Sphingosine-1-phosphate Phosphatase 1 Research Articles

BCL2L13 protein prevents apoptosis in acute myeloid leukemia cells

Abstract Objectives The B cell lymphoma-2 (BCL-2) protein group is essential for regulating apoptosis. BCL2L13 harbors all BH domains present in the BCL-2 protein family in addition to a BHNo domain comprising 250 amino acids at its C-terminal. BCL2L13 is highly expressed in AML and is involved in apoptosis. We investigated the possibility of discovering BCL2L13 as a treatment target for acute leukemia. Methods We reduced the expression of BCL2L13 in Mono Mac 6 (MM6) cells using shRNA and overexpressed BCL2L13 in THP-1 cells. MM6 and THP-1 cells were treated with staurosporine (STS) to confirm the role of BCL2L13 in apoptosis. Results In acute myeloid leukemia (AML) cells, BCL2L13 is involved in inhibiting apoptosis as evidenced by its strikingly augmented expression in these cells. BCL-2 was downregulated and cleaved Caspase 3, and sphingosine-1-phosphate phosphatase 1 (SGPP1) was upregulated when MM6 cells with knockdown BCL2L13 were treated with STS, which significantly increased their apoptosis. Furthermore, mitochondrial membrane potential decreased in MM6 cells in response to the downregulation of BCL2L13. In BCL2L13-depleted MM6 cells, the amount of cytochrome c increased in the cytoplasm. The THP-1 cells overexpressing BCL2L13 and treated with STS showed a significant decrease in the expression of genes related to apoptosis induction, resulting in decreased apoptosis. Conclusion BCL2L13 inhibits apoptosis in AML, and BCL2L13-specific inhibition might serve as a new strategy for treating this condition.

Downregulation of Lipid Phosphate Phosphatase 3 Correlates With Tumor-Infiltrating Immune Cells in Oral Cancer.

BackgroundSphingosine-1-phosphate (S1P) is a potent oncogenic lipid. Intracellular levels of S1P are tightly regulated by eight S1P-metabolizing enzymes. S1P synthesis is catalyzed by two sphingosine kinases, i.e., sphingosine kinase 1 (SphK1) and sphingosine kinase 2 (SphK2). Five lipid phosphatases (two S1P phosphatases and lipid phosphate phosphatases (LPPs) 1, 2, and 3) reversibly convert S1P back to sphingosine. Previously, we have determined the mRNA expression profile of eight S1P-metabolizing enzymes in tumor tissues and adjacent normal tissues from oral squamous cell carcinoma (OSCC) patients. Except for SphK1, the role of S1P-metabolizing enzymes in OSCC has been poorly studied.MethodsWe have determined the protein expression of four S1P-metabolizing enzymes (SphK1, SphK2, sphingosine-1-phosphate phosphatase 1 (SGPP1), and lipid phosphate phosphatase 3 (LPP3)) by immunohistochemistry (IHC) in tumor tissues of 46 OSCC patients. Six subjects with non-dysplastic oral mucosa were also included in the study. The immunoreactivity score (IRS) was calculated for each protein in every subject. Further, we determined the associations of expression of S1P-metabolizing enzymes with clinicopathological features of OSCC patients.ResultsWe demonstrate the low IRS for SphK2 and LPP3 in OSCC tumors. Importantly, expression of SphK2 and LPP3 was downregulated in malignant epithelial cells compared to non-malignant mucosa. Further, LPP3 expression negatively correlated with tumor‑node‑metastasis (TNM) staging of patients (r = −0.307, p = 0.043). Importantly, expression of LPP3 in tumors was found to be an independent predictor of perinodal extension (b = −0.440, p = 0.009), lymphovascular invasion (b = −0.614, p < 0.001), lymph node ratio (b = 0.336, p = 0.039), and TNM staging (b = −0.364, p = 0.030).ConclusionTaken together, our data show that expression of SphK2 and LPP3 is decreased compared to normal mucosa. Thus, the S1P signaling pathway could represent a potential therapeutic target.

MicroRNA-656-3p inhibits colorectal cancer cell migration, invasion, and chemo-resistance by targeting sphingosine-1-phosphate phosphatase 1

ABSTRACT Colorectal cancer presents high rates of recurrence and metastasis, and the occurrence and progression and mechanism of its invasion and metastasis are not fully understood. The expression of miR-656-3p in patient samples and 10 cell lines were measured. Bioinformatic databases were used to predict miRNAs. Protein expressions were examined using Western blot. Transwell assay was used to measure cell migration and invasion. Transplanted tumor model in nude mice was established. Removal of the miR-656-3p by specific knocking-down of this gene promoted the chemo-resistance of colorectal cancer cells. Critically, we identified sphingosine-1-phosphate phosphatase 1 (SGPP1) as a downsteam target of the miR-656-3p, which we first obtained from 199 potential target genes from Targetscan, 200 genes from miRDB and 200 genes from DIANA, respectively. Then, we identified the interaction between SGPP1 and the miR-656-3p on 3’ UTR of SGPP1 gene. Knockdown of SGPP1 greatly suppressed the tumor growth in vivo and epithelial mesenchymal transition process. miR-656-3p could regulate cell proliferation and chemoresistance in the colorectal cancer that associate to downstream target with SGPP1. Along with its downstream molecule, we would like to predict that the SGPP1 associated miR-656-3p could be used to develop early for early diagnostics for CRC oncogenesis.

Sevoflurane Suppresses the Proliferation, Migration and Invasion of Colorectal Cancer Through Regulating Circ_0000423/miR-525-5p/SGPP1 Network.

Sevoflurane (SEV) has been shown to inhibit the malignant progression in many cancers, including colorectal cancer (CRC). However, it is not clear whether SEV regulates the progression of CRC by mediating the circular RNA (circRNA) axis. Different concentrations of SEV were used to treat CRC cells. Cell proliferation, migration and invasion were determined by cell counting kit 8 assay, colony formation assay and transwell assay. The expression of circ_0000423, microRNA (miR)-525-5p and sphingosine-1-phosphate phosphatase 1 (SGPP1) mRNA was measured by quantitative real-time PCR. Cell apoptosis was assessed using flow cytometry, and protein expression was measured by western blot analysis. Dual-luciferase reporter assay and RIP assay were performed to confirm the interactions among circ_0000423, miR-525-5p and SGPP1. Animal experiments were performed to explore the effect of SEV and circ_0000423 on CRC tumorigenesis. SEV could inhibit CRC cell proliferation, migration and invasion. Circ_0000423 was upregulated in CRC and its expression could be reduced by SEV. Overexpressed circ_0000423 reversed the inhibitory effect of SEV on CRC cell proliferation, migration and invasion and the promotion effect on cell apoptosis. MiR-525-5p could be sponged by circ_0000423, and its overexpression also abolished the regulation of circ_0000423 on the progression of SEV-treated CRC cells. In addition, SGPP1 was confirmed to be a target of miR-525-5p, and its expression was positively regulated by circ_0000423. MiR-525-5p inhibitor promoted CRC cell progression under the treatment of SEV, while these effects could be overturned by SGPP1 silencing. Furthermore, the inhibition effect of SEV on CRC tumorigenesis also could be abolished by overexpressing circ_0000423. Our results showed that SEV inhibited CRC progression through the regulation of circ_0000423/miR-525-5p/SGPP1 axis. The online version contains supplementary material available at 10.1007/s12195-021-00717-5.

Circular Network of Coregulated Sphingolipids Dictates Chronic Hypoxia Damage in Patients With Tetralogy of Fallot.

Background: Tetralogy of Fallot (TOF) is the most common cyanotic heart disease. However, the association of cardiac metabolic reprogramming changes and underlying molecular mechanisms in TOF-related chronic myocardial hypoxia damage are still unclear.Methods: In this study, we combined microarray transcriptomics analysis with liquid chromatography tandem-mass spectrometry (LC–MS/MS) spectrum metabolomics analysis to establish the metabolic reprogramming that occurs in response to chronic hypoxia damage. Two Gene Expression Omnibus (GEO) datasets, GSE132176 and GSE141955, were downloaded to analyze the metabolic pathway in TOF. Then, a metabolomics analysis of the clinical samples (right atrial tissue and plasma) was performed. Additionally, an association analysis between differential metabolites and clinical phenotypes was performed. Next, four key genes related to sphingomyelin metabolism were screened and their expression was validated by real-time quantitative PCR (QT-PCR).Results: The gene set enrichment analysis (GSEA) showed that sphingolipid metabolism was downregulated in TOF and the metabolomics analysis showed that multiple sphingolipids were dysregulated. Additionally, genes related to sphingomyelin metabolism were identified. We found that four core genes, UDP-Glucose Ceramide Glucosyltransferase (UGCG), Sphingosine-1-Phosphate Phosphatase 2 (SGPP2), Fatty Acid 2-Hydroxylase (FA2H), and Sphingosine-1-Phosphate Phosphatase 1 (SGPP1), were downregulated in TOF.Conclusion: Sphingolipid metabolism was downregulated in TOF; however, the detailed mechanism needs further investigation.

Sevoflurane suppresses cell viability and invasion and promotes cell apoptosis in colon cancer by modulating exosome‑mediated circ‑HMGCS1 via the miR‑34a‑5p/SGPP1 axis.

As a novel halogenated hydroxyl ether-inhaled general anesthetic, sevoflurane has been reported to affect the progression of diverse human cancers. In the present study, we aimed to explore the functions and underlying mechanisms of sevoflurane in colon cancer. MTT assay, flow cytometric analysis and Transwell assay were conducted to evaluate cell viability, apoptosis and invasion, respectively. Western blot analysis was performed to determine the protein level of sphingosine-1-phosphate phosphatase 1 (SGPP1). The morphology and size of exosomes were analyzed by TEM and NTA. The levels of circular RNA 3-hydroxy-3-methylglutaryl-CoA synthase 1 (circ-HMGCS1), microRNA (miR)-34a-5p and SGPP1 mRNA were examined by RT-qPCR. Dual-luciferase reporter and RNA RIP assays were utilized to explore the interaction between miR-34a-5p and circ-HMGCS1 or SGPP1. A murine xenograft model was established to investigate the effect of circ-HMGCS1 in vivo. As a result, it was determined that sevoflurane suppressed cell viability and invasion and induced apoptosis in colon cancer in a dose-dependent way. Exosomal circ-HMGCS1 was increased in the serums and cells of colon cancer patients. Circ-HMGCS1 was downregulated by sevoflurane treatment in colon cancer cells and circ-HMGCS1 overexpression could restore the effect of sevoflurane on colon cancer cell development. miR-34a-5p was a target of circ-HMGCS1 and miR-34a-5p inhibition reversed the effect of circ-HMGCS1 silencing on colon cancer cell progression. Moreover, circ-HMGCS1 knockdown suppressed SGPP1 expression via sponging miR-34a-5p. Knockdown of circ-HMGCS1 blocked tumor growth in vivo. In conclusion, sevoflurane inhibited colon cancer progression by modulating the exosome-transmitted circ-HMGCS1/miR-34a-5p/SGPP1 axis.

The effects of miR-25 and the potential molecular mechanisms on migration and invasion of triple negative breast cancer

Objective To explore the expression of tiny RNA-25 (microRNA-25, miR-25) in the plasma、tissues of triple-negative breast cancer (TNBC) patients and cell lines, to investigate the potential molecular mechanisms of miR-25 on migration and invasion of TNBC. Methods Real-time fluorescent quantitative PCR was used to detect the expression of miR-25 in the plasma of TNBC patients. Linked omics web platform was used to analyse miR-25 level in samples of TNBC and non-TNBC. Real-time fluorescent quantitative PCR was also used to detect the miR-25 level in TNBC cell lines. The wound healing and transwell assay was applied to assess the effects on migration and invasion of TNBC cell lines which transfected with miR-25 inhibitor or the negative control. The luciferase reporter assay was used to validate the relationship between miR-25 and the sphingosine-1-phosphate phosphatase 1 (SGPP1) in HEK293T cell. The wound healing and transwell assay was used to detect the migration and invasion ability of TNBC cell lines when cotransfected with pCMV6-SGPP1 and miR-25. Furthermore, Western blot was performed to detect the SGPP1 level in TNBC cell lines. Results The expression of miR-25 was significantly elevated in the plasma of 86 TNBC patients compared with the healthy controls (P value was 0.031). LinkedOmics web platform analysis showed that miR-25 expression was significantly higher in TNBC samples than in non-TNBC samples with Luminal A or Luminal B (P value was<0.001 and 0.006). The level of miR-25 was also elevated in TNBC cell lines HS578T, HCC1806, MDA-MB-231 and BT549(P value was 0.006, 0.01, 0.029 and 0.046). The MDA-MB-231 and HS578T cells which transfected with miR-25 inhibitor exhibited a significant slower wound healing rate than control (P value was 0.035 and 0.001). At the same time, when transfected with miR-25 inhibitor, MDA-MB-231 and HS578T both exhibited a decreased invasion ability compared with the control group(P value was 0.002 and 0.001). LinkedOmics web platform analysis showed that sphingosine-1-phosphate phosphatase 1 (SGPP1) gene level was negatively correlated with miR-25 in the tissues of TNBC patients (P value was 0.037). The luciferase reporter assay validated that SGPP1 was a directed target of miR-25. The western blot assay indicated that the SGPP1 level was increased in MDA-MB-231 and HS578T after transfection with miR-25 inhibitor. Over-expression of SGPP1 could abrogate the positive effects of miR-25 on migration and invasion when pCMV6-SGPP1 was cotransfected with miR-25 (P value was all 0.002). Conclusions MiR-25 was elevated in both plasma and tissues of TNBC patients and also increased in TNBC cell lines. Transfection of MDA-MB-231 and HS578T cells with miR-25 inhibitor resulted in reduced migration and invasion. Moreover, SGPP1 was identified as a novel target of miR-25. The ability of miR-25 to promote TNBC cell migration and invasion is attributable to its effect on SGPP1 suppression. Key words: Triple negative breast neoplasms; microRNAs; Membrane proteins; Phosphoric monoester hydrolases; Neoplasm metastasis

Corrigendum] Inhibition of sphingosine-1-phosphate phosphatase1 promotes cancer cells migration in gastric cancer: Clinical implications.

We have recently noticed an accidental error in part of a figure which appeared in the above‑mentioned article. In Fig.3A, the image for the HGC27‑pEF, 15h panel was mistakenly replicated as the HGC27‑KD, 0h panel in the same figure, and the AGS‑pEF, 15h and AGS KD, 0h panels were mistakenly switched with each other. We have reviewed the original files and the individual figures for the submitted composite figure, and realized that the error occurred when we produced the composite figure by marrying the individual images to the final figure. The same image was accidentally pasted twice without us being fully aware of the error. We have identified all the original images, and the corrected version of Fig. 3 is shown below. We regret that this error occurred, and thank the Editor for affording us the opportunity to publish this Corrigendum. [the original article was published in the Oncology Reports 34: 1977-1987, 2015; DOI: 10.3892/or.2015.4162].

Lysosomotropic agents selectively target chronic lymphocytic leukemia cells due to altered sphingolipid metabolism.

Lysosome membrane permeabilization (LMP) mediates cell death in a variety of cancer cells. However, little is known about lysosomes and LMP in chronic lymphocytic leukemia (CLL). Owing to drug resistance and toxicity in CLL patients, better treatment strategies are required. Our results show that CLL cells were sensitive to the lysosomotropic agent siramesine. Furthermore, this drug was more effective in CLL cells, regardless of prognostic factors, compared with normal B cells. Siramesine caused LMP, lipid peroxidation and transcription factor EB nuclear translocation followed by mitochondrial membrane potential loss and reactive oxygen species release. Siramesine-induced cell death was blocked by lipid antioxidants, but not by soluble antioxidants or protease inhibitors. To determine whether CLL cells had altered lysosomes, we investigated sphingolipid metabolism as the lysosome is a hub for lipid metabolism. We found that CLL cells had more lysosomes, increased sphingosine-1-phosphate phosphatase 1 (SPP1) expression, and increased levels of sphingosine compared with normal B cells. Raising sphingosine levels increased LMP and cell death in CLL cells, but not in normal B cells. Together, these results show that excess sphingosine in CLL cells could contribute to their sensitivity toward LMP. Thus, targeting the lysosome could be a novel therapeutic strategy in CLL.

Inhibition of sphingosine-1-phosphate phosphatase 1 promotes cancer cells migration in gastric cancer: Clinical implications.

Sphingosine-1-phosphate (S1P) plays an important role in regulating many biological processes. Sphingosine-1-phosphate phosphatase 1 (SGPP1) can dephosphorylate S1P into sphingosine and tip the balance of sphingosine-S1P. Increased levels of sphingosine leads to a decrease in the ability of cell invasion as well as an increase in the ability of cell apoptosis. However, little is known regarding the effects of SGPP1 in gastric cancer. The present study examined the function of SGPP1 on gastric cancer cell lines as well as its clinical relevance in gastric cancer progression. Using immunohistochemistry and RT-qPCR techniques, the clinical significance of SGPP1 expression was analyzed in 288 paraffin-embedded gastric tissue specimens and 219 fresh gastric tissues, respectively. Transgenes encoding ribozymes to specifically target human SGPP1 (pEF-SGPP1) was constructed. Human gastric cancer cell lines (AGS and HGC27) were transfected with pEF-SGPP1 transgene and examined by functional analysis. SGPP1 was downregulated in gastric cancer tissues, compared with adjacent normal gastric tissues (p=0.034). SGPP1 mRNA levels in gastric cancer tissues were significantly decreased when compared with their adjacent non-cancerous tissues (p<0.001). Weakly expressed SGPP1 was positively correlated with the lymph node metastasis (p=0.005) and distant metastasis (p=0.031). Kaplan-Meier survival curves revealed that patients with SGPP1 positive expression had a significant increase in overall survival (OS) (p=0.034) and progression-free survival (PFS) (p=0.041). Multivariate analysis indicated the expression of SGPP1 was an independent prognostic factor in gastric cancer patients (p=0.041). In vitro experiments showed that knockdown of SGPP1 resulted in an increase in the invasion (2-fold) and migration (5-fold) of AGS and HGC27. The two gastric cancer cells transfected with pEF-SGPP1 exhibited a slower rate of growth with less adhesion. Thus, our findings provided evidence that SGPP1 may serve as a prognostic biomarker for patients with advanced gastric cancers.

886 SPHINGOSINE KINASE 1 KNOCK DOWN IMPROVES ERECTILE FUNCTION IN STREPTOZOTOCIN- INDUCED DIABETIC RATS AND DECREASES RHO-KINASE ACTIVITY IN VITRO

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research II1 Apr 2010886 SPHINGOSINE KINASE 1 KNOCK DOWN IMPROVES ERECTILE FUNCTION IN STREPTOZOTOCIN- INDUCED DIABETIC RATS AND DECREASES RHO-KINASE ACTIVITY IN VITRO Guillermo Villegas, Moses Tar, Arnold Melman, and Michael DiSanto Guillermo VillegasGuillermo Villegas More articles by this author , Moses TarMoses Tar More articles by this author , Arnold MelmanArnold Melman More articles by this author , and Michael DiSantoMichael DiSanto More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1642AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that is mainly formed by phosphorylation of sphingosine by sphingosine kinase 1 (SPHK1) and can be reverted to sphingosine by sphingosine-1-phosphate phosphatase 1 (SPP1). Our recent studies show that S1P plays a mayor role in the Ca+2 sensitization pathway and contractility of corpus cavernosum smooth muscle (CCSM). Furthermore we demonstrated that SPHK1 expression is elevated in Type 1 diabetic patients with impotence while SPHK1 overexpression in vivo induces erectile dysfunction (ED) in normal rats and upregulates the expression of Rho-kinase (ROCK). Our goals were a) to asses if knocking down expression of SPHK1 in CCSM of diabetic (DM) rats improves erectile function and therefore confirm the determinant role of S1P in the etiology of DM-induced ED b) To examine if SPHK1 expression modulates smooth muscle calcium sensitization by increasing or decreasing ROCK activity and c) to determine whether ROCK is modulated directly by SPHK1 or by S1P. METHODS 1) SPHK1 mRNA was silenced to prevent the synthesis of S1P by using small hairpin RNA (sh) cloned into a shRNA expression vector (pSiren/SPHK1) 2) Human SPP1 and SPHK1 were cloned into the pVax vector to overexpress these proteins 3) For in vivo studies, 2 month strepotozotocin induced diabetic rats (STZ) with ED were injected in the CC with 100 μg pSiren/SPHK1 or pSiren/Luciferase (MOC) and erectile function assessed via cavernous nerve stimulation-induced increase in intracavernous pressure (ICP) one week post-injection 4) For the in vitro studies rat and human primary CCSM cells were transfected with either pSiren/SPHK1, pVax/SPP1 or pVax/SPHK1. In addition S1P receptors were blocked with suramin or N-ethylmaleimide. Cells were harvested at 48 hours, lysed for protein and RNA extraction and analyzed by real time PCR and Western blot. RESULTS a) pSiren/SPHK1 injected STZ rats showed an improved erectile function especially at lower stimulations compared to MOC b) Transfection of CCSM cells with either SPHK1 shRNA or SPP1 significant decreased ROCK activity and c) Blocking of the S1P receptors also downregulated ROCK activity. CONCLUSIONS We showed that in STZ rats, treatment of CC with SPHK1 shRNA improves erectile function confirming the direct role S1P plays in DM-induced ED. Our results further suggest that S1P mediates Ca+2 sensitization of CCSM cells by modulating ROCK activity and that the mechanism involves S1P and not SPHK1. Bronx, NY© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e346-e347 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Guillermo Villegas More articles by this author Moses Tar More articles by this author Arnold Melman More articles by this author Michael DiSanto More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Vascular Endothelium As a Contributor of Plasma Sphingosine 1-Phosphate

Sphingosine 1-phosphate (S1P), an abundant lipid mediator in plasma, regulates vascular and immune cells by activating S1P receptors. In this report, we investigated the mechanisms by which high plasma S1P levels are maintained in mice. We found that plasma S1P turns over rapidly with a half-life of approximately 15 minutes, suggesting the existence of a high-capacity biosynthetic source(s). Transplantation of bone marrow from wild-type to Sphk1(-/-)Sphk2(+/-) mice restored plasma S1P levels, suggesting that hematopoietic cells are capable of secreting S1P into plasma. However, plasma S1P levels were not appreciably altered in mice that were thrombocytopenic, anemic, or leukopenic. Surprisingly, reconstitution of Sphk1(-/-)Sphk2(+/-) bone marrow cells into wild-type hosts failed to reduce plasma S1P, suggesting the existence of an additional, nonhematopoietic source for plasma S1P. Adenoviral expression of Sphk1 in the liver of Sphk1(-/-) mice restored plasma S1P levels. In vitro, vascular endothelial cells, but not hepatocytes, secreted S1P in a constitutive manner. Interestingly, laminar shear stress downregulated the expression of S1P lyase (Sgpl) and S1P phosphatase-1 (Sgpp1) while concomitantly stimulating S1P release from endothelial cells in vitro. Modulation of expression of endothelial S1P lyase with small interfering RNA and adenoviral expression altered S1P secretion, suggesting an important role played by this enzyme. These data suggest that the vascular endothelium, in addition to the hematopoietic system, is a major contributor of plasma S1P.