Cellular concentrations of plasmalogen species containing a polyunsaturated fatty acid significantly increase under hypoxia in human colorectal cancer, Caco2 cells.

Plasmalogen localized in the raft of mammalian cell membranes plays a role in the storage of polyunsaturated fatty acid (PUFA), and exists to a higher extent in malignant cells that survive, and even grow in hypoxic conditions. The biosynthesis of plasmalogen in mammalian cells has been reported to depend on aerobic conditions. Using liquid chromatography-tandem mass spectrometry, we found that the intracellular concentration of plasmalogen species containing a PUFA at the sn-2-position did not change for two days from the start of hypoxic culture in human colorectal cancer-derived Caco2 cells. At the third day of hypoxia, Caco2 cells showed the average increase rate of 2.6 times in ethanolamine plasmalogen and 2.9 times in choline plasmalogen depending on the molecular species compared with those in the second day of hypoxia. In normoxic culture, there was little quantitative change in any species of both ethanolamine and choline plasmalogens for three days. The up-regulations of mRNA of Ca2+-independent phospholipase A2ß and cytoplasmic phospholipase A2γ as well as the down-regulation of lysoplasmalogenase observed in hypoxia were suggested to be responsible for the increase of plasmalogen in Caco2 cells under hypoxia.

CEBPγ facilitates lamellipodia formation and cancer cell migration through CERS6 upregulation.

Ceramide synthase 6 (CERS6) promotes lung cancer metastasis by stimulating cancer cell migration. To examine the underlying mechanisms, we performed luciferase analysis of the CERS6 promoter region and identified the Y-box as a cis-acting element. As a parallel analysis of database records for 149 non-small-cell lung cancer (NSCLC) cancer patients, we screened for trans-acting factors with an expression level showing a correlation with CERS6 expression. Among the candidates noted, silencing of either CCAAT enhancer-binding protein γ (CEBPγ) or Y-box binding protein 1 (YBX1) reduced the CERS6 expression level. Following knockdown, CEBPγ and YBX1 were found to be independently associated with reductions in ceramide-dependent lamellipodia formation as well as migration activity, while only CEBPγ may have induced CERS6 expression through specific binding to the Y-box. The mRNA expression levels of CERS6, CEBPγ, and YBX1 were positively correlated with adenocarcinoma invasiveness. YBX1 expression was observed in all 20 examined clinical lung cancer specimens, while 6 of those showed a staining pattern similar to that of CERS6. The present findings suggest promotion of lung cancer migration by possible involvement of the transcription factors CEBPγ and YBX1.

CERS6 required for cell migration and metastasis in lung cancer.

Sphingolipids constitute a class of bio-reactive molecules that transmit signals and exhibit a variety of physical properties in various cell types, though their functions in cancer pathogenesis have yet to be elucidated. Analyses of gene expression profiles of clinical specimens and a panel of cell lines revealed that the ceramide synthase gene CERS6 was overexpressed in non-small-cell lung cancer (NSCLC) tissues, while elevated expression was shown to be associated with poor prognosis and lymph node metastasis. NSCLC profile and in vitro luciferase analysis results suggested that CERS6 overexpression is promoted, at least in part, by reduced miR-101 expression. Under a reduced CERS6 expression condition, the ceramide profile became altered, which was determined to be associated with decreased cell migration and invasion activities in vitro. Furthermore, CERS6 knockdown suppressed RAC1-positive lamellipodia/ruffling formation and attenuated lung metastasis efficiency in mice, while forced expression of CERS6 resulted in an opposite phenotype in examined cell lines. Based on these findings, we consider that ceramide synthesis by CERS6 has important roles in lung cancer migration and metastasis.

Vaticanol C, a phytoalexin, induces apoptosis of leukemia and cancer cells by modulating expression of multiple sphingolipid metabolic enzymes.

Resveratrol (RSV) has recently attracted keen interest because of its pleiotropic effects. It exerts a wide range of health-promoting effects. In addition to health-promoting effects, RSV possesses anti-carcinogenic activity. However, a non-physiological concentration is needed to achieve an anti-cancer effect, and its in vivo bioavailability is low. Therefore, the clinical application of phytochemicals requires alternative candidates that induce the desired effects at a lower concentration and with increased bioavailability. We previously reported a low IC50 of vaticanol C (VTC), an RSV tetramer, among 12 RSV derivatives (Ito T. et al, 2003). However, the precise mechanism involved remains to be determined. Here, we screened an in-house chemical library bearing RSV building blocks ranging from dimers to octamers for cytotoxic effects in several leukemia and cancer cell lines and their anti-cancer drug-resistant sublines. Among the compounds, VTC exhibited the highest cytotoxicity, which was partially inhibited by a caspase 3 inhibitor, Z-VAD-FMK. VTC decreased the expression of sphingosine kinase 1, sphingosine kinase 2 and glucosylceramide synthase by transcriptional or post-transcriptional mechanisms, and increased cellular ceramides/dihydroceramides and decreased sphingosine 1-phosphate (S1P). VTC-induced sphingolipid rheostat modulation (the ratio of ceramide/S1P) is thought to be involved in cellular apoptosis. Indeed, exogenous S1P addition modulated VTC cytotoxicity significantly. A combination of SPHK1, SPHK2, and GCS chemical inhibitors induced sphingolipid rheostat modulation, cell growth suppression, and cytotoxicity similar to that of VTC. These results suggest the involvement of sphingolipid metabolism in VTC-induced cytotoxicity, and indicate VTC is a promising prototype for translational research.

Oxidized Perilla and Linseed Oils Induce Neuronal Apoptosis by Caspase-Dependent and -Independent Pathways.

Alpha-linolenic acid (ALA), a polyunsaturated fatty acid, is involved in bioregulatory functions. In recent years, the health-promoting effects of vegetable-derived edible oils rich in ALA have attracted attention. ALA has a variety of physiological effects such as anti-arteriosclerotic and antiallergic properties, but is prone to oxidation. Therefore, safety concerns exist with regard to adverse effects on humans induced by its oxides. However, the effects on neuronal cells induced by oxidized ALA-rich oils, such as perilla and linseed oils, have not been fully investigated. This information is very important from the viewpoint of food safety. In this study, we investigated the effects of oxidized perilla and linseed oils, which are rich in ALA, on the toxicity of neuronal SH-SY5Y cells. Perilla and linseed oils were significantly oxidized compared with other edible vegetable oils. These oxidized oils induce neuronal cell death and apoptosis via caspase-dependent and -independent pathways through reactive oxygen species (ROS) generation. Furthermore, they suppressed neurite outgrowth. These results suggest that oxidized perilla and linseed oils have the potential to cause neuronal loss and ROS-mediated apoptosis, and thus may affect the onset and progression of neurodegenerative disorders and other diseases.

Inhibition of mast cell degranulation by melanin.

Melanin is a dark naturally occurring pigment produced in nature and in many organisms. Although several reports have demonstrated applications for melanins in various therapeutic treatments, to date, no research has examined the anti-allergic effect of melanin. In this study, we for the first time found that solubilized or synthesized soluble melanin acts as a potent inhibitor of the degranulation of mast cells. We found that squid-ink-derived melanin significantly inhibited antigen-IgE-FcεRI-mediated degranulation of the mucosal mast cell line RBL-2H3. A homogenized melanin nanoparticle prepared by laser ablation also clearly suppressed antigen-induced mast cell degranulation. We also successfully solubilized synthetic melanin in a neutral biochemical buffer and found that it also significantly inhibited IgE-sensitized mast cells. The anti-degranulation activity of synthesized melanin was abolished in the melanin fraction below 50-kD molecular weight. All melanins used in this study did not exert significant cell death. Signal transduction analysis revealed that melanin suppressed antigen-triggered phosphorylation of signaling molecules as well as calcium influx. Transmission electron microscopy revealed that homogenized melanin nanoparticles partially attached to the cell surface and some nanoparticles were internalized to the cell. Flow cytometry revealed that the number of FcεRI-bound IgE molecules was decreased by melanin. Fluorescence recovery after photobleaching analysis indicated that melanin attenuated both plasma membrane and cytoplasmic fluidity, implying that melanin increased their viscosities. In vivo experiments using passive systemic anaphylaxis (PSA) and passive cutaneous anaphylaxis (PCA) mouse models demonstrated that oral administration of melanin accelerated the recovery of decreased body temperature after antigen infection in PSA, and combination sensitization of IgE with melanin attenuated antigen-induced extravasation in PCA. These findings indicated that melanin exhibits preventative effects against IgE-mast cell-mediated anaphylaxis. This study provides the first evidence that homogenized melanin may be a potential therapeutic agent for diseases involving mast cells.

Myonectin Is an Exercise-Induced Myokine That Protects the Heart From Ischemia-Reperfusion Injury.

RATIONALE: Physical exercise provides benefits for various organ systems, and some of systemic effects of exercise are mediated through modulation of muscle-derived secreted factors, also known as myokines. Myonectin/C1q (complement component 1q)/TNF (tumor necrosis factor)-related protein 15/erythroferrone is a myokine that is upregulated in skeletal muscle and blood by exercise. OBJECTIVE: We investigated the role of myonectin in myocardial ischemic injury. METHODS AND RESULTS: Ischemia-reperfusion in myonectin-knockout mice led to enhancement of myocardial infarct size, cardiac dysfunction, apoptosis, and proinflammatory gene expression compared with wild-type mice. Conversely, transgenic overexpression of myonectin in skeletal muscle reduced myocardial damage after ischemia-reperfusion. Treadmill exercise increased circulating myonectin levels in wild-type mice, and it reduced infarct size after ischemia-reperfusion in wild-type mice, but not in myonectin-knockout mice. Treatment of cultured cardiomyocytes with myonectin protein attenuated hypoxia/reoxygenation-induced apoptosis via S1P (sphingosine-1-phosphate)-dependent activation of cAMP/Akt cascades. Similarly, myonectin suppressed inflammatory response to lipopolysaccharide in cultured macrophages through the S1P/cAMP/Akt-dependent signaling pathway. Moreover, blockade of S1P-dependent pathway reversed myonectin-mediated reduction of myocardial infarct size in mice after ischemia-reperfusion. CONCLUSIONS: These data indicate that myonectin functions as an endurance exercise-induced myokine which ameliorates acute myocardial ischemic injury by suppressing apoptosis and inflammation in the heart, suggesting that myonectin mediates some of the beneficial actions of exercise on cardiovascular health.

BCL2 inhibitor ABT-199 and JNK inhibitor SP600125 exhibit synergistic cytotoxicity against imatinib-resistant Ph+ ALL cells.

Imatinib (IMT), a specific tyrosine kinase inhibitor (TKI), has drastically changed the treatment strategy for Ph+ ALL (Philadelphia chromosome-positive acute lymphoblastic leukemia). However, TKI resistance remains a serious problem for patient prognosis. Here, a Ph+ ALL cell line NphA2 and the IMT-resistant subline NphA2/STIR were analyzed to identify a potential novel treatment strategy. We also examined other Ph+ ALL cells, MR87 and its IMT-resistant subline, MR87/STIR. IMT induced apoptosis of NphA2 and MR87 but had no effect on resistant sublines. Increased phosphorylated ERK and BCL2, but not BCL-XL, were observed in NphA2/STIR compared with NphA2. NphA2/STIR but not NphA2 was moderately sensitive to U0126, an ERK inhibitor. Interestingly, SP600125, a JNK inhibitor, was potent in cell growth inhibition and apoptosis induction of both parental and IMT-resistant NphA2 and MR87 cells. Moreover, NphA2 and MR87 and their IMT-resistant sublines were sensitive to ABT-199, a specific BCL2 inhibitor. The combination of SP600125 and ABT-199 synergistically suppressed both parental and IMT-resistant cells, including one with T315I mutation, suggesting that Ph+ ALL exhibits high sensitivity to ABT-199 and SP600125 regardless of TKI resistance. This combination might be a possible therapeutic strategy for Ph+ ALL in the future.

Molecular hydrogen modulates gene expression via histone modification and induces the mitochondrial unfolded protein response.

Molecular hydrogen (H2) is a biologically active gas that is used medically to ameliorate various systemic pathological conditions. H2 also regulates gene expression involved in intracellular signaling and metabolic pathways. However, it is unclear whether H2 affects gene expression directly or through indirect effects as a consequence of health improvement. Therefore, we attempted to identify genes that exhibit similar changes in expression in response to H2 by employing DNA microarrays and gene set enrichment analysis to analyze RNA from liver and lung of rats and mice with or without dietary stress. We found that H2 activated the expression of sets of genes regulated by histone H3K27 methylation status. H2 also modified the expression of many genes regulated by a wide variety of signaling pathways. RT-qPCR showed that H2 up-regulated expression of Kcnc3, a H3K27-regulated gene, in organs such as liver, lung, kidney and brain. Furthermore, using immunohistochemistry and immunoblot analysis, we observed changes in H3K27 methylation status in the liver of mice and rats administered H2. Moreover, we showed that H2 simultaneously induced the H3K27 demethylase, Jmjd3, and mitochondrial unfolded protein response (mtUPR)-related genes. Recently, alteration of mitochondrial function was shown to cause induction of H3K27 demethylase or chromatin restructuring, followed by mtUPR activation through the alteration of H3K27 or H3K9 methylation states. Taken together, our study suggests that H2 can induce beneficial effects through mtUPR activation via epigenetic histone modification and by modification of gene expression.

Modulation of the sphingolipid rheostat is involved in paclitaxel resistance of the human prostate cancer cell line PC3-PR.

Taxoids are anti-cancer drugs frequently used to treat solid tumors, but they are sometimes ineffective and tumors may become resistant to their action. Here, we examined the involvement of sphingolipid metabolic enzymes in paclitaxel (PTX) resistance using a human prostate cancer cell line, PC3, and its PTX-resistant subline, PC3-PR. PTX (20 nM) suppressed cell proliferation and increased various ceramide species in PC3, but not PC3-PR, cells. PC3-PR contained higher S1P levels than did PC3, regardless of PTX treatment. Western blotting revealed that PC3-PR cells expressed higher levels of sphingosine kinase 1 (SPHK1) and glucosylceramide synthase (GCS) but lower levels of acid sphingomyelinase (ASMase) and neutral sphingomyelinase 2 than did PC3 cells. Inhibition of SPHK1 using siRNA or a pharmacological inhibitor decreased S1P levels in PC3-PR cells and inhibited proliferation in the presence or absence of PTX, suggesting that SPHK1 is at least partially responsible for PTX resistance. Similarly, GCS inhibitors (PDMP and PPMP) increased cellular ceramides and suppressed the proliferation of PC3-PR. However, inhibition of proteasome function or histone deacetylase activity increased SMase and ceramide levels and suppressed PC3-PR proliferation. These results suggest that modulation of metabolic enzyme expression and alteration of the sphingolipid rheostat protects cancer cells against PTX.

Mechanism of paclitaxel resistance in a human prostate cancer cell line, PC3-PR, and its sensitization by cabazitaxel.

Paclitaxel (PTX) is a microtubule-targeting drug widely used for the treatment of a variety of cancers. However, drug resistance can emerge after a series of treatments, and this can seriously affect the patient's prognosis. Here, we analyzed the mechanism of PTX resistance using a human prostate cancer cell line, PC3, and its PTX-resistant subline, PC3-PR. Compared with PC3, PC3-PR exhibited some unique phenotypes that might be associated with PTX resistance, including decreased expression of acetylated α-tubulin and the cell cycle regulator p21, and increased expression of ßIII tubulin, histone deacetylase 6 (HDAC6), and the anti-apoptotic protein Bcl2. The drug exporters MDR1 and MRP1 were not involved in PTX resistance. Although cabazitaxel (CTX), a novel taxoid, has been reported to overcome PTX resistance, its mechanism of action is unknown. We found that treatment of PC3-PR cells with CTX induced expression of acetylated α-tubulin and p21, but not the related regulators p27, p15, and p16 or the Bcl2 family proteins. The pan-HDAC inhibitors trichostatin A and suberanilohydroxamic acid and the HDAC6-specific inhibitor tubacin inhibited PC3-PR proliferation and increased expression of p21 and acetylated α-tubulin in a manner similar to CTX. Our data shed light on the cellular response to PTX and CTX.

Characterization of gene expression profiling of mouse tissues obtained during the postmortem interval.

Attempts to establish a tissue bank from autopsy samples have led to uncovering of the secrets of many diseases. Here, we examined the length of time that the RNA from postmortem tissues is available for microarray analysis and reported the gene expression profile for up- and down-regulated genes during the postmortem interval. We extracted RNA from fresh-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) brains and livers of three different groups of mice: 1) mice immediately after death, 2) mice that were stored at room temperature for 3h after death, and 3) mice that were stored at 4°C for 18h after death, as this storage resembles the human autopsy process in Japan. The RNA quality of the brain and the liver was maintained up to 18h during the postmortem interval. Based on the microarray analysis, we selected genes that were altered by >1.3-fold or <0.77-fold and classified these genes using hierarchical cluster analysis following DAVID gene ontology analysis. These studies revealed that cytoskeleton-related genes were enriched in the set of up-regulated genes, while serine protease inhibitors were enriched in the set of down-regulated genes. Interestingly, although the RNA quality was maintained due to high RNA integrity number (RIN) values, up-regulated genes were not validated by quantitative PCR, suggesting that these genes may become fragmented or modified by an unknown mechanism. Taken together, our findings suggest that under typical autopsy conditions, gene expression profiles that reflect disease pathology can be examined by understanding comprehensive recognition of postmortem fluctuation of gene expression.

Resveratrol-induced transcriptional up-regulation of ASMase (SMPD1) of human leukemia and cancer cells.

Resveratrol (RSV) is a plant-derived phytoalexin present in plants, whose pleiotropic effects for health benefits have been previously reported. Its anti-cancer activity is among the current topics for novel cancer treatment. Here, effects of RSV on cell proliferation and the sphingolipid metabolism of K562, a human leukemia cell line, were analyzed. Some experiments were also performed in HCT116, a human colon cancer cell line. RSV inhibited cell proliferation of both cell lines. Increased cellular ceramide and decreased sphingomyelin and S1P by RSV were observed in RSV-treated K562 cells. Further analysis revealed that acid sphingomyelinase mRNA and enzyme activity levels were increased by RSV. Desipramine, a functional ASMase inhibitor, prevented RSV-induced ceramide increase. RSV increased ATF3, EGR1, EGR3 proteins and phosphorylated c-Jun and FOXO3. However, co-transfection using these transcription factor expression vectors and ASMase promoter reporter vector revealed positive effects of EGR1 and EGR3 but not others. Electrophoresis mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay demonstrated the direct binding of EGR1/3 transcription factors with ASMase 5'-promoter. These results indicate that increased EGR1/3 and ASMase expression play an important role in cellular ceramide increase by RSV treatment.

C1q/TNF-related protein-1 functions to protect against acute ischemic injury in the heart.

Obesity is associated with an increased risk of cardiovascular disease. C1q/TNF-related protein (CTRP)-1 is a poorly characterized adipokine that is up-regulated in association with ischemic heart disease. We investigated the role of CTRP1 in myocardial ischemia injury. CTRP1-knockout mice showed increased myocardial infarct size, cardiomyocyte apoptosis, and proinflammatory gene expression after I/R compared with wild-type (WT) mice. In contrast, systemic delivery of CTRP1 attenuated myocardial damage after I/R in WT mice. Treatment of cardiomyocytes with CTRP1 led to reduction of hypoxia-reoxygenation-induced apoptosis and lipopolysaccharide-stimulated expression of proinflammatory cytokines, which was reversed by inhibition of sphingosine-1-phosphate (S1P) signaling. Treatment of cardiomyocytes with CTRP1 also resulted in the increased production of cAMP, which was blocked by suppression of S1P signaling. The antiapoptotic and anti-inflammatory actions of CTRP1 were cancelled by inhibition of adenylyl cyclase or knockdown of adiponectin receptor 1. Furthermore, blockade of S1P signaling reversed CTRP1-mediated inhibition of myocardial infarct size, apoptosis, and inflammation after I/R in vivo. These data indicate that CTRP1 protects against myocardial ischemic injury by reducing apoptosis and inflammatory response through activation of the S1P/cAMP signaling pathways in cardiomyocytes, suggesting that CTRP1 plays a crucial role in the pathogenesis of ischemic heart disease.

Targeting ceramide synthase 6-dependent metastasis-prone phenotype in lung cancer cells.

Sphingolipids make up a family of molecules associated with an array of biological functions, including cell death and migration. Sphingolipids are often altered in cancer, though how these alterations lead to tumor formation and progression is largely unknown. Here, we analyzed non-small-cell lung cancer (NSCLC) specimens and cell lines and determined that ceramide synthase 6 (CERS6) is markedly overexpressed compared with controls. Elevated CERS6 expression was due in part to reduction of microRNA-101 (miR-101) and was associated with increased invasion and poor prognosis. CERS6 knockdown in NSCLC cells altered the ceramide profile, resulting in decreased cell migration and invasion in vitro, and decreased the frequency of RAC1-positive lamellipodia formation while CERS6 overexpression promoted it. In murine models, CERS6 knockdown in transplanted NSCLC cells attenuated lung metastasis. Furthermore, combined treatment with l-α-dimyristoylphosphatidylcholine liposome and the glucosylceramide synthase inhibitor D-PDMP induced cell death in association with ceramide accumulation and promoted cancer cell apoptosis and tumor regression in murine models. Together, these results indicate that CERS6-dependent ceramide synthesis and maintenance of ceramide in the cellular membrane are essential for lamellipodia formation and metastasis. Moreover, these results suggest that targeting this homeostasis has potential as a therapeutic strategy for CERS6-overexpressing NSCLC.

Hypoxia remodels the composition of the constituent ceramide species of HexCer and Hex2Cer with phytosphingosine and hydroxy fatty acids in human colon cancer LS174T cells.

Oxygen-requiring enzymes, such as Δ4-desaturase (dihydroceramide desaturase), sphingolipid Δ4-desaturase/C-4-hydroxylase, and fatty acid 2-hydroxylase are involved in ceramide synthesis. We prepared free ceramides, sphingomyelins and glycosphingolipids (GSLs) from cancer cells cultivated under conditions of normoxia and hypoxia, and analyzed these compounds using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Human colon cancer LS174T cells were employed because these cells highly express hydroxyl fatty acids and phytosphingosine (t18:0) which are expected to be greatly influenced by changes in oxygen levels. As expected, the populations of dihydro-species of free ceramide and sphingomyelin with C16:0 non-hydroxy fatty acid were elevated, and the populations of HexCers and Hex2Cers, composed of C16:0 or C16:0 hydroxy fatty acid (C16:0h), and sphingosine (d18:1) or t18:0, were decreased under hypoxia. However, appreciable populations of HexCer and Hex2Cer species of C24:0 or C24:0h and t18:0 remained. These results suggest that the individual species of GSLs with fatty acids possessing different alkyl chain lengths, either non-hydroxy fatty acids or hydroxyl fatty acids, may be metabolized individually.

Increased acid ceramidase expression depends on upregulation of androgen-dependent deubiquitinases, USP2, in a human prostate cancer cell line, LNCaP.

Acid ceramidase (ACDase) metabolizes ceramide to sphingosine, leading to sphingosine 1-phosphate production. Reportedly, ACDase has been upregulated in prostate cancer. However, its regulatory mechanism remains unclear. LNCaP (androgen-sensitive prostate cancer cell line) but not PC3 and DU-145, (androgen-unresponsive cell lines) exhibited the highest ACDase protein. Among three cell lines, ASAH1 mRNA level was not correlated with ACDase protein expression, and the 5'-promoter activity did not show androgen dependency, suggesting the post-transcriptional regulation of ACDase in LNCaP cells. Based on these results, LNCaP was analysed further. Casodex, androgen receptor antagonist, and charcoal-stripped FCS (CS-FCS) decreased ACDase protein and activity, whereas dihydrotestosterone in CS-FCS culture increased ACDase protein and enzyme activity. MG132, a proteasome inhibitor, prevented the decrease of ACDase protein when cultured in CS-FCS, suggesting the involvement of ubiquitin/proteasome system. Reportedly, USP2, a deubiquitinase, plays an important role in LNCaP cells. USP2 siRNA decreased ACDase protein, whereas USP2 overexpression increased ACDase protein of LNCaP cells. However, SKP2, an ubiquitin E3 ligase known to be active in prostate cancer, did not affect androgen-dependent ACDase expression in LNCaP cells. Thus, ACDase regulation by androgen in androgen-sensitive LNCaP cells is mainly due to its prolonged protein half-life by androgen-stimulated USP2 expression.

Increased SPHK2 Transcription of Human Colon Cancer Cells in Serum-Depleted Culture: The Involvement of CREB Transcription Factor.

Sphingosine kinases (SPHK) are important to determine cells' fate by producing sphingosine 1-phosphate. Reportedly, exogenous SPHK2 overexpression induces cell cycle arrest or cell death. However, the regulatory mechanism of SPHK2 expression has not been fully elucidated. Here, we analyzed this issue using human colon cancer cell lines under various stress conditions. Serum depletion (FCS(-)) but not hypoxia and glucose depletion increased mRNA, protein and enzyme activity of SPHK2 but not SPHK1. In HCT116 cells mostly used, SPHK2 activity was predominant over SPHK1, and serum depletion increased both nuclear and cytoplasmic SPHK2 activity. Based on previous reports analyzing cellular response after serum depletion, the temporal changes of intracellular signaling molecules and candidate transcription factors for SPHK2 were examined using serum-depleted HCT116 cells, and performed transfection experiments with siRNA or cDNA of candidate transcription factors. Results showed that the rapid and transient JNK activation followed by CREB activation was the major regulator of increased SPHK2 transcription in FCS(-) culture. EMSA and ChIP assay confirmed the direct binding of activated CREB to the CREB binding site of 5' SPHK2 promoter region. Colon cancer cells examined continued to grow in FCS(-) culture, although mildly, while hypoxia and glucose depletion suppressed cell proliferation or induced cell death, suggesting the different role of SPHK2 in different stress conditions. Because of the unique relationship observed after serum depletion, we examined effects of siRNA for SPHK2, and found the role of SPHK2 as a growth or survival factor but not a cell proliferation inhibitor in FCS(-) culture.