RESUMO
To better understand the role of sphingolipids in the multifactorial process of inflammatory bowel disease (IBD), we elucidated the role of CerS4 in colitis and colitis-associated cancer (CAC). For this, we utilized the azoxymethane/dextran sodium sulphate (AOM/DSS)-induced colitis model in global CerS4 knockout (CerS4 KO), intestinal epithelial (CerS4 Vil/Cre), or T-cell restricted knockout (CerS4 LCK/Cre) mice. CerS4 KO mice were highly sensitive to the toxic effect of AOM/DSS, leading to a high mortality rate. CerS4 Vil/Cre mice had smaller tumors than WT mice. In contrast, CerS4 LCK/Cre mice frequently suffered from pancolitis and developed more colon tumors. In vitro, CerS4-depleted CD8+ T-cells isolated from the thymi of CerS4 LCK/Cre mice showed impaired proliferation and prolonged cytokine production after stimulation in comparison with T-cells from WT mice. Depletion of CerS4 in human Jurkat T-cells led to a constitutively activated T-cell receptor and NF-κB signaling pathway. In conclusion, the deficiency of CerS4 in T-cells led to an enduring active status of these cells and prevents the resolution of inflammation, leading to a higher tumor burden in the CAC mouse model. In contrast, CerS4 deficiency in epithelial cells resulted in smaller colon tumors and seemed to be beneficial. The higher tumor incidence in CerS4 LCK/Cre mice and the toxic effect of AOM/DSS in CerS4 KO mice exhibited the importance of CerS4 in other tissues and revealed the complexity of general targeting CerS4.
Assuntos
Azoximetano/efeitos adversos , Neoplasias Associadas a Colite/patologia , Neoplasias do Colo/patologia , Sulfato de Dextrana/efeitos adversos , Esfingosina N-Aciltransferase/genética , Linfócitos T/metabolismo , Animais , Neoplasias Associadas a Colite/induzido quimicamente , Neoplasias Associadas a Colite/genética , Neoplasias Associadas a Colite/imunologia , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Células Jurkat , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Especificidade de Órgãos , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Carga TumoralRESUMO
Ceramide synthase 5 is one of six enzymes that catalyze the production of ceramides from sphingosine or sphinganine. Ceramides are important components of cell membranes and act as signaling molecules. Previously it has been shown that ceramide synthase 6 and 2 influence colitis in several animal models with sometimes opposite effects. Here, we investigated the disease course of dextran sodium sulfate-induced acute colitis and azoxymethane/dextran sodium sulfate-induced colitis-associated colon cancer in mice with global ceramide synthase 5 knockout (CerS5-ko) or with ceramide synthase 5 knockout restricted to the colon epithelium (CerS5fl/fl VilCre). We monitored disease development and analyzed colon barrier function as well as the immune cell status in these mice. CerS5-ko mice but not CerS5fl/fl-VilCre mice were more susceptible to acute and chronic inflammation. However, the cell barrier function of colon epithelial cells was not disturbed by downregulation of ceramide synthase 5. Instead, untreated CerS5-ko mice displayed reduced numbers of CD3+ immune cells in the spleen, colon, and blood, especially of intraepithelial CD8+ T-cells, which was not obvious in CerS5fl/fl Vil Cre mice. Reduced T-cell number in colon tissue of CerS5-ko mice was accompanied by a reduced expression of IL-1ß, IFNγ, and IL-4. In vitro investigations revealed that knockdown of ceramide synthase 5 in T-cells impaired T-cell activation. In summary, we show that CerS5-ko mice were more susceptible to dextran sodium sulfate-induced colitis and azoxymethane/dextran sodium sulfate-induced colitis-associated colon cancer. A reduced number of T-cells in the colon epithelium that was already the case in untreated CerS5-ko mice might have contributed to this effect.
RESUMO
The factors that contribute to the development of ulcerative colitis (UC), are still not fully identified. Disruption of the colon barrier is one of the first events leading to invasion of bacteria and activation of the immune system. The colon barrier is strongly influenced by sphingolipids. Sphingolipids impact cell-cell contacts and function as second messengers. We collected blood and colon tissue samples from UC patients and healthy controls and investigated the sphingolipids and other lipids by LC-MS/MS or LC-QTOFMS. The expression of enzymes of the sphingolipid pathway were determined by RT-PCR and immunohistochemistry. In inflamed colon tissue, the de novo-synthesis of sphingolipids is reduced, whereas lactosylceramides are increased. Reduction of dihydroceramides was due to posttranslational inhibition rather than altered serine palmitoyl transferase or ceramide synthase expression in inflamed colon tissue. Furthermore, in human plasma from UC-patients, several sphinglipids change significantly in comparison to healthy controls. Beside sphingolipids free fatty acids, lysophosphatidylcholines and triglycerides changed significantly in the blood of colitis patients dependent on the disease severity. Our data indicate that detraction of the sphingolipid de novo synthesis in colon tissue might be an important trigger for UC. Several lipids changed significantly in the blood, which might be used as biomarkers for disease control; however, diet-related variabilities need to be considered.
RESUMO
Resistance against chemotherapy is a life-threatening complication in colon cancer therapy. To increase response rate, new additional targets that contribute to chemoresistance are still needed to be explored. Ceramides, which belong to the group of sphingolipids, are well-known regulators of cell death and survival, respectively. Here, we show that in human wild-type (wt) p53 HCT-116 colon cancer cells treatment with oxaliplatin or 5-fluorouracil (5-FU) leads to a strong increase in ceramide synthase 5 (CerS5) expression and C16:0-ceramide levels, which was not shown in HCT-116 lacking p53 expression (HCT-116 p53-/-). The increase in CerS5 expression occurs by stabilizing CerS5 mRNA at the 3'-UTR. By contrast, in the p53-deficient cells CerS2 expression and CerS2-related C24:0- and C24:1-ceramide levels were elevated which is possibly related to enhanced polyadenylation of the CerS2 transcript in these cells. Stable knockdown of CerS5 expression using CerS5-targeting shRNA led to an increased sensitivity of HCT-116 p53wt cells, but not of p53-/- cells, to oxaliplatin and 5-FU. Enhanced sensitivity was accompanied by an inhibition of autophagy and inhibition of mitochondrial respiration in these cells. However, knockdown of CerS2 had no significant effects on chemosensitivity of both cell lines. In conclusion, in p53wt colon cancer cells chemosensitivity against oxaliplatin or 5-FU could be enhanced by downregulation of CerS5 expression leading to reduced autophagy and mitochondrial respiration.
Assuntos
Autofagia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Esfingosina N-Aciltransferase/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Autofagia/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Regulação para Baixo/efeitos dos fármacos , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Oxaliplatina/farmacologia , Esfingolipídeos/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
R-flurbiprofen is the non-COX-inhibiting enantiomer of flurbiprofen and is not converted to S-flurbiprofen in human cells. Nevertheless, it reduces extracellular prostaglandin E2 (PGE2) in cancer or immune cell cultures and human extracellular fluid. Here, we show that R-flurbiprofen acts through a dual mechanism: (i) it inhibits the translocation of cPLA2α to the plasma membrane and thereby curtails the availability of arachidonic acid and (ii) R-flurbiprofen traps PGE2 inside of the cells by inhibiting multidrug resistance-associated protein 4 (MRP4, ABCC4), which acts as an outward transporter for prostaglandins. Consequently, the effects of R-flurbiprofen were mimicked by RNAi-mediated knockdown of MRP4. Our data show a novel mechanism by which R-flurbiprofen reduces extracellular PGs at physiological concentrations, particularly in cancers with high levels of MRP4, but the mechanism may also contribute to its anti-inflammatory and immune-modulating properties and suggests that it reduces PGs in a site- and context-dependent manner.
Assuntos
Dinoprostona/metabolismo , Flurbiprofeno/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Células A549 , Ácido Araquidônico/metabolismo , Western Blotting , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Flurbiprofeno/química , Expressão Gênica/efeitos dos fármacos , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Fosfolipases A2 do Grupo IV/metabolismo , Células HeLa , Humanos , Interleucina-1beta/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estereoisomerismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Ceramides (Cer) are mediators of inflammatory processes. In a chronic experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), we observed a significant elevation of C16-Cer and its synthesizing enzyme, ceramide synthase(CerS)6, in the lumbar spinal cord. In the present study, we have confirmed that C16-Cer and CerS6 are also upregulated in the lumbar spinal cord in a spontaneous relapse-remitting EAE model, using SJL mice overexpressing a transgenic T cell receptor (TCR1640). CerS6 was found to be expressed in macrophages, T cells and B cells in EAE lesions. In macrophages, we demonstrated that interferon gamma (IFN-γ)-induced CerS6 upregulation was amplified by 17ß-estradiol, an action that was further accompanied by increased upregulation of tumor necrosis factor alpha (TNF-α). Accordingly, CerS6 and TNF-α expression was upregulated predominantly in the spinal cord in female TCR1640 mice, which usually develop the relapse-remitting form of EAE, while male TCR1640 mice showed an attenuated regulation of CerS6 and TNF-α and exhibit mostly chronic disease progression. Furthermore, expression of TNFR2, one of two receptors of TNF-α, which is linked to neuroprotection and remyelination, was also upregulated to a greater extent during EAE in female TCR1640 mice in comparison to male TCR1640 mice. Taken together, our results confirm the upregulation of CerS6 and C16-Cer in an adjuvant-independent, physiological EAE model and further suggest an anti-inflammatory role of CerS6 in the regulation of the disease course in female TCR1640 mice via TNF-α/TNFR2.
Assuntos
Encefalomielite Autoimune Experimental/enzimologia , Caracteres Sexuais , Esfingosina N-Aciltransferase/biossíntese , Animais , Encefalomielite Autoimune Experimental/fisiopatologia , Feminino , Humanos , Masculino , CamundongosRESUMO
Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated inflammatory autoimmune disease model of multiple sclerosis (MS). The inflammatory process is initiated by activation and proliferation of T cells and monocytes and by their subsequent migration into the central nervous system (CNS), where they induce demyelination and neurodegeneration. Prostaglandin E2 (PGE2) - synthesized by cyclooxygenase 2 (COX-2) - has both pro- and anti-inflammatory potential, which is translated via four different EP receptors. We hypothesized that PGE2 synthesized in the preclinical phase by peripheral immune cells exerts pro-inflammatory properties in the EAE model. To investigate this, we used a bone marrow transplantation model, which enables PGE2 synthesis or EP receptor expression to be blocked specifically in peripheral murine immune cells. Our results reveal that deletion of COX-2 or its EP4 receptor in bone marrow-derived cells leads to a significant delay in the onset of EAE. This effect is due to an impaired preclinical inflammatory process indicated by a reduced level of the T cell activating interleukin-6 (IL-6), reduced numbers of T cells and of the T cell secreted interleukin-17 (IL-17) in the blood of mice lacking COX-2 or EP4 in peripheral immune cells. Moreover, mice lacking COX-2 or EP4 in bone marrow-derived cells show a reduced expression of matrix metalloproteinase 9 (MMP9), which results in decreased infiltration of monocytes and T cells into the CNS. In conclusion, our data demonstrate that PGE2 synthesized by monocytes in the early preclinical phase promotes the development of EAE in an EP4 receptor dependent manner.
Assuntos
Células da Medula Óssea/imunologia , Dinoprostona/fisiologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Monócitos/imunologia , Receptores de Prostaglandina E Subtipo EP4/fisiologia , Transdução de Sinais/imunologia , Animais , Células da Medula Óssea/patologia , Dinoprostona/biossíntese , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/patologia , Receptores de Prostaglandina E Subtipo EP4/biossínteseRESUMO
Ceramides with different N-acyl chains can act as second messengers in various signaling pathways. They are involved in cell processes such as apoptosis, differentiation and inflammation. Ceramide synthases (CerS) are key enzymes in the biosynthesis of ceramides and dihydroceramides. Six isoenzymes (CerS1-6) catalyze the N-acylation of the sphingoid bases, albeit with strictly acyl-Coenzyme A (CoA) chain length specificity. We analyzed the mRNA expression, the protein expression, the specific activity of the CerS, and acyl-CoA, dihydroceramide and ceramide levels in different tissues by LC-MS/MS. Our data indicate that each tissue express a distinct composition of CerS, whereby the CerS mRNA expression levels do not correlate with the respective protein expression levels in the tissues. Furthermore, we found a highly significant negative correlation between the protein expression level of CerS6 and the C16:0-acyl-CoA amounts as well as between the protein expression of CerS2 and C24:0-acyl-CoA amounts. These data indicate that in mouse tissues low substrate availability is compensated by higher CerS protein expression level and vice versa. Apart from the expression level and the specific activity of the CerS, other enzymes of the sphingolipid pathway also influence the composition of ceramides with distinct chain lengths in each cell. Acyl-CoA availability seems to be less important for ceramide composition and might be compensated for by CerS expression/activity.
Assuntos
Ceramidas/metabolismo , Especificidade de Órgãos , Acil Coenzima A/metabolismo , Animais , Ceramidas/biossíntese , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos/genética , Oxirredutases/genética , Oxirredutases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estatísticas não ParamétricasRESUMO
Ceramides are mediators of apoptosis and inflammatory processes. In an animal model of multiple sclerosis (MS), the experimental autoimmune encephalomyelitis (EAE) model, we observed a significant elevation of C(16:0)-Cer in the lumbar spinal cord of EAE mice. This was caused by a transiently increased expression of ceramide synthase (CerS) 6 in monocytes/macrophages and astroglia. Notably, this corresponds to the clinical finding that C(16:0)-Cer levels were increased 1.9-fold in cerebrospinal fluid of MS patients. NO and TNF-α secreted by IFN-γ-activated macrophages play an essential role in the development of MS. In murine peritoneal and mouse-derived RAW 264.7 macrophages, IFN-γ-mediated expression of inducible NO synthase (iNOS)/TNF-α and NO/TNF-α release depends on upregulation of CerS6/C(16:0)-Cer. Downregulation of CerS6 by RNA interference or endogenous upregulation of C(16:0)-Cer mediated by palmitic acid in RAW 264.7 macrophages led to a significant reduction or increase in NO/TNF-α release, respectively. EAE/IFN-γ knockout mice showed a significant delay in disease onset accompanied by a significantly less pronounced increase in CerS6/C(16:0)-Cer, iNOS, and TNF-α compared with EAE/wild-type mice. Treatment of EAE mice with l-cycloserine prevented the increase in C(16:0)-Cer and iNOS/TNF-α expression and caused a remission of the disease. In conclusion, CerS6 plays a critical role in the onset of MS, most likely by regulating NO and TNF-α synthesis. CerS6 may represent a new target for the inhibition of inflammatory processes promoting MS development.
Assuntos
Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/imunologia , Proteínas de Membrana/fisiologia , Esfingosina N-Aciltransferase/fisiologia , Adulto , Idoso , Animais , Células Cultivadas , Encefalomielite Autoimune Experimental/patologia , Humanos , Inflamação/enzimologia , Inflamação/imunologia , Inflamação/patologia , Interferon gama/fisiologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana/líquido cefalorraquidiano , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , Esfingosina N-Aciltransferase/biossíntese , Esfingosina N-Aciltransferase/líquido cefalorraquidiano , Adulto JovemRESUMO
Ceramide synthases (CerSs) are key enzymes in the biosynthesis of ceramides and display a group of at least six different isoenzymes (CerS1-6). Ceramides itself are bioactive molecules. Ceramides with different N-acyl side chains (C(14:0)-Cer - C(26:0)-Cer) possess distinct roles in cell signaling. Therefore, the selective inhibition of specific CerSs which are responsible for the formation of a specific ceramide holds promise for a number of new clinical treatment strategies, e.g., cancer. Here, we identified four of hitherto unknown functional inhibitors of CerSs derived from the FTY720 (Fingolimod) lead structure and showed their inhibitory effectiveness by two in vitro CerS activity assays. Additionally, we tested the substances in two cell lines (HCT-116 and HeLa) with different ceramide patterns. In summary, the in vitro activity assays revealed out that ST1058 and ST1074 preferentially inhibit CerS2 and CerS4, while ST1072 inhibits most potently CerS4 and CerS6. Importantly, ST1060 inhibits predominately CerS2. First structure-activity relationships and the potential biological impact of these compounds are discussed.
Assuntos
Ceramidas/antagonistas & inibidores , Oxirredutases/antagonistas & inibidores , Propilenoglicóis/farmacologia , Esfingosina/análogos & derivados , Sobrevivência Celular/efeitos dos fármacos , Ceramidas/biossíntese , Relação Dose-Resposta a Droga , Cloridrato de Fingolimode , Células HeLa , Humanos , Concentração Inibidora 50 , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Oxirredutases/metabolismo , Propilenoglicóis/síntese química , Transdução de Sinais/efeitos dos fármacos , Esfingosina/síntese química , Esfingosina/farmacologia , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
BACKGROUND: Molecular descriptors are essential for many applications in computational chemistry, such as ligand-based similarity searching. Spherical harmonics have previously been suggested as comprehensive descriptors of molecular structure and properties. We investigate a spherical harmonics descriptor for shape-based virtual screening. METHODOLOGY/PRINCIPAL FINDINGS: We introduce and validate a partially rotation-invariant three-dimensional molecular shape descriptor based on the norm of spherical harmonics expansion coefficients. Using this molecular representation, we parameterize molecular surfaces, i.e., isosurfaces of spatial molecular property distributions. We validate the shape descriptor in a comprehensive retrospective virtual screening experiment. In a prospective study, we virtually screen a large compound library for cyclooxygenase inhibitors, using a self-organizing map as a pre-filter and the shape descriptor for candidate prioritization. CONCLUSIONS/SIGNIFICANCE: 12 compounds were tested in vitro for direct enzyme inhibition and in a whole blood assay. Active compounds containing a triazole scaffold were identified as direct cyclooxygenase-1 inhibitors. This outcome corroborates the usefulness of spherical harmonics for representation of molecular shape in virtual screening of large compound collections. The combination of pharmacophore and shape-based filtering of screening candidates proved to be a straightforward approach to finding novel bioactive chemotypes with minimal experimental effort.
Assuntos
Inibidores de Ciclo-Oxigenase/análise , Inibidores de Ciclo-Oxigenase/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Interface Usuário-Computador , Animais , Ciclo-Oxigenase 2/sangue , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/sangue , Inibidores de Ciclo-Oxigenase/química , Ensaios Enzimáticos , Humanos , Ligantes , Curva ROC , Padrões de Referência , OvinosRESUMO
Ceramides serve as bioactive molecules with important roles in cell proliferation and apoptosis. Ceramides (Cer) with different N-acyl side chains (C(14:0)-Cer-C(26:0)-Cer) possess distinctive roles in cell signaling and are differentially expressed in HCT-116 colon cancer cells. Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, exhibiting antiproliferative effects, activates the sphingolipid pathway. To elucidate the mechanism, HCT-116 cells were treated with 50µM celecoxib leading to a significant increase of C(16:0)-Cer. Interestingly, 50µM celecoxib resulted in a 2.8-fold increase of ceramide synthase (CerS) activity as measured by a cell-based activity assay. siRNA against several CerSs revealed that CerS6 was predominantly responsible for the increase of C(16:0)-Cer in HCT-116 cells. Moreover, the silencing of CerS6 partially protected HCT-116 cells from the toxic effects induced by celecoxib. Treatment of cells with celecoxib and fumonisin B1 (inhibitor of CerSs) or myriocin (inhibitor of l-serine palmitoyl transferase) or desipramine (inhibitor of acid sphingomyelinase and acid ceramidase) revealed that the increase of C(16:0)-Cer results predominantly from activation of the salvage pathway. Using the nude mouse model we demonstrated that celecoxib induces also in vivo a significant increase of C(16:0)-Cer in stomach, small intestine and tumor tissue. In conclusion, celecoxib causes a specific increase of C(16:0)-Cer by activating CerS6 and the salvage pathway, which contribute to the toxic effects of celecoxib.
Assuntos
Ceramidas/biossíntese , Proteínas de Membrana/metabolismo , Pirazóis/farmacologia , Esfingosina N-Aciltransferase/metabolismo , Sulfonamidas/farmacologia , Animais , Celecoxib , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Células HCT116 , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Nus , Pirazóis/toxicidade , Esfingosina N-Aciltransferase/antagonistas & inibidores , Esfingosina N-Aciltransferase/fisiologia , Sulfonamidas/toxicidade , Distribuição Tecidual/efeitos dos fármacos , Distribuição Tecidual/fisiologiaRESUMO
Celecoxib is a selective cyclooxygenase-2-(COX-2)-inhibitor used to treat inflammation and pain and prevents colorectal cancer in patients at high doses by affecting several non-COX-2 proteins. However, celecoxib concentrations appropriate to inhibit proliferation or to induce apoptosis in cell culture (up to 100 microM) clearly exceed those in human plasma (up to 10 microM). Therefore, we speculated that celecoxib might accumulate in human cells, which may facilitate the drug's interaction with non-COX-2 proteins. Determination of intracellular celecoxib concentrations by liquid chromatography tandem mass spectrometry gave five- to tenfold higher levels as compared to other coxibs (etoricoxib, valdecoxib, lumiracoxib, and rofecoxib) in different tumor cell types, including human HCA-7 and HCT-116 colon carcinoma cells, BL-41 B lymphocytes, Mono Mac 6 monocytes, and in mouse NIH-3T3 non-tumor fibroblasts. This intracellular accumulation of celecoxib was due to an integration of the drug into cellular phospholipid membranes as demonstrated by nuclear Overhauser spectroscopy/nuclear magnetic resonance. Consequently, celecoxib disturbed the plasma membrane integrity of HCT-116 cells and displayed an increased COX-2-inhibitory potency in HCA-7 cells. The use of other coxibs demonstrated that intracellular accumulation is peculiar of celecoxib. Accumulation of celecoxib in human cells may provide a novel molecular basis for the ability of the drug to interact with non-COX-2 targets in vivo despite comparatively low plasma concentrations.
Assuntos
Membrana Celular/metabolismo , Inibidores de Ciclo-Oxigenase 2/metabolismo , Membranas Intracelulares/metabolismo , Pirazóis/metabolismo , Sulfonamidas/metabolismo , Animais , Transporte Biológico/fisiologia , Celecoxib , Linhagem Celular , Inibidores de Ciclo-Oxigenase 2/química , Humanos , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Fosfolipídeos/metabolismo , Pirazóis/química , Sulfonamidas/químicaRESUMO
Several in vitro studies have correlated dysfunction of the sphingolipid-signaling pathway with promotion of tumor cell growth as well as progression and resistance of tumors to chemotherapeutic agents. As ceramides (Cer) constitute the structural backbones of all sphingolipids, we investigated the endogenous ceramide levels in 43 malignant breast tumors and 21 benign breast biopsies and compared them with those of normal tissues using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The total ceramide levels in malignant tumor tissue samples were statistically significantly elevated when compared with normal tissue samples. Upregulation of the total ceramide level averaged 12-fold and 4-fold higher than normal tissue samples, for malignant tumors and benign tissues, respectively. Specifically, the levels of C(16:0)-Cer, C(24:1)-Cer and C(24:0)-Cer were significantly raised in malignant tumors as compared with benign and normal tissue. The augmentation of the various ceramides could be assigned to an increase of the messenger RNA levels of ceramide synthases (CerS) LASS2 (longevity assurance), LASS4 and LASS6. Notably, elevated levels of C(16:0)-Cer were associated with a positive lymph node status, indicating a metastatic potential for this ceramide. Moreover, the levels of C(18:0)-Cer and C(20:0)-Cer were significantly higher in estrogen receptor (ER) positive tumor tissues as compared with ER negative tumor tissues. In conclusion, progression in breast cancer is associated with increased ceramide levels due to an upregulation of specific LASS genes.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Ceramidas/metabolismo , Oxirredutases/metabolismo , Mama/enzimologia , Mama/metabolismo , Doenças Mamárias/metabolismo , Doenças Mamárias/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Divisão Celular , Feminino , Humanos , Proteínas de Membrana/genética , Oxirredutases/genética , RNA Mensageiro/genética , Receptores de Estrogênio/metabolismo , Valores de Referência , Esfingolipídeos/metabolismo , Esfingosina N-Aciltransferase , Regulação para CimaRESUMO
Sphingolipids such as ceramides (Cers) play important roles in cell proliferation, apoptosis, and cell cycle regulation. An increased Cer level is linked to the cytotoxic effects of several chemotherapeutics. Various selective cyclooxygenase-2 (COX-2) inhibitors induce anti-proliferative effects in tumor cells. We addressed the possible interaction of the selective COX-2 inhibitors, coxibs, with the sphingolipid pathway as an explanation of their anti-proliferative effects. Sphingolipids were measured using liquid chromatography tandem mass spectrometry. Treatment of various cancer cell lines with celecoxib significantly increased sphinganine, C(16:0)-, C(24:0)-, C(24:1)-dihydroceramide (dhCer) and led to a depletion of C(24:0)-, C(24:1)-Cer in a time- and concentration-dependent manner, whereas other coxibs had no effect. Using (13)C,(15)N-labeled l-serine, we demonstrated that the augmented dhCers after celecoxib treatment originate from de novo synthesis. Celecoxib inhibited the dihydroceramide desaturase (DEGS) in vivo with an IC(50) of 78.9 +/- 1.5 muM and increased total Cer level about 2-fold, indicating an activation of sphingolipid biosynthesis. Interestingly, inhibition of the sphingolipid biosynthesis by specific inhibitors of l-serine palmitoyltransferase diminished the anti-proliferative potency of celecoxib. In conclusion, induction of de novo synthesis of sphingolipids and inhibition of DEGS contribute to the anti-proliferative effects of celecoxib.
Assuntos
Inibidores de Ciclo-Oxigenase 2/farmacologia , Ciclo-Oxigenase 2/metabolismo , Pirazóis/farmacologia , Esfingolipídeos/metabolismo , Sulfonamidas/farmacologia , Apoptose , Celecoxib , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Humanos , Concentração Inibidora 50 , Lipídeos/química , Modelos Biológicos , Modelos Químicos , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/químicaRESUMO
Dimethylcelecoxib (DMC), a derivative of celecoxib, has been developed to distinguish between the COX-dependent and COX-independent anti-carcinogenic effects of celecoxib. Although DMC has been shown to have no COX-inhibitory activity, it is important to ensure that DMC has no other influence on prostaglandin production. Interestingly, in this study we show that DMC inhibits PGE(2) production in vitro in the low micromolar range in different cancer cell lines. This effect can be at least partly explained by our findings that DMC inhibits microsomal prostaglandin E synthase-1 (mPGES-1) activity in a cell-free assay. Moreover, it prevents mPGES-1 up-regulation after stimulation of HeLa cells with IL-1beta and TNFalpha. Conversely, DMC has no effect on the expression levels of COX-1, COX-2, cytosolic PGES (cPGES) or mPGES-2 in these cells. However, in the cell-free assay DMC inhibits mPGES-1 to a maximum of 65% only and concentrations needed for inhibition of mPGES-1 activity are about 10-fold higher than needed for inhibition of PGE(2) production in cell culture. This suggests that DMC also has an impact on other proteins involved in PGE(2) production. In cell culture experiments the anti-proliferative effect of DMC, measured by the WST-1 assay, seems not to be dependent on PGE(2) inhibition, as DMC was equally effective in unstimulated HeLa cells as well as in stimulated HeLa cells, and the addition of external PGE(2) did not reverse the anti-proliferative effect of DMC in HCA-7 cells. We conclude that DMC is not a suitable non-prostaglandin-inhibiting control substance for research purposes.
Assuntos
Dinoprostona/biossíntese , Antagonistas de Prostaglandina/farmacologia , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Western Blotting , Proliferação de Células/efeitos dos fármacos , Células HeLa , Humanos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
S-ibuprofen which inhibits the cyclooxygenase-1/-2 and R-ibuprofen which shows no COX-inhibition at therapeutic concentrations have anti-carcinogenic effects in human colon cancer cells; however, the molecular mechanisms for these effects are still unknown. Using HCT-116 colon carcinoma cell lines, expressing either the wild-type form of p53 (HCT-116 p53(wt)) or being p(HCT-116 p53(-/-)), we demonstrated that both induction of a cell cycle block and apoptosis after S- and R-ibuprofen treatment is in part dependent on p53. Also in the in vivo nude mice model HCT-116 p53(-/-) xenografts were less sensitive for S- and R-ibuprofen treatment than HCT-116 p53(wt) cells. Furthermore, results indicate that induction of apoptosis in HCT-116 p53(wt) cells after ibuprofen treatment is in part dependent on a signalling pathway including the neutrophin receptor p75(NTR), p53 and Bax.
Assuntos
Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/patologia , Proliferação de Células/efeitos dos fármacos , Ibuprofeno/administração & dosagem , Proteínas do Tecido Nervoso/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Anti-Inflamatórios não Esteroides/administração & dosagem , Relação Dose-Resposta a Droga , HumanosRESUMO
PAM (Protein Associated with Myc) is an almost ubiquitously expressed protein that is one of the most potent inhibitors of adenylyl cyclase activity known so far. Here we show that PAM is localized at the endoplasmic reticulum in HeLa cells and that upon serum treatment PAM is recruited to the plasma membrane, causing an inhibition of adenylyl cyclase activity. We purified the serum factor that induced PAM translocation and identified it as sphingosine-1-phosphate (S1P). Within 15 min after incubation with S1P, PAM appeared at the plasma membrane and was detectable for up to 120 min. Sphingosine-1-phosphate induced adenylyl cyclase inhibition in two phases: an initial (1-10 min) and a late (20-240 min) phase. The initial adenylyl cyclase inhibition was Gi-mediated and PAM independent. In the late phase, adenylyl cyclase inhibition was PAM dependent and attenuated cyclic AMP (cAMP) signaling by various cAMP-elevating signals. This makes PAM the longest lasting nontranscriptional regulator of adenylyl cyclase activity known to date and presents a novel mechanism for the temporal regulation of cAMP signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , AMP Cíclico/metabolismo , Lisofosfolipídeos/farmacologia , Oxigenases de Função Mista/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Inibidores de Adenilil Ciclases , Adenilil Ciclases/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Oxigenases de Função Mista/genética , Toxina Pertussis/farmacologia , Proteína Quinase C/metabolismo , Transporte Proteico/efeitos dos fármacos , Soro , Fosfolipases Tipo C/metabolismo , Ubiquitina-Proteína LigasesRESUMO
PAM (protein associated with Myc) is a potent inhibitor of adenylyl cyclases (ACs) which is primarily expressed in neurones. Here we describe that PAM is highly expressed in dorsal horn neurones and motoneuron of the spinal cord, as well as in neurones of dorsal root ganglia in adult rats. PAM mRNA expression is differentially regulated during development in both spinal cord and dorsal root ganglia of rats, being strongest during the major respective synaptogenic periods. In adult rats, PAM expression was up-regulated in the spinal cord after peripheral nociceptive stimulation using zymosan and formalin injection, suggesting a role for PAM in spinal nociceptive processing. Since PAM inhibited Galphas-stimulated AC activity in dorsal root ganglia as well as spinal cord lysates, we hypothesized that PAM may reduce spinal nociceptive processing by inhibition of cAMP-dependent signalling. Accordingly, intrathecal treatment with antisense but not sense oligonucleotides against PAM increased basal and Galphas-stimulated AC activity in the spinal cord and enhanced formalin-induced nociceptive behaviour in adult rats. Taken together our findings demonstrate that PAM is involved in spinal nociceptive processing.