OBJECTIVE: To investigate the effect of PPP2R5C to the activity of Molt-4 cells in childhood acute T lymphocytic leukemia and its mechanism. METHODS: The small interfering RNA (siRNA) technology targeting PPP2R5C gene was used to down-regulate the expression of PPP2R5C in Molt-4 cells. At the same time, a blank control group, a negative control group and a 17-DMAG group were set up. The cells in the negative control group were transfected with siRNA-NC, the cells in 17-DMAG group were treated with the HSP90 inhibitor 17-DMAG at a final concentration of 6.4 μmol/L for 48 h. Real-time fluorescent quantitative PCR (RT-qPCR) and Western blot were used to detect transfection efficiency; CCK-8 method was used to detect the proliferation activity of the cells in each group, EdU was used to detect the proliferation level of the cells in each group, flow cytometry was used to detect the cell cycle distribution ratio of the cells in each group, Annexin V-FITC/PI staining was used to detect the apoptosis of the cell, RT-qPCR and Western blot were used to detect the expression changes of heat shock protein 90 (HSP90) and glucocorticoid receptor (GR) of the cells in each group. RESULTS: After Molt-4 cells were transfected with siRNA-PPP2R5C, the expression of PPP2R5C mRNA and protein in the cells were down-regulated significantly compared with those in the blank control group and the si-NC group (P<0.05); compared with cells in the blank control group and the si-NC group, the proliferation activity of the cells in the siRNA-PPP2R5C group and the 17-DMAG group significantly decreased (P<0.05), and the rate of EdU positive cells was significantly reduced (P<0.05); the proportion of the cells in G1 phase decreased while the proportion of the cells in G₂ phase increased (P<0.05), the apoptosis rate of the cells also increased significantly (P<0.05); in addition, the expression of PPP2R5C mRNA and protein of the cells in siRNA-PPP2R5C group was significantly down-regulated compared with those in the blank control group and si-NC group (P<0.05). The expressions of PPP2R5C mRNA and protein in the 17-DMAG group were also significantly down-regulated compared with those in the blank control group and si-NC group (P<0.05). CONCLUSION Down-regulation of PPP2R5C gene expression can inhibit Molt-4 cell activity in childhood acute T lymphocytic leukemia, block the cells in G₂ phase, and promote cell apoptosis, the mechanism may be related to the inhibition of HSP90-GR signaling pathway.
Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; Child ; HSP90 Heat-Shock Proteins ; Humans ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; RNA, Small Interfering ; Receptors, Glucocorticoid

Objective: To investigate the effects and mechanism of negative pressure microenvironment on the neogenesis of human umbilical vein endothelial cells (HUVECs). Methods: The experimental research methods were adopted. The third to the fifth passage of HUVECs in the logarithmic growth stage were used for the subsequent experiments. Three batches of cells were taken, with each batch of cells being divided into normal control group and negative pressure treatment alone group (both routinely cultured for 24 h), and 17-allylamino-17-demethoxy-geldanamycin (17-AAG) alone group and 17-AAG+negative pressure treatment group (both cultured with 17-AAG for 24 h). In addition, the intermittent negative pressure suction, with the negative pressure value of -5.33 kPa (suction for 30 s, pause for 10 s) was continuously applied for 8 h on cells in the two negative pressure treatment groups using an automatic three-dimensional cell gradient negative pressure loading device designed and developed by ourselves. After the treatment of the first batch of cells, the cell proliferation level was detected by cell counting kit 8 method at 0 (immediately), 24, 48, and 72 h of culture, with the number of samples being 6. After the treatment of the second batch of cells, the scratch experiment was performed. At 12 h after scratching, the cell migration was observed under an inverted phase contrast microscope and the cell migration rate was calculated, with the number of samples being 3. After the treatment of the third batch of cells, the tubule formation experiment was conducted. After 6 h of culture, the tubulogenesis was observed under an inverted phase contrast microscope and the total tubule length and the number of branch nodes of cells were calculated, with the number of samples being 3. The cells were taken and divided into normal control group, negative pressure treatment alone group, and 17-AAG+negative pressure treatment group. The cells were treated the same as in the previous corresponding group. After the treatment, Western blotting was used to detect the protein expressions of heat shock protein 90 (HSP90), caveolin 1, endothelial nitric oxide synthase (eNOS), and eNOS phosphorylation site 1177 in the cells, and the eNOS phosphorylation site 1177/eNOS ratio was calculated, with the number of samples being 3; co-immunoprecipitation (co-precipitating HSP90 and caveolin 1, caveolin 1 and eNOS) and Western blotting were used to detect the protein expressions of caveolin 1 and eNOS in the cells, with the number of samples being 3; the protein co-localization of HSP90 and caveolin 1 and that of caveolin 1 and eNOS in the cells was assessed by immunofluorescence double staining. The molecular docking prediction of caveolin 1 and eNOS was processed by HADDOCK 2.4 protein-protein docking program. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, and least significant difference method. Results: Compared with that in normal control group, the cell proliferation level in 17-AAG alone group was significantly decreased at culture hour of 24, 48, and 72 after the treatment (P<0.01), while the cell proliferation level in negative pressure treatment alone group was significantly increased at culture hour of 24, 48, and 72 after the treatment (P<0.01). Compared with that in 17-AAG alone group, the cell proliferation level in 17-AAG+negative pressure treatment group was significantly increased at culture hour of 48 and 72 after the treatment (P<0.05 or P<0.01). Compared with that in negative pressure treatment alone group, the cell proliferation level in 17-AAG+negative pressure treatment group was significantly decreased at culture hour of 24, 48, and 72 after the treatment (P<0.01). At 12 h after scratching, compared with (39.9±2.7)% in normal control group, the cell migration rate in 17-AAG alone group was significantly decreased ((10.7±2.7)%, P<0.01), while the cell migration rate in negative pressure treatment alone group was significantly increased ((61.9±2.4)%, P<0.01). Compared with those in 17-AAG alone group, the cell migration rate in 17-AAG+negative pressure treatment group was significantly increased ((37.7±3.7)%, P<0.01). Compared with that in negative pressure treatment alone group, the cell migration rate in 17-AAG+negative pressure treatment group was significantly decreased (P<0.01). At culture hour of 6 after the treatment, compared with those in normal control group, the total length of the tube formed by the cells in 17-AAG alone group was significantly shortened (P<0.05) and the number of branch nodes was significantly reduced (P<0.05), while the total length of the tube formed by the cells in negative pressure treatment alone group was significantly prolonged (P<0.01) and the number of branch nodes was dramatically increased (P<0.01). Compared with that in 17-AAG alone group, the number of branch nodes of the tube formed by the cells was significantly increased in 17-AAG+negative pressure treatment group (P<0.05). Compared with those in negative pressure treatment alone group, the total length of the tube formed by the cells in 17-AAG+negative pressure treatment group was significantly shortened (P<0.01) and the number of branch nodes was significantly reduced (P<0.01). Western blotting detection showed that after treatment, the overall comparison of eNOS and caveolin 1 protein expressions among the three groups of cells showed no statistically significant differences (P>0.05). The expression of HSP90 protein and the eNOS phosphorylation site 1177/eNOS ratio in the cells of negative pressure treatment alone group were significantly increased (P<0.01) compared with those in normal control group. Compared with those in negative pressure treatment alone group, the HSP90 protein expression and the eNOS phosphorylation site 1177/eNOS ratio in the cells of 17-AAG+negative pressure treatment group were significantly decreased (P<0.01). Co-immunoprecipitation and Western blotting detection after the treatment showed that compared with those in normal control group, the expression of caveolin 1 protein in the cells of negative pressure treatment alone group was significantly increased (P<0.01), while the protein expression of eNOS was significantly decreased (P<0.05). Compared with those in negative pressure treatment alone group, the expression of caveolin 1 protein in the cells of 17-AAG+negative pressure treatment group was significantly decreased (P<0.01), while the protein expression of eNOS was significantly increased (P<0.01). After the treatment, compared with those in normal control group, the co-localization of HSP90 and caveolin 1 protein in the cells of negative pressure treatment alone group was significantly increased, while the co-localization of caveolin 1 and eNOS protein was significantly decreased. Compared with those in negative pressure treatment alone group, the co-localization of HSP90 and caveolin 1 protein in the cells of 17-AAG+negative pressure treatment group was significantly decreased, while the co-localization of caveolin 1 and eNOS protein was significantly increased. Molecular docking prediction suggested that caveolin 1 interacted strongly with eNOS and inhibited the 1177 site phosphorylation of eNOS. Conclusions: The negative pressure microenvironment may inhibit the binding of caveolin 1 to eNOS by promoting the binding of HSP90 to caveolin 1 in HUVECs, so as to relieve the inhibition of 1177 site phosphorylation of eNOS by caveolin 1, thereby promoting the proliferation, migration, and tubulogenesis of HUVECs, and ultimately promoting the neogenesis of HUVECs.
Caveolin 1/metabolism* ; Cells, Cultured ; HSP90 Heat-Shock Proteins/metabolism* ; Human Umbilical Vein Endothelial Cells/metabolism* ; Humans ; Molecular Docking Simulation ; Phosphorylation

OBJECTIVE: To investigate the expression of HSP90 in bone marrow samples of multiple myeloma (MM) patients and explore its clinical significance. METHODS: Maxvision immunohistochemistry technique was used to detect the protein expression level of HSP90 76 MM patients and 29 normal healthy donors. The clinical characteristics of the patients were collected, and the correlation between the HSP90 expression and the clinical characteristics was analyzed. RESULTS: The count of MM patients with positive HSP90 protein was significantly higher than that of normal healthy donor, and there were no significant correlation between HSP90 expression and age, sex, hemoglobin (Hb), creatinine (CREA), blood calcium, lactate dehydrogenase (LDH), bone marrow plasma cell proportion and MM subtypes (P>0.05), but HSP90 expression was correlated with β CONCLUSION HSP90 protein was over-expressed in MM patients, and was correlated with β
Bone Marrow ; HSP90 Heat-Shock Proteins ; Humans ; Multiple Myeloma ; Prognosis ; beta 2-Microglobulin

This study was aimed to investigate the regulatory mechanism of heat shock protein 90 (Hsp90) on transcription factor EB (TFEB) during autophagy in liver cancer cells. Human hepatocellular carcinoma cell line HepG2 was treated with Hsp90 N- and C-terminal inhibitors (STA9090 and Novobiocin), respectively. Western blot and RT-PCR were used to detect the expression levels of TFEB and autophagy-related proteins. Chromatin immunoprecipitation (ChIP) assay was used to observe the ability of Hsp90α binding to the TFEB proximal promoter region. The double-luciferase gene reporter experiment was used to determine the activity of TFEB promoter. The results showed that hypoxia induced up-regulation of TFEB protein and mRNA expression levels in the HepG2 cells. The protein expression levels of TFEB, LC3 and P62 were down-regulated significantly by either STA9090 or Novobiocin, under both normoxic and hypoxic conditions. Transfection of Hsp90α-overexpressing plasmids up-regulated TFEB protein levels in either wild-type or Hsp90α knockout HepG2 cells. Hsp90 bound to the TFEB proximal promoter region and was involved in regulating TFEB transcriptional process. Whereas both STA9090 and Novobiocin inhibited Hsp90 to bind to the TFEB proximal promoter region, and decreased the activity of TFEB promoter. These results suggest that Hsp90 promotes TFEB transcription in human hepatocellular carcinoma cells by binding to the proximal promoter region, thereby up-regulating the expression levels of autophagy-related proteins.
Autophagy ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ; metabolism ; Carcinoma, Hepatocellular ; metabolism ; pathology ; HSP90 Heat-Shock Proteins ; metabolism ; Hep G2 Cells ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Promoter Regions, Genetic

PURPOSE: The mechanisms underlying repolarization abnormalities during pregnancy are not fully understood. Although maternal serotonin (5-hydroxytryptamine, 5-HT) production is an important determinant for normal fetal development in mice, its role in mothers remains unclear. We evaluated the role of serotonin in ventricular repolarization in mice hearts via 5Htr3 receptor (Htr3a) and investigated the mechanism of QT-prolongation during pregnancy. MATERIALS AND METHODS: We measured current amplitudes and the expression levels of voltage-gated K⁺ (Kv) channels in freshly-isolated left ventricular myocytes from wild-type non-pregnant (WT-NP), late-pregnant (WT-LP), and non-pregnant Htr3a homozygous knockout mice (Htr3a(−/−)-NP). RESULTS: During pregnancy, serotonin and tryptophan hydroxylase 1, a rate-limiting enzyme for the synthesis of serotonin, were markedly increased in hearts and serum. Serotonin increased Kv current densities concomitant with the shortening of the QT interval in WT-NP mice, but not in WT-LP and Htr3a(−/−)-NP mice. Ondansetron, an Htr3 antagonist, decreased Kv currents in WT-LP mice, but not in WT-NP mice. Kv4.3 directly interacted with Htr3a, and this binding was facilitated by serotonin. Serotonin increased the trafficking of Kv4.3 channels to the cellular membrane in WT-NP. CONCLUSION: Serotonin increases repolarizing currents by augmenting Kv currents. Elevated serotonin levels during pregnancy counterbalance pregnancy-related QT prolongation by facilitating Htr3-mediated Kv currents.
*Action Potentials/drug effects ; Animals ; Cell Membrane/drug effects/metabolism ; Disease Models, Animal ; Electrocardiography ; Female ; HSC70 Heat-Shock Proteins/metabolism ; HSP90 Heat-Shock Proteins/metabolism ; Heart Ventricles/drug effects/*metabolism ; Mice, Inbred C57BL ; Mice, Knockout ; Myocytes, Cardiac/drug effects/metabolism ; Potassium Channels/metabolism ; Pregnancy ; Rabbits ; Rats, Sprague-Dawley ; Receptors, Serotonin, 5-HT3/metabolism ; Serotonin/*metabolism ; Serotonin 5-HT3 Receptor Agonists/pharmacology

To determine heat-shock protein (Hsp)90 expression is connected with cellular apoptotic response to heat stress and its mechanism, chicken (Gallus gallus) primary myocardial cells were treated with the Hsp90 promoter, aspirin, and its inhibitor, geldanamycin (GA), before heat stress. Cellular viability, heat-stressed apoptosis and reactive oxygen species level under different treatments were measured, and the expression of key proteins of the signaling pathway related to Hsp90 and their colocalization with Hsp90 were detected. The results showed that aspirin treatment increased the expression of protein kinase B (Akt), the signal transducer and activator of transcription (STAT)-3 and p-IKKα/β and the colocalization of Akt and STAT-3 with Hsp90 during heat stress, which was accompanied by improved viability and low apoptosis. GA significantly inhibited Akt expression and p-IKKα/β level, but not STAT-3 quantity, while the colocalization of Akt and STAT-3 with Hsp90 was weakened, followed by lower cell viability and higher apoptosis. Aspirin after GA treatment partially improved the stress response and apoptosis rate of tested cells caused by the recovery of Akt expression and colocalization, rather than the level of STAT-3 (including its co-localization with Hsp90) and p-IKKα/β. Therefore, Hsp90 expression has a positive effect on cellular capacity to resist heat-stressed injury and apoptosis. Moreover, inhibition of Hsp90 before stress partially attenuated its positive effects.
Apoptosis* ; Aspirin ; Cell Survival ; Chickens* ; Heat Stress Disorders ; Heat-Shock Proteins* ; Hot Temperature* ; HSP90 Heat-Shock Proteins ; In Vitro Techniques* ; Proto-Oncogene Proteins c-akt ; Reactive Oxygen Species ; Transducers

Protein palmitoylation, one of post-translation modifications, refers to the addition of saturated 16-carbon palmitic acid to cysteine residues via the thioester bond. It plays key roles in various functional activities, such as the interaction, stability and location of proteins. Heat shock protein 90 (Hsp90), an important molecular chaperone, has been reported to be involved in sperm capacitation. However, it remains unclear whether protein palmitoylation exists in sperm and whether Hsp90 in sperm is palmitoylated under different physiological conditions. In this study, we examined whether the protein palmitoylation is present in mouse cauda epididymis sperm using acyl-biotin exchange method, predicted the potential palmitoylated sites of Hsp90 by the software CSS-Palm 4.0 and detected the palmitoylated Hsp90 in the mouse sperm from caput epididymis and cauda epididymis by immunoprecipitation. We found that some proteins, approximately 50, 65, 72, 85 and 130 kDa, were palmitoylated in mouse cauda epididymis sperm. Five sites in two Hsp90 isoforms were predicted to be palmitoylated. The results also showed that Hsp90 in mouse sperm was palmitoylated and its palmitoylation level was involved in different physiological conditions: the palmitoylation level of cauda epididymis sperm was higher than that of caput epididymis sperm; and the palmitoylation level after capacitation was much higher than that before capacitation. In conclusion, this study reveals that protein palmitoylation is present in mouse sperm and the palmitoylated Hsp90 is associated with different physiological conditions in sperm.
Animals ; Epididymis ; HSP90 Heat-Shock Proteins ; metabolism ; Lipoylation ; Male ; Mice ; Palmitic Acid ; chemistry ; Sperm Capacitation ; Spermatozoa ; metabolism

The protective effect of aspirin during exposure to heat stress in broiler chickens was investigated. We assayed pathological damage, expression and distribution of Hsp90 protein and hsp90 mRNA expression in chicken heart tissues after oral administration of aspirin following exposure to high temperature for varying times. Heat stress induced increases in plasma aspartate aminotransferase, creatine kinase and lactate dehydrogenase activities while causing severe heart damage, which was characterized by granular and vacuolar degeneration, nuclear shrinkage and even myocardium fragmentation in cardiac muscle fibers. After aspirin administration, myocardial cells showed fewer pathological lesions than broilers treated with heat alone. A high positive Hsp90 signal was always detected in the nuclei of myocardial cells from broilers treated with aspirin, while in myocardial cells treated with heat alone, Hsp90 in the nuclei decreased, as did that in the cytoplasm. Aspirin induced rapid and significant synthesis of Hsp90 before and at the initial phase of heat stress, and significant expression of hsp90 mRNA was stimulated throughout the experiment when compared with cells exposed to heat stress alone. Thus, specific pre-induction of Hsp90 in cardiovascular tissue was useful for resisting heat stress damage because it produced stable damage-related enzymes and fewer pathologic changes.
Animals ; Anti-Inflammatory Agents, Non-Steroidal/pharmacology ; Aspirin/*pharmacology ; Cell Nucleus/genetics ; Chickens ; Gene Expression Regulation/*drug effects ; HSP90 Heat-Shock Proteins/*genetics ; Hot Temperature ; Myocytes, Cardiac/*drug effects/enzymology/pathology ; Stress, Physiological/*drug effects

OBJECTIVETo investigate the apoptotic effects of Hsp90 selective inhibitor 17-AAG on human leukemia HL-60 and NB4 cells and analyse its possible mechanism.

METHODSCCK-8 assay was used to quantify the growth inhibition of cells after exposure to 17-AAG for 24 hours. Flow cytometrve with annexin V/propidium iodide staining was used to detect apoptosis of leukemia cells. Then Western blot was used to detect the activation of apoptosis related protein caspase-3 and PARP level. Gene expression profile of NB4 cells treated with 17-AAG was analyzed with real-time PCR arrays.

RESULTSThe inhibition of leukemia cell proliferation displayed a dose-dependent manner. Annexin V assay, cell cycle analysis and activation of PARP demonstrate that 17-AAG induced apoptosis leukemia cells. Real-time PCR array analysis showed that expression of 56 genes significantly up-regulated and expression of 23 genes were significantly down-regulated after 17-AAG treatment.

CONCLUSIONThe 17-AAG can inhibit the proliferation and induce the apoptosis of leukemia cells. After leukemia cells are treated with 17-AAG, the significant changes of apoptosis-related genes occured, and the cell apoptosis occurs via activating apoptosis related signaling pathway.

Apoptosis ; Benzoquinones ; pharmacology ; Caspase 3 ; metabolism ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; HL-60 Cells ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; Humans ; Lactams, Macrocyclic ; pharmacology ; Leukemia ; metabolism ; Poly(ADP-ribose) Polymerases ; metabolism ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Transcriptome

UNLABELLEDObjective：To explore the influence of co-inhibiting mTORC2 and HSP90 on the proliferation and apoptosis of multiple myeloma(MM) cell line U266.

METHODSDuring culture, the human MM cell line U266 were treated with 20 nmol/L of rapamycin, 600 nmol/L 17-AAG, 20 nmol/L of rapamycin + 600 nmol/L 17-AGG and phosphate-buffered saline (PBS), then the growth inhibition rate, morphologic changes, apoptosis rate and the expression of caspase 3 and ATK protein in U266 cells were compared and analyzed.

RESULTSThe rapamycin and 17-AAG both could inhibit the growth of U266 cells, while the inhibitory effect of rapamycin in combination with 17-AAG on growth of U266 cells was significantly higher them that of rapamycin and 17-AAG alone and control (PBS); the apoptosis rate of U266 cells treated with rapamycin, 17-AAG and their combination was higher than that of control PBS groups, and the efficacy of 2 drug conbination was higher than that of control PBS group, and the efficacy of 2 drug combination was superior to single drug. The expression levels of caspase 3 and ATK in U266 cells treated with rapamycin, 17-AAG and their combination were higher and lower than those in control group respectively, and the efficacy of 2 drug combination was superior to signle drug. There were significant difference between them (P<0.05).

CONCLUSIONThe co-inhibition of mTORC2 and HSP90 can suppress the proliferation and induce the apoptosis of MM cells.

Apoptosis ; Benzoquinones ; Caspase 3 ; Cell Line, Tumor ; Cell Proliferation ; HSP90 Heat-Shock Proteins ; Humans ; Lactams, Macrocyclic ; Mechanistic Target of Rapamycin Complex 2 ; Multiple Myeloma ; Multiprotein Complexes ; Sirolimus ; TOR Serine-Threonine Kinases