Downregulating sphingosine kinase-1 for cancer therapy

Background: The sphingolipids ceramide and sphingosine 1-phosphate (S1P) are key regulators of cell death and proliferation. The subtle balance between their intracellular levels is governed mainly by sphingosine kinase-1, which produces the pro-survival S1P. Sphingosine kinase-1 is an oncogene; is overexpressed in many tumors; protects cancer cells from apoptosis in vitro and in vivo; and its activity is decreased by anticancer therapies. Hence, sphingosine kinase-1 appears to be a target of interest for therapeutic manipulation. Objective: This review considers recent developments regarding the involvement of sphingosine kinase-1 as a therapeutic target for cancer, and describes the pharmacological tools currently available. Results/conclusion: The studies described provide strong evidence that strategies to kill cancer cells via sphingosine kinase-1 inhibition are valid and could have a favorable therapeutic index.     

Renal mesangial cells: moving on sphingosine kinase-1

Sphingosine kinase-1 (SphK1) catalyses the phosphorylation of sphingosine to sphingosine-1-phosphate (S1P), which acts on at least five specific G-protein-coupled receptors and also intracellularly. SphK1 has been implicated in cell proliferation, cancer growth, chemoresistance, immune cell functions and cell migration. In this issue of the British Journal of Pharmacology, Klawitter et al. demonstrate that extracellular nucleotides stimulate the migration of renal mesangial cells. The nucleotides furthermore upregulated SphK1 expression and activity, and this enzyme was required for nucleotide-induced migration. Together with previous findings, these data raise exciting questions: by which mechanism does SphK1 regulate migration in mesangial cells, how is the interplay of purinoceptors and S1P receptors organized in these cells, and how would SphK1-deficient mice respond to kidney damage?     

Inhibition of sphingosine kinase-2 ablates androgen resistant prostate cancer proliferation and survival

BackgroundEndogenous sphingolipid signaling has been shown to play an important role in prostate cancer endocrine resistance.MethodsThe novel SphK2 inhibitor, ABC294640, was used to explore SphK signaling in androgen resistant prostate cancer cell death signaling.ResultsIt dose-dependently decreased PC-3 and LNCaP cell viability, IC₅₀ of 28 ± 6.1 μM (p < 0.05) and 25 ± 4.0 μM (p < 0.05), respectively. ABC294640 was more potent in long-term clonogenic survival assays; IC₅₀ of 14 ± 0.4 μM (p < 0.05) in PC-3 cells and 12 ± 0.9 μM (p < 0.05) in LNCaP cells. Intrinsic apoptotic assays failed to demonstrate increased caspase-9 activity. Ki-67 staining demonstrated decreased proliferation by 50 ± 8.4% (p < 0.01) in PC-3 cells.ConclusionsSphK2 inhibition decreases androgen resistant prostate cancer viability, survival, and proliferation independently of the intrinsic apoptotic pathway. Findings are in contrast to recent observations of ABC29460 acting dependently on the intrinsic pathway in other endocrine resistant cancer cell lines.     

Loss of sphingosine kinase-1 in carcinoma cells increases formation of reactive oxygen species and sensitivity to doxorubicin-induced DNA damage

BACKGROUND AND PURPOSE

Sphingosine kinases (SK) catalyse the formation of sphingosine 1-phosphate, which is a key lipid mediator regulating cell responses such as proliferation, survival and migration. Here we have investigated the effect of targeted inhibition of SK-1 on cell damage and elucidated the mechanisms involved.

EXPERIMENTAL APPROACH

Three human carcinoma cell lines (colon HCT-116, breast MDA-MB-231, lung NCI-H358) were used, which were either transduced with shRNA constructs to deplete SK-1, or treated with a SK-1 inhibitor. Cell growth and viability were assayed by [³H]thymidine incorporation and colony formation. Reactive oxygen species (ROS) were measured by fluorescence and apoptosis by annexin V with flow cytometry. Proteins were analysed by Western blotting. DNA damage was induced by doxorubicin.

KEY RESULTS

Knock-down of SK-1 by shRNA strongly inhibited DNA synthesis and colony formation of carcinoma cells. SK-1 knock-down (SK-1kd) cells revealed dysfunctional extracellular signal-regulated protein kinase and PKB/Akt cascades, and contained increased levels of ROS. After SK-1kd, treatment with doxorubicin increased DNA damage, measured by histone-2AX phosphorylation. Similar effects were found in cells with a SK-1 inhibitor and doxorubicin. The increased damage response in SK-1kd cells was accompanied by greater reduction of DNA synthesis and colony formation, and by more pronounced apoptosis. Addition of a NADPH oxidase inhibitor reduced the increased apoptosis in doxorubicin-treated SK-1kd cells.

CONCLUSIONS AND IMPLICATIONS

SK-1kd in carcinoma cells triggered oxidative stress by increasing intracellular Ros production. Targeted inhibition of SK-1 represents a promising approach to sensitize cells to DNA damage and facilitate apoptosis upon doxorubicin treatment.     

Ginsenoside compound K inhibits angiogenesis via regulation of sphingosine kinase-1 in human umbilical vein endothelial cells

Ginsenoside compound K (CK) is a metabolite of the protopanaxadiol-type saponins of Panax ginseng C.A. Meyer (Araliaceae), has long been used to treat against the development of cancer, inflammation, allergies, and diabetes. This study examined the anti-angiogenic properties of CK against sphingosine 1-phosphate (S1P)-induced cell migration via regulation of sphingosine kinase 1 (SPHK1) in human umbilical vein endothelial cells (HUVEC). Studies on S1P-induced cell migration, expression of SPHK1 and MMPs and analysis of sphingolipid metabolites by LC–MS/MS were examined after the treatment of CK (2.5, 5, 10 μg/mL) in HUVEC. S1P produced by SPHK1 is also involved in cell growth, migration, and protection of apoptosis; therefore, we sought to investigate whether ginsenosides are able to regulate SPHK1. For this purpose, we developed an inhibitory assay of SPHK1 activity and an analytical method for detection of S1P and other sphingolipid metabolites in HUVEC. Ginsenoside CK inhibited 100 nM S1P-induced cell migrations in a dose-dependent manner. Among tested ginsenosides, CK exclusively inhibited S1P production, SPHK1 activity and SPHK1 expression in HUVEC, whereas expression of the pro-apoptotic sphingolipids, sphingosine and ceramide, was increased in response to CK. The major subspecies of the increased ceramide was C24:0-ceramide. CK also disrupted the sphingolipid rheostat, which ultimately influences cell fate, and dose-dependently inhibited HUVEC migration by reducing expression of metalloproteinases (MMPs). Ginsenoside CK acts as a unique HUVEC migration inhibitor by regulating MMP expression, as well as the activity of SPHK1 and its related sphingolipid metabolites.     

Extracellular nucleotides induce migration of renal mesangial cells by upregulating sphingosine kinase-1 expression and activity

BACKGROUND AND PURPOSE: Extracellular nucleotides act as potent mitogens for renal mesangial cells (MC). In this study we determined whether extracellular nucleotides trigger additional responses in MCs and the mechanisms involved. EXPERIMENTAL APPROACH: MC migration was measured after nucleotide stimulation in an adapted Boyden-chamber. Sphingosine kinase-1 (SK-1) protein expression was detected by Western blot analysis and mRNA expression quantified by real-time PCR. SK activity was measured by an in vitro kinase assay using sphingosine as substrate. KEY RESULTS: Nucleotide stimulation caused biphasic activation of SK-1, but not SK-2. The first peak occurred after minutes of stimulation and was followed by a second delayed peak after 4-24 h of stimulation. The delayed activation of SK-1 is due to increased SK-1 mRNA steady-state levels and de novo synthesis of SK-1 protein, and depends on PKC and the classical MAPK cascade. To see whether nucleotide-stimulated cell responses require SK-1, we selectively depleted SK-1 from cells by using small-interference RNA (siRNA). MC migration is highly stimulated by ATP and UTP; this is mimicked by exogenously added S1P. Depletion of SK-1 by siRNA drastically reduced the effect of ATP and UTP on cell migration but not on cell proliferation. Furthermore, MCs isolated from SK-1-deficient mice were completely devoid of nucleotide-induced migration. CONCLUSIONS AND IMPLICATIONS: These data show that extracellular nucleotides besides being mitogenic also trigger MC migration and this cell response critically requires SK-1 activity. Thus, pharmacological intervention of SK-1 may have impacts on situations where MC migration is important such as during inflammatory kidney diseases.     

Membrane lesions in cultured mouse neuroblastoma cells exposed to metal compounds

Tritiated 2-deoxy-D-glucose (dGlc) was rapidly taken up into cultured mouse neuroblastoma C1300 cells (clone 41A₃). Upon perfusion the pre-loaded cultures slowly released radioactivity as [³H]2-deoxy-D-glucose-6-phosphate ([³H]dGlc-6-P) (rate const. = 0.017 min^?1) from a pool corresponding to 74% (t1/2 = 41 min) of the total radioactivity incorporated. Destruction of the plasma membrane of the cells by means of Triton X-100 (1.0%) resulted in a rapid and total release of the radioactivity. CH₃HgCl, HgCl, (C₂H₅)₃SnCl and K₂Cr₂O₇ all caused an increase in the passive cell membrane permeability to [³H]dGlc-6-P. A membrane toxic concentration (MTC) was defined as the concentration of the tested metal compound giving rise to an increase in the relative efflux from 1.0 to 1.2 during 60 min perfusion. Using this MTC-value, the membrane toxicity of the compounds could be ranked in the following order: CH₃HgCl (MTC = 9 × 10^?7 M >; HgCl (MTC = 6 × 10^?6 M) > (C₂H₅)₃SnCl (MTC = 3 × 10^?4 M) > K₂Cr₂O₇ (MTC = 7 × 10^?4 M. Since this differential toxicity is in accordance with other reports it is concluded that 2-deoxy-D-glucose (dGlc) may be used together with 41A₃ cells to screen metal compounds for their membrane toxicity.     

Release of heat shock proteins from human neuroblastoma cells exposed to acrylamide

When human neuroblastoma cells (SH-SY5Y) were exposed to 0.5 - 5 mM acrylamide for 18 hr, the levels of heat shock proteins (HSPs) of 90, 70 and 27 kDa (Hsp90, Hsp70, and Hsp27, respectively) were elevated in the incubation media depending on the dose of acrylamide whereas only the Hsp70 level increased within cells. U0126, a specific inhibitor of extracellular signal-regulated protein kinase kinase and a potent suppressor of the cytotoxicity of acrylamide, suppressed the increase in the levels of all HSPs in the incubation media but not their expression within cells. Total protein concentrations in the incubation media increased depending on the dose of acrylamide, and this increase was associated with the increasing number of bands detected by silver staining after SDS-polyacrylamide gel electrophoresis. One of the clearest bands was identified as Hsp90 by peptide mass fingerprinting. Thus, acrylamide causes release of proteins, including that of HSPs, from SH-SY5Y cells. HSP in extracellular fluid may be a good indicator of cytotoxicity of acrylamide.     

The effects of acute pesticide exposure on neuroblastoma cells chronically exposed to diazinon

Speculation about potential neurotoxicity due to chronic exposure to low doses of organophosphate (OP) pesticides is not yet supported by experimental evidence. The objective of this work was to use a cell culture model of chronic OP exposure to determine if such exposure can alter the sensitivity of nerve cells to subsequent acute exposure to OPs or other compounds. NB2a neuroblastoma cells were grown in the presence of 25 microM diazinon for 8 weeks. The OP was then withdrawn and the cells were induced to differentiate in the presence of various other pesticides or herbicides, including OPs and OP-containing formulations. The resulting outgrowth of neurite-like structures was measured by light microscopy and quantitative image analysis and the IC(50) for each OP or formulation was calculated. The IC(50) values in diazinon-pre-exposed cells were compared with the equivalent values in cells not pre-exposed to diazinon. The IC(50) for inhibition of neurite outgrowth by acute application of diazinon, pyrethrum, glyphosate or a commercial formulation of glyphosate was decreased by between 20 and 90% after pre-treatment with diazinon. In contrast, the IC(50) for pirimiphos methyl was unaffected and those for phosmet or chlorpyrifos were increased by between 1.5- and 3-fold. Treatment of cells with chlorpyrifos or with a second glyphosate-containing formulation led to the formation of abnormal neurite-like structures in diazinon-pre-exposed cells. The data support the view that chronic exposure to an OP may reduce the threshold for toxicity of some, but by no means all, environmental agents.     

Development of amidine-based sphingosine kinase 1 nanomolar inhibitors and reduction of sphingosine 1-phosphate in human leukemia cells

Sphingosine 1-phosphate (S1P) is a bioactive lipid that has been identified as an accelerant of cancer progression. The sphingosine kinases (SphKs) are the sole producers of S1P, and thus, SphK inhibitors may prove effective in cancer mitigation and chemosensitization. Of the two SphKs, SphK1 overexpression has been observed in a myriad of cancer cell lines and tissues and has been recognized as the presumptive target over that of the poorly characterized SphK2. Herein, we present the design and synthesis of amidine-based nanomolar SphK1 subtype-selective inhibitors. A homology model of SphK1, trained with this library of amidine inhibitors, was then used to predict the activity of additional, more potent, inhibitors. Lastly, select amidine inhibitors were validated in human leukemia U937 cells, where they significantly reduced endogenous S1P levels at nanomolar concentrations.     

Tetrandrine selectively protects against amyloid-beta protein - but not against MPTP-induced cytotoxicity in SK-N-SH neuroblastoma cells

The evidence for loss of Ca2+ homeostasis due to neuronal degeneration is considerable and rapidly increasing. In this study, we try to evaluate the protective effect of tetrandrine (TET), an alkaloid isolated from the Chinese medicinal herb Radix Stephania tetrandrae S., on amyloid-beta protein (Abeta) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced cell death in SK-N-SH neuroblastoma cells. Both compounds reduced cell viability in a concentration-dependent manner after 72 h in culture. Cell proliferation in the presence of 20 microM Abeta or 0.4 mM MPTP was reduced to 58.3 +/- 4.9 or 54.9 +/- 5.5 %, respectively. TET (0.1, 0.5 and 1 microM) alone had no significant effect on cell survival; however, it prevented Abeta-induced cell death in a concentration-dependent manner. In contrast, TET failed to counteract MPTP-induced cytotoxicity. Also, an L-type calcium channel blocker, nimodipine, solely reversed Abeta-induced cell death. On the other hand, ELISA determination of mono-/oligo-nucleosomes accumulation showed that the mode of cell death evoked by Abeta was necrosis while that evoked by MPTP was presumably apoptosis. These results suggest that TET may mitigate the harmful effects of Abeta on cell survival, probably by interfering via the necrotic signal related to Ca2+ overloading through the L-type calcium channel.     

Autophagy counteracts apoptosis in human multiple myeloma cells exposed to oridonin in vitro via regulating intracellular ROS and SIRT1

Aim:

To explore the mechanisms underlying the oridonin-induced apoptosis and autophagy in human multiple myeloma cells in vitro.

Methods:

Human multiple myeloma RPMI8266 cells were used. The cell viability was assessed using MTT assay. Morphological changes of apoptosis and autophagy were observed under transmission electron microscope. TUNEL and annexin V-FITC/PI dual staining assays were used to measure apoptosis. Autophagy was analyzed using Western blot analysis and immunofluorescence staining with a QDs_605 nm-Anti-LC3 fluorescent probe. Intracellular ROS was estimated with flow cytometry using DCFH-DA fluorescent probe. Protein levels of active caspase 3, Beclin 1 and SIRT1 were determined with Western blot analysis.

Results:

Exposure to oridonin (1-64 μmol/L) inhibited the proliferation of RPMI8266 cells in a concentration-dependent manner with an IC₅₀ value of 6.74 μmol/L. Exposure to oridonin (7 μmol/L) simultaneously induced caspase 3-mediated apoptosis and Beclin 1-dependent autophagy of RPMI8266 cells. Both the apoptosis and autophagy were time-dependent, and apoptosis was the main effector pathway of cell death. Exposure to oridonin (7 μmol/L) increased intracellular ROS and reduced SIRT1 nuclear protein in a time-dependent manner. The blockade of intracellular generation of ROS by NAC (5 mmol/L) abrogated apoptosis, autophagy and the decrease of SIRT1 in the cells exposed to oridonin (7 μmol/L). The inhibition of autophagy by 3-MA (5 mmol/L) sensitized the cells to oridonin-induced apoptosis, which was accompanied by increased intracellular ROS and decreased SIRT1.

Conclusion:

Oridonin simultaneously induces apoptosis and autophagy of human multiple myeloma RPMI8266 cells via regulation of intracellular ROS generation and SIRT1 nuclear protein. The cytotoxicity of oridonin is mainly mediated through the apoptotic pathway, whereas the autophagy protects the cells from apoptosis.     

Activation of c-Jun N-terminal kinase in A549 lung carcinoma cells by sodium dichromate: role of dissociation of apoptosis signal regulating kinase-1 from its physiological inhibitor thioredoxin

Changes in the components of the Jun N-terminal kinase (JNK) signalling pathway were investigated in human A549 lung carcinoma cells treated with sodium dichromate. Sodium dichromate (100 microM, 0-6h) failed to activate nuclear factor kappa B (NF-kappaB) as determined by a lack of nuclear translocation of p65 but resulted in Jun N-terminal kinase activation as assessed by phospho-Jun N-terminal kinase Western blotting in a dose-dependent (>25 microM) and time-dependent (>1h) manner. In addition, c-Jun, a downstream target of Jun N-terminal kinase signalling was also activated with a similar dose- and time-dependency at the level of both protein expression and degree of phosphorylation. In contrast, sodium dichromate treatment had no effect on levels of phospho-p38. Immunoprecipitation demonstrated that apoptosis signal regulating kinase-1 (ASK-1), an upstream activator of Jun N-terminal kinase was dissociated from its inhibitory partner thioredoxin (Trx) in response to sodium dichromate (100 microM, 4h) treatment. This treatment was also associated with a transient (2h) increase in cytosolic levels of thioredoxin but no nuclear translocation of thioredoxin was observed. In conclusion, sodium dichromate had a stimulatory effect on the Jun N-terminal kinase signalling pathway in A549 cells, resulting in activation of downstream effector molecules. We hypothesise that dissociation of apoptosis signal regulating kinase-1 from thioredoxin may be at least partially responsible for Jun N-terminal kinase activation.     

Atrial natriuretic peptide binds to ANP-R1 receptors in neuroblastoma cells or is degraded extracellularly at the Ser-Phe bond.

ANP-R1 receptors for atrial natriuretic peptide (ANP) showed the following rank order of affinity in intact human neuroblastoma cells NB-OK-1: human ANP-(99-126) approximately human ANP-(102-126) approximately rat ANP-(99-126) (K1 17-32 pM) > human ANP-(103-126) > porcine brain natriuretic peptide (BNP). Analogues truncated at the C-terminal extremity or devoid of a disulphide bridge, such as rat ANP-(103-123), rat C-ANP-(102-121), rat ANP-(111-126), rat ANP-(99-109) and rat [desCys105,Cys121]ANP-(104-126) and chicken C-type natriuretic peptide, were not recognized. The occupancy of these high affinity ANP-R1 receptors led to marked cyclic GMP accumulation in the presence of 3-isobutyl 1-methylxanthine. An ectoenzymic activity, partly shed in the incubation medium, provoked the stepwise release of Phe-Arg-[125I]Tyr, Arg-[125I]Tyr and [125I]Tyr from rat [125I]ANP-(99-126), at an optimal pH of 7.0. Its inhibition by 1,10-phenanthroline, EDTA and bacitracin but not by thiorphan suggests the contribution of at least one neutral metalloendopeptidase, distinct from EC 3.4.24.11, for which ANP showed high affinity.     

Targeting amyloid-beta by glucagon-like peptide -1 (GLP-1) in Alzheimer's disease and diabetes

Introduction: Epidemiological evidence suggests an association between type 2 diabetes (T2DM) and Alzheimer's disease (AD), in that one disease increases the risk of the other. T2DM and AD share several molecular processes which underlie the tissue degeneration in either disease. Disturbances in insulin signaling may be the link between the two conditions. Drugs originally developed for T2DM are currently being considered as possible novel agents in the treatment of AD.

Areas covered: This review discusses the potential role of glucagon-like peptide -1 (GLP-1) treatment in AD. GLP-1 receptors are expressed in areas of the brain important to memory and learning, and GLP-1 has growth-factor-like properties similar to insulin. A key neuropathological feature of AD is the accumulation of amyloid-beta (Aβ). In preclinical studies, GLP-1 and longer lasting analogues have been shown to have both neuroprotective and neurotrophic effects, and to protect synaptic activity in the brain from Aβ toxicity.

Expert opinion: A convincing amount of evidence has shown a beneficial effect of GLP-1 agonist treatment on cognitive function, memory and learning in experimental models of AD. GLP-1 analogues may therefore be the new therapeutic agent of choice for intervention in AD.     

Targeting amyloid-beta by glucagon-like peptide -1 (GLP-1) in Alzheimer's disease and diabetes

INTRODUCTION: Epidemiological evidence suggests an association between type 2 diabetes (T2DM) and Alzheimer's disease (AD), in that one disease increases the risk of the other. T2DM and AD share several molecular processes which underlie the tissue degeneration in either disease. Disturbances in insulin signaling may be the link between the two conditions. Drugs originally developed for T2DM are currently being considered as possible novel agents in the treatment of AD. AREAS COVERED: This review discusses the potential role of glucagon-like peptide -1 (GLP-1) treatment in AD. GLP-1 receptors are expressed in areas of the brain important to memory and learning, and GLP-1 has growth-factor-like properties similar to insulin. A key neuropathological feature of AD is the accumulation of amyloid-beta (Aβ). In preclinical studies, GLP-1 and longer lasting analogues have been shown to have both neuroprotective and neurotrophic effects, and to protect synaptic activity in the brain from Aβ toxicity. EXPERT OPINION: A convincing amount of evidence has shown a beneficial effect of GLP-1 agonist treatment on cognitive function, memory and learning in experimental models of AD. GLP-1 analogues may therefore be the new therapeutic agent of choice for intervention in AD.     

"Death and survival of neuronal cells exposed to Alzheimer's disease-relevant insults"

The mechanism for neuronal cell death by familial Alzheimer's disease (FAD) genes turned out to consist of various elemental combinations by the different types of cytotoxicity. We therefore tried a new approach toward identifying novel anti-AD suppressors of neuronal death, termed disease-based death trap screening. The identified genes were classified into three categories. In the first category, there were known anti-cell death genes; in the second, there were known genes with unknown function; and in the third, there were novel genes. The cDNA that encodes a 24-residue peptide, termed HN, is in the third category. HN protects neuronal death caused not only by all known kinds of FAD genes (mutant APP, PS1 and PS2), but also by anti-APP antibody and Abeta peptides, but not by long polyQ or SOD1 mutants. HN is a secretory peptide and exerts its protective function from the outside of the cell. The function of HN strictly depends on the structure. C8A-HN lost the activity and S14G-HN had approximately 1000 times increased potency of its action. Immunoblot analysis detected 3-kDa HN immunoreactive peptide in the testis and the colon in 3-week-old mice and only in the testis in 12-week-old mice. Notably, no HN immunoreactivity was detected in the brain. However, in an AD brain, not in an age-matched control, HN immunoreactivity was detected in neurons in the occipital lobe and in reactive glias in the hippocampal sections. The specific binding for HN exists on the neuronal cells and the rescue action of HN is specifically inhibited by genistein but not by wortmannin, suggesting that HN acts through the neuronal surface receptor linked to certain tyrosine kinases, but different from typical receptor tyrosine kinases. This study will provide a new insight into the research of AD.