RESUMO
Heat shock proteins (HSPs) are available and/or induced for the survival of all organisms, including eukaryotic, prokaryotic, and plants, from higher temperature stresses. They are the chaperone proteins that protect all cells against heat, as the name implies. In addition to thermal stress, they also protect them from chemical, physical, and other stresses, including exposure to oxidative stress, nutritional deficiencies, ultraviolet radiation, ethanol, viral infection, ischemia-reperfusion injury, and cancer-related stresses. They are classified based on their molecular weights in kDa, such as HSP90 and HSP70. In our label-free, high-throughput, quantitative LC-MS/MS-based proteomic studies of MDA-MB-231, human, triple-negative breast cancer cells, treated with electrical pulses (EP) and cisplatin (CsP), we identified a number of HSPs, such as HSP90AA1, and others to be significantly downregulated in EP + CsP, compared to CsP alone. This indicates that cells will undergo apoptotic cell death and hence could cause effective cancer cure/treatment. Considering that over 2 million new cases and over 600,000 deaths in 2020, of which ~ 15% are TNBC, heat shock proteins could be the untapped resources, available for the next biomarkers and/or inhibitors for new/additional therapies.
Assuntos
Cisplatino/farmacologia , Regulação para Baixo/efeitos dos fármacos , Eletroquimioterapia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP90/biossíntese , Proteínas de Neoplasias/biossíntese , Neoplasias de Mama Triplo Negativas , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Type 2 DCs (DC2s) comprise the majority of conventional DCs within most tumors; however, little is known about their ability to initiate and sustain antitumor immunity, as most studies have focused on antigen cross-presenting DC1s. Here, we report that DC2 infiltration identified by analysis of multiple human cancer data sets showed a significant correlation with survival across multiple human cancers, with the benefit being seen in tumors resistant to cytotoxic T cell control. Characterization of DC subtype infiltration into an immunotherapy-resistant model of breast cancer revealed that impairment of DC1s through 2 unique models resulted in enhanced DC2 functionality and improved tumor control. BATF3 deficiency depleted intratumoral DC1s, which led to increased DC2 lymph node migration and CD4+ T cell activation. Enhancing DC2 stimulatory potential by genetic deletion of Hsp90b1 (encoding molecular chaperon GP96) led to a similar enhancement of T cell immunity and improved survival in a spontaneous breast cancer model. These data highlight the therapeutic and prognostic potential of DC2s within checkpoint blockade-resistant tumors.
Assuntos
Células Dendríticas/imunologia , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP90/genética , Imunidade Celular , Imunoterapia/métodos , Melanoma Experimental/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Apresentação Cruzada , Células Dendríticas/patologia , Proteínas de Choque Térmico HSP90/biossíntese , Ativação Linfocitária , Melanoma Experimental/genética , Melanoma Experimental/terapia , Camundongos Transgênicos , Neoplasias Experimentais , RNA Neoplásico/genética , Linfócitos T Citotóxicos/patologiaRESUMO
Heat stress-induced decline in milk production and mammary glands dysfunction are economically important challenges facing the dairy industry, especially in summer. Choline is an organic water-soluble compound that can regulate a series of vital biological process, including cellular structural integrity and oxidative stress. However, it is unclear whether choline plays an anti-apoptosis and antioxidant effect in heat stress-induced mammary epithelial cells. This study aimed to determine the antioxidant effect of choline on heat stress-induced apoptosis and oxidative stress and its underlying molecular mechanism in bovine mammary epithelial cells (MAC-T cells). The MAC-T cells were divided into four treatment groups: control (37â), choline (37â), heat stress (HS, 42â), and HS + choline. The results showed that heat stress up-regulated the HSP70 and HSP90 expression both in mRNA and protein, enhanced ROS accumulation, increased malondialdehyde (MDA) content, reduced the superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity, significantly increased the expression of caspase-3 and upregulated the ratio of Bax/Bcl-2 and ultimately lead to oxidative stress and apoptosis in MAC-T cells. However, choline pretreatment reversed the above phenomenon compared with the HS group. The HS + choline group inhibited heat stress-induced phosphorylation of PERK, nuclear translocation of Nrf-2 and the protein expression of GRP78. In addition, the ratio of Bax/Bcl-2 and the expression of caspase-3 were significantly reduced in HS + choline group, thereby reduced the HS-induced oxidative stress and apoptosis in MAC-T cells. In conclusion, choline attenuates heat stress-induced oxidative stress and apoptosis of MAC-T cells by modulating PERK/Nrf-2 pathway.
Assuntos
Apoptose/efeitos dos fármacos , Colina/farmacologia , Resposta ao Choque Térmico/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , eIF-2 Quinase/metabolismo , Animais , Antioxidantes/farmacologia , Caspase 3/metabolismo , Bovinos , Linhagem Celular , Chaperona BiP do Retículo Endoplasmático , Células Epiteliais/metabolismo , Feminino , Glutationa Peroxidase/metabolismo , Proteínas de Choque Térmico HSP72/biossíntese , Proteínas de Choque Térmico HSP90/biossíntese , Proteínas de Choque Térmico/metabolismo , Glândulas Mamárias Animais/metabolismo , Leite/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Linfócitos T/efeitos dos fármacosRESUMO
In spite of the abundance of antifungal therapies, 75% of women in the world suffer from the second most common cause of vaginal infection named vulvovaginal candidiasis. This complication is characterized with overgrowth of Candida albicans. The low efficacy and side effects of current antifungal therapies have convinced the researchers to look for a non-antibiotic based treatment such as cold atmospheric plasmas (CAP). The aim of this research was to evaluate the effects of CAP on C. albicans growth, ergosterol and biofilm formation. In addition, antibiotic resistance, phospholipase and proteinase activity, and structural properties were examined with different exposure duration. Putative critical effect of CAP on the expression of HSP90 as a target of anti-fungal therapy was investigated. ROS production in C. albicans exposed to CAP was assessed. For this purpose, C. albicans subjected to 0, 90, 120, 150, 180 and 210 s of He/O2 (2%), and non-treated cells as control were examined in terms of the mentioned virulence factors. The results showed that CAP had a significant effect on inhibition of C. albicans growth, Inhibition of biofilm formation, ergosterol content, and fluconazole and amphotericin B antibiotic sensitivity were significant in 210 s treatment group. This effect was validated based on changes of the cell architecture and morphology given the microscopy imaging results. The expression of HSP90 in both C. albicans ATCC 10231 and C. albicans PFCC 9362 was inhibited in 210 s of exposition. CAP exposition induced intracellular ROS, which may cause membrane damage and cell death in C. albicans. Taken together, the potential of CAP for therapeutic purposes in C. albicans-induced fungal infections is supported.
Assuntos
Biofilmes/efeitos dos fármacos , Candida albicans , Proteínas Fúngicas/biossíntese , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/biossíntese , Gases em Plasma/farmacologia , Fatores de Virulência/biossíntese , Biofilmes/crescimento & desenvolvimento , Candida albicans/patogenicidade , Candida albicans/fisiologiaRESUMO
Untranslated regions (UTRs) of the transcripts play significant roles in translation regulation and continue to raise many intriguing questions in our understanding of cellular stress physiology. Internal ribosome entry site (IRES) mediated alternative translation initiations are emerging as unique mechanisms. Present study is aimed to indentify a functional short 92 base pair length putative sequence located at the 5' untranslated region of bovine heat shock protein 90 AA1 (Hsp90AA1) may interact with ribosomal as well as eukaryotic initiation factor binding site. Here we have predicted both the two and three dimensional structures of bovine Hsp90AA1 IRES (MF400854) element with their respective free energy. Molecular interactions between bovine RPS5 and IRES have been determined after the preparation of docking complex of IRES bound RPS5. Structure of bovine ribosomal translational initiation factor (TIF) has also been determined and docked with IRES. Molecular interaction between bovine TIF and IRES was analyzed from the complex structure. We further detected the relative expression efficiency of the viral (original) in relation with Hsp90AA1 IRES-driven GFP expression, which revealed that efficiency under the control of identified bovine Hsp90AA1 IRES was slightly lower than viral origin. It was also noted that identified bovine HSP90 IRES may increase the expression level of GFP under in vitro heat stressed condition.
Assuntos
Regiões 5' não Traduzidas , Proteínas de Choque Térmico HSP90 , Conformação de Ácido Nucleico , Ribossomos , Animais , Bovinos , Linhagem Celular , Proteínas de Choque Térmico HSP90/biossíntese , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/genética , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Heat shock protein 90 (HSP90) plays essential roles in the normal physiology and comprises four distinct domains, including NH2-terminal (N), charged linker region (LR), middle (M), and COOH-terminal (C) domains, all of which regulate HSP90 biological functions. We reported herein detailed protocols to produce recombinant full-length (FL) and all these four domains of human HSP90 from Escherichia coli. cDNAs encoding FL, N, LR, M and C domains of human HSP90α were amplified and cloned into pET-32b(+) expression vector. All HSP90 constructs were expressed as soluble Trx-His-S tagged proteins after induction with 0.25 mM isopropyl-ß-d-thiogalactopyranoside (IPTG) at 18 °C overnight and further purified by affinity chromatography using nickel-nitrilotriacetic acid (Ni-NTA) resin. The enterokinase (EK) digestion was optimized for efficient cleavage of the Trx-His-S tag from each HSP90 construct by varying concentrations of EK (0.5-1 U) and urea (0-3 M). Each HSP90 construct was highly purified and approximately 0.1-1 mg proteins were obtained from 100 ml of bacterial culture. All the purified HSP90 constructs were successfully confirmed by tandem mass spectrometry (nanoLC-ESI-ETD MS/MS) and their secondary structure was quantified using attenuated total reflection - Fourier-transform infrared (ATR-FTIR) spectroscopy. Our expression and purification protocols would facilitate further structural and functional studies of human HSP90.
Assuntos
Proteínas de Choque Térmico HSP90/biossíntese , Proteínas Recombinantes/biossíntese , Clonagem Molecular , Escherichia coli/genética , Proteínas de Choque Térmico HSP90/isolamento & purificação , Humanos , Domínios Proteicos , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
A tight quality control system over protein folding, turnover and synthesis, involving molecular chaperones and co-chaperones, maintains the balance of cardiac proteins. Various cardiac pathologies, including myocardial infarction, increase stresses and post-translational modifications favoring misfolding due to an overwhelmed quality control system. The toxic nature of accumulated misfolded proteins further worsens the condition. The important molecular chaperones which act as quality control proteins are involved in protecting the heart, these include heat shock protein70 (HSP70) and HSP90. Here, we review the emerging roles of heat shock proteins in the maintenance of cardiac cell populations in experimental models of ischemia/reperfusion (I/R) injury. Furthermore, we discuss the expression of HSP70 and HSP90 with therapeutic and diagnostic considerations. Although there is only a partial understanding of these important HSPs in I/R injury, there is an immense therapeutic potential of modulating these HSPs to counteract the imbalance between misfolding and endogenous protein quality control systems.
Assuntos
Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP90/biossíntese , Infarto do Miocárdio/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Animais , Humanos , Camundongos , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologiaRESUMO
Muscle-specific RING-finger proteins (MuRFs) are E3 ubiquitin ligases that play important roles in protein quality control in skeletal and cardiac muscles. Here we characterized murf gene expression and protein localization in zebrafish embryos. We found that the zebrafish genome contains six murf genes, including murf1a, murf1b, murf2a, murf2b, murf3 and a murf2-like gene that are specifically expressed in skeletal and cardiac muscles of zebrafish embryos. To analyze the subcellular localization, we generated transgenic zebrafish models expressing MurF1a-GFP or MuRF2a-GFP fusion proteins. MuRF1a-GFP and MuRF2a-GFP showed distinct patterns of subcellular localization. MuRF1a-GFP displayed a striated pattern of localization in myofibers, whereas MuRF2a-GFP mainly exhibited a random pattern of punctate distribution. The MuRF1a-GFP signal appeared as small dots aligned along the M-lines of the sarcomeres in skeletal myofibers. To determine whether knockdown of smyd1b or hsp90α1 that increased myosin protein degradation could alter murf gene expression or MuRF protein localization, we knocked down smyd1b or hsp90α1 in wild type, Tg(ef1a:MurF1a-GFP) and Tg(ef1a:MuRF2a-GFP) transgenic zebrafish embryos. Knockdown of smyd1b or hsp90α1 had no effect on murf gene expression. However, the sarcomeric distribution of MuRF1a-GFP was abolished in the knockdown embryos. This was accompanied by an increased random punctate distribution of MuRF1a-GFP in muscle cells of zebrafish embryos. Collectively, these studies demonstrate that MuRFs are specifically expressed in developing muscles of zebrafish embryos. The M-line localization MuRF1a is altered by sarcomere disruption in smyd1b or hsp90α1 knockdown embryos.
Assuntos
Animais Geneticamente Modificados , Embrião não Mamífero/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Choque Térmico HSP90 , Histona-Lisina N-Metiltransferase , Modelos Biológicos , Proteínas Recombinantes de Fusão , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Animais Geneticamente Modificados/embriologia , Animais Geneticamente Modificados/genética , Proteínas de Choque Térmico HSP90/biossíntese , Proteínas de Choque Térmico HSP90/genética , Histona-Lisina N-Metiltransferase/biossíntese , Histona-Lisina N-Metiltransferase/genética , Músculo Esquelético/embriologia , Proteínas Recombinantes de Fusão/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/genéticaRESUMO
AIMS: HSP90, as a molecular chaperone, has numerous substrate proteins, including HIF-1α and p-AKT, but the relationships among HSP90, HIF-1α and p-AKT have not been investigated in NPC. We examined and analysed the correlation between expression of HSP90, HIF-1α and p-AKT and clinicopathological features of NPC. METHODS: We collected 445 cases of NPC and 54 cases of non-cancerous nasopharyngeal epithelia tissues, detected expression of HSP90, HIF-1α and p-AKT proteins in these tissues by immunohistochemistry. RESULTS: The results indicated that overexpression of HSP90, HIF-1α and p-AKT in NPC was significantly higher than that in non-cancerous nasopharyngeal epithelia (P < 0.05). The overexpression of HIF-1α in primary NPC was significantly lower than that in matched lymph node metastatic NPC (P = 0.024) or recurrent NPC (P = 0.039). The overexpression of HSP90 (P < 0.001) and HIF-1α (P = 0.031) was evidently higher in late stage NPC. NPC patients with lymph node metastasis (LNM) had a higher overexpression rate of HSP90 (P < 0.001) than those without LNM. Increased HSP90 expression was positively associated with HIF-1α expression (r = 0.367, P < 0.001) and p-AKT (r = 0.142, P = 0.003) expression in NPC. Furthermore, HIF-1α was also related to p-AKT expression (r = 0.114, P = 0.017). The overall survival rate for NPC patients with up-regulated HSP90 was significantly lower than those with down-regulated HSP90 (P < 0.001), as was found with raised HIF-1α (P = 0.036) and increased p-AKT (P = 0.044). Multivariate Cox regression analysis further identified that HSP90 and HIF-1α were independent poor prognostic factors for NPC. CONCLUSIONS: Taken together, elevated HSP90 was associated with expression of HIF-1α and p-AKT in NPC. Furthermore, high expression of HSP90 and HIF-1α could be used as a novel independent poor prognostic biomarker for patients with NPC.
Assuntos
Biomarcadores Tumorais/análise , Proteínas de Choque Térmico HSP90/biossíntese , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/patologia , Adulto , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Masculino , Pessoa de Meia-Idade , Carcinoma Nasofaríngeo/mortalidade , Neoplasias Nasofaríngeas/mortalidade , Prognóstico , Proteínas Proto-Oncogênicas c-akt/biossínteseRESUMO
OBJECTIVES: Heat shock protein 90 (HSP90) contributes to cutaneous vasodilatation during exercise in the heat through nitric oxide (NO) synthase (NOS)-dependent mechanisms in young adults. We hypothesized that similar responses would be observed in older middle-aged adults. METHODS: In nineteen habitually active older middle-aged (56 ± 5 years) men (n = 9) and women (n = 10), cutaneous vascular conductance (CVC) was measured at four forearm skin sites continuously treated with (a) lactated Ringers solution (Control), (b) 10 mmol/L L-NAME (NOS inhibitor), (c) 178 µmol/L geldanamycin (HSP90 inhibitor), or (d) 10 mmol/L L-NAME and 178 µmol/L geldanamycin combined. Participants rested in an upright semi-recumbent position in the heat (35°C) for 70 minutes, followed by a 50-minute bout of moderate-intensity cycling (~55% peak oxygen uptake) and a 30-minute recovery period in the heat. RESULTS: In both men and women, we observed no significant effects of HSP90 inhibition on CVC throughout rest, exercise, and recovery in the heat (all P > 0.27). Conversely, NOS inhibition and dual NOS and HSP90 inhibition attenuated CVC relative to Control throughout the protocol (all P ≤ 0.05). CONCLUSIONS: While NOS mediates cutaneous vasodilatation during rest, exercise, and recovery in the heat, HSP90 does not measurably influence this response in habitually active older middle-aged men or women under these conditions.
Assuntos
Proteínas de Choque Térmico HSP90/biossíntese , Transtornos de Estresse por Calor/metabolismo , Transtornos de Estresse por Calor/fisiopatologia , Pele , Vasodilatação , Idoso , Feminino , Antebraço/irrigação sanguínea , Antebraço/patologia , Antebraço/fisiopatologia , Transtornos de Estresse por Calor/tratamento farmacológico , Transtornos de Estresse por Calor/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Pele/irrigação sanguínea , Pele/patologia , Pele/fisiopatologiaRESUMO
Salinomycin is a polyether ionophore antibiotic having anti-tumorigenic property in various types of cancer. Elevated thymidine phosphorylase (TP) levels, a key enzyme in the pyrimidine nucleoside salvage pathway, are associated with an aggressive disease phenotype and poor prognoses. Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone that is responsible for the stabilization and maturation of many oncogenic proteins. In this study, we report whether Hsp90 inhibitor 17-AAG could enhance salinomycin-induced cytotoxicity in NSCLC cells through modulating TP expression in two non-small-cell lung cancer (NSCLC) cell lines, A549 and H1975. We found that salinomycin increased TP expression in a MKK3/6-p38 MAPK activation manner. Knockdown of TP using siRNA or inactivation of p38 MAPK by pharmacological inhibitor SB203580 enhanced the cytotoxic and growth inhibition effects of salinomycin. In contrast, enforced expression of MKK6E (a constitutively active form of MKK6) reduced the cytotoxicity and cell growth inhibition of salinomycin. Moreover, Hsp90 inhibitor 17-AAG enhanced cytotoxicity and cell growth inhibition of salinomycin in NSCLC cells, which were associated with down-regulation of TP expression and inactivation of p38 MAPK. Together, the Hsp90 inhibition induced TP down-regulation involved in enhancing the salinomycin-induced cytotoxicity in A549 and H1975 cells.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/enzimologia , Citotoxinas/toxicidade , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Neoplasias Pulmonares/enzimologia , Piranos/toxicidade , Timidina Fosforilase/antagonistas & inibidores , Células A549 , Antineoplásicos/toxicidade , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica , Proteínas de Choque Térmico HSP90/biossíntese , Humanos , Timidina Fosforilase/biossíntese , Timidina Fosforilase/genéticaRESUMO
Essential for cell survival, the 90 kD Heat Shock Proteins (HSP90) are molecular chaperons required for conformational stabilization and trafficking of numerous client proteins. Functional HSP90 is required for the stability of AKT, a serine-threonine kinase phosphorylated in response to growth factor stimulation. AKT plays a crucial regulatory role in differentiation, cell cycle, transcription, translation, metabolism and apoptosis. Acute promyelocytic leukaemia (APL) is characterized by the presence of the promyelocytic leukaemia/retinoic acid receptor alpha (PML/RARA) fusion protein, which deregulates expression of several genes involved in differentiation and apoptosis. Here, we report inhibition of HSP90AA1 and HSP90AB1 isomer transcription in blasts isolated from patients with APL, associated with reduction of HSP90 protein expression and loss of control on AKT protein phosphorylation. We show that in vitro treatment of PML/RARA expressing cells with all-trans retinoic acid (ATRA) up-regulates HSP90 expression and stabilizes AKT. Addition of the HSP90-inhibitor 17-(allylamino)-17-demethoxygeldanamycin in combination with ATRA, blocks upregulation of AKT protein, indicating that HSP90 is necessary for ATRA action on AKT. This is the first report proving that expression of HSP90 isomers are directly and differentially repressed by PML/RARA, with critical results on cellular homeostasis of target proteins, such as AKT, in APL blasts.
Assuntos
Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Leucemia Promielocítica Aguda/tratamento farmacológico , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Benzoquinonas/farmacologia , Células HEK293 , Proteínas de Choque Térmico HSP90/biossíntese , Proteínas de Choque Térmico HSP90/genética , Histonas/genética , Histonas/metabolismo , Humanos , Lactamas Macrocíclicas/farmacologia , Leucemia Promielocítica Aguda/patologia , Proteínas de Fusão Oncogênica/biossíntese , Proteínas de Fusão Oncogênica/genética , Regiões Promotoras Genéticas , Proteína da Leucemia Promielocítica/biossíntese , Proteína da Leucemia Promielocítica/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptor alfa de Ácido Retinoico/biossíntese , Receptor alfa de Ácido Retinoico/genética , Transfecção , Tretinoína/farmacologia , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacosRESUMO
Bactrocera cucurbitae (melon flies) are prominent invasive pests in southern China. To screen for a stable reference gene in melon flies suitable for comparing tissue samples subjected to different conditions in four categories (temperature, insect stage, days of age and gender), the expression of 12 candidate reference genes under different treatment conditions was analyzed by real-time fluorescent quantitative PCR. The results obtained from a comprehensive analysis with geNorm, NormFinder, BestKeeper and RefFinder software showed that the most stable reference gene was RPL60, and the least stable reference gene was actin-5. We used a heat shock protein gene (HSP-90) to verify the results, and the conclusion was consistent. When the reference gene RPL60 was used as the reference gene, the relative expression of HSP-90 was essentially constant with the prolongation of treatment time. When actin-5 was used, HSP-90 expression changed markedly with treatment time. The results of this study can be used for further research on gene expression inBactrocera cucurbitae.
Assuntos
Actinas/genética , Regulação da Expressão Gênica , Genes de Insetos , Proteínas de Choque Térmico HSP90/genética , Proteínas de Insetos/genética , Reação em Cadeia da Polimerase em Tempo Real , Tephritidae/genética , Actinas/biossíntese , Animais , Proteínas de Choque Térmico HSP90/biossíntese , Proteínas de Insetos/biossíntese , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Tephritidae/metabolismoRESUMO
Invasive fungal infections especially in immunocompromised patients represent a dominating cause of mortality. The most commonly used antifungal agents can be divided into three broad categories, including triazoles, echinocandins and polyenes. Antifungal resistance is on the increase, posing a growing threat to the stewardship of immunocompromised patients with fungal infections. The paucity of currently available antifungals leads to the rapid emergence of drug resistance and thus aggravates the refractoriness of invasive fungal infections. Therefore, deep exploration into mechanisms of drug resistance and search for new antifungal targets are required. This review highlights the therapeutic strategies targeting Hsp90, calcineurin, trehalose biosynthesis and sphingolipids biosynthesis, in an attempt to provide clinical evidence for overcoming drug resistance and to form the rationale for combination therapy of conventional antifungals and agents with novel mechanisms of action. What's more, this review also gives a concise introduction of three new-fashioned antifungals, including carboxymethyl chitosan, silver nanoparticles and chromogranin A-N46.
Assuntos
Antifúngicos/farmacologia , Farmacorresistência Fúngica/efeitos dos fármacos , Fungos/efeitos dos fármacos , Calcineurina/biossíntese , Biologia Computacional , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/biossíntese , Humanos , Testes de Sensibilidade Microbiana , Esfingolipídeos/antagonistas & inibidores , Esfingolipídeos/biossíntese , Trealose/antagonistas & inibidores , Trealose/biossínteseRESUMO
Up-regulation of heat shock protein 90 (HSP90) gene takes place in lung cancer cells. Therefore, targeting HSP90 in lung cancer may be promising step in lung cancer therapy. The present study aimed to evaluate the efficiency of implantable 17-dimethylaminoethylamino-17-demethoxy geldanamycin (17-DMAG)-loaded Poly(caprolactone)-poly(ethylene glycol) (PCL/PEG) nanofibers to increase the anti-cancer effects via inhibition of HSP90 expression and telomerase activity. For this purpose, 17-DMAG-loaded PCL/PEG nanofibers were successfully fabricated via electrospinning and characterized using FE-SEM and FTIR. Colorimetric MTT assay was used to determine the drug cytotoxicity. Also, the expression levels of HSP90 mRNA in the A549 cells treated with the nanofibers were assessed using Quantitative Real-Time PCR. The effect of free 17-DMAG and 17-DMAG-loaded PCL/PEG nanofiber treatment on telomerase activity was monitored by TRAP assay. MTT assay confirmed that loading of 17-DMAG into PCL/PEG nanofiber enhanced dramatically cytotoxicity in the lung cancer cells. This finding was associated with reduction of HSP90 mRNA expression and telomerase activity in the cells seeded on 17-DMAG-loaded PCL/PEG nanofibers in relative to free 17-DMAG. In conclusion, the findings demonstrated that 17-DMAG-loaded PCL/PEG nanofibers are more effectual than free 17-DMAG against A549 lung cancer cells via modulation of Hsp90 expression and inhibition of telomerase activity. Hence, the implantable 17-DMAG-loaded nanofibrous scaffolds might be an excellent tool for efficiently killing of the lung residual cancer cells and avoid the local cancer recurrence.
Assuntos
Benzoquinonas/administração & dosagem , Portadores de Fármacos/administração & dosagem , Inibidores do Crescimento/administração & dosagem , Proteínas de Choque Térmico HSP90/biossíntese , Lactamas Macrocíclicas/administração & dosagem , Neoplasias Pulmonares/metabolismo , Nanofibras/administração & dosagem , Células A549 , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos/métodos , Implantes de Medicamento , Expressão Gênica , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Neoplasias Pulmonares/tratamento farmacológicoRESUMO
BACKGROUND/AIMS: Uncaria rhynchophylla, known as "Gou-teng", is a traditional Chinese medicine (TCM) used to extinguish wind, clear heat, arrest convulsions, and pacify the liver. Although U. rhynchophylla has a long history of being often used to treat central nervous system (CNS) diseases, its efficacy and potential mechanism are still uncertain. This study investigated neuroprotective effect and the underlying mechanism of U. rhynchophylla extract (URE) in MPP+-induced SH-SY5Y cells and MPTP-induced mice. METHODS: MPP+-induced SH-SY5Y cells and MPTP-induced mice were used to established Parkinson's disease (PD) models. Quantitative proteomics and bioinformatics were used to uncover proteomics changes of URE. Western blotting was used to validate main differentially expressed proteins and test HSP90 client proteins (apoptosis-related, autophagy-related, MAPKs, PI3K, and AKT proteins). Flow cytometry and JC-1 staining assay were further used to confirm the effect of URE on MPP+-induced apoptosis in SH-SY5Y cells. Gait analysis was used to detect the behavioral changes in MPTP-induced mice. The levels of dopamine (DA) and their metabolites were examined in striatum (STR) by HPLC-EC. The positive expression of tyrosine hydroxylase (TH) was detected by immunohischemical staining and Western blotting. RESULTS: URE dose-dependently increased the cell viability in MPP+-induced SH-SY5Y cells. Quantitative proteomics and bioinformatics results confirmed that HSP90 was an important differentially expressed protein of URE. URE inhibited the expression of HSP90, which further reversed MPP+-induced cell apoptosis and autophagy by increasing the expressions of Bcl-2, Cyclin D1, p-ERK, p-PI3K p85, PI3K p110α, p-AKT, and LC3-I and decreasing cleaved caspase 3, Bax, p-JNK, p-p38, and LC3-II. URE also markedly decreased the apoptotic ratio and elevated mitochondrial transmembrane potential (DΨm). Furthermore, URE treatment ameliorated behavioral impairments, increased the contents of DA and its metabolites and elevated the positive expressions of TH in SN and STR as well as the TH protein. CONCLUSIONS: URE possessed the neuroprotective effect in vivo and in vitro, regulated MAPK and PI3K-AKT signal pathways, and inhibited the expression of HSP90. U. rhynchophylla has potentials as therapeutic agent in PD treatment.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Choque Térmico HSP90/biossíntese , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Transtornos Parkinsonianos , Uncaria/química , Animais , Linhagem Celular Tumoral , Medicamentos de Ervas Chinesas/química , Humanos , Camundongos , Fármacos Neuroprotetores/química , Transtornos Parkinsonianos/tratamento farmacológico , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologia , ProteômicaRESUMO
Heat shock protein 90 (Hsp90) plays a vital role in the progress of malignant disease and elevated Hsp90 expression has been reported in pancreatic cancer. In this study, we radiolabeled a dimeric Sansalvamide A derivative (Di-San A1) with 64Cu, and evaluated the feasibility of using 64Cu-Di-San A1 for PET imaging of Hsp90 expression in a mouse model of pancreatic cancer. A macrocyclic chelator NOTA (1,4,7-triazacyclononane-1,4,7-trisacetic acid) was conjugated to Di-San A1. 64Cu-Di-San A1 was successfully prepared in a radiochemical yield > 97% with a radiochemical purity > 98%. 64Cu-Di-San A1 is stable in PBS and mouse serum with > 92% of parent probe intact after 4 h incubation. The cell binding and uptake revealed that 64Cu-Di-San A1 binds to Hsp90-positive PL45 pancreatic cancer cells, and the binding can be effectively blocked by an Hsp90 inhibitor (17AAG). For microPET study, 64Cu-Di-San A1 shows good in vivo performance in terms of tumor uptake in nude mice bearing PL45 tumors. The Hsp90-specific tumor activity accumulation of 64Cu-Di-San A1 was further demonstrated by significant reduction of PL45 tumor uptake with a pre-injected blocking dose of 17AAG. The ex vivo PET imaging and biodistribution results were consistent with the quantitative analysis of PET imaging, demonstrating good tumor-to-muscle ratio (5.35 ± 0.46) of 64Cu-Di-San A1 at 4 h post-injection in PL45 tumor mouse xenografts. 64Cu-Di-San A1 allows PET imaging of Hsp90 expression in PL45 tumors, which may provide a non-invasive method to quantitatively characterize Hsp90 expression in pancreatic cancer.
Assuntos
Cobre/farmacologia , Depsipeptídeos/farmacologia , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP90/biossíntese , Proteínas de Neoplasias/biossíntese , Neoplasias Pancreáticas , Tomografia por Emissão de Pósitrons , Animais , Linhagem Celular Tumoral , Cobre/química , Depsipeptídeos/química , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/metabolismo , Multimerização ProteicaRESUMO
Dasatinib and radotinib are oral BCR-ABL tyrosine kinase inhibitors that were developed as drugs for the treatment of chronic myeloid leukemia. We report here that the c-KIT (CD117) targeting with dasatinib and radotinib promotes acute myeloid leukemia (AML) cell death, and c-KIT endocytosis is essential for triggering c-KIT-positive AML cell death by dasatinib and radotinib during the early stages. In addition, dasatinib and radotinib reduce heat shock protein 90ß (HSP90ß) expression and release Apaf-1 in c-KIT-positive AML cells. Finally, this activates a caspase-dependent apoptotic pathway in c-KIT-positive AML cells. Moreover, the inhibition of c-KIT endocytosis by dynamin inhibitor (DY) reversed cell viability and c-KIT expression by dasatinib and radotinib. HSP90ß expression was recovered by DY in c-KIT-positive AML cells as well. Furthermore, the effect of radotinib on c-KIT and HSP90ß showed the same pattern in a xenograft animal model using HEL92.1.7 cells. Therefore, dasatinib and radotinib promote AML cell death by targeting c-KIT. Taken together, these results indicate that dasatinib and radotinib treatment have a potential role in anti-leukemic therapy on c-KIT-positive AML cells.
Assuntos
Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Dasatinibe/farmacologia , Sistemas de Liberação de Medicamentos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas Proto-Oncogênicas c-kit/análise , Pirazinas/farmacologia , Animais , Fator Apoptótico 1 Ativador de Proteases/genética , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Linhagem Celular Tumoral , Feminino , Proteínas de Choque Térmico HSP90/biossíntese , Proteínas de Choque Térmico HSP90/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The mitochondrial proteins TRAP1 and HTRA2 have previously been shown to be phosphorylated in the presence of the Parkinson's disease kinase PINK1 but the downstream signalling is unknown. HTRA2 and PINK1 loss of function causes parkinsonism in humans and animals. Here, we identified TRAP1 as an interactor of HTRA2 using an unbiased mass spectrometry approach. In our human cell models, TRAP1 overexpression is protective, rescuing HTRA2 and PINK1-associated mitochondrial dysfunction and suggesting that TRAP1 acts downstream of HTRA2 and PINK1. HTRA2 regulates TRAP1 protein levels, but TRAP1 is not a direct target of HTRA2 protease activity. Following genetic screening of Parkinson's disease patients and healthy controls, we also report the first TRAP1 mutation leading to complete loss of functional protein in a patient with late onset Parkinson's disease. Analysis of fibroblasts derived from the patient reveal that oxygen consumption, ATP output and reactive oxygen species are increased compared to healthy individuals. This is coupled with an increased pool of free NADH, increased mitochondrial biogenesis, triggering of the mitochondrial unfolded protein response, loss of mitochondrial membrane potential and sensitivity to mitochondrial removal and apoptosis. These data highlight the role of TRAP1 in the regulation of energy metabolism and mitochondrial quality control. Interestingly, the diabetes drug metformin reverses mutation-associated alterations on energy metabolism, mitochondrial biogenesis and restores mitochondrial membrane potential. In summary, our data show that TRAP1 acts downstream of PINK1 and HTRA2 for mitochondrial fine tuning, whereas TRAP1 loss of function leads to reduced control of energy metabolism, ultimately impacting mitochondrial membrane potential. These findings offer new insight into mitochondrial pathologies in Parkinson's disease and provide new prospects for targeted therapies.
Assuntos
Proteínas de Choque Térmico HSP90/genética , Metformina/uso terapêutico , Mitocôndrias/efeitos dos fármacos , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Trifosfato de Adenosina/metabolismo , Apoptose/efeitos dos fármacos , Estudos de Casos e Controles , Células Cultivadas , Fibroblastos/metabolismo , Proteínas de Choque Térmico HSP90/biossíntese , Serina Peptidase 2 de Requerimento de Alta Temperatura A , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mutação , NAD/metabolismo , Biogênese de Organelas , Consumo de Oxigênio , Doença de Parkinson/genética , Proteínas Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina Endopeptidases/metabolismoRESUMO
Based on the breast cancer cells and the vascular endothelial cells are both estrogen-sensitive, we proposed a close reciprocity existed between them in the tumor microenvironment, via shared molecular mechanism affected by environmental endocrine disruptors (EDCs). In this study, bisphenol A (BPA), via triggering G-protein estrogen receptor (GPER), stimulated cell proliferation and migration of bovine vascular endothelial cells (BVECs) and breast cancer cells (SkBr-3 and MDA-MB-231) and enhanced tumor growth in vivo. Moreover, the expression of both hypoxia inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) were up-regulated in a GPER-dependent manner by BPA treatment under hypoxic condition, and the activated GPER induced the HIF-1α expression by competitively binding to caveolin-1 (Cav-1) and facilitating the release of heat shock protein 90 (HSP90). These findings show that in a hypoxic microenvironment, BPA promotes HIF-1α and VEGF expressions through a shared GPER/Cav-1/HSP90 signaling cascade. Our observations provide a probable hypothesis that the effects of BPA on tumor development are copromoting relevant biological responses in both vascular endothelial and breast cancer cells.