RESUMO
Inhibition of the mitochondrial metabolism offers a promising therapeutic approach for the treatment of cancer. Here, we identify the mycotoxin viriditoxin (VDT), derived from the endophytic fungus Cladosporium cladosporioides, as an interesting candidate for leukemia and lymphoma treatment. VDT displayed a high cytotoxic potential and rapid kinetics of caspase activation in Jurkat leukemia and Ramos lymphoma cells in contrast to solid tumor cells that were affected to a much lesser extent. Most remarkably, human hematopoietic stem and progenitor cells and peripheral blood mononuclear cells derived from healthy donors were profoundly resilient to VDT-induced cytotoxicity. Likewise, the colony-forming capacity was affected only at very high concentrations, which provides a therapeutic window for cancer treatment. Intriguingly, VDT could directly activate the mitochondrial apoptosis pathway in leukemia cells in the presence of antiapoptotic Bcl-2 proteins. The mitochondrial toxicity of VDT was further confirmed by inhibition of mitochondrial respiration, breakdown of the mitochondrial membrane potential (ΔΨm), the release of mitochondrial cytochrome c, generation of reactive oxygen species (ROS), processing of the dynamin-like GTPase OPA1 and subsequent fission of mitochondria. Thus, VDT-mediated targeting of mitochondrial oxidative phosphorylation (OXPHOS) might represent a promising therapeutic approach for the treatment of leukemia and lymphoma without affecting hematopoietic stem and progenitor cells.
Assuntos
Leucemia , Linfoma , Micotoxinas , Humanos , Micotoxinas/metabolismo , Leucócitos Mononucleares/metabolismo , Apoptose , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Linfoma/tratamento farmacológico , Linfoma/metabolismo , Potencial da Membrana MitocondrialRESUMO
Ferroptosis is a form of regulated cell death that emerges to be relevant for therapy-resistant and dedifferentiating cancers. Although several lines of evidence suggest that ferroptosis is a type of autophagy-dependent cell death, the underlying molecular mechanisms remain unclear. Fin56, a type 3 ferroptosis inducer, triggers ferroptosis by promoting glutathione peroxidase 4 (GPX4) protein degradation via a not fully understood pathway. Here, we determined that Fin56 induces ferroptosis and autophagy in bladder cancer cells and that Fin56-triggered ferroptosis mechanistically depends on the autophagic machinery. Furthermore, we found that autophagy inhibition at different stages attenuates Fin56-induced oxidative stress and GPX4 degradation. Moreover, we investigated the effects of Fin56 in combination with Torin 2, a potent mTOR inhibitor used to activate autophagy, on cell viability. We found that Fin56 synergizes with Torin 2 in cytotoxicity against bladder cancer cells. Collectively, our findings not only support the concept that ferroptosis is a type of autophagy-dependent cell death but imply that the combined application of ferroptosis inducers and mTOR inhibitors is a promising approach to improve therapeutic options in the treatment of bladder cancer.
Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Ferroptose/efeitos dos fármacos , Inibidores de MTOR/farmacologia , Naftiridinas/farmacologia , Oximas/farmacologia , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Neoplasias da Bexiga Urinária/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Estresse Oxidativo/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Neoplasias da Bexiga Urinária/patologiaRESUMO
The protein kinase TBK1 is a central regulator of innate immune responses and autophagy, and ablation of either function has been linked to neuroinflammatory or degenerative diseases. Autophagy is an intracellular process that recycles old or damaged proteins and organelles. In recent years, the TBK1-dependent regulation of autophagy pathways has been characterized. However, the autophagy-dependent regulation of TBK1 activity awaits further clarification. Here, we observed that TBK1 is recruited to SQSTM1/p62-containing aggregates via the selective autophagy receptor TAX1BP1. In these aggregates, TBK1 phosphorylates SQSTM1/p62 at serine 403 and thus presumably regulates the efficient engulfment and clearance of these structures. We found that TBK1 activation is strongly increased if FIP200, a component of the autophagy-inducing ULK1 complex, is not present or cannot bind to TAX1BP1. Given our collective findings, we hypothesize that FIP200 ensures the inducible activation of TBK1 at SQSTM1/p62 condensates.
Assuntos
Proteínas Relacionadas à Autofagia/genética , Imunidade Inata/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Proteína Sequestossoma-1/genética , Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Humanos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Fosforilação/genética , Transdução de Sinais/genéticaRESUMO
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
Macroautophagy/autophagy and necroptosis represent two opposing cellular s tress responses. Whereas autophagy primarily fulfills a cyto-protective function, necroptosis is a form of regulated cell death induced via death receptors. Here, we aimed at investigating the molecular crosstalk between these two pathways. We observed that RIPK3 directly associates with AMPK and phosphorylates its catalytic subunit PRKAA1/2 at T183/T172. Activated AMPK then phosphorylates the autophagy-regulating proteins ULK1 and BECN1. However, the lysosomal degradation of autophagosomes is blocked by TNF-induced necroptosis. Specifically, we observed dysregulated SNARE complexes upon TNF treatment; e.g., reduced levels of full-length STX17. In summary, we identified RIPK3 as an AMPK-activating kinase and thus a direct link between autophagy- and necroptosis-regulating kinases.Abbreviations: ACACA/ACC: acetyl-CoA carboxylase alpha; AMPK: AMP-activated protein kinase; ATG: autophagy-related; BECN1: beclin 1; GFP: green fluorescent protein; EBSS: Earle's balanced salt solution; Hs: Homo sapiens; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MLKL: mixed lineage kinase domain like pseudokinase; Mm: Mus musculus; MTOR: mechanistic target of rapamycin kinase; MVB: multivesicular body; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PLA: proximity ligation assay; PRKAA1: protein kinase AMP-activated catalytic subunit alpha 1; PRKAA2: protein kinase AMP-activated catalytic subunit alpha 2; PRKAB2: protein kinase AMP-activated non-catalytic subunit beta 2; PRKAG1: protein kinase AMP-activated non-catalytic subunit gamma 1; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; RIPK1: receptor interacting serine/threonine kinase 1; RIPK3: receptor interacting serine/threonine kinase 3; SNAP29: synaptosome associated protein 29; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SQSTM1/p62: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; STX7: syntaxin 7; STX17: syntaxin 17; TAX1BP1: Tax1 binding protein 1; TNF: tumor necrosis factor; ULK1: unc-51 like autophagy activating kinase 1; VAMP8: vesicle associated membrane protein 8; WT: wild-type.
Assuntos
Proteínas Quinases Ativadas por AMP , Autofagia , Necroptose , Proteína Serina-Treonina Quinases de Interação com Receptores , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Autofagia/fisiologia , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Fibroblastos/metabolismo , Camundongos , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
A new epidithiodiketopiperazine (ETP), pretrichodermamide G (1), along with three known (epi)dithiodiketopiparazines (2-4) were isolated from cultures of Trichoderma harzianum and Epicoccum nigrum, endophytic fungi associated with medicinal plants Zingiber officinale and Salix sp., respectively. The structure of the new compound (1) was established on the basis of spectroscopic data, including 1D/2D NMR and HRESIMS. The isolated compounds were investigated for their antifungal, antibacterial and cytotoxic potential against a panel of microorganisms and cell lines. Pretrichodermamide A (2) displayed antimicrobial activity towards the plant pathogenic fungus Ustilago maydis and the human pathogenic bacterium Mycobacterium tuberculosis with MIC values of 1 mg/mL (2 mM) and 25 µg/mL (50 µM), respectively. Meanwhile, epicorazine A (3) exhibited strong to moderate cytotoxicity against L5178Y, Ramos, and Jurkat J16 cell lines with IC50 values ranging from 1.3 to 28 µM. Further mechanistic studies indicated that 3 induces apoptotic cell death.
Assuntos
Ascomicetos/química , Dicetopiperazinas/química , Dicetopiperazinas/farmacologia , Hypocreales/química , Antibacterianos/química , Antibacterianos/farmacologia , Antifúngicos/química , Antifúngicos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Basidiomycota/efeitos dos fármacos , Endófitos/química , Humanos , Células Jurkat , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Plantas Medicinais/microbiologia , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Fermentation of the endophytic fungus Aplosporella javeedii on solid rice medium in presence of either 3.5% NaNO3 or 3.5% monosodium glutamate caused a significant change of the fungal metabolite pattern compared to fungal controls grown only on rice. Chemical investigation of the former fungal extracts yielded 11 new lactam derivatives, aplosporellins A-K (2-12), in addition to the known compound, pramanicin A (1). All of these compounds were not detected when the fungus was grown on rice medium without these activators thereby indicating the power of this OSMAC approach. The structures of the new compounds were elucidated by one- and two- dimensional NMR spectroscopy, DFT-NMR calculations and by mass spectrometry as well as by comparison with the literature whereas the absolute configuration of the lactam core was determined by TDDFT-ECD and OR calculations. Pramanicin A (1) showed strong cytotoxicity against human lymphoma (Ramos) and leukemia (Jurkat J16) cells with IC50 values of 4.7 and 4.4 µM, respectively. Mechanistic studies indicated that 1 activates caspase-3 and induces apoptotic cell death.
RESUMO
Epigenetics refers to heritable changes in gene expression and chromatin structure without change in a DNA sequence. Several epigenetic modifications and respective regulators have been reported. These include DNA methylation, chromatin remodeling, histone post-translational modifications, and non-coding RNAs. Emerging evidence has revealed that epigenetic dysregulations are involved in a wide range of diseases including cancers. Therefore, the reversible nature of epigenetic modifications concerning activation or inhibition of enzymes involved could be promising targets and useful tools for the elucidation of cellular and biological phenomena. In this review, emphasis is laid on natural products that inhibit DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) making them promising candidates for the development of lead structures for anticancer-drugs targeting epigenetic modifications. However, most of the natural products targeting HDAC and/or DNMT lack isoform selectivity, which is important for determining their potential use as therapeutic agents. Nevertheless, the structures presented in this review offer the well-founded basis that screening and chemical modifications of natural products will in future provide not only leads to the identification of more specific inhibitors with fewer side effects, but also important features for the elucidation of HDAC and DNMT function with respect to cancer treatment.
RESUMO
Five sesterterpenes (1-5) including two new compounds (1 and 2), as well as a new (6) and a known macrolide (7) were isolated from the endophytic fungus Aplosporella javeedii. The structures of the new compounds were elucidated by analysis of their 1D and 2D NMR and HRMS data as well as by comparison with the literature. Compound 4 and its acetyl derivatives 4a, 4b, 4c which were prepared by acetylation of 4 exhibited moderate cytotoxicity against the mouse lymphoma cell line L5178Y with IC50 values ranging from 6.2 to 12.8 µM, respectively. Moreover, 4a and 4c exhibited also cytotoxicity against human leukemia (Jurkat J16) and lymphoma (Ramos) cell lines. Compound 7 showed strong cytotoxicity against the L5178Y cell line, as well as against human Jurkat J16 and Ramos cells with IC50 values of 0.4, 5.8, and 4.4 µM, respectively. Mechanistic studies indicated that 7 induces apoptotic cell death. In addition, compounds 3, 4 and 7 showed low antibacterial activities against Mycobacterium tuberculosis H37Rv and compound 6 against Staphylococcus aureus, respectively, with MICs of 100 µM. Preliminary structure-activity relationships are discussed.
Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Ascomicetos/química , Macrolídeos/farmacologia , Sesterterpenos/farmacologia , Animais , Antibacterianos/isolamento & purificação , Antineoplásicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Brassicaceae/microbiologia , Linhagem Celular Tumoral , China , Endófitos/química , Humanos , Macrolídeos/isolamento & purificação , Camundongos , Estrutura Molecular , Sesterterpenos/isolamento & purificação , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
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
Didymellanosine (1), the first analogue of the decahydrofluorene-class of natural products bearing a 13-membered macrocyclic alkaloid conjugated with adenosine, and a new benzolactone derivative, ascolactone C (4) along with eight known compounds (2, 3, 5-10), were isolated from a solid rice fermentation of the endophytic fungus Didymella sp. IEA-3B.1 derived from the host plant Terminalia catappa. In addition, ascochitamine (11) was obtained when (NH4)2SO4 was added to rice medium and is reported here for the first time as a natural product. Didymellanosine (1) displayed strong activity against the murine lymphoma cell line L5178Y, Burkitt's lymphoma B cells (Ramos) and adult lymphoblastic leukemia T cells (Jurkat J16), with IC50 values of 2.0, 3.3 and 4.4 µM, respectively. When subjected to a NFκB inhibition assay, didymellanosine (1) moderately blocked NFκB activation in the triple-negative breast cancer cell line MDA-MB 231. In an antimicrobial assay, ascomylactam C (3) was the most active compound when tested against a panel of Gram-positive bacteria including drug-resistant strains with MICs of 3.1-6.3 µM, while 1 revealed weaker activity. Interestingly, both compounds were also found active against Gram-negative Acinetobacter baumannii with MICs of 3.1 µM, in the presence of a sublethal concentration (0.1 µM) of colistin.
RESUMO
There is a variety of antineoplastic drugs that are based on natural compounds from ecological niches with high evolutionary pressure. We used two cell lines (Jurkat J16 and Ramos) in a screening to assess 300 different naturally occurring compounds with regard to their antineoplastic activity. The results of the compounds 4,6-dibromo-2-(2',4'-dibromophenoxy)phenol (P01F03), 4,5,6-tribromo-2-(2',4'-dibromophenoxy)phenol (P01F08), and 5-epi-nakijinone Q (P03F03) prompted us to perform further research. Using viability and apoptosis assays on the cell lines of primary human leukemic and normal hematopoietic cells, we found that P01F08 induced apoptosis in the cell lines at IC50 values between 1.61 and 2.95 µM after 72 h. IC50 values of peripheral blood mononuclear cells (PBMNCs) from healthy donors were higher, demonstrating that the cytotoxicity in the cell lines reached 50%, while normal PBMNCs were hardly affected. The colony-forming unit assay showed that the hematopoietic progenitor cells were not significantly affected in their growth by P01F08 at a concentration of 3 µM. P01F08 showed a 3.2-fold lower IC50 value in primary leukemic cells [acute myeloid leukemia (AML)] compared to the PBMNC of healthy donors. We could confirm the antineoplastic effect of 5-epi-nakijinone Q (P03F03) on the cell lines via the induction of apoptosis but noted a similarly strong cytotoxic effect on normal PBMNCs.
Assuntos
Antineoplásicos/uso terapêutico , Fenol/uso terapêutico , Adulto , Idoso , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Células HL-60 , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Células Jurkat , Leucemia Mieloide Aguda/tratamento farmacológico , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Células THP-1RESUMO
Two azaphilone pigments (1 and 2), two dihydrobenzofurans (3 and 4), two macrodiolides (5 and 6), and a dimeric alkyl aromatic constituent (7) were isolated from the goose dung-derived fungus Coniella fragariae. Compounds 1-3 proved to be new natural products. Coniellins H and I (1 and 2) feature a tetracyclic core and an aldehyde group at C-5, which is unusual for azaphilone derivatives. The X-ray structure of pyrenophorin (5) is reported for the first time. Pyrenophorin (5) showed strong cytotoxicity against several cancer cell lines with IC50 values ranging from 0.07 to 7.8⯵M.
Assuntos
Ascomicetos/química , Benzopiranos/farmacologia , Pigmentos Biológicos/farmacologia , Animais , Benzofuranos/isolamento & purificação , Benzopiranos/isolamento & purificação , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Fezes/microbiologia , Gansos/microbiologia , Alemanha , Humanos , Estrutura Molecular , Mar do Norte , Pigmentos Biológicos/isolamento & purificaçãoRESUMO
3-(Hetero)aryl substituted 7-azaindoles possessing multikinase inhibitor activity are readily accessed in a one-pot Masuda borylation-Suzuki coupling sequence. Several promising derivatives were identified as apoptosis inducers and, emphasizing the multikinase inhibition potential, as sphingosine kinase 2 inhibitors. Our measurements provide additional insights into the structure-activity relationship of meriolin derivatives, suggesting derivatives bearing a pyridine moiety with amino groups in 2-position as most active anticancer compounds and thus as highly promising candidates for future in vivo studies.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Compostos Bicíclicos Heterocíclicos com Pontes/síntese química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células Jurkat , Estrutura Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-AtividadeRESUMO
Targeting of oncogene-driven replicative stress as therapeutic option for high-risk medullobastoma was assessed using a panel of medulloblastoma cells differing in their c-Myc expression [i.e. group SHH (c-Myc low) vs. group 3 (c-Myc high)]. High c-Myc levels were associated with hypersensitivity to pharmacological Chk1 and ATR inhibition but not to CDK inhibition nor to conventional (genotoxic) anticancer therapeutics. The enhanced sensitivity of group 3 medulloblastoma cells to Chk1 inhibitors likely results from enhanced damage to intracellular organelles, elevated replicative stress and DNA damage and activation of apoptosis/necrosis. Furthermore, Chk1 inhibition differentially affected c-Myc expression and functions. In c-Myc high cells, Chk1 blockage decreased c-Myc and p-GSK3α protein and increased p21 and GADD45A mRNA expression. By contrast, c-Myc low cells revealed increased p-GSK3ß protein and CHOP and DUSP1 mRNA levels. Inhibition of Chk1 sensitized medulloblastoma cells to additional replication stress evoked by cisplatin independent of c-Myc. Importantly, Chk1 inhibition only caused minor toxicity in primary rat neurons in vitro. Collectively, targeting of ATR/Chk1 effectively triggers death in high-risk medulloblastoma, potentiates the anticancer efficacy of cisplatin and is well tolerated in non-cancerous neuronal cells.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Meduloblastoma/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Caenorhabditis elegans , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Dano ao DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Proteínas Hedgehog/metabolismo , Humanos , Meduloblastoma/genética , Meduloblastoma/patologia , Neurônios/efeitos dos fármacos , Cultura Primária de Células , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tiofenos/farmacologia , Tiofenos/uso terapêutico , Testes de Toxicidade , Ureia/análogos & derivados , Ureia/farmacologia , Ureia/uso terapêuticoRESUMO
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
Investigation of the sponge Clathria basilana collected in Indonesia afforded five new peptides, including microcionamides C (1) and D (2), gombamides B (4), C (5), and D (6), and an unusual amide, (E)-2-amino-3-methyl-N-styrylbutanamide (7), along with 11 known compounds, among them microcionamide A (3). The structures of the new compounds were elucidated by one- and two-dimensional NMR spectroscopy as well as by high-resolution mass spectrometry. The absolute configurations of the constituent amino acid residues in 1-7 were determined by Marfey's analysis. Microcionamides A, C, and D (1-3) showed in vitro cytotoxicity against lymphoma (Ramos) and leukemia cell lines (HL-60, Nomo-1, Jurkat J16), as well as against a human ovarian carcinoma cell line (A2780) with IC50 values ranging from 0.45 to 28 µM. Mechanistic studies showed that compounds 1-3 rapidly induce apoptotic cell death in Jurkat J16 and Ramos cells and that 1 and 2 potently block autophagy upon starvation conditions, thereby impairing pro-survival signaling of cancer cells. In addition, microcionamides C and A (1 and 3) inhibited bacterial growth of Staphylococcus aureus and Enterococcus faecium with minimal inhibitory concentrations between 6.2 and 12 µM. Mechanistic studies indicate dissipation of the bacterial membrane potential.
Assuntos
Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/isolamento & purificação , Poríferos/química , Animais , Antineoplásicos/química , Ensaios de Seleção de Medicamentos Antitumorais , Enterococcus faecium/efeitos dos fármacos , Indonésia , Biologia Marinha , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos Cíclicos/farmacologia , Staphylococcus aureus/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
We investigated cytotoxic mechanisms evoked by the spongean alkaloids aaptamine (Aa) and aeroplysinin-1 (Ap), applied alone and in combination with daunorubicin, employing acute myeloid leukemia (AML) cells. Aa and Ap reduced the viability of AML cells in a dose dependent manner with IC50 of 10-20 µM. Ap triggered apoptotic cell death more efficiently than Aa. Both alkaloids increased the protein level of S139-phosphorylated H2AX (γH2AX), which however was independent of the induction of DNA damage. Expression of the senescence markers p21 and p16 was increased, while the phosphorylation level of p-Chk-2 was reduced following Aa treatment. As a function of dose, Aa and Ap protected or sensitized AML cells against daunorubicin. Protection by Aa was paralleled by reduced formation of ROS and lower level of DNA damage. Both Aa and Ap attenuated daunorubicin-stimulated activation of the DNA damage response (DDR) as reflected on the levels of γH2AX, p-Kap-1 and p-Chk-1. Specifically Ap restored the decrease in S10 phosphorylation of histone H3 resulting from daunorubicin treatment. The cytoprotective effects of Aa and Ap were independent of daunorubicin import/export. Both Aa and Ap abrogated daunorubicin-induced accumulation of cells in S-phase. Inhibition of DNA synthesis was specific for Ap. The data show that Aa and Ap have both congruent and agent-specific pleiotropic effects that are preferential for anticancer drugs. Since Ap showed a broader spectrum of anticancer activities, this compound is suggested as novel lead compound for forthcoming in vivo studies elucidating the usefulness of spongean alkaloids in AML therapy.