RESUMEN
In this work, we report the scalable and modular synthesis of a library of 55 monomeric and dimeric flavonoids including 14 8,8'-biflavones. The sterically demanding tetra-ortho-substituted axis of an acetophenone dimer key intermediate was constructed in a regioselective manner using Fe-mediated oxidative coupling. This step was systematically optimized and performed on up to multigram scale. The biological activities of this compound library were evaluated, including cytotoxicity against healthy and malignant human cell lines, antimicrobial activity against the apicomplexan parasite Toxoplasma gondii, and antioxidant capacity. A marked increase in activity for the 8,8'-dimeric structures compared to that of their monomeric counterparts was observed. Several biflavones were identified with high selectivity indices (low cytotoxicity and high antiprotozoal activity), showing that this class of natural products may serve as lead structures for further investigations.
RESUMEN
BACKGROUND: The bacterial secondary metabolite prodigiosin has been shown to exert anticancer, antimalarial, antibacterial and immunomodulatory properties. With regard to cancer, it has been reported to affect cancer cells but not non-malignant cells, rendering prodigiosin a promising lead compound for anticancer drug discovery. However, a direct protein target has not yet been experimentally identified. METHODS: We used mass spectrometry-based thermal proteome profiling in order to identify target proteins of prodigiosin. For target validation, we employed a genetic knockout approach and electron microscopy. RESULTS: We identified the Golgi stacking protein GRASP55 as target protein of prodigiosin. We show that prodigiosin treatment severely affects Golgi morphology and functionality, and that prodigiosin-dependent cytotoxicity is partially reduced in GRASP55 knockout cells. We also found that prodigiosin treatment results in decreased cathepsin activity and overall blocks autophagic flux, whereas co-localization of the autophagosomal marker LC3 and the lysosomal marker LAMP1 is clearly promoted. Finally, we observed that autophagosomes accumulate at GRASP55-positive structures, pointing towards an involvement of an altered Golgi function in the autophagy-inhibitory effect of this natural compound. CONCLUSION: Taken together, we propose that prodigiosin affects autophagy and Golgi apparatus integrity in an interlinked mode of action involving the regulation of organelle alkalization and the Golgi stacking protein GRASP55. Video Abstract.
Asunto(s)
Aparato de Golgi , Prodigiosina , Humanos , Prodigiosina/farmacología , Prodigiosina/metabolismo , Aparato de Golgi/metabolismo , Lisosomas/metabolismo , Autofagosomas/metabolismo , AutofagiaRESUMEN
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.
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Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Ferroptosis/efectos de los fármacos , Inhibidores mTOR/farmacología , Naftiridinas/farmacología , Oximas/farmacología , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Proteolisis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Neoplasias de la Vejiga Urinaria/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Estrés Oxidativo/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
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.
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Proteínas Relacionadas con la Autofagia/genética , Inmunidad Innata/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinasas/genética , Proteína Sequestosoma-1/genética , Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Humanos , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Fosforilación/genética , Transducción de Señal/genéticaRESUMEN
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.
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Ensayos Analíticos de Alto Rendimiento/métodos , Floroglucinol/análogos & derivados , Pironas/uso terapéutico , Autofagia , Humanos , Floroglucinol/farmacología , Floroglucinol/uso terapéutico , Pironas/farmacologíaRESUMEN
Cisplatin-based treatment is the standard of care therapy for urothelial carcinomas. However, complex cisplatin resistance mechanisms limit the success of this approach. Both apoptosis and autophagy have been shown to contribute to this resistance. Prodigiosin, a secondary metabolite from various bacteria, exerts different biological activities including the modulation of these two cellular stress response pathways. We analyzed the effect of prodigiosin on protein levels of different autophagy- and apoptosis-related proteins in cisplatin-sensitive and -resistant urothelial carcinoma cells (UCCs). Furthermore, we investigated the effect on cell viability of prodigiosin alone or in combination with cisplatin. We made use of four different pairs of cisplatin-sensitive and -resistant UCCs. We found that prodigiosin blocked autophagy in UCCs and re-sensitized cisplatin-resistant cells to apoptotic cell death. Furthermore, we found that prodigiosin is a potent anticancer agent with nanomolar IC50 values in all tested UCCs. In combination studies, we observed that prodigiosin sensitized both cisplatin-sensitive and -resistant urothelial carcinoma cell lines to cisplatin treatment with synergistic effects in most tested cell lines. These effects of prodigiosin are at least partially mediated by altering lysosomal function, since we detected reduced activities of cathepsin B and L. We propose that prodigiosin is a promising candidate for the therapy of cisplatin-resistant urothelial carcinomas, either as a single agent or in combinatory therapeutic approaches.
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Antineoplásicos/química , Productos Biológicos/química , Prodigiosina/química , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Productos Biológicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Prodigiosina/farmacologíaRESUMEN
Malignant melanoma is an aggressive type of cancer and the deadliest form of skin cancer. Even though enormous efforts have been undertaken, in particular the treatment options against the metastasizing form are challenging and the prognosis is generally poor. A novel therapeutical approach is the application of secondary plant constituents occurring in food and food products. Herein, the effect of the dietary chalcone cardamonin, inter alia found in Alpinia species, was tested using human malignant melanoma cells. These data were compared to cardamonin treated normal melanocytes and dermal fibroblasts representing healthy cells. To investigate the impact of cardamonin on tumor and normal cells, it was added to monolayer cell cultures and cytotoxicity, proliferation, tumor invasion, and apoptosis were studied with appropriate cell biological and biochemical methods. Cardamonin treatment resulted in an apoptosis-mediated increase in cytotoxicity towards tumor cells, a decrease in their proliferation rate, and a lowered invasive capacity, whereas the viability of melanocytes and fibroblasts was hardly affected at such concentrations. A selective cytotoxic effect of cardamonin on melanoma cells compared to normal (healthy) cells was shown in vitro. This study along with others highlights that dietary chalcones may be a valuable tool in anticancer therapies which has to be proven in the future in vivo.