RESUMO
Autophagy is an intracellular recycling pathway with implications for intracellular homeostasis and cell survival. Its pharmacological modulation can aid chemotherapy by sensitizing cancer cells toward approved drugs and overcoming chemoresistance. Recent translational data on autophagy modulators show promising results in reducing tumor growth and metastasis, but also reveal a need for more specific compounds and novel lead structures. Here, we searched for such autophagy-modulating compounds in a flow cytometry-based high-throughput screening of an in-house natural compound library. We successfully identified novel inducers and inhibitors of the autophagic pathway. Among these, we identified arzanol as an autophagy-modulating drug that causes the accumulation of ATG16L1-positive structures, while it also induces the accumulation of lipidated LC3. Surprisingly, we observed a reduction of the size of autophagosomes compared to the bafilomycin control and a pronounced accumulation of p62/SQSTM1 in response to arzanol treatment in HeLa cells. We, therefore, speculate that arzanol acts both as an inducer of early autophagosome biogenesis and as an inhibitor of later autophagy events. We further show that arzanol is able to sensitize RT-112 bladder cancer cells towards cisplatin (CDDP). Its anticancer activity was confirmed in monotherapy against both CDDP-sensitive and -resistant bladder cancer cells. We classified arzanol as a novel mitotoxin that induces the fragmentation of mitochondria, and we identified a series of targets for arzanol that involve proteins of the class of mitochondria-associated quinone-binding oxidoreductases. Collectively, our results suggest arzanol as a valuable tool for autophagy research and as a lead compound for drug development in cancer therapy.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Floroglucinol/análogos & derivados , Pironas/uso terapêutico , Autofagia , Humanos , Floroglucinol/farmacologia , Floroglucinol/uso terapêutico , Pironas/farmacologiaRESUMO
Autophagy, apoptosis, and necroptosis are stress responses governing the ultimate fate of a cell. However, the crosstalk between these cellular stress responses is not entirely understood. Especially, it is not clear whether the autophagy-initiating kinase ULK1 and the cell-death-regulating kinase RIPK1 are involved in this potential crosstalk. Here, we identify RIPK1 as a substrate of ULK1. ULK1-dependent phosphorylation of RIPK1 reduces complex IIb/necrosome assembly and tumor necrosis factor (TNF)-induced cell death, whereas deprivation of ULK1 enhances TNF-induced cell death. We observe that ULK1 phosphorylates multiple sites of RIPK1, but it appears that especially phosphorylation of S357 within the intermediate domain of RIPK1 mediates this cell-death-inhibiting effect. We propose that ULK1 is a regulator of RIPK1-mediated cell death.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Autofagia , Morte Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HEK293 , Humanos , Fosforilação , Transdução de SinaisRESUMO
Mitochondria are cellular organelles with crucial functions in the generation and distribution of ATP, the buffering of cytosolic Ca2+ and the initiation of apoptosis. Compounds that interfere with these functions are termed mitochondrial toxins, many of which are derived from microbes, such as antimycin A, oligomycin A, and ionomycin. Here, we identify the mycotoxin phomoxanthone A (PXA), derived from the endophytic fungus Phomopsis longicolla, as a mitochondrial toxin. We show that PXA elicits a strong release of Ca2+ from the mitochondria but not from the ER. In addition, PXA depolarises the mitochondria similarly to protonophoric uncouplers such as CCCP, yet unlike these, it does not increase but rather inhibits cellular respiration and electron transport chain activity. The respiration-dependent mitochondrial network structure rapidly collapses into fragments upon PXA treatment. Surprisingly, this fragmentation is independent from the canonical mitochondrial fission and fusion mediators DRP1 and OPA1, and exclusively affects the inner mitochondrial membrane, leading to cristae disruption, release of pro-apoptotic proteins, and apoptosis. Taken together, our results suggest that PXA is a mitochondrial toxin with a novel mode of action that might prove a useful tool for the study of mitochondrial ion homoeostasis and membrane dynamics.
Assuntos
Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Micotoxinas/toxicidade , Xantonas/toxicidade , Animais , Ascomicetos/metabolismo , Cálcio/metabolismo , Linhagem Celular , Transporte de Elétrons/efeitos dos fármacos , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Camundongos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Micotoxinas/metabolismo , Xantonas/metabolismoRESUMO
Macroautophagy/autophagy is an evolutionarily conserved cellular process whose induction is regulated by the ULK1 protein kinase complex. The subunit ATG13 functions as an adaptor protein by recruiting ULK1, RB1CC1 and ATG101 to a core ULK1 complex. Furthermore, ATG13 directly binds both phospholipids and members of the Atg8 family. The central involvement of ATG13 in complex formation makes it an attractive target for autophagy regulation. Here, we analyzed known interactions of ATG13 with proteins and lipids for their potential modulation of ULK1 complex formation and autophagy induction. Targeting the ATG101-ATG13 interaction showed the strongest autophagy-inhibitory effect, whereas the inhibition of binding to ULK1 or RB1CC1 had only minor effects, emphasizing that mutations interfering with ULK1 complex assembly do not necessarily result in a blockade of autophagy. Furthermore, inhibition of ATG13 binding to phospholipids or Atg8 proteins had only mild effects on autophagy. Generally, the observed phenotypes were more severe when autophagy was induced by MTORC1/2 inhibition compared to amino acid starvation. Collectively, these data establish the interaction between ATG13 and ATG101 as a promising target in disease-settings where the inhibition of autophagy is desired.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Motivos de Aminoácidos , Sequência de Aminoácidos , Aminoácidos/deficiência , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Autofagossomos/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Fibroblastos/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos Knockout , Mutação/genética , Ligação Proteica , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Cisplatin-based regimens are routinely employed for the treatment of urothelial carcinoma. However, therapeutic success is hampered by the primary presence of or the development of cisplatin resistance. This chemoresistance is executed by multiple cellular pathways. In recent years, the cellular process of autophagy has been identified as a prosurvival pathway of cancer cells. On the one hand, autophagy enables cancer cells to survive conditions of low oxygen or nutrient supply, frequently found in tumors. On the other hand, autophagy supports chemoresistance of cancer cells. Here, we aimed at investigating the involvement of autophagy for cisplatin resistance in different urothelial carcinoma cell lines. MATERIALS & METHODS: We analyzed the expression levels of different autophagy-related proteins in cisplatin-sensitive and cisplatin-resistant urothelial carcinoma cell lines. Furthermore, we performed cell viability assays and caspase activity assays with cells treated with cisplatin, non-specific or specific autophagy inhibitors (chloroquine, 3-methyladenine, SAR405) or combinations thereof. RESULTS: We found that autophagy-related proteins are up-regulated in different cisplatin-resistant urothelial carcinoma cells compared to the sensitive parental cell lines. Furthermore, inhibition of autophagy, in general, or of the autophagy-inducing class III PtdIns3K complex, in particular, sensitized both sensitive and resistant urothelial carcinoma cells to cisplatin-induced cytotoxic effects. CONCLUSION: We propose that targeting the autophagic machinery might represent a suitable approach to complement or even increase cisplatin efficacy in order to overcome cisplatin resistance in urothelial carcinoma.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Autofagia/efeitos dos fármacos , Carcinoma de Células de Transição/tratamento farmacológico , Classe III de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Neoplasias da Bexiga Urinária/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma de Células de Transição/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Regulação para Cima , Neoplasias da Bexiga Urinária/patologiaRESUMO
The stress-responsive mitochondrial sirtuin SIRT4 controls cellular energy metabolism in a NAD+-dependent manner and is implicated in cellular senescence and aging. Here we reveal a novel function of SIRT4 in mitochondrial morphology/quality control and regulation of mitophagy. We report that moderate overexpression of SIRT4, but not its enzymatically inactive mutant H161Y, sensitized cells to mitochondrial stress. CCCP-triggered dissipation of the mitochondrial membrane potential resulted in increased mitochondrial ROS levels and autophagic flux, but surprisingly led to increased mitochondrial mass and decreased Parkin-regulated mitophagy. The anti-respiratory effect of elevated SIRT4 was accompanied by increased levels of the inner-membrane bound long form of the GTPase OPA1 (L-OPA1) that promotes mitochondrial fusion and thereby counteracts fission and mitophagy. Consistent with this, upregulation of endogenous SIRT4 expression in fibroblast models of senescence either by transfection with miR-15b inhibitors or by ionizing radiation increased L-OPA1 levels and mitochondrial fusion in a SIRT4-dependent manner. We further demonstrate that SIRT4 interacts physically with OPA1 in co-immunoprecipitation experiments. Overall, we propose that the SIRT4-OPA1 axis is causally linked to mitochondrial dysfunction and altered mitochondrial dynamics that translates into aging-associated decreased mitophagy based on an unbalanced mitochondrial fusion/fission cycle.
Assuntos
Envelhecimento/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mitofagia/fisiologia , Sirtuínas/metabolismo , Envelhecimento/patologia , Células Cultivadas , Senescência Celular/fisiologia , Células HEK293 , Humanos , Mitocôndrias/patologia , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
AIM: To develop a safe and efficient method for targeted, anti-apoptotic gene therapy of corneal endothelial cells (CECs). MATERIALS & METHODS: Magnetofection (MF), a combination of lipofection with magnetic nanoparticles (MNPs; PEI-Mag2, SO-Mag5, PalD1-Mag1), was tested in human CECs and in explanted human corneas. Effects on cell viability and function were investigated. Immunocompatibility was assessed in human peripheral blood mononuclear cells. RESULTS: Silica iron-oxide MNPs (SO-Mag5) combined with X-tremeGENE-HP achieved high transfection efficiency in human CECs and explanted human corneas, without altering cell viability or function. Magnetofection caused no immunomodulatory effects in human peripheral blood mononuclear cells. Magnetofection with anti-apoptotic P35 gene effectively blocked apoptosis in CECs. CONCLUSION: Magnetofection is a promising tool for gene therapy of corneal endothelial cells with potential for targeted on-site delivery.
Assuntos
Endotélio Corneano/metabolismo , Magnetismo/métodos , Nanopartículas de Magnetita/química , Dióxido de Silício/química , Transfecção/métodos , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular , DNA/administração & dosagem , DNA/genética , Humanos , Subunidade alfa de Receptor de Interleucina-2/análise , Lectinas Tipo C/análise , Leucócitos Mononucleares/citologia , Campos Magnéticos , Plasmídeos/administração & dosagem , Plasmídeos/genética , Proteínas Virais/genéticaRESUMO
Autophagy represents an intracellular degradation process which is involved in both regular cell homeostasis and disease settings. In recent years, the molecular machinery governing this process has been elucidated. The ULK1 kinase complex consisting of the serine/threonine protein kinase ULK1 and the adapter proteins ATG13, RB1CC1, and ATG101, is centrally involved in the regulation of autophagy initiation. This complex is in turn regulated by the activity of different nutrient- or energy-sensing kinases, including MTOR, AMPK, and AKT. However, next to phosphorylation processes it has been suggested that ubiquitination of ULK1 positively influences ULK1 function. Here we report that the inhibition of deubiquitinases by the compound WP1130 leads to increased ULK1 ubiquitination, the transfer of ULK1 to aggresomes, and the inhibition of ULK1 activity. Additionally, WP1130 can block the autophagic flux. Thus, treatment with WP1130 might represent an efficient tool to inhibit the autophagy-initiating ULK1 complex and autophagy.
Assuntos
Autofagia/efeitos dos fármacos , Cianoacrilatos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Agregados Proteicos/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Piridinas/farmacologia , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Ubiquitinação/efeitos dos fármacosRESUMO
Autophagy describes an intracellular process responsible for the lysosome-dependent degradation of cytosolic components. The ULK1/2 complex comprising the kinase ULK1/2 and the accessory proteins ATG13, RB1CC1, and ATG101 has been identified as a central player in the autophagy network, and it represents the main entry point for autophagy-regulating kinases such as MTOR and AMPK. It is generally accepted that the ULK1 complex is constitutively assembled independent of nutrient supply. Here we report the characterization of the ATG13 region required for the binding of ULK1/2. This binding site is established by an extremely short peptide motif at the C terminus of ATG13. This motif is mandatory for the recruitment of ULK1 into the autophagy-initiating high-molecular mass complex. Expression of a ULK1/2 binding-deficient ATG13 variant in ATG13-deficient cells resulted in diminished but not completely abolished autophagic activity. Collectively, we propose that autophagy can be executed by mechanisms that are dependent or independent of the ULK1/2-ATG13 interaction.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Autofagia , Mutação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Motivos de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Estabilidade Enzimática , Fibroblastos/metabolismo , Proteínas de Choque Térmico/metabolismo , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Peptídeos/metabolismo , Fagossomos/metabolismo , Ligação Proteica , Proteólise , Proteína Sequestossoma-1RESUMO
Mangrove associated endophytes are treasure chests for bioprospecting especially in light of the need for new anticancer leads that are necessary to overcome drug resistance of cancer cells. This review highlights the potent anti-tumour compound phomoxanthone A (PXA), which represents a tetrahydroxanthone atropisomer derived from the mangrove-associated fungus Phomopsis longicolla. PXA displayed strong anti-tumour activity when tested against a panel of solid (including cisplatin resistant) tumour cell lines or of blood cancer cell lines with IC50 values in the submicromolar range whereas it was up to 100 folds less active against peripheral blood mononuclear cells (PBMC) from healthy donors. The anti-tumour activity of PXA was demonstrated to be due to an induction of caspase 3 dependent apoptosis. At the same time PXA was shown to activate immune cells such as murine T-lymphocytes, NK cells and macrophages which might help in fighting resistance during cancer chemotherapy. Structure activity studies that involved several naturally occurring as well as semisynthetic derivatives of PXA indicated the position of the biaryl linkage and the acetyl substituents as structural features that are important for the activity of this natural product.
Assuntos
Fungos/química , Neoplasias/tratamento farmacológico , Áreas Alagadas , Xantonas/uso terapêutico , Animais , Anticarcinógenos/química , Anticarcinógenos/farmacologia , Anticarcinógenos/uso terapêutico , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Xantonas/química , Xantonas/farmacologiaRESUMO
A novel macrolide, callyspongiolide, whose structure was determined by comprehensive analysis of the NMR and HRMS spectra, was isolated from the marine sponge Callyspongia sp. collected in Indonesia. The compound features a carbamate-substituted 14-membered macrocyclic lactone ring with a conjugated structurally unprecedented diene-ynic side chain terminating at a brominated benzene ring. Callyspongiolide showed strong cytotoxicity against human Jurkat J16 T and Ramos B lymphocytes.
Assuntos
Linfócitos B/efeitos dos fármacos , Callyspongia/química , Macrolídeos/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Células Jurkat , Macrolídeos/química , Macrolídeos/isolamento & purificação , Conformação Molecular , Relação Estrutura-AtividadeRESUMO
Four tetrahydroxanthone dimers (1-4) and four biogenetically related monomers (5-8), including the new derivatives 4-6, were isolated from the endophyte Phomopsis longicolla. The absolute configurations of 2-4 were established for the first time by TDDFT electronic circular dichroism calculations, and that of phomoxanthone A (1) was revised by X-ray crystallography. Phomoxanthone A (1) showed the strongest pro-apoptotic activity when tested against a panel of human cancer cell lines, including cisplatin-resistant cells, whereas it was up to 100-fold less active against healthy blood cells. It was also the most potent activator of murine T lymphocytes, NK cells, and macrophages, suggesting an activation of the immune system in parallel to its pro-apoptotic activity. This dual effect in combating cancer cells could help in fighting resistance during chemotherapy. Preliminary structure-activity studies of isolated compounds and derivatives obtained by semisynthesis (9a-11) hinted at the location of the biaryl axis and the presence of acetyl groups as important structural elements for the biological activity of the studied tetrahydroxanthones.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ascomicetos/química , Xantonas/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Apoptose/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dimerização , Relação Dose-Resposta a Droga , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Modelos Moleculares , Estrutura Molecular , Teoria Quântica , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Xantonas/química , Xantonas/isolamento & purificaçãoRESUMO
Gold(I) complexes of imidazole and thiazole-based diphos type ligands were prepared and their potential as chemotherapeutics investigated. Depending on the ligands employed and the reaction conditions complexes [L(AuCl)(2)] and [L(2)Au]X (X = Cl, PF(6)) are obtained. The ligands used are diphosphanes with azoyl substituents R(2)P(CH(2))(2)PR(2) {R = 1-methylimidazol-2-yl (1), 1-methylbenzimidazol-2-yl (4), thiazol-2-yl (5) and benzthiazol-2-yl (6)} as well as the novel ligands RPhP(CH(2))(2)PRPh {R = 1-methylimidazol-2-yl (3)} and R(2)P(CH(2))(3)PR(2) {R = 1-methylimidazol-2-yl (2)}. The cytotoxic activity of the complexes was assessed against three human cancer cell lines and a rat hepatoma cell line and correlated to the lipophilicity of the compounds. The tetrahedral gold complexes [(3)(2)Au]PF(6) and [(5)(2)Au]PF(6) with intermediate lipophilicity (logD(7.4) = 0.21 and 0.25) showed significant cytotoxic activity in different cell lines. Both compounds induce apoptosis and inhibit the enzymes thioredoxin reductase and glutathione reductase.