A facile stable-isotope dilution method for determination of sphingosine phosphate lyase activity.

A new technique for quantifying sphingosine phosphate lyase activity in biological samples is described. In this procedure, 2-hydrazinoquinoline is used to convert (2E)-hexadecenal into the corresponding hydrazone derivative to improve ionization efficiency and selectivity of detection. Combined utilization of liquid chromatographic separation and multiple reaction monitoring-mass spectrometry allows for simultaneous quantification of the substrate S1P and product (2E)-hexadecenal. Incorporation of (2E)- d5-hexadecenal as an internal standard improves detection accuracy and precision. A simple one-step derivatization procedure eliminates the need for further extractions. Limits of quantification for (2E)-hexadecenal and sphingosine-1-phosphate are 100 and 50fmol, respectively. The assay displays a wide dynamic detection range useful for detection of low basal sphingosine phosphate lyase activity in wild type cells, SPL-overexpressing cell lines, and wild type mouse tissues. Compared to current methods, the capacity for simultaneous detection of sphingosine-1-phosphate and (2E)-hexadecenal greatly improves the accuracy of results and shows excellent sensitivity and specificity for sphingosine phosphate lyase activity detection.

Sphingosine-1-phosphate lyase downregulation promotes colon carcinogenesis through STAT3-activated microRNAs.

Growing evidence supports a link between inflammation and cancer; however, mediators of the transition between inflammation and carcinogenesis remain incompletely understood. Sphingosine-1-phosphate (S1P) lyase (SPL) irreversibly degrades the bioactive sphingolipid S1P and is highly expressed in enterocytes but downregulated in colon cancer. Here, we investigated the role of SPL in colitis-associated cancer (CAC). We generated mice with intestinal epithelium-specific Sgpl1 deletion and chemically induced colitis and tumor formation in these animals. Compared with control animals, mice lacking intestinal SPL exhibited greater disease activity, colon shortening, cytokine levels, S1P accumulation, tumors, STAT3 activation, STAT3-activated microRNAs (miRNAs), and suppression of miR-targeted anti-oncogene products. This phenotype was attenuated by STAT3 inhibition. In fibroblasts, silencing SPL promoted tumorigenic transformation through a pathway involving extracellular transport of S1P through S1P transporter spinster homolog 2 (SPNS2), S1P receptor activation, JAK2/STAT3-dependent miR-181b-1 induction, and silencing of miR-181b-1 target cylindromatosis (CYLD). Colon biopsies from patients with inflammatory bowel disease revealed enhanced S1P and STAT3 signaling. In mice with chemical-induced CAC, oral administration of plant-type sphingolipids called sphingadienes increased colonic SPL levels and reduced S1P levels, STAT3 signaling, cytokine levels, and tumorigenesis, indicating that SPL prevents transformation and carcinogenesis. Together, our results suggest that dietary sphingolipids can augment or prevent colon cancer, depending upon whether they are metabolized to S1P or promote S1P metabolism through the actions of SPL.