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The heat shock protein 90 (Hsp90) protein family is a cluster of highly conserved molecules that play an important role in maintaining cellular homeostasis. Hsp90 and its co-chaperones regulate a variety of pathways and cellular functions, such as cell growth, cell cycle control and apoptosis. Hsp90 is closely associated with the occurrence and development of tumors and other diseases, making it an attractive target for cancer therapeutics. Inhibition of Hsp90 expression can affect multiple oncogenic pathways simultaneously. Most Hsp90 small molecule inhibitors are in clinical trials due to their low efficacy, toxicity or drug resistance, but they have obvious synergistic anti-tumor effect when used with histone deacetylase (HDAC) inhibitors, tubulin inhibitors or topoisomerase II (Topo II) inhibitors. To address this issue, the design of Hsp90 dual-target inhibitors can improve efficacy and reduce drug resistance, making it an effective tumor treatment strategy. In this paper, the domain and biological function of Hsp90 are briefly introduced, and the design, discovery and structure-activity relationship of Hsp90 dual inhibitors are discussed, in order to provide reference for the discovery of novel Hsp90 dual inhibitors and clinical drug research from the perspective of medicinal chemistry.
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Metastasis and resistance are main causes to affect the outcome of the current anticancer therapies. Heat shock protein 90 (Hsp90) as an ATP-dependent molecular chaperone takes important role in the tumor metastasis and resistance. Targeting Hsp90 and downregulating its expression show promising in inhibiting tumor metastasis and resistance. In this study, a redox-responsive dual-drug nanocarrier was constructed for the effective delivery of a commonly used chemotherapeutic drug PTX, and a COA-modified 4-arm PEG polymer (4PSC) was synthesized. COA, an active component in oleanolic acid that exerts strong antitumor activity by downregulating Hsp90 expression, was used as a structural and functional element to endow 4PSC with redox responsiveness and Hsp90 inhibitory activity. Our results showed that 4PSC/PTX nanomicelles efficiently delivered PTX and COA to tumor locations without inducing systemic toxicity. By blocking the Hsp90 signaling pathway, 4PSC significantly enhanced the antitumor effect of PTX, inhibiting tumor proliferation and invasiveness as well as chemotherapy-induced resistance in vitro. Remarkable results were further confirmed in vivo with two preclinical tumor models. These findings demonstrate that the COA-modified 4PSC drug delivery nanosystem provides a potential platform for enhancing the efficacy of chemotherapies.
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【Objective】 To investigate the effects of formononetin (FMN) on cardiomyocyte apoptosis and HSP90/AKT in rats with dilated cardiomyopathy-mediated heart failure. 【Methods】 Echocardiography, ELISA, histological staining, and TUNEL staining were used to observe the protective effect of different doses of FMN on dilated cardiomyopathy-mediated heart failure in rats and the apoptosis of cardiomyocytes. The potential targets of formononetin on dilated cardiomyopathy-mediated heart failure were obtained from TCMSP, DisGeNet, GeneCards, and other databases, the key targets were obtained according to the protein-protein interaction (PPI) network, and the key targets were verified by molecular docking. Western blotting was used to further verify the regulatory role of key targets in the treatment of dilated cardiomyopathy-mediated heart failure with formononetin. 【Results】 Formononetin could reduce the levels of LVIDS, LVIDD, NT-pro BNP, cTn-T, CK, CK-MB, and LDH in rats with dilated cardiomyopathy-mediated heart failure, increase the levels of EF and FS, and reduce the apoptosis of cardiomyocytes. FMN had a strong binding effect on 10 key targets (AKT1, HSP90AA1, CASP3, MAPK1, MMP9, SRC, ALB, HRAS, IGF1, and EGFR) screened by network pharmacology, with HSP90AA1 and AKT1 having the strongest binding effect. Formononetin decreased the expression of HSP90, AKT and downstream CASP3 protein, but increased the expression of p-AKT in myocardial tissue. 【Conclusion】 Formononetin may inhibit the expression of HSP90, promote phosphorylation of AKT to p-AKT, and inhibit the expression of CASP3, thereby reducing the apoptosis of cardiomyocytes and improving myocardial tissue damage, so as to achieve the purpose of treating dilated cardiomyopathy-mediated heart failure.
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Hepatitis B virus （HBV） has the characteristics of wide transmission， a high chronic infection rate， and a low cure rate， and improving the cure rate of HBV may help to improve the long－term prognosis of patients. Heat shock protein 90 （Hsp90） is a chaperone protein widely present in organisms. In recent years， more and more studies have shown that Hsp90 is associated with HBV infection and plays an important role in HBV replication. It can not only interact with specific proteins of the virus to promote its replication， but also interact with the host’s own proteins to perform its function. This article reviews the role of Hsp90 in HBV replication in recent studies， so as to provide new theoretical guidance and directions for the development of new anti－HBV drugs targeting Hsp90 and the prevention and treatment of HBV infection in the future.
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Background: Heat shock proteins (Hsps), expression of which are induced by thermal treatment, function in the protection of kidneys by suppressing apoptosis and maintaining renal tubular viability. Moreover, recently, it has been indicated that the expression of Hsps can be a therapeutic target for autosomal dominant polycystic kidney disease (ADPKD). We investigated the effect of dry sauna therapy on ADPKD model mice. Methods and Results: The mice (male DBA/2FG-pcy mice) were categorized into three groups: controls, TS: pcy mice subjected to prolonged sauna with administered water containing 4% sucrose, SW: pcy mice administered water containing 4% sucrose. The TS group was subjected to sauna sessions twice a week for four weeks. The TS group attained and were maintained at rectal temperatures of approximately 39.0°C, until they were carefully removed from the far infrared-ray device. After 4 weeks of sauna treatment, creatinine and blood-urea-nitrogen (BUN) levels determined by an enzymatic method. The heat shock protein (HSP) or cell growth and size related proteins were analyzed by western blotting. The TS group exhibited marginally higher creatinine and BUN levels than did the control and SW groups, however, the differences were not significant. However, cyst enlargement in the TS group reduced significantly compared to that of the control group. HSP90 expression was slightly decreased in the TS and SW groups relative to the control group (p < 0.01 or p < 0.001, vs. control), as was Erk expression, which is linked to cyst development and proliferation (p < 0.05, TS vs. control). Hsp27 expression and phosphorylation level in the SW group were comparable with that of the control group. However, the TS group had increased levels of Hsp27 and phosphorylation (NS). The expression of pro-caspase-3 in the TS group was marginally lower than that in the control group. However, the activity of caspase-3 in all groups showed no differences. Conclusion: The findings of this study indicated that 4 weeks of sauna treatment could cause transient dehydration and related renal dysfunction and led to the risk of stimulating cyst growth by increased Hsp27 expression. Moreover, we concluded that prevention of dehydration and cyst growth could be suppressed by taking an appropriate amount of water directly after sauna treatment.
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Objective:To investigate the potential molecular mechanisms of liver cancer cell-derived secretory autophagosomes, extracellular vesicles expressing LC3B (LC3B + EVs), in promoting the exhaustion of CD8 + T cells. Methods:The proportions of LC3B + EVs and PD-1 + CD8 + T cells in peripheral blood and ascites of liver cancer patients were measured by flow cytometry. Spearman correlation test was used to analyze the correlation between the proportions of LC3B + EVs and PD-1 + CD8 + T cells. Peripheral blood mononuclear cells (PBMCs) from healthy donors were treated with LC3B + EVs or heat shock protein 90α (HSP90α) blocking antibody-pretreated LC3B + EVs for 72 h in the presence of αCD3/CD28 antibodies and IL-2 in vitro. The proportions of PD-1 + CD8 + T and IFN-γ + CD8 + T cells and the concentrations of IL-2, TNF-α and IFN-γ in the supernatants were all detected by flow cytometry. Results:The proportions of LC3B + EVs and HSP90α + LC3B + EVs in plasma and ascites from liver cancer patients were significantly higher than those in healthy control group and non-cancerous ascites group. The level of plasma LC3B + EVs, especially HSP90α + LC3B + EVs, was significantly correlated with the percentage of exhausted PD-1 + CD8 + T cells. In addition, LC3B + EVs from human liver cancer cells up-regulated the percentage of exhausted CD8 + T cells in vitro. However, LC3B + EVs pretreated with HSP90α blocking antibody could significantly inhibit LC3B + EVs-induced CD8 + T cell exhaustion. Conclusions:Liver cancer cell-derived LC3B + EVs could effectively induce CD8 + T cell exhaustion mainly through the membrane-bound HSP90α.
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Objective To investigate the influence of preoperative serum heat shock protein 90α (HSP90α) level on the survival time of patients with hepatocellular carcinoma treated by transarterial chemoembolization (TACE). Methods A retrospective analysis was performed for the clinical data of 97 patients with hepatocellular carcinoma who received TACE alone in Department of Hepatobiliary Surgery, The Affiliated Hospital of Southwest Medical University, from January 1, 2019 to June 1, 2020. With the median of serum HSP90α level as the cut-off value, the patients were divided into high-level group with 48 patients (HSP90α > 135 ng/L) and low-level group with 49 patients (HSP90α ≤135 ng/L). The chi-square test was used for comparison of categorical data between groups. The Kaplan-Meier method was used to calculate the median survival time, and the log-rank test was used for comparison between groups. The log-rank univariate analysis and multivariate Cox regression analysis were used to explore the influencing factors for the survival time of patients after surgery. Results There were significant differences between the high-level group and the low-level group in Child-Pugh class ( χ 2 =19.356, P <0.01), tumor necrosis ( χ 2 =9.964, P =0.002), BCLC staging ( χ 2 =22.356, P <0.01), and ECOG score ( χ 2 =6.644, P <0.05). The high-level group had a significantly shorter median survival time than the low-level group ( χ 2 =15.551, P <0.01). HSP90α level (hazard ratio [ HR ]=1.690, P <0.05) and BCLC staging ( HR =2.373, P <0.05) were independent influencing factors for the survival time of patients with hepatocellular carcinoma after TACE. Conclusion Preoperative serum HSP90α level is an independent influencing factor for the survival time of patients with hepatocellular carcinoma after TACE, and it is expected to become one of the potential indicators for evaluating the prognosis of patients with hepatocellular carcinoma treated by TACE.
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Objective:To study the expressions of heat shock protein (HSP) 90α and HSP90β in colorectal cancer and paracancer tissues, and to investigate the relationships between HSP90α, HSP90β and clinicopathological features of colorectal cancer patients, and to analyze their correlation.Methods:The tumor tissues and paracancer tissues of 117 patients with colorectal cancer were selected from the Department of Gastrointestinal Surgery, Third Affiliated Hospital of Shandong First Medical University from January 2016 to December 2020. The expression levels of HSP90α and HSP90β were detected by immunohistochemistry, and the relationships between the two proteins and clinicopathological features and the correlation of their expressions were analyzed.Results:The positive expression rates of HSP90α in colorectal cancer tissues and paracancer tissues were 74.4% (87/117) and 12.0% (14/117) , and there was a statistically significant difference ( χ2=92.83, P<0.001) . The positive expression rate of HSP90β in colorectal cancer tissues and paracancer tissues was 61.5% (72/117) and 10.3% (12/117) , and there was a statistically significant difference ( χ2=66.86, P<0.001) . The expression of HSP90α was correlated with tumor location ( χ2=8.67, P=0.003) , vascular invasion ( χ2=8.68, P=0.003) , lymph node metastasis ( χ2=8.52, P=0.004) , T stage ( χ2=21.07, P<0.001) , N stage ( χ2=11.94, P=0.003) , M stage ( χ2=5.37, P=0.020) , pathological stage ( χ2=25.64, P<0.001) . The expression of HSP90β was correlated with lymph node metastasis ( χ2=4.03, P=0.045) , T stage ( χ2=11.09, P=0.007) , N stage ( χ2=6.56, P=0.038) , M stage ( χ2=12.43, P<0.001) , pathological stage ( χ2=17.34, P=0.001) . There was a positive correlation between the expressions of the two proteins in colorectal cancer tissues ( r=0.42, P<0.001) . Conclusion:The expressions of HSP90α and HSP90β in colorectal cancer tissues are significantly higher than those in paracancer tissues, and they are related to lymph node metastasis and pathological stage. There is a positive correlation between the two proteins, which may be involved in the occurrence and development of colorectal cancer and are expected to become new tumor markers.
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SUMMARY: Diabetes is a metabolic disorder characterized by high blood sugar levels and it causes complications in many systems, including the reproductive system. As a result of diabetic conditions, one of the mechanisms that can cause repression of reproductive activity is testicular oxidant stress. The identification of diabetes on the cell signaling molecules axis is still under discussion. The aim of this study was to determine the effect of Transforming Growth Factor (TGFβ), Nuclear Factor kappa B (NF-kB), Heat-schock 90β (HSP90β) signal pathways and E-cadherin cell adhesion molecule on infertility in diabetic rat testicular tissue. In our study, includes histological, molecular and biochemical analysis of testicular tissue removed at the end of the 2 weeks experiment period. A total of 14 adult male rats were divided as control and diabetes. No intervention was given to 7 male rats in the control group. For the diabetic group, 7 male rats were injected by intraperitoneal with a single dose of 55 mg/kg streptozotocin (STZ). TGFβ, NF-kB, HSP90β and E-cadherin proteins were immunohistochemically studied to investigate possible tissue damage, inflammatory process, cell stabilization and integrity due to diabetes. In order to determine oxidant stress, lipid peroxidation product malondialdehyde (MDA), glutathione (GSH) and glutathione peroxidase (GPx) analyzes were performed. Fibrosis, inflammatory changes and loss of spermatogenetic series are prominent findings in the diabetic group. On analysis of all the samples with immunostaining, in the diabetic group, TGFβ and NF-kB immunoexpression significantly increased, while Hsp90β and E-cadherin immunoexpression significantly decreased compared with control groups. Experimental diabetes was found to cause fibrosis, inflammation, disrupting cell adhesion and stabilization in testicular tissue. These results suggest that cellular therapy studies are needed for possible damage.
RESUMEN: La diabetes es una enfermedad metabólica caracterizada por niveles altos de azúcar en sangre y causa complicaciones en muchos sistemas, incluido el sistema reproductivo. Como resultado de las condiciones diabéticas, uno de los mecanismos que puede causar alteraciones en la actividad reproductiva es el estrés oxidativo testicular. La identificación de la diabetes en el eje de las moléculas de señalización celular aún está en discusión. El objetivo de este estudio fue determinar el efecto del factor de crecimiento transformante (TGFβ), el factor nuclear kappa B (NF-kB), las vías de señalización de Heat-Schock 90b (HSP90β) y la molécula de adhesión celular de E-cadherina sobre la infertilidad en testículo de rata diabética. Al término de dos semanas se realizaron análisis histológico, molecular y bioquímico del tejido testicular extraído. Las 7 ratas macho del grupo control no fueron intervenidas. Para el grupo de diabéticos, 7 ratas macho fueron inyectadas por vía intraperitoneal con una dosis única de 55 mg / kg de estreptozotocina (STZ). Se estudiaron inmunohistoquímicamente las proteínas TGFβ, NF-kB, HSP90β y E-cadherina para investigar el posible daño tisular, el proceso inflamatorio, la estabilización celular y la integridad debido a la diabetes. Para determinar el estrés oxidativo, se realizaron análisis del producto de peroxidación lipídica malondialdehído (MDA), glutatión (GSH) y glutatión peroxidasa (GPx). La fibrosis, los cambios inflamatorios y la pérdida de series espermatogenéticas son hallazgos destacados en el grupo de ratas diabéticas. En el análisis de todas las muestras con inmunotinción, en el grupo diabético, la inmunoexpresión de TGFβ y NF-kB aumentó significativamente, mientras que la inmunoexpresión de Hsp90β y e-cadherina disminuyó significativamente en comparación con los grupos control. Se encontró que la diabetes experimental causa fibrosis, inflamación, alteración de la adhesión celular y estabilización en el tejido testicular. Estos resultados sugieren que son necesarios estudios de terapia celular para verificar posibles daños.
Subject(s)
Animals , Male , Rats , Testis/pathology , Diabetes Mellitus, Experimental/metabolism , Testis/metabolism , Immunohistochemistry , Transforming Growth Factors/metabolism , Cadherins/metabolism , NF-kappa B/metabolism , HSP90 Heat-Shock Proteins/metabolismABSTRACT
Background: Heat shock proteins (Hsps), expression of which are induced by thermal treatment, function in the protection of kidneys by suppressing apoptosis and maintaining renal tubular viability. Moreover, recently, it has been indicated that the expression of Hsps can be a therapeutic target for autosomal dominant polycystic kidney disease (ADPKD). We investigated the effect of dry sauna therapy on ADPKD model mice. Methods and Results: The mice (male DBA/2FG-pcy mice) were categorized into three groups: controls, TS: pcy mice subjected to prolonged sauna with administered water containing 4% sucrose, SW: pcy mice administered water containing 4% sucrose. The TS group was subjected to sauna sessions twice a week for four weeks. The TS group attained and were maintained at rectal temperatures of approximately 39.0°C, until they were carefully removed from the far infrared-ray device. After 4 weeks of sauna treatment, creatinine and blood-urea-nitrogen (BUN) levels determined by an enzymatic method. The heat shock protein (HSP) or cell growth and size related proteins were analyzed by western blotting. The TS group exhibited marginally higher creatinine and BUN levels than did the control and SW groups, however, the differences were not significant. However, cyst enlargement in the TS group reduced significantly compared to that of the control group. HSP90 expression was slightly decreased in the TS and SW groups relative to the control group (p < 0.01 or p < 0.001, vs. control), as was Erk expression, which is linked to cyst development and proliferation (p < 0.05, TS vs. control). Hsp27 expression and phosphorylation level in the SW group were comparable with that of the control group. However, the TS group had increased levels of Hsp27 and phosphorylation (NS). The expression of pro-caspase-3 in the TS group was marginally lower than that in the control group. However, the activity of caspase-3 in all groups showed no differences. Conclusion: The findings of this study indicated that 4 weeks of sauna treatment could cause transient dehydration and related renal dysfunction and led to the risk of stimulating cyst growth by increased Hsp27 expression. Moreover, we concluded that prevention of dehydration and cyst growth could be suppressed by taking an appropriate amount of water directly after sauna treatment.
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The 90-kiloDalton (kD) heat shock protein (Hsp90) is a ubiquitous, ATP-dependent molecular chaperone whose primary function is to ensure the proper folding of several hundred client protein substrates. Because many of these clients are overexpressed or become mutated during cancer progression, Hsp90 inhibition has been pursued as a potential strategy for cancer as one can target multiple oncoproteins and signaling pathways simultaneously. The first discovered Hsp90 inhibitors, geldanamycin and radicicol, function by competitively binding to Hsp90's N-terminal binding site and inhibiting its ATPase activity. However, most of these N-terminal inhibitors exhibited detrimental activities during clinical evaluation due to induction of the pro-survival heat shock response as well as poor selectivity amongst the four isoforms. Consequently, alternative approaches to Hsp90 inhibition have been pursued and include C-terminal inhibition, isoform-selective inhibition, and the disruption of Hsp90 protein-protein interactions. Since the Hsp90 protein folding cycle requires the assembly of Hsp90 into a large heteroprotein complex, along with various co-chaperones and immunophilins, the development of small molecules that prevent assembly of the complex offers an alternative method of Hsp90 inhibition.
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Abstract Purpose To investigate whether heat shock protein 90 (HSP90) is involved in complement regulation in ischemic postconditioning (IPC). Methods The left coronary artery of rats underwent 30 min of occlusion, followed by 120 min of reperfusion and treatment with IPC via 3 cycles of 30s reperfusion and 30s occlusion. The rats were injected intraperitoneally with 1 mg/kg HSP90 inhibitor geldanamycin (GA) after anesthesia. Eighty rats were randomly divided into four groups: sham, ischemia-reperfusion (I/R), IPC and IPC + GA. Myocardial infarct size, apoptosis index and the expression of HSP90, C3, C5a, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1β and c-Jun N-terminal kinase (JNK) were assessed. Results Compared with the I/R injury, the IPC treatment significantly reduced infarct size, release of troponin T, creatine kinase-MB, and lactate dehydrogenase, and cardiomyocyte apoptosis. These beneficial effects were accompanied by a decrease in TNF-α, IL-1β, C3, C5a and JNK expression levels. However, all these effects were abrogated by administration of the HSP90 inhibitor GA. Conclusion HSP90 exerts a profound effect on IPC cardioprotection, and may be linked to the inhibition of the complement system and JNK, ultimately attenuating I/R-induced myocardial injury and apoptosis.
Subject(s)
Animals , Rats , Complement System Proteins/metabolism , Myocardial Reperfusion Injury/metabolism , Benzoquinones/pharmacology , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Lactams, Macrocyclic/pharmacology , JNK Mitogen-Activated Protein Kinases/metabolism , Myocardial Infarction/metabolism , RNA, Messenger/metabolism , Random Allocation , Tumor Necrosis Factor-alpha/metabolism , Rats, Sprague-Dawley , Inflammation Mediators , Creatine Kinase, MB Form/metabolism , Ischemic Postconditioning/methodsABSTRACT
This paper was to investigate the effect of total flavonoids of Lichi Semen(TFL) on carbon tetrachloride(CCl_4)-induced liver fibrosis in rats, analyze and predict its mechanism of action and potential quality markers(Q-marker). Firstly, male SD rats were taken and injected subcutaneously with a 40% CCl_4-vegetable oil solution twice a week for 8 consecutive weeks to establish a rat model of liver fibrosis. The rats with liver fibrosis were randomly divided into model group, silybin group(43.19 mg·kg~(-1)), Fuzheng Huayu Capsules group(462.75 mg·kg~(-1)), and TFL groups(100 mg·kg~(-1) and 25 mg·kg~(-1)), with normal rats as a blank group, 10 rats in each group. Except for the blank group, the rats in the other groups were subcutaneously injected with 40% CCl_4-vegetable oil solution of a maintenance dose, once a week. The rats in various treatment groups received corresponding doses of drugs, while the rats in the blank group and model group received the same volume of normal saline once a day for 4 weeks. At the end of the experiment, blood was collected from the abdominal aorta and the liver tissues were collected. The levels of total bilirubin(TBiL), direct bilirubin(DBiL), indirect bilirubin(IBiL), alanine aminotransferase(ALT), and aspartate aminotransferase(AST) in serum were detected by using an automatic biochemical detector. Masson staining was used to observe the histopathological changes of rat liver. Then, the chemical compositions of TFL were collected, and the action targets of these chemical compositions were predicted through SWISS database and reverse molecular docking server(DRAR-CPI). After screening of disease targets of liver fibrosis by Gene Cards database, the protein-protein interaction was analyzed with use of STRING database, and GO(gene ontology) analysis and KEGG(Kyoto encyclopedia of genes and genomes) enrich analysis were also carried out. Moreover, an iTRAQ proteomics technology was used to determine protein expression in liver tissues of rats in TFL, model and blank groups to verify the targets. Furthermore, Cytoscape software was used to establish and visualize the network of chemical components, targets and pathways, and predict the potential Q-marker of TFL. The results showed that the levels of TBiL, DBiL, IBiL, ALT, and AST in the model group were significantly higher than those in the blank normal group(P<0.05), and the above levels in the treatment groups were lower than those in the model group, but with no significant differences. Masson staining showed that the liver damage and the degree of fibrosis were severe in the model group, and were relieved to different degrees in the treatment groups. Then, 74 chemical components were screened, which could act on 865 targets such as EGFR and SRC, participating in the regulation of cancer pathways, PI3 K-Akt signaling pathway, HIF-1 signaling pathway and other signaling pathways closely related to liver fibrosis. Pinocembrin, quercetin, epicatechin, procyanidin A2, naringenin, nobiletin, phlorizin and rutin showed the highest correlation with liver fibrosis-related targets and pathways. Proteomics results showed that a total of 18 proteins among the 45 proteins predicted by internet pharmacology were identified, among which 6 proteins were significantly expressed, including 5 up-regulated proteins and 1 down-regulated protein. The protein expression of ALB, PLG, HSP90 AA1, EGFR and MAP2 K1 was significantly returned to a normal state in the TFL treatment groups. In conclusion, TFL may demonstrate the anti-hepatic fibrosis and potential hepatoprotective effects by regulating the expression of ALB, PLG, HSP90 AA1, EGFR and MAP2 K1, which may be associated with the regulation of multiple signaling pathways related to liver fibrosis such as PI3 K-Akt pathway. Pinocembrin, quercetin, epicatechin, procyanidin A2, naringenin, nobiletin, phlorizin and rutin could be regarded as potential Q-markers of TFL for quality control.
Subject(s)
Animals , Male , Rats , Carbon Tetrachloride , Flavonoids , Liver/pathology , Liver Cirrhosis , Molecular Docking Simulation , Rats, Sprague-Dawley , SemenABSTRACT
A redução da reatividade vascular à fenilefrina (PE) em aorta de ratas espontaneamente hipertensas (SHR) ao final da prenhez é dependente de maior produção e/ou maior biodisponibilidade de óxido nítrico (NO), consequente do aumento da fosforilação da enzima óxido nítrico sintase endotelial (eNOS) via PI3K/Akt. A glicosilação do tipo N-acetil-glucosamina (O-GlcNAc) é uma modificação pós-traducional que compete com a fosforilação pelos mesmos sítios de ligação nas proteínas. A O-GlcNAcilação da eNOS em serina1177 leva a redução da sua atividade enquanto a fosforilação leva a sua ativação. Além destes mecanismos, a interação da eNOS com outras proteínas é capaz de regular positiva ou negativamente a sua atividade. O objetivo deste trabalho foi analisar possíveis alterações nos mecanismos de modificação pós-traducional que controlam a ativação da eNOS os quais poderiam contribuir para maior ativação e maior biodisponibilidade de NO observada em artérias de ratas prenhes. Foram avaliados o conteúdo proteico O-GlcNAc e também expressão das enzimas que participam desta modificação, O-GlcNAc transferase (OGT) e O-GlcNAcase (OGA) por Western Blotting e a atividade da OGA por ensaio bioquímico em aorta e em artéria mesentérica (2º ou 3º ramo) de ratas não prenhes (NP) e prenhes (P), normotensas (Wistar) e SHR. Ensaios de Western Blotting foram realizados também para análise da expressão das seguintes proteínas: Cav-1, p-Cav-1, CaM e Hsp90. Realizamos a contagem do número de cavéolas endoteliais da aorta e da artéria mesentérica na presença ou ausência da metil-ß-ciclodextrina (dextrina, 10 mmol/L) por microscopia eletrônica. Em estudos funcionais, avaliamos a participação da enzima OGA, pela inibição com PugNAc (100 µmol/L) e das cavéolas, utilizando um desorganizador de cavéolas, a dextrina (10 ou 20 mmol/L), na menor reatividade vascular à PE observada em aortas de ratas P. Observamos que o conteúdo de proteínas O-GlcNAciladas estava diminuído em aorta e em leito mesentérico de ratas Wistar P e SHR P. Apesar da expressão da OGT e da OGA não estar alterada, a atividade da OGA foi aumentada em aorta e leito mesentérico de ratas Wistar P, mas, encontra-se diminuída em aorta e aumentada em leito mesentérico de SHP P. A incubação com PugNAc reverteu a reduzida reatividade à PE em aorta e artéria mesentérica de ratas Wistar P mas este efeito não foi observado em vasos SHR P, demonstrando que a OGA parece ter um papel importante na redução da O-GlcNAcilação de proteínas vasculares em Wistar P. Em vasos incubados com PugNAc, a remoção do endotélio ou a incubação com L-NAME, não alterou significativamente a reatividade à PE. Juntos estes resultados sugerem que a maior atividade da eNOS observada em vasos de Wistar P, fica prejudicada na presença do PugNAc, e depende da atividade da OGA. Como não houve alteração da resposta contrátil à PE em vasos de SHR P incubados com PugNAc, possivelmente um mecanismo diferente, envolvendo a menor atividade da OGT, ocorre nestas artérias para a redução da O-GlcNAcilação da eNOS. A desorganização das cavéolas por meio da dextrina causou aumento de contração à PE e redução de potência da ACh em aortas de Wistar NP e SHR NP, porém não houve alteração em aortas de ratas Wistar P e SHR P. A dextrina não alterou o número de cavéolas em artérias de Wistar P e SHR P quando comparado com ratas NP. SHR NP apresentam um reduzido número de cavéolas das aortas em relação a Wistar NP bem como expressão reduzida de Cav-1, p-Cav-1 e CaM. A prenhez não foi capaz de alterar a expressão da Cav-1, CaM e Hsp90 em aorta e leito mesentérico de ratas normotensas e hipertensas. Estes resultados sugerem que a prenhez não altera a expressão das proteínas Cav-1, CaM e Hsp90 e possivelmente a interação com a eNOS em aorta e artérias mesentéricas de ratas normotensas e hipertensas. Em conclusão, entre os mecanismos estudados de modificação pós-traducional da eNOS, a redução da O-GlcNAcilação da eNOS, por mecanismos que envolvem a atividade da OGA e possivelmente da OGT, favoreceria a fosforilação da eNOS e consequente maior biodisponibilidade de NO, contribuindo desta forma para modulação da resposta contrátil da PE nas artérias de ratas P(AU)
Reduction of vascular reactivity to phenylephrine (PE) in aorta of spontaneously hypertensive rats (SHR) at the end of pregnancy is dependent on higher production and/or higer bioavailability of nitric oxide (NO), as a consequence of increased endothelial nitric oxide synthase enzyme (eNOS) phosphorylation, by PI3K/Akt. Glycosylation with O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification that competes with phosphorylation by the same binding sites in proteins. O-GlcNAcylation of eNOS on serine site leads to a reduction in its activity while eNOS phosphorylation leads to its activation. In addition to these mechanisms, the interaction of eNOS with other proteins is able to regulate positively or negatively its activity. The objective of this study was to analyze possible changes in the mechanisms of post-translational modification that control the eNOS activation, which could contribute to its the greater activation and greater bioavailability of NO observed in arteries of pregnant rats. The O-GlcNAc-protein content and also the enzymes expression that participate in this modification, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) was assessed by Western Blotting, and OGA activity were evaluated by biochemical assay in the aorta and in the artery mesenteric (2nd or 3rd branch) of non-pregnant (NP) and pregnant (P), normotensive rats (Wistar) and SHR. Western Blotting assays were also performed for expression analysis of the following proteins: Cav-1, p-Cav-1, CaM and Hsp90. We performed the counting of the number of endothelial caveolae in the aorta and the mesenteric artery in the presence or absence of methyl-ß-cyclodextrin (dextrin, 10 mmol/L) by electronic microscopy. In functional studies, we evaluated the participation of the OGA enzyme, by inhibition with PugNAc (100 µmol/L) and of the caveolae, using a caveolae disassembler, dextrin (10 or 20 mmol/L), in the reduced vascular reactivity observed in aortas or mesenteric arteries of P rats. We observed that the content of O-GlcNAcylated proteins was decreased in the aorta and in the mesenteric bed of Wistar P and SHR P rats. Although OGT and OGA expression is not altered, OGA activity was increased in the aorta and mesenteric bed of Wistar P rats but was decreased in the aorta and increased in the mesenteric bed of SHP P. Incubation with PugNAc reversed the reduced reactivity to PE in the aorta and mesenteric artery of Wistar P but this effect was not observed in SHR P arteries, demonstrating that OGA appears to play an important role in reducing O-GlcNAcylation of vascular proteins in Wistar P. In arteries incubated with PugNAc, endothelial removal or incubation with L-NAME did not significantly alter reactivity to PE. Together, these results suggest that the greater eNOS activity observed in Wistar P vessels was impaired in the presence of PugNAc, and it depends on OGA activity. As there was no change in the contractile response to PE in SHR P arteries incubated with PugNAc, possibly a different mechanism, involving the lower activity of OGT, occurs in these vessels for the reduction of O-GlcNAcylation of eNOS. Dextrin caused increased contraction of PE and decreased ACh potency in Wistar NP and SHR NP aortas, but there was no change in aortas of Wistar P and SHR P. Dextrin did not alter the number of cavelae in Wistar P and SHR P arteries compared to NP rats. SHR NP showed a lower number of caveolae than to NP Wistar as well reduced expression of Cav-1 and CaM. Pregnancy was not able to alter the expression of Cav-1, CaM and Hsp90 in the aorta and mesenteric bed of normotensive and hypertensive rats. These results suggest that pregnancy does not alter the expression of Cav-1, CaM and Hsp90 proteins and possibly interaction with eNOS in the aorta and mesenteric arteries of normotensive and hypertensive rats. In conclusion, among the studied mechanisms of post-translational modification of eNOS, the reduction of O-GlcNAcylation of eNOS, by mechanisms that involve OGA activity and possibly OGT, would favor eNOS phosphorylation and consequent greater NO bioavailability, contributing in this way for modulation of the contractile response to PE in the arteries of P rats(AU)
Subject(s)
Animals , Female , Pregnancy , Aorta , Nitric Oxide Synthase , Hypertension , Glycosylation , Calmodulin , Rats, Wistar , HSP90 Heat-Shock Proteins , Caveolin 1 , Mesenteric ArteriesABSTRACT
Objective The objectives of this study were to screen and identify monoclonal antibodies against hepatoma stem cells by screening for hepatoma spheroid cells,and to provide candidate therapeutic monoclonal antibodies for targeting cancer stem cells to treat hepatic cancer. Methods Hepatic cancer stem cells were enriched by serum-free suspension culture. Immunofluores-cence,cisplatin resistance assay, Real -time qPCR, subcutaneous tumor formation in nude mice, and other methods were used to screen and identify anti-hepatocarcinoma stem cell monoclonal antibodies. Immunohistochemistry was used to identify the expression of antigen recognized by monoclonal antibody in liver cancer tissues. The antigen was identified by mass spectrometry. Results MH-CC97L cells were able to form cell spheres in serum -free suspension culture and were labeled with PKH26 dye. Flow cytometry showed that the expression of CD90 + in MHCC97L spheroid cells was 3. 4 times higher than that in the parental cells. In the inhibition experiment of serum-free spheroid,6 monoclonal antibodies significantly inhibited MHCC97L cells in serum-free medium,and in-hibitory rates were 54. 67% ,50. 33% ,45. 73% ,42. 26% ,39. 11% ,and 37. 63% ,respectively. The results of immunofluorescence showed that monoclonal antibodies 28C10 and CD90 were colocalized in MHCC97L cells. The results of real-time qPCR showed that the expression of Sox-2 and Oct-4 in MHCC97L 28C10 + cells was significantly higher than those of MHCC97L 28C10 - cells. Flow cytometry showed that the ratio of 28C10 + in MHCC97L cells and its sphere cells were 7. 98% and 10. 7% ,respectively. The ratio of 28C10 + cells was increased by 1. 34 times. The in vitro globing ability and invasive ability of 28C10 + cells obtained by flow cytometry were significantly higher than those of 28C10 - cells. The results of CCK-8 assay showed that 28C10 + cells were resistance to cispla-tin in 28C10 - cells,which are 1. 96 g/ml and 1. 16 g/ml,respectively. Tumorigenic assay showed that 28C10 + cells were inoculated subcutaneously with 2×104 cells into the nude mice,and tumors were formed in 2 months,with 40% of tumor formation rate. Another nude mouse that did not form a tumor had formed a lung metastasis(1/5). Immunohistochemistry showed that the target antigen posi-tive rate of monoclonal antibody 28C10 in hepatic cancer tissues was about 72. 0% (77/107),while it was lowly expressed in adjacent tissues,and the difference was significant. Mass spectrometry showed that the antigen recognized by 28C10 was HSP90α. Conclusion The MHCC97L spheroid cell model is successfully used to identify a monoclonal antibody that specifically recognizes hepatoma stem cells,which provides a foundation for antibody therapy targeting hepatic cancer stem cells.
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HSP90 is a molecular chaperone that increases the stability of client proteins. Cancer cells show higher HSP90 expression than normal cells because many client proteins play an important role in the growth and survival of cancer cells. HSP90 inhibitors mainly bind to the ATP binding site of HSP90 and inhibit HSP90 activity, and these inhibitors can be distinguished as ansamycin and non-ansamycin depending on the structure. In addition, the histone deacetylase inhibitors inhibit the activity of HSP90 through acetylation of HSP90. These HSP90 inhibitors have undergone or are undergoing clinical trials for the treatment of cancer. On the other hand, recent studies have reported that various reagents induce cleavage of HSP90, resulting in reduced HSP90 client proteins and growth suppression in cancer cells. Cleavage of HSP90 can be divided into enzymatic cleavage and non-enzymatic cleavage. Therefore, reagents inducing cleavage of HSP90 can be classified as another class of HSP90 inhibitors. We discuss that the cleavage of HSP90 can be another mechanism in the cancer treatment by HSP90 inhibition.
Subject(s)
Acetylation , Adenosine Triphosphate , Binding Sites , Drug Therapy , Hand , Heat-Shock Proteins , Histone Deacetylase Inhibitors , Hot Temperature , Indicators and Reagents , Molecular Chaperones , RifabutinABSTRACT
Objective To explore the specific mechanism of ginsenoside Rh2(S) inducing the apoptosis of leukemia cells. Methods The effect of Rh2(S) on proliferation of leukemia cells K562 and KG1a was measured by cell counting kit-8 assay (CCK-8 assay). The growth states of cells were observed under the inverted phase microscope, and cell cycle distribution, and apoptosis were determined by flow cytometry (FCM). The expression levels of HDAC6, HSP90, Akt, GSK-3β, β-catenin, and cell cycle apoptosis related proteins were ascertained by Western blotting. Results The results of CCK-8 showed that Rh2(S) had the most obvious inhibitory effect on the proliferation of leukemia cells. Rh2(S) significantly induced apoptosis and led to cell cycle arrest at G0/G1 phase of leukemia cells by FCM. While the microscope observation showed that the number of cells was decreased and normal cell morphology changed. Rh2(S) decreased the expression of Bcl-2, Cyclin D1, HDAC6, HSP90, p-Akt, and β-catenin and increased the expression of Bax, Ac-α-tubulin, and GSK-3β by Western blotting. Conclusion Rh2(S) can effectively inhibit the proliferation and promote the apoptosis of K562 and KG1a cells, its specific mechanism may relate to inhibitting the expression of HDAC6, resulting in a decline in the expression of HSP90, so as to further inhibit the activity of Akt activation of GSK-3, and finally inhibit Wnt/β-catenin pathway.
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Aim To estimate the inhibition of curcumin derivative C1209 on the proliferation of chronic myeloid leukemia (CML) cells involving the disruption Hsp90 chaperon function. Methods The fluorescence spec-trum experiment was applied to examine the affinity be-tween different C1209 concentrations and Hsp90, NH-sp90,MHsp90,CHsp90;fluorescence intensities were recorded in the range of 290~510 nm at 293 K,303 K and 310 K,respectively;a colorimetric assay for in-organic phosphate based on the formation of a phospho-molybdate complex and subsequent reaction with mala-chite green was used to examine the inhibitory effects of C1209 on the activity of Hsp90 ATPase. MTT assay and CFSE were used for K562 and K562/G01cell pro-liferation determination in vitro by C1209. Western blot was used to detect the client proteins and the mo-lecular chaperone of Hsp90 level. Results The disso-ciation constant KDvalues of C1209 was (14.733 ± 0.713) μmol·L-1. The interaction between C1209 and Hsp90 was driven mainly by electrostatic interac-tion. C1209 showed the strongest affinity with CHsp90. When the concentration of ATP was 1mmol· L-1,the inhibition of Hsp90 ATPase activity of C1209 with the IC50value was 11.4 μmol·L-1; C1209 showed inhibition of K562 and K562/G01cells in dose-dependent proliferation and the IC50value was 1.14 μmol·L-1and 0.56 μmol·L-1, respectively after 24 h incubation. C1209 affected the molecular chaper-one functions of Hsp90 and down-regulated Bcr-Abl, Akt, MEK, ERK, c-Raf, p-Akt, p-MEK and p-ERK protein levels which were client proteins of Hsp90 in K562 and K562/G01cells. Conclusions Curcumin derivative C1209 is Hsp90 inhibitor;C1209 has signif-icant inhibitory effects on proliferation of K562 and K562 / G01, which may be related to C1209 affecting the molecular chaperone functions of Hsp90 and down-regulating client proteins of Hsp90 level.
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Objective To explore the expression and meaning of heat shock protein 90 (Hsp90) in pancreatic cancer and its correlation with the occurrence,development and metastasis of the tumor tissues and its influence on prognosis. Methods Via immunohistochemical staining, this study used the commercial tissue chip to determine the expression of Hsp90 in 63 pancreatic cancer resection specimens and their para-cancerous counterparts, which were pathologically confirmed from January 2009 to August 2013. We also analyzed the correlation with patient's clinical parameters by using one-way ANOVA or t test. Results Hsp90 expression in pancreatic cancer tissues was significantly higher than tissue adjacent to carcinoma, and it was associated with histological grade, lymph node metastasis,clinical and TNM phase of pancreatic cancer (P<0.05). Conclusions Hsp90 is highly expressed in pancreatic cancer tissues,and its expression is associated with invasion and metastasis of pancreatic cancer. There-fore,Hsp90 may serves as a prognostic indicator of pancreatic cancer while its high expression may represent poor prognosis of pancreatic cancer patients.
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To explore the prognostic value of heat shock protein-90α (HSP-90α) plasma levels on breast cancer and non-breast malignant tumors, monitoring the response of chemotherapy, and the predictive value of cancer recurrence and metastasis. Methods: A total of 615 female patients were enrolled between June 2016 and September 2016 in Cancer Hospital, Chinese Academy of Medical Sciences, who were divided into the examination (n=389) and control (n=216) groups. The former group consisted of static (n=289) and dynamic (n=110) groups, which were analyzed by stages, histological and molecular type, and so on. The latter group in-cluded healthy people (n=103), and those with breast benign tumors (n=51) and non-breast malignant tumors (n=62). In all the plasma samples, HSP-90α was detected using a double-antibody enzyme-linked immunosorbent assay. The receiving-operating characteristic curve was used to analyze the effectiveness of plasma HSP-90α in the diagnosis of breast cancer. Wilcoxon＇s rank test and the Kruskal-Wallis test were used to analyze the association between clinical characteristics and levels of plasma HSP-90α. Results: The levels of plasma HSP-90α were significantly higher in patients with breast cancer than in healthy controls (P<0.001). When the cut-off value was set as 59.7 ng/mL for the diagnosis of breast cancer and 43.22 ng/mL for disease recurrence, the areas under the curve were 0.834 and 0.877, sensitivities were 90.3% and 95.7%, and specificities were 78.6% and 74.5%, respectively. The levels of plasma HSP-90α sig-nificantly decreased after achieving a response to neoadjuvant chemotherapy or surgery (P<0.05). Conclusions: Plasma HSP-90α has good clinical value in the diagnosis and monitoring of response and recurrence in breast cancer.