RESUMEN
BACKGROUND: Low targeting efficacy and high toxicity continue to be challenges in Oncology. A promising strategy is the glycosylation of chemotherapeutic agents to improve their pharmacodynamics and anti-tumoral activity. Herein, we provide evidence of a novel approach using diglycosidases from fungi of the Hypocreales order to obtain novel rutinose-conjugates therapeutic agents with enhanced anti-tumoral capacity. RESULTS: Screening for diglycosidase activity in twenty-eight strains of the genetically related genera Acremonium and Sarocladium identified 6-O-α-rhamnosyl-ß-glucosidase (αRßG) of Sarocladium strictum DMic 093557 as candidate enzyme for our studies. Biochemically characterization shows that αRßG has the ability to transglycosylate bulky OH-acceptors, including bioactive compounds. Interestingly, rutinoside-derivatives of phloroglucinol (PR) resorcinol (RR) and 4-methylumbelliferone (4MUR) displayed higher growth inhibitory activity on pancreatic cancer cells than the respective aglycones without significant affecting normal pancreatic epithelial cells. PR exhibited the highest efficacy with an IC50 of 0.89 mM, followed by RR with an IC50 of 1.67 mM, and 4MUR with an IC50 of 2.4 mM, whereas the respective aglycones displayed higher IC50 values: 4.69 mM for phloroglucinol, 5.90 mM for resorcinol, and 4.8 mM for 4-methylumbelliferone. Further, glycoconjugates significantly sensitized pancreatic cancer cells to the standard of care chemotherapy agent gemcitabine. CONCLUSIONS: αRßG from S. strictum transglycosylate-based approach to synthesize rutinosides represents a suitable option to enhance the anti-proliferative effect of bioactive compounds. This finding opens up new possibilities for developing more effective therapies for pancreatic cancer and other solid malignancies.
Asunto(s)
Antineoplásicos , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Antineoplásicos/farmacología , Antineoplásicos/química , Línea Celular Tumoral , Hypocreales/metabolismo , Rutina/farmacología , Rutina/química , Acremonium , Gemcitabina , Disacáridos/farmacología , Disacáridos/químicaRESUMEN
BACKGROUND & AIMS: Reaching efficacious drug delivery to target cells/tissues represents a major obstacle in the current treatment of solid malignancies including hepatocellular carcinoma (HCC). In this study, we developed a pipeline to selective add complex-sugars to the aglycone 4-methylumbelliferone (4MU) to help their bioavailability and tumour cell intake. METHODS: The therapeutic efficacy of sugar-modified rutinosyl-4-methylumbelliferone (4MUR) and 4MU were compared in vitro and in an orthotopic HCC model established in fibrotic livers. The mechanistic bases of its selective target to liver tumour cells were evaluated by the interaction with asialoglycoprotein receptor (ASGPR), the mRNA expression of hyaluronan synthases (HAS2 or HAS3) and hyaluronan deposition. RESULTS: 4MUR showed a significant antiproliferative effect on liver tumoural cells as compared to non-tumoural cells in a dose-dependent manner. Further analysis showed that 4MUR is incorporated mostly into HCC cells by interaction with ASGPR, a receptor commonly overexpressed in HCC cells. 4MUR-treatment decreased the levels of HAS2 and HAS3 and the cytoplasmic deposition of hyaluronan. Moreover, 4MUR reduced CFSC-2G activation, hence reducing the fibrosis. In vivo efficacy showed that 4MUR treatment displayed a greater tumour growth inhibition and increased survival in comparison to 4MU. 4MUR administration was associated with a significant reduction of liver fibrosis without any signs of tissue damage. Further, 60% of 4MUR treated mice did not present macroscopically tumour mass post-treatment. CONCLUSION: Our results provide evidence that 4MUR may be used as an effective HCC therapy, without damaging non-tumoural cells or other organs, most probably due to the specific targeting.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animales , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Hialuronano Sintasas , Himecromona/farmacología , Himecromona/uso terapéutico , Cirrosis Hepática/patología , Neoplasias Hepáticas/patología , RatonesRESUMEN
Cancer constitutes the second leading cause of death globally and is considered to have been responsible for an estimated 9.6 million fatalities in 2018. Although treatments against gastrointestinal tumors have recently advanced, those interventions can only be applied to a minority of patients at the time of diagnosis. Therefore, new therapeutic options are necessary for advanced stages of the disease. Glycosylation of antitumor agents, has been found to improve pharmacokinetic parameters, reduce side effects, and expand drug half-life in comparison with the parent compounds. In addition, glycosylation of therapeutic agents has been proven to be an effective strategy for their targeting tumor tissue, thereby reducing the doses of the glycodrugs administered to patients. This review focusses on the effect of the targeting properties of glycosylated antitumor agents on gastrointestinal tumors.
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We have elucidated a novel mechanism through which the autophagy-specific class III phosphatidylinositol 3-kinase (PtdIns3K) complex can be recruited to the PAS in mammalian cells, through the interaction between BECN1 and the vacuole membrane protein 1 (VMP1), an integral autophagosomal membrane protein. This interaction involves the binding between the C-terminal 20 amino acids of the VMP1 hydrophilic domain, which we have named the VMP1 autophagy-related domain (VMP1-AtgD), and the BH3 domain of BECN1. The association between these two proteins allows the formation of the autophagy-specific PtdIns3K complex, which activity favors the generation of phosphatidylinositol-3-phosphate (PtdIns3P) and the subsequent association of the autophagy-related (ATG) proteins, including ATG16L1, with the phagophore membranes. Therefore, VMP1 regulates the PtdIns3K activity on the phagophore membrane through its interaction with BECN1. Our data provide a novel model describing one of the key steps in phagophore assembly site (PAS) formation and autophagy regulation, and positions VMP1 as a new interactor of the autophagy-specific PtdIns3K complex in mammalian cells.
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Autofagia , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Activación Enzimática , Humanos , Modelos Biológicos , Unión ProteicaRESUMEN
The Vacuole Membrane Protein 1 -VMP1- is a pancreatitis-associated transmembrane protein whose expression triggers autophagy in several human diseases. In the current study, we unveil the mechanism through which this protein induces autophagosome formation in mammalian cells. We show that VMP1 autophagy-related function requires its 20-aminoacid C-terminus hydrophilic domain (VMP1-AtgD). This is achieved through its direct binding to the BH3 motif of Beclin 1 leading to the formation of a complex with the Class III phosphatidylinositol-3 kinase (PI3K) hVps34, a key positive regulator of autophagy, at the site where autophagosomes are generated. This interaction also concomitantly promotes the dissociation of Bcl-2, an autophagy inhibitor, from Beclin 1. Moreover, we show that the VMP1-Beclin 1-hVps34 complex favors the association of Atg16L1 and LC3 with the autophagosomal membranes. Collectively, these findings reveal that VMP1 expression recruits and activates the Class III PI3K complex at the site of autophagosome formation during mammalian autophagy.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia/fisiología , Proteínas de la Membrana/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/química , Beclina-1 , Línea Celular , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Humanos , Proteínas de la Membrana/química , Ratones , Complejos Multiproteicos/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismoRESUMEN
Autophagy is an evolutionarily conserved degradation process of cytoplasmic cellular constituents. It has been suggested that autophagy plays a role in tumor promotion and progression downstream oncogenic pathways; however, the molecular mechanisms underlying this phenomenon have not been elucidated. Here, we provide both in vitro and in vivo evidence of a novel signaling pathway whereby the oncogene KRAS induces the expression of VMP1, a molecule needed for the formation of the authophagosome and capable of inducing autophagy, even under nutrient-replete conditions. RNAi experiments demonstrated that KRAS requires VMP1 to induce autophagy. Analysis of the mechanisms identified GLI3, a transcription factor regulated by the Hedgehog pathway, as an effector of KRAS signaling. GLI3 regulates autophagy as well as the expression and promoter activity of VMP1 in a Hedgehog-independent manner. Chromatin immunoprecipitation assays demonstrated that GLI3 binds to the VMP1 promoter and complexes with the histone acetyltransferase p300 to regulate promoter activity. Knockdown of p300 impaired KRAS- and GLI3-induced activation of this promoter. Finally, we identified the PI3K-AKT1 pathway as the signaling pathway mediating the expression and promoter activity of VMP1 upstream of the GLI3-p300 complex. Together, these data provide evidence of a new regulatory mechanism involved in autophagy that integrates this cellular process into the molecular network of events regulating oncogene-induced autophagy.
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Autofagia , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas de la Membrana/biosíntesis , Neoplasias/mortalidad , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Proteínas ras/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Regulación Neoplásica de la Expresión Génica/genética , Células HeLa , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Proteínas de la Membrana/genética , Ratones , Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Elementos de Respuesta/genética , Proteína Gli3 con Dedos de Zinc , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismo , Proteínas ras/genéticaRESUMEN
Autophagy is an evolutionarily preserved degradation process of cytoplasmic cellular constituents and plays important physiological roles in human health and disease. It has been proposed that autophagy plays an important role both in tumor progression and in promotion of cancer cell death, although the molecular mechanisms responsible for this dual action of autophagy in cancer have not been elucidated. Pancreatic ductal adenocarcinoma is one of the most aggressive human malignancies with 2-3% five-year survival rate. Its poor prognosis has been attributed to the lack of specific symptoms and early detection tools, and its relatively refractory to traditional cytotoxic agents and radiotherapy. Experimental evidence pointed at autophagy as a pancreatic cancer cell mechanism to survive under adverse environmental conditions, or as a defective programmed cell death mechanism that favors pancreatic cancer cell resistance to treatment. Here, we consider several phenotypical alterations that have been related to increase or decrease the autophagic process in pancreatic tumor cells. We specially review autophagy as a cell death mechanism in response to chemotherapeutic drugs.
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Autofagia , Carcinoma Ductal Pancreático/patología , Neoplasias Pancreáticas/patología , Animales , Autofagia/efectos de los fármacos , Capecitabina , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapéutico , Resistencia a Antineoplásicos , Fluorouracilo/análogos & derivados , Fluorouracilo/uso terapéutico , Humanos , Pronóstico , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/fisiología , GemcitabinaRESUMEN
Autophagy has recently elicited significant attention as a mechanism that either protects or promotes cell death, although different autophagy pathways, and the cellular context in which they occur, remain to be elucidated. We report a thorough cellular and biochemical characterization of a novel selective autophagy that works as a protective cell response. This new selective autophagy is activated in pancreatic acinar cells during pancreatitis-induced vesicular transport alteration to sequester and degrade potentially deleterious activated zymogen granules. We have coined the term "zymophagy" to refer to this process. The autophagy-related protein VMP1, the ubiquitin-protease USP9x, and the ubiquitin-binding protein p62 mediate zymophagy. Moreover, VMP1 interacts with USP9x, indicating that there is a close cooperation between the autophagy pathway and the ubiquitin recognition machinery required for selective autophagosome formation. Zymophagy is activated by experimental pancreatitis in genetically engineered mice and cultured pancreatic acinar cells and by acute pancreatitis in humans. Furthermore, zymophagy has pathophysiological relevance by controlling pancreatitis-induced intracellular zymogen activation and helping to prevent cell death. Together, these data reveal a novel selective form of autophagy mediated by the VMP1-USP9x-p62 pathway, as a cellular protective response.