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1.
Mol Cell ; 57(5): 860-872, 2015 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-25702873

RESUMO

During apoptosis, the mitochondrial outer membrane is permeabilized, leading to the release of cytochrome c that activates downstream caspases. Mitochondrial outer membrane permeabilization (MOMP) has historically been thought to occur synchronously and completely throughout a cell, leading to rapid caspase activation and apoptosis. Using a new imaging approach, we demonstrate that MOMP is not an all-or-nothing event. Rather, we find that a minority of mitochondria can undergo MOMP in a stress-regulated manner, a phenomenon we term "minority MOMP." Crucially, minority MOMP leads to limited caspase activation, which is insufficient to trigger cell death. Instead, this caspase activity leads to DNA damage that, in turn, promotes genomic instability, cellular transformation, and tumorigenesis. Our data demonstrate that, in contrast to its well-established tumor suppressor function, apoptosis also has oncogenic potential that is regulated by the extent of MOMP. These findings have important implications for oncogenesis following either physiological or therapeutic engagement of apoptosis.


Assuntos
Apoptose/fisiologia , Dano ao DNA , Instabilidade Genômica , Membranas Mitocondriais/fisiologia , Animais , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Western Blotting , Caspases/metabolismo , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p19/deficiência , Inibidor de Quinase Dependente de Ciclina p19/genética , Relação Dose-Resposta a Droga , Embrião de Mamíferos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HCT116 , Células HeLa , Histonas/metabolismo , Humanos , Células MCF-7 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Nitrofenóis/farmacologia , Permeabilidade , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Estaurosporina/farmacologia , Sulfonamidas/farmacologia , Fatores de Tempo
2.
Blood ; 123(18): 2806-15, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24622325

RESUMO

All 6 human prosurvival Bcl-2 proteins can drive cancer development and contribute to therapy resistance. However, their relative abilities to protect cells against cancer therapy were not examined previously. We report that Bcl-2, Bcl-xL, or Bcl-w consistently protected leukemic cells better than Bcl-B, Bfl-1, or Mcl-1 against a wide variety of anticancer regimens. Current thinking would attribute this to differences in their ability to bind to BH3-only proteins, Bax, and Bak. To address this, we established the first complete, quantitative cellular interaction profile of all human prosurvival Bcl-2 proteins with all their proapoptotic relatives. Binding was unexpectedly promiscuous, except for Bad and Noxa, and did not explain the differential antiapoptotic capacity of the Bcl-2 proteins. Rather, Bcl-B, Bfl-1, or Mcl-1 proved less potent due to steady-state or drug-induced proteasomal degradation. All 6 Bcl-2 proteins similarly protected against the diverse anticancer regimens when expressed at equal protein levels, in agreement with their broad interaction profile. Therefore, clinical diagnostics should include all family members and should be performed at the protein rather than at the messenger RNA level. In drug development, targeting the ubiquitination machinery of prosurvival Bcl-2 proteins will complement and potentially improve on targeting Bcl-2 protein interactions with BH3 mimetics.


Assuntos
Apoptose , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Antígenos de Histocompatibilidade Menor , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Estabilidade Proteica , Proteólise , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
3.
Sci Rep ; 13(1): 5333, 2023 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-37005459

RESUMO

The majority of proteins in mammalian cells are modified by covalent attachment of an acetyl-group to the N-terminus (Nt-acetylation). Paradoxically, Nt-acetylation has been suggested to inhibit as well as to promote substrate degradation. Contrasting these findings, proteome-wide stability measurements failed to detect any correlation between Nt-acetylation status and protein stability. Accordingly, by analysis of protein stability datasets, we found that predicted Nt-acetylation positively correlates with protein stability in case of GFP, but this correlation does not hold for the entire proteome. To further resolve this conundrum, we systematically changed the Nt-acetylation and ubiquitination status of model substrates and assessed their stability. For wild-type Bcl-B, which is heavily modified by proteasome-targeting lysine ubiquitination, Nt-acetylation did not correlate with protein stability. For a lysine-less Bcl-B mutant, however, Nt-acetylation correlated with increased protein stability, likely due to prohibition of ubiquitin conjugation to the acetylated N-terminus. In case of GFP, Nt-acetylation correlated with increased protein stability, as predicted, but our data suggest that Nt-acetylation does not affect GFP ubiquitination. Similarly, in case of the naturally lysine-less protein p16, Nt-acetylation correlated with protein stability, regardless of ubiquitination on its N-terminus or on an introduced lysine residue. A direct effect of Nt-acetylation on p16 stability was supported by studies in NatB-deficient cells. Together, our studies argue that Nt-acetylation can stabilize proteins in human cells in a substrate-specific manner, by competition with N-terminal ubiquitination, but also by other mechanisms that are independent of protein ubiquitination status.


Assuntos
Lisina , Proteoma , Animais , Humanos , Lisina/metabolismo , Proteoma/metabolismo , Acetilação , Processamento de Proteína Pós-Traducional , Ubiquitinação , Mamíferos/metabolismo
4.
Nat Commun ; 14(1): 4253, 2023 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-37474523

RESUMO

Immune checkpoint blockade therapy is beneficial and even curative for some cancer patients. However, the majority don't respond to immune therapy. Across different tumor types, pre-existing T cell infiltrates predict response to checkpoint-based immunotherapy. Based on in vitro pharmacological studies, mouse models and analyses of human melanoma patients, we show that the cytokine GDF-15 impairs LFA-1/ß2-integrin-mediated adhesion of T cells to activated endothelial cells, which is a pre-requisite of T cell extravasation. In melanoma patients, GDF-15 serum levels strongly correlate with failure of PD-1-based immune checkpoint blockade therapy. Neutralization of GDF-15 improves both T cell trafficking and therapy efficiency in murine tumor models. Thus GDF-15, beside its known role in cancer-related anorexia and cachexia, emerges as a regulator of T cell extravasation into the tumor microenvironment, which provides an even stronger rationale for therapeutic anti-GDF-15 antibody development.


Assuntos
Melanoma , Linfócitos T , Humanos , Camundongos , Animais , Linfócitos T/patologia , Antígeno-1 Associado à Função Linfocitária , Células Endoteliais/patologia , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Melanoma/patologia , Imunoterapia , Microambiente Tumoral
5.
FASEB J ; 23(1): 224-31, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18794335

RESUMO

CYP3A4 is an important xenobiotic metabolizing enzyme. We previously found that CYP2C55 is highly up-regulated in Cyp3a(-/-) mice. Here, we have further investigated the mechanism of regulation of CYP2C55 and other detoxifying systems in Cyp3a(-/-) mice. Induction studies with prototypical inducers demonstrated an important role for the nuclear receptors PXR and CAR in the up-regulation of CYP2C55. Subsequent diet-switch experiments revealed that food-derived xenobiotics are primarily responsible for the increased induction of CYP2C55, as well as of several other primary detoxifying systems in Cyp3a(-/-) mice. Our data suggest that CYP3A normally metabolizes food-derived activators of PXR and/or CAR, explaining the increased levels of such activators in Cyp3a(-/-) mice and subsequent up-regulation of a range of detoxifying systems. Interestingly, our studies with tissue-specific CYP3A4 transgenic Cyp3a(-/-) mice revealed that not only hepatic but also intestinal expression of CYP3A4 could reduce the hepatic expression of detoxifying systems to near wild-type levels. Apparently, intestinal CYP3A4 can limit the hepatic exposure to food-derived activators of nuclear receptors, thereby regulating the expression of a range of detoxifying systems in the liver. This broad biological effect further emphasizes the importance of intestinal CYP3A activity and could have profound implications for the prediction of drug exposure.


Assuntos
Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Intestinos/enzimologia , Fígado/enzimologia , Animais , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Família 2 do Citocromo P450 , Dieta , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Camundongos , Camundongos Knockout , Receptor de Pregnano X , Receptores de Esteroides/genética
6.
Clin Cancer Res ; 15(6): 2031-8, 2009 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-19276268

RESUMO

PURPOSE: Various proapoptotic agents are currently being explored to improve the outcome of radiotherapy. We have evaluated whether APO010-a novel recombinant ligand of the Fas/CD95 death receptor-enhanced the cytotoxic effect of radiation on lymphoid and solid tumor cell types. EXPERIMENTAL DESIGN: A Bcl-2-overexpressing T-leukemic cell line (Jurkat), a colon carcinoma cell line (HCT116), and a mesothelioma cell line were used as model systems in vitro and in a subcutaneous transplant setting in immunodeficient mice. Sensitivity to single and combined treatment was read out by apoptosis hallmarks and clonogenic survival in vitro, and by tumor growth delay using bioluminescence and palpation in vivo. RESULTS: Whereas the three cell lines resisted apoptosis induction by irradiation and APO010 alone, combined treatment greatly enhanced their apoptotic response. In clonogenic survival assays, APO010 reduced the outgrowth of Jurkat-Bcl-2 and HCT116 cells and sensitized the mesothelioma cell line to radiation. In vivo, systemic treatment with APO010 alone caused tumor growth delay in Jurkat-Bcl-2 and HCT116 cells. However, APO010 did not improve the efficacy of radiotherapy in any of the model systems at the selected single dose, which had moderate and reversible systemic toxicity. CONCLUSIONS: Although APO010 and radiation had a clear combined cytotoxic effect on tumor cells in vitro, a combined therapeutic effect was not achieved on the same cells subcutaneously grafted in mice, at APO010 doses approximating the maximally tolerable level. These findings suggest that it will be difficult to identify a therapeutic window for this combined modality approach in a clinical setting.


Assuntos
Adiponectina/farmacologia , Proteína Ligante Fas/farmacologia , Neoplasias/terapia , Proteínas Recombinantes de Fusão/farmacologia , Adiponectina/toxicidade , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Peso Corporal/efeitos dos fármacos , Peso Corporal/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Terapia Combinada , Proteína Ligante Fas/toxicidade , Células HCT116 , Humanos , Células Jurkat , Camundongos , Camundongos Endogâmicos BALB C , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Drug Metab Dispos ; 37(12): 2305-13, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19752211

RESUMO

CYP3A4 is an important determinant of drug-drug interactions. In this study, we evaluated whether cytochrome P450 3A knockout mice [Cyp3a(-/-)] and CYP3A4 transgenic (CYP3A4-Tg) mice can be used to study drug-drug interactions in the liver and intestine. Triazolam was used as a probe drug because it is a highly specific CYP3A substrate and not a P-glycoprotein substrate. Triazolam metabolism was profoundly reduced in Cyp3a(-/-) mice both in vitro and in vivo. In vitro studies revealed clear species differences in humans and mice, but triazolam metabolism in microsomes derived from CYP3A4-Tg "humanized" mice closely resembled that in human microsomes. It is interesting to note that studies with tissue-specific CYP3A4-Tg mice revealed that intestinal CYP3A4 has a major impact on oral triazolam exposure, whereas the effect of hepatic CYP3A4 was limited. To mimic a drug-drug interaction, we coadministered triazolam with the prototypical CYP3A inhibitor ketoconazole, which increased triazolam exposure in all the CYP3A-proficient mouse strains but not in Cyp3a(-/-) mice. We further found that the anticancer drug gefitinib is a potent stimulator of 1'-OH triazolam formation in vitro. It is noteworthy that we could also show in vivo stimulation of triazolam metabolism by gefitinib, resulting in a lower oral triazolam exposure. To our knowledge, this is the first in vivo example of direct stimulation of CYP3A4 activity after oral drug administration. Overall, this study illustrates how Cyp3a(-/-) and CYP3A4-Tg mice can be used to study drug-drug interactions. The data clarify that for drugs that are not P-glycoprotein substrates, intestinal metabolism also can be more important than hepatic metabolism after oral administration.


Assuntos
Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Intestinos/enzimologia , Fígado/enzimologia , Triazolam/farmacocinética , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Citocromo P-450 CYP3A/genética , Inibidores do Citocromo P-450 CYP3A , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/deficiência , Sistema Enzimático do Citocromo P-450/genética , Interações Medicamentosas , Ativadores de Enzimas/administração & dosagem , Inibidores Enzimáticos/administração & dosagem , Gefitinibe , Humanos , Hidroxilação , Intestinos/efeitos dos fármacos , Cetoconazol/administração & dosagem , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microssomos/enzimologia , Quinazolinas/administração & dosagem , Especificidade da Espécie , Especificidade por Substrato , Triazolam/administração & dosagem
9.
Nat Biotechnol ; 34(11): 1095-1098, 2016 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-27824845
10.
Cancer Res ; 69(23): 8996-9002, 2009 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19920203

RESUMO

Docetaxel is one of the most widely used anticancer drugs. A major problem with docetaxel treatment, however, is the considerable interpatient variability in docetaxel exposure. Another disadvantage of the drug is that it has a very low oral bioavailability and can therefore only be administered i.v. The drug-metabolizing enzyme cytochrome P450 3A (CYP3A) and the drug transporter P-glycoprotein (P-gp; MDR1) are considered to be major determinants of docetaxel pharmacokinetics. It has been hypothesized that CYP3A and P-gp work synergistically in limiting the systemic exposure to many orally ingested drugs. However, it has been difficult to examine this interplay in vivo. We therefore generated mice lacking all CYP3A and P-gp genes. Although missing two primary detoxification systems, Cyp3a/Mdr1a/1b(-/-) mice are viable, fertile, and without spontaneous abnormalities. When orally challenged with docetaxel, a disproportionate (>70-fold) increase in systemic exposure was observed compared with the increases in single Cyp3a(-/-) (12-fold) or Mdr1a/1b(-/-) (3-fold) mice. Unexpectedly, although CYP3A and P-gp collaborated extremely efficiently in lowering docetaxel exposure, their individual efficacy was not dependent on activity of the other protein. On reflection, this absence of functional synergism makes biological sense, as synergism would conflict with a robust detoxification defense. Importantly, the disproportionate increase in docetaxel exposure in Cyp3a/Mdr1a/1b(-/-) mice resulted in dramatically altered and lethal toxicity, with severe intestinal lesions as a major cause of death. Simultaneous inhibition of CYP3A/P-gp might thus be a highly effective strategy to improve oral drug bioavailability but with serious risks when applied to drugs with narrow therapeutic windows.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/deficiência , Citocromo P-450 CYP3A/deficiência , Enteropatias/induzido quimicamente , Taxoides/farmacocinética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Administração Oral , Animais , Antineoplásicos/sangue , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidade , Disponibilidade Biológica , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Docetaxel , Enteropatias/sangue , Enteropatias/metabolismo , Masculino , Camundongos , Camundongos Knockout , Taxoides/sangue , Taxoides/toxicidade
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