishment via de novo synthesis. Therefore, despite their initial decrease due to increased conversion to S1P, ceramides eventually reach cell death-inducing levels, making transformed pre-neoplastic keratinocytes die even before they are extruded, thereby abrogating the normally barrier-preserving mode of apical live cell extrusion. Our results offer an in vivo perspective of the dynamics of sphingolipid homeostasis and its relevance for epithelial cell survival versus cell death, linking apical cell extrusion and apoptosis. Implications for human carcinomas and their treatments are discussed.

Adipocyte sphingosine kinase 1 regulates histone modifiers to disrupt circadian function.

Circadian rhythms align biological functions with the 24-hour day-night cycle, but modern artificial light disrupts these patterns, contributing to health issues like obesity and cardiovascular disease. The circadian clock operates through a transcriptional-translational feedback loop involving core components such as BMAL1 and CLOCK. Recent research has shown circadian variations in sphingolipid metabolism, specifically sphingosine-1-phosphate (S1P), which plays crucial signaling roles. This study investigates the sphingolipid enzyme, sphingosine kinase 1 (SphK1), which converts sphingosine to S1P, as a circadian-regulated gene in adipocytes. We find that SphK1 expression and activity follow a circadian rhythm, regulated by BMAL1 and CLOCK binding to its promoter. Adipocyte-specific SphK1 knockout mice exhibit disrupted circadian rhythms, and impaired adipocyte function. Additionally, SphK1 deficiency leads to reduced histone acetylation and altered histone deacetylase (HDAC) localization, affecting gene regulation. These results highlight the critical role of SphK1 in linking lipid metabolism with circadian biology.

Thymoquinone, artemisinin, and thymol attenuate proliferation of lung cancer cells as Sphingosine kinase 1 inhibitors

Sphingosine-1-phosphate (S1P) formed via catalytic actions of sphingosine kinase 1 (SphK1) behaves as a pro-survival substance and activates downstream target molecules associated with various pathologies, including initiation, inflammation, and progression of cancer. Here, we aimed to investigate the SphK1 inhibitory potentials of thymoquinone (TQ), Artemisinin (AR), and Thymol (TM) for the therapeutic management of lung cancer. We implemented docking, molecular dynamics (MD) simulations, enzyme inhibition assay, and fluorescence measurement studies to estimate binding affinity and SphK1 inhibitory potential of TQ, AR, and TM. We further investigated the anti-cancer potential of these compounds on non–small cell lung cancer (NSCLC) cell lines (H1299 and A549), followed by estimation of mitochondrial ROS, mitochondrial membrane potential depolarization, and cleavage of DNA by comet assay. Enzyme activity and fluorescence binding studies suggest that TQ, AR, and TM significantly inhibit the activity of SphK1 with IC50 values of 35.52 µM, 42.81 µM, and 53.68 µM, respectively, and have an excellent binding affinity. TQ shows cytotoxic effect and anti-proliferative potentials on H1299 and A549 with an IC50 value of 27.96 µM and 54.43 µM, respectively. Detection of mitochondrial ROS and mitochondrial membrane potential depolarization shows promising TQ-induced oxidative stress on H1299 and A549 cell lines. Comet assay shows promising TQ-induced oxidative DNA damage. In conclusion, TQ, AR, and TM act as potential inhibitors for SphK1, with a strong binding affinity. In addition, the cytotoxicity of TQ is linked to oxidative stress due to mitochondrial ROS generation. Overall, our study suggests that TQ is a promising inhibitor of SphK1 targeting lung cancer therapy.

Dihydromyricetin ameliorates diabetic renal fibrosis via regulating SphK1 to suppress the activation of NF-κB pathway

Dihydromyricetin (DHM) is a flavonoid from vine tea with broad pharmacological benefits, which improve inflammation by blocking the NF-κB pathway. A growing body of research indicates that chronic kidney inflammation is vital to the pathogenesis of diabetic renal fibrosis. Sphingosine kinase-1 (SphK1) is a key regulator of diabetic renal inflammation, which triggers the NF-κB pathway. Hence, we evaluated whether DHM regulates diabetic renal inflammatory fibrosis by acting on SphK1. Here, we demonstrated that DHM effectively suppressed the synthesis of fibrotic and inflammatory adhesion factors like ICAM-1, and VCAM-1 in streptozotocin-treated high-fat diet-induced diabetic mice and HG-induced glomerular mesangial cells (GMCs). Moreover, DHM significantly suppressed NF-κB pathway activation and reduced SphK1 activity and protein expression under diabetic conditions. Mechanistically, the results of molecular docking, molecular dynamics simulation, and cellular thermal shift assay revealed that DHM stably bound to the binding pocket of SphK1, thereby reducing sphingosine-1-phosphate content and SphK1 enzymatic activity, which ultimately inhibited NF-κB DNA binding, transcriptional activity, and nuclear translocation. In conclusion, our data suggested that DHM inhibited SphK1 phosphorylation to prevent NF-κB activation thus ameliorating diabetic renal fibrosis. This supported the clinical use and further drug development of DHM as a potential candidate for treating diabetic renal fibrosis.

Role of Sphingosine Kinase 1 in Glucolipotoxicity-Induced Early Activation of Autophagy in INS-1 Pancreatic β Cells.

Insulin-producing pancreatic β cells play a crucial role in the regulation of glucose homeostasis, and their failure is a key event for diabetes development. Prolonged exposure to palmitate in the presence of elevated glucose levels, termed gluco-lipotoxicity, is known to induce β cell apoptosis. Autophagy has been proposed to be regulated by gluco-lipotoxicity in order to favor β cell survival. However, the role of palmitate metabolism in gluco-lipotoxcity-induced autophagy is presently unknown. We therefore treated INS-1 cells for 6 and 24 h with palmitate in the presence of low and high glucose concentrations and then monitored autophagy. Gluco-lipotoxicity induces accumulation of LC3-II levels in INS-1 at 6 h which returns to basal levels at 24 h. Using the RFP-GFP-LC3 probe, gluco-lipotoxicity increased both autophagosomes and autolysosmes structures, reflecting early stimulation of an autophagy flux. Triacsin C, a potent inhibitor of the long fatty acid acetyl-coA synthase, completely prevents LC3-II formation and recruitment to autophagosomes, suggesting that autophagic response requires palmitate metabolism. In contrast, etomoxir and bromo-palmitate, inhibitors of fatty acid mitochondrial β-oxidation, are unable to prevent gluco-lipotoxicity-induced LC3-II accumulation and recruitment to autophagosomes. Moreover, bromo-palmitate and etomoxir potentiate palmitate autophagic response. Even if gluco-lipotoxicity raised ceramide levels in INS-1 cells, ceramide synthase 4 overexpression does not potentiate LC3-II accumulation. Gluco-lipotoxicity also still stimulates an autophagic flux in the presence of an ER stress repressor. Finally, selective inhibition of sphingosine kinase 1 (SphK1) activity precludes gluco-lipotoxicity to induce LC3-II accumulation. Moreover, SphK1 overexpression potentiates autophagic flux induced by gluco-lipotxicity. Altogether, our results indicate that early activation of autophagy by gluco-lipotoxicity is mediated by SphK1, which plays a protective role in β cells.

Mitochondrial oxidative damage reprograms lipid metabolism of renal tubular epithelial cells in the diabetic kidney

The functional and structural changes in the proximal tubule play an important role in the occurrence and development of diabetic kidney disease (DKD). Diabetes-induced metabolic changes, including lipid metabolism reprogramming, are reported to lead to changes in the state of tubular epithelial cells (TECs), and among all the disturbances in metabolism, mitochondria serve as central regulators. Mitochondrial dysfunction, accompanied by increased production of mitochondrial reactive oxygen species (mtROS), is considered one of the primary factors causing diabetic tubular injury. Most studies have discussed how altered metabolic flux drives mitochondrial oxidative stress during DKD. In the present study, we focused on targeting mitochondrial damage as an upstream factor in metabolic abnormalities under diabetic conditions in TECs. Using SS31, a tetrapeptide that protects the mitochondrial cristae structure, we demonstrated that mitochondrial oxidative damage contributes to TEC injury and lipid peroxidation caused by lipid accumulation. Mitochondria protected using SS31 significantly reversed the decreased expression of key enzymes and regulators of fatty acid oxidation (FAO), but had no obvious effect on major glucose metabolic rate-limiting enzymes. Mitochondrial oxidative stress facilitated renal Sphingosine-1-phosphate (S1P) deposition and SS31 limited the elevated Acer1, S1pr1 and SPHK1 activity, and the decreased Spns2 expression. These data suggest a role of mitochondrial oxidative damage in unbalanced lipid metabolism, including lipid droplet (LD) formulation, lipid peroxidation, and impaired FAO and sphingolipid homeostasis in DKD. An in vitro study demonstrated that high glucose drove elevated expression of cytosolic phospholipase A2 (cPLA2), which, in turn, was responsible for the altered lipid metabolism, including LD generation and S1P accumulation, in HK-2 cells. A mitochondria-targeted antioxidant inhibited the activation of cPLA2f isoforms. Taken together, these findings identify mechanistic links between mitochondrial oxidative metabolism and reprogrammed lipid metabolism in diabetic TECs, and provide further evidence for the nephroprotective effects of SS31 via influencing metabolic pathways.

Targeting the SphK1/S1P/PFKFB3 axis suppresses hepatocellular carcinoma progression by disrupting glycolytic energy supply that drives tumor angiogenesis

BackgroundHepatocellular carcinoma (HCC) remains a leading life-threatening health challenge worldwide, with pressing needs for novel therapeutic strategies. Sphingosine kinase 1 (SphK1), a well-established pro-cancer enzyme, is aberrantly overexpressed in a multitude of malignancies, including HCC. Our previous research has shown that genetic ablation of Sphk1 mitigates HCC progression in mice. Therefore, the development of PF-543, a highly selective SphK1 inhibitor, opens a new avenue for HCC treatment. However, the anti-cancer efficacy of PF-543 has not yet been investigated in primary cancer models in vivo, thereby limiting its further translation.MethodsBuilding upon the identification of the active form of SphK1 as a viable therapeutic target in human HCC specimens, we assessed the capacity of PF-543 in suppressing tumor progression using a diethylnitrosamine-induced mouse model of primary HCC. We further delineated its underlying mechanisms in both HCC and endothelial cells. Key findings were validated in Sphk1 knockout mice and lentiviral-mediated SphK1 knockdown cells.ResultsSphK1 activity was found to be elevated in human HCC tissues. Administration of PF-543 effectively abrogated hepatic SphK1 activity and significantly suppressed HCC progression in diethylnitrosamine-treated mice. The primary mechanism of action was through the inhibition of tumor neovascularization, as PF-543 disrupted endothelial cell angiogenesis even in a pro-angiogenic milieu. Mechanistically, PF-543 induced proteasomal degradation of the critical glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, thus restricting the energy supply essential for tumor angiogenesis. These effects of PF-543 could be reversed upon S1P supplementation in an S1P receptor-dependent manner.ConclusionsThis study provides the first in vivo evidence supporting the potential of PF-543 as an effective anti-HCC agent. It also uncovers previously undescribed links between the pro-cancer, pro-angiogenic and pro-glycolytic roles of the SphK1/S1P/S1P receptor axis. Importantly, unlike conventional anti-HCC drugs that target individual pro-angiogenic drivers, PF-543 impairs the PFKFB3-dictated glycolytic energy engine that fuels tumor angiogenesis, representing a novel and potentially safer therapeutic strategy for HCC.

SKI-349, a Sphingosine Kinases 1/2 Inhibitor, Suppresses Cell Viability, Invasion, and AKT/mTOR Signaling Pathway, and Shows Synergistic Cytotoxic Effects with Sorafenib in Hepatocellular Carcinoma.

SKI-349 is a novel sphingosine kinases (SPHK) inhibitor with anti-tumor effects. This study aimed to assess the effect of SKI-349 on cell biological behaviors, downstream pathways, and its synergistic effect with sorafenib in hepatocellular carcinoma (HCC). HCC cell lines (Huh7 and Hep3B) were treated with SKI-349 at concentrations of 1, 2, 4, or 8 μM. Then, SPHK1/2 activity, cell viability, proliferation, apoptosis, invasion, and protein expressions of phosphorylated-protein kinase B (p-AKT), AKT, phosphorylated-mammalian target of rapamycin (p-mTOR) and mTOR were detected. Combination index values of SKI-349 (0, 1, 2, 4, or 8 μM) and sorafenib (0, 2.5, 5, 10, or 20 μM) were calculated. SKI-349 decreased the relative SPHK1 and SPHK2 activity compared with blank control in a dose-dependent manner in the Huh7 and Hep3B cell lines. Meanwhile, SKI-349 reduced cell viability, 5-ethynyl-2'-deoxyuridine (EdU) positive cells, and invasive cells, while it increased apoptotic cells compared to blank control in a dose-dependent manner in Huh7 and Hep3B cell lines. Based on the western blot assay, SKI-349 decreased the ratio of p-AKT to AKT and that of p-mTOR to mTOR compared with blank control in a dose-dependent manner in the Huh7 and Hep3B cell lines. Additionally, SKI-349 combined with sorafenib declined cell viability with concentration gradient effects compared to SKI-349 sole treatment, and they had synergistic cytotoxic effects in Huh7 and Hep3B cell lines. SKI-349 suppresses SPHK1 and SPHK2 activity, cell viability, invasion, and AKT/mTOR signaling pathway, as well as exhibits a synergistic cytotoxic effect with sorafenib in HCC.

Targeting SphK1/2 by SKI-178 inhibits prostate cancer cell growth

Sphingosine kinases (SphK), including SphK1 and SphK2, are important enzymes promoting progression of prostate cancer. SKI-178 is a novel and highly potent SphK1/2 dual inhibitor. We here tested the potential anti-prostate cancer cell activity of SKI-178. Bioinformatics analyses and results from local tissues demonstrated that that both SphK1 and SphK2 are upregulated in human prostate cancer tissues. Ectopic overexpression of SphK1 and SphK2, by lentiviral constructs, promoted primary prostate cancer cell proliferation and migration. In primary human prostate cancer cells and immortalized cell lines, SKI-178 potently inhibited cell viability, proliferation, cell cycle progression and cell migration, causing robust cell death and apoptosis. SKI-178 impaired mitochondrial functions, causing mitochondrial depolarization, reactive oxygen species production and ATP depletion.SKI-178 potently inhibited SphK activity and induced ceramide production, without affecting SphK1/2 expression in prostate cancer cells. Further, SKI-178 inhibited Akt-mTOR activation and induced JNK activation in prostate cancer cells. Contrarily, a constitutively-active Akt1 construct or the pharmacological JNK inhibitors attenuated SKI-178-induced cytotoxicity in prostate cancer cells. In vivo, daily intraperitoneal injection of a single dose of SKI-178 potently inhibited PC-3 xenograft growth in nude mice. SphK inhibition, ceramide production, ATP depletion and lipid peroxidation as well as Akt-mTOR inactivation and JNK activation were detected in PC-3 xenograft tissues with SKI-178 administration. Together, targeting SphK1/2 by SKI-178 potently inhibited prostate cancer cell growth in vitro and in vivo.

Neurite outgrowth induced by stimulation of angiotensin II AT2 receptors in SH-SY5Y neuroblastoma cells involves c-Src activation

Neuroblastoma, the most common extracranial solid tumor occurring in childhood, originates from the aberrant proliferation of neural crest cells. Accordingly, the mechanism underling neuronal differentiation could provide new strategies for neuroblastoma treatment. It is well known that neurite outgrowth could be induced by Angiotensin II (Ang II) AT2 receptors; however, the signaling mechanism and its possible interaction with NGF (neural growth factor) receptors remain unclear. Here, we show that Ang II and CGP42112A (AT2 receptor agonist) promote neuronal differentiation by inducing neurite outgrowth and βIII-tubulin expression in SH-SY5Y neuroblastoma cells. In addition, we demonstrate that treatment with PD123319 (AT2 receptor antagonist) reverts Ang II or CGP42112A-induced differentiation. By using specific pharmacological inhibitors we established that neurite outgrowth induced by CGP42112A requires the activation of MEK (mitogen-activated protein kinase kinase), SphK (sphingosine kinase) and c-Src but not PI3K (phosphatidylinositol 3-kinase). Certainly, CGP42112A stimulated a rapid and transient (30 s, 1 min) phosphorylation of c-Src at residue Y416 (indicative of activation), following by a Src deactivation as indicated by phosphorylation of Y527. Moreover, inhibition of the NGF receptor tyrosine kinase A (TrkA) reduced neurite outgrowth induced by Ang II and CGP42112A. In summary, we demonstrated that AT2 receptor-stimulated neurite outgrowth in SH-SY5Y cells involves the induction of MEK, SphK and c-Src and suggests a possible transactivation of TrkA. In that regard, AT2 signaling pathway is a key player in neuronal differentiation and might be a potential target for therapeutic treatments.

MiR-542-5p targets c-myc and negates the cell proliferation effect of SphK1 in intestinal epithelial cells.

Intestinal epithelial barrier defects occur commonly during a variety of pathological conditions, though their underlying mechanisms are not completely understood. Sphingosine-1-phosphate (S1P) has been shown to be a critical regulator of proliferation and of maintenance of an intact intestinal epithelial barrier, as is also sphingosine kinase 1 (SphK1), the rate-limiting enzyme for S1P synthesis. SphK1 has been shown to modulate its effect on intestinal epithelial proliferation through increased levels of c-myc. We conducted genome-wide profile analysis to search for differential microRNA expression related to overexpressed SphK1 demonstrating adjusted expression of microRNA 542-5p (miR-542-5p). Here, we show that miR-542-5p is regulated by SphK1 activity and is an effector of c-myc translation that ultimately serves as a critical regulator of the intestinal epithelial barrier. miR-542-5p directly regulates c-myc translation through direct binding to the c-myc mRNA. Exogenous S1P analogs administered in vivo protect murine intestinal barrier from damage due to mesenteric ischemia reperfusion, and damaged intestinal tissue had increased levels of miR-542-5p. These results indicate that miR-542-5p plays a critical role in the regulation of S1P-mediated intestinal barrier function, and may highlight a novel role in potential therapies.

Targeting sphingosine kinase 1/2 by a novel dual inhibitor SKI-349 suppresses non-small cell lung cancer cell growth

Sphingosine kinase 1 (SphK1) and sphingosine kinase (SphK2) are both important therapeutic targets of non-small cell lung cancer (NSCLC). SKI-349 is a novel, highly efficient and small molecular SphK1/2 dual inhibitor. Here in primary human NSCLC cells and immortalized cell lines, SKI-349 potently inhibited cell proliferation, cell cycle progression, migration and viability. The dual inhibitor induced mitochondrial depolarization and apoptosis activation in NSCLC cells, but it was non-cytotoxic to human lung epithelial cells. SKI-349 inhibited SphK activity and induced ceramide accumulation in primary NSCLC cells, without affecting SphK1/2 expression. SKI-349-induced NSCLC cell death was attenuated by sphingosine-1-phosphate and by the SphK activator K6PC-5, but was potentiated by the short-chain ceramide C6. Moreover, SKI-349 induced Akt-mTOR inactivation, JNK activation, and oxidative injury in primary NSCLC cells. In addition, SKI-349 decreased bromodomain-containing protein 4 (BRD4) expression and downregulated BRD4-dependent genes (Myc, cyclin D1 and Klf4) in primary NSCLC cells. At last, SKI-349 (10 mg/kg) administration inhibited NSCLC xenograft growth in nude mice. Akt-mTOR inhibition, JNK activation, oxidative injury and BRD4 downregulation were detected in SKI-349-treated NSCLC xenograft tissues. Taken together, targeting SphK1/2 by SKI-349 potently inhibits NSCLC cell growth in vitro and in vivo.

Sphingosine 1‐Phosphate Prevents Human Embryonic Stem Cell Death Following Ischemic Injury

AbstractCell therapy based on humanembryonic stem cells (hESCs) is a potential treatment for several human diseases caused by ischemic processes. Efficiency and feasibility of these therapies rely on understanding stem cell biology and the interaction and survival of these cells within the injured tissue. hESCs are an important source of diffusible bioactive paracrine modulators and the targets for different signaling molecules that prime cellular tissue repair. Notably, sphingosine 1‐phosphate (S1P) is an emerging bioactive lipid that activates G protein‐coupled receptors, known as S1P receptors (S1PR1–5), promoting cell survival, differentiation, and migration. It is shown that S1P increases cell viability and prevents death in about 50% after the ischemic insult. S1P‐mediated protectiveeffect is attributed to the modulation of S1PR3 and S1PR4 expression, which have been associated with the reperfusion injury salvage kinase/survivoractivating factor enhancement (RISK/SAFE) pathway. Involvement of the SAFE pathway is further verified by applying Janus Kinase (JAK2) and signal transducer and activation of transcription 3 pathway inhibitors, which prevents the S1P protective effect. An increase in sphingosine kinase (SphK) activity is also observed after S1P pretreatment. These results provide evidence for an S1P‐dependent, SphK positive feedback that stimulates hESCs to deliver large amounts of S1P. Thus, S1P protects hESC under ischemic conditions through the different receptors.Practical Applications: It is considered that S1P can be used as an adjuvant to pretreat hESCs prior to the administration in cell therapy‐based protocols for different diseases.

Tumor Necrosis Factor-α Induces a Preeclamptic-like Phenotype in Placental Villi via Sphingosine Kinase 1 Activation.

Preeclampsia (PE) involves inadequate placental function. This can occur due to elevated pro-inflammatory tumor necrosis factor-α (TNF-α). In other tissues, TNF-α signals via sphingosine kinase 1 (SphK1). SphK1 hinders syncytial formation. Whether this occurs downstream of TNF-α signaling is unclear. We hypothesized that placental SphK1 levels are higher in PE and elevated TNF-α decreases syncytial function, increases syncytial shedding, and increases cytokine/factor release via SphK1 activity. Term placental biopsies were analyzed for SphK1 using immunofluorescence and qRT-PCR. Term placental explants were treated after 4 days of culture, at the start of syncytial regeneration, with TNF-α and/or SphK1 inhibitors, PF-543. Syncytialization was assessed by measuring fusion and chorionic gonadotropin release. Cell death and shedding were measured by lactate dehydrogenase release and placental alkaline phosphatase-positive shed particles. Forty-two cytokines were measured using multiplex assays. Placental SphK1 was increased in PE. Increased cell death, shedding, interferon-α2, IFN-γ-induced protein 10, fibroblast growth factor 2, and platelet-derived growth factor-AA release induced by TNF-α were reversed upon SphK1 inhibition. TNF-α increased the release of 26 cytokines independently of SphK1. TNF-α decreased IL-10 release and inhibiting SphK1 reversed this effect. Inhibiting SphK1 alone decreased TNF-α release. Hence, SphK1 partially mediates the TNF-α-induced PE placental phenotype, primarily through cell damage, shedding, and specific cytokine release.

The anti-osteosarcoma cell activity by the sphingosine kinase 1 inhibitor SKI-V

Sphingosine kinase 1 (SphK1) expression and activity are elevated in human osteosarcoma (OS) and is a promising target of therapy. SKI-V is a non-competitive and highly-efficient non-lipid SphK1 inhibitor. The potential anti-OS cell activity by the SphK1 inhibitor was studied here. In primary OS cells and immortalized cell lines, SKI-V robustly suppressed cell survival, growth and proliferation as well as cell mobility, and inducing profound OS cell death and apoptosis. The SphK1 inhibitor was however non-cytotoxic nor pro-apoptotic in human osteoblasts. SKI-V robustly inhibited SphK1 activation and induced accumulation of ceramides, without affecting SphK1 expression in primary OS cells. The SphK1 activator K6PC-5 or sphingosine-1-phosphate partially inhibited SKI-V-induced OS cell death. We showed that SKI-V concurrently blocked Akt-mTOR activation in primary OS cells. A constitutively-active Akt1 (ca-Akt1, S473D) construct restored Akt-mTOR activation and mitigated SKI-V-mediated cytotoxicity in primary OS cells. In vivo, daily injection of SKI-V potently suppressed OS xenograft tumor growth in nude mice. In SKI-V-administrated OS xenograft tissues, SphK1 inhibition, ceramide increase and Akt-mTOR inhibition were detected. Together, SKI-V exerts significant anti-OS activity by inhibiting SphK1 and Akt-mTOR cascades in OS cells.

Sphingosine Kinase-1 Is Overexpressed and Correlates with Hypoxia in Osteosarcoma: Relationship with Clinicopathological Parameters.

Simple SummaryHypoxia has been recognized as a hallmark of solid tumors and a negative prognostic factor for response to therapeutics and survival of patients. Studies have demonstrated that the Sphingosine kinase-1/Sphingosine 1-Phosphate (SphK1/S1P) signaling pathway regulates the expression of the HIF-1 transcription factor in a number of solid tumor models, but no data are available in osteosarcoma characterized by hypoxia. The objectives of the present study were (i) to assess the contribution of SphK1/S1P signaling in regulating HIF-1α expression under hypoxia in various osteosarcoma cell models, (ii) quantify SphK1 enzymatic activity in biopsies of osteosarcoma, and (iii) examine the relationship between SphK1, S1P receptor 1 (S1P1) and hypoxia (GLUT-1) in 130 cases of osteosarcoma by immunohistochemistry. Our data suggest that the SphK1/S1P signaling might represent a potential target to investigate in osteosarcoma patients, considering that fingolimod, which inhibits SphK1 and the S1P1 receptor, is now reconsidered for repurposing in cancer.The Sphingosine kinase-1/Sphingosine 1-Phosphate (SphK1/S1P) signaling pathway is overexpressed in various cancers, and is instrumental for the adaptation to hypoxia in a number of solid tumor models, but no data are available in osteosarcoma. Here we report that SphK1 and the S1P1 receptor are involved in HIF-1α accumulation in hypoxic osteosarcoma cells. FTY720 (Fingolimod), which targets SphK1 and S1P1, prevented HIF-1α accumulation, and also inhibited cell proliferation in both normoxia and hypoxia unlike conventional chemotherapy. In human biopsies, a significant increase of SphK1 activity was observed in cancer compared with normal bones. In all sets of TMA samples (130 cases of osteosarcoma), immunohistochemical analysis showed the hypoxic marker GLUT-1, SphK1 and S1P1 were expressed in tumors. SphK1 correlated with the GLUT-1 suggesting that SphK1 is overexpressed and correlates with intratumoral hypoxia. No correlation was found between GLUT-1 or SphK1 and response to chemotherapy, but a statistical difference was found with increased S1P1 expression in patients with poor response in long bone osteosarcomas. Importantly, multivariate analyses showed that GLUT-1 was associated with an increased risk of death in flat bone, whereas SphK1 and S1P1 were associated with an increased risk of death in long bones.

Arsenic trioxide inhibits the activity of SphK1 by decreasing the level of phosphatidylserine and phosphatidic acid in the human gastric cancer cell line MGC-803

Sphingosine kinase 1 (SphK1) is an important synthetase during the synthesis of sphingosine-1-phosphate (S1P) from sphingosine (Sph). Previous studies demonstrated that arsenic trioxide (As2O3) could reduce the level of S1P in human gastric cancer cell line MGC-803, indicating that As2O3 may inhibit the activity of SphK1. In this study, the effect of As2O3 on the SphK1 activation pathway was investigated. Western blot and quantitative real-time PCR analysis were used to evaluate the changes in protein and mRNA levels. The multi-dimensional mass spectrometry-based shotgun lipidomics method (MDMS-SL) was used for the quantitative detection of phosphatidylserine (PS) and phosphatidic acid (PA). The results revealed that As2O3 did not affect the protein and mRNA expression of SphK1 in the MGC-803 cells. However, As2O3 increased the levels of p-ERK1/2 and CIB1 in the SphK1 activation pathway and decreased the levels of PS and PA in the MGC-803 cells. The outcomes suggested that As2O3 may enhance the activity of SphK1 by increasing the levels of p-ERK1/2 and CIB1 and decrease the activity of SphK1 by decreasing the levels of PS and PA. It was suggested that the inhibition effect is stronger and resulting in an overall decrease in the activity of SphK1.

Arsenic trioxide inhibits the activity of SphK1 by decreasing the level of phosphatidylserine and phosphatidic acid in the human gastric cancer cell line MGC-803

Sphingosine kinase 1 (SphK1) is an important synthetase during the synthesis of sphingosine-1-phosphate (S1P) from sphingosine (Sph). Previous studies demonstrated that arsenic trioxide (As2O3) could reduce the level of S1P in human gastric cancer cell line MGC-803, indicating that As2O3 may inhibit the activity of SphK1. In this study, the effect of As2O3 on the SphK1 activation pathway was investigated. Western blot and quantitative real-time PCR analysis were used to evaluate the changes in protein and mRNA levels. The multi-dimensional mass spectrometry-based shotgun lipidomics method (MDMS-SL) was used for the quantitative detection of phosphatidylserine (PS) and phosphatidic acid (PA). The results revealed that As2O3 did not affect the protein and mRNA expression of SphK1 in the MGC-803 cells. However, As2O3 increased the levels of p-ERK1/2 and CIB1 in the SphK1 activation pathway and decreased the levels of PS and PA in the MGC-803 cells. The outcomes suggested that As2O3 may enhance the activity of SphK1 by increasing the levels of p-ERK1/2 and CIB1 and decrease the activity of SphK1 by decreasing the levels of PS and PA. It was suggested that the inhibition effect is stronger and resulting in an overall decrease in the activity of SphK1.

The sphingosine kinase inhibitor SKI-V suppresses cervical cancer cell growth.

Overexpression and/or overactivation of sphingosine kinase 1/2 (SphK1/2) is important for tumorigenesis and progression of cervical cancer. The current study examined the potential activity and signaling mechanisms of SKI-V, a non-lipid small molecule SphK inhibitor, against cervical cancer cells. In different primary and immortalized cervical cancer cells, SKI-V exerted significant anti-cancer activity by inhibiting cell viability, colony formation, proliferation, cell cycle progression and cell migration. Significant apoptosis activation was detected in SKI-V-treated cervical cancer cells. Significantly, SKI-V also provoked programmed necrosis cascade in cervical cancer cells, as it induced mitochondrial p53-cyclophilin-D-adenine nucleotide translocator-1 (ANT1) complexation, mitochondrial membrane potential collapse, reactive oxygen species production and the release of lactate dehydrogenase into the medium. Further, SKI-V blocked SphK activation and induced ceramide accumulation in primary cervical cancer cells, without affecting SphK1/2 expression. SKI-V-induced cytotoxicity in cervical cancer cells was largely inhibited by sphingosine-1-phosphate or the SphK1 activator K6PC-5, but was sensitized by adding the short-chain ceramide C6. Moreover, SKI-V inhibited Akt-mTOR (mammalian target of rapamycin) activation in primary cervical cancer cells, and its cytotoxicity was mitigated by a constitutively-active Akt. In vivo, daily intraperitoneal injection of SKI-V significantly inhibited subcutaneous primary cervical cancer xenograft growth in nude mice. Together, the SphK inhibitor SKI-V suppresses cervical cancer growth in vitro and in vivo.

APC/PAR1 confers endothelial anti-apoptotic activity via a discrete, β-arrestin-2–mediated SphK1-S1PR1-Akt signaling axis

Endothelial dysfunction is associated with vascular disease and results in disruption of endothelial barrier function and increased sensitivity to apoptosis. Currently, there are limited treatments for improving endothelial dysfunction. Activated protein C (aPC), a promising therapeutic, signals via protease-activated receptor-1 (PAR1) and mediates several cytoprotective responses, including endothelial barrier stabilization and anti-apoptotic responses. We showed that aPC-activated PAR1 signals preferentially via β-arrestin-2 (β-arr2) and dishevelled-2 (Dvl2) scaffolds rather than G proteins to promote Rac1 activation and barrier protection. However, the signaling pathways utilized by aPC/PAR1 to mediate anti-apoptotic activities are not known. aPC/PAR1 cytoprotective responses also require coreceptors; however, it is not clear how coreceptors impact different aPC/PAR1 signaling pathways to drive distinct cytoprotective responses. Here, we define a β-arr2-mediated sphingosine kinase-1 (SphK1)-sphingosine-1-phosphate receptor-1 (S1PR1)-Akt signaling axis that confers aPC/PAR1-mediated protection against cell death. Using human cultured endothelial cells, we found that endogenous PAR1 and S1PR1 coexist in caveolin-1 (Cav1)-rich microdomains and that S1PR1 coassociation with Cav1 is increased by aPC activation of PAR1. Our study further shows that aPC stimulates β-arr2-dependent SphK1 activation independent of Dvl2 and is required for transactivation of S1PR1-Akt signaling and protection against cell death. While aPC/PAR1-induced, extracellular signal-regulated kinase 1/2 (ERK1/2) activation is also dependent on β-arr2, neither SphK1 nor S1PR1 are integrated into the ERK1/2 pathway. Finally, aPC activation of PAR1-β-arr2-mediated protection against apoptosis is dependent on Cav1, the principal structural protein of endothelial caveolae. These studies reveal that different aPC/PAR1 cytoprotective responses are mediated by discrete, β-arr2-driven signaling pathways in caveolae.