Objective:To investigate the effect of phosphorylated HSP27 on the proliferation and metastasis of nasopharyngeal carcinoma and its molecular mechanism. Methods:①Western blot assay was used to detect the expression levels of HSP27 and p-HSP27 in CNE1 and CNE2 cells of nasopharyngeal carcinoma. Inhibited the phosphorylation of HSP27, Transwell assay detected the metastasis ability of nasopharyngeal carcinoma cells. ②Nasopharyngeal carcinoma cell lines（CNE2-OE1 and CNE2-OE2） overexpressing phosphorylated and dephosphorylated mutants of HSP27 were synthesized, empty vector transfected CNE2 cells（CNE2-NC） were used as controls. The proliferation ability of the three groups of cells was detected by CCK8, and the expression levels of CyclinD1, Bax and Bcl-2 were detected by Western blot. Transwell was used to detect the migration and invasion ability of cells, and Western blot was used to detect the expression levels of E-cadherin, Vimentin, MMP2 and MMP9. Results:The expression level of HSP27 in CNE2 was higher than that of CNE1 cells, while the expression level of p-HSP27 was opposite. After inhibition of HSP27 phosphorylation, the invasion and migration ability of CNE1 cells decreased significantly, with no significant change in CNE2 cells. Compared with CNE2-NC, the growth rate of CNE2-OE1 decreased, and the growth rate of CNE2-OE2 increased. The expression level of CyclinD1 was down-regulated in CNE2-OE1 and higher in CNE2-OE2. The expression level of Bax in CNE2-OE1 was increased, while the expression of Bcl-2 was decreased. There was no significant change in the expression of Bax and Bcl-2 in CNE2-OE2. Compared with CNE2-NC, the migration ability of CNE2-OE1 was enhanced and the invasion ability was weakened, while the migration ability of CNE2-OE2 was weakened and the invasion ability was enhanced. There was no significant difference in the expression levels of E-cadherin was decreased in CNE2-OE1 and increased in CNE2-OE2. There was no significant difference in the expression levels of Vimentin among the three groups. The expression levels of MMP2 and MMP9 were up-regulated in CNE2-OE2, and slightly down-regulated in CNE2-OE1. Conclusion:HSP27 and p-HSP27 were differentially expressed in different nasopharyngeal carcinoma cells, and the metastasis ability of nasopharyngeal carcinoma was not only related to the expression level of HSP27, but also related to the level of p-HSP27. The p-HSP27 inhibited CNE 2 cell proliferation and promoted their apoptosis. As p-HSP27 plays different roles in different stages of nasopharyngeal carcinoma metastasis, it can increase the migration ability of CNE2 cells and reduce its invasion ability. p-HSP27 may induce EMT changes in CNE2 by down-regulating E-cadherin, thus promoting CNE2 migration, and may inhibit CNE2 invasion by down-regulating MMPs expression.
Humans ; Cell Proliferation ; Cell Line, Tumor ; Nasopharyngeal Carcinoma/pathology* ; Nasopharyngeal Neoplasms/pathology* ; Cell Movement ; HSP27 Heat-Shock Proteins/metabolism* ; Phosphorylation ; Neoplasm Metastasis ; Matrix Metalloproteinase 9/metabolism* ; Neoplasm Invasiveness ; Matrix Metalloproteinase 2/metabolism* ; Heat-Shock Proteins/metabolism* ; Cyclin D1/metabolism* ; Molecular Chaperones/metabolism* ; Cadherins/metabolism*

Objective: To investigate the effect of acetyl-CoA carboxylase 1 (ACC1) knockdown on the migration of esophageal squamous cell carcinoma (ESCC) KYSE-450 cell and underlying mechanism. Methods: Lentiviral transfection was conducted to establish sh-NC control cell and ACC1 knocking down cell (sh-ACC1). Human siRNA HSP27 and control were transfected by Lipo2000 to get si-HSP27 and si-NC. The selective acetyltransferase P300/CBP inhibitor C646 was used to inhibit histone acetylation and DMSO was used as vehicle control. Transwell assay was performed to detect cell migration. The expression of HSP27 mRNA was examined by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and the expressions of ACC1, H3K9ac, HSP27 and epithelial-mesenchymal transition-related proteins E-cadherin and Vimentin were detected by western blot. Results: The expression level of ACC1 in sh-NC group was higher than that in sh-ACC1 group (P<0.01). The number of cell migration in sh-NC group was (159.00±24.38), lower than (361.80±26.81) in sh-ACC1 group (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-NC group were statistically significant compared with sh-AAC1 group (P<0.05). The migrated cell number in sh-NC+ si-NC group was (189.20±16.02), lower than (371.60±38.40) in sh-ACC1+ si-NC group (P<0.01). The migrated cell number in sh-NC+ si-NC group was higher than that in sh-NC+ si-HSP27 group (152.40±24.30, P<0.01), and the migrated cell number in sh-ACC1+ si-NC group was higher than that in sh-ACC1+ si-HSP27 group (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-NC+ si-NC group were significantly different from those in sh-ACC1+ si-NC and sh-NC+ si-HSP27 groups (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-ACC1+ si-NC group were significantly different from those in sh-ACC1+ si-HSP27 group (P<0.01). After 24 h treatment with C646 at 20 μmmo/L, the migrated cell number in sh-NC+ DMSO group was (190.80±11.95), lower than (395.80±17.10) in sh-ACC1+ DMSO group (P<0.01). The migrated cell number in sh-NC+ DMSO group was lower than that in sh-NC+ C646 group (256.20±23.32, P<0.01). The migrated cell number in sh-ACC1+ DMSO group was higher than that in sh-ACC1+ C646 group (87.80±11.23, P<0.01). The protein expressions of H3K9ac, HSP27, E-cadherin and Vimentin in sh-NC+ DMSO group were significantly different from those in sh-ACC1+ DMSO group and sh-NC+ C646 group (P<0.01). The protein expression levels of H3K9ac, HSP27, E-cadherin and Vimentin in sh-ACC1+ DMSO group were significantly different from those in sh-ACC1+ C646 group (P<0.01). Conclusion: Knockdown of ACC1 promotes the migration of KYSE-450 cell by up-regulating HSP27 and increasing histone acetylation.
Humans ; Esophageal Neoplasms/pathology* ; Esophageal Squamous Cell Carcinoma/genetics* ; Vimentin/metabolism* ; Dimethyl Sulfoxide ; HSP27 Heat-Shock Proteins/metabolism* ; Histones/metabolism* ; Cadherins/metabolism* ; Cell Movement ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Epithelial-Mesenchymal Transition/genetics* ; Gene Expression Regulation, Neoplastic

OBJECTIVES: The pathogenesis of androgenetic alopecia (AGA) is related to the level of androgen and its metabolic pathways. The binding of androgen and androgen receptor (AR) depends on the assistance of heat shock protein 27 (HSP27). HSP27 combined with microRNAs (miR)-1 can regulate AR levels. However, it is not clear whether HSP27 and miR-1 jointly participate in the pathogenesis of AGA. This study aims to investigate the role of AR up-regulation in the pathogenesis of AGA and underlying mechanisms. METHODS: A total of 46 male AGA patients (AGA group), who admitted to the First Affiliated Hospital of Guangzhou Medical University from September 2019 to February 2020, and 52 healthy controls admitted to the same period were enrolled in this study. Serum levels of dihydrotestosterone (DHT) and HSP27 in patients and healthy controls were measured by ELISA. Western blotting was used to detect the protein expression of HSP27 and AR in scalp tissues of patients and the healthy controls. The levels of HSP27, AR, and miR-1 were analyzed using real-time PCR. Human dermal papilla cells were transfected with HSP27 siRNA to inhibit the expression of HSP27. MiR-1 and miR-1 inhibitors were transfected simultaneously or separately into cells and then the changes in AR protein expression were detected. RESULTS: The levels of DHT and HSP27 in the AGA group were (361.4±187.7) pg/mL and (89.4±21.8) ng/mL, respectively, which were higher than those in the control group [(281.8±176.6) pg/mL and (41.2±13.7) ng/mL, both P<0.05]. However, there was no significant difference in serum HSP27 and AR levels among AGA patients with different degrees of hair loss (P>0.05). Correlation analysis showed that there was a positive correlation between HSP27 level and DHT level in the AGA patients (P<0.05). The level of HSP27 mRNA in scalp tissue was negatively correlated with that of miR-1 mRNA (P<0.05). Compared with the control group, the levels of HSP27 protein, AR protein, HSP27 mRNA, and AR mRNA in scalp tissues of AGA group were significantly increased (P<0.05). The up-regulation of HSP27 in scalp tissues of AGA patients was closely related to the increased levels of AR. However, the level of miR-1 in scalp tissues of AGA patients was significantly down-regulated, contrary to the expression of AR (P<0.05). Further in cell studies showed that inhibition of HSP27 or miR-1 expression in human dermal papilla cells could inhibit the expression of AR, and inhibition of both HSP27 and miR-1 expression was found to have an accumulative effect on AR, with statistically significant differences (all P<0.05). CONCLUSIONS HSP27 could combine with miR-1 to up-regulate AR levels, which is closely related to the development of AGA.
Alopecia/pathology* ; HSP27 Heat-Shock Proteins/metabolism* ; Humans ; Male ; MicroRNAs/genetics* ; RNA, Messenger ; Receptors, Androgen/metabolism* ; Up-Regulation

Objective: To study the effect of anti-fibrotic tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) on phosphorylated heat shock protein 27 (P-HSP27) and zinc finger family transcriptional repressor 1 (SNAI1) expression to explore the anti-silicosis fibrosis effect of Ac-SDKP. Methods: In December 2014, the rat silicosis animal model was prepared by one-time bronchial infusion of silicon dioxide (SiO(2)) dust. 80 SPF healthy adult Wistar rats were selected, and the rats were divided into 8 groups according to the random number table method, 10 in each group. Model control group for 4 weeks (feeding for 4 weeks) , model control group for 8 weeks (feeding for 8 weeks) : bronchial perfusion with normal saline 1.0 ml per animal. Silicosis model group for 4 weeks (feeding for 4 weeks) and silicosis model group for 8 weeks (feeding for 8 weeks) : bronchial perfusion of 50 mg/ml SiO(2) suspension 1.0 ml per animal. Ac-SDKP administration group for 4 weeks (feeding for 4 weeks) , Ac-SDKP administration group for 8 weeks (feeding for 8 weeks) : Ac-SDKP 800 μg·kg(-1)·d(-1) was administered by intraperitoneal pump. Ac-SDKP preventive treatment group: 48 h after Ac-SDKP 800 μg·kg(-1)·d(-1) administration, bronchial perfusion of SiO(2) suspension 1.0 ml per animal, raised for 8 weeks. Ac-SDKP anti-fibrosis treatment group: after bronchial perfusion of 1.0 ml of SiO(2) suspension for 4 weeks, Ac-SDKP 800 μg·kg(-1)·d(-1) was administered for 4 weeks. Western blotting was used to detect the expression of P-HSP27, SNAI1, α-smooth muscle actin (α-SMA) , and collage typeⅠ and Ⅲ in each group. The expression of P-HSP27 and SNAI1 was detected by immunohistochemistry, and the co-localized expression of P-HSP27 and α-SMA was detected by laser confocal microscopy. Results: Compared with the model control group, the expressions of P-HSP27, SNAI1, α-SMA, and collage typeⅠ and Ⅲ in the silicosis fibrosis area of the rats in the silicosis model group were enhanced, and the differences were statistically significant (P<0.05) . After Ac-SDKP intervention, compared with silicosis model group for 8 weeks, the expressions of P-HSP27, SNAI1 α-SMA, and collage typeⅠ and Ⅲ in the Ac-SDKP preventive and anti-fibrosis treatment groups were significantly decreased, and the differences were statistically significant (P<0.05) . However, the expressions of P-HSP27 SNAI1, and collage typeⅠ and Ⅲ between the Ac-SDKP administration group and the model control group did not change significantly, and the differences were not statistically significant (P>0.05) . Laser confocal results showed that the positive cells expressing P-HSP27 and α-SMA in the lung tissue of the silicosis model group were more than those in the model control group. Compared with the silicosis model group, the Ac-SDKP prevention and anti-fibrosis treatment groups expressing the positive cells of P-HSP27 and α-SMA decreased. Compared with the model control group for 8 weeks, there were some double-positive cells expressing P-HSP27 and α-SMA in the nodules of the silicosis model group for 8 weeks. Conclusion: Ac-SDKP may play an anti-silicic fibrosis effect by regulating the P-HSP27/SNAI1 pathway.
Animals ; HSP27 Heat-Shock Proteins ; Oligopeptides ; Rats ; Rats, Wistar ; Silicon Dioxide ; Silicosis/metabolism*

BACKGROUND/AIMS: Several lines of evidence from epidemiologic and laboratory studies have shown that the consumption of Artemisia or green tea extracts (MPGT) is inversely associated with the risk of alcohol-induced damage and other chronic diseases. Supported by previous studies showing that the combined extract of Artemisia and green tea, MPGT, exerted significantly either antioxidative or anti-inflammatory actions against Helicobacter pylori-associated gastric diseases, it was hypothesized that MPGT can offer protection against alcoholic gastritis. METHODS: Ethanol was administered to induce gastric damage in Wistar rats, which had been pretreated with various doses of MPGT, to measure the rescuing action of a MPGT pretreatment against ethanol-induced gastric damage. In addition, the molecular mechanisms for the preventive effects were examined. RESULTS: The MPGT pretreatment (100, 300, and 500 mg/kg) alleviated the ethanol-induced gastric damage, which was evidenced by the significant decrease in calcium-dependent phospholipase A2, MAPKs, and NF-κB levels compared to ethanol alone. Furthermore, the MPGT pretreatment preserved 15-prostaglandin dehydrogenase, whereas cyclooxygenase-2 was decreased significantly. All of these biochemical changes led to the significant alleviation of alcohol-associated gastric mucosal damage. Ethanol significantly increased the TUNEL positivity in the stomach, but MPGT decreased the apoptotic index significantly, which was associated with significantly lower pathological scores of ethanol-induced mucosal ulcerations. The significant protective changes observed alcoholic gastritis with MPGT were related to the increased expression of cytoprotective genes, such as heat-shock protein (HSP)27, HSP60, and PDGF. CONCLUSIONS: The efficient anti-inflammatory, anti-apoptotic, and regenerative actions of MPGT make it a potential nutrient phytoceutical to rescue the stomach from alcoholic gastritis.
Alcoholics* ; Artemisia* ; Chronic Disease ; Cyclooxygenase 2 ; Ethanol ; Gastritis* ; Heat-Shock Proteins* ; Helicobacter ; HSP27 Heat-Shock Proteins* ; Humans ; In Situ Nick-End Labeling ; Oxidoreductases ; Phospholipases A2 ; Rats, Wistar ; Stomach ; Stomach Diseases ; Tea* ; Ulcer

Ferroptosis is a newly recognized type of cell death that results from iron-dependent lipid peroxidation and is different from other types of cell death, such as apoptosis, necrosis, and autophagic cell death. This type of cell death is characterized by mitochondrial shrinkage with an increased mitochondrial membrane density and outer mitochondrial membrane rupture. Ferroptosis can be induced by a loss of activity of system Xc− and the inhibition of glutathione peroxidase 4, followed by the accumulation of lipid reactive oxygen species (ROS). In addition, inactivation of the mevalonate and transsulfuration pathways is involved in the induction of ferroptosis. Moreover, nicotinamide adenine dinucleotide phosphate oxidase and p53 promote ferroptosis by increasing ROS production, while heat shock protein beta-1 and nuclear factor erythroid 2-related factor 2 inhibit ferroptosis by reducing iron uptake. This article outlines the molecular mechanisms and signaling pathways of ferroptosis regulation, and explains the roles of ferroptosis in human disease.
Apoptosis ; Autophagy ; Cell Death* ; Glutathione Peroxidase ; HSP27 Heat-Shock Proteins ; Humans ; Iron ; Lipid Peroxidation ; Mevalonic Acid ; Mitochondrial Membranes ; NADP ; Necrosis ; Oxidoreductases ; Reactive Oxygen Species ; Rupture

Ferroptosis is a newly recognized type of cell death that results from iron-dependent lipid peroxidation and is different from other types of cell death, such as apoptosis, necrosis, and autophagic cell death. This type of cell death is characterized by mitochondrial shrinkage with an increased mitochondrial membrane density and outer mitochondrial membrane rupture. Ferroptosis can be induced by a loss of activity of system Xc− and the inhibition of glutathione peroxidase 4, followed by the accumulation of lipid reactive oxygen species (ROS). In addition, inactivation of the mevalonate and transsulfuration pathways is involved in the induction of ferroptosis. Moreover, nicotinamide adenine dinucleotide phosphate oxidase and p53 promote ferroptosis by increasing ROS production, while heat shock protein beta-1 and nuclear factor erythroid 2-related factor 2 inhibit ferroptosis by reducing iron uptake. This article outlines the molecular mechanisms and signaling pathways of ferroptosis regulation, and explains the roles of ferroptosis in human disease.
Apoptosis ; Autophagy ; Cell Death ; Glutathione Peroxidase ; HSP27 Heat-Shock Proteins ; Humans ; Iron ; Lipid Peroxidation ; Mevalonic Acid ; Mitochondrial Membranes ; NADP ; Necrosis ; Oxidoreductases ; Reactive Oxygen Species ; Rupture

The effects of storage temperatures, repeated freeze-thaw cycles, or delays in separating plasma or serum from blood samples are largely unknown for heat shock protein 27 (HSP27). We evaluated (1) the imprecision of the HSP27 assay used in this study; (2) the in vitro stability of HSP27 in blood samples stored at 4℃ for up to 6 hr with immediate and delayed serum/plasma separation from cells; and (3) the in vitro stability of HSP27 in blood samples stored at -80℃ after repeated freeze-thaw cycles. The ELISA to detect HSP27 in this study showed a within-run CV of <9% and a total CV of <15%. After 4-6 hr of storage at 4℃, HSP27 concentrations remained stable when using serum tubes irrespective of sample handling, but HSP27 concentrations decreased by 25-45% when using EDTA plasma tubes. Compared with baseline HSP27, one freeze-thaw cycle had no effect on serum concentrations. However, plasma concentrations increased by 3.1-fold after one freeze-thaw cycle and by 7.3-fold after five freeze-thaw cycles. In conclusion, serum is an appropriate biological sample type for use in epidemiological and large-scale clinical studies.
*Enzyme-Linked Immunosorbent Assay ; Freezing ; HSP27 Heat-Shock Proteins/*blood ; Humans ; Protein Stability ; Reproducibility of Results ; Specimen Handling ; Temperature ; Time Factors

OBJECTIVEThis research aimed to observe the expression of heat shock protein 27 (HSP27) in human gingival fibroblasts (HGFs) cell injury induced by different concentrations of cigarette smoke extract (CSE).

METHODSThe third to eighth generations of cultured HGFs were treated with serially diluted CSE of different concentrations (0, 2.5%, 5.0%, 12.5%, 25.0%, 50.0%). Wound-healing assay was performed to determine the migration of HGFs, and Western blot was used to determine the expression of HSP27.

RESULTSThe migration capability of HGFs weakened with the increase of CSE concentration. HSP27 expression was negative in normal HGFs but positive in CSE-intervened HGFs in a concentration-dependent manner.

CONCLUSIONHSP27 concentration increased in the CSE-induced injury of HGFs. This finding suggests that HSP27 plays an important role in CSE-induced epithelial injury.

PURPOSE: Heat shock proteins (HSPs) are highly expressed during stress responses and cellular adaptation to environmental changes. One such protein is HSP27, a 27kDa protein that prevents cell death induced by many pro-apoptotic agents. Therefore, the aim of this study was to investigate the correlation between HSP27 expression and apoptosis induced by doxazosin treatment in prostate cancer cell line PC-3. MATERIALS AND METHODS: RT-PCR, Western blotting, and immunocytochemical staining were performed to determine whether HSP27 mRNA and protein are expressed in PC-3 cells. Next, to investigate the effects of doxazosin on apoptosis and HSP27 protein expression in PC-3 cells, the cells were stained using a TUNEL kit (to detect apoptotic cells) and with HSP27 antibody (to assess HSP27 protein expression) 6, 12, 24, and 48h after treatment with 25microM doxazosin. In addition, to determine whether HSP27 mRNA interference accelerates doxazosin-induced apoptosis of PC-3, we knocked down HSP27 with siRNA and then evaluated the rate of apoptosis after doxazosin treatment. RESULTS: HSP27 mRNA and protein were expressed in PC-3 cells. Furthermore, HSP27 mRNA and protein levels increased until 12 hours after 25microM doxazosin treatment, whereas the rate of apoptosis did not increased dramatically. After 12 hours, HSP27 expression decreased and then apoptosis was accelerated. In addition, siRNA-mediated knockdown of HSP27 induce higher apoptosis rate of PC-3 cells even before 12hrs after doxazosin treatment. CONCLUSIONS: By inhibiting apoptosis, HSP27 expression might play an important role in inhibiting progression to castration-refractory prostate cancer and resistance to anti-cancer treatment.
Apoptosis* ; Blotting, Western ; Cell Death ; Cell Line* ; Doxazosin ; Heat-Shock Proteins* ; Hot Temperature* ; HSP27 Heat-Shock Proteins* ; In Situ Nick-End Labeling ; Prostate* ; Prostatic Neoplasms* ; RNA, Messenger ; RNA, Small Interfering