Expressions of Sphingosine-1-phosphate (S1P) Receptors, Sphingosine Kinases in Malignant Bone and Soft Tissue Tumors, and The role of Sphingosine Kinase-1 in Growth of MFH Cell Lines

Sphingolipids are ubiquitous components of cell membranes. Their metabolites ceramide, sphingosine, and sphingosine-1-phosphate (S1P) have important physiological functions, including regulation of cell growth and survival. S1P is generated by phosphorylation of sphingosine catalyzed by sphingosine kinase-1 (SPHK1). The purpose of this study is to explore the roles of S1P, S1P receptors, and sphingosine kinases in malignant musculoskeletal tumors. Twenty-one tumor samples (7 liposarcomas, 3 chondrosarcomas, 6 osteosarcomas, 5 MFH) obtained at open biopsy, and four human MFH cell lines (Nara H, Nara F, TNMY1, GBS-1) were used. We examined the mRNA expression of S1P receptors by RT-PCR, and the expression levels of SPHK by Real-time PCR. We used 4 MFH cell lines to analyze SPHK1 proteins by Western blotting. SPHK1 siRNA was transfected into MFH cell lines by lipofection method. Cell proliferation (control and transfected with siRNA) was assayed using WST-8 (Cell Counting Kit-8) assay. All high grade malignant tumors expressed S1P1, S1P2, S1P3 receptors, whereas the expression of S1P1 receptor was detected in 50% of low-grade malignant tumors, S1P2 receptor in 30%, and S1P3 in 50%. No statistically significant difference was found in the expression level of SPHK1 between high-grade and low-grade malignant tumors by Real-time PCR. By results of Western blotting, proteins of SPHK1 were expressed in all MFH cell lines. In MFH cell lines, transfection with SPHK1 siRNA oligonucleotides resulted in approximately 50 to 80% suppression of SPHK1 mRNA expression as determined by real-time PCR. Down-regulation of SPHK1 with small interfering RNA significantly reduced SPHK1 protein levels by Western blotting. Knock down of SPHK1 expression significantly decreased cell proliferation of all MFH cells. These results suggest that the expression of S1P receptors may play an important role for cell proliferation and may correlate with histologic grade in malignant bone and soft tissue tumors, and that SPHK1 may be one of essential molecules for cell proliferation in MFH cell lines.

Sphingosine Kinase-1/sphingosine 1-phosphate pathway in diabetic nephropathy

Objective Diabetic nephropathy （DN） is the major cause of end-stage renal disease worldwide and its prevalence continues to increase.Currently,therapies for DN provide only partial renoprotection; hence new targets for therapeutic intervention need to be identified.In this review,we summarized the new target,sphingosine kinase-1/sphingosine 1-phosphate （SphK1/S1P） pathway,explored its potential therapeutic role in the prevention and treatment of DN.Data sources Most relevant articles were mainly identified by searching PubMed in English.Study selection Mainly original articles and critical review articles by major pioneer investigators in this field were selected to be reviewed.Results SphK1/S1P pathway can be activated by hyperglycemia,advanced glycation end products,and many proinflammatory cytokines,which leads to fibronectin,transforming growth factor-31 up-regulation and AP-1 activation.And then it could promote glomerular mesangial cells proliferation and extracellular matrix accumulation,mediating the initiation and progression of diabetic renal fibrosis.Conclusions SphK1/S1P pathway is closely correlated with the pathogenesis of DN.The results suggest that SphK1/ S1P pathway as a new target for clinically improving DN in future is of great prospect.

Research Progress on Targets and Selective Inhibitors of Polo-like Kinase-1(PLK-1)

In this paper,the biological function of PLK-1,the correlation between PLK-1 and tumors,and the latest research progress on PLK-1 inhibitors under study are reviewed,in order to provide references for the research and development of PLK-1 inhibitors.

MiRNA-145-5p inhibits gastric cancer progression via the serpin family E member 1-extracellular signal-regulated kinase-1/2 axis

BACKGROUND MicroRNAs(miRNAs)regulate gene expression and play a critical role in cancer physiology.However,there is still a limited understanding of the function and regulatory mechanism of miRNAs in gastric cancer(GC).AIM To investigate the role and molecular mechanism of miRNA-145-5p(miR145-5p)in the progression of GC.METHODS Real-time polymerase chain reaction(RT-PCR)was used to detect miRNA expression in human GC tissues and cells.The ability of cancer cells to migrate and invade was assessed using wound-healing and transwell assays,respectively.Cell proliferation was measured using cell counting kit-8 and colony formation assays,and apoptosis was evaluated using flow cytometry.Expression of the epithelial-mesenchymal transition(EMT)-associated protein was determined by Western blot.Targets of miR-145-5p were predicated using bioinformatics analysis and verified using a dual-luciferase reporter system.Serpin family E member 1(SERPINE1)expression in GC tissues and cells was evaluated using RT-PCR and immunohistochemical staining.The correlation between SERPINE1 expression and overall patient survival was determined using Kaplan-Meier plot analysis.The association between SERPINE1 and GC progression was also tested.A rescue experiment of SERPINE1 overexpression was conducted to verify the relationship between this protein and miR-145-5p.The mechanism by which miR-145-5p influences GC progression was further explored by assessing tumor formation in nude mice.RESULTS GC tissues and cells had reduced miR-145-5p expression and SERPINE1 was identified as a direct target of this miRNA.Overexpression of miR-145-5p was associated with decreased GC cell proliferation,invasion,migration,and EMT,and these effects were reversed by forcing SERPINE1 expression.Kaplan-Meier plot analysis revealed that patients with higher SERPINE1 expression had a shorter survival rate than those with lower SERPINE1 expression.Nude mouse tumorigenesis experiments confirmed that miR-145-5p targets SERPINE1 to regulate extracellular signal-regulated kinase-1/2(ERK1/2).CONCLUSION This study found that miR-145-5p inhibits tumor progression and is expressed in lower amounts in patients with GC.MiR-145-5p was found to affect GC cell proliferation,migration,and invasion by negatively regulating SERPINE1 levels and controlling the ERK1/2 pathway.

Unveiling the biological role of sphingosine-1-phosphate receptor modulators in inflammatory bowel diseases

Inflammatory bowel disease(IBD)is chronic inflammation of the gastrointestinal tract that has a high epidemiological prevalence worldwide.The increasing disease burden worldwide,lack of response to current biologic therapeutics,and treatment-related immunogenicity have led to major concerns regarding the clinical management of IBD patients and treatment efficacy.Understanding disease pathogenesis and disease-related molecular mechanisms is the most important goal in developing new and effective therapeutics.Sphingosine-1-phosphate(S1P)receptor(S1PR)modulators form a class of oral small molecule drugs currently in clinical development for IBD have shown promising effects on disease improvement.S1P is a sphingosine-derived phospholipid that acts by binding to its receptor S1PR and is involved in the regulation of several biological processes including cell survival,differentiation,migration,proliferation,immune response,and lymphocyte trafficking.T lymphocytes play an important role in regulating inflammatory responses.In inflamed IBD tissue,an imbalance between T helper(Th)and regulatory T lymphocytes and Th cytokine levels was found.The S1P/S1PR signaling axis and metabolism have been linked to inflammatory responses in IBD.S1P modulators targeting S1PRs and S1P metabolism have been developed and shown to regulate inflammatory responses by affecting lymphocyte trafficking,lymphocyte number,lymphocyte activity,cytokine production,and contributing to gut barrier function.

Research progress of sphingosine 1-phosphate and its signal transduction in central nervous system diseases

Sphingosine 1-phosphate(S1P),as a sphingolipid metabolite,has become a key substance in regulating various physiological processes,involved in differentiation,proliferation,migration,morphogenesis,cytoskeleton formation,adhesion,apoptosis,etc.process.Sphingosine 1-phosphate can not only activate the S1P-S1PR signaling pathway by binding to the corresponding receptors on the cell membrane,but also play a role in the cell.In recent years,studies have found that there is a certain relationship between its level changes and the occurrence and development of central nervous system diseases.This article reviews the latest knowledge of sphingosine-1-phosphate in the occurrence and treatment of nervous system diseases,and further clarifies its molecular mechanism in the treatment and development of central nervous system diseases.

山姜素调节VEGF/SphK1/S1P信号通路对膝骨关节炎大鼠血管生成的影响

目的探讨山姜素(APT)调节血管内皮生长因子/鞘氨醇激酶1/1磷酸鞘氨醇(VEGF/SphK1/S1P)信号通路对膝骨关节炎(KOA)大鼠血管生成的影响。方法采用改良的Videman法构建KOA大鼠模型,将90只大鼠分为对照组(Control组)、模型组(Model组)、山姜素低剂量组(L-APT组)、山姜素高剂量组(H-APT组)、山姜素高剂量组+慢病毒阴性对照组(APT+NC组)、山姜素高剂量组+过表达SphK1慢病毒组(APT+SphK1组),每组15只。HE染色观察大鼠软骨组织病理变化;酶联免疫吸附试验测定软骨组织白细胞介素(IL)-1β、肿瘤坏死因子α(TNF-α)、IL-6、基质金属蛋白酶-13(MMP-13)水平;TUNEL检测软骨组织细胞凋亡情况;免疫组化检测血管内皮生长因子(VEGF)、CD31蛋白表达情况;Western blot检测血管内皮生长因子受体2(VEGFR2)、磷酸化VEGFR2(p-VEGFR2)、SphK1、S1P蛋白水平。结果与Control组比较,Model组大鼠出现病理损伤,细胞凋亡率、IL-1β、TNF-α、IL-6、MMP-13、VEGF阳性表达、CD31阳性表达和p-VEGFR2、SphK1、S1P蛋白表达水平增加(P<0.05);与Model组比较,L-APT组、H-APT组病理损伤明显减轻,细胞凋亡率、IL-1β、TNF-α、IL-6、MMP-13、VEGF阳性表达、CD31阳性表达和pVEGFR2、SphK1、S1P蛋白表达水平降低(P<0.05);与APT+NC组比较,APT+SphK1组软骨组织病理损伤加重,细胞凋亡率、IL-1β、TNF-α、IL-6、MMP-13、VEGF阳性表达、CD31阳性表达和p-VEGFR2、SphK1、S1P蛋白表达水平增加(P<0.05)。结论APT通过抑制VEGF/SphK1/S1P信号通路抑制KOA大鼠血管生成。

阿芬太尼调节SphK1/S1P信号通路保护心肌缺血再灌注损伤大鼠

[目的]探究阿芬太尼对心肌缺血再灌注损伤(MIRI)大鼠的作用及在该过程中对鞘氨醇激酶1(SphK1)/鞘氨醇-1-磷酸(S1P)信号通路的调节机制。[方法]将SPF级SD雄性大鼠随机分为假手术组、模型组、阳性药物组(复方丹参组)和阿芬太尼低剂量组、阿芬太尼高剂量组、阿芬太尼高剂量+SphK1激动剂组(阿芬太尼+PMA组),每组20只。除假手术组,其余组均利用结扎左前降支冠状动脉后再灌注复制MIRI模型。全自动生物化学分析仪检测血清乳酸脱氢酶(LDH)、肌酸激酶(CK)和谷草转氨酶(AST)的活性;TTC检测大鼠心肌梗死面积;HE染色观察大鼠心肌组织形态学特征;TUNEL染色检测大鼠心肌细胞凋亡;ELISA检测血清肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、白细胞介素1β(IL-1β)及S1P的水平;试剂盒检测心肌组织中丙二醛(MDA)含量和超氧化物歧化酶(SOD)的活性;Western blot检测心肌组织SphK1蛋白表达。[结果]相较于假手术组,模型组大鼠心肌组织病理损伤严重,血清中心肌损伤标志物LDH、CK和AST的活性,心肌梗死面积和心肌细胞凋亡率,TNF-α、IL-6、IL-1β、MDA、S1P水平及SphK1蛋白表达均升高,SOD活性降低(P<0.05);相较于模型组,阳性药物组和阿芬太尼低、高剂量组大鼠心肌组织损伤减轻,血清中心肌损伤标志物LDH、CK和AST的活性,心肌梗死面积和心肌细胞凋亡率,TNF-α、IL-6、IL-1β、MDA、S1P水平及SphK1蛋白表达均降低,SOD活性升高(P<0.05)。SphK1激动剂可逆转高剂量阿芬太尼对上述指标的影响(P<0.05)。[结论]阿芬太尼对MIRI大鼠发挥保护作用,其机制可能与抑制SphK1/S1P信号通路有关。

和血柔肝方含药血清通过鞘氨醇激酶-1对肝星状细胞增殖与凋亡的作用

目的:和血柔肝方(HXRGF)含药血清调节鞘氨醇激酶-1(SphK1)对肝星状细胞(HSC)增殖与凋亡的作用。方法:采用Resistin处理HSC-T6细胞,建立纤维化HSC模型,采用LV-rSphK1-OE、LV-rSphk1-shRNA慢病毒方法构建SphK1过表达、低表达稳转细胞株,运用实时定量PCR、WB检测TGF-β、α-SMA、Col I、FN、Smad、SphK1mRNA及蛋白的表达、CCK8实验检测细胞活力、AnnexinV-APC/7-AAD流式细胞学检测细胞凋亡情况。结果:与对照组比较,MF-HSC-T6细胞中可见TGF-β、α-SMA、Col I、FN、SphK1mRNA及蛋白表达水平升高,Smad mRNA水平降低(P<0.01);HSC-T6 SphK1过表达细胞株中SphK1及相关纤维化标志物的表达水平升高(P<0.01);HSC-T6 SphK1低表达细胞株中TGF-β、α-SMA、Col I、FN mRNA等纤维化相关标志物降低,Smad mRNA水平升高(P<0.01)。与模型组比较,中药复方HXRGF含药血清干预后,可见MF-HSC T6 SphK1mRNA及蛋白表达水平降低,并同时伴有α-SMA、Col I、FN表达水平的下降(P<0.01),HSC细胞增殖率下降(110.85%);shRNA-SphK1转染组细胞增殖率下降,凋亡率(12.14%)升高(P<0.01);HSC-T6 SphK1低表达组细胞凋亡率(16.48%)升高(P<0.01)。结论:SphK1可作为肝纤维化潜在治疗靶点。HXRGF干预通过抑制SphK1表达,下调TGF-β、α-SMA等促纤维化因子的表达水平,抑制活化的HSC增殖,诱导HSC凋亡,缓解肝纤维化。

白藜芦醇通过调控microRNA-506/sphingosine kinase 1抑制非小细胞肺癌细胞的增殖及促进其凋亡

目的:探讨白藜芦醇对A549细胞增殖、细胞周期及凋亡的影响及机制。方法:实验分为空白对照组、白藜芦醇干预组,miR-506-inhibitor组及白藜芦醇干预miR-506-inhibitor组。CCK-8法检测A549细胞增殖;流式细胞术检测A549的细胞周期及凋亡;qRT-PCR检测miR-506表达及SPHK1的mRNA表达;Western blot法检测SPHK1的蛋白表达。结果:白藜芦醇抑制A549细胞的增殖,使G0-G1期的细胞数量增加,S期的细胞数量减少并且诱导A549细胞的凋亡。白藜芦醇上调miR-506的表达及下调SPHK1的表达。结论:白藜芦醇通过调控miR-506/SPHK1轴从而抑制A549细胞的增殖并且诱导其凋亡。

Sphingosine kinase 1 is upregulated with lysophosphatidic acid receptor 2 in human colorectal cancer

AIM: To examine the expression of SphK1, an oncogenic kinase that produces sphingosine 1-phosphate (S1P), and its correlation with the expression of LPAR2, a major lysophosphatidic acid (LPA) receptor overexpressed in various cancers, in human colorectal cancer.METHODS: Real-time reverse-transcription polymerase chain reaction was used to measure the mRNA expression of SphK1, LPAR2, and the three major S1P receptors in 27 colorectal cancer samples and corresponding normal tissue samples. We also examined the correlation between the expression of SphK1 and LPAR2.RESULTS: Colorectal cancer tissue in 22 of 27 patients had higher levels of SphK1 mRNA than in normal tissue. In two-thirds of the samples, SphK1 mRNA expression was more than two-fold higher than in normal tissue. Consistent with previous reports, LPAR2 mRNA expression in 20 of 27 colorectal cancer tissue samples was higher compared to normal tissue samples. Expression profiles of all three major S1P receptors, S1PR1, S1PR2, and S1PR3, varied without any trend, with no significant difference in expression between cancer and normal tissues. A highly significant positive correlation was found between SphK1 and LPAR2 expression [Pearson&#x02019;s correlation coefficient (r) = 0.784 and P &#x0003c; 0.01]. The mRNA levels of SphK1 and LPAR2 did not correlate with TNM stage.CONCLUSION: Our findings suggest that S1P and LPA may play important roles in the development of colorectal cancer via the upregulation of SphK1 and LPAR2, both of which could serve as new therapeutic targets in the treatment of colorectal cancer.

Inhibition of sphingosine kinase 1 ameliorates acute liver failure by reducing high-mobility group box 1 cytoplasmic translocation in liver cells

AIM: To determine the therapeutic potential of sphingosine kinase 1(Sphk1) inhibition and its underlying mechanism in a well-characterized mouse model of D-galactosamine(D-Gal N)/lipopolysaccharide(LPS)-induced acute liver failure(ALF).METHODS: Balb/c mice were randomly assigned to different groups,with ALF induced by intraperitoneal injection of D-Ga IN(600 mg/kg) and LPS(10 μg/kg). The Kaplan-Meier method was used for survival analysis. Serum alanine aminotransferase(ALT) and aspartate aminotransferase(AST) levels at different time points within one week were determined using a multi-parametric analyzer. Serum high-mobility group box 1(HMGB1),tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,IL-6,IL-10,and sphingosine-1-phosphate were detected by enzyme-linked immunosorbent assay. Hepatic morphological changes at 36 h after acute liver injury induction were assessed by hematoxylin and eosin staining. HMGB1 expression in hepatocytes and cytoplasmic translocation were detected by immunohistochemistry. Expression of Sphk1 in liver tissue and peripheral blood mononuclear cells(PBMCs) was analyzed by Western blot.RESULTS: The expression of Sphk1 in liver tissue and PBMCs was upregulated in Gal N/LPS-induced ALF. Upregulated Sphk1 expression in liver tissue was mainly caused by Kupffer cells,the resident macrophages of the liver. The survival rates of mice in the N,Ndimethylsphingosine(DMS,a specific inhibitor of Sph K1) treatment group were significantly higher than that of the control group(P < 0.001). DMS treatment significantly decreased the levels of serum ALT and AST at 6,12,and 24 h compared with that of the control group(P < 0.01 for all). Serum HMGB1 levels at 6,12,and 24 h,as well as serum TNF-α,IL-6,and IL-1β levels at 12 h,were significantly lower in the DMS treatment group than in the control group(P < 0.01 for all). Furthermore,hepatic inflammation,necrosis,and HMGB1 cytoplasm translocation in liver cells were significantly decreased in the DMS treatment group compared to the control group(43.72% ± 5.51% vs 3.57% ± 0.83%,χ2 = 12.81,P < 0.01).CONCLUSION: Inhibition of Sph K1 ameliorates ALF by reducing HMGB1 cytoplasmic translocation in liver cells,and so might be a potential therapeutic strategy for this disease.

血清1-磷酸鞘氨醇联合透明质酸检测对结直肠癌的诊断及预后价值

目的:观察分析结直肠癌(CRC)患者血清1-磷酸鞘氨醇(S1P)和透明质酸(HA)的水平变化及相关性,探讨其对结直肠癌诊断和预后评估的临床价值。方法:收集47例结直肠癌患者(结直肠癌组)、30例肠道良性疾病患者(良性疾病组)和38名健康的体检者(健康对照组)的临床资料,并检测所有受试者的血清S1P和HA水平。利用Spearman相关性分析评估两者相关性。采用受试者工作特征(ROC)曲线评估S1P、HA、癌胚抗原(CEA)和糖类抗原19-9(CA19-9)单独和联合诊断CRC的效能。结果:血清S1P水平在健康对照组、良性疾病组及结直肠癌组中逐次升高(P<0.05)。结直肠癌组血清HA水平高于健康对照组(P<0.05),然而良性疾病组的血清HA水平与结直肠癌组和健康对照组之间的差异无统计学意义(P>0.05)。根据Spearman相关性分析结果,结直肠癌组血清S1P水平和HA水平之间存在正相关关系(r=0.406,P<0.05)。ROC曲线结果显示,S1P、HA、CEA、CA19-9单独和联合诊断CRC的曲线下面积分别为0.871、0.642、0.694、0.593及0.916。术后CRC患者血清HA水平显著降低(P<0.001),但术后血清S1P水平与术前差异无统计学意义(P>0.05)。结论:血清S1P与HA结合检测在结直肠癌的辅助诊断及预后评估中具有一定的临床价值。

Sphingosine kinase 1 knockout alleviates hepatic ischemia/reperfusion injury by attenuating inflammation and oxidative stress in mice

Background: Hepatic ischemia/reperfusion(I/R) injury remains a significant problem in clinical practice. Sphingosine kinase 1(Sph K1) phosphorylates sphingosine to sphingosine-1-phosphate(S1 P) which participates in multiple bioactive processes. However, little is known about the role of Sph K1 in hepatic I/R injury. This study aimed to investigate the effect of Sph K1 knockout on liver I/R injury and to explore underlying mechanisms. Methods: Sph K1 knockout and wild type mice were subjected to 70% partial hepatic I/R. Serum alanine aminotransferase was determined to indicate the degree of liver damage. Hematoxylin-eosin staining and TUNEL assay were used to assess histological changes and hepatocellular apoptosis, respectively. Immunohistochemistry was performed to detect the expression and translocation of phosphorylated p65 and signal transducer and activator of transcription 3(STAT3). Western blotting was used to determine the expression of S1 P receptor 1(S1 PR1), phosphorylated p65 and STAT3. Real-time PCR was used to demonstrate the changes of proinflammatory cytokines. Oxidative stress markers were also determined through biochemical assays. Results: Sph K1 knockout significantly ameliorated I/R-induced liver damage, mitigated liver tissue necrosis and apoptosis compared with wild type control. I/R associated inflammation was alleviated in Sph K1 knockout mice as demonstrated by attenuated expression of S1 PR1 and reduced phosphorylation of nuclear factor kappa B p65 and STAT3. The proinflammatory cytokines interleukin-1 β, interleukin-6 and tumor necrosis factor-α were also inhibited by Sph K1 genetic deletion. The oxidative stress markers were lower in Sph K1 knockout mice after I/R injury than wild type mice. Conclusions: Knockout of Sph K1 significantly alleviated damage after hepatic I/R injury, possibly through inhibiting inflammation and oxidative stress. Sph K1 may be a novel and potent target in clinical practice in I/R-related liver injury.

Roles of sphingosine 1-phosphate on tumorigenesis

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid with a variety of biological activities.It is generated from the conversion of ceramide to sphingosine by ceramidase and the subsequent conversion of sphingosine to S1P,which is catalyzed by sphingosine kinases.Through increasing its intracellular levels by sphingolipid metabolism and binding to its cell surface receptors,S1P regulates several physiological and pathological processes,including cell proliferation,migration,angiogenesis and autophagy.These processes are responsible for tumor growth,metastasis and invasion and promote tumor survival.Since ceramide and S1P have distinct functions in regulating in cell fate decision,the balance between the ceramide/sphingosine/S1P rheostat becomes a potent therapeutic target for cancer cells.Herein,we summarize our current understanding of S1P signaling on tumorigenesis and its potential as a target for cancer therapy.

Role of sphingosine kinase and sphingosine-1-phosphate in inflammatory arthritis

The importance of sphingosine kinase(SphK)and sphingosine-1-phosphate(S1P)in inflammation has been extensively demonstrated.As an intracellular second messenger,S1P plays an important role in calcium signaling and mobilization,and cell proliferation and survival.Activation of various plasma membrane receptors,such as the formyl methionyl leucyl phenylalanine receptor,C5a receptor,and tumor necrosis factor α receptor,leads to a rapid increase in intracellular S1P level via SphK stimulation.SphK and S1P are implicated in various chronic autoimmune conditions such as rheumatoid arthritis, primary Sjgren's syndrome,and inflammatory bowel disease.Recent studies have demonstrated the important role of SphK and S1P in the development of arthritis by regulating the pro-inflammatory responses.These novel pathways represent exciting potential therapeutic targets.

KAI1 inhibits HGF-induced invasion of pancreatic cancer by sphingosine kinase activity

BACKGROUND:KAI1/CD82 has been reported to attenuate the process of metastases in a variety of tumors;however,its mechanism of action in invasion has not been fully elucidated. The present study aimed to investigate the importance of KAI1 in invasion and its correlation with activation of sphingosine kinase(SPK)in human pancreatic cancer PANC1 and Miapaca-2 cell lines. METHODS:The expression of KAI1 in PANC1 and Miapaca-2 cells,which was mediated by recombinant adenovirus(Ad-KAI1), was assessed by a flow cytometer and Western blotting.After successful infection was established,in vitro growth curve and invasive ability in Boyden Chamber assay were studied.The presence of KAI1 correlating with c-Met and SPK was detected by co-immunoprecipitation and[γ-32P]ATP incorporation. RESULTS:KAI1 genes had no significant effects on the curve representing cell growth.After infection with the KAI1 gene,decreased invasive ability in the Boyden Chamber assay was observed in PANC1 and Miapaca-2 cells that were induced by hepatocyte growth factor.Over-expression of KAI1 in the cells led to the deactivation of SPK and a decreased level of intracellular sphingosine-1-phosphate.No correlation was observed between c-Met and KAI1 during co-immunoprecipitation. CONCLUSION:The results of this study for the first time demonstrated a regulatory role for KAI1 in SPK activation, which leads to decreased invasive ability in disease progression of human pancreatic cancer.

Regulatory role of sphingosine kinase and sphingosine-1-phosphate receptor signaling in progenitor/stem cells

Balanced sphingolipid signaling is important for the maintenance of homeostasis. Sphingolipids were demonstrated to function as structural components, second messengers, and regulators of cell growth and survival in normal and disease-affected tissues. Particularly, sphingosine kinase 1 (SphK1) and its product sphingosine-1-phosphate (S1P) operate as mediators and facilitators of proliferation-linked signaling. Unlimited proliferation (selfrenewal) within the regulated environment is a hallmark of progenitor/stem cells that was recently associated with the S1P signaling network in vasculature, nervous,muscular, and immune systems. S1P was shown to regulate progenitor-related characteristics in normal and cancerstemcells(CSCs) viaG-protein coupled receptorsS1Pn(n=1 to 5). The SphK/S1P axis is crucially involved in the regulation of embryonic development of vasculature and the nervous system, hematopoietic stem cell migration, regeneration of skeletal muscle, and development of multiple sclerosis. The ratio of the S1P receptor expression, localization, and specific S1P receptoractivated downstream effectors influenced the rate of selfrenewal and should be further explored as regeneration related targets. Considering malignant transformation,it is essential to control the level of self-renewal capacity.Proliferation of the progenitor cell should be synchronized with differentiation to provide healthy lifelong function of blood, immune systems, and replacement of damaged ordead cells. The differentiation-related role of SphK/S1P remains poorly assessed. A few pioneering investigations exploredpharmacologicaltoolsthattargetsphingolipid signaling and can potentially confine and direct self-renewal towards normal differentiation. Further investigation is required to test the role of the SphK/S1P axis in regulation of self-renewal and differentiation.

STAT3 and sphingosine-1-phosphate in inflammation-associated colorectal cancer

Accumulated evidences have demonstrated that signal transducer and activator of transcription 3(STAT3)is a critical link between inflammation and cancer.Multiple studies have indicated that persistent activation of STAT3 in epithelial/tumor cells in inflammation-associated colorectal cancer(CRC)is associated with sphingosine-1-phosphate(S1P)receptor signaling.In inflammatory response whereby interleukin(IL)-6 production is abundant,STAT3-mediated pathways were found to promote the activation of sphingosine kinases(SphK1and SphK2)leading to the production of S1P.Reciprocally,S1P encourages the activation of STAT3 through a positive autocrine-loop signaling.The crosstalk between IL-6,STAT3 and sphingolipid regulated pathways may play an essential role in tumorigenesis and tumor progression in inflamed intestines.Therapeutics targeting both STAT3 and sphingolipid are therefore likely to contribute novel and more effective therapeutic strategies against inflammation-associated CRC.

抑制S1P/S1PR2介导的周细胞脱失可缓解NPSLE小鼠血脑屏障功能障碍

目的:探讨在MRL/lpr小鼠模型中鞘氨醇-1-磷酸(S1P)/S1P受体2(S1PR2)信号通路的异常激活引起周细胞丢失并进一步影响神经精神性系统性红斑狼疮(NPSLE)小鼠血脑屏障功能障碍的病理机制。方法:采用旷场实验、新物体识别实验和强迫游泳实验评估6周龄开始的雌性MRL/lpr小鼠行为学改变,将神经行为学与基线比改变大于20%者定义为NPSLE小鼠。将NPSLE小鼠随机分为NPSLE-S1P拮抗组、NPSLE-S1PR2阻断组和NPSLE生理盐水处理组,每组6只。另将行为学无异常改变的6只小鼠作为对照组。NPSLE-S1P拮抗组给予S1P拮抗剂FTY720(2 mg/kg,灌胃),NPSLE-S1PR2阻断组给予S1PR2阻断剂JTE-013(8 mg/kg,腹腔注射),NPSLE生理盐水处理组和对照组给予等体积的生理盐水。每周给药3次,持续3周。通过旷场实验、新物体识别实验和强迫游泳实验评估小鼠的行为学改变。采用酶联免疫吸附实验(ELISA)检测血清中S1P、白细胞介素6(IL-6)和干扰素α(IFN-α)水平;伊文思蓝法评估血脑屏障通透性改变;苏木精-伊红(HE)染色观察脑组织炎症情况;尼氏染色观察脑神经元受损情况;免疫荧光染色观察微血管中周细胞标志物神经胶质抗原2(NG2)和内皮细胞标志物CD31的表达;Western blot检测脑组织中S1P、S1PR2、血小板源性生长因子受体β(PDGFR-β)和闭锁小带蛋白1(ZO-1)的蛋白表达水平;RT-qPCR检测S1PR2和PDGFR-β的mRNA表达水平。结果:与模型组比较,S1P拮抗组和S1PR2阻断组小鼠水中潜伏和静止时间显著缩短(P<0.05),中央区探索距离显著增加(P<0.05),脑室炎性渗出和神经元受损减少,周细胞(NG2,绿色)和内皮细胞(CD31,红色)脱失情况减少,血清S1P、IL-6和IFN-α水平显著下降(P<0.05),S1PR2的mRNA表达水平显著下降(P<0.05),PDGFR-β的mRNA表达水平显著升高(P<0.05),S1P和S1PR2蛋白表达水平显著下降(P<0.05),PDGFR-β和ZO-1蛋白表达水平显著升高(P<0.05)。结论:抑制S1P/S1PR2途径介导的周细胞脱失可缓解NPSLE小鼠的神经行为症状,降低血脑屏障通透性,并减轻炎症反应。