RÉSUMÉ
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.
Sujet(s)
Récidive tumorale locale , Tumeurs , Proteasome endopeptidase complex , Ubiquitine , Humains , Proteasome endopeptidase complex/métabolisme , Tumeurs/anatomopathologie , Tumeurs/métabolisme , Tumeurs/génétique , Ubiquitine/métabolisme , Récidive tumorale locale/anatomopathologie , Animaux , Ubiquitin-protein ligases/métabolisme , Ubiquitin-protein ligases/génétique , Cellules souches tumorales/anatomopathologie , Cellules souches tumorales/métabolisme , Microenvironnement tumoralRÉSUMÉ
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.
Sujet(s)
Protéines de liaison à l'ADN , Facteurs de transcription , Animaux , Humains , Souris , Lignée cellulaire tumorale , Protéines de liaison à l'ADN/métabolisme , Résistance aux substances , Étoposide/pharmacologie , Facteurs de transcription de choc thermique/métabolisme , Réaction de choc thermique , Facteurs de transcription/métabolisme , Macrophages associés aux tumeurs/métabolismeRÉSUMÉ
BACKGROUND: Cancer recurrence is regulated by a variety of factors, among which is the material of dying tumor cells; it is suggested that remaining after anti-cancer therapy tumor cells receive a signal from proteins called damage-associated molecular patterns (DAMPs), one of which is heat shock protein 70 (Hsp70). METHODS: Two models of tumor repopulation were employed, based on minimal population of cancer cells and application of conditioned medium (CM). To deplete the CMs of Hsp70 affinity chromatography on ATP-agarose and immunoprecipitation were used. Cell proliferation and the dynamics of cell growth were measured using MTT assay and xCELLigence technology; cell growth markers were estimated using qPCR and with the aid of ELISA for prostaglandin E detection. Immunoprecipitation followed by mass-spectrometry was employed to identify Hsp70-binding proteins and protein-protein interaction assays were developed to reveal the above protein complexes. RESULTS: It was found that CM of dying tumor cells contains tumor regrowth-initiating factors and the removal of one of them, Hsp70, caused a reduction in the relapse-activating capacity. The pull out of Hsp70 alone using ATP-agarose had no effect on repopulation, while the immunodepletion of Hsp70 dramatically reduced its repopulation activity. Using proteomic and immunochemical approaches, we showed that Hsp70 in conditioned medium binds and binds another abundant alarmin, the High Mobility Group B1 (HMGB1) protein; the complex is formed in tumor cells treated with anti-cancer drugs, persists in the cytosol and is further released from dying tumor cells. Recurrence-activating power of Hsp70-HMGB1 complex was proved by the enhanced expression of proliferation markers, Ki67, Aurka and MCM-10 as well as by increase of prostaglandin E production and autophagy activation. Accordingly, dissociating the complex with Hsp70 chaperone inhibitors significantly inhibited the pro-growth effects of the above complex, in both in vitro and in vivo tumor relapse models. CONCLUSIONS: These data led us to suggest that the abundance of the Hsp70-HMGB1 complex in the extracellular matrix may serve as a novel marker of relapse state in cancer patients, while specific targeting of the complex may be promising in the treatment of cancers with a high risk of recurrence.
Sujet(s)
Protéine HMGB1 , Protéines du choc thermique HSP70 , Humains , Alarmines , Protéine HMGB1/métabolisme , Milieux de culture conditionnés , Protéomique , Maladie chronique , Récidive , ProstaglandinesRÉSUMÉ
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.
Sujet(s)
Ascomycota , Infections à staphylocoques , Staphylococcus aureus , Anthraquinones/pharmacologieRÉSUMÉ
The amyloid concept of Alzheimer's disease (AD) assumes the ß-amyloid peptide (Aß) as the main pathogenic factor, which injures neural and other brain cells, causing their malfunction and death. Although Aß has been documented to exert its cytotoxic effect in a solitary manner, there is much evidence to claim that its toxicity can be modulated by other proteins. The list of such Aß co-factors or interactors includes tau, APOE, transthyretin, and others. These molecules interact with the peptide and affect the ability of Aß to form oligomers or aggregates, modulating its toxicity. Thus, the list of potential substances able to reduce the harmful effects of the peptide should include ones that can prevent the pathogenic interactions by specifically binding Aß and/or its partners. In the present review, we discuss the data on Aß-based complexes in AD pathogenesis and on the compounds directly targeting Aß or the destructors of its complexes with other polypeptides.
RÉSUMÉ
Tumor recurrence is a paradoxical function of a machinery, whereby a small proportion of the cancer cell population enters a resistant, dormant state, persists long-term in this condition, and then transitions to proliferation. The dormant phenotype is typical of cancer stem cells, tumor-initiating cells, disseminated tumor cells, and drug-tolerant persisters, which all demonstrate similar or even equivalent properties. Cancer cell dormancy and its conversion to repopulation are regulated by several protein signaling systems that inhibit or induce cell proliferation and provide optimal interrelations between cancer cells and their special niche; these systems act in close connection with tumor microenvironment and immune response mechanisms. During dormancy and reawakening periods, cell proteostasis machineries, autophagy, molecular chaperones, and the unfolded protein response are recruited to protect refractory tumor cells from a wide variety of stressors and therapeutic insults. Proteostasis mechanisms functionally or even physically interfere with the main regulators of tumor relapse, and the significance of these interactions and implications in the tumor recurrence phases are discussed in this review.
Sujet(s)
Récidive tumorale locale , Transduction du signal , Humains , Récidive tumorale locale/anatomopathologie , Chaperons moléculaires , Autophagie , Microenvironnement tumoral , Réponse aux protéines mal repliéesRÉSUMÉ
Alzheimer's disease (AD) is the most common cause of age-related dementia. Neuronal calcium homeostasis impairment may contribute to AD. Here we demonstrated that voltage-gated calcium (VGC) entry and store-operated calcium (SOC) entry regulated by calcium sensors of intracellular calcium stores STIM proteins are affected in hippocampal neurons of the 5xFAD transgenic mouse model. We observed excessive SOC entry in 5xFAD mouse neurons, mediated by STIM1 and STIM2 proteins with increased STIM1 contribution. There were no significant changes in cytoplasmic calcium level, endoplasmic reticulum (ER) bulk calcium levels, or expression levels of STIM1 or STIM2 proteins. The potent inhibitor BTP-2 and the FDA-approved drug leflunomide reduced SOC entry in 5xFAD neurons. In turn, excessive voltage-gated calcium entry was sensitive to the inhibitor of L-type calcium channels nifedipine but not to the T-type channels inhibitor ML218. Interestingly, the depolarization-induced calcium entry mediated by VGC channels in 5xFAD neurons was dependent on STIM2 but not STIM1 protein in cells with replete Ca2+ stores. The result gives new evidence on the VGC channel modulation by STIM2. Overall, the data demonstrate the changes in calcium signaling of hippocampal neurons of the AD mouse model, which precede amyloid plaque accumulation or other signs of pathology manifestation.
Sujet(s)
Maladie d'Alzheimer , Calcium , Animaux , Souris , Calcium/métabolisme , Molécule-1 d'interaction stromale/génétique , Molécule-1 d'interaction stromale/métabolisme , Molécule-2 d'interaction stromale/métabolisme , Signalisation calcique/physiologie , Canaux calciques de type L/métabolisme , Modèles animaux de maladie humaineRÉSUMÉ
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.
Sujet(s)
Maladie d'Alzheimer , Souris , Animaux , Maladie d'Alzheimer/traitement médicamenteux , Maladie d'Alzheimer/métabolisme , Peptides bêta-amyloïdes/métabolisme , Neurones/métabolisme , Vieillissement/métabolisme , Chaperons moléculaires/métabolisme , Souris transgéniques , Modèles animaux de maladie humaineRÉSUMÉ
Despite significant progress in the diagnosis and treatment of colorectal cancer, drug resistance continues to be a major limitation of therapy. In this regard, studies aimed at creating combination therapy are gaining popularity. One of the most promising adjuvants are inhibitors of the proteostasis system, chaperone machinery, and autophagy. The main HSP regulator, HSF1, is overactivated in cancer cells and autophagy sustains the survival of malignant cells. In this work, we focused on the selection of combination therapy for the treatment of rectal cancer cells obtained from patients after tumor biopsy without prior treatment. We characterized the migration, proliferation, and chaperone status in the resulting lines and also found them to be resistant to a number of drugs widely used in the clinic. However, these cells were sensitive to the autophagy inhibitor, chloroquine. For combination therapy, we used an HSF1 activity inhibitor discovered earlier in our laboratory, the cardenolide CL-43, which has already been proven as an auxiliary component of combined therapy in established cell lines. CL-43 effectively suppressed HSF1 activity and Hsp70 expression in all investigated cells. We tested the autophagy inhibitor, chloroquine, in combination with CL-43. Our results indicate that the use of an inhibitor of HSF1 activity in combination with an autophagy inhibitor results in effective cancer cell death, therefore, this therapeutic approach may be a promising treatment regimen for certain patients.
RÉSUMÉ
Six new polyketides acrucipentyns A-F (1-6) were isolated from the alga-derived fungus Asteromyces cruciatus KMM 4696. Their structures were established based on spectroscopic methods. The absolute configurations of acrucipentyn A was assigned by the modified Mosher's method and ROESY data analysis. Acrucipentyns A-E were identified to be the very first examples of chlorine-containing asperpentyn-like compounds. The cytotoxic and antimicrobial activities of the isolated compounds were examined. Acrucipentyns A-F were found as antimicrobial agents, which inhibited sortase A enzyme activity, bacterial growth and biofilm formation of Staphylococcus aureus and decreased LDH release from human keratinocytes HaCaT in S. aureus skin infection in an in vitro model.
RÉSUMÉ
The recovery period after traumatic brain injury (TBI) is often complicated by secondary damage that may last for days or even months after trauma. Two proteins, Hsp70 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were recently described as modulating post-traumatic processes, and in this study, we test them as targets for combination therapy using an inhibitor of GAPDH aggregation (derivative of hydrocortisone RX624) and an inducer of Hsp70 synthesis (the pyrrolylazine derivative PQ-29). The protective effect of the combination on C6 rat glioblastoma cells treated with the cerebrospinal fluid of traumatized animals resulted in an increase in the cell index and in a reduced level of apoptosis. Using a rat weight drop model of TBI, we found that the combined use of both drugs prevented memory impairment and motor deficits, as well as a reduction of neurons and accumulation of GAPDH aggregates in brain tissue. In conclusion, we developed and tested a new approach to the treatment of TBI based on influencing distinct molecular mechanisms in brain cells.
RÉSUMÉ
These data are related to our previous paper "Synthesis and approbation of new neuroprotective chemicals of pyrrolyl- and indolylazine classes in a cell model of Alzheimer's disease" (Dutysheva et al., 2021), in which we demonstrate neuroprotective abilities of pyrrolyl- and indolylazines in a cell model of Alzheimer's disease. Using a novel procedure of photocatalysis we have synthesized a group of new compounds. The current article presents nuclear magnetic resonance spectra including heteronuclear single quantum coherence spectra of chemicals synthesized by us. The effect of new compounds have on heat shock proteins genes expression in reprogrammed human neurons are presented. We also presented data that verify neuronal phenotype of reprogrammed cells.
RÉSUMÉ
Currently, the success of targeted anticancer therapies largely depends on the correct understanding of the dormant state of cancer cells, since it is increasingly regarded to fuel tumor recurrence. The concept of cancer cell dormancy is often considered as an adaptive response of cancer cells to stress, and, therefore, is limited. It is possible that the cancer dormant state is not a privilege of cancer cells but the same reproductive survival strategy as diapause used by embryonic stem cells (ESCs). Recent advances reveal that high autophagy and mTOR pathway reduction are key mechanisms contributing to dormancy and diapause. ESCs, sharing their main features with cancer stem cells, have a delicate balance between the mTOR pathway and autophagy activity permissive for diapause induction. In this review, we discuss the functioning of the mTOR signaling and autophagy in ESCs in detail that allows us to deepen our understanding of the biology of cancer cell dormancy.
RÉSUMÉ
The release of Hsp70 chaperone from tumor cells is found to trigger the full-scale anti-cancer immune response. Such release and the proper immune reaction can be induced by the delivery of recombinant Hsp70 to a tumor and we sought to explore how the endogenous Hsp70 can be transported to extracellular space leading to the burst of anti-cancer activity. Hsp70 transport mechanisms were studied by analyzing its intracellular tracks with Rab proteins as well as by using specific inhibitors of membrane domains. To study Hsp70 forms released from cells we employed the assay consisting of two affinity chromatography methods. Hsp70 content in culture medium and extracellular vesicles (EVs) was measured with the aid of ELISA. The properties and composition of EVs were assessed using nanoparticle tracking analysis and immunoblotting. The activity of immune cells was studied using an assay of cytotoxic lymphocytes, and for in vivo studies we employed methods of affinity separation of lymphocyte fractions. Analyzing B16 melanoma cells treated with recombinant Hsp70 we found that the chaperone triggered extracellular transport of its endogenous analog in soluble and enclosed in EVs forms; both species efficiently penetrated adjacent cells and this secondary transport was corroborated with the strong increase of Natural Killer (NK) cell toxicity towards melanoma. When B16 and CT-26 colon cancer cells before their injection in animals were treated with Hsp70-enriched EVs, a powerful anti-cancer effect was observed as shown by a two-fold reduction in tumor growth rate and elevation of life span. We found that the immunomodulatory effect was due to the enhancement of the CD8-positive response and anti-tumor cytokine accumulation; supporting this there was no delay in CT-26 tumor growth when Hsp70-enriched EVs were grafted in nude mice. Importantly, pre-treatment of B16 cells with Hsp70-bearing EVs resulted in a decline of arginase-1-positive macrophages, showing no generation of tumor-associated macrophages. In conclusion, Hsp70-containing EVs generated by specifically treated cancer cells give a full-scale and effective pattern of anti-tumor immune responses.
Sujet(s)
Immunité acquise , Vésicules extracellulaires , Protéines du choc thermique HSP70/pharmacologie , Animaux , Carcinomes/immunologie , Lignée cellulaire tumorale , Tumeurs du côlon/immunologie , Cellules HEK293 , Humains , Cellules tueuses naturelles/immunologie , Mélanome expérimental/immunologie , SourisRÉSUMÉ
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.
Sujet(s)
Antibactériens/pharmacologie , Cérébrosides/pharmacologie , Glycosphingolipides/pharmacologie , Kératinocytes/effets des médicaments et des substances chimiques , Penicillium , Dermatoses bactériennes/prévention et contrôle , Infections à staphylocoques/prévention et contrôle , Staphylococcus aureus/effets des médicaments et des substances chimiques , Organismes aquatiques , Cellules HaCaT/effets des médicaments et des substances chimiques , HumainsRÉSUMÉ
Neuronal cell death at late stages of Alzheimer's disease (AD) causes the release of cytosolic proteins. One of the most abundant such proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forms stable aggregates with extracellular amyloid-ß (Aß). We detect these aggregates in cerebrospinal fluid (CSF) from AD patients at levels directly proportional to the progressive stages of AD. We found that GAPDH forms a covalent bond with Q15 of Aß that is mediated by transglutaminase (tTG). The Q15A substitution weakens the interaction between Aß and GAPDH and reduces Aß-GAPDH cytotoxicity. Lentivirus-driven GAPDH overexpression in two AD animal models increased the level of apoptosis of hippocampal cells, neural degeneration, and cognitive dysfunction. In contrast, in vivo knockdown of GAPDH reversed these pathogenic abnormalities suggesting a pivotal role of GAPDH in Aß-stimulated neurodegeneration. CSF from animals with enhanced GAPDH expression demonstrates increased cytotoxicity in vitro. Furthermore, RX-624, a specific GAPDH small molecular ligand reduced accumulation of Aß aggregates and reversed memory deficit in AD transgenic mice. These findings argue that extracellular GAPDH compromises Aß clearance and accelerates neurodegeneration, and, thus, is a promising pharmacological target for AD.
RÉSUMÉ
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.
Sujet(s)
Glycémie , Transition épithélio-mésenchymateuse , Glucose/métabolisme , Protéines du choc thermique HSP70/métabolisme , Marqueurs biologiques , Cadhérines/métabolisme , Lignée cellulaire tumorale , Transformation cellulaire néoplasique/génétique , Transformation cellulaire néoplasique/métabolisme , Transition épithélio-mésenchymateuse/effets des médicaments et des substances chimiques , Glucose/pharmacologie , Humains , Hyperglycémie/étiologie , Hyperglycémie/métabolisme , Liaison aux protéines , Facteurs de transcription de la famille Snail/métabolismeRÉSUMÉ
One of the major causes of neurodegeneration in the pathogenesis of Alzheimer's disease is the accumulation of cytotoxic amyloid species within the intercellular compartments of the brain. The efficacy of the anti-proteotoxic mechanism based on the molecular chaperones Hsp70 and Hsp90 in numerous types of neurons is often low, while its pharmacological enhancement has been shown to ameliorate the physiological and cognitive functions of the brain. Suggesting that the chemicals able to induce heat shock protein synthesis and therefore rescue neural cells from cytotoxicity associated with amyloid, we have synthesized a group of pyrrolyl- and indolylazines that cause the accumulation of heat shock proteins, using a novel method of photocatalysis that is employed in green chemistry. The selected compounds were tested in a cell model of Alzheimer's disease and demonstrated a pronounced neuroprotective effect. These substances increased the survival of neurons, blocked the activation of ß-galactosidase, and prevented apoptosis in neurons cultured in the presence of ß-amyloid.
Sujet(s)
Maladie d'Alzheimer/traitement médicamenteux , Hydrazines/pharmacologie , Neurones/effets des médicaments et des substances chimiques , Neuroprotecteurs/pharmacologie , Pyrroles/pharmacologie , Maladie d'Alzheimer/métabolisme , Maladie d'Alzheimer/anatomopathologie , Peptides bêta-amyloïdes/antagonistes et inhibiteurs , Peptides bêta-amyloïdes/métabolisme , Apoptose/effets des médicaments et des substances chimiques , Cellules cultivées , Relation dose-effet des médicaments , Humains , Hydrazines/synthèse chimique , Hydrazines/composition chimique , Structure moléculaire , Neurones/métabolisme , Neurones/anatomopathologie , Neuroprotecteurs/synthèse chimique , Neuroprotecteurs/composition chimique , Pyrroles/synthèse chimique , Pyrroles/composition chimique , Relation structure-activitéRÉSUMÉ
Plant-derived extracellular vesicles (EVs) gain more and more attention as promising carriers of exogenous bioactive molecules to the human cells. Derived from various edible sources, these EVs are remarkably biocompatible, biodegradable and highly abundant from plants. In this work, EVs from grapefruit juice were isolated by differential centrifugation followed by characterization of their size, quantity and morphology by nanoparticle tracking analysis, dynamic light scattering, atomic force microscopy and cryo-electron microscopy (Cryo-EM). In Cryo-EM experiments, we visualized grapefruit EVs with the average size of 41 ± 13 nm, confirmed their round-shaped morphology and estimated the thickness of their lipid bilayer as 5.3 ± 0.8 nm. Further, using cell culture models, we have successfully demonstrated that native grapefruit-derived extracellular vesicles (GF-EVs) are highly efficient carriers for the delivery of the exogenous Alexa Fluor 647 labeled bovine serum albumin (BSA) and heat shock protein 70 (HSP70) into both human peripheral blood mononuclear cells and colon cancer cells. Interestingly, loading to plant EVs significantly ameliorated the uptake of exogenous proteins by human cells compared to the same proteins without EVs. Most importantly, we have confirmed the functional activity of human recombinant HSP70 in the colon cancer cell culture upon delivery by GF-EVs. Analysis of the biodistribution of GF-EVs loaded with 125I-labeled BSA in mice demonstrated a significant uptake of the grapefruit-derived extracellular vesicles by the majority of organs. The results of our study indicate that native plant EVs might be safe and effective carriers of exogenous proteins into human cells.
Sujet(s)
Citrus paradisi/composition chimique , Vésicules extracellulaires/composition chimique , Nanocapsules/composition chimique , Cellules cultivées , Vésicules extracellulaires/ultrastructure , Cellules HCT116 , Protéines du choc thermique HSP70/administration et posologie , Humains , Agranulocytes/métabolisme , Nanocapsules/ultrastructure , Sérumalbumine bovine/administration et posologieRÉSUMÉ
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.