Heat Shock Protein HSP27 and the Status of the Glutathione System in Dexamethasone-Induced Apoptosis of Jurkat Tumor Cells.

We studied the effect of the HSP27 inhibitor, 5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl)-isoxazole, at a final concentration of 0.1 µM and/or the apoptosis inducer dexamethasone at a final concentration of 10 µM on the content of hydroxyl radical, reduced and oxidized glutathione, HSP27, activity of glutathione reductase, glutathione peroxidase, caspase-3, and the number of Annexin+ Jurkat tumor cells. The involvement of HSP27 in apoptosis of Jurkat tumor cells was demonstrated. Simultaneous exposure to the HSP27 inhibitor and dexamethasone resulted in an increase in the level of HSP27 against the background of developing oxidative stress (increase in the concentration of hydroxyl radicals and changes in the state of the glutathione system).

Induction of heat shock protein expression in SP2/0 transgenic cells and its effect on the production of monoclonal antibodies.

The objective of the current investigation was to evaluate the induction of heat shock proteins (HSPs) in SP2/0 transgenic cells and the effect of these proteins on the production of monoclonal antibodies (mAbs). The SP2/0 cell line expressing the PSG-026 antibody, a biosimilar candidate of golimumab, the culture parameters, and the target protein expression were not justified for industrial production and were used for the experiments. Paracetamol and heat shock were used as chemical and physical inducers of HSPs, respectively. The results showed that paracetamol and heat shock increased the expression of HSP70 and HSP27 at the mRNA and protein levels. The expression of HSPs was greater in paracetamol-treated cells than in heat shock-treated cells. Paracetamol treatment at concentrations above 0.5 mM significantly reduced cell viability and mAb expression. However, treatment with 0.25 mM paracetamol results in delayed cell death and increased mAb production. Heat shock treatment at 45°C for 30 minutes after enhanced mAb expression was applied after pre-treatment with paracetamol. In bioreactor cultures, pretreatment of cells with paracetamol improved cell viability and shortened the lag phase, resulting in increased cell density. The production of mAbs in paracetamol-treated cultures was markedly greater than that in the control. Analysis of protein quality and charge variants revealed no significant differences between paracetamol-treated and control cultures, indicating that the induction of HSPs did not affect protein aggregation or charge variants. These findings suggest that inducing and manipulating HSP expression can be a valuable strategy for improving recombinant protein production in biopharmaceutical processes.

Exosomal HSPB1, interacting with FUS protein, suppresses hypoxia-induced ferroptosis in pancreatic cancer by stabilizing Nrf2 mRNA and repressing P450.

Ferroptosis is a new type of programmed cell death, which has been involved in the progression of tumours. However, the regulatory network of ferroptosis in pancreatic cancer is still largely unknown. Here, using datasets from GEO and TCGA, we screened HSPB1, related to the P450 monooxygenase signalling, a fuel of ferroptosis, to be a candidate gene for regulating pancreatic cancer cell ferroptosis. We found that HSPB1 was enriched in the exosomes derived from human pancreatic cancer cell lines SW1990 and Panc-1. Then, hypoxic SW1990 cells were incubated with exosomes alone or together with HSPB1 siRNA (si-HSPB1), and we observed that exosomes promoted cell proliferation and invasion and suppressed ferroptosis, which was reversed by si-HSPB1. Moreover, we found a potential binding affinity between HSPB1 and FUS, verified their protein interaction by using dual-colour fluorescence colocalization and co-IP assays, and demonstrated the promoting effect of FUS on oxidative stress and ferroptosis in hypoxic SW1990 cells. Subsequently, FUS was demonstrated to bind with and stabilize the mRNA of Nrf2, a famous anti-ferroptosis gene that negatively regulates the level of P450. Furthermore, overexpressing FUS and activating the Nrf2/HO-1 pathway (using NK-252) both reversed the inhibitory effect of si-HSPB1 on exosome functions. Finally, our in vivo studies showed that exosome administration promote tumour growth in nude mice of xenotransplantation, which was able to be eliminated by knockdown of HSPB1. In conclusion, exosomal HSPB1 interacts with the RNA binding protein FUS and decreases FUS-mediated stability of Nrf2 mRNA, thus suppressing hypoxia-induced ferroptosis in pancreatic cancer.

Overexpression of HSP27 accelerates stress-induced gastric ulcer healing via the CXCL12/CXCR4 axis.

Chronic stress often triggers gastrointestinal complications, including gastric injury and ulcers. Understanding the role of heat shock protein 27 (HSP27) in stress-induced gastric ulcers could unveil novel therapeutic targets. Here, we established a stress-induced gastric ulcer rat model using water immersion restraint stress and administered adenovirus-packaged HSP27 overexpression vector. Gastric ulcer severity was scored, and mucosal changes were assessed. Gastric epithelial and endothelial cells were treated with lipopolysaccharide and transfected with HSP27 overexpression vectors to evaluate cell viability, migration and angiogenesis. Expression levels of HSP27, C-X-C motif chemokine ligand 12 (CXCL12) and C-X-C motif chemokine receptor 4 (CXCR4) were measured in tissues and cells. HSP27 expression was initially low during stress-induced gastric ulceration but increased during ulcer healing. HSP27 overexpression accelerated ulcer healing in rats, promoting gastric epithelial cell proliferation and migration and gastric endothelial cell angiogenesis through the CXCL12/CXCR4 axis. Inhibitor IT1t reversed the effects of HSP27 overexpression on cell proliferation, migration and angiogenesis. In summary, HSP27 overexpression facilitated ulcer healing, which was partially mediated by the CXCL12/CXCR4 axis.

Distinct induction pathways of heat shock protein 27 in human keratinocytes: Heat stimulation or capsaicin through phosphorylation of heat shock factor 1 at serine 326 and/or suppression of ΔNp63.

Epidermal keratinocytes, forming the outermost layer of the human body, serve as a crucial barrier against diverse external stressors such as ultraviolet radiation. Proper keratinocyte differentiation and effective responses to external stimuli are pivotal for maintaining barrier integrity. Heat is one such stimulus that triggers the synthesis of heat shock proteins (HSPs) when cells are exposed to temperatures above 42 °C. Additionally, activation of the transient receptor potential cation channel subfamily V member 1 (TRPV1) occurs at 42 °C. Here, we explore the interplay between TRPV1 signaling and HSP induction in human keratinocytes. Both heat and capsaicin, a TRPV1 agonist, induce expression of HSP27, HSP70, and HSP90 in keratinocytes. Interestingly, pharmacological inhibition of TRPV1 attenuates heat-induced HSP27 expression, but not that of HSP70 or HSP90. Furthermore, both heat and capsaicin stimulation result in distinct phosphorylation patterns of heat shock factor 1 (HSF1), with phosphorylation at serine 326 being a common feature. Notably, genetic manipulation to mimic dephosphorylation of HSF1 at serine 326 reduces HSP27 levels. Additionally, ΔNp63, a key regulator of epidermal differentiation, negatively modulates HSP27 expression independently of HSF1 phosphorylation status. While heat stimulation has no effect on ΔNp63 expression, capsaicin reduces its levels. The precise role of TRPV1 signaling in keratinocytes warrants further investigation for a comprehensive understanding of its impact on barrier function.

HSP27 promotes vasculogenic mimicry formation in human salivary adenoid cystic carcinoma via the AKT-MMP-2/9 pathway.

PURPOSE: To explore the role and mechanism of heat shock protein 27 (HSP27) in SACC VM formation. STUDY DESIGN: Immunohistochemistry and double staining with cluster of differentiation 31 (CD31) and periodic acid-Schiff (PAS) were used to detect HSP27 expression and VM in 70 SACC tissue samples separately. Quantitative real-time polymerase chain reaction (qRT-PCR), western blot analysis, and immunofluorescence were used to detect gene and protein expression. HSP27 in SACC cells were overexpression or downregulated by transfecting HSP27 or short hairpin RNA target HSP27 (sh-HSP27). The migration and invasion abilities of SACC cells were detected using wound healing and Transwell invasion assays. The VM formation ability of the cells in vitro was detected using a Matrigel 3-dimensional culture. RESULTS: HSP27 expression was positively correlated with VM formation and affected the prognosis of patients. In vitro, HSP27 upregulation engendered VM formation and the invasion and migration of SACC cells. Mechanistically, HSP27 upregulation increased Akt phosphorylation and subsequently increased downstream matrix metalloproteinase 2 and 9 expressions. CONCLUSION: HSP27 may plays an important role in VM formation in SACC via the AKT-MMP-2/9 signalling pathway.

Rapamycin protects mouse skin from ultraviolet B-induced photodamage by modulating Hspb2-mediated autophagy and apoptosis.

BACKGROUND: Continuous exposure to UVB is the main extrinsic cause of skin photodamage, which is associated with oxidative stress, DNA damage, apoptosis and degradation of collagen. Rapamycin, a mechanistic target inhibitor of rapamycin complex 1 (mTORC1), has been shown to play a crucial role anti-tumor and aging retardation, but its mechanism of action in UVB-induced photodamage still remains unknown. In this study, we investigated the role of rapamycin and Hspb2 (also known as Hsp27) in UVB-induced photodamage in mice. METHODS AND RESULTS: We constructed skin acute photodamage models on the ears of WT and Hspb2 KO mice, respectively, and administered rapamycin treatment. Histological results showed that knockout of the hspb2 exacerbated the skin damage, as evidenced by thickening of the epidermis, breakage and disruption of collagen fibers and reduction in their number, which is reversed by rapamycin treatment. In addition, hspb2 knockout promoted UVB-induced apoptosis and reduced autophagy levels, with a significant increase in p53 levels and Bax/Bcl-2 ratio, a reduction in LC3II/I ratio and an increase in p62 levels in the KO mice compared to those in WT mice after the same dose of UVB irradiation. Rapamycin was also found to inhibit collagen degradation induced by hspb2 knockdown through activation of the TGF-ß/Smad signaling pathway. CONCLUSIONS: Rapamycin can alleviate skin photodamage from Hspb2 knockout to some extent. It may be a potential therapeutic drug for skin photodamage. In this study, we investigated the role of rapamycin and Hspb2 in UVB-induced photodamage in mice. Histological results showed that knockout of the hspb2 exacerbated the skin damage, as evidenced by thickening of the epidermis, breakage and disruption of collagen fibers and reduction in their number, which is reversed by rapamycin treatment. In addition, hspb2 knockout promoted UVB-induced apoptosis and reduced autophagy levels. Rapamycin was also found to inhibit collagen degradation induced by hspb2 knockdown through activation of the TGF-ß/Smad signaling pathway. We conclude that rapamycin and Hspb2 exert a synergistic protective effect in skin photodamage.

The pathogenicity and immune effects of different generations of Mycoplasma synoviae on chicken embryos.

1. Mycoplasma synoviae (MS) is the primary causative agent of synovitis in avian species. In order to investigate the pathogenicity and immunological responses associated with MS in specific pathogen-free chicken embryos, a series of generations (F1, F95, F120, F160 and F200) of MS were introduced into 7-day-old SPF chicken embryos and subsequent mortality rates were recorded and analysed2. Reverse transcription-quantitative polymerase chain reaction was performed to detect expression of heat shock proteins HSP27, HSP40, HSP60, HSP70 and HSP90 and inflammatory factors interleukin (IL)-1ß, caspase-1 and IL-18 in the tracheal tissue.3. The results showed that the mortality rate of SPF chicken embryos decreased with an increase in the number of passages, with the highest being 80% (8/10) for F1 generation and the lowest being 10% (1/10) for F200. The expression of HSP27, IL-1ß, HSP40, caspase-1, HSP70 and HSP90 showed a significant downregulation trend with an increase in the generation (except IL-18; P < 0.05). The HSP60 expression was significantly upregulated with increasing generations (P < 0.05).4. A relationship between pathogenicity and the number of passages was observed and the decrease in pathogenicity appeared to be associated with HSP and genes related to inflammatory factors. The present work offers a scientific foundation for screening potential MS strains that might be employed to develop attenuated vaccines.

Mechanistic insights into the anti-restenotic effects of HSP27 and HO1 modulated by reconstituted HDL on neointimal hyperplasia.

High-density lipoprotein (HDL) therapy has demonstrated beneficial effects in acute stroke and acute myocardial infarction models by reducing infarct size. In this study, we investigated the inhibitory effects of reconstituted HDL (rHDL) on neointimal hyperplasia and elucidated its underlying mechanism using a balloon injury rat model. Our finding revealed a significant 37% reduction in the intima to media ratio in the arteries treated with 80 mg/kg rHDL compared to those subjected to injury alone (p < 0.05), indicating a specific inhibition of neointimal hyperplasia. In vivo analysis further supported the positive effects of rHDL by demonstrating a reduction in smooth muscle cell (SMC) proliferation and an increase in endothelial cell (EC) proliferation. Additionally, rHDL treatment led to decreased infiltration of leukocytes and downregulated the expression of matrix metallopeptidase 9 (MMP9) in the neointimal area. Notably, rHDL administration resulted in decreased expression of VCAM1 and HIF1α, alongside increased expression of heme oxygenase 1 (HO1) and heat shock protein 27 (HSP27). Overexpression of HSP27 and HO1 effectively inhibited SMC proliferation. Moreover, rHDL-mediated suppression of injury-induced HIF1α coincided with upregulation of HSP27. Interestingly, HSP27 and HO1 had varying effects on the expression of chemokine receptors and rHDL did not exert significant effect on chemokine receptor expression in THP1 cells. These findings underscore the distinct roles of HSP27 and HO1 as potential regulatory factors in the progression of restenosis. Collectively, our study demonstrates that rHDL exerts a potent anti-neointimal hyperplasia effect by reducing leukocytes infiltration and SMC proliferation while promoting EC proliferation.

The heat shock protein Hsp27 controls mitochondrial function by modulating ceramide generation.

Sphingolipids have key functions in membrane structure and cellular signaling. Ceramide is the central molecule of the sphingolipid metabolism and is generated by ceramide synthases (CerS) in the de novo pathway. Despite their critical function, mechanisms regulating CerS remain largely unknown. Using an unbiased proteomics approach, we find that the small heat shock protein 27 (Hsp27) interacts specifically with CerS1 but not other CerS. Functionally, our data show that Hsp27 acts as an endogenous inhibitor of CerS1. Wild-type Hsp27, but not a mutant deficient in CerS1 binding, inhibits CerS1 activity. Additionally, silencing of Hsp27 enhances CerS1-generated ceramide accumulation in cells. Moreover, phosphorylation of Hsp27 modulates Hsp27-CerS1 interaction and CerS1 activity in acute stress-response conditions. Biologically, we show that Hsp27 knockdown impedes mitochondrial function and induces lethal mitophagy in a CerS1-dependent manner. Overall, we identify an important mode of CerS1 regulation and CerS1-mediated mitophagy through protein-protein interaction with Hsp27.

Heat shock protein 27 regulates myogenic and self-renewal potential of bovine satellite cells under heat stress.

While satellite cells play a key role in the hypertrophy, repair, and regeneration of skeletal muscles, their response to heat exposure remains poorly understood, particularly in beef cattle. This study aimed to investigate the changes in the transcriptome, proteome, and proliferation capability of bovine satellite cells in response to different levels of heat stress (HS) and exposure times. Satellite cells were isolated from 3-mo-old Holstein bulls (body weight: 77.10 ± 2.02 kg) and subjected to incubation under various temperature conditions: 1) control (38 °C; CON), 2) moderate (39.5 °C; MHS), and extreme (41 °C; EHS) for different durations ranging from 0 to 48 h. Following 3 h of exposure to extreme heat (EHS), satellite cells exhibited significantly increased gene expression and protein abundance of heat shock proteins (HSPs; HSP70, HSP90, HSP20) and paired box gene 7 (Pax7; P < 0.05). HSP27 expression peaked at 3 h of EHS and remained elevated until 24 h of exposure (P < 0.05). In contrast, the expression of myogenic factor 5 (Myf5) and paired box gene 3 (Pax3) was decreased by EHS compared to the control at 3 h of exposure (P < 0.05). Notably, the introduction of HSP27 small interference RNA (siRNA) transfection restored Myf5 expression to control levels, suggesting an association between HSP27 and Myf5 in regulating the self-renewal properties of satellite cells upon heat exposure. Immunoprecipitation experiments further confirmed the direct binding of HSP27 to Myf5, supporting its role as a molecular chaperone for Myf5. Protein-protein docking algorithms predicted a high probability of HSP27-Myf5 interaction as well. These findings indicate that extreme heat exposure intrinsically promotes the accumulation of HSPs and modulates the early myogenic regulatory factors in satellite cells. Moreover, HSP27 acts as a molecular chaperone by binding to Myf5, thereby regulating the division or differentiation of satellite cells in response to HS. The results of this study provide a better understanding of muscle physiology in heat-stressed cells, while unraveling the intricate molecular mechanisms that underlie the HS response in satellite cells.

This study aimed to elucidate the response of bovine satellite cells to heat exposure. Satellite cells were isolated from Holstein bulls and subjected to varying temperatures. Transcriptional, proteomic, and proliferative changes were assessed. Following extreme heat exposure, cells exhibited upregulated expression of heat shock proteins (HSPs; HSP70, HSP90, HSP20) and paired box gene 7 (Pax7). Conversely, the expression of myogenic factor 5 (Myf5) and paired box gene 3 (Pax3), key regulators of myogenesis, decreased under conditions of extreme heat. Notably, downregulation of HSP27 expression using siRNA restored Myf5 expression to normal levels, implying an association between HSP27 and Myf5 in the modulation of satellite cell properties during heat exposure. Our results validated the direct binding of HSP27 to Myf5, substantiating its role as a molecular chaperone. These findings underscore the elevation of HSPs, and alteration of early myogenic regulatory factors implicated in muscle development upon exposure to extreme heat. HSP27 functions as a molecular chaperone by engaging with Myf5, thereby influencing the division or differentiation of satellite cells during heat stress (HS). This study contributes to the advancement of our comprehension regarding the muscular physiology of heat-stressed animals, while clarifying the intricate molecular mechanisms governing the response of satellite cells to HS.

Targeting Heat Shock Protein 27 and Fatty Acid Oxidation Augments Cisplatin Treatment in Cisplatin-Resistant Ovarian Cancer Cell Lines.

Most ovarian cancer patients develop recurrent cancers which are often resistant to commonly employed chemotherapy agents, such as cisplatin. We have previously shown that the inhibition of heat shock protein 27 (HSP27) or fatty acid oxidation (FAO) sensitizes cisplatin-resistant ovarian cancer cell lines to cisplatin and dual inhibition of both HSP27 and FAO induces substantial cell death in vitro. However, it is unclear how HSP27 and FAO promote cisplatin resistance, and if dual inhibition of both HSP27 and FAO would augment cisplatin treatment in vivo. Here we showed that HSP27 knockdown in two cisplatin-resistant ovarian cancer cell lines (A2780CIS and PEO4) resulted in more ROS production upon cisplatin treatment. HSP27-knockdown cancer cells exhibited decreased levels of reduced glutathione (GSH) and glucose6phosphate dehydrogenase (G6PD), a crucial pentose phosphate pathway enzyme. ROS depletion with the compound N-acetyl cysteine (NAC) attenuated cisplatin-induced upregulation of HSP27, FAO, and markers of apoptosis and ferroptosis in cisplatin-resistant ovarian cancer cell lines. Finally, inhibition of HSP27 and FAO with ivermectin and perhexiline enhanced the cytotoxic effect of cisplatin in A2780CIS xenograft tumors in vivo. Our results suggest that two different cisplatin-resistant ovarian cancer cell lines upregulate HSP27 and FAO to deplete cisplatin-induced ROS to attenuate cisplatin's cytotoxic effect.

O-GlcNAc Modification Alters the Chaperone Activity of HSP27 Charcot-Marie-Tooth Type 2 (CMT2) Variants in a Mutation-Selective Fashion.

Increased O-GlcNAc is a common feature of cellular stress, and the upregulation of this dynamic modification is associated with improved survival under these conditions. Likewise, the heat shock proteins are also increased under stress and prevent protein misfolding and aggregation. We previously linked these two phenomena by demonstrating that O-GlcNAc directly increases the chaperone of certain small heat shock proteins, including HSP27. Here, we examine this linkage further by exploring the potential function of O-GlcNAc on mutants of HSP27 that cause a heritable neuropathy called Charcot-Marie-Tooth type 2 (CMT2) disease. Using synthetic protein chemistry, we prepared five of these mutants bearing an O-GlcNAc at the major site of modification. Upon subsequent biochemical analysis of these proteins, we found that O-GlcNAc has different effects, depending on the location of the individual mutants. We believe that this has important implications for O-GlcNAc and other PTMs in the context of polymorphisms or diseases with high levels of protein mutation.

Upregulation of Heat-Shock Protein (hsp)-27 in a Patient with Heterozygous SPG11 c.1951C>T and SYNJ1 c.2614G>T Mutations Causing Clinical Spastic Paraplegia.

We report a 49-year-old patient suffering from spastic paraplegia with a novel heterozygous mutation and analyzed the levels of heat shock proteins (hsp)-27, dopamine (DA), and its metabolites in their cerebrospinal fluid (CSF). The hsp27 protein concentration in the patient's CSF was assayed by an ELISA kit, while DA levels and its metabolites in the CSF, 3,4-dihydroxyphenylacetic acid (DOPAC), Cys-DA, and Cys-DOPA were measured by HPLC. Whole exome sequencing demonstrated SPG-11 c.1951C>T and novel SYNJ1 c.2614G>T mutations, both heterozygous recessive. The patient's DA and DOPAC levels in their CSF were significantly decreased (53.0 ± 6.92 and 473.3 ± 72.19, p < 0.05, respectively) while no differences were found in their Cys-DA. Nonetheless, Cys-DA/DOPAC ratio (0.213 ± 0.024, p < 0.05) and hsp27 levels (1073.0 ± 136.4, p < 0.05) were significantly higher. To the best of our knowledge, the c.2614G>T SYNJ1 mutation has not been previously reported. Our patient does not produce fully functional spatacsin and synaptojanin-1 proteins. In this line, our results showed decreased DA and DOPAC levels in the patient's CSF, indicating loss of DAergic neurons. Many factors have been described as being responsible for the increased cys-DA/DOPAC ratio, such as MAO inhibition and decreased antioxidant activity in DAergic neurons which would increase catecholquinones and consequently cysteinyl-catechols. In conclusion, haploinsufficiency of spatacsin and synaptojanin-1 proteins might be the underlying cause of neurodegeneration produced by protein trafficking defects, DA vesicle trafficking/recycling processes, autophagy dysfunction, and cell death leading to hsp27 upregulation as a cellular mechanism of protection and/or to balance impaired protein trafficking.

The potential mediation of hypoxia-inducible factor-1α in heat shock protein 27 translocations, caspase-3 and calpain activities and yak meat tenderness during postmortem aging.

The present study aimed to characterize the influence of hypoxia-inducible factor-1α on heat shock protein 27 and cytochrome c translocation, yak meat microstructure destruction, and endogenous enzymes activities, refining the understanding of the tenderization process after slaughter. Postmortem yak longissimus thoracis et lumborum muscles were incubated with 0.9% saline or hypoxia-inducible factor-1α stabilizer dimethyloxaloylglycine at 4 °C for 6, 12, 24, 72, and 120 h. Results showed that hypoxia-inducible factor-1α activation promoted heat shock protein 27 migration and cytochrome c release, facilitating (P < 0.05) caspase-3 activity by mediating the heat shock protein 27/caspase-3 interaction but did not exert (P > 0.05) significant effects on the calpain-1 activity. Additionally, hypoxia-inducible factor-1α activation contributed to the mitochondrial apoptosis cascade, leading to a higher (P < 0.01) apoptosis rate. Therefore, these observations indicate that hypoxia-inducible factor-1α affects caspase-3 activity and tenderness of postmortem muscle through distinct regulatory mechanisms, possibly, in part, with heat shock protein 27 and cytochrome c mediation.

Small molecule heat shock protein 27 inhibitor J2 decreases ovarian cancer cell proliferation via induction of apoptotic pathways.

Heat shock protein 27 (Hsp27) is an important member of the chaperone protein family and its overexpression promotes cancer cell survival. Here, we investigated the apoptosis inducer role of the J2 compound (Hsp27 inhibitor) in human ovarian cancer cell lines (SKOV3 and OVCAR-3). Cell proliferation was measured by MTT assay. The parameters of J2-Hsp27 interaction were determined with molecular docking calculation. The inhibitory effect of the J2 compound on Hsp27 chaperone activity was investigated by luciferase activity assay. Finally, the apoptotic inducer role of the J2 compound on SKOV3 and OVCAR-3 cells was determined by RT-PCR and caspase-3 activity assay. J2 compound decreased SKOV3 and OVCAR-3 cell proliferation in a dose-dependent manner at 48 h with IC50 values of 17.34 µM and 12.63 µM, respectively. J2 inhibited the refolding process of denatured luciferase as an Hsp27 inhibitor. Molecular docking calculation was carried out to determine the interaction between Hsp27 and J2. The results indicated that J2 selectively binds to the phosphorylation site of the Hsp27 and inhibits the phosphorylation process of Hsp27. To determine the apoptotic potential of the J2 compound against ovarian cancer cells, the mRNA expression levels of apoptotic and antiapoptotic markers (Bax, Bcl-2, Bcl-xL, Cyt-c, p53, Apaf-1, Cas-3, Cas-8, Cas-9, TNF-α, DAXX, and Ask-1) were measured using RT-PCR. While J2 increased the expressions of apoptotic genes, it decreased the expressions of anti-apoptotic genes. Further, the J2 compound increased Cas-3 activity in SKOV3 and OVCAR-3 at 5.52 and 4.12 folds, respectively. These results confirm that J2 has great potential and significance in the stimulation of apoptosis in ovarian cancer cells as an Hsp27 inhibitor.

HSPB1 facilitates chemoresistance through inhibiting ferroptotic cancer cell death and regulating NF-κB signaling pathway in breast cancer.

Chemoresistance is one of the major causes of therapeutic failure and poor prognosis for breast cancer patients, especially for triple-negative breast cancer patients. However, the underlying mechanism remains elusive. Here, we identified novel functional roles of heat shock protein beta-1 (HSPB1), regulating chemoresistance and ferroptotic cell death in breast cancer. Based on TCGA and GEO databases, HSPB1 expression was upregulated in breast cancer tissues and associated with poor prognosis of breast cancer patients, which was considered an independent prognostic factor for breast cancer. Functional assays revealed that HSPB1 could promote cancer growth and metastasis in vitro and in vivo. Furthermore, HSPB1 facilitated doxorubicin (DOX) resistance through protecting breast cancer cells from drug-induced ferroptosis. Mechanistically, HSPB1 could bind with Ikß-α and promote its ubiquitination-mediated degradation, leading to increased nuclear translocation and activation of NF-κB signaling. In addition, HSPB1 overexpression led to enhanced secretion of IL6, which further facilitated breast cancer progression. These findings revealed that HSPB1 upregulation might be a key driver to progression and chemoresistance through regulating ferroptosis in breast cancer while targeting HSPB1 could be an effective strategy against breast cancer.

CD93 Ameliorates Diabetic Wounds by Promoting Angiogenesis via the p38MAPK/MK2/HSP27 Axis.

OBJECTIVE: Diabetic wounds are a complication of diabetes mellitus, which is characterised by microcirculation dysfunction caused by decreased local blood supply and insufficient metabolic exchange. Clinically, in addition to glycaemic control, the most important treatment for diabetic wounds is to promote local angiogenesis, which accelerates wound healing. The authors previous study demonstrated that CD93, which is specifically expressed on vascular endothelial cells (ECs), redundantly regulates angiogenesis in zebrafish, suggesting that CD93 is a potential angiogenic molecule. However, the role of CD93 in diabetic wounds has not yet been elucidated. METHODS: The angiogenic effects of CD93 were studied from four aspects: exogenous, endogenous, in vitro, and in vivo. CD93 recombinant protein was used in microvascular ECs and in mice to observe angiogenesis in vitro and in vivo. The wound model was established in CD93-/- and wild type diabetic mice, and the degree of wound healing as well as the amount and maturity of neovascularisation were investigated. The possible mechanism of CD93 in angiogenesis was determined by CD93 overexpression in cultured ECs. RESULTS: CD93 recombinant protein was found to exogenously promote tube formation and sprouting of ECs. It also recruited cells to promote the formation of vascular like structures in subcutaneous tissue and accelerated wound healing by optimising angiogenesis and re-epithelisation. Furthermore, CD93 deficiency was observed to delay wound repair, characterised by reduced neovascularisation, vascular maturity, and re-epithelisation level. Mechanically, CD93 activated the p38MAPK/MK2/HSP27 signalling pathway, positively affecting the angiogenic functions of ECs. CONCLUSION: This study demonstrated that CD93 promotes angiogenesis both in vitro and in vivo and that its angiogenic role in vitro is mediated by the p38MAPK/MK2/HSP27 signalling pathway. It was also found that CD93 exerts beneficial effects on wound healing in diabetic mice by promoting angiogenesis and re-epithelisation.

High HSPB1 expression predicts poor clinical outcomes and correlates with breast cancer metastasis.

BACKGROUND: Heat shock protein beta-1 (HSPB1) is a crucial biomarker for pathological processes in various cancers. However, the clinical value and function of HSPB1 in breast cancer has not been extensively explored. Therefore, we adopted a systematic and comprehensive approach to investigate the correlation between HSPB1 expression and clinicopathological features of breast cancer, as well as determine its prognostic value. We also examined the effects of HSPB1 on cell proliferation, invasion, apoptosis, and metastasis. METHODS: We investigated the expression of HSPB1 in patients with breast cancer using The Cancer Genome Atlas and immunohistochemistry. Chi-squared test and Wilcoxon signed-rank test were used to examine the relationship between HSPB1 expression and clinicopathological characteristics. RESULTS: We observed that HSPB1 expression was significantly correlated with the stage N, pathologic stages, as well as estrogen and progesterone receptors. Furthermore, high HSPB1 expression resulted in a poor prognosis for overall survival, relapse-free survival, and distant metastasis-free survival. Multivariable analysis showed that patients with poor survival outcomes had higher tumor, node, metastasis, and pathologic stages. Pathway analysis of HSPB1 and the altered neighboring genes suggested that HSPB1 is involved in the epithelial-to-mesenchymal transition. Functional analysis revealed showed that transient knockdown of HSPB1 inhibited the cell migration/invasion ability and promoted apoptosis. CONCLUSIONS: HSPB1 may be involved in breast cancer metastasis. Collectively, our study demonstrated that HSPB1 has prognostic value for clinical outcomes and may serve as a therapeutic biomarker for breast cancer.

[Knockdown of ACC1 promotes migration of esophageal cancer cell].

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.