Development of an advanced liquid chromatography-tandem mass spectrometry measurement system for simultaneous sphingolipid analysis.

Mass spectrometry-based lipidomics approaches offer valuable tools for the detection and quantification of various lipid species, including sphingolipids. The present study aimed to develop a new method to simultaneously detect various sphingolipid species that applies to diverse biological samples. We developed and validated a measurement system by employing a single-column liquid chromatography-mass spectrometry system utilizing a normal-phase separation mode with positive ionization. The measurement system provided precision with a coefficient of variant below 20% for sphingolipids in all types of samples, and we observed good linearity in diluted serum samples. This system can measure the following sphingolipids: sphingosine 1-phosphate (S1P), sphingosine (Sph), dihydroS1P (dhS1P), dihydroSph (dhSph), ceramide 1-phosphate (Cer1P), hexosylceramide (HexCer), lactosylceramide (LacCer), dh-ceramide, deoxy-ceramide, deoxy-dh-ceramide, and sphingomyelin (SM). By measuring these sphingolipids in cell lysates where S1P lyase expression level was modulated, we could observe significant and dynamic modulations of sphingolipids in a comprehensive manner. Our newly established and validated measurement system can simultaneously measure many kinds of sphingolipids in biological samples. It holds great promise as a valuable tool for laboratory testing applications to detect overall modulations of sphingolipids, which have been proposed to be involved in pathogenesis processes in a series of elegant basic research studies.

Cerebrospinal Fluid Lysophosphatidylcholine Species for Distinguishing Narrowing of the Lumbar Spine.

BACKGROUND: Reoperation, sometimes multiple, is common with progressively worse outcomes in patients with degenerative lumbar spine diseases. Lysophosphatidylcholine (LPC), a precursor of lysophosphatidic acid, in the cerebrospinal fluid (CSF) is a possible biomarker for neuropathic pain and discriminating neuropathic pain caused by lumbar spinal canal stenosis (LSCS) from other etiologies. This study aimed to explore this possible use of LPC species in the CSF. METHODS: Patients with LSCS (n = 137) and persistent spinal pain syndrome (n = 22) were subjected in this multi-site observational study. The CSF was collected by lumbar puncture. Using liquid chromatography-tandem mass spectrometry, we measured 6 LPC species, (16:0), (18:0), (18:1), (18:2), (20:4), and (22:6), in the CSF. We compared the LPC values between the groups and determined the cutoff levels that could efficiently discriminate the groups with high accuracy. RESULTS: The levels of all measured LPC species were significantly higher in the LSCS group than the persistent spinal pain syndrome group. Four LPC species demonstrated more than 0.80 area under the curve obtained from the receiver operating characteristic curve analysis. Although the specificity of cutoff levels for the 6 LPC species was low to moderate, their sensitivity was consistently high. CONCLUSIONS: The existing diagnostic protocols combining physical examinations and morphological imaging studies for lumbar spinal pain have limited sensitivity. Measuring LPC species in the CSF is a promising objective laboratory test and could be suitable for detecting the presence of lumbar spinal stenosis and can help indications for surgery.

Understanding the modulations of glycero-lysophospholipids in an elastase-induced murine emphysema model.

BACKGROUND: Increasing evidence indicates that bioactive lipid mediators are involved in chronic obstructive pulmonary disease (COPD) pathogenesis. Recently, glycero-lysophospholipids, such as lysophosphatidic acid (LysoPA) and lysophosphatidylserine (LysoPS), have been recognized as significant inflammation-related lipid mediators. However, their association with COPD remains unclear. METHODS: We used an elastase-induced murine emphysema model to analyze the levels of lysophospholipids and diacyl-phospholipids in the lungs. Additionally, we assessed the expression of LysoPS-related genes and published data on smokers. RESULTS: In the early phase of an elastase-induced murine emphysema model, the levels of LysoPS and its precursor (phosphatidylserine [PS]) were significantly reduced, without significant modulations in other glycero-lysophospholipids. Additionally, there was an upregulation in the expression of lysoPS receptors, specifically GPR34, observed in the lungs of a cigarette smoke-exposed mouse model and the alveolar macrophages of human smokers. Elastase stimulation induces GPR34 expression in a human macrophage cell line in vitro. CONCLUSIONS: Elastase-induced lung emphysema affects the LysoPS/PS-GPR34 axis, and cigarette smoking or elastase upregulates GPR34 expression in alveolar macrophages. This novel association may serve as a potential pharmacological target for COPD treatment.

Apolipoprotein M bound sphingosine 1-phosphate suppresses NETosis through activating S1P1 and S1P4.

The pleiotropic effects of high-density lipoprotein (HDL), including its protective properties against sepsis, are attributed to the sphingosine 1-phosphate and apolipoprotein M (ApoM) that are carried on the lipoproteins. In this study, we attempted to elucidate the possible mechanisms underlying the sepsis coagulopathic state by considering the modulation of NETosis. Our results revealed that in a lipopolysaccharide-induced sepsis mouse model, the levels of NETosis markers, such as plasma DNA and histone, were elevated in ApoM-knockout (KO) mice and attenuated in ApoM-overexpressing mice. In ApoM-KO mice, the survival rate decreased and the occurrence rates of coagulopathy and organ injury increased following the administration of histone. Treatment with a conditioned medium of ApoM-overexpressing cells attenuated the observed NETosis in HL-60S cells that differentiated into neutrophils and were inhibited through the suppression of S1P1 or S1P4. The attenuation of PKCδ and PKCα/ß by S1P1 and S1P4 activation may also be involved. In ApoM-overexpressing mice, coagulopathy and organ injuries were attenuated following an injection of histone; these effects were partially inhibited by S1P1, 3, S1P4, or S1P1 antagonists. Furthermore, the exogenous administration of ApoM protected ApoM-KO mice that were challenged with histone from developing NETosis. In conclusion, the ApoM/S1P axis protects against NETosis through the attenuation of PKC activation by S1P1 and S1P4. The development of drugs targeting the ApoM/S1P axis may be beneficial for the treatment of pathological conditions involving uncontrolled NETosis, such as sepsis.

The Lysophospholipase PNPLA7 Controls Hepatic Choline and Methionine Metabolism.

The in vivo roles of lysophospholipase, which cleaves a fatty acyl ester of lysophospholipid, remained unclear. Recently, we have unraveled a previously unrecognized physiological role of the lysophospholipase PNPLA7, a member of the Ca2+-independent phospholipase A2 (iPLA2) family, as a key regulator of the production of glycerophosphocholine (GPC), a precursor of endogenous choline, whose methyl groups are preferentially fluxed into the methionine cycle in the liver. PNPLA7 deficiency in mice markedly decreases hepatic GPC, choline, and several metabolites related to choline/methionine metabolism, leading to various symptoms reminiscent of methionine shortage. Overall metabolic alterations in the liver of Pnpla7-null mice in vivo largely recapitulate those in methionine-deprived hepatocytes in vitro. Reduction of the methyl donor S-adenosylmethionine (SAM) after methionine deprivation decreases the methylation of the PNPLA7 gene promoter, relieves PNPLA7 expression, and thereby increases GPC and choline levels, likely as a compensatory adaptation. In line with the view that SAM prevents the development of liver cancer, the expression of PNPLA7, as well as several enzymes in the choline/methionine metabolism, is reduced in human hepatocellular carcinoma. These findings uncover an unexplored role of a lysophospholipase in hepatic phospholipid catabolism coupled with choline/methionine metabolism.

Modulations of bioactive lipids and their receptors in postmortem Alzheimer's disease brains.

Background: Analyses of brain samples from Alzheimer's disease (AD) patients may be expected to help us improve our understanding of the pathogenesis of AD. Bioactive lipids, including sphingolipids, glycerophospholipids, and eicosanoids/related mediators have been demonstrated to exert potent physiological actions and to be involved in the pathogenesis of various human diseases. In this cross-sectional study, we attempted to elucidate the associations of these bioactive lipids with the pathogenesis/pathology of AD through postmortem studies of human brains. Methods: We measured the levels of glycerophospholipids, sphingolipids, and eicosanoids/related mediators in the brains of patients with AD (AD brains), patients with Cerad score B (Cerad-b brains), and control subjects (control brains), using a liquid chromatography-mass spectrometry method; we also measured the mRNA levels of specific receptors for these bioactive lipids in the same brain specimens. Results: The levels of several species of sphingomyelins and ceramides were higher in the Cerad-b and AD brains. Levels of several species of lysophosphatidic acids (LPAs), lysophosphatidylcholine, lysophosphatidylserine, lysophosphatidylethanolamine (LPE), lysophosphatidylinositol, phosphatidylcholine, phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol, and phosphatidylglycerol were especially high in the Cerad-b brains, while those of lysophosphatidylglycerol (LPG) were especially high in the AD brains. Several eicosanoids, including metabolites of prostaglandin E2, oxylipins, metabolites of epoxide, and metabolites of DHA and EPA, such as resolvins, were also modulated in the AD brains. Among the lipid mediators, the levels of S1P2, S1P5, LPA1, LPA2, LPA6, P2Y10, GPR174, EP1, DP1, DP2, IP, FP, and TXA2r were lower in the AD and/or Cerad-b brains. The brain levels of ceramides, LPC, LPI, PE, and PS showed strong positive correlations with the Aß contents, while those of LPG showed rather strong positive correlations with the presence of senile plaques and neurofibrillary tangles. A discriminant analysis revealed that LPG is especially important for AD and the LPE/PE axis is important for Cerad-b. Conclusions: Comprehensive lipidomics, together with the measurement of lipid receptor expression levels provided novel evidence for the associations of bioactive lipids with AD, which is expected to facilitate future translational research and reverse translational research.

Sphingosine 1-phosphate lyase facilitates cancer progression through converting sphingolipids to glycerophospholipids.

BACKGROUND: In addition to potent agonist properties for sphingosine 1-phosphate (S1P) receptors, intracellularly, S1P is an intermediate in metabolic conversion pathway from sphingolipids to glycerolysophospholipids (glyceroLPLs). We hypothesized that this S1P metabolism and its products might possess some novel roles in the pathogenesis of cancer, where S1P lyase (SPL) is a key enzyme. METHODS: The mRNA levels of sphingolipid-related and other cancer-related factors were measured in human hepatocellular carcinoma (HCC), colorectal cancer, and esophageal cancer patients' tumours and in their adjacent non-tumour tissues. Phospholipids (PL) and glyceroLPLs were measured by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In-vitro experiments were performed in Colon 26 cell line with modulation of the SPL and GPR55 expressions. Xenograft model was used for determination of the cancer progression and for pharmacological influence. RESULTS: Besides high SPL levels in human HCC and colon cancer, SPL levels were specifically and positively linked with levels of glyceroLPLs, including lysophosphatidylinositol (LPI). Overexpression of SPL in Colon 26 cells resulted in elevated levels of LPI and lysophosphatidylglycerol (LPG), which are agonists of GPR55. SPL overexpression-enhanced cell proliferation was inhibited by GPR55 silencing. Conversely, inhibition of SPL led to the opposite outcome and reversed by adding LPI, LPG, and metabolites generated during S1P degradation, which is regulated by SPL. The xenograft model results suggested the contribution of SPL and glyceroLPLs to tumour progression depending on levels of SPL and GPR55. Moreover, the pharmacological inhibition of SPL prevented the progression of cancer. The underlying mechanisms for the SPL-mediated cancer progression are the activation of p38 and mitochondrial function through the LPI, LPG-GPR55 axis and the suppression of autophagy in a GPR55-independent manner. CONCLUSION: A new metabolic pathway has been proposed here in HCC and colon cancer, SPL converts S1P to glyceroLPLs, mainly to LPI and LPG, and facilitates cancer development.

Inhibition of autotaxin activity ameliorates neuropathic pain derived from lumbar spinal canal stenosis.

Lumbar spinal canal stenosis (LSS) or mechanical compression of dorsal root ganglion (DRG) is one of the causes of low back pain and neuropathic pain (NP). Lysophosphatidic acid (LPA) is a potent bioactive lipid mediator that is produced mainly from lysophosphatidylcholine (LPC) via autotaxin (ATX) and is known to induce NP via LPA1 receptor signaling in mice. Recently, we demonstrated that LPC and LPA were higher in cerebrospinal fluid (CSF) of patients with LSS. Based on the possible potential efficacy of the ATX inhibitor for NP treatment, we used an NP model with compression of DRG (CD model) and investigated LPA dynamics and whether ATX inhibition could ameliorate NP symptoms, using an orally available ATX inhibitor (ONO-8430506) at a dose of 30 mg/kg. In CD model, we observed increased LPC and LPA levels in CSF, and decreased threshold of the pain which were ameliorated by oral administration of the ATX inhibitor with decreased microglia and astrocyte populations at the site of the spinal dorsal horn projecting from injured DRG. These results suggested possible efficacy of ATX inhibitor for the treatment of NP caused by spinal nerve root compression and involvement of the ATX-LPA axis in the mechanism of NP induction.

Suppression of sphingosine 1-phosphate lyase retards the liver regeneration in mice after partial hepatectomy.

BACKGROUND: Liver regeneration is an extremely complicated process that is regulated by a number of signaling pathways. Sphingosine 1-phosphate (S1P), a potent bioactive lipid mediator playing crucial roles in various cellular responses through its receptors, has been attracting attention in the fields of hepatology, where S1P lyase (SPL), an irreversibly degrading enzyme of S1P, reportedly has a stimulatory role in growth of hepatocellular carcinoma (HCC). AIM OF THE STUDY: To examine whether SPL might play a stimulatory role in liver regeneration. METHOD: Using in-vivo siRNA technology, we inhibited SPL expression. Seventy percent of the liver was resected in mice as partial hepatectomy (PH). Liver tissue samples were collected and mRNA expression level of the SPL, IHC of the proliferating cell nuclear antigen (PCNA), protein levels of various proliferation factors and lipid measurements were performed in different groups. RESULTS: The mRNA levels of SPL increased in PH mice on the third day after PH surgery. When we suppressed the expression of SPL by in-vivo siRNA, we observed a significant decline of the PCNA positive cell numbers. Furthermore, the Cyclin D1 expressions and phosphorylation of ERK also were decreased in the siSPL injected PH group. CONCLUSION: We verified the importance of the SPL in liver regeneration, using the mice PH model. SPL might be a potential target to facilitate liver regeneration.

Possible involvement of PS-PLA1 and lysophosphatidylserine receptor (LPS1) in hepatocellular carcinoma.

Lysophosphatidylserine (LysoPS) is a lysophospholipid, its generating enzyme, phosphatidylserine-specific phospholipase A1 (PS-PLA1), reportedly plays roles in stomach and colon cancers. Here, we examined the potential roles of LysoPS in hepatocellular carcinoma (HCC). The ninety-seven HCC patients who underwent surgical treatment were enrolled in this study and approved by the institutional review board. Among LysoPS-related enzymes and receptors, increased PS-PLA1 or LysoPS receptor 1 (LPS1) mRNA was observed in HCC tissues compared to non-HCC tissues. PS-PLA1 mRNA in HCC was associated with no clinical parameters, while LPS1 mRNA in HCC was correlated inversely with tumor differentiation. Furthermore, higher serum PS-PLA1 was observed in HCC patients compared to healthy control and correlated with PS-PLA1 mRNA in non-HCC tissues and with serum AST or ALT. Additionally, serum levels of PS-PLA1 were higher in HCC patients with HCV-related liver injury than in those with HBV or non-HBV-, non-HCV-related liver diseases. In conclusion, among LysoPS-related enzymes and receptors, PS-PLA1 and LPS1 mRNA were increased in HCC. Based on the correlation between the serum PS-PLA1 and the mRNA level of PS-PLA1 in non-HCC tissues, the liver may be the main source of serum PS-PLA1, and serum PS-PLA1 levels may be a useful marker for liver injury.

Identification of novel biomarkers of hepatocellular carcinoma by high-definition mass spectrometry: Ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry and desorption electrospray ionization mass spectrometry imaging.

RATIONALE: Hepatocellular carcinoma (HCC) is a highly malignant disease for which the development of prospective or prognostic biomarkers is urgently required. Although metabolomics is widely used for biomarker discovery, there are some bottlenecks regarding the comprehensiveness of detected features, reproducibility of methods, and identification of metabolites. In addition, information on localization of metabolites in tumor tissue is needed for functional analysis. Here, we developed a wide-polarity global metabolomics (G-Met) method, identified HCC biomarkers in human liver samples by high-definition mass spectrometry (HDMS), and demonstrated localization in cryosections using desorption electrospray ionization MS imaging (DESI-MSI) analysis. METHODS: Metabolic profiling of tumor (n = 38) and nontumor (n = 72) regions in human livers of HCC was performed by an ultrahigh-performance liquid chromatography quadrupole time-of-flight MS (UHPLC/QTOFMS) instrument equipped with a mixed-mode column. The HCC biomarker candidates were extracted by multivariate analyses and identified by matching values of the collision cross section and their fragment ions on the mass spectra obtained by HDMS. Cryosections of HCC livers, which included both tumor and nontumor regions, were analyzed by DESI-MSI. RESULTS: From the multivariate analysis, m/z 904.83 and m/z 874.79 were significantly high and low, respectively, in tumor samples and were identified as triglyceride (TG) 16:0/18:1(9Z)/20:1(11Z) and TG 16:0/18:1(9Z)/18:2(9Z,12Z) using the synthetic compounds. The TGs were clearly localized in the tumor or nontumor areas of the cryosection. CONCLUSIONS: Novel biomarkers for HCC were identified by a comprehensive and reproducible G-Met method with HDMS using a mixed-mode column. The combination analysis of UHPLC/QTOFMS and DESI-MSI revealed that the different molecular species of TGs were associated with tumor distribution and were useful for characterizing the progression of tumor cells and discovering prospective biomarkers.

Alteration of the lysophosphatidic acid and its precursor lysophosphatidylcholine levels in spinal cord stenosis: A study using a rat cauda equina compression model.

Cauda equina compression (CEC) is a major cause of neurogenic claudication and progresses to neuropathic pain (NP). A lipid mediator, lysophosphatidic acid (LPA), is known to induce NP via the LPA1 receptor. To know a possible mechanism of LPA production in neurogenic claudication, we determined the levels of LPA, lysophosphatidylcholine (LPC) and LPA-producing enzyme autotaxin (ATX), in the cerebrospinal fluid (CSF) and spinal cord (SC) using a CEC as a possible model of neurogenic claudication. Using silicon blocks within the lumbar epidural space, we developed a CEC model in rats with motor dysfunction. LPC and LPA levels in the CSF were significantly increased from day 1. Importantly, specific LPA species (16:0, 18:2, 20:4) were upregulated, which have been shown to produce by ATX detected in the CSF, without changes on its level. In SC, the LPC and LPA levels did not change, but mass spectrometry imaging analysis revealed that LPC was present in a region where the silicon blocks were inserted. These results propose a model for LPA production in SC and CSF upon neurogenic claudication that LPC produced locally by tissue damages is converted to LPA by ATX, which then leak out into the CSF.

Lysophosphatidic acids and their substrate lysophospholipids in cerebrospinal fluid as objective biomarkers for evaluating the severity of lumbar spinal stenosis.

Lysophospholipids (LPLs) are known to have potentially important roles in the initiation and maintenance of neuropathic pain in animal models. This study investigated the association between the clinical severity of lumbar spinal stenosis (LSS) and the cerebrospinal fluid (CSF) levels of LPLs, using human samples. We prospectively identified twenty-eight patients with LSS and fifteen controls with idiopathic scoliosis or bladder cancer without neurological symptoms. We quantified LPLs from CSF using liquid chromatography-tandem mass spectrometry. We assessed clinical outcome measures of LSS (Neuropathic Pain Symptom Inventory (NPSI) and Zurich Claudication Questionnaire (ZCQ)) and categorized patients into two groups according to their severity. Five species of lysophosphatidic acid (LPA), nine species of lysophosphatidylcholine (LPC), and one species of lysophosphatidylinositol (LPI) were detected. The CSF levels of all species of LPLs were significantly higher in LSS patients than controls. Patients in the severe NPSI group had significantly higher LPL levels (three species of LPA and nine species of LPC) than the mild group. Patients in the severe ZCQ group also had significantly higher LPL levels (four species of LPA and nine species of LPC). This investigation demonstrates a positive correlation between the CSF levels of LPLs and the clinical severity of LSS. LPLs are potential biomarkers for evaluating the severity of LSS.

Lysophosphatidic acid is associated with neuropathic pain intensity in humans: An exploratory study.

The underlying mechanisms of neuropathic pain remain to be elucidated. Basic animal research has suggested that lysophosphatidic acids, which are bioactive lipids produced by autotaxin from lysophosphatidylcholine, may play key roles in the initiation and maintenance of neuropathic pain. Here, we investigated the clinical relevance of lysophosphatidic acids signaling on neuropathic pain in humans. Eighteen patients who had been diagnosed with neuropathic pain with varied etiologies participated in the study. Cerebrospinal fluid samples were obtained by lumbar puncture and the concentrations of 12 species of lysophosphatidic acids and lysophosphatidylcholine, autotaxin, and the phosphorylated neurofilament heavy subunit were measured. Pain symptoms were assessed using an 11-point numeric rating scale and the Neuropathic Pain Symptom Inventory regarding intensity and descriptive dimensions of neuropathic pain. The total lysophosphatidic acids were significantly associated with both pain intensity and symptoms. 18:1 and 20:4 lysophosphatidic acids in particular demonstrated the most correlations with dimensions of pain symptoms. Autotaxin and the phosphorylated neurofilament heavy subunit showed no association with pain symptoms. In conclusions, lysophosphatidic acids were significantly associated with pain symptoms in neuropathic pain patients. These results suggest that lysophosphatidic acids signaling might be a potential therapeutic target for neuropathic pain.

Evidence Suggests Sphingosine 1-Phosphate Might Be Actively Generated, Degraded, and Transported to Extracellular Spaces With Increased S1P2 and S1P3 Expression in Colon Cancer.

BACKGROUND: A pivotal role of sphingosine 1-phosphate (S1P) in cancer has been suggested based on the ceramide-S1P rheostat theory that the intracellular balance between prosurvival S1P and proapoptotic ceramide determines cell fate. Upregulation of S1P-generating sphingosine kinases (SKs) and downregulation of S1P-degrading S1P lyase (SPL) might increase intracellular S1P levels to exert a prosurvival effect in cancer in general, such as colon cancer. However, we recently observed a distinct S1P metabolism in hepatocellular carcinoma tissues that increased SPL mRNA levels with reduced S1P levels. Thus, we investigated S1P metabolism in colon cancer. PATIENTS AND METHODS: We enrolled 26 consecutive colon cancer patients, who had undergone surgical treatment. RESULTS: Not only SK, but also SPL, mRNA levels were increased in colon cancer tissues compared with the adjacent nontumorous tissues. Furthermore, the mRNA levels of another S1P degrading enzyme, S1P phosphatase 1, S1P transporters, spinster homolog 2, adenosine triphosphate-binding cassette subfamily C member 1, and S1P receptors, S1P2 and S1P3 were also increased, but the S1P levels were not increased in the colon cancer tissues. The reduction of SPL expression by silencing led to reduced proliferation and invasion, and overexpression of SPL caused enhanced proliferation in colon cancer cell lines. CONCLUSION: In human colon cancer tissues, mRNA levels of S1P-generating and S1P-degrading enzymes, transporters from inside to outside the cells, and S1P receptors, S1P2 and S1P3 were elevated, suggesting active S1P metabolism and movement. This altered S1P metabolism might play a role in colon cancer pathophysiology.

Higher LPA2 and LPA6 mRNA Levels in Hepatocellular Carcinoma Are Associated with Poorer Differentiation, Microvascular Invasion and Earlier Recurrence with Higher Serum Autotaxin Levels.

Hepatocellular carcinoma (HCC) commonly develops in patients with liver fibrosis; in these patients, the blood levels of lysophosphatidic acid (LPA) and its generating enzyme autotaxin (ATX) increase with the liver fibrosis stage. We aimed to examine the potential relevance of ATX and LPA in HCC. Fifty-eight HCC patients who underwent surgical treatment were consecutively enrolled in the study. Among the LPA receptors in HCC, higher LPA2 mRNA levels correlated with poorer differentiation, and higher LPA6 mRNA levels correlated with microvascular invasion, which suggested a higher malignant potential of HCC with increased LPA2 and LPA6 expression. In patients with primary HCC, neither LPA2 nor LPA6 mRNA levels were associated with recurrence. However, when serum ATX levels were combined for analysis as a surrogate for plasma LPA levels, the cumulative intra-hepatic recurrence rate was higher in patients in whom both serum ATX levels and LPA2 or LPA6 mRNA levels were higher than the median. However, the mRNA level of phosphatidic acid-selective phospholipase A1ɑ, another LPA-generating enzyme, in HCC patients was not associated with pathological findings or recurrence, even in combination with the expression of LPA receptors. Higher LPA2 mRNA levels were associated with poorer differentiation, and higher LPA6 levels were associated with microvascular invasion in HCC; both became a risk factor for recurrence after surgical treatment when combined with increased serum ATX levels. ATX and LPA receptors merit consideration as therapeutic targets of HCC.

Sphingosine kinase-1, S1P transporter spinster homolog 2 and S1P2 mRNA expressions are increased in liver with advanced fibrosis in human.

The role of sphingosine 1-phosphate (S1P) in liver fibrosis or inflammation was not fully examined in human. Controversy exists which S1P receptors, S1P1 and S1P3 vs S1P2, would be importantly involved in its mechanism. To clarify these matters, 80 patients who received liver resection for hepatocellular carcinoma and 9 patients for metastatic liver tumor were enrolled. S1P metabolism was analyzed in background, non-tumorous liver tissue. mRNA levels of sphingosine kinase 1 (SK1) but not SK2 were increased in livers with fibrosis stages 3-4 compared to those with 0-2 and to normal liver. However, S1P was not increased in advanced fibrotic liver, where mRNA levels of S1P transporter spinster homolog 2 (SPNS2) but not S1P-degrading enzymes were enhanced. Furthermore, mRNA levels of S1P2 but not S1P1 or S1P3 were increased in advanced fibrotic liver. These increased mRNA levels of SK1, SPNS2 and S1P2 in fibrotic liver were correlated with α-smooth muscle actin mRNA levels in liver, and with serum ALT levels. In conclusion, S1P may be actively generated, transported to outside the cells, and bind to its specific receptor in human liver to play a role in fibrosis or inflammation. Altered S1P metabolism in fibrotic liver may be their therapeutic target.

Increased mRNA Levels of Sphingosine Kinases and S1P Lyase and Reduced Levels of S1P Were Observed in Hepatocellular Carcinoma in Association with Poorer Differentiation and Earlier Recurrence.

Although sphingosine 1-phosphate (S1P) has been reported to play an important role in cancer pathophysiology, little is known about S1P and hepatocellular carcinoma (HCC). To clarify the relationship between S1P and HCC, 77 patients with HCC who underwent surgical treatment were consecutively enrolled in this study. In addition, S1P and its metabolites were quantitated by LC-MS/MS. The mRNA levels of sphingosine kinases (SKs), which phosphorylate sphingosine to generate S1P, were increased in HCC tissues compared with adjacent non-HCC tissues. Higher mRNA levels of SKs in HCC were associated with poorer differentiation and microvascular invasion, whereas a higher level of SK2 mRNA was a risk factor for intra- and extra-hepatic recurrence. S1P levels, however, were unexpectedly reduced in HCC compared with non-HCC tissues, and increased mRNA levels of S1P lyase (SPL), which degrades S1P, were observed in HCC compared with non-HCC tissues. Higher SPL mRNA levels in HCC were associated with poorer differentiation. Finally, in HCC cell lines, inhibition of the expression of SKs or SPL by siRNA led to reduced proliferation, invasion and migration, whereas overexpression of SKs or SPL enhanced proliferation. In conclusion, increased SK and SPL mRNA expression along with reduced S1P levels were more commonly observed in HCC tissues compared with adjacent non-HCC tissues and were associated with poor differentiation and early recurrence. SPL as well as SKs may be therapeutic targets for HCC treatment.

Increased serum autotaxin levels in hepatocellular carcinoma patients were caused by background liver fibrosis but not by carcinoma.

BACKGROUND: Controversy exists as to whether autotaxin (ATX) may be importantly associated with pathophysiology of hepatocellular carcinoma (HCC). METHODS: We evaluated serum ATX levels and its mRNA expression in consecutive 148 HCC patients treated with radiofrequency ablation (RFA) and 30 patients with hepatic resection. RESULTS: Although increased serum ATX levels were observed in almost all the patients treated with RFA, they were not reduced after RFA. Furthermore, serum ATX levels were associated not with tumor burden but with the parameters predicting for liver fibrosis, such as liver stiffness values. Then, in surgically-treated patients, there was no significant correlation between serum ATX levels and ATX mRNA expression levels in HCC tissues. Notably, ATX mRNA expression levels in HCC tissues were not higher than those in peri-tumorous tissues. Finally, serum ATX levels in surgically-treated HCC patients were rather correlated with ATX mRNA expression levels in peri-tumorous tissues as well as with liver fibrosis stage. CONCLUSION: The increase in serum ATX levels in HCC patients may not be caused by abundant ATX production in HCC tissues but by fibrosis in the background livers.