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’s correlation coefficient (r) = 0.784 and P < 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.

Inhibitory Effect of Resveratrol on LPS-induced Glomerular Mesangial Cells Proliferation and TGF-β1 Expression via Sphingosine Kinase 1 Pathway

Objective To elucidate the renoprotective effect of resveratrol(RSV)on sphingosine kinase 1(SphK1)signaling pathway and expression of its downstream molecules including activator protein 1(AP-1)and transformation growth factor-β1(TGF-β1)in lipopolysaccharide(LPS)-induced glomerular mesangial cells(GMCs).Methods The rat GMCs line(HBZY-1)were cultured and randomly divided into 5 groups,including control,LPS(100 ng/mL),and 5,10,20µmol/L RSV-treated groups.In addition,SphK1 inhibitor(SK-II)was used as positive control.GMCs were pretreated with RSV for 2 h and treated with LPS for another 24 h.GMCs proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)assay.The proteins expression of SphK1,p-c-Jun and TGF-β1 in GMCs were detected by Western blot,and DNA-binding activity of AP-1 was performed by electrophoretic mobility shift assay(EMSA).The binding activity between RSV and SphK1 protein was detected by AutoDock Vina and visualized by Discovery Studio 2016.Results LPS could obviously stimulate GMCs proliferation,elevate SphK1,p-c-Jun and TGF-β1 expression levels and increase the DNA-binding activity of AP-1(P<0.05 or P<0.01),whereas these effects were significantly blocked by RSV pretreatment.It was also suggested that the effect of RSV was similar to SK-II(P>0.05).Moreover,RSV exhibited good binding affinity towards SphK1,with docking scores of−8.1 kcal/moL and formed hydrogen bonds with ASP-178 and LEU-268 in SphK1.Conclusion RSV inhibited LPS-induced GMCs proliferation and TGF-β1 expression,which may be independent of its hypoglycemic effect on preventing the development of mesangial cell fibrosis and closely related to the direct inhibition of SphK1 pathway.

C5a/C5a R pathway is essential for up-regulating Sph K1 expression through p38-MAPK activation in acute liver failure

AIM To investigate the role of the complement 5a(C5a)/C5 a receptor(C5a R) pathway in the pathogenesis of acute liver failure(ALF) in a mouse model.METHODS BALB/c mice were randomly assigned to different groups, and intraperitoneal injections of lipopolysaccharide(LPS)/D-galactosamine(D-Gal N)(600 mg/kg and 10 μg/kg) were used to induce ALF. The KaplanMeier method was used for survival analysis. Serum alanine aminotransferase(ALT) levels, at different time points within a 1-wk period, were detected with a biochemistry analyzer. Pathological examination of liver tissue was performed 36 h after ALF induction. Serum complement 5(C5), C5 a, tumor necrosis factor-α(TNF-α), interleukin(IL)-1β, IL-6, high-mobility group protein B1(HMGB1) and sphingosine-1-phosphatelevels were detected by enzyme-linked immunosorbant assay. Hepatic morphological changes at 36 h after ALF induction were assessed by hematoxylin and eosin staining. Expression of C5 a R, sphingosine kinase 1(Sph K1), p38-MAPK and p-p38-MAPK in liver tissue, peripheral blood mononuclear cells(PBMCs) and peritoneal exudative macrophages(PEMs) of mice or RAW 264.7 cells was analyzed by western blotting. C5 a R m RNA levels were detected by quantitative real-time PCR.RESULTS Activation of C5 and up-regulation of C5 a R were observed in liver tissue and PBMCs of mice with ALF. Blockade of C5 a R with a C5 a R antagonist(C5a Ra C5 a Ra) significantly reduced the levels of serum ALT, inflammatory cytokines(TNF-α, IL-1β and IL-6) and HMGB1, as well as the liver tissue damage, but increased the survival rates(P < 0.01 for all). Blockade of C5 a R decreased Sph K1 expression in both liver tissue and PBMCs significantly at 0.5 h after ALF induction. C5 a Ra pretreatment significantly downregulated the phosphorylation of p38-MAPK in liver tissues of ALF mice and C5 a stimulated PEMs or RAW 264.7 cells. Moreover, inhibition of p38-MAPK activity with SB203580 reduced Sph K1 protein production significantly in PEMs after C5 a stimulation.CONCLUSION The C5a/C5 a R pathway is essential for up-regulating Sph K1 expression through p38 MAPK activation in ALF in mice, which provides a potential immunotherapeutic strategy for ALF in patients.

A novel oncolytic adenovirus inhibits hepatocellular carcinoma growth

Objective: To evaluate the inhibitory role of a novel oncolytic adenovirus(OA), GP73-SphK1 sR-Ad5, on the growth of hepatocellular carcinoma(HCC). Methods: GP73-SphK1 sR-Ad5 was constructed by integrating Golgi protein 73(GP73) promoter and sphingosine kinase 1(SphK1)-short hairpin RNA(shRNA) into adenovirus serotype 5(Ad5), and transfecting into HCC Huh7 cells and normal human liver HL-7702 cells. The expression of SphK1 and adenovirus early region 1(E1 A) was detected by quantitative real-time PCR(qRT-PCR) and western blot, respectively. Cell viability was detected by methylthiazolyldiphenyl-tetrazolium bromide(MTT) assay, and apoptotic rate was determined by flow cytometry. An Huh7 xenograft model was established in mice injected intratumorally with GP73-SphK 1 sR-Ad5. Twenty days after injection, the tumor volume and weight, and the survival time of the mice were recorded. The histopathological changes in tumor tissues were observed by hematoxylin-eosin(HE) staining and transmission electron microscopy(TEM). Results: Transfection of GP73-SphK1 sR-Ad5 significantly upregulated E1 A and downregulated SphK1 in Huh7 cells, but not in HL7702 cells. GP73-SphK1 sR-Ad5 transfection significantly decreased the viability and increased the apoptotic rate of Huh7 cells, but had no effect on HL7702 cells. Intratumoral injection of GP73-SphK1 sR-Ad5 into the Huh7 xenograft mouse model significantly decreased tumor volume and weight, and prolonged survival time. It also significantly decreased the tumor infiltration area and blood vessel density, and increased the percentages of cells with nucleus deformation and cells with condensed chromatin in tumor tissues. Conclusions: GP73-SphK1 sR-Ad5 serves as a novel OA and can inhibit HCC progression with high specificity and efficacy.