RESUMO
B13 is an acid ceramidase (ACDase) inhibitor. The two chiral centers of this aromatic amido alcohol lead to four stereoisomers, yet we have little knowledge about its erythro- enantiomers, (1R, 2S) and (1S, 2R). In this paper, for the first time, the synthesis of two erythro- enantiomers is described, and the compounds are evaluated along with two threo- enantiomers, (1R, 2R) and (1S, 2S). The key metabolites and sphingolipid (SL) profile of the full set of B13 stereoisomers in MCF7 breast carcinoma cells are presented. The results demonstrated that the erythro- enantiomers were more effective than the threo- enantiomers on growth inhibition in MCF7 cells, although there were no statistically significant differences within the threo- and erythro- series. Measurement of intracellular levels of the compounds indicated that the erythro- seemed a little more cell permeable than the threo- enantiomers; also, the (1R, 2S) isomer with the same stereo structure as natural ceramide (Cer) could be hydrolyzed and phosphorylated in MCF7 cells. Furthermore, we also observed the formation of C16 homologs from the full set of B13 isomers within the cells, indicating the occurrence of de-acylation and re-acylation of the amino group of the aromatic alcohol. Moreover, the decrease in the Cer/Sph ratio suggests that the growth inhibition from (1R, 2S) isomer is not because of the inhibition of ceramidases. Taken together, (1R, 2S) could be developed as a substitute of natural Cer.
Assuntos
Amidas/farmacologia , Antineoplásicos/farmacologia , Propanolaminas/farmacologia , Esfingolipídeos/antagonistas & inibidores , Amidas/síntese química , Amidas/química , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células MCF-7 , Estrutura Molecular , Propanolaminas/síntese química , Propanolaminas/química , Esfingolipídeos/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
The bioactive sphingolipid ceramide affects immune responses although its effect on antigen (Ag) processing and delivery by HLA class II to CD4+T-cells remains unclear. Therefore, we examined the actions of a novel cell-permeable acid ceramidase (AC) inhibitor [(1R,2R) N myristoylamino-(4'-nitrophenyl)-propandiol-1,3] on antigen presentation and inflammatory cytokine production by Ag-presenting cells (APCs) such as B-cells, macrophages, and dendritic cells. We found that AC inhibition in APCs perturbed Ag-processing and presentation via HLA-DR4 (MHC class II) proteins as measured by coculture assay and T-cell production of IL-2. Mass spectral analyses showed that B13 treatment significantly raised levels of four types of ceramides in human B-cells. B13 treatment did not alter Ag internalization and class II protein expression, but significantly inhibited lysosomal cysteinyl cathepsins (B, S and L) and thiol-reductase (GILT), HLA class II Ag-processing, and generation of functional class II-peptide complexes. Ex vivo Ag presentation assays showed that inhibition of AC impaired primary and recall CD4+T-cell responses and cytokine production in response against type II collagen. Further, B13 delayed onset and reduced severity of inflamed joints and cytokine production in the collagen-induced arthritis mouse model in vivo. These findings suggest that inhibition of AC in APCs may dysregulate endolysosomal proteases and HLA class II-associated self-antigen presentation to CD4+T-cells, attenuating inflammatory cytokine production and suppressing host autoimmune responses.
Assuntos
Ceramidase Ácida/imunologia , Apresentação de Antígeno/imunologia , Artrite Experimental/imunologia , Doenças Autoimunes/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Catepsinas/imunologia , Linhagem Celular , Antígeno HLA-DR4/imunologia , Humanos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos DBARESUMO
Our recent investigation uncovered that the acid ceramidase inhibitor LCL521 enhances the direct tumor cell killing effect of photodynamic therapy (PDT) treatment. The present study aimed at elucidating the mechanisms underlying this effect. Exposing mouse squamous cell carcinoma SCCVII cells treated with temoporfin-based PDT to LCL521 (rising ceramide concentration) produced a much greater decrease in cell survival than comparable exposure to the sphingosine kinase-1 inhibitor PF543 (that reduces sphingosine-1-phosphate concentration). This is consistent with recognizing the rising levels of pro-apoptotic sphingolipid ceramide as being more critical in promoting the death of PDT-treated cells than the reduction in the availability of pro-survival acting sphingosine-1 phosphate. This pro-apoptotic impact of LCL521, which was suppressed by the apoptosis inhibitor bongkrekic acid, involves the interaction with the cellular stress signaling network. Hence, inhibiting the key elements of these pathways markedly influenced the adjuvant effect of LCL521 on the PDT response. Particularly effective was the inositol-requiring element-1 (IRE1) kinase inhibitor STF-083010 that dramatically enhanced the killing of cells treated with PDT plus LCL521. An important role in the survival of these cells was exhibited by master transcription factors STAT3 and HIF-1α. The STAT3 inhibitor NSC 74859 was especially effective in further reducing the cell survival rates, suggesting its possible exploitation for therapeutic gain. An additional finding in this study is that LCL521-promoted PDT-mediated cell killing through ceramide-mediated lethal effects is extended to the interaction with other cancer treatment modalities with a rapid cellular stress impact such as photothermal therapy (PTT) and cryoablation therapy (CAT).
Assuntos
Acetatos/farmacologia , Aminas/farmacologia , Antineoplásicos/farmacologia , Ceramidases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Hipertermia Induzida , Fotoquimioterapia , Acetatos/síntese química , Acetatos/química , Aminas/síntese química , Aminas/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ceramidases/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Camundongos , Células Tumorais CultivadasRESUMO
Numerous genetic alterations have been identified during prostate cancer progression. The influence of environmental factors, particularly the diet, on the acceleration of tumor progression is largely unknown. Expression levels and/or activity of Src kinase are highly elevated in numerous cancers including advanced stages of prostate cancer. In this study, we demonstrate that high-fat diets (HFDs) promoted pathological transformation mediated by the synergy of Src and androgen receptor in vivo. Additionally, a diet high in saturated fat significantly enhanced proliferation of Src-mediated xenograft tumors in comparison with a diet high in unsaturated fat. The saturated fatty acid palmitate, a major constituent in a HFD, significantly upregulated the biosynthesis of palmitoyl-CoA in cancer cells in vitro and in xenograft tumors in vivo. The exogenous palmitate enhanced Src-dependent mitochondrial ß-oxidation. Additionally, it elevated the amount of C16-ceramide and total saturated ceramides, increased the level of Src kinase localized in the cell membrane, and Src-mediated downstream signaling, such as the activation of mitogen-activated protein kinase and focal adhesion kinase. Our results uncover how the metabolism of dietary palmitate cooperates with elevated Src kinase in the acceleration of prostate tumor progression.
Assuntos
Palmitatos/administração & dosagem , Neoplasias da Próstata/etiologia , Quinases da Família src/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Dieta Hiperlipídica/efeitos adversos , Progressão da Doença , Células HEK293 , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Células PC-3 , Palmitatos/metabolismo , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologiaRESUMO
The function of acid ceramidase (ACDase), whose congenital deficiency leads to Farber disease, has been recognized to be vital to tumor cell biology, and inhibition of its activity may be beneficial in cancer therapy. Therefore, manipulation of the activity of this enzyme may have significant effect, especially on cancer cells. LCL521, Di-DMG-B13, is a lysosomotropic inhibitor of ACDase. Here we define complexities in the actions of LCL521 on ACDase. Systematic studies in MCF7 cells showed dose and time divergent action of LCL521 on ACDase protein expression and sphingolipid levels. Low dose of LCL521 (1⯵M) effectively inhibited ACDase in cells, but the effects were transient. A higher dose of LCL521 (10⯵M) caused a profound decrease of sphingosine and increase of ceramide, but additionally affected the processing and regeneration of the ACDase protein, with biphasic and reversible effects on the expression of ACDase, which paralleled the long term changes of cellular sphingosine and ceramide. Finally, the higher concentrations of LCL521 also inhibited Dihydroceramide desaturase (DES-1). In summary, LCL521 exhibits significant effects on ACDase in a dose and time dependent manner, but dose range and treatment time need to be paid attention to specify its future exploration on ACDase targeted cancer treatment.
Assuntos
Acetatos/farmacologia , Ceramidase Ácida/antagonistas & inibidores , Aminas/farmacologia , Inibidores Enzimáticos/farmacologia , Esfingolipídeos/antagonistas & inibidores , Ceramidase Ácida/metabolismo , Relação Dose-Resposta a Droga , Humanos , Células MCF-7 , Estrutura Molecular , Esfingolipídeos/metabolismo , Relação Estrutura-Atividade , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Acute kidney injury (AKI), resulting from chemotherapeutic agents such as cisplatin, remains an obstacle in the treatment of cancer. Cisplatin-induced AKI involves apoptotic and necrotic cell death, pathways regulated by sphingolipids such as ceramide and glucosylceramide. Results from this study indicate that C57BL/6J mice treated with cisplatin had increased ceramide and hexosylceramide levels in the renal cortex 72 h following cisplatin treatment. Pretreatment of mice with inhibitors of acid sphingomyelinase and de novo ceramide synthesis (amitriptyline and myriocin, respectively) prevented accumulation of ceramides and hexosylceramide in the renal cortex and protected from cisplatin-induced AKI. To determine the role of ceramide metabolism to hexosylceramides in kidney injury, we treated mice with a potent and highly specific inhibitor of glucosylceramide synthase, the enzyme responsible for catalyzing the glycosylation of ceramides to form glucosylceramides. Inhibition of glucosylceramide synthase attenuated the accumulation of the hexosylceramides and exacerbated ceramide accumulation in the renal cortex following treatment of mice with cisplatin. Increasing ceramides and decreasing glucosylceramides in the renal cortex sensitized mice to cisplatin-induced AKI according to markers of kidney function, kidney injury, inflammation, cell stress, and apoptosis. Under conditions of high ceramide generation, data suggest that metabolism of ceramides to glucosylceramides buffers kidney ceramides and helps attenuate kidney injury.-Dupre, T. V., M. A. Doll, P. P. Shah, C. N. Sharp, D. Siow, J. Megyesi, J. Shayman, A. Bielawska, J. Bielawski, L. J. Beverly, M. Hernandez-Corbacho, C. J. Clarke, A. J. Snider, R. G. Schnellmann, L. M. Obeid, Y. A. Hannun, and L. J. Siskind. Inhibiting glucosylceramide synthase exacerbates cisplatin-induced acute kidney injury. J. Lipid Res 2017. 58: 1439-1452.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Cisplatino/efeitos adversos , Inibidores Enzimáticos/farmacologia , Glucosiltransferases/antagonistas & inibidores , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/fisiopatologia , Animais , Ceramidas/metabolismo , Córtex Renal/irrigação sanguínea , Córtex Renal/efeitos dos fármacos , Córtex Renal/metabolismo , Masculino , Camundongos , Ratos , Traumatismo por Reperfusão/metabolismoRESUMO
Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid metabolite which has been implicated in many diseases including cancer and inflammatory diseases. Recently, sphingosine kinase 1 (SK1), one of the isozymes which generates S1P, has been implicated in the development and progression of inflammatory bowel disease (IBD). Based on our previous work, we set out to determine the efficacy of a novel SK1 selective inhibitor, LCL351, in a murine model of IBD. LCL351 selectively inhibits SK1 both in vitro and in cells. LCL351, which accumulates in relevant tissues such as colon, did not have any adverse side effects in vivo. In mice challenged with dextran sodium sulfate (DSS), a murine model for IBD, LCL351 treatment protected from blood loss and splenomegaly. Additionally, LCL351 treatment reduced the expression of pro-inflammatory markers, and reduced neutrophil infiltration in colon tissue. Our results suggest inflammation associated with IBD can be targeted pharmacologically through the inhibition and degradation of SK1. Furthermore, our data also identifies desirable properties of SK1 inhibitors.
Assuntos
Colite/tratamento farmacológico , Colite/imunologia , Sulfato de Dextrana/efeitos adversos , Guanidinas/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Esfingosina/farmacologia , Células A549 , Quimiocina CXCL1/genética , Quimiocina CXCL2/genética , Colite/induzido quimicamente , Colite/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Guanidinas/uso terapêutico , Humanos , Esfingosina/uso terapêutico , Fator de Necrose Tumoral alfa/genéticaRESUMO
Th1 pro-inflammatory cytokines, i.e., TNF-α and IFN-γ, in combination are known to induce cell death in several cell types, including oligodendrocytes, but the mechanism of their synergistic cytotoxicity is unclear. Although ceramide (Cer) has been implicated in cytokine- and stress-induced cell death, its intracellular levels alone cannot explain cytokine synergy. We considered the possibility that Cer released as part of extracellular vesicles may contribute to cytokine-induced synergistic cell death. Using a human oligodendroglioma (HOG) cell line as a model, here we show that exosomes derived from TNF-α-treated "donor" cells, while being mildly toxic to fresh cultures (similar to individual cytokines), induce enhanced cell death when added to IFN-γ-primed target cultures in a fashion resembling the effect of cytokine combination. Further, the sphingolipid profiles of secreted exosomes, as determined by HPLC-MS/MS, revealed that the treatment with the cytokines time-dependently induced the formation and exosomal release, in particular of C16-, C24-, and C24:1-Cer species; C16-, C24-, and C24:1-dihydroCer species; and C16-, C24-, and C24:1-SM species. Finally, exogenous C6-Cer or C16-Cer mimicked and enhanced the cytotoxic effects of the cytokines upon HOG cells, thereby supporting the cell death-signaling role of extracellular Cer.
Assuntos
Ceramidas/metabolismo , Interferon gama/metabolismo , Oligodendroglioma/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Morte Celular/genética , Linhagem Celular Tumoral , Ceramidas/química , Ceramidas/genética , Cromatografia Líquida de Alta Pressão , Exossomos , Vesículas Extracelulares/metabolismo , Humanos , Interferon gama/administração & dosagem , Interferon gama/genética , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Oligodendroglioma/patologia , Esfingolipídeos/química , Esfingolipídeos/metabolismo , Espectrometria de Massas em Tandem , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/genéticaRESUMO
A family of six ceramide synthases with distinct but overlapping substrate specificities is responsible for generation of ceramides with acyl chains ranging from â¼14-26 carbons. Ceramide synthase 6 (CerS6) preferentially generates C14- and C16-ceramides, and we have previously shown that down-regulation of this enzyme decreases apoptotic susceptibility. In this study, we further evaluated how increased CerS6 expression impacts sphingolipid composition and metabolism. Overexpression of CerS6 in HT29 colon cancer cells resulted in increased apoptotic susceptibility and preferential generation of C16-ceramide, which occurred at the expense of very long chain, saturated ceramides. These changes were also reflected in sphingomyelin composition. HT-CerS6 cells had increased intracellular levels of sphingosine, which is generated by ceramidases upon hydrolysis of ceramide. qRT-PCR analysis revealed that only expression of acid ceramidase (ASAH1) was increased. The increase in acid ceramidase was confirmed by expression and activity analyses. Pharmacological inhibition of JNK (SP600125) or curcumin reduced transcriptional up-regulation of acid ceramidase. Using an acid ceramidase promoter driven luciferase reporter plasmid, we demonstrated that CerS1 has no effect on transcriptional activation of acid ceramidase and that CerS2 slightly but significantly decreased the luciferase signal. Similar to CerS6, overexpression of CerS3-5 resulted in an â¼2-fold increase in luciferase reporter gene activity. Exogenous ceramide failed to induce reporter activity, while a CerS inhibitor and a catalytically inactive mutant of CerS6 failed to reduce it. Taken together, these results suggest that increased expression of CerS6 can mediate transcriptional activation of acid ceramidase in a JNK-dependent manner that is independent of CerS6 activity.
Assuntos
Ceramidase Ácida/metabolismo , Apoptose/efeitos dos fármacos , Ceramidas/farmacologia , Neoplasias do Colo/patologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas de Membrana/metabolismo , Esfingosina N-Aciltransferase/metabolismo , Ceramidase Ácida/genética , Antimetabólitos Antineoplásicos/farmacologia , Western Blotting , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Citometria de Fluxo , Fluoruracila/farmacologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas de Membrana/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esfingolipídeos/metabolismo , Esfingosina N-Aciltransferase/genética , Células Tumorais CultivadasRESUMO
Acid ceramidase has been identified as a promising target for cancer therapy. One of its most effective inhibitors, LCL521, was examined as adjuvant to photodynamic therapy (PDT) using mouse squamous cell carcinoma SCCVII model of head and neck cancer. Lethal effects of PDT, assessed by colony forming ability of in vitro treated SCCVII cells, were greatly enhanced when combined with 10 µM LCL521 treatment particularly when preceding PDT. When PDT-treated SCCVII cells are used to vaccinate SCCVII tumor-bearing mice (PDT vaccine protocol), adjuvant LCL521 treatment (75 mg/kg) resulted in a marked retardation of tumor growth. This effect can be attributed to the capacity of LCL521 to effectively restrict the activity of two main immunoregulatory cell populations (Tregs and myeloid-derived suppressor cells, MDSCs) that are known to hinder the efficacy of PDT vaccines. The therapeutic benefit with adjuvant LCL521 was also achieved with SCCVII tumors treated with standard PDT when using immunocompetent mice but not with immunodeficient hosts. The interaction of LCL521 with PDT-based antitumor mechanisms is dominated by immune system contribution that includes overriding the effects of immunoregulatory cells, but could also include a tacit contribution from boosting direct tumor cell kill.
Assuntos
Ceramidase Ácida/antagonistas & inibidores , Vacinas Anticâncer , Inibidores Enzimáticos/farmacologia , Fotoquimioterapia , Animais , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Terapia Combinada , Modelos Animais de Doenças , Humanos , Imunomodulação , Camundongos , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/terapia , Fenótipo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
BACKGROUND: We previously reported that dietary glucosylceramides show cancer-prevention activity in a mouse xenograft model of human head and neck cancer cells (SCCKN). However, the mechanism was unclear. Ceramides, metabolites of glucosylceramides, induce apoptotic cell death in various malignancies. Here, we investigated the inhibitory effects of dietary glucosylceramides on tumor growth in vivo and in vitro. METHODS: SCCKN were subcutaneously inoculated into the right flanks of NOD/SCID mice. Mice were treated with or without dietary glucosylceramides (300 mg/kg) daily for 14 consecutive days after confirmation of tumor progression. Microvessel areas around the tumor were assessed by hematoxylin-eosin staining and immunohistochemistry of CD31, and, as markers for angiogenesis, protein levels of VEGF, VEGF receptor-2, and HIF-1α were assessed by Western blotting. Mass spectrometry was performed to measure the levels of sphingolipids in mouse serum after treatment with dietary glucosylceramides. RESULTS: Oral administration of glucosylceramides significantly decreased SCCKN growth in the xenograft model with inhibition of angioinvasion. In tumor-invasive areas, VEGF and HIF-1α in the tumor cells, and VEGF receptor-2 in endothelial cells decreased after treatment with dietary glucosylceramides. Dietary glucosylceramides increased serum levels of sphingosine-based ceramides as compared to the control. In SCCKN and UVâ2 cells, C6-ceramide suppressed the expressions of VEGF, VEGF receptor-2, and HIF-1α in vitro. CONCLUSION: These results suggest that dietary glucosylceramides trigger the de novo pathway of ceramide synthesis, indicating that sphingosine-based ceramide suppresses the growth of head and neck tumors through the inhibition of pro-angiogenic signals such as VEGF, VEGF receptor-2, and HIF-1α.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Carcinoma de Células Escamosas/dietoterapia , Glucosilceramidas/administração & dosagem , Neoplasias de Cabeça e Pescoço/dietoterapia , Neovascularização Patológica/dietoterapia , Administração Oral , Animais , Carcinoma de Células Escamosas/metabolismo , Ceramidas/biossíntese , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neovascularização Patológica/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Ceramide is a bioeffector that mediates various cellular processes, including apoptosis. However, the mechanism underlying ceramide function in apoptosis is apparently cell type-dependent and is not well-understood. We aimed at identifying molecular targets of ceramide in metastatic human colon and breast cancer cells, and determining the efficacy of ceramide analog in suppression of colon and breast cancer metastasis. METHODS: The activity of and mechanism underlying ceramide as a cytotoxic agent, and as a sensitizer for Fas-mediated apoptosis was analyzed in human cell lines established from primary or metastatic colon and breast cancers. The efficacy of ceramide analog LCL85 in suppression of metastasis was examined in preclinical mouse tumor models. RESULTS: Exposure of human colon carcinoma cells to ceramide analog LCL85 results in apoptosis in a dose-dependent manner. Interestingly, a sublethal dose of LCL85 increased C16 ceramide content and overcame tumor cell resistance to Fas-mediated apoptosis. Subsequently, treatment of tumor cells with exogenous C16 ceramide resulted in increased tumor cell sensitivity to Fas-mediated apoptosis. LCL85 resembles Smac mimetic BV6 in sensitization of colon carcinoma cells to Fas-mediated apoptosis by inducing proteasomal degradation of cIAP1 and xIAP proteins. LCL85 also decreased xIAP1 and cIAP1 protein levels and sensitized metastatic human breast cancer cells to Fas-mediated apoptosis. Silencing xIAP and cIAP1 with specific siRNAs significantly increased the metastatic human colon carcinoma cell sensitivity to Fas-mediated apoptosis, suggesting that IAP proteins mediate apoptosis resistance in metastatic human colon carcinoma cells and ceramide induces IAP protein degradation to sensitize the tumor cells to apoptosis induction. Consistent with its apoptosis sensitization activity, subtoxic doses of LCL85 suppressed colon carcinoma cell metastatic potential in an experimental lung metastasis mouse model, as well as breast cancer growth and spontaneous lung metastasis in an orthotopic breast cancer mouse model. CONCLUSION: We have identified xIAP and cIAP1 as molecular targets of ceramide and determined that ceramide analog LCL85 is an effective sensitizer in overcoming resistance of human cell lines established from metastatic colon and breast cancers to apoptosis induction to suppress metastasis in vivo.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Ceramidas/farmacologia , Neoplasias do Colo/tratamento farmacológico , Proteínas Inibidoras de Apoptose/metabolismo , Propanolaminas/farmacologia , Compostos de Piridínio/farmacologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/secundário , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/secundário , Progressão da Doença , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Proteínas Inibidoras de Apoptose/genética , Camundongos , Camundongos Endogâmicos BALB C , Terapia de Alvo Molecular , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Transfecção , Ubiquitina-Proteína Ligases , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor fas/metabolismoRESUMO
The sphingolipid ceramide modulates stress-induced cell death and apoptosis. We have shown that ceramide generated via de novo sphingolipid biosynthesis is required to initiate apoptosis after photodynamic therapy (PDT). The objective of this study was to define the role of ceramide synthase (CERS) in PDT-induced cell death and apoptosis using fumonisin B1 (FB), a CERS inhibitor. We used the silicon phthalocyanine Pc4 for PDT, and SCC17B cells, as a clinically-relevant model of human head and neck squamous carcinoma. zVAD-fmk, a pan-caspase inhibitor, as well as FB, protected cells from death after PDT. In contrast, ABT199, an inhibitor of the anti-apoptotic protein Bcl2, enhanced cell killing after PDT. PDT-induced accumulation of ceramide in the endoplasmic reticulum and mitochondria was inhibited by FB. PDT-induced Bax translocation to the mitochondria and cytochrome c release were also inhibited by FB. These novel data suggest that PDT-induced cell death via apoptosis is CERS/ceramide-dependent.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fumonisinas/farmacologia , Indóis/química , Compostos de Organossilício/química , Oxirredutases/antagonistas & inibidores , Clorometilcetonas de Aminoácidos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Ceramidas/análise , Ceramidas/metabolismo , Citocromos c/metabolismo , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/uso terapêutico , Fumonisinas/uso terapêutico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Espectrometria de Massas , Mitocôndrias/química , Mitocôndrias/metabolismo , Oxirredutases/metabolismo , Fotoquimioterapia , Proteína X Associada a bcl-2/metabolismoRESUMO
Acid ceramidase (ACDase) is being recognized as a therapeutic target for cancer. B13 represents a moderate inhibitor of ACDase. The present study concentrates on the lysosomal targeting of B13 via its N,N-dimethylglycine (DMG) esters (DMG-B13 prodrugs). Novel analogs, the isomeric mono-DMG-B13, LCL522 (3-O-DMG-B13·HCl) and LCL596 (1-O-DMG-B13·HCl) and di-DMG-B13, LCL521 (1,3-O, O-DMG-B13·2HCl) conjugates, were designed and synthesized through N,N-dimethyl glycine (DMG) esterification of the hydroxyl groups of B13. In MCF7 cells, DMG-B13 prodrugs were efficiently metabolized to B13. The early inhibitory effect of DMG-B13 prodrugs on cellular ceramidases was ACDase specific by their lysosomal targeting. The corresponding dramatic decrease of cellular Sph (80-97% Control/1h) by DMG-B13 prodrugs was mainly from the inhibition of the lysosomal ACDase.
Assuntos
Ceramidase Ácida/antagonistas & inibidores , Amidas/química , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Nitrobenzenos/química , Pró-Fármacos/síntese química , Propanolaminas/química , Ceramidase Ácida/genética , Ceramidase Ácida/metabolismo , Amidas/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Ésteres , Células HeLa , Humanos , Lisossomos/enzimologia , Células MCF-7 , Nitrobenzenos/metabolismo , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Propanolaminas/metabolismo , Ligação ProteicaRESUMO
Ceramide is a bioactive sphingolipid involved in regulation of numerous cell signaling pathways. Evidence is accumulating that differences in ceramide structure, such as N-acyl chain length and desaturation of sphingoid base, determine the biological activities of ceramide. Using synthetic (R)-2'-hydroxy-C16-ceramide, which is the naturally occurring stereoisomer, we demonstrate that this ceramide has more potent pro-apoptotic activity compared to its (S) isomer or non-hydroxylated C16-ceramide. Upon exposure to (R)-2'-hydroxy-ceramide, C6 glioma cells rapidly underwent apoptosis as indicated by caspase-3 activation, PARP cleavage, chromatin condensation, and annexin V stain. A 2D gel proteomics analysis identified 28 proteins whose levels were altered during the initial 3 h of exposure. Using the list of 28 proteins, we performed a software-assisted pathway analysis to identify possible signaling events that would result in the observed changes. The result indicated that Akt and MAP kinase pathways are among the possible pathways regulated by (R)-2'-hydroxy-ceramide. Experimental validation confirmed that 2'-hydroxy-ceramide significantly altered phosphorylation status of Akt and its downstream effector GSK3ß, as well as p38, ERK1/2, and JNK1/2 MAP kinases. Unexpectedly, robust phosphorylation of Akt was observed within 1 h of exposure to 2'-hydroxy-ceramide, followed by dephosphorylation. Phosphorylation status of MAPKs showed a complex pattern, in which rapid phosphorylation of ERK1/2 was followed by dephosphorylation of p38 and ERK1/2 and phosphorylation of the 46 kDa isoform of JNK1/2. These data indicate that (R)-2'-hydroxy-ceramide regulates multiple signaling pathways by affecting protein kinases and phosphatases with kinetics distinct from that of the extensively studied non-hydroxy-ceramide or its unnatural stereoisomer.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ceramidas/farmacologia , Proteoma/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Glioma , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , RatosRESUMO
The sphingolipid, ceramide, self-assembles in the mitochondrial outer membrane (MOM), forming large channels capable of translocating proteins. These channels are believed to be involved in protein release from mitochondria, a key decision-making step in cell death. Synthetic analogs of ceramide, bearing modifications in each of the major structural features of ceramide were used to probe the molecular basis for the stability of ceramide channels. Channel stability and mitochondrial permeabilization were disrupted by methylation of the C1-hydroxyl group whereas modifications of the C3 allylic hydroxyl group were well tolerated. A change in chirality at C2 that would influence the orientation of the C1-hydroxyl group resulted in a strong reduction of channel-forming ability. Similarly, methylation of the amide nitrogen is also detrimental to channel formation. Many changes in the degree, location and nature of the unsaturation of ceramide had little effect on mitochondrial permeabilization. Competition experiments between ceramide and analogs resulted in synergy with structures compatible with the ceramide channel model and antagonism with incompatible structures. The results are consistent with ceramide channels being highly organized structures, stabilized by specific inter-molecular interactions, similar to the interactions responsible for protein folding.
Assuntos
Ceramidas/química , Canais Iônicos/química , Mitocôndrias Hepáticas/química , Membranas Mitocondriais/química , Modelos Moleculares , Animais , Cavalos , Masculino , Permeabilidade , Ratos , Ratos Sprague-DawleyRESUMO
Treatment of pancreatic cancer that cannot be surgically resected currently relies on minimally beneficial cytotoxic chemotherapy with gemcitabine. As the fourth leading cause of cancer-related death in the United States with dismal survival statistics, pancreatic cancer demands new and more effective treatment approaches. Resistance to gemcitabine is nearly universal and appears to involve defects in the intrinsic/mitochondrial apoptotic pathway. The bioactive sphingolipid ceramide is a critical mediator of apoptosis initiated by a number of therapeutic modalities. It is noteworthy that insufficient ceramide accumulation has been linked to gemcitabine resistance in multiple cancer types, including pancreatic cancer. Taking advantage of the fact that cancer cells frequently have more negatively charged mitochondria, we investigated a means to circumvent resistance to gemcitabine by targeting delivery of a cationic ceramide (l-t-C6-CCPS [LCL124: ((2S,3S,4E)-2-N-[6'-(1â³-pyridinium)-hexanoyl-sphingosine bromide)]) to cancer cell mitochondria. LCL124 was effective in initiating apoptosis by causing mitochondrial depolarization in pancreatic cancer cells but demonstrated significantly less activity against nonmalignant pancreatic ductal epithelial cells. Furthermore, we demonstrate that the mitochondrial membrane potentials of the cancer cells were more negative than nonmalignant cells and that dissipation of this potential abrogated cell killing by LCL124, establishing that the effectiveness of this compound is potential-dependent. LCL124 selectively accumulated in and inhibited the growth of xenografts in vivo, confirming the tumor selectivity and therapeutic potential of cationic ceramides in pancreatic cancer. It is noteworthy that gemcitabine-resistant pancreatic cancer cells became more sensitive to subsequent treatment with LCL124, suggesting that this compound may be a uniquely suited to overcome gemcitabine resistance in pancreatic cancer.
Assuntos
Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Ceramidas/farmacologia , Mitocôndrias/metabolismo , Neoplasias Pancreáticas/patologia , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzimidazóis , Western Blotting , Carbocianinas , Linhagem Celular Tumoral , Ceramidas/metabolismo , Cromatografia Líquida de Alta Pressão , Corantes , Citocromos c/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Feminino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Nus , Consumo de Oxigênio/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Análise Espectral , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
The present study demonstrates the important structural features of ceramide required for proper regulation, binding and identification by both pro-apoptotic and anti-apoptotic Bcl-2 family proteins. The C-4=C-5 trans-double bond has little influence on the ability of Bax and Bcl-xL to identify and bind to these channels. The stereochemistry of the headgroup and access to the amide group of ceramide is indispensible for Bax binding, indicating that Bax may interact with the polar portion of the ceramide channel facing the bulk phase. In contrast, Bcl-xL binding to ceramide channels is tolerant of stereochemical changes in the headgroup. The present study also revealed that Bcl-xL has an optimal interaction with long-chain ceramides that are elevated early in apoptosis, whereas short-chain ceramides are not well regulated. Inhibitors specific for the hydrophobic groove of Bcl-xL, including 2-methoxyantimycin A3, ABT-737 and ABT-263 provide insights into the region of Bcl-xL involved in binding to ceramide channels. Molecular docking simulations of the lowest-energy binding poses of ceramides and Bcl-xL inhibitors to Bcl-xL were consistent with the results of our functional studies and propose potential binding modes.
Assuntos
Apoptose , Ceramidas/farmacologia , Canais Iônicos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Compostos de Anilina/farmacologia , Animais , Compostos de Bifenilo/farmacologia , Caspases/metabolismo , Simulação por Computador , Citocromos c/metabolismo , Canais Iônicos/efeitos dos fármacos , Masculino , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Modelos Moleculares , Nitrofenóis/farmacologia , Oxirredução , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Ratos Sprague-Dawley , Sulfonamidas/farmacologia , Proteína X Associada a bcl-2/genéticaRESUMO
The current study sought to assess cognitive and emotional functions among children and adolescents with atrioventricular reentry tachycardia (AVRT) and atrioventricular nodal reentry tachycardia (AVNRT). 113 patients (62 girls and 51 boys ages, 9-18 years) scheduled for radiofrequency ablation due to AVRT or AVNRT underwent neuropsychologic examination. The study excluded patients who had experienced cardiac arrest, congenital heart defects, neurologic disorders, or other diseases affecting cognitive or emotional development. Standardized tests for examining verbal and visual memory as well as visual-spatial functioning were performed. For patients exhibiting deficits in two or more tests, a diagnosis of "cognitive deficits" was determined. Levels of anxiety were tested using the State-Trait Anxiety Inventory. Cognitive deficits were found in 47.8 % of the patients. The age at first arrhythmia attack was related to memory dysfunction. The mean age at which the first symptoms occurred was significantly lower for patients with deficits (8.3 years) than for patients who had no deficit (10.2 years) (t = 2.15; p = 0.03). Boys exhibited a significantly higher level of trait anxiety than girls (t = 3.42; p = 0.0009). A significant negative correlation was found between anxiety and the age at appearance of the first symptoms (r = -0.26; p = 0.005). These findings led us to conclude that cognitive and emotional developments can be negatively affected by AVNRT and AVRT, particularly if tachycardia appears early in life.
Assuntos
Transtornos Cognitivos/etiologia , Transtornos Cognitivos/psicologia , Emoções , Átrios do Coração/fisiopatologia , Taquicardia por Reentrada no Nó Atrioventricular/complicações , Taquicardia por Reentrada no Nó Atrioventricular/psicologia , Taquicardia Supraventricular/complicações , Taquicardia Supraventricular/psicologia , Síndrome de Wolff-Parkinson-White/complicações , Síndrome de Wolff-Parkinson-White/psicologia , Adolescente , Distribuição de Qui-Quadrado , Criança , Transtornos Cognitivos/fisiopatologia , Eletrocardiografia , Feminino , Humanos , Masculino , Testes Neuropsicológicos , Fatores Sexuais , Taquicardia por Reentrada no Nó Atrioventricular/fisiopatologia , Taquicardia Supraventricular/fisiopatologia , Síndrome de Wolff-Parkinson-White/fisiopatologiaRESUMO
Transferrin (Tf) endocytosis and recycling are essential for iron uptake and the regulation of cell proliferation. Tf and Tf receptor (TfR) complexes are internalized via clathrin-coated pits composed of a variety of proteins and lipids and pass through early endosomes to recycling endosomes. We investigated the role of sphingomyelin (SM) synthases (SMS1 and SMS2) in clathrin-dependent trafficking of Tf and cell proliferation. We employed SM-deficient lymphoma cells that lacked SMSs and that failed to proliferate in response to Tf. Transfection of SMS1, but not SMS2, enabled these cells to incorporate SM into the plasma membrane, restoring Tf-mediated proliferation. SM-deficient cells showed a significant reduction in clathrin-dependent Tf uptake compared with the parental SM-producing cells. Both SMS1 gene transfection and exogenous short-chain SM treatment increased clathrin-dependent Tf uptake in SM-deficient cells, with the Tf being subsequently sorted to Rab11-positive recycling endosomes. We observed trafficking of the internalized Tf to late/endolysosomal compartments, and this was not dependent on the clathrin pathway in SM-deficient cells. Thus, SMS1-mediated SM synthesis directs Tf-TfR to undergo clathrin-dependent endocytosis and recycling, promoting the proliferation of lymphoma cells.