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1.
J Lipid Res ; 61(11): 1464-1479, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32769146

RESUMO

Multiple sclerosis (MS) is a CNS disease characterized by immune-mediated demyelination and progressive axonal loss. MS-related CNS damage and its clinical course have two main phases: active and inactive/progressive. Reliable biomarkers are being sought to allow identification of MS pathomechanisms and prediction of its course. The purpose of this study was to identify sphingolipid (SL) species as candidate biomarkers of inflammatory and neurodegenerative processes underlying MS pathology. We performed sphingolipidomic analysis by HPLC-tandem mass spectrometry to determine the lipid profiles in post mortem specimens from the normal-appearing white matter (NAWM) of the normal CNS (nCNS) from subjects with chronic MS (active and inactive lesions) as well as from patients with other neurological diseases. Distinctive SL modification patterns occurred in specimens from MS patients with chronic inactive plaques with respect to NAWM from the nCNS and active MS (Ac-MS) lesions. Chronic inactive MS (In-MS) lesions were characterized by decreased levels of dihydroceramide (dhCer), ceramide (Cer), and SM subspecies, whereas levels of hexosylceramide and Cer 1-phosphate (C1P) subspecies were significantly increased in comparison to NAWM of the nCNS as well as Ac-MS plaques. In contrast, Ac-MS lesions were characterized by a significant increase of major dhCer subspecies in comparison to NAWM of the nCNS. These results suggest the existence of different SL metabolic pathways in the active versus inactive phase within progressive stages of MS. Moreover, they suggest that C1P could be a new biomarker of the In-MS progressive phase, and its detection may help to develop future prognostic and therapeutic strategies for the disease.


Assuntos
Esclerose Múltipla/metabolismo , Esfingolipídeos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença Crônica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/diagnóstico , Esfingolipídeos/análise
2.
J Biol Chem ; 294(2): 502-519, 2019 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-30420430

RESUMO

Formation of membrane pores/channels regulates various cellular processes, such as necroptosis or stem cell niche signaling. However, the roles of membrane lipids in the formation of pores and their biological functions are largely unknown. Here, using the cellular stress model evoked by the sphingolipid analog drug FTY720, we show that formation of ceramide-enriched membrane pores, referred to here as ceramidosomes, is initiated by a receptor-interacting Ser/Thr kinase 1 (RIPK1)-ceramide complex transported to the plasma membrane by nonmuscle myosin IIA-dependent trafficking in human lung cancer cells. Molecular modeling/simulation coupled with site-directed mutagenesis revealed that Asp147 or Asn169 of RIPK1 are key for ceramide binding and that Arg258 or Leu293 residues are involved in the myosin IIA interaction, leading to ceramidosome formation and necroptosis. Moreover, generation of ceramidosomes independently of any external drug/stress stimuli was also detected in the plasma membrane of germ line stem cells in ovaries during the early stages of oogenesis in Drosophila melanogaster Inhibition of ceramidosome formation via myosin IIA silencing limited germ line stem cell signaling and abrogated oogenesis. In conclusion, our findings indicate that the RIPK1-ceramide complex forms large membrane pores we named ceramidosomes. They further suggest that, in addition to their roles in stress-mediated necroptosis, these ceramide-enriched pores also regulate membrane integrity and signaling and might also play a role in D. melanogaster ovary development.


Assuntos
Membrana Celular/metabolismo , Ceramidas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Motores Moleculares/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Necrose/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Células A549 , Animais , Linhagem Celular , Membrana Celular/patologia , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Humanos , Neoplasias Pulmonares/patologia , Simulação de Acoplamento Molecular , Necrose/patologia , Oogênese , Ovário/crescimento & desenvolvimento
3.
Cytokine ; 135: 155219, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32738771

RESUMO

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 DBA
4.
FASEB J ; 33(6): 7647-7666, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30917007

RESUMO

The su(var)3-9, enhancer of zeste, trithorax (SET)/inhibitor 2 of protein phosphatase 2A (PP2A) oncoprotein binds and inhibits PP2A, composed of various isoforms of scaffolding, regulatory, and catalytic subunits. Targeting SET with a sphingolipid analog drug fingolimod (FTY720) or ceramide leads to the reactivation of tumor suppressor PP2A. However, molecular details of the SET-FTY720 or SET-ceramide, and mechanism of FTY720-dependent PP2A activation, remain unknown. Here, we report the first in solution examination of the SET-FTY720 or SET-ceramide complexes by NMR spectroscopy. FTY720-ceramide binding resulted in chemical shifts of residues residing at the N terminus of SET, preventing its dimerization or oligomerization. This then released SET from PP2ACα, resulting in PP2A activation, while monomeric SET remained associated with the B56γ. Our data also suggest that the PP2A holoenzyme, composed of PP2A-Aß, PP2A-B56γ, and PP2ACα subunits, is selectively activated in response to the formation of the SET-FTY720 complex in A549 cells. Various PP2A-associated downstream effector proteins in the presence or absence of FTY720 were then identified by stable isotope labeling with amino cells in cell culture, including tumor suppressor nonmuscle myosin IIA. Attenuation of FTY720-SET association by point mutations of residues that are involved in FTY720 binding or dephosphorylation of SET at Serine 171, enhanced SET oligomerization and the formation of the SET-PP2A inhibitory complex, leading to resistance to FTY720-dependent PP2A activation.-De Palma, R. M., Parnham, S. R., Li, Y., Oaks, J. J., Peterson, Y. K., Szulc, Z. M., Roth, B. M., Xing, Y., Ogretmen, B. The NMR-based characterization of the FTY720-SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET-PP2A interaction.


Assuntos
Cloridrato de Fingolimode/farmacologia , Histona-Lisina N-Metiltransferase/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Proteína Fosfatase 2/metabolismo , Moduladores do Receptor de Esfingosina 1 Fosfato/farmacologia , Células A549 , Dimerização , Humanos , Ligação Proteica
5.
Photochem Photobiol Sci ; 19(9): 1145-1151, 2020 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-32821888

RESUMO

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 Cultivadas
6.
Blood ; 128(15): 1944-1958, 2016 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-27540013

RESUMO

Signaling pathways regulated by mutant Fms-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD), which mediate resistance to acute myeloid leukemia (AML) cell death, are poorly understood. Here, we reveal that pro-cell death lipid ceramide generation is suppressed by FLT3-ITD signaling. Molecular or pharmacologic inhibition of FLT3-ITD reactivated ceramide synthesis, selectively inducing mitophagy and AML cell death. Mechanistically, FLT3-ITD targeting induced ceramide accumulation on the outer mitochondrial membrane, which then directly bound autophagy-inducing light chain 3 (LC3), involving its I35 and F52 residues, to recruit autophagosomes for execution of lethal mitophagy. Short hairpin RNA (shRNA)-mediated knockdown of LC3 prevented AML cell death in response to FLT3-ITD inhibition by crenolanib, which was restored by wild-type (WT)-LC3, but not mutants of LC3 with altered ceramide binding (I35A-LC3 or F52A-LC3). Mitochondrial ceramide accumulation and lethal mitophagy induction in response to FLT3-ITD targeting was mediated by dynamin-related protein 1 (Drp1) activation via inhibition of protein kinase A-regulated S637 phosphorylation, resulting in mitochondrial fission. Inhibition of Drp1 prevented ceramide-dependent lethal mitophagy, and reconstitution of WT-Drp1 or phospho-null S637A-Drp1 but not its inactive phospho-mimic mutant (S637D-Drp1), restored mitochondrial fission and mitophagy in response to crenolanib in FLT3-ITD+ AML cells expressing stable shRNA against endogenous Drp1. Moreover, activating FLT3-ITD signaling in crenolanib-resistant AML cells suppressed ceramide-dependent mitophagy and prevented cell death. FLT3-ITD+ AML drug resistance is attenuated by LCL-461, a mitochondria-targeted ceramide analog drug, in vivo, which also induced lethal mitophagy in human AML blasts with clinically relevant FLT3 mutations. Thus, these data reveal a novel mechanism which regulates AML cell death by ceramide-dependent mitophagy in response to FLT3-ITD targeting.


Assuntos
Benzimidazóis/farmacologia , Ceramidas , Resistencia a Medicamentos Antineoplásicos , Leucemia Mieloide Aguda , Mitofagia , Mutação , Piperidinas/farmacologia , RNA Interferente Pequeno/farmacologia , Transdução de Sinais , Tirosina Quinase 3 Semelhante a fms , Animais , Ceramidas/genética , Ceramidas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Dinaminas , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mitofagia/efeitos dos fármacos , Mitofagia/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo
7.
Artigo em Inglês | MEDLINE | ID: mdl-28377281

RESUMO

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/genética
8.
J Lipid Res ; 57(11): 2028-2039, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27623848

RESUMO

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/genética
9.
Int J Cancer ; 139(6): 1372-8, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27136745

RESUMO

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/metabolismo
10.
Nat Chem Biol ; 8(10): 831-8, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22922758

RESUMO

Mechanisms by which autophagy promotes cell survival or death are unclear. We provide evidence that C(18)-pyridinium ceramide treatment or endogenous C(18)-ceramide generation by ceramide synthase 1 (CerS1) expression mediates autophagic cell death, independent of apoptosis in human cancer cells. C(18)-ceramide-induced lethal autophagy was regulated via microtubule-associated protein 1 light chain 3 ß-lipidation, forming LC3B-II, and selective targeting of mitochondria by LC3B-II-containing autophagolysosomes (mitophagy) through direct interaction between ceramide and LC3B-II upon Drp1-dependent mitochondrial fission, leading to inhibition of mitochondrial function and oxygen consumption. Accordingly, expression of mutant LC3B with impaired ceramide binding, as predicted by molecular modeling, prevented CerS1-mediated mitochondrial targeting, recovering oxygen consumption. Moreover, knockdown of CerS1 abrogated sodium selenite-induced mitophagy, and stable LC3B knockdown protected against CerS1- and C(18)-ceramide-dependent mitophagy and blocked tumor suppression in vivo. Thus, these data suggest a new receptor function of ceramide for anchoring LC3B-II autophagolysosomes to mitochondrial membranes, defining a key mechanism for the induction of lethal mitophagy.


Assuntos
Autofagia , Ceramidas/farmacologia , Mitofagia/efeitos dos fármacos , Fagossomos/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Lipídeos/química , Microscopia Confocal
11.
Bioorg Med Chem ; 22(24): 6933-44, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25456083

RESUMO

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 Proteica
12.
iScience ; 27(6): 109860, 2024 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-38779482

RESUMO

Mechanisms by which Porphyromonas gingivalis (P. gingivalis) infection enhances oral tumor growth or resistance to cell death remain elusive. Here, we determined that P. gingivalis infection mediates therapeutic resistance via inhibiting lethal mitophagy in cancer cells and tumors. Mechanistically, P. gingivalis targets the LC3B-ceramide complex by associating with LC3B via bacterial major fimbriae (FimA) protein, preventing ceramide-dependent mitophagy in response to various therapeutic agents. Moreover, ceramide-mediated mitophagy is induced by Annexin A2 (ANXA2)-ceramide association involving the E142 residue of ANXA2. Inhibition of ANXA2-ceramide-LC3B complex formation by wild-type P. gingivalis prevented ceramide-dependent mitophagy. Moreover, a FimA-deletion mutant P. gingivalis variant had no inhibitory effects on ceramide-dependent mitophagy. Further, 16S rRNA sequencing of oral tumors indicated that P. gingivalis infection altered the microbiome of the tumor macroenvironment in response to ceramide analog treatment in mice. Thus, these data provide a mechanism describing the pro-survival roles of P. gingivalis in oral tumors.

13.
J Proteome Res ; 12(10): 4366-75, 2013 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-23987666

RESUMO

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 , Ratos
14.
Biochim Biophys Acta ; 1818(5): 1291-301, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22365970

RESUMO

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-Dawley
15.
J Pharmacol Exp Ther ; 344(1): 167-78, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23086228

RESUMO

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'-(1″-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 , Gencitabina
16.
Biochem J ; 445(1): 81-91, 2012 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-22494048

RESUMO

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/genética
17.
Mult Scler Relat Disord ; 71: 104565, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36821978

RESUMO

BACKGROUND: The disturbed metabolism of ceramide (Cer) is supposed to evoke the autoimmune response, contributing to MS pathology. OBJECTIVES: To determine levels of anti-Cer immunoglobulins G (IgGs) in the CSF and serum of subjects with various phenotypes of MS, and to investigate relationships between levels of anti-Cer antibodies and MS-related variables. METHODS: IgGs isolated from serum and the CSF of 68 MS patients and appropriate controls were examined for their reactivity to Cer subspecies. Their levels were compared between the studied groups and compartments, and analyzed with regard to clinical variables. RESULTS: Increased levels of anti-C16:0-, C18:0-, C18:1-, C24:0- and C24:1-Cer IgGs were detected in the CSF and serum of MS patients in comparison with controls. For IgGs against particular Cer subspecies, correlations were found between their CSF and serum level, as well as with the Link index. Serum and the CSF anti-Cer IgGs differed between patients with clinically isolated syndrome (CIS) and relapsing-remitting MS from those with progressive MS. No correlations were found between anti-Cer IgGs and other MS-related clinical variables. CONCLUSION: Patients with MS have shown altered panels of anti-Cer IgGs in the CSF and serum, which might suggest a relevant, though limited role of Cer as a target for autoimmune humoral response. Utility of antibodies against Cer subspecies as potential markers for MS activity and progression deserves further investigations.


Assuntos
Doenças Desmielinizantes , Esclerose Múltipla , Humanos , Ceramidas , Autoimunidade , Imunoglobulina G
18.
Aging Cell ; 22(10): e13954, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37614052

RESUMO

The metabolic consequences of mitophagy alterations due to age-related stress in healthy aging brains versus neurodegeneration remain unknown. Here, we demonstrate that ceramide synthase 1 (CerS1) is transported to the outer mitochondrial membrane by the p17/PERMIT transporter that recognizes mislocalized mitochondrial ribosomes (mitoribosomes) via 39-FLRN-42 residues, inducing ceramide-mediated mitophagy. P17/PERMIT-CerS1-mediated mitophagy attenuated the argininosuccinate/fumarate/malate axis and induced d-glucose and fructose accumulation in neurons in culture and brain tissues (primarily in the cerebellum) of wild-type mice in vivo. These metabolic changes in response to sodium-selenite were nullified in the cerebellum of CerS1to/to (catalytically inactive for C18-ceramide production CerS1 mutant), PARKIN-/- or p17/PERMIT-/- mice that have dysfunctional mitophagy. Whereas sodium selenite induced mitophagy in the cerebellum and improved motor-neuron deficits in aged wild-type mice, exogenous fumarate or malate prevented mitophagy. Attenuating ceramide-mediated mitophagy enhanced damaged mitochondria accumulation and age-dependent sensorimotor abnormalities in p17/PERMIT-/- mice. Reinstituting mitophagy using a ceramide analog drug with selenium conjugate, LCL768, restored mitophagy and reduced malate/fumarate metabolism, improving sensorimotor deficits in old p17/PERMIT-/- mice. Thus, these data describe the metabolic consequences of alterations to p17/PERMIT/ceramide-mediated mitophagy associated with the loss of mitochondrial quality control in neurons and provide therapeutic options to overcome age-dependent sensorimotor deficits and related disorders like amyotrophic lateral sclerosis (ALS).


Assuntos
Malatos , Mitofagia , Camundongos , Animais , Ceramidas/metabolismo , Neurônios Motores/metabolismo , Fumaratos , Ubiquitina-Proteína Ligases
19.
bioRxiv ; 2023 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-37961314

RESUMO

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy requiring urgent treatment advancements. Ceramide is a cell death-promoting signaling lipid that plays a central role in therapy-induced cell death. Acid ceramidase (AC), a ceramide-depleting enzyme, is overexpressed in AML and promotes leukemic survival and drug resistance. The ceramidase inhibitor B-13 and next-generation lysosomal-localizing derivatives termed dimethylglycine (DMG)-B-13 prodrugs have been developed but remain untested in AML. Here, we report the in vitro anti-leukemic efficacy and mechanism of DMG-B-13 prodrug, LCL-805, across AML cell lines and primary patient samples. LCL-805 inhibited AC enzymatic activity, increased total ceramides, and reduced sphingosine levels. A median EC50 value of 11.7 µM was achieved for LCL-805 in cell viability assays across 32 human AML cell lines. As a single agent tested across a panel of 71 primary AML patient samples, a median EC50 value of 15.8 µM was achieved. Exogenous ceramide supplementation with C6-ceramide nanoliposomes, which is entering phase I/II clinical trial for relapsed/refractory AML, significantly enhanced LCL-805 killing. Mechanistically, LCL-805 antagonized Akt signaling and led to iron-dependent cell death distinct from canonical ferroptosis. These findings elucidated key factors involved in LCL-805 cytotoxicity and demonstrated the potency of combining AC inhibition with exogenous ceramide.

20.
Cancers (Basel) ; 15(24)2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-38136410

RESUMO

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy requiring urgent treatment advancements. Ceramide is a cell-death-promoting signaling lipid that plays a central role in therapy-induced cell death. We previously determined that acid ceramidase (AC), a ceramide-depleting enzyme, is overexpressed in AML and promotes leukemic survival and drug resistance. The ceramidase inhibitor B-13 and next-generation lysosomal-localizing derivatives termed dimethylglycine (DMG)-B-13 prodrugs have been developed but remain untested in AML. Here, we report the in vitro anti-leukemic efficacy and mechanism of DMG-B-13 prodrug LCL-805 across AML cell lines and primary patient samples. LCL-805 inhibited AC enzymatic activity, increased total ceramides, and reduced sphingosine levels. A median EC50 value of 11.7 µM was achieved for LCL-805 in cell viability assays across 32 human AML cell lines. As a single agent tested across a panel of 71 primary AML patient samples, a median EC50 value of 15.8 µM was achieved. Exogenous ceramide supplementation with C6-ceramide nanoliposomes, which is entering phase I/II clinical trial for relapsed/refractory AML, significantly enhanced LCL-805 killing. Mechanistically, LCL-805 antagonized Akt signaling and led to iron-dependent cell death distinct from canonical ferroptosis. These findings elucidated key factors involved in LCL-805 cytotoxicity and demonstrated the potency of combining AC inhibition with exogenous ceramide.

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