RESUMEN
Ischemic stroke is a life-threatening brain disease with the leading cause of disability and mortality worldwide. Heat-shock protein A12A (HSPA12A) is recognized as a neuroprotective target for treating ischemic stroke; however, its regulatory mechanism has been not fully elucidated yet. Human brain microvascular endothelial cells (hBMECs) were induced by oxygen-glucose deprivation/reoxygenation (OGD/R) to mimic ischemic stroke. Gain- and loss-of-function experiments were conducted to explore the regulation of HSAPA12 and PGC-1α. Cell viability, apoptosis, and permeability were assessed by CCK-8, TUNEL, and transendothelial electrical resistance (TEER) assays, respectively. The expression of HSPA12A and corresponding proteins was measured by western blot. Cell immunofluorescence was adopted to evaluate ZO-1 expression. THP-1 cells were applied to adhere hBMECs in vitro to simulate leukocyte adhesion in the brain. HSPA12A was downregulated in OGD/R-treated hBMECs. HSPA12A overexpression significantly suppressed OGD/R-induced cell viability loss and apoptosis in hBMECs. Meanwhile, HSPA12A overexpression attenuated blood-brain barrier (BBB) integrity in OGD/R-induced hBMECs, evidenced by the restored TEER value and the upregulated ZO-1, occludin, and claudin-5. HSPA12A also restricted OGD/R-induced attachment of THP-1 cells to hBMECs, accompanied with downregulating ICAM-1 and VCAM-1. Additionally, OGD/R-caused downregulation of PGC-1α/SIRT3 in hBMECs was partly restored by HSPA12A overexpression. Furthermore, the above effects of HSPA12A on OGD/R-induced hBMECs injury were partly reversed by PGC-1α knockdown. HSPA12A plays a protective role against OGD/R-induced hBMECs injury by upregulating PGC-1α, providing a potential neuroprotective role of HSPA12A in ischemic stroke.
Asunto(s)
Encefalopatías , Accidente Cerebrovascular Isquémico , Sirtuina 3 , Humanos , Oxígeno/metabolismo , Oxígeno/farmacología , Células Endoteliales , Sirtuina 3/genética , Sirtuina 3/metabolismo , Sirtuina 3/farmacología , Glucosa/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/farmacología , Encéfalo/metabolismo , Encefalopatías/metabolismo , Apoptosis , Accidente Cerebrovascular Isquémico/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/farmacologíaRESUMEN
Hsp70 is the main molecular chaperone responsible for cellular proteostasis under normal conditions and for restoring the conformation or utilization of proteins damaged by stress. Increased expression of endogenous Hsp70 or administration of exogenous Hsp70 is known to exert neuroprotective effects in models of many neurodegenerative diseases. In this study, we have investigated the effect of exogenous Hsp70 on recovery from peripheral nerve injury in a model of sciatic nerve transection in rats. It was shown that recombinant Hsp70 after being added to the conduit connecting the ends of the nerve at the site of its extended severance, migrates along the nerve into the spinal ganglion and is retained there at least three days. In animals with the addition of recombinant Hsp70 to the conduit, a decrease in apoptosis in the spinal ganglion cells after nerve rupture, an increase in the level of PTEN-induced kinase 1 (PINK1), an increase in markers of nerve tissue regeneration and a decrease in functional deficit were observed compared to control animals. The obtained data indicate the possibility of using recombinant Hsp70 preparations to accelerate the recovery of patients after neurotrauma.
Asunto(s)
Fármacos Neuroprotectores , Humanos , Ratas , Animales , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Fármacos Neuroprotectores/metabolismo , Axotomía , Neuronas/metabolismo , Nervio Ciático/lesiones , Apoptosis , Proteínas HSP70 de Choque Térmico/farmacología , Proteínas HSP70 de Choque Térmico/metabolismo , Ganglios Espinales/metabolismo , Regeneración NerviosaRESUMEN
Polaprezinc (PZ) plays a role in the protection of gastric mucosa and inhibiting Helicobacter pylori (H. pylori) growth in vitro. The objective of this study was to determine the protective effects of PZ on human gastric epithelial cells (GES-1) against H. pylori-induced damage, while also examining heat shock protein 70 (HSP70) as a potential underlying factor in this protection. Our findings revealed that PZ exerted bactericidal effects against H. pylori strains. We also observed that PZ mitigated the H. pylori-induced damage to GES-1 cells by increasing cell viability, reducing LDH release, and decreasing the secretion of pro-inflammatory factors such as MCP-1 and IL-6. Co-culturing PZ with GES-1 cells significantly up-regulated the GES-1 HSP70 expression in both a time and dose-dependent manner. Pre-incubating (for 12 h) or co-culturing (for 24 h) GES-1 cells with PZ reversed the down-regulation of HSP70 in GES-1 cells caused by H. pylori infection. However, when quercetin was used to inhibit the up-regulation of HSP70 in GES-1 cells, the protective effect of PZ on GES-1 cells was significantly reduced. Based on the results of this study, PZ exhibits a protective role on GES-1 cells against H. pylori injury, as well as a direct bactericidal effect on H. pylori. HSP70 is involved in the PZ-driven host cell protection against H. pylori injury. These findings provide insight into alternative strategies for H. pylori treatment.
Asunto(s)
Infecciones por Helicobacter , Helicobacter pylori , Compuestos Organometálicos , Humanos , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/farmacología , Citoprotección , Compuestos Organometálicos/metabolismo , Compuestos Organometálicos/farmacología , Células Epiteliales/metabolismo , Infecciones por Helicobacter/metabolismo , Mucosa GástricaRESUMEN
Receptor-interacting protein kinase 1 (RIPK1) contributes to necroptosis. Our previous study showed that pharmacological or genetic inhibition of RIPK1 protects against ischemic stroke-induced astrocyte injury. In this study, we investigated the molecular mechanisms underlying RIPK1-mediated astrocyte injury in vitro and in vivo. Primary cultured astrocytes were transfected with lentiviruses and then subjected to oxygen and glucose deprivation (OGD). In a rat model of permanent middle cerebral artery occlusion (pMCAO), lentiviruses carrying shRNA targeting RIPK1 or shRNA targeting heat shock protein 70.1B (Hsp70.1B) were injected into the lateral ventricles 5 days before pMCAO was established. We showed that RIPK1 knockdown protected against OGD-induced astrocyte damage, blocked the OGD-mediated increase in lysosomal membrane permeability in astrocytes, and inhibited the pMCAO-induced increase in astrocyte lysosome numbers in the ischemic cerebral cortex; these results suggested that RIPK1 contributed to the lysosomal injury in ischemic astrocytes. We revealed that RIPK1 knockdown upregulated the protein levels of Hsp70.1B and increased the colocalization of Lamp1 and Hsp70.1B in ischemic astrocytes. Hsp70.1B knockdown exacerbated pMCAO-induced brain injury, decreased lysosomal membrane integrity and blocked the protective effects of the RIPK1-specific inhibitor necrostatin-1 on lysosomal membranes. On the other hand, RIPK1 knockdown further exacerbated the pMCAO- or OGD-induced decreases in the levels of Hsp90 and the binding of Hsp90 to heat shock transcription factor-1 (Hsf1) in the cytoplasm, and RIPK1 knockdown promoted the nuclear translocation of Hsf1 in ischemic astrocytes, resulting in increased Hsp70.1B mRNA expression. These results suggest that inhibition of RIPK1 protects ischemic astrocytes by stabilizing lysosomal membranes via the upregulation of lysosomal Hsp70.1B; the mechanism underlying these effects involves decreased Hsp90 protein levels, increased Hsf1 nuclear translocation and increased Hsp70.1B mRNA expression.
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Astrocitos , Isquemia Encefálica , Ratas , Animales , Ratas Sprague-Dawley , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/farmacología , Infarto de la Arteria Cerebral Media/metabolismo , Lisosomas/metabolismo , ARN Interferente Pequeño/farmacología , ARN Mensajero/metabolismo , Glucosa/metabolismo , Isquemia Encefálica/metabolismoRESUMEN
Ubiquitin proteasome activity is suppressed in enzalutamide resistant prostate cancer cells, and the heat shock protein 70/STIP1 homology and U-box-containing protein 1 (HSP70/STUB1) machinery are involved in androgen receptor (AR) and AR variant protein stabilization. Targeting HSP70 could be a viable strategy to overcome resistance to androgen receptor signaling inhibitor (ARSI) in advanced prostate cancer. Here, we showed that a novel HSP70 allosteric inhibitor, JG98, significantly suppressed drug-resistant C4-2B MDVR and CWR22Rv1 cell growth, and enhanced enzalutamide treatment. JG98 also suppressed cell growth in conditional reprogramed cell cultures (CRCs) and organoids derived from advanced prostate cancer patient samples. Mechanistically, JG98 degraded AR/AR-V7 expression in resistant cells and promoted STUB1 nuclear translocation to bind AR-V7. Knockdown of the E3 ligase STUB1 significantly diminished the anticancer effects and partially restored AR-V7 inhibitory effects of JG98. JG231, a more potent analog developed from JG98, effectively suppressed the growth of the drug-resistant prostate cancer cells, CRCs, and organoids. Notably, the combination of JG231 and enzalutamide synergistically inhibited AR/AR-V7 expression and suppressed CWR22Rv1 xenograft tumor growth. Inhibition of HSP70 using novel small-molecule inhibitors coordinates with STUB1 to regulate AR/AR-V7 protein stabilization and ARSI resistance.
Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración , Receptores Androgénicos , Masculino , Humanos , Receptores Androgénicos/metabolismo , Antagonistas de Andrógenos , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Línea Celular Tumoral , Nitrilos/farmacología , Antagonistas de Receptores Androgénicos , Andrógenos/farmacología , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/farmacología , Resistencia a Antineoplásicos , Ubiquitina-Proteína LigasasRESUMEN
BACKGROUND: Postoperative cognitive dysfunction (POCD) is a severe postoperative neurological sequela in elderly patients, and there is currently no standard treatment for POCD. In this study, whether recombinant human heat shock protein 70 (rHsp70) could alleviate sevoflurane-induced cognitive impairment in aged mice is investigated. METHODS: To determine the prophylactic effect of rHsp70 in sevoflurane-induced cognitive dysfunction, aged mice were pretreated with different concentrations of rHsp70 (29.4, 58.8, and 117.6 µg/kg; intranasal injected; N = 12) every day for 1 week; then, 3% sevoflurane was utilized to anesthetize the aged mice. Cognitive function, neurotoxicity, and serum and hippocampal Hsp70 levels in aged mice undergoing sevoflurane anesthesia were assessed by the Morris water maze test and enzyme-linked immunosorbent assay. The effects of rHsp70 on inflammatory response were assessed by proinflammatory cytokine production and nuclear factor-κB (NF-κB) activation assays. RESULTS: We found that aged mice exposed to sevoflurane showed reduced learning and memory ability and reduced Hsp70 expression, which were both restored by rHsp70 pretreatment. RHsp70 also reversed sevoflurane-induced up-regulated Bax and Bcl-2 expression and interleukin-1, IL-6, and monocyte chemoattractant protein-1 overproduction. Finally, rHsp70 pretreatment suppressed sevoflurane-induced NF-κB activation. Our study indicated that rHsp70 was sufficient to suppress sevoflurane-induced cognitive decline and neurotoxicity. CONCLUSION: Our important finding warrants further study on the clinical application of rHsp70 in elderly patients undergoing anesthesia.
Asunto(s)
Anestesia , Anestésicos por Inhalación , Disfunción Cognitiva , Anciano , Animales , Humanos , Ratones , Disfunción Cognitiva/inducido químicamente , Disfunción Cognitiva/tratamiento farmacológico , Disfunción Cognitiva/prevención & control , Hipocampo/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/farmacología , FN-kappa B/metabolismo , Sevoflurano/farmacologíaRESUMEN
Hesperetin, a predominant flavonoid found in citrus fruits, has received considerable attention for its potential anticancer activity through the reduction of cell viability and the induction of apoptosis. Several effector mechanisms have been demonstrated underlying the antitumor properties of hesperetin but its specific mechanisms have not yet been fully elucidated. In the present study, how hesperetin affects the proliferation of A549 cells and the related cell proliferation regulatory mechanisms, were inevstigated. To elucidate the mechanisms underlying the effects of hesperetin on A549 cells, MTT assay, colony formation assay, flow cytometry, immunoblotting, reverse transcriptionquantitative PCR and JC1 staining were performed. The data revealed that hesperetin inhibited cell proliferation and induced apoptosis in these cells. Hesperetin also decreased the level of heat shock protein 70 (Hsp70), a negative regulator of the mitochondrial apoptosis pathway, often overexpressed in various cancer cells and suspected to contribute to tumor development. Hesperetininduced Hsp70 suppression was associated with reduced cytosolic Bax and increased mitochondrial Bax levels, leading to the enhancement of the mitochondrial apoptotic cascade. The Hsp70 overexpressioninduced reduction in the level of hesperetininduced apoptosis provides evidence to hesperetininduced apoptosis being mediated by affecting Hsp70. Furthermore, it was demonstrated that hesperetin reduced Hsp70 expression by inducing a proteasomemediated degradation via the upregulation of E3ligase, Cterminus of Hsp70interacting protein (CHIP). The present study highlighted the importance of the Bax activationtriggered mitochondriamediated pathway for hesperetininduced apoptosis and demonstrated a novel mechanism of how Hsp70 played a critical role in the negative regulation of this apoptotic network in cancer cells.
Asunto(s)
Proteínas HSP70 de Choque Térmico , Complejo de la Endopetidasa Proteasomal , Células A549 , Apoptosis , Flavonoides/farmacología , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/farmacología , Hesperidina , Humanos , Ligasas/farmacología , Proteína X Asociada a bcl-2/metabolismoRESUMEN
As an environmental stress factor, ultraviolet-B (UV-B) radiation directly affects insect growth, development, and reproduction. Heat shock protein 70s kDa (Hsp70s) plays an important role in the environmental adaptation of insects. To determine the role of MpHsp70s in the UV-B tolerance of Myzus persicae (Sulzer), we identified the complete complementary DNA sequences of seven MpHsp70s. They were found to be ubiquitously expressed during different developmental stages and were highly expressed in second-instar nymphs and wingless adults. The expression levels of the MpHsp70s were significantly upregulated when exposed to different durations of UV-B stress. Nanocarrier-mediated dsMpHsp70 suppressed the expression of the MpHsp70s and reduced the body length, weight, survival rate, and fecundity of M. persicae under UV-B exposure. When the combinational RNAi approach was adopted, the effects on the survival rate and fecundity were greater under UV-B stress, except for MpHsc70-4. These results suggest that MpHsp70s are essential for the resistance of M. persicae to UV-B stress.
Asunto(s)
Áfidos , Animales , Áfidos/genética , ADN Complementario , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/farmacología , Ninfa , ReproducciónRESUMEN
Previous studies have indicated a role for molecular chaperone heat shock protein 70 (Hsp70) in the development of behavioural sensitization to morphine in rodents, suggesting that Hsp70 expression following morphine exposure is involved in molecular changes that may underlie addiction vulnerability. The current study was carried out to investigate the role of Hsp70 in the positive reinforcing properties of morphine using conditioned place preference (CPP) in male rats. An unbiased CPP procedure of three phases (pre-conditioning: d1-d3; conditioning: d4-d6; and testing: d7) was used. During the conditioning phase, morphine injections (5 mg/kg, subcutaneously) were administered to induce significant place preference. To explore the effect of Hsp70 on the development and expression of morphine CPP, Hsp70 inhibitors (PES, KNK437 and methylene blue) were administered into the lateral ventricle prior to either morphine conditioning sessions or a morphine challenge on the test day. Furthermore, Hsp70 expression within the mesocorticolimbic system was measured after the treatment with KNK437, a transcriptional inhibitor. We found that PES and KNK437, respectively, injected intracerebroventricularly dose-dependently attenuated both the development and expression of morphine CPP. Methylene blue treatment demonstrated an attenuation of the development, but had no effect on the expression of morphine CPP. Following KNK437 treatment, Hsp70 expression was significantly inhibited in the shell of nucleus accumbens (NAc) during both the development and expression of morphine CPP. The findings suggest that Hsp70 in the NAc shell plays an important role in the reinforcing effects of morphine and may be involved in the development of morphine dependence.
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Proteínas HSP70 de Choque Térmico , Morfina , Animales , Condicionamiento Clásico , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/farmacología , Masculino , Azul de Metileno/farmacología , Chaperonas Moleculares/farmacología , Morfina/farmacología , RatasRESUMEN
To understand the effects of the toxic marine dinoflagellate, Gymnodinium catenatum, on the brine shrimp, Artemia franciscana, we examined the acute toxicity and swimming behavior parameters such as swimming speed, swimming distance, and swimming path trajectory with transcriptional regulation of heat shock protein (hsp) genes in response to G. catenatum exposure. Mortality was not observed in response to G. catenatum. In the case of swimming behavior parameters, swimming speed and swimming distance were significantly decreased (P < 0.05) for 5 min at three concentrations (240, 360, and 600 cells/mL) of G. catenatum, whereas no significant change in swimming path trajectory was observed, suggesting that G. catenatum has potential adverse effects on the swimming behavior of A. franciscana. Additionally, the four A. franciscana-hsp genes (hsp26, hsp40, hsp70, and hsp90) were upregulated in response to G. catenatum. In particular, A. franciscana-hsp40 was significantly upregulated in response to 600 cells/mL G. catenatum, suggesting that A. franciscana-hsp genes are highly associated with cellular defense mechanisms and that A. franciscana-hsp40 is a potential biomarker for G. catenatum exposure. Overall, this study improves our understanding of the effects of G. catenatum on the swimming behavior and cellular defense mechanisms of A. franciscana.
Asunto(s)
Artemia , Dinoflagelados , Animales , Dinoflagelados/genética , Dinoflagelados/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/farmacología , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/farmacología , NataciónRESUMEN
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.
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Inmunidad Adaptativa , Vesículas Extracelulares , Proteínas HSP70 de Choque Térmico/farmacología , Animales , Carcinoma/inmunología , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Células HEK293 , Humanos , Células Asesinas Naturales/inmunología , Melanoma Experimental/inmunología , RatonesRESUMEN
Although the connection between heat shock protein 70 (HSP70) and vestibular migraine is not clear, HSP70 is neuroprotective in other scenarios. This study aimed to investigate the potential of exogenous HSP70 for treating migraine-like symptoms in a mouse model of nitroglycerin (NTG)-induced migraine. HSP70 levels were assessed in patients with vestibular migraine and healthy individuals by ELISA. Migraine was induced in mice by NTG, and HSP70 expression was examined in the trigeminal nucleus caudalis (TNC) tissue of mice treated with NTG and NTG together with exogenous HSP70. The effects of exogenous HSP70 on migraine-like symptoms were assessed through behavioral assays. Finally, the impact of HSP70 on oxidative stress and NF-κB signaling in mice with migraine was investigated. Serum HSP70 in patients with vestibular migraine was significantly lower than that of healthy individuals. NTG administration significantly suppressed HSP70 expression in mouse TNC tissue, which was reversed by exogenous HSP70. HSP70 alleviated NTG-induced mechanical hypersensitivity, light aversion, and anxiety-like behavior. Finally, exogenous HSP70 suppressed NTG-induced oxidative stress and NF-κB signaling. Our study suggests that exogenous HSP70 may be a potential therapy for alleviating migraine symptoms and our promising finding warrants further investigation of HSP70 for clinical application.NEW & NOTEWORTHY The study suggests that exogenous HSP70 may be a potential therapy for alleviating migraine symptoms and our promising finding warrants further investigation of HSP70 for clinical application.
Asunto(s)
Proteínas HSP70 de Choque Térmico/sangre , Proteínas HSP70 de Choque Térmico/farmacología , Trastornos Migrañosos/sangre , Trastornos Migrañosos/tratamiento farmacológico , Nitroglicerina/farmacología , Vasodilatadores/farmacología , Adulto , Animales , Modelos Animales de Enfermedad , Femenino , Proteínas HSP70 de Choque Térmico/administración & dosificación , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Trastornos Migrañosos/inducido químicamente , Proteínas Recombinantes , Adulto JovenRESUMEN
Cancer cells have developed numerous strategies to maintain their proliferative capacity and to withstand different kinds of stress. The mitochondrial stress70 protein named glucose regulated protein 75 (GRP75), also known as mortalin, is an intriguing cancer prosurvival factor. It is constitutively expressed in normal tissues but is upregulated in many tumors, and was shown to be a cancer prognostic biomarker. Mortalin is an inhibitor of complementdependent cytotoxicity (CDC) and may therefore protect cells from antibodybased immunotherapy. To target mortalin for cancer therapy, our laboratory designed several mortalin mimetic peptides with sequences predicted to be involved in mortalin binding to its client proteins. The peptides were synthesized with a Cterminal transactivator of transcription sequence. By using cell death methodologies, the mechanism of action of the mortalin mimetic peptides on cancer cells was studied. Two peptides in particular, MotP2 and MotP7, were found to be highly toxic to lymphoma and ovarian, breast and prostate carcinoma cells. The analysis of their mode of action revealed that they may induce, within minutes, plasma membrane perturbations and mitochondrial stress. Furthermore, MotP2 and MotP7 activated necrotic cell death, leading to plasma membrane perforation, mitochondrial inner membrane depolarization and decrease in ATP level. In addition, MotP7, but not MotP2, required extracellular calcium ions to fully mediate cell death and was partially inhibited by plasma membrane cholesterol. At subtoxic concentrations, the two peptides moderately inhibited cancer cell proliferation and blocked cell cycle at G2/M. Both peptides may bind intracellularly to mortalin and/or a mortalinbinding protein, hence knocking down mortalin expression reduced cell death. Combining treatment with MotP2 or MotP7 and CDC resulted in increased cell death. This study identified highly cytotoxic mortalin mimetic peptides that may be used as monotherapy or combined with complementactivating antibody therapy to target mortalin for precision cancer therapy.
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Proteínas del Sistema Complemento/inmunología , Proteínas HSP70 de Choque Térmico/farmacología , Proteínas Mitocondriales/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Péptidos/farmacología , Peptidomiméticos/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/inmunología , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/inmunología , Neoplasias/metabolismo , Neoplasias/patología , Péptidos/química , Unión ProteicaRESUMEN
BACKGROUND: MAGE family genes have been studied as targets for tumor immunotherapy for a long time. Here, we combined MAGE1-, MAGE3- and MAGEn-derived peptides as a cancer vaccine and tested whether a new combination nanoemulsion-encapsulated vaccine could be used to inhibit the growth of tumor cells in humanized SCID mice. METHODS: The nanoemulsion-encapsulated complex protein vaccine (MAGE1, MAGE3, and MAGEn/HSP70 fusion protein; M1M3MnH) was prepared using a magnetic ultrasonic technique. After screening, human PBMCs were injected into SCID mice to mimic the human immune system. Then, the humanized SCID mice were challenged with M3-HHCC cells and immunized with nanoemulsion-encapsulated MAGE1-MAGE3-MAGEn/HSP70 [NE(M1M3MnH)] or M1M3MnH. The cellular immune responses were detected by IFN-γ ELISPOT and cytotoxicity assays. Therapeutic and tumor challenge experiments were also performed. RESULTS: The results showed that the immune responses elicited by NE(M1M3MnH) were apparently stronger than those elicited by M1M3MnH, NE(-) or PBS, suggesting that this novel nanoemulsion carrier induces potent antitumor immunity against the encapsulated antigens. The results of the therapeutic and tumor challenge experiments also indicated that the new vaccine had a definite effect on SCID mice bearing human hepatic cancer. CONCLUSION: Our study indicated that the combination of several tumor antigen-derived peptides may be a relatively good strategy for peptide-based cancer immunotherapy. These results suggest that the complex nanoemulsion vaccine could have broader applications for both therapy and prevention mediated by antitumor effects in the future.
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Antígenos de Neoplasias/farmacología , Vacunas contra el Cáncer/farmacología , Proteínas HSP70 de Choque Térmico/farmacología , Antígenos Específicos del Melanoma/farmacología , Proteínas de Neoplasias/farmacología , Animales , Antígenos de Neoplasias/inmunología , Emulsiones , Femenino , Proteínas HSP70 de Choque Térmico/inmunología , Humanos , Antígenos Específicos del Melanoma/inmunología , Ratones , Ratones SCID , Nanopartículas , Proteínas de Neoplasias/inmunología , Neoplasias Experimentales , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Heat shock protein 70 (Hsp70) has been shown to exert cardioprotection. Intracellular calcium ([Ca2+]i) overload induced by p38 mitogen-activated protein kinase (p38 MAPK) activation contributes to cardiac ischemia/reperfusion (I/R) injury. However, whether Hsp70 interacts with p38 MAPK signaling is unclear. Therefore, this study investigated the regulation of p38 MAPK by Hsp70 in I/R-induced cardiac injury. METHODS: Neonatal rat cardiomyocytes were subjected to oxygen-glucose deprivation for 6 h followed by 2 h reoxygenation (OGD/R), and rats underwent left anterior artery ligation for 30 min followed by 30 min of reperfusion. The p38 MAPK inhibitor (SB203580), Hsp70 inhibitor (Quercetin), and Hsp70 short hairpin RNA (shRNA) were used prior to OGD/R or I/R. Cell viability, lactate dehydrogenase (LDH) release, serum cardiac troponin I (cTnI), [Ca2+]i levels, cell apoptosis, myocardial infarct size, mRNA level of IL-1ß and IL-6, and protein expression of Hsp70, phosphorylated p38 MAPK (p-p38 MAPK), sarcoplasmic/endoplasmic reticulum Ca2+-ATPase2 (SERCA2), phosphorylated signal transducer and activator of transcription3 (p-STAT3), and cleaved caspase3 were assessed. RESULTS: Pretreatment with a p38 MAPK inhibitor, SB203580, significantly attenuated OGD/R-induced cell injury or I/R-induced myocardial injury, as evidenced by improved cell viability and lower LDH release, resulted in lower serum cTnI and myocardial infarct size, alleviation of [Ca2+]i overload and cell apoptosis, inhibition of IL-1ß and IL-6, and modulation of protein expressions of p-p38 MAPK, SERCA2, p-STAT3, and cleaved-caspase3. Knockdown of Hsp70 by shRNA exacerbated OGD/R-induced cell injury, which was effectively abolished by SB203580. Moreover, inhibition of Hsp70 by quercetin enhanced I/R-induced myocardial injury, while SB203580 pretreatment reversed the harmful effects caused by quercetin. CONCLUSIONS: Inhibition of Hsp70 aggravates [Ca2+]i overload, inflammation, and apoptosis through regulating p38 MAPK signaling during cardiac I/R injury, which may help provide novel insight into cardioprotective strategies.
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Proteínas HSP70 de Choque Térmico/uso terapéutico , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Proteínas HSP70 de Choque Térmico/farmacología , Masculino , Ratas , Ratas Sprague-Dawley , Transducción de SeñalRESUMEN
Natural killer (NK) cell therapy is one of the most promising treatments for Glioblastoma Multiforme (GBM). However, this emerging technology is limited by the availability of sufficient numbers of fully functional cells. Here, we investigated the efficacy of NK cells that were expanded and treated by interleukin-2 (IL-2) and heat shock protein 70 (HSP70), both in vitro and in vivo. Proliferation and cytotoxicity assays were used to assess the functionality of NK cells in vitro, after which treated and naïve NK cells were administrated intracranially and systemically to compare the potential antitumor activities in our in vivo rat GBM models. In vitro assays provided strong evidence of NK cell efficacy against C6 tumor cells. In vivo tracking of NK cells showed efficient homing around and within the tumor site. Furthermore, significant amelioration of the tumor in rats treated with HSP70/Il-2-treated NK cells as compared to those subjected to nontreated NK cells, as confirmed by MRI, proved the efficacy of adoptive NK cell therapy. Moreover, results obtained with systemic injection confirmed migration of activated NK cells over the blood brain barrier and subsequent targeting of GBM tumor cells. Our data suggest that administration of HSP70/Il-2-treated NK cells may be a promising therapeutic approach to be considered in the treatment of GBM.
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Barrera Hematoencefálica/efectos de los fármacos , Glioblastoma/patología , Proteínas HSP70 de Choque Térmico/farmacología , Interleucina-2/farmacología , Animales , Línea Celular Tumoral , Técnicas de Cocultivo , Glioblastoma/metabolismo , Inmunofenotipificación , Células Asesinas Naturales/inmunología , Masculino , RatasRESUMEN
Neuroinflammation plays a key role in the pathogenesis of neurodegenerative diseases. Microglial cells are the main immune cells of the central nervous system. On exposure to lipopolysaccharides (LPS, components of the cell wall of Gram-negative enterobacteria), microglia is activated to produce reactive oxygen species (ROS), cytokines, and inflammatory mediators, which may cause neuron death. Exogenous recombinant human heat shock protein 70 (HSP70) was tested for effect on the activation of human microglial and neuroblastoma cells in response to LPS from Escherichia coli. Experiments included cell cultivation separately and transferring the conditioned medium from A-172 microglial cells to SK-N-SH neuroblastoma cells to simulate the effect of microglia treated with LPS and/or HSP70. The levels of ROS, TNFα, and apoptosis in LPS-treated cells were estimated in the presence or absence of HSP70. HSP70 was found to reduce the LPS-induced ROS generation, TNFα production, apoptosis, and necrosis, in both separate cell cultures and neuroblastoma cells grown in the conditioned medium from microglial cells. Signaling pathways involving protein kinases p38MAPK, JNK, and PI3K were demonstrated to play an important role in HSP70-mediated protection of microglial and neuroblastoma cells from LPS-induced apoptosis and ROS production.
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Medios de Cultivo Condicionados/química , Proteínas HSP70 de Choque Térmico/farmacología , Lipopolisacáridos/toxicidad , Neuroblastoma/tratamiento farmacológico , Neuroprotección/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Transducción de Señal/efectos de los fármacos , Apoptosis/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Humanos , Lipopolisacáridos/inmunología , Microglía/efectos de los fármacos , Microglía/inmunología , Microglía/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
OBJECTIVE: To address whether a targeted modulation of the abnormal expression of Hsp70 and autoantibodies against this molecule in systemic lupus erythematosus can influence disease. METHODS: Lupus-prone (NZB × NZW)F1 mice that had been DNA-vaccinated with plasmids encoding Hsp70 and controls were monitored for lupus disease parameters including anti-double stranded DNA (anti-dsDNA) autoantibodies and cytokines using enzyme-linked immunosorbent assay, and for kidney function and pathology. The phenotypic and numerical changes in relevant immune cells were evaluated by flow cytometry, and cell function was assessed. RESULTS: Mice that had been DNA-vaccinated with Hsp70 displayed marked suppression of anti-dsDNA antibody production, reduced renal disease, and antiinflammatory responses that are associated with a significantly extended survival, compared to controls. These protective effects in Hsp70-vaccinated mice were associated with an induction of tolerogenic immune responses and an expansion of functional Treg cells. CONCLUSION: DNA vaccination with Hsp70 suppresses murine lupus by inducing tolerogenic immune responses and antiinflammatory immune responses associated with reduced disease manifestations and increased mouse survival.
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Anticuerpos Antinucleares/efectos de los fármacos , Autoanticuerpos/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/farmacología , Lupus Eritematoso Sistémico/prevención & control , Vacunas de ADN/farmacología , Animales , Anticuerpos Antinucleares/inmunología , Autoanticuerpos/inmunología , ADN/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Lupus Eritematoso Sistémico/inmunología , Ratones , Ratones Endogámicos NZBRESUMEN
AIMS: The present study was aimed to investigate the neuroprotective effect of HSP70 against neuroinflammation in a rotenone-induced Parkinson's disease model. MATERIALS AND METHODS: In the present study, SH-SY5Y cells were treated with HSP70 (5-20â¯mg/L) for 72â¯h. Cell viability, reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), levels of oxidative markers, mitochondrial fragmentation, apoptosis, and mRNA and protein expressions of signal transducer and activator of transcription (STAT)-3 and nuclear factor-kappa B (NF-κB) were assessed. KEY FINDINGS: Cells treated with 5, 10, 15, and 20â¯mg/L of HSP70 exhibited increased, by 61.7%, 70.3%, 84.6%, and 96.7%, respectively, in cell viability. ROS and lipid peroxidation levels decreased following treatment with HSP70, and reductions in glutathione (GSH), catalase, glutathione peroxidase (Gpx), and superoxide dismutase (SOD) levels were reversed following treatment with HSP70. Additionally, MMP levels were reduced by 29.7, 46.4, 79.5, and 125.2 relative units following treatment with 5-20â¯mg/L of HSP70, respectively. HSP70 treatment also decreased levels of fragmented mitochondria and apoptosis, and mRNA and protein expressions of NF-κB and STAT3 were reduced by >25%. SIGNIFICANCE: Taken together, these findings indicate that supplementation with HSP70s recovered cell viability and MMP and reduced levels of ROS, apoptosis, and mitochondrial fragmentation. Additionally, supplementation with HSP70 significantly reduced the expressions of STAT3 and NF-κB.
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Regulación hacia Abajo/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/farmacología , Inflamación/tratamiento farmacológico , FN-kappa B/genética , Fármacos Neuroprotectores/farmacología , Enfermedad de Parkinson Secundaria/tratamiento farmacológico , Factor de Transcripción STAT3/genética , Apoptosis/efectos de los fármacos , Línea Celular , Humanos , Inflamación/genética , Enfermedad de Parkinson Secundaria/genética , ARN Mensajero/genética , RotenonaRESUMEN
NK cells (natural killer cells) being a part of the innate immune system have been shown to be involved in immunoregulation of autoimmune diseases. Previously we have shown that HINT1/Hsp70 treatment induced regulatory NK cells ameliorating experimental autoimmune encephalomyelitis (EAE) course and CD4+ T cells proliferation. NK cells were isolated from mice treated with HINT1/Hsp70 and co-cultured with proteolipid protein (PLP)-stimulated CD4+ T cells isolated from EAE mice. Cell proliferation was assessed by thymidine uptake, cytotoxicity by lactate dehydrogenase (LDH) release assay and fluorescence activated cell sorting (FACS) analysis, protein expression by Western blot, mRNA by quantitative RT-PCR. Gene related to anergy in lymphocytes (GRAIL) expression was downregulated by specific siRNA and GRAIL overexpression was induced by pcDNA-GRAIL transfection. HINT1/Hsp70 pretreatment of EAE SJL/J mice ameliorated EAE course, suppressed PLP-induced T cell proliferation by enhancing T cell expression of GRAIL as GRAIL downregulation restored T cell proliferation. HINT1/Hsp70 treatment induced immunoregulatory NK cells which inhibited PLP-stimulated T cell proliferation not depending on T cell necrosis and apoptosis. This immunoregulatory NK cell function depended on NK cell expression of GRAIL as GRAIL downregulation diminished inhibition of NK cell suppression of T cell proliferation. Similarly GRAIL overexpression in NK cells induced their regulatory function. HINT1/Hsp70 treatment generated regulatory NK cells characterized by expression of GRAIL.