Synergistic effects of vemurafenib and fingolimod (FTY720) in vemurafenib­resistant melanoma cell lines.

Vemurafenib, a selective inhibitor of mutated BRAF, is used to treat late­stage melanoma. However, resistance to vemurafenib is urgently required as it can have fatal consequences. Fingolimod (FTY720), a sphingosine­1­phosphate receptor modulator, has been used for the treatment of several malignant neoplasms in clinical trials. The present study investigated the effects of FTY720 and vemurafenib combination treatment on cell death induction, and defined the molecular mechanisms in vemurafenib­resistant melanoma cells. The combination treatment with FTY720 and vemurafenib reduced cell viability, and the expression of apoptosis­associated cleaved poly (adenosine diphosphate­ribose) polymerase (PARP) was increased when compared with treatment with vemurafenib alone in WM­115 cells, a vemurafenib­resistant human melanoma cell line. In addition, the protein expression of phosphorylated extracellular signal­related kinase (ERK) in WM­115 cells was decreased by this combination treatment. Vemurafenib­resistant SK­Mel­28 cells (R­SK­Mel) were established by culturing SK­Mel­28 cells, which are the most sensitive to vemurafenib, in the presence of vemurafenib. Similar to WM­155 cells, the viability of R­SK­Mel cells was reduced and the expression of cleaved PARP was increased by the combination treatment with FTY720 and vemurafenib. In addition, the expression of phosphorylated ERK and Akt was also reduced by this treatment. These results suggested that FTY720 and vemurafenib synergistically induced cell death by downregulating proliferation and survival signalling pathways in vemurafenib­resistant melanoma cells.

Phospholipase D1-regulated autophagy supplies free fatty acids to counter nutrient stress in cancer cells.

Cancer cells utilize flexible metabolic programs to maintain viability and proliferation under stress conditions including nutrient deprivation. Here we report that phospholipase D1 (PLD1) participates in the regulation of metabolic plasticity in cancer cells. PLD1 activity is required for cancer cell survival during prolonged glucose deprivation. Blocking PLD1 sensitizes cancer cells to glycolysis inhibition by 2-deoxy-D-glucose (2-DG) and results in decreased autophagic flux, enlarged lysosomes, and increased lysosomal pH. Mechanistically, PLD1-regulated autophagy hydrolyzes bulk membrane phospholipids to supply fatty acids (FAs) for oxidation in mitochondria. In low glucose cultures, the blockade of fatty acid oxidation (FAO) by PLD1 inhibition suppresses adenosine triphosphate (ATP) production and increases reactive oxygen species (ROS), leading to cancer cell death. In summary, our findings reveal a novel role of PLD1 in sustaining cancer cell survival during metabolic stress, and suggest PLD1 as a potential target for anticancer metabolism therapy.

FTY720 and cisplatin synergistically induce the death of cisplatin-resistant melanoma cells through the downregulation of the PI3K pathway and the decrease in epidermal growth factor receptor expression.

Sphingosine kinase (SK), a key enzyme in sphingosine-1-phosphate (S1P) synthesis, is known to be overexpressed in various types of cancer cells. The effects of anticancer agents on SK1/S1P signaling have not yet been fully assessed in melanoma cells. In the present study, we investigated the effects of the combination of FTY720, an S1P receptor antagonist, and cisplatin, a DNA-damaging agent, on the induction of the death of human melanoma cells, as well as the molecular mechanisms involved. The viability of various human melanoma cell lines was examined following treatment with anticancer drugs. The cisplatin-resistant SK-Mel-28 and cisplatin-sensitive A375 cell lines were selected for this analysis. Protein expression and apoptotic rates were evaluated by western blot analysis following treatment with cisplatin and/or FTY720. Following treatment with a combination of FTY720 and cisplatin, cell viability significantly decreased and the expression of apoptosis-associated cleaved poly(ADP-ribose) polymerase (PARP) was significantly higher in comparison to treatment with cisplatin alone in the SK-Mel-28 cells. In addition, the combination of FTY720 and cisplatin reduced the protein expression of SK1 and the phosphorylation levels of phosphoinositide 3-kinase (PI3K), Akt and mTOR in the SK-Mel-28 cells; the expression of epidermal growth factor receptor (EGFR) was also markedly reduced. These findings suggest that FTY720 and cisplatin synergistically induce cell death through the downregulation of the PI3K/Akt/mTOR pathway and the decrease in EGFR expression in SK-Mel-28 cells. Thus, the combination of FTY720 and cisplatin may have therapeutic potential for chemotherapy-resistant melanoma, and the effects are likely exerted through the downregulation of S1P signaling.

The importance of interaction with membrane lipids through the pleckstrin homology domain of the guanine nucleotide exchange factor for rho family small guanosine triphosphatase, FLJ00018.

FLJ00018, a heterotrimeric guanosine 5'-triphosphate (GTP)-binding protein (G protein) Gßγ subunit-activated guanine nucleotide exchange factor for Rho family small GTPases, regulates cellular responses, including cell morphological changes and gene transcriptional regulation, and targets the cellular membranes. FLJ00018 contains a Dbl homology (DH) domain in addition to a pleckstrin homology (PH) domain. Here we show that the PH domain of FLJ00018 is required for FLJ00018-induced, serum response element-dependent gene transcription. Although the PH domain of KIAA1415/P-Rex1, another Gßγ subunit-activated guanine nucleotide exchange factor for Rho family small GTPases, binds to phosphatidylinositol 3,4,5-triphosphate and phosphatidylinositol 3,4-bisphosphate, the PH domain of FLJ00018 binds to polyphosphoinositides including phosphatidylinositol 4,5-bisphosphate, and phosphatidic acid. These results suggest that FLJ00018 is targeted via its PH domain to cellular membranes.

Sphingosine kinase 1 plays a role in the upregulation of CD44 expression through extracellular signal-regulated kinase signaling in human colon cancer cells.

Our previous study has shown that the activity and expression of sphingosine kinase (SPHK) regulated the sensitivity of human colon cancer cells to the chemotherapeutic oxaliplatin (L-OHP). In addition, the cancer stem cell marker CD44 increases cell resistance to anticancer drugs. Here, we use colon cancer cell lines to examine the relationship between SPHK1 activity and CD44 expression.CD44 expression was measured by western blotting and quantitative PCR in two human colon cancer cell lines: L-OHP-resistant RKO and L-OHP-sensitive HCT116. The regulation of CD44 by SPHK1 was examined by either blocking or overexpressing SPHK1 and by using an L-OHP-resistant HCT116 clone (HCT116-R).The levels of SPHK1, CD44, phosphorylated-Akt, and phosphorylated-extracellular signal-regulated kinase (ERK) were much higher in the RKO cells than in the HCT116 cells. The treatment of RKO cells with the SPHK inhibitor or SPHK1 silencing by RNA interference suppressed CD44 protein expression. SPHK1 and CD44 levels were much higher in HCT116-R cells compared with the parental HCT116 cells. Transfection of HCT116 cells with SPHK1 cDNA enhanced the expression of both CD44 and phosphorylated-ERK. The increase in the CD44 protein level was abolished by the inhibition of ERK phosphorylation. Treatment of RKO cells with the sphingosine-1-phosphate (S1P)2 receptor antagonist suppressed ERK phosphorylation and the expression of CD44 mRNA and protein. Exogenous stimulation with S1P increased ERK phosphorylation and CD44 protein expression in HCT116 cells, but treatment with an MEK inhibitor and S1P2 receptor antagonist blocked this effect.These findings indicate that SPHK1 and its product, S1P, contribute toward the regulation of CD44 protein expression through the ERK signaling pathway through S1P2 in human colon cancer cells.

Transcriptional regulation of neutral sphingomyelinase 2 in all-trans retinoic acid-treated human breast cancer cell line, MCF-7.

Effects of all-trans retinoic acid (ATRA) on sphingomyelinase expression were examined using MCF-7 (ATRA-sensitive) and MDA-MB-231 (ATRA-resistant) breast cancer cells. Increased NSMase activity, NSMase2 mRNA and protein were observed in ATRA-treated MCF-7 but not in ATRA-treated MDA-MB-231. Increased NSMase2 mRNA of ATRA-treated MCF-7 was mostly due to enhanced transcription. Promoter analysis revealed the important 5'-promoter region of NSMase2 between -148 and -42 bp containing three Sp1 sites but no retinoic acid responsive elements. Experiments using mutated Sp1 sites of the NSMase2 promoter, Mithramycin A (a Sp inhibitor) and Sp family over-expression demonstrated the importance of Sp family protein and the three Sp1 sites for ATRA-induced NSMase2 transcription of MCF-7 cells. Although no quantitative change of bound Sp1 on NSMase2 promoter region after ATRA treatment was detected, Sp1 phosphorylation (activation) by ATRA was observed. Interestingly, PKCδ was involved in ATRA-induced increased NSMase2 transcription. ATRA-induced PKCδ phosphorylation and then activated PKCδ phosphorylated Sp1. Chromatin immunoprecipitation (ChIP) assay showed Sp1, RARα and RXRα complex formation in MCF-7 cells regardless of ATRA treatment and ATRA-induced acetylated histone H3 of the 5'-promoter. Thus, NSMase2 mRNA expression enhanced by ATRA was due to increased transcription via phosphorylated Sp1 caused by PKCδ activation, followed by chromatin remodelling with histone H3 acetylation.

Role of down-regulated neutral ceramidase during all-trans retinoic acid-induced neuronal differentiation in SH-SY5Y neuroblastoma cells.

Neutral ceramidase (NCDase) is considered to be a critical enzyme for controlling the turnover of ceramide, an important bioactive lipid, which determines cell's fate. All-trans retinoic acid (ATRA) has been reported to induce neuronal differentiation and cell-cycle arrest [Lopez-Carballo, Moreno, Masia, Perez, and Barettino (Activation of the phosphatidylinositol 3-kinase/Akt signalling pathway by retinoic acid is required for neural differentiation of SH-SY5Y human neuroblastoma cells. J Biol Chem 2002:277:25297-304.)]. In this study, we observed that ATRA-induced cellular ceramide accumulation, cell-growth arrest and differentiation accompanied with down-regulation of NCDase in SH-SY5Y cells, without a decrease in sphingosine or sphingosine 1-phosphate. We examined whether the down-regulation of NCDase was involved in the increase in ceramide and cell differentiation. ATRA was found to down-regulate mRNA, protein and the enzyme activity of NCDase. Interestingly, GATA-2 was also decreased with ATRA treatment, and experiments using its expression vector and siRNA and chromatin immunoprecipitation assay demonstrated GATA-2 acted as transcription-factor of NCDase gene expression. By establishing stable transfectants with decreased NCDase expression and activity, we clarified the significance of NCDase down-regulation for ATRA-induced neuronal differentiation. Those sub-clones showed both increased cellular ceramide and reduced cell growth as well as neuronal differentiation phenotypes. These results demonstrate that down-regulation of NCDase through ATRA-induced GATA-2 decrease plays an important role in induction of ceramide accumulation and neuronal differentiation in SH-SY5Y cells.

The pivotal role of intracellular calcium in oxaliplatin-induced inhibition of neurite outgrowth but not cell death in differentiated PC12 cells.

The antineoplastic efficacy of oxaliplatin, a widely used anticancer drug, is restricted by its adverse effects such as peripheral neuropathy. Infusing a combination of calcium gluconate and magnesium sulfate (Ca/Mg) suppresses the acute neurotoxic side effects of oxaliplatin, although the mechanism is unclear. To elucidate the molecular mechanisms of oxaliplatin-induced neurotoxicity and the effects of Ca/Mg against this toxicity, we examined the effect of Ca/Mg on oxaliplatin-induced inhibition of neurite outgrowth in PC12 cells, a commonly used neuronal cell model. Oxaliplatin and oxalate suppressed nerve growth factor (NGF)-induced neurite outgrowth and reduced the NGF-mediated increase in the intracellular calcium concentration [Ca(2+)](i). A calcium-chelating agent, BAPTA/AM, also exhibited similar inhibitory effects on neurite outgrowth and [Ca(2+)](i). The addition of Ca/Mg attenuated these inhibitions induced by oxaliplatin and oxalate. The NGF-induced upregulation of growth-associated protein-43 (GAP-43) was suppressed by oxaliplatin and oxalate. Oxaliplatin, but not oxalate, suppressed NGF-stimulated extracellular signal-regulated kinase activation, and this inhibition was not affected by Ca/Mg. Ca/Mg did not modify the oxaliplatin-induced loss of cell viability or apoptosis in PC12 or HCT-116 cells, a human colorectal cancer cell line. These results suggest that the inhibition of neurite outgrowth but not tumor cell death induced by oxaliplatin is partly associated with reductions in [Ca(2+)](i) and GAP-43 expression, and this inhibition was suppressed by the addition of Ca/Mg. Therefore, it may be assumed that Ca/Mg is useful for protecting against oxaliplatin-induced neurotoxicity without reducing the antitumor activity of oxaliplatin.

Gs and Gq signalings regulate hPEM-2-induced cell responses in Neuro-2a cells.

Rho family GTPase-specific guanine nucleotide exchange factors of the Dbl family regulate a variety of cellular events including cytoskeletal arrangement, signal transduction, and gene expression through activation of Rho family GTPases. In this study, we show that hPEM-2 is a downstream effector of G(s) and G(q) signaling in Neuro-2a neuroblastoma cells. Co-expression with hPEM-2 and GTPase-deficient (constitutively active) mutants of Gαs (Gα(s)Q213L) or Gα(q) (Gα(q)Q209L), but not other GTPase-deficient mutants of Gα subunit and Gßγ subunits, activated serum response element (SRE)-dependent gene transcription, which is known to be induced by Rho family activation. Although a dominant negative mutant of Rac1 strongly blocks Gα(s)Q213L or Gα(q)Q209L/hPEM-2 activated SRE-dependent gene transcription, those of Cdc42 or RhoA are marginally affected. A PKA inhibitor, H-89, attenuated Gα(s)/hPEM-2-activated SRE-dependent gene transcription. And a dominant negative mutant of c-Src and an Src inhibitor attenuated Gα(q)Q209L/hPEM-2-activated SRE-dependent gene transcription. Experiments using hPEM-2 deletion mutants indicate that some regions of hPEM-2 play an important role in enhancing SRE activation by G(s) and G(q) signalings. These results reveal that G(s) and G(q) signalings regulate hPEM-2 functions through PKA and c-Src in Neuro-2a neuroblastoma cells, respectively.

Sphingosine kinase 1/S1P pathway involvement in the GDNF-induced GAP43 transcription.

Glial cell line-derived neurotrophic factor (GDNF) is important for the development and maintenance of dopamine neurons (Lin et al. [1993] Science 260: 1130-1132). GDNF is neuroprotective in animal models of Parkinson disease, where dopamine neurons show selective degeneration. We previously reported GDNF-induced SPHK1 gene expression in a neuroblastoma cell line, TGW (Murakami et al. [2007] J Neurochem 102: 1585-1594). In the present study, we focused on the regulatory mechanism of GAP43 (GDNF-induced neuronal phenotype) transcription to further elucidate physiological roles of GDNF-induced SPHK1 expression and activity. Stable wild-type (SPHK1-WT) but not dominant-negative SPHK1 (SPHK1-DN) overexpression increased both control- and GDNF-induced GAP43 expression. SPHK1-WT cells showed enhanced GDNF-induced sphingosine 1-phosphate (S1P) secretion compared with mock- and SPHK1-DN cells. Exogenous S1P also increased GAP43 expression. In TGW cells, PD98059, a MEK inhibitor, but not SB203580 (a p38 MAPK inhibitor) and LY294002 (a PI3K inhibitor) inhibited GDNF-induced GAP43 expression, suggesting the MEK/ERK pathway has a major role in GDNF-induced GAP43 transcription. A G-protein-coupled receptor inhibitor, pertussis toxin, and S1P(1) and S1P(3) receptor antagonists (VPC23019 and CAY10444) also inhibited ERK activation. Moreover, both S1P1 and S1P3 were serine-phosphorylated by GDNF, suggesting their activated states. C/EBPß transcription factor was induced by GDNF, and DNA pull-down and chromatin immunoprecipitation assays revealed the C/EBP binding site between -131 bp and -98 bp from the first exon of GAP43. Taken together, our results showed that in TGW cells, GDNF increased SPHK1 transcription, leading to the production and secretion of S1P. Through MEK/ERK pathway, S1P stimulates GAP43 transcription with increased binding of C/EBPß to the 5'-promoter.

Involvement of the aldo-keto reductase, AKR1B10, in mitomycin-c resistance through reactive oxygen species-dependent mechanisms.

The human aldo-keto reductase (AKR) 1B10 is suggested as a tumor marker in various solid tumors. Using colon cancer cells, we found that AKR1B10 was induced with acquisition of resistance to the anticancer drug mitomycin-c (MMC). In the resistant cells, treatment with an AKR1B10 inhibitor decreased their MMC tolerance. In the nonresistant cells, overexpression and silencing of AKR1B10 decreased and increased, respectively, susceptibility to cytotoxic effects of MMC and 4-hydroxy-2-nonenal, which was formed as a product of lipid peroxidation by MMC treatment. These results suggest a role of AKR1B10 in the development of MMC resistance, which may be mediated by its ability to detoxify cytotoxic aldehydes including 4-hydroxy-2-nonenal.

Heterogeneous sphingosine-1-phosphate lyase gene expression and its regulatory mechanism in human lung cancer cell lines.

The role of sphingolipid metabolic pathway has been recognized in determining cellular fate. Although sphingolipid degradation has been extensively studied, gene expression of human sphingosine 1-phosphate lyase (SPL) catalyzing sphingosine 1-phosphate (S1P) remains to be determined. Among 5 human lung cancer cell lines examined, SPL protein levels paralleled the respective mRNA and enzyme activities. Between H1155 and H1299 cells used for further experiments, higher cellular S1P was observed in H1155 with higher SPL activity compared with H1299 with low SPL activity. GATA-4 has been reported to affect SPL transcription in Dictyostelium discoideum. GATA-4 was observed in H1155 but not in other cell lines. Overexpression of GATA-4 in H1299 increased SPL expression. However, promoter analysis of human SPL revealed that the most important region was located between -136bp and -88bp from the first exon, where 2 Sp1 sites exist but no GATA site. DNA pull-down assay of H1155 showed increased DNA binding of Sp1 and GATA-4 within this promoter region compared with H1299. Electrophoresis mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) assay, reporter assay using mutated binding motif, and mithramycin A, a specific Sp1 inhibitor, suggest the major role of Sp1 in SPL transcription and no direct binding of GATA-4 with this 5' promoter region. The collaborative role of GATA-4 was proved by showing coimmunoprecipitation of Sp1 and GATA-4 using GST-Sp1 and overexpressed GATA-4. Thus, high SPL transcription of H1155 cells was regulated by Sp1 and GATA-4/Sp1 complex formation, both of which bind to Sp1 sites of the 5'-SPL promoter.

Acid sphingomyelinase regulates glucose and lipid metabolism in hepatocytes through AKT activation and AMP-activated protein kinase suppression.

Acid sphingomyelinase (ASM) regulates the homeostasis of sphingolipids, including ceramides and sphingosine-1-phosphate (S1P). Because sphingolipids regulate AKT activation, we investigated the role of ASM in hepatic glucose and lipid metabolism. Initially, we overexpressed ASM in the livers of wild-type and diabetic db/db mice by adenovirus vector (Ad5ASM). In these mice, glucose tolerance was improved, and glycogen and lipid accumulation in the liver were increased. Using primary cultured hepatocytes, we confirmed that ASM increased glucose uptake, glycogen deposition, and lipid accumulation through activation of AKT and glycogen synthase kinase-3ß. In addition, ASM induced up-regulation of glucose transporter 2 accompanied by suppression of AMP-activated protein kinase (AMPK) phosphorylation. Loss of sphingosine kinase-1 (SphK1) diminished ASM-mediated AKT phosphorylation, but exogenous S1P induced AKT activation in hepatocytes. In contrast, SphK1 deficiency did not affect AMPK activation. These results suggest that the SphK/S1P pathway is required for ASM-mediated AKT activation but not for AMPK inactivation. Finally, we found that treatment with high-dose glucose increased glycogen deposition and lipid accumulation in wild-type hepatocytes but not in ASM(-/-) cells. This result is consistent with glucose intolerance in ASM(-/-) mice. In conclusion, ASM modulates AKT activation and AMPK inactivation, thus regulating glucose and lipid metabolism in the liver.

Mechanism of increased PLD1 gene expression during early adipocyte differentiation process of mouse cell line 3T3-L1.

A mouse cell line 3T3-L1 is differentiated into adipocytes when treated with an inducer cocktail (IDX) (insulin, dexametahsone, and a cAMP phosphodiesterase inhibitor of isobutyl-methylxanthine (IBMX)). Here, we report that PLD1, but not PLD2, mRNA and protein increased during the early differentiation process. Our analysis shows that IDX resulted in a sequential induction of C/EBPbeta, PLD1, and C/EBPalpha which is a key transcription factor of late adipocyte differentiation. Among the three inducers, IBMX + any other inducer induced mild adipocyte differentiation, whereas insulin + dexamethasone did not. IBMX increased PLD1 but not PLD2 mRNA. Forskolin, an adenylate cyclase activator, and dbcAMP also increased PLD1 mRNA, suggesting the cellular cAMP as the inducer of both adipocyte differentiation and PLD1 transcription. We focused on the regulatory mechanism of PLD1 transcription during this differentiation process. IDX or a combination of inducers including IBMX increased PLD1 promoter activity, which is consistent with mRNA analysis. Promoter analysis identified two adjacent C/EBP motifs located between -338 and -231 bp from the first exon as the IBMX responsive elements. Furthermore, overexpression of C/EBPbeta, but not C/EBPalpha, increased PLD1 mRNA and PLD1 5' promoter activity. EMSA and chromatin immunoprecipitation assay confirmed the direct binding of C/EBPbeta, but not C/EBPalpha, to these C/EBP motifs of PLD1 5' promoter. Our results show that PLD1 is a target gene of C/EBPbeta through the increased cellular cAMP during early adipocyte differentiation of 3T3-L1 cells.

ATRA inhibits ceramide kinase transcription in a human neuroblastoma cell line, SH-SY5Y cells: the role of COUP-TFI.

Ceramide is the central lipid in the sphingolipid metabolism. Ceramide kinase (CERK) and its product, ceramide 1-phosphate, have been implicated in various cellular functions. However, the regulatory mechanism of CERK gene expression remains to be determined. Here, we examined CERK mRNA level during all-trans retinoic acid (ATRA)-induced differentiation of a human neuroblastoma cell line, SH-SY5Y. ATRA reduced CERK mRNA and protein levels. Over-expression and small interfering RNA (siRNA) of CERK revealed that CERK is inhibitory against ATRA-induced neuronal differentiation and cell growth arrest. ATRA inhibited the transcriptional activity of 5'-promoter of CERK. Truncation and mutation study suggests that ATRA-responsible region was mainly located in the tandem retinoic acid responsive elements (RARE) between -40 bp and the first exon. The electrophoresis mobility shift assay revealed that ATRA produced two retarded bands, which were erased by antibody against chicken ovalbumin upstream promoter transcription factor I (COUP-TFI), RARalpha, and RXRalpha, respectively. DNA pull-down assay confirmed increased binding of these transcription factors to RARE. Transient expression of RAR, RXR, and COUP-TFI and siRNA transfection of these genes revealed that COUP-TFI inhibited CERK mRNA. Furthermore, chromatin immunoprecipitation assay showed the recruitment of co-repressors as well as three transcription factors. These results suggest that COUP-TFI was the ATRA-responsive suppressive transcription factor of CERK gene transcription.

Mutated ras induced PLD1 gene expression through increased Sp1 transcription factor.

The underlying mechanisms of oncogene-induced phospholipase D (PLD) activation have not been fully elucidated. The effect of the mutated-ras on PLD mRNA was examined using colon cancer cell lines as well as mock- and mutated ras-transfected NIH3T3 cells. Ras-mutation and activation were correlated, and cells with enhanced ras-activation showed increased PLD1 mRNA and protein. Analysis of the 5' PLD1 promoter using a representative cell line, DLD-1 and also mutated ras-NIH3T3, showed one Sp1-site as the important ras-responsible motif. Spl inhibition with mithramycin A and Spl siRNA inhibited PLD1 protein expression and its promoter activity. Sp1 but not Sp3 protein level and increased Sp1-motif binding activity were correlated with ras activation. Furthermore, overexpression of Sp1 in drosophila SL2 cells lacking Sp family proteins increased PLD1 promoter activity. EMSA and chromatin immunoprecipitation assay confirmed the importance of Sp1 protein binding to the Sp1-motif in ras-induced PLD1 mRNA expression.

Transcriptional regulation of neutral sphingomyelinase 2 gene expression of a human breast cancer cell line, MCF-7, induced by the anti-cancer drug, daunorubicin.

Mg(2+)-dependent neutral SMases (NSMases) have emerged as prime candidates for stress-induced ceramide production. Among isoforms identified, previous reports have suggested the importance of NSMase2. However, its activation mechanism has not been precisely reported. Here, we analyzed the mechanism of NSMase2 gene expression by the anti-cancer drug, daunorubicin (DA). DA increased cellular ceramides (C16, C18 and C24) and NSMase activity of a human breast cancer cell line, MCF-7. DA remarkably increased the NSMase2 message and protein, whereas little change in NSMase1 and NSMase3 mRNAs and only a mild increase in acid SMase mRNA were observed. Overexpression and a knock down of NSMase2 indicated that NSMase2 played a role in DA-induced cell death. NSMase2 promoter analysis revealed that three Sp1 motifs located between -148 and -42bp upstream of the first exon were important in basic as well as in DA-induced promoter activity. Consistently, luciferase vectors containing three consensus Sp1-motifs but not its mutated form showed DA-induced transcriptional activation. DA-treated MCF-7 showed increased Sp3 protein. In SL2 cells lacking Sp family proteins, both Sp1 and Sp3 overexpression increased NSMase promoter activity. Increased binding of Sp family proteins by DA to three Sp1 motifs was shown by electrophoresis mobility shift and ChIP assays.

Sphingosine kinase isoforms regulate oxaliplatin sensitivity of human colon cancer cells through ceramide accumulation and Akt activation.

The relationship between sphingosine kinase (SPHK), cellular ceramide concentration and chemosensitivity was investigated in human colon cancer cell lines. Among nine colon cancer cell lines, SPHK1 and SPHK2 activity and protein expression was highest in RKO cells and lowest in HCT116 cells. A viability assay revealed that HCT116 cells were sensitive to the effects of oxaliplatin (l-OHP), whereas RKO cells were resistant to those of l-OHP. Treatment with 5microg/ml l-OHP induced a marked time-dependent increase in various ceramides (C16, C24, C24:1) in HCT116 cells but not in RKO cells, as indicated by liquid chromatography/mass spectrometry. The increase in ceramide and caspase activation induced by l-OHP in the sensitive HCT116 cells was abolished by pretreatment with a neutral sphingomyelinase inhibitor, suggesting that the ceramide formation was due to the activation of neutral, rather than acid, sphingomyelinase. In contrast, in l-OHP-resistant RKO cells, treatment with an SPHK inhibitor or SPHK1 and SPHK2 silencing by RNA interference suppressed cell viability and increased caspase activity and cellular ceramide formation after l-OHP treatment. The elevated ceramide formation induced by SPHK inhibition and l-OHP was inhibited by fumonisin B1 but not myriocin, suggesting that ceramide formation was through the salvage pathway. Endogenous phosphorylated Akt levels were much higher in the resistant RKO cells than in the sensitive HCT116 cells. Either SPHK1 or SPHK2 silencing in RKO cells decreased phosphorylated Akt levels and increased p53 and p21 protein levels as well as poly(ADP-ribose) polymerase cleavage in response to l-OHP treatment. These findings indicate that SPHK isoforms and neutral sphingomyelinase contribute to the regulation of chemosensitivity by controlling ceramide formation and the downstream Akt pathway in human colon cancer cells.

Nicorandil, an adenosine triphosphate-sensitive potassium channel opener, inhibits muscarinic acetylcholine receptor-mediated activation of extracellular signal-regulated kinases in PC12 cells.

BACKGROUND: Nicorandil, an adenosine triphosphate-sensitive potassium channel opener, is reported to have an antinociceptive effect by hyperpolarization through the K(+) channel. The activation of extracellular signal-regulated kinase (ERK), a family of mitogen-activated protein kinases, plays an important role in synaptic plasticity and noxious stimulation in the dorsal root ganglion, and spinal neurons have been reported to induce its activation. To understand the biological mechanisms of nicorandil, we examined the effects of nicorandil on muscarinic acetylcholine (ACh) receptor-mediated activation of ERK in a neuronal model cell, rat pheochromocytoma PC12 cells. METHODS: PC12 cells were stimulated with ACh in the presence or absence of nicorandil, and phosphorylation of ERK was examined by a Western blot analysis. We also examined the effects of nicorandil on the ERK activation induced by 4beta-phorbol 12-myristate 13-acetate, an activator of protein kinase C, or ionomycin, a calcium ionophore. Intracellular Ca(2+) increase was visualized in fluo-3-loaded PC12 cells using fluorescence microscopy. RESULTS: Nicorandil inhibited ACh-induced ERK activation in a concentration-dependent manner. The inhibition was abolished by glibenclamide, an adenosine triphosphate-sensitive potassium channel blocker. Nicorandil suppressed the ERK activation induced by ionomycin but not 4beta-phorbol 12-myristate 13-acetate. Pretreatment of PC12 cells with nicorandil reduced the intracellular Ca(2+) concentration stimulated by ACh. CONCLUSIONS: Nicorandil inhibits muscarinic activation of the ERK signaling pathway by reducing the intracellular Ca(2+) concentration.

Depolarization-induced differentiation of PC12 cells is mediated by phospholipase D2 through the transcription factor CREB pathway.

The present study examined the role of phospholipase D2 (PLD2) in the regulation of depolarization-induced neurite outgrowth and the expression of growth-associated protein-43 (GAP-43) and synapsin I in rat pheochromocytoma (PC12) cells. Depolarization of PC12 cells with 50 mmol/L KCl increased neurite outgrowth and elevated mRNA and protein expression of GAP-43 and synapsin I. These increases were suppressed by inhibition of Ca2+-calmodulin-dependent protein kinase II (CaMKII), PLD, or mitogen-activated protein kinase kinase (MEK). Knockdown of PLD2 by small interfering RNA (siRNA) suppressed the depolarization-induced neurite outgrowth, and the increase in GAP-43 and synapsin I expression. Depolarization evoked a Ca2+ rise that activated various signaling enzymes and the cAMP response element-binding protein (CREB). Silencing CaMKIIdelta by siRNA blocked KCl-induced phosphorylation of proline-rich protein tyrosine kinase 2 (Pyk2), Src kinase, and extracellular signal-regulated kinase (ERK). Inhibition of Src or MEK abolished phosphorylation of ERK and CREB. Furthermore, phosphorylation of Pyk2, ERK, and CREB was suppressed by the PLD inhibitor, 1-butanol and transfection of PLD2 siRNA, whereas it was enhanced by over-expression of wild-type PLD2. Depolarization-induced PLD2 activation was suppressed by CaMKII and Src inhibitors, but not by MEK or protein kinase A inhibitors. These results suggest that the signaling pathway of depolarization-induced PLD2 activation was downstream of CaMKIIdelta and Src, and upstream of Pyk2(Y881) and ERK/CREB, but independent of the protein kinase A. This is the first demonstration that PLD2 activation is involved in GAP-43 and synapsin I expression during depolarization-induced neuronal differentiation in PC12 cells.