RESUMO
Human corneal fibrosis can lead to opacity and ultimately partial or complete vision loss. Currently, corneal transplantation is the only treatment for severe corneal fibrosis and comes with the risk of rejection and donor shortages. Sphingolipids (SPLs) are known to modulate fibrosis in various tissues and organs, including the cornea. We previously reported that SPLs are tightly related to both, transforming growth factor beta (TGF-ß) signaling and corneal fibrogenesis. The aim of this study was to investigate the effects of sphingosine-1-phosphate (S1P) and S1P inhibition on specific TGF-ß and SPL family members in corneal fibrosis. Healthy human corneal fibroblasts (HCFs) were isolated and cultured in EMEM + FBS + VitC (construct medium) on 3D transwells for 4 weeks. The following treatments were prepared in a construct medium: 0.1 ng/mL TGF-ß1 (ß1), 1 µM sphingosine-1-phosphate (S1P), and 5 µM Sphingosine kinase inhibitor 2 (I2). Five groups were tested: (1) control (no treatment); rescue groups; (2) ß1/S1P; (3) ß1/I2; prevention groups; (4) S1P/ß1; and (5) I2/ß1. Each treatment was administered for 2 weeks with one treatment and switched to another for 2 weeks. Using Western blot analysis, the 3D constructs were examined for the expression of fibrotic markers, SPL, and TGF-ß signaling pathway members. Scratch assays from 2D cultures were also utilized to evaluate cell migration We observed reduced fibrotic expression and inactivation of latent TGF-ß binding proteins (LTBPs), TGF-ß receptors, Suppressor of Mothers Against Decapentaplegic homologs (SMADs), and SPL signaling following treatment with I2 prevention and rescue compared to S1P prevention and rescue, respectively. Furthermore, we observed increased cell migration following stimulation with I2 prevention and rescue groups, with decreased cell migration following stimulation with S1P prevention and rescue groups after 12 h and 18 h post-scratch. We have demonstrated that I2 treatment reduced fibrosis and modulated the inactivation of LTBPs, TGF-ß receptors, SPLs, and the canonical downstream SMAD pathway. Further investigations are warranted in order to fully uncover the potential of utilizing SphK I2 as a novel therapy for corneal fibrosis.
Assuntos
Córnea , Fibrose , Lisofosfolipídeos , Transdução de Sinais , Esfingosina , Fator de Crescimento Transformador beta , Humanos , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Esfingosina/farmacologia , Lisofosfolipídeos/metabolismo , Lisofosfolipídeos/farmacologia , Córnea/metabolismo , Córnea/patologia , Córnea/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Células Cultivadas , Esfingolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Doenças da Córnea/metabolismo , Doenças da Córnea/patologia , Doenças da Córnea/tratamento farmacológicoRESUMO
Corneal haze brought on by fibrosis due to insult can lead to partial or complete vision loss. Currently, corneal transplantation is the gold standard for treating severe corneal fibrosis, which comes with the risk of rejection and the issue of donor tissue shortages. Sphingolipids (SPLs) are known to be associated with fibrosis in various tissues and organs, including the cornea. We previously reported that SPLs are tightly related to Transforming Growth Factor ß (TGF-ß) signaling and corneal fibrogenesis. This study aimed to elucidate the interplay of SPLs, specifically sphingosine-1-phosphate (S1P) signaling, and its' interactions with TGF-ß signaling through detailed analyses of the corresponding downstream signaling targets in the context of corneal fibrosis, in vitro. Healthy human corneal fibroblasts (HCFs) were isolated, plated on polycarbonate membranes, and stimulated with a stable Vitamin C derivative. The 3D constructs were treated with either 5 µM sphingosine-1-phosphate (S1P), 5 µM SPHK I2 (I2; inhibitor of sphingosine kinase 1, one of the two enzymes responsible for generating S1P in mammalian cells), 0.1 ng/mL TGF-ß1, or 0.1 ng/mL TGF-ß3. Cultures with control medium-only served as controls. All 3D constructs were examined for protein expression of fibrotic markers, SPLs, TGF-ßs, and relevant downstream signaling pathways. This data revealed no significant changes in any LTBP (latent TGF-ß binding proteins) expression when stimulated with S1P or I2. However, LTBP1 was significantly upregulated via stimulation of TGF-ß1 and TGF-ß3, whereas LTBP2 was significantly upregulated only with TGF-ß3 stimulation. Significant downregulation of TGF-ß receptor II (TGF-ßRII) following S1P stimulation but significant upregulation following I2 stimulation was observed. Following TGF-ß1, S1P, and I2 stimulation, phospho-SMAD2 (pSMAD2) was significantly downregulated. Furthermore, I2 stimulation led to significant downregulation of SMAD4. Adhesion/proliferation/transcription regulation targets, SRC, FAK, and pERK 1/2 were all significantly downregulated by exogenous S1P, whereas I2 only significantly downregulated FAK. Exogenous TGF-ß3 caused significant upregulation of AKT. Interestingly, both I2 and TGF-ß3 caused significant downregulation of JNK expression. Lastly, TGF-ß1 led to significant upregulation of sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate receptor 3 (S1PR3), whereas TGF-ß3 caused significant upregulation of only SphK1. Together with previously published work from our group and others, S1P inhibition exhibits great potential as an efficacious anti-fibrotic modality in human corneal stromal ECM. The current findings shed further light on a very complex and rather incompletely investigated mechanism, and cement the intricate crosstalk between SPLs and TGF-ß in corneal fibrogenesis. Future studies will dictate the potential of utilizing SPLs/TGF-ß signaling modulators as novel therapeutics in corneal fibrosis.
Assuntos
Esfingolipídeos , Fator de Crescimento Transformador beta , Animais , Humanos , Esfingolipídeos/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Substância Própria/metabolismo , Fator de Crescimento Transformador beta3 , Transdução de Sinais , Lisofosfolipídeos/farmacologia , Lisofosfolipídeos/metabolismo , Esfingosina/farmacologia , Esfingosina/metabolismo , Fibrose , Mamíferos , Proteínas de Ligação a TGF-beta LatenteRESUMO
Multiple myeloma (MM) remains an incurable disease and there is an unmet medical need for novel therapeutic drugs that do not share similar mechanisms of action with currently available agents. Sphingosine kinase 2 (SK2) is an innovative molecular target for anticancer therapy. We previously reported that treatment with SK2 inhibitor opaganib inhibited myeloma tumor growth in vitro and in vivo in a mouse xenograft model. In the current study, we performed a phase I study of opaganib in patients with relapsed/refractory multiple myeloma (RRMM). Thirteen patients with RRMM previously treated with immunomodulatory agents and proteasome inhibitors were enrolled and treated with single-agent opaganib at three oral dosing regimens (250 mg BID, 500 mg BID, or 750 mg BID, 28 days as a cycle). Safety and maximal tolerated dose (MTD) were determined. Pharmacokinetics, pharmacodynamics, and correlative studies were also performed. Opaganib was well tolerated up to a dose of 750 mg BID. The most common possibly related adverse event (AE) was decreased neutrophil counts. There were no serious AEs considered to be related to opaganib. MTD was determined as at least 750 mg BID. On an intent-to-treat basis, one patient (7.7%) in the 500 mg BID dose cohort showed a very good partial response, and one other patient (7.7%) achieved stable disease for 3 months. SK2 is an innovative molecular target for antimyeloma therapy. The first-in-class SK2 inhibitor opaganib is generally safe for administration to RRMM patients, and has potential therapeutic activity in these patients. Clinicaltrials.gov: NCT02757326.
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Mieloma Múltiplo , Humanos , Animais , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/uso terapêutico , Inibidores de Proteassoma/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , DexametasonaRESUMO
The glutamate excitotoxicity has been suggested as a factor involved in the loss of retinal neuronal cells, including retinal ganglion cell (RGC), in various retinal degenerative diseases including ischemia-reperfusion injury, diabetic retinopathy, and glaucoma. Excitotoxic RGC death is caused not only by direct damage to RGCs but also by indirect damage due to the inflammation of retinal glial cells. Sphingosine 1-phosphate (S1P) and ceramides are bioactive sphingolipids which have been shown to possess important physiological roles in cellular survival and apoptosis, and the balance between S1P and ceramide, sphingolipid rheostat, has been suggested to be important for determining cellular fate. Therefore, we conducted the present study to clarify the neuroprotective role of sphingolipid rheostat in excitotoxic RGC death in vivo and in vitro. Acute RGC death was induced by intravitreal N-methyl-d-aspartate (NMDA) injection in the mouse. The mRNA expression of sphingosine kinase (SphK1/SphK2) was examined by quantitative real-time polymerase chain reaction (qRT-PCR). The expressions of SphK1/2, S1P, S1P-receptor (S1PR), glial fibrillary acidic protein (GFAP), Iba1, and CD31 were examined by immunostaining. Retinal sphingolipids and ceramides were quantified by liquid chromatography with tandem mass spectrometry. The neuroprotective effect of the sphingosine kinase inhibitor (SKI) on RGC death was assessed by RGC count and Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Further, the in vitro effect of SKI was investigated using rat primary cultured RGCs and glial cells. In addition, MG5 cells and A1 cells, which were mouse microglia and astrocyte cell-line, were also used. The expression of cleaved-caspase-3, GFAP, and Iba1 in RGCs, primary glial cells, MG5 cells, and A1 cells was assessed by immunostaining. NMDA injection resulted in mRNA upregulation of SphK1; however, SphK2 was reduced in the mouse retina. SphKs, S1P, S1PR1, S1PR2, and GFAP expression increased in the early-stage NMDA group, whereas S1P and GFAP were higher in the late-stage NMDA + SKI group. In the NMDA group, S1P expression was lower whereas sphingosine, C20, C22, and C24 ceramides showed higher levels. The proportion of very-long-chain ceramide was elevated in the NMDA group but reduced in the NMDA + SKI group. SKI treatment significantly increased RGC survival in retinal wholemount analysis and decreased apoptosis in the ganglion cell layer and inner nuclear layer. In vitro, SKI suppressed excitotoxic RGC death, cleaved-caspase-3 expression, and activated glial cells. The findings in the present study provide the first evidence demonstrating the involvement of sphingolipid rheostat in the neuroprotection against excitotoxic RGC death. Therefore, regulation of sphingolipid rheostat might serve as a potential therapy for retinal degenerative disease.
Assuntos
Morte Celular/efeitos dos fármacos , Ceramidas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Pró-Proteína Convertases/metabolismo , Degeneração Retiniana/prevenção & controle , Células Ganglionares da Retina/patologia , Serina Endopeptidases/metabolismo , Esfingolipídeos/farmacologia , Animais , Contagem de Células , Células Cultivadas , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ratos , Ratos Wistar , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismoRESUMO
Our sphingosine kinase inhibitor (SKI) optimization studies originated with the optimization of the SKI-I chemotype by replacement of the substituted benzyl rings with substituted phenyl rings giving rise to the discovery of SKI-178. We have recently reported that SKI-178 is a dual-targeted inhibitor of both sphingosine kinase isoforms (SphK1/2) and a microtubule disrupting agent (MDA). In mechanism-of-action studies, we have shown that these two separate actions synergize to induce cancer cell death in acute myeloid leukemia (AML) cell and animal models. Owning to the effectiveness of SKI-178, we sought to further refine the chemotype while maintaining "on-target" SKI and MDA activities. Herein, we modified the "linker region" between the substituted phenyl rings of SKI-178 through a structure guided approach. These studies have yielded the discovery of an SKI-178 congener, SKI-349, with log-fold enhancements in both SphK inhibition and cytotoxic potency. Importantly, SKI-349 also demonstrates log-fold improvements in therapeutic efficacy in a retro-viral transduction model of MLL-AF9 AML as compared to previous studies with SKI-178. Together, our results strengthen the hypothesis that simultaneous targeting of the sphingosine kinases (SphK1/2) and the induction of mitotic spindle assembly checkpoint arrest, via microtubule disruption, might be an effective therapeutic strategy for hematological malignancies including AML.
Assuntos
Antineoplásicos/farmacologia , Desenvolvimento de Medicamentos , Inibidores Enzimáticos/farmacologia , Microtúbulos/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Microtúbulos/metabolismo , Estrutura Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Polimerização/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Sickle cell disease is a destructive genetic disorder characterized by the formation of fibrils of deoxygenated hemoglobin, leading to the red blood cell (RBC) morphology changes that underlie the clinical manifestations of this disease. Using cryogenic soft X-ray tomography (SXT), we characterized the morphology of sickled RBCs in terms of volume and the number of protrusions per cell. We were able to identify statistically a relationship between the number of protrusions and the volume of the cell, which is known to correlate to the severity of sickling. This structural polymorphism allows for the classification of the stages of the sickling process. Recent studies have shown that elevated sphingosine kinase 1 (Sphk1)-mediated sphingosine 1-phosphate production contributes to sickling. Here, we further demonstrate that compound 5C, an inhibitor of Sphk1, has anti-sickling properties. Additionally, the variation in cellular morphology upon treatment suggests that this drug acts to delay the sickling process. SXT is an effective tool that can be used to identify the morphology of the sickling process and assess the effectiveness of potential therapeutics.
Assuntos
Anemia Falciforme/enzimologia , Eritrócitos/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Tomografia por Raios X/métodos , Animais , Extensões da Superfície Celular/efeitos dos fármacos , Extensões da Superfície Celular/metabolismo , Eritrócitos/efeitos dos fármacos , Humanos , Camundongos , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Sphingosine kinase inhibitor (SKI) II has been reported as a dual inhibitor of sphingosine kinases (SKs) 1 and 2 and has been extensively used to prove the involvement of SKs and sphingosine-1-phosphate (S1P) in cellular processes. Dihydroceramide desaturase (Des1), the last enzyme in the de novo synthesis of ceramide (Cer), regulates the balance between dihydroceramides (dhCers) and Cers. Both SKs and Des1 have interest as therapeutic targets. Here we show that SKI II is a noncompetitive inhibitor (Ki = 0.3 µM) of Des1 activity with effect also in intact cells without modifying Des1 protein levels. Molecular modeling studies support that the SKI II-induced decrease in Des1 activity could result from inhibition of NADH-cytochrome b5 reductase. SKI II, but not the SK1-specific inhibitor PF-543, provoked a remarkable accumulation of dhCers and their metabolites, while both SKI II and PF-543 reduced S1P to almost undetectable levels. SKI II, but not PF543, reduced cell proliferation with accumulation of cells in the G0/G1 phase. SKI II, but not PF543, induced autophagy. These overall findings should be taken into account when using SKI II as a pharmacological tool, as some of the effects attributed to decreased S1P may actually be caused by augmented dhCers and/or their metabolites.
Assuntos
Autofagia/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Oxirredutases/antagonistas & inibidores , Oxirredutases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fase G1/efeitos dos fármacos , Células HeLa , Humanos , Metanol , Pirrolidinas/farmacologia , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Sulfonas/farmacologiaRESUMO
BACKGROUND AND OBJECTIVE: Colorectal cancer (CRC) is the fourth leading cause of cancer- related death globally, with a high incidence rate in economically fast-growing countries. Sphingosine- 1-phosphate (S1P) is a bioactive lipid mediator that plays critical roles in cancer cell proliferation, migration, and angiogenesis converted by the isoforms of sphingosine kinase (SK1 and SK2). SK1 is highly expressed in colorectal cancer; its inhibitors suppress the formation of S1P and increase ceramide levels having a pro-apoptotic function. RB005 is a selective SK1 inhibitor and a structural analog of PP2A activator FTY720. The purpose of this study is to test whether RB005, an SK1 inhibitor, can be used as an anticancer agent by inhibiting the growth of colon cancer cells. METHODS: We performed MTT and colony-forming assay using colon cancer cell lines HT29 and HCT116 cells to examine the cell toxicity effect of RB005. To determine whether apoptosis of RB005 in colon cancer cell line is due to SK1 inhibition or other mechanisms due to its structural similarity with FTY720, we conducted LC/MS, siRNA knockdown, and PP2A activity experiments. RESULTS: RB005 notably inhibited CRC cell growth and proliferation compared to PF543 and ABC294640 by inducing the mitochondria-mediated intrinsic apoptotic pathway. Apoptotic cell death is caused by increased mitochondrial permeability Initiated by the activation of pro-apoptotic protein BAX, increased ceramides, and activation of PP2A. Also, RB005 treatment in HT29 cells did not change the expression level of SK1, but strikingly decreased SK1 activity and S1P levels. All these events of cell death and apoptosis were less effective when SK1 was knocked down by siRNA. CONCLUSION: This result indicates that RB005 shows the in-vitro anti-CRC effect by inhibiting SK1 activity and PP2A activation, increasing proapoptotic ceramide levels following the activation of the intrinsic apoptotic pathway.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Apoptose , Ceramidas/metabolismo , Ceramidas/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Cloridrato de Fingolimode/farmacologia , Humanos , RNA Interferente Pequeno/genéticaRESUMO
The bioactive sphingolipids ceramide and sphingosine-1-phosphate (S1P) are involved in the regulation of cell homeostasis and activity ranging from apoptosis to proliferation. We recently described that the two compounds ceranib-2 (inhibiting acid ceramidase) and SKI-II [inhibiting the sphingosine kinases 1 and - 2 (SphK1/2)] reduce mTORC1 activity and measles virus (MV) replication in human primary peripheral blood lymphocytes (PBL) by about one log step. We now further investigated whether mTORC1 downstream signaling and viral protein expression may be affected by ceranib-2 and/or SKI-II. Western blot analyses showed that in uninfected cells the phosphorylation of the eukaryotic initiation factor 4E (eIF4E) was reduced by both inhibitors. Interestingly, MV infection led to an increase of rpS6 protein levels and phosphorylation of eIF4E. Treatment with both inhibitors reduced the rpS6 protein expression, and in addition, SKI-II reduced rpS6 phosphorylation. The phosphorylation of eIF4E was slightly reduced by both inhibitors. In addition, SKI-II led to reduced levels of IKK in MV-infected cells. Both inhibitors reduced the expression of viral proteins and the titers of newly synthesized MV by approximately one log step. As expected, SKI-II and rapamycin reduced also the virally encoded GFP expression; however, ceranib-2 astonishingly led to increased levels of GFP fluorescence. Our findings suggest that the inhibitors ceranib-2 and SKI-II act via differential mechanisms on MV replication. The observed effects on mTORC1 downstream signaling, predominantly the reduction of rpS6 levels by both inhibitors, may affect the translational capacity of the cells and contribute to the antiviral effect in human primary PBL.
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Sphingosine kinases (SPHKs) are conserved lipid enzymes that catalyze the formation of sphingosine-1-phosphate (S1P) through ATP-dependent phosphorylation of sphingosine. Two distinct SPHK isoforms, namely SPHK1 and SPHK2, have been identified to date, and the former has been implicated for its oncogenic roles in cancer development and progression. While SPHK1 signaling axis has been extensively studied in non-stem breast cancer cells, recent evidence has emerged to suggest a role of SPHK1 in regulating cancer stem cells (CSCs). With the clinical implications of CSCs in disease relapse and metastasis, it is believed that therapeutic approaches that can eradicate both non-stem cancer cells and CSCs could be a key to cancer cure. In this review, we first explore the oncogenic functions of sphingosine kinase 1 in human cancers and summarize current research findings of SPHK1 signaling with a focus on breast cancer. We also discuss the therapeutic potentials and perspectives of targeting SPHK1 signaling in breast cancer and cancer stem cells. We aim to offer new insights and inspire future studies looking further into the regulatory functions of SPHK1 in CSC-driven tumorigenesis, uncovering novel therapeutic avenues of using SPHK1-targeted therapy in the treatment of CSC-enriched refractory cancers.
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Aim: Previously, we have shown that inhibition of SphK by the SphK inhibitor-II (SKI II) prevents lipopolysaccharide-induced preterm birth in mice. The aim of this study was to develop a vaginal self-nanoemulsifying drug-delivery system (SNEDDS) for SKI II. Materials & methods: A SKI II-loaded SNEDDS was characterized and tested in a murine preterm birth model. Results: The SNEDDS immediately formed a gel and then slowly emulsified to nanoglobules with over 500-fold enhancement of SKI II solubility at vaginal pH. Intravaginal administration of the SKI II SNEDDS significantly decreased lipopolysaccharide-induced preterm birth in mice. Conclusion: A vaginal nanoformulation of SKI II represents a novel, noninvasive approach to prevent preterm birth.
Assuntos
Emulsões/química , Inibidores Enzimáticos/química , Lipopolissacarídeos/metabolismo , Nanocápsulas/química , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Nascimento Prematuro/tratamento farmacológico , Animais , Disponibilidade Biológica , Composição de Medicamentos , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Emulsões/farmacologia , Inibidores Enzimáticos/metabolismo , Feminino , Humanos , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Gravidez , Solubilidade , VaginaRESUMO
As structural membrane components and signaling effector molecules sphingolipids influence a plethora of host cell functions, and by doing so also the replication of viruses. Investigating the effects of various inhibitors of sphingolipid metabolism in primary human peripheral blood lymphocytes (PBL) and the human B cell line BJAB we found that not only the sphingosine kinase (SphK) inhibitor SKI-II, but also the acid ceramidase inhibitor ceranib-2 efficiently inhibited measles virus (MV) replication. Virus uptake into the target cells was not grossly altered by the two inhibitors, while titers of newly synthesized MV were reduced by approximately 1 log (90%) in PBL and 70-80% in BJAB cells. Lipidomic analyses revealed that in PBL SKI-II led to increased ceramide levels, whereas in BJAB cells ceranib-2 increased ceramides. SKI-II treatment decreased sphingosine-1-phosphate (S1P) levels in PBL and BJAB cells. Furthermore, we found that MV infection of lymphocytes induced a transient (0.5-6 h) increase in S1P, which was prevented by SKI-II. Investigating the effect of the inhibitors on the metabolic (mTORC1) activity we found that ceranib-2 reduced the phosphorylation of p70 S6K in PBL, and that both inhibitors, ceranib-2 and SKI-II, reduced the phosphorylation of p70 S6K in BJAB cells. As mTORC1 activity is required for efficient MV replication, this effect of the inhibitors is one possible antiviral mechanism. In addition, reduced intracellular S1P levels affect a number of signaling pathways and functions including Hsp90 activity, which was reported to be required for MV replication. Accordingly, we found that pharmacological inhibition of Hsp90 with the inhibitor 17-AAG strongly impaired MV replication in primary PBL. Thus, our data suggest that treatment of lymphocytes with both, acid ceramidase and SphK inhibitors, impair MV replication by affecting a number of cellular activities including mTORC1 and Hsp90, which alter the metabolic state of the cells causing a hostile environment for the virus.
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Mastocytosis is a disorder resulting from an abnormal mast cell (MC) accumulation in tissues that is often associated with the D816V mutation in KIT, the tyrosine kinase receptor for stem cell factor. Therapies available to treat aggressive presentations of mastocytosis are limited, thus exploration of novel pharmacological targets that reduce MC burden is desirable. Since increased generation of the lipid mediator sphingosine-1-phosphate (S1P) by sphingosine kinase (SPHK) has been linked to oncogenesis, we studied the involvement of the two SPHK isoforms (SPHK1 and SPHK2) in the regulation of neoplastic human MC growth. While SPHK2 inhibition prevented entry into the cell cycle in normal and neoplastic human MCs with minimal effect on cell survival, SPHK1 inhibition caused cell cycle arrest in G2/M and apoptosis, particularly in D816V-KIT MCs. This was mediated via activation of the DNA damage response (DDR) cascade, including phosphorylation of the checkpoint kinase 2 (CHK2), CHK2-mediated M-phase inducer phosphatase 3 depletion, and p53 activation. Combination treatment of SPHK inhibitors with KIT inhibitors showed greater growth inhibition of D816V-KIT MCs than either inhibitor alone. Furthermore, inhibition of SPHK isoforms reduced the number of malignant bone marrow MCs from patients with mastocytosis and the growth of D816V-KIT MCs in a xenograft mouse model. Our results reveal a role for SPHK isoforms in the regulation of growth and survival in normal and neoplastic MCs and suggest a regulatory function for SPHK1 in the DDR in MCs with KIT mutations. The findings also suggest that targeting the SPHK/S1P axis may provide an alternative to tyrosine kinase inhibitors, alone or in combination, for the treatment of aggressive mastocytosis and other hematological malignancies associated with the D816V-KIT mutation.
Assuntos
Neoplasias Hematológicas/metabolismo , Mastócitos/fisiologia , Mastocitose/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Isoformas de Proteínas/metabolismo , Adamantano/análogos & derivados , Adamantano/farmacologia , Animais , Apoptose , Carcinogênese , Proliferação de Células , Sobrevivência Celular , Reparo do DNA , Neoplasias Hematológicas/genética , Humanos , Hidrazinas/farmacologia , Mastocitose/genética , Camundongos , Camundongos Knockout , Mutação/genética , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Pirazóis/farmacologia , Piridinas/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
AIM: To further characterize the selectivity, mechanism-of-action and therapeutic efficacy of the novel small molecule inhibitor, SKI-178. METHODS: Using the state-of-the-art Cellular Thermal Shift Assay (CETSA) technique to detect "direct target engagement" of proteins intact cells, in vitro and in vivo assays, pharmacological assays and multiple mouse models of acute myeloid leukemia (AML). RESULTS: Herein, we demonstrate that SKI-178 directly target engages both Sphingosine Kinase 1 and 2. We also present evidence that, in addition to its actions as a Sphingosine Kinase Inhibitor, SKI-178 functions as a microtubule network disrupting agent both in vitro and in intact cells. Interestingly, we separately demonstrate that simultaneous SphK inhibition and microtubule disruption synergistically induces apoptosis in AML cell lines. Furthermore, we demonstrate that SKI-178 is well tolerated in normal healthy mice. Most importantly, we demonstrate that SKI-178 has therapeutic efficacy in several mouse models of AML. CONCLUSION: SKI-178 is a multi-targeted agent that functions both as an inhibitor of the SphKs as well as a disruptor of the microtubule network. SKI-178 induced apoptosis arises from a synergistic interaction of these two activities. SKI-178 is safe and effective in mouse models of AML, supporting its further development as a multi-targeted anti-cancer therapeutic agent.