RESUMO
It is well established that lysosomes play an active role during the execution of cell death. A range of stimuli can lead to lysosomal membrane permeabilization (LMP), thus inducing programmed cell death without involvement of the classical apoptotic programme. However, these lysosomal pathways of cell death have mostly been described in vitro or under pathological conditions. Here we show that the physiological process of post-lactational regression of the mammary gland is accomplished through a non-classical, lysosomal-mediated pathway of cell death. We found that, during involution, lysosomes in the mammary epithelium undergo widespread LMP. Furthermore, although cell death through LMP is independent of executioner caspases 3, 6 and 7, it requires Stat3, which upregulates the expression of lysosomal proteases cathepsin B and L, while downregulating their endogenous inhibitor Spi2A (ref. 8). Our findings report a previously unknown, Stat3-regulated lysosomal-mediated pathway of cell death under physiological circumstances. We anticipate that these findings will be of major importance in the design of treatments for cancers such as breast, colon and liver, where cathepsins and Stat3 are commonly overexpressed and/or hyperactivated respectively.
Assuntos
Regulação da Expressão Gênica , Lisossomos/metabolismo , Neoplasias/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Catepsina B/metabolismo , Catepsina L/metabolismo , Catepsinas/metabolismo , Morte Celular , Permeabilidade da Membrana Celular , Cruzamentos Genéticos , Feminino , Imuno-Histoquímica/métodos , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in cancer development through stimulation of cell survival, proliferation, migration, and angiogenesis. Irreversible degradation of S1P is catalyzed by S1P lyase (SPL). The human SGPL1 gene that encodes SPL maps to a region often mutated in cancers. To investigate the effect of SPL deficiency on cell survival and transformation, the susceptibility to anticancer drugs of fibroblasts generated from SPL-deficient mouse embryos (Sgpl1(-/-)) was compared with that of cells from heterozygous (Sgpl1(+/-)) or wild-type (Sgpl1(+/+)) embryos. First, loss of SPL caused resistance to the toxic effects of etoposide and doxorubicin. Interestingly, heterozygosity for the Sgpl1 gene resulted in partial resistance to apoptosis. Secondly, doxorubicin-induced apoptotic signaling was strongly inhibited in Sgpl1(-/-) cells (phosphatidylserine externalization, caspase activation, and cytochrome c release). This was accompanied by a strong increase in Bcl-2 and Bcl-xL protein content. Whereas correction of SPL deficiency in Sgpl1(-/-) cells led to downregulation of antiapoptotic proteins, Bcl-2 and Bcl-xL small interfering RNA-mediated knockdown in SPL-deficient cells resulted in increased sensitivity to doxorubicin, suggesting that Bcl-2 upregulation mediates SPL protective effects. Moreover, SPL deficiency led to increased cell proliferation, anchorage-independent cell growth, and formation of tumors in nude mice. Finally, transcriptomic studies showed that SPL expression is downregulated in human melanoma cell lines. Thus, by affecting S1P metabolism and the expression of Bcl-2 members, the loss of SPL enhances cell resistance to anticancer regimens and results in an increased ability of cells to acquire a transformed phenotype and become malignant.
Assuntos
Aldeído Liases/deficiência , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteína bcl-X/biossíntese , Aldeído Liases/biossíntese , Aldeído Liases/genética , Aldeído Liases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Autofagia/efeitos dos fármacos , Autofagia/genética , Linhagem Celular Tumoral , Regulação para Baixo , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Dosagem de Genes , Humanos , Melanoma/genética , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Regulação para Cima , Proteína bcl-X/genéticaRESUMO
Much is known about the pathways that control the biosynthesis, transport and degradation of sphingolipids. During the last two decades, considerable progress has been made regarding the roles this complex group of lipids play in maintaining membrane integrity and modulating responses to numerous signals. Further novel insights have been provided by the analysis of newly discovered genetic diseases in humans as well as in animal models harboring mutations in the genes whose products control sphingolipid metabolism and action. Through the description of the phenotypic consequences of genetic defects resulting in the loss of activity of the many proteins that synthesize, transport, bind, or degrade sphingolipids, this review summarizes the (patho)physiological functions of these lipids.
Assuntos
Erros Inatos do Metabolismo Lipídico/metabolismo , Esfingolipídeos/metabolismo , Animais , Galactosilceramidas/fisiologia , Gangliosídeos/fisiologia , Glucosilceramidas/fisiologia , Humanos , Lactosilceramidas/fisiologia , Lisofosfolipídeos/metabolismo , Camundongos , Transporte Proteico , Receptores de Lisoesfingolipídeo/deficiência , Esfingomielina Fosfodiesterase/deficiência , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Sulfoglicoesfingolipídeos/metabolismoRESUMO
The Nuclear Factor-kappaB (NF-kappaB) family of transcription factors are ubiquitously expressed and control a wide range of cellular responses, including apoptosis, proliferation, differentiation, inflammation and immunity. Here, we investigated the function of the NF-kappaB upstream regulator IkappaB kinase 2/beta (IKK2) in apoptosis regulation in the normal physiological setting of regressing mammary gland. Conditional deletion of the gene encoding IKK2 resulted, surprisingly, in delayed apoptosis and remodelling, and abrogation of caspase 3 cleavage. This failure to induce involution was associated with reduced expression, within 24 hours of involution, of the death receptor (DR) ligand TNF and its receptor TNFR1, which are known NF-kappaB targets. This was associated with elevated levels of active AKT and phosphorylated FOXO3a. Furthermore, we show that expression of TWEAK, another DR ligand, is dramatically downregulated, even in heterozygous IKK2 mammary glands. Unlike other DR ligands, the TWEAK promoter has six consensus FOXO-binding sites, further suggesting that it is differentially regulated. Interestingly, a cleaved form of TWEAK is upregulated during involution. This unexpected function of the IKK2/NF-kappaB pathway as a regulator of TWEAK expression and inducer of apoptosis has significant consequences for future therapeutic approaches for cancer and inflammatory diseases.
Assuntos
Quinase I-kappa B/fisiologia , Fatores de Necrose Tumoral/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose , Citocina TWEAK , Células Epiteliais/metabolismo , Feminino , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Quinase I-kappa B/genética , Ligantes , Glândulas Mamárias Animais/citologia , Camundongos , Regiões Promotoras Genéticas , Transdução de Sinais , Fatores de Necrose Tumoral/genéticaRESUMO
Tamoxifen is a selective estrogen receptor modulator widely used for the prophylactic treatment of breast cancer. In addition to the estrogen receptor (ER), tamoxifen binds with high affinity to the microsomal antiestrogen binding site (AEBS), which is involved in ER-independent effects of tamoxifen. In the present study, we investigate the modulation of the biosynthesis of cholesterol in tumor cell lines by AEBS ligands. As a consequence of the treatment with the antitumoral drugs tamoxifen or PBPE, a selective AEBS ligand, we show that tumor cells produced a significant concentration- and time-dependent accumulation of cholesterol precursors. Sterols have been purified by HPLC and gas chromatography, and their chemical structures determined by mass spectrometric analysis. The major metabolites identified were 5alpha-cholest-8-en-3beta-ol for tamoxifen treatment and 5alpha-cholest-8-en-3beta-ol and cholesta-5,7-dien-3beta-ol, for PBPE treatment, suggesting that these AEBS ligands affect at least two enzymatic steps: the 3beta-hydroxysterol-Delta8-Delta7-isomerase and the 3beta-hydroxysterol-Delta7-reductase. Steroidal antiestrogens such as ICI 182,780 and RU 58,668 did not affect these enzymatic steps, because they do not bind to the AEBS. Transient co-expression of human 3beta-hydroxysterol-Delta8-Delta7-isomerase and 3beta-hydroxysterol-Delta7-reductase and immunoprecipitation experiments showed that both enzymes were required to reconstitute the AEBS in mammalian cells. Altogether, these data provide strong evidence that the AEBS is a hetero-oligomeric complex including 3beta-hydroxysterol-Delta8-Delta7-isomerase and the 3beta-hydroxysterol-Delta7-reductase as subunits that are necessary and sufficient for tamoxifen binding in mammary cells. Furthermore, because selective AEBS ligands are antitumoral compounds, these data suggest a link between cholesterol metabolism at a post-lanosterol step and tumor growth control. These data afford both the identification of the AEBS and give new insight into a novel molecular mechanism of action for drugs of clinical value.