RESUMEN
Macrophages constitute a major part of tumor microenvironment, and most of existing data demonstrate their ruling role in the development of anti-drug resistance of cancer cell. One of the most powerful protection system is based on heat shock proteins whose synthesis is triggered by activated Heat Shock Factor-1 (HSF1); the inhibition of the HSF1 with CL-43 sensitized A549 lung cancer cells to the anti-cancer effect of etoposide. Notably, analyzing A549 tumor xenografts in mice we observed nest-like pattern of co-localization of A549 cells demonstrating enhanced expression of HSF1 with macrophages, and decided to check whether the above arrangement has a functional value for both cell types. It was found that the incubation of A549 or DLD1 colon cancer cells with either human monocytes or THP1 monocyte-like cells activated HSF1 and increased resistance to etoposide. Importantly, the same effect was shown when primary cultures of colon tumors were incubated with THP1 cells or with human monocytes. To prove that HSF1 is implicated in enhanced resistance caused by monocytic cells, we generated an A549 cell subline devoid of HSF1 which did not respond to incubation with THP1 cells. The pharmacological inhibition of HSF1 with CL-43 also abolished the effect of THP1 cells on primary tumor cells, highlighting a new target of tumor-associated macrophages in a cell proteostasis mechanism.
Asunto(s)
Proteínas de Unión al ADN , Factores de Transcripción , Animales , Humanos , Ratones , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Resistencia a Medicamentos , Etopósido/farmacología , Factores de Transcripción del Choque Térmico/metabolismo , Respuesta al Choque Térmico , Factores de Transcripción/metabolismo , Macrófagos Asociados a Tumores/metabolismoRESUMEN
Proteostasis mechanisms, such as proteotoxic-stress response and autophagy, are increasingly recognized for their roles in influencing various cancer hallmarks such as tumorigenesis, drug resistance, and recurrence. However, the precise mechanisms underlying their coordination remain not fully elucidated. The aim of this study is to investigate the molecular interplay between Hsp70 and autophagy in lung adenocarcinoma cells and elucidate its impact on the outcomes of anticancer therapies in vitro. For this purpose, we utilized the human lung adenocarcinoma A549 cell line and genetically modified it by knockdown of Hsp70 or HSF1, and the H1299 cell line with knockdown or overexpression of Hsp70. In addition, several treatments were employed, including treatment with Hsp70 inhibitors (VER-155008 and JG-98), HSF1 activator ML-346, or autophagy modulators (SAR405 and Rapamycin). Using immunoblotting, we found that Hsp70 negatively regulates autophagy by directly influencing AMPK activation, uncovering a novel regulatory mechanism of autophagy by Hsp70. Genetic or chemical Hsp70 overexpression was associated with the suppression of AMPK and autophagy. Conversely, the inhibition of Hsp70, genetically or chemically, resulted in the upregulation of AMPK-mediated autophagy. We further investigated whether Hsp70 suppression-mediated autophagy exhibits pro-survival- or pro-death-inducing effects via MTT test, colony formation, CellTiter-Glo 3D-Spheroid viability assay, and Annexin/PI apoptosis assay. Our results show that combined inhibition of Hsp70 and autophagy, along with cisplatin treatment, synergistically reduces tumor cell metabolic activity, growth, and viability in 2D and 3D tumor cell models. These cytotoxic effects were exerted by substantially potentiating apoptosis, while activating autophagy via rapamycin slightly rescued tumor cells from apoptosis. Therefore, our findings demonstrate that the combined inhibition of Hsp70 and autophagy represents a novel and promising therapeutic approach that may disrupt the capacity of refractory tumor cells to withstand conventional therapies in NSCLC.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Autofagia , Carcinoma de Pulmón de Células no Pequeñas , Proteínas HSP70 de Choque Térmico , Neoplasias Pulmonares , Humanos , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Autofagia/efectos de los fármacos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Línea Celular Tumoral , Proteínas Quinasas Activadas por AMP/metabolismo , Células A549 , Factores de Transcripción del Choque Térmico/metabolismo , Factores de Transcripción del Choque Térmico/genética , Sirolimus/farmacología , Apoptosis/efectos de los fármacos , Nucleósidos de Purina/farmacología , Isoxazoles , ResorcinolesRESUMEN
New anthraquinone derivatives acruciquinones A-C (1-3), together with ten known metabolites, were isolated from the obligate marine fungus Asteromyces cruciatus KMM 4696. Acruciquinone C is the first member of anthraquinone derivatives with a 6/6/5 backbone. The structures of isolated compounds were established based on NMR and MS data. The absolute stereoconfigurations of new acruciquinones A-C were determined using ECD and quantum chemical calculations (TDDFT approach). A plausible biosynthetic pathway of the novel acruciquinone C was proposed. Compounds 1-4 and 6-13 showed a significant antimicrobial effects against Staphylococcus aureus growth, and acruciquinone A (1), dendryol B (4), coniothyrinone B (7), and ω-hydroxypachybasin (9) reduced the activity of a key staphylococcal enzyme, sortase A. Moreover, the compounds, excluding 4, inhibited urease activity. We studied the effects of anthraquinones 1, 4, 7, and 9 and coniothyrinone D (6) in an in vitro model of skin infection when HaCaT keratinocytes were cocultivated with S. aureus. Anthraquinones significantly reduce the negative impact of S. aureus on the viability, migration, and proliferation of infected HaCaT keratinocytes, and acruciquinone A (1) revealed the most pronounced effect.
Asunto(s)
Ascomicetos , Infecciones Estafilocócicas , Staphylococcus aureus , Antraquinonas/farmacologíaRESUMEN
The risk of progression of most sporadic neurodegenerative diseases, including Alzheimer's disease, increases with age. Traditionally, this is associated with a decrease in the efficiency of cell protection systems, in particular, molecular chaperones. Thus, the development of small molecules able to induce the synthesis of chaperones is a promising therapeutic approach to prevent neural diseases associated with ageing. Here, we describe a new compound IA-50, belonging to the class of indolylazines and featured by a low size of topological polar surface area, the property related to substances with potentially high membrane-penetrating activity. We also estimated the absorption, distribution, metabolism and excretion characteristics of IA-50 and found the substance to fit the effective drug criteria. The new compound was found to induce the synthesis and accumulation of Hsp70 in normal and aged neurons and in the hippocampi of young and old mice. The transgenic model of Alzheimer's disease, based on 5xFAD mice, confirmed that the injection of IA-50 prevented the formation of ß-amyloid aggregates, loss of hippocampal neurons and the development of memory impairment. These data indicate that this novel substance may induce the expression of chaperones in neural cells and brain tissues, suggesting its possible application in the therapy of ageing-associated disorders.
Asunto(s)
Enfermedad de Alzheimer , Ratones , Animales , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Neuronas/metabolismo , Envejecimiento/metabolismo , Chaperonas Moleculares/metabolismo , Ratones Transgénicos , Modelos Animales de EnfermedadRESUMEN
Cerebrosides are glycosylated sphingolipids, and in mammals they contribute to the pro-/anti-inflammatory properties and innate antimicrobial activity of the skin and mucosal surfaces. Staphylococcus aureus infection can develop, not only from minor scratches of the skin, but this pathogen can also actively promote epithelial breach. The effect of cerebroside flavuside B from marine sediment-derived fungus Penicillium islandicum (Aniva Bay, the Sea of Okhotsk) on viability, apoptosis, total caspase activity, and cell cycle in human epidermal keratinocytes HaCaT line co-cultivated with S. aureus, as well as influence of flavuside B on LPS-treated HaCaT cells were studied. Influence of flavuside B on bacterial growth and biofilm formation of S. aureus and its effect on the enzymatic activity of sortase A was also investigated. It was found S. aureus co-cultivated with keratinocytes induces caspase-depended apoptosis and cell death, arrest cell cycle in the G0/G1 phase, and increases in cellular immune inflammation. Cerebroside flavuside B has demonstrated its antimicrobial and anti-inflammatory properties, substantially eliminating all the negative consequences caused by co-cultivation of keratinocytes with S. aureus or bacterial LPS. The dual action of flavuside B may be highly effective in the treatment of bacterial skin lesions and will be studied in the future in in vivo experiments.
Asunto(s)
Antibacterianos/farmacología , Cerebrósidos/farmacología , Glicoesfingolípidos/farmacología , Queratinocitos/efectos de los fármacos , Penicillium , Enfermedades Cutáneas Bacterianas/prevención & control , Infecciones Estafilocócicas/prevención & control , Staphylococcus aureus/efectos de los fármacos , Organismos Acuáticos , Células HaCaT/efectos de los fármacos , HumanosRESUMEN
Hypoxia, which commonly accompanies tumor growth, depending on its strength may cause the enhancement of tumorigenicity of cancer cells or their death. One of the proteins targeted by hypoxia is glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and we demonstrated here that hypoxia mimicked by treating C6 rat glioblastoma cells with cobalt chloride caused an up-regulation of the enzyme expression, while further elevation of hypoxic stress caused the enzyme aggregation concomitantly with cell death. Reduction or elevation of GAPDH performed with the aid of specific shRNAs resulted in the augmentation of the tumorigenicity of C6 cells or their sensitization to hypoxic stress. Another hypoxia-regulated protein, Hsp70 chaperone, was shown to prevent the aggregation of oxidized GAPDH and to reduce hypoxia-mediated cell death. In order to release the enzyme molecules from the chaperone, we employed its inhibitor, derivative of colchicine. The compound was found to substantially increase aggregation of GAPDH and to sensitize C6 cells to hypoxia both in vitro and in animals bearing tumors with distinct levels of the enzyme expression. In conclusion, blocking the chaperonic activity of Hsp70 and its interaction with GAPDH may become a promising strategy to overcome tumor resistance to multiple environmental stresses and enhance existing therapeutic tools.
Asunto(s)
Hipoxia de la Célula , Glioblastoma/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Animales , Línea Celular , Cobalto , Glioblastoma/fisiopatología , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Células HEK293 , Humanos , Oxidación-Reducción , Agregado de Proteínas , Unión Proteica , RatasRESUMEN
Hyperglycemia may contribute to the progression of carcinomas by triggering epithelial-to-mesenchymal transition (EMT). Some proteostasis systems are involved in metastasis; in this paper, we sought to explore the mechanism of Hsp70 chaperone in EMT. We showed that knockdown of Hsp70 reduced cell migration capacity concomitantly with levels of mRNA of the Slug, Snail, and Twist markers of EMT, in colon cancer cells incubated in high glucose medium. Conversely, treatment of cells with Hsp70 inducer U-133 were found to elevate cell motility, along with the other EMT markers. To prove that inhibiting Hsp70 may reduce EMT efficiency, we treated cells with a CL-43 inhibitor of the HSF1 transcription factor, which lowered Hsp70 and HSF1 content in the control and induced EMT in carcinoma cells. Importantly, CL-43 reduced migration capacity, EMT-linked transcription factors, and increased content of epithelial marker E-cadherin in colon cancer cells of three lines, including one derived from a clinical sample. To prove that Hsp70 chaperone should be targeted when inhibiting the EMT pathway, we treated cancer cells with 2-phenylethynesulfonamide (PES) and demonstrated that the compound inhibited substrate-binding capacity of Hsp70. Furthermore, PES suppressed EMT features, cell motility, and expression of specific transcription factors. In conclusion, the Hsp70 chaperone machine efficiently protects mechanisms of the EMT, and the safe inhibitors of the chaperone are needed to hamper metastasis at its initial stage.
Asunto(s)
Glucemia , Transición Epitelial-Mesenquimal , Glucosa/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Biomarcadores , Cadherinas/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Glucosa/farmacología , Humanos , Hiperglucemia/etiología , Hiperglucemia/metabolismo , Unión Proteica , Factores de Transcripción de la Familia Snail/metabolismoRESUMEN
Defective pluripotent cells are removed from embryos prior to differentiation, presumably due to upregulation of the p53 pathway. However, the mechanism underlying p53 protein activation is still unknown. Embryonic stem cells (ESCs), corresponding to cells of the preimplantation blastocyst, likely have similar mechanisms for abnormal cell elimination. Using a mouse ESC cell line with inducible ulk1 gene expression, we showed that Ulk1 upregulation is accompanied by p53 phosphorylation on Ser15. ESCs tolerated the activated p53 and did not undergo apoptosis or cell cycle blockade upon Ulk1 overexpression. However, massive cell death was observed after retinoic acid treatment, suggesting a role of Ulk1-induced p53 activation in the elimination of defective pluripotent cells prior to differentiation.
Asunto(s)
Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Células Madre Embrionarias de Ratones/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Autofagia/fisiología , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Muerte Celular , Línea Celular , Proliferación Celular , Doxiciclina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Fosforilación/efectos de los fármacos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/fisiología , Serina/metabolismo , Tretinoina/farmacologíaRESUMEN
Cancer cells are known to contain high levels of the heat shock protein 70 kDa (Hsp70), which mediates increased cell proliferation, escape from programmed cell death, enhanced invasion, and metastasis. A part of Hsp70 molecules may release from cancer cells and affect the behavior of adjacent stromal cells. To explore the effects of Hsp70 on the status of monocytes/macrophages in the tumor locale, we incubated human carcinoma cells of three distinct lines with normal and reduced content of Hsp70 with THP1 monocytes. Using two methods, we showed that the cells with knock-down of Hsp70 released a lower amount of protein in the extracellular medium. Three cycles of the co-cultivation of cancer and monocytic cells led to the secretion of several cytokines typical of the tumor microenvironment (TME) and to pro-cancer activation of the monocytes/macrophages as established by elevation of F4/80 and arginase-1 markers. Unexpectedly, the efficacy of epithelial-mesenchymal transition and resistance of carcinoma cells to anticancer drugs after incubation with monocytic cells were more pronounced in cells with lower Hsp70, e.g., releasing less Hsp70 into the extracellular milieu. These data suggest that Hsp70 released from tumor cells into the TME is able, together with the development of an anti-cancer immune response, to limit the conversion of a considerable part of monocytic cells to the pro-tumor phenotype.
Asunto(s)
Carcinogénesis/inmunología , Proteínas HSP70 de Choque Térmico/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Microambiente Tumoral , Células A549 , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular , Técnicas de Cocultivo , Transición Epitelial-Mesenquimal , Humanos , Inmunidad , Macrófagos/patología , Monocitos/patologíaRESUMEN
Massive neuronal death caused by a neurodegenerative pathology or damage due to ischaemia or traumatic brain injury leads to the appearance of cytosolic proteins in the extracellular space. We found that one of the most abundant cellular polypeptides, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), appearing in the medium of dying cells or body fluids is able to form aggregates that are cytotoxic to adjacent cells. Since we previously showed that the hydrocortisone derivative RX624 can inhibit the ability of GAPDH to transport the enzyme complex with polyglutamine and reduce the cytotoxicity of the complex, we explored the effects of GAPDH on SH-SY5Y neuroblastoma cells. We found that the latter treated with particular forms of GAPDH molecules die with a high efficiency, suggesting that the exogenous enzyme does kill adjacent cells. RX624 prevented the interaction of exogenous GAPDH with the cell membrane and reduced the level of death by more than 10%. We also demonstrated the efficiency of RX624 treatment in a rat model of traumatic brain injury. The chemical blocked the formation of GAPDH aggregates in the brain, inhibited the cytotoxic effects of cerebrospinal fluid and rescued the motor function of injured rats. Importantly, RX624 treatment of rats had a similar effect as the intracranial injection of anti-GAPDH antibodies.
Asunto(s)
Lesiones Traumáticas del Encéfalo/patología , Lesiones Traumáticas del Encéfalo/terapia , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Terapia Molecular Dirigida , Animales , Lesiones Traumáticas del Encéfalo/líquido cefalorraquídeo , Lesiones Traumáticas del Encéfalo/fisiopatología , Línea Celular Tumoral , Membrana Celular/metabolismo , Modelos Animales de Enfermedad , Humanos , Actividad Motora , Conejos , Ratas WistarRESUMEN
The Hsp70 chaperone binds and inhibits proteins implicated in apoptotic signaling including Caspase-3. Induction of apoptosis is an important mechanism of anti-cancer drugs, therefore Hsp70 can act as a protective system in tumor cells against therapeutic agents. In this study we present an assessment of candidate compounds that are able to dissociate the complex of Hsp70 with Caspase-3, and thus sensitize cells to drug-induced apoptosis. Using the PASS program for prediction of biological activity we selected a derivative of benzodioxol (BT44) that is known to affect molecular chaperones and caspases. Drug affinity responsive target stability and microscale thermophoresis assays indicated that BT44 bound to Hsp70 and reduced the chaperone activity. When etoposide was administered, heat shock accompanied with an accumulation of Hsp70 led to an inhibition of etoposide-induced apoptosis. The number of apoptotic cells increased following BT44 administration, and forced Caspase-3 processing. Competitive proteinâ»protein interaction and immunoprecipitation assays showed that BT44 caused dissociation of the Hsp70â»Caspase-3 complex, thus augmenting the anti-tumor activity of etoposide and highlighting the potential role of molecular separators in cancer therapy.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Etopósido/farmacología , Proteínas HSP70 de Choque Térmico/metabolismo , Neoplasias/metabolismo , Apoptosis/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Proteínas HSP70 de Choque Térmico/genética , Humanos , Unión ProteicaRESUMEN
Huntington's disease (HD) has been recently shown to have a horizontally transmitted, prion-like pathology. Thus, the migration of polyglutamine-containing aggregates to acceptor cells is important for the progression of HD. These aggregates contain glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which increases their intracellular transport and their toxicity. Here, we show that RX624, a derivative of hydrocortisone that binds to GAPDH, prevents the formation of aggregates of GAPDH-polyglutamine excreted into the culture medium by PC-12 rat cells expressing mutant huntingtin. RX624 was previously shown to be unable to penetrate cells and, thus, its principal therapeutic action might be the inhibition of polyglutamine-GAPDH complex aggregation in the extracellular matrix. The administration of RX624 to SH-SY5Y acceptor cells that incubated in conditioned medium from PC-12 cells expressing mutant huntingtin caused an approximately 20% increase in survival. This suggests that RX624 might be useful as a drug against polyglutamine pathologies, and that is could be administered exogenously without affecting target cell physiology. This protective effect was validated by the similar effect of an anti-GAPDH specific antibody.
Asunto(s)
Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Hidrocortisona/administración & dosificación , Neuronas/metabolismo , Agregado de Proteínas/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Líquido Extracelular , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/antagonistas & inhibidores , Humanos , Hidrocortisona/análogos & derivados , Hidrocortisona/farmacocinética , Neuronas/citología , Neuronas/efectos de los fármacos , PéptidosRESUMEN
The common feature of Huntington disease is the accumulation of oligomers or aggregates of mutant huntingtin protein (mHTT), which causes the death of a subset of striatal neuronal populations. The cytotoxic species can leave neurons and migrate to other groups of cells penetrating and damaging them in a prion-like manner. We hypothesized that the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), previously shown to elevate the aggregation of mHTT, is associated with an increased efficiency of intercellular propagation of mHTT. GAPDH, on its own or together with polyglutamine species, was shown to be released into the extracellular milieu mainly from dying cells as assessed by a novel enzyme immunoassay, western blotting, and ultrafiltration. The conditioned medium of cells with growing GAPDH-polyQ aggregates was toxic to naïve cells, whereas depletion of the aggregates from the medium lowered this cytotoxicity. The GAPDH component of the aggregates was found to increase their toxicity by two-fold in comparison with polyQ alone. Furthermore, GAPDH-polyQ complexes were shown to penetrate acceptor cells and to increase the capacity of polyQ to prionize its intracellular homolog containing a repeat of 25 glutamine residues. Finally, inhibitors of intracellular transport showed that polyQ-GAPDH complexes, as well as GAPDH itself, penetrated cells using clathrin-mediated endocytosis. This suggested a pivotal role of the enzyme in the intercellular transmission of Huntington disease pathogenicity. In conclusion, GAPDH occurring in complexes with polyglutamine strengthens the prion-like activity and toxicity of the migrating aggregates. Aggregating polygluatmine tracts were shown to release from the cells over-expressing mutant huntingtin in a complex with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The enzyme enhances the intracellular transport of aggregates to healthy cells, prionization of normal cellular proteins and finally cell death, thus demonstrating the pivotal role of GAPDH in the horizontal transmission of neurodegeneration.
Asunto(s)
Transporte Biológico/fisiología , Muerte Celular/efectos de la radiación , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Enfermedad de Huntington/metabolismo , Neuronas/metabolismo , Péptidos/farmacología , Animales , Células Cultivadas , Citoplasma/metabolismo , Neuronas/efectos de los fármacos , RatasRESUMEN
The Hsp70 chaperone is known to elicit cytoprotective activity and this protection has a negative impact in anti-tumor therapy. In cancer cells subjected to oxidative stress Hsp70 may bind damaged polypeptides and proteins involved in apoptosis signaling. Since one of the important targets of oxidative stress is glyceraldehyde-3-phospate dehydrogenase (GAPDH) we suggested that Hsp70 might elicit its protective effect by binding GAPDH. Microscopy data show that in C6 rat glioma cells subjected to hydrogen peroxide treatment a considerable proportion of the GAPDH molecules are denatured and according to dot ultrafiltration data they form SDS-insoluble aggregates. Using two newly developed assays we show that Hsp70 can bind oxidized GAPDH in an ATP-dependent manner. Pharmacological up- or down-regulation of Hsp70 with the aid of U133 echinochrome or triptolide, respectively, reduced or increased the number of C6 glioma cells containing GAPDH aggregates and dying due to treatment with hydrogen peroxide. Using immunoprecipitation we found that Hsp70 is able to sequester aggregation-prone GAPDH and this may explain the anti-oxidative power of the chaperone. The results of this study led us to conclude that in cancer cells constantly exposed to conditions of oxidative stress, the protective power of Hsp70 should be abolished by specific inhibitors of Hsp70 expression.
Asunto(s)
Adenosina Trifosfato/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patología , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Estrés Oxidativo , Animales , Apoptosis , Línea Celular Tumoral , Supervivencia Celular , Chaperonas Moleculares/metabolismo , RatasRESUMEN
Recombinant HSP70 chaperone exerts a profound anticancer effect when administered intratumorally. This action is based on the ability of HSP70 to penetrate tumor cells and extract its endogenous homolog. To enhance the efficacy of HSP70 cycling, we employed phloretin, a flavonoid that enhances the pore-forming activity of the chaperone on artificial membranes. Phloretin increased the efficacy of HSP70 penetration in B16 mouse melanoma cells and K-562 human erythroblasts; this was accompanied with increased transport of the endogenous HSP70 to the plasma membrane. Importantly, treatment with HSP70 combined with phloretin led to the elevation of cell sensitivity to cytotoxic lymphocytes by 16-18 % compared to treatment with the chaperone alone. The incubation of K-562 cells with biotinylated HSP70 and phloretin increased the amount of the chaperone released from cells, suggesting that chaperone cycling could trigger a specific anti-tumor response. We studied the effect of the combination of HSP70 and phloretin using B16 melanoma and a novel method of HSP70-gel application. We found that the addition of phloretin to the gel reduced tumor weight almost fivefold compared with untreated mice, while the life span of the animals extended from 25 to 39 days. The increased survival was corroborated by the activation of innate and adaptive immunity; interestingly, HSP70 was more active in induction of CD8+ cell-mediated toxicity and γIFN production while phloretin contributed largely to the CD56+ cell response. In conclusion, the combination of HSP70 with phloretin could be a novel treatment for efficient immunotherapy of intractable cancers such as skin melanoma.
Asunto(s)
Proteínas HSP70 de Choque Térmico/inmunología , Inmunoterapia/métodos , Melanoma/inmunología , Microscopía Confocal/métodos , Floretina/inmunología , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Neoplasias Cutáneas , Melanoma Cutáneo MalignoRESUMEN
Superparamagnetic iron-oxide based contrast agents can provide important diagnostic information regarding the assessment of cardiac inflammatory diseases. The aim of the study was to analyze whether nanoparticles conjugated to recombinant 70-kDa heat shock protein (Hsp70-SPION) can be applied for the detection of acute myocardium infarct by MRI. Cellular experiments demonstrated increased CD40-mediated uptake of Hsp70-SPIONs in comparison to non-conjugated SPIONs. Following induction of an acute infarct in rats by ligation of the left anterior descending artery SPIONs and Hsp70-SPION conjugates were injected intravenously on day 4. The animals underwent sequential MRI that showed the presence of the particles in the infarcted zone. Subsequent biodistribution analyses with the help of method on non-linear magnetic response indicated the preferential accumulation of the Hsp70-SPIONs in the heart tissue that was further confirmed with histological analyses. The study demonstrated that an acute infarct can be visualized by MRI using Hsp70-functionalized SPION conjugates. FROM THE CLINICAL EDITOR: Superparamagnetic iron oxides nanoparticles (SPIONs) have been studied extensively as a contrast agent for MRI. Their tissue specificity can be further enhanced by conjugation with various ligands. In this study, the authors conjugated superparamagnetic nanoparticles to 70-kDa heat shock protein (Hsp70-SPION) to investigate the feasibility for the detection of acute myocardium infarct. The positive findings would suggest that this approach might be used clinically in the future.
Asunto(s)
Medios de Contraste/química , Compuestos Férricos/química , Proteínas HSP70 de Choque Térmico/química , Imagen por Resonancia Magnética/métodos , Nanopartículas de Magnetita/química , Infarto del Miocardio/diagnóstico por imagen , Animales , Medios de Contraste/farmacocinética , Compuestos Férricos/farmacocinética , Proteínas HSP70 de Choque Térmico/farmacocinética , Nanopartículas de Magnetita/análisis , Masculino , Miocardio/patología , Ratas Wistar , Distribución TisularRESUMEN
Chaperone Hsp70 can activate adaptive immunity suggesting its possible application as an antitumor vaccine. To assess the therapeutic capacity of Hsp70 we administered purified chaperone into a C6 glioblastoma brain tumor and explored the viability and tumor size as well as interferon gamma (IFNγ) production and cytotoxicity of lymphocytes in the treated animals. Targeted intratumoral injection of Hsp70 resulted in its distribution within the area of glioblastoma, and caused significant inhibition of tumor progression as confirmed by magnetic resonance imaging. The delay in tumor growth corresponded to the prolonged survival of tumor-bearing animals of up to 31 days versus 20 days in control. Continuous administration of Hsp70 with an osmotic pump increased survival even further (39 days). Therapeutic efficacy was associated with infiltration to glioblastoma of NK cells (Ly-6c+) and T lymphocytes (CD3+, CD4+ and CD8+) as well as with an increase in the activity of NK cells (granzyme B production) and CD8+ T lymphocytes as shown by IFNγ ELISPOT assay. Furthermore, we found that Hsp70 treatment caused concomitantly, with a tenfold elevated IFNγ production, an increase in anti-C6 tumor cytotoxicity of lymphocytes. In conclusion, continuous intratumoral delivery of Hsp70 demonstrates high therapeutic potential and therefore could be applied in the treatment of glioblastoma.
Asunto(s)
Neoplasias Encefálicas/terapia , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/administración & dosificación , Glioblastoma/terapia , Proteínas HSP70 de Choque Térmico/metabolismo , Inmunoterapia , Animales , Apoptosis , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/metabolismo , Proliferación Celular , Sistemas de Liberación de Medicamentos , Citometría de Flujo , Glioblastoma/inmunología , Glioblastoma/metabolismo , Proteínas HSP70 de Choque Térmico/administración & dosificación , Humanos , Técnicas para Inmunoenzimas , Inyecciones Intralesiones , Interferón gamma/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Ratas , Linfocitos T Citotóxicos/inmunología , Células Tumorales CultivadasRESUMEN
Tumor recurrence is a mechanism triggered in sparse populations of cancer cells that usually remain in a quiescent state after strict stress and/or therapeutic factors, which is affected by a variety of autocrine and microenvironmental cues. Despite thorough investigations, the biology of dormant and/or cancer stem cells is still not fully elucidated, as for the mechanisms of their reawakening, while only the major molecular patterns driving the relapse process have been identified to date. These molecular patterns profoundly interfere with the elements of cellular proteostasis systems that support the efficiency of the recurrence process. As a major proteostasis machinery, we review the role of the ubiquitin-proteasome system (UPS) in tumor cell dormancy and reawakening, devoting particular attention to the functions of its components, E3 ligases, deubiquitinating enzymes and proteasomes in cancer recurrence. We demonstrate how UPS components functionally or mechanistically interact with the pivotal proteins implicated in the recurrence program and reveal that modulators of the UPS hold promise to become an efficient adjuvant therapy for eradicating refractory tumor cells to impede tumor relapse.
Asunto(s)
Recurrencia Local de Neoplasia , Neoplasias , Complejo de la Endopetidasa Proteasomal , Ubiquitina , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Neoplasias/patología , Neoplasias/metabolismo , Neoplasias/genética , Ubiquitina/metabolismo , Recurrencia Local de Neoplasia/patología , Animales , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/metabolismo , Microambiente TumoralRESUMEN
The key feature of polyglutamine aggregates accumulating in the course of Huntington disease (HD) is their resistance to protein denaturants, and to date only chaperones are proved to prevent mutant protein aggregation. It was suggested that expanded polyglutamine chains (polyQ) of mutant huntingtin are cross-linked to other proteins such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Here we clarify the roles of GAPDH and molecular chaperone Hsp70 in the formation of sodium dodecyl sulfate (SDS)-insoluble polyQ aggregates. First, the addition of pure GAPDH was found to enhance the aggregation of polyQ in a cell-free model of HD. Secondly, the immunodepletion of GAPDH dose-dependently decreased polyQ aggregation. Finally, siRNA-mediated inhibition of GAPDH protein in SK-N-SH neuroblastoma cells has also reduced the aggregation of cellular polyQ. Regulated over-expression of Hsp70 decreased the amount of GAPDH associated with SDS-insoluble polyQ aggregates. Physical association of Hsp70 and GAPDH in SK-N-SH cells was shown by reciprocal immunoprecipitation and confocal microscopy. Pure Hsp70 dose-dependently inhibited the formation of polyQ aggregates in cell-free model of HD by sequestering both GAPDH and polyQ. We demonstrated that Hsp70 binds to polyQ in adenosine triphosphate-dependent manner, which suggests that Hsp70 exerts a chaperoning activity in the course of this interaction. Binding of Hsp70 to GAPDH was nicotinamide adenine dinucleotide-dependent suggesting another type of association. Based on our findings, we conclude that Hsp70 protects cells in HD by removing/sequestering two intrinsic components of protein aggregates: the polyQ itself and GAPDH. We propose that GAPDH might be an important target for pharmacological treatment of HD and other polyglutamine expansion-related diseases.
Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Enfermedad de Huntington/metabolismo , Péptidos/metabolismo , Línea Celular Tumoral , Expresión Génica , Regulación de la Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Humanos , Enfermedad de Huntington/genética , Unión Proteica , SolubilidadRESUMEN
PURPOSE: Heat shock protein 70 (HSPA family) is a multi-functional protein which protects individual cells from proteotoxic shock and the whole organism from microbial, viral and oncogenic pathogens. These diverse functions may depend upon 'chaperone' activity that allows Hsp70 to regulate the mechanism of damaged protein recovery or utilisation inside a cell and to be a potent adjuvant, stimulating immune activity against a variety of viral or tumour antigens. The aim of this review is to present recent data on specific roles of intracellular and extracellular Hsp70 in cancerous tissue. CONCLUSION: The data presented in this paper show that endogenous Hsp70 protects cancer cells of different origins from a variety of cytotoxic threats including cancer cell therapeutics. In contrast, however, Hsp70 released from stressed cancer cells can serve as a danger signal or may recruit cells responsible for the generation of innate and adaptive immune responses against tumour cells.