Tacrolimus reverses pemphigus vulgaris serum-induced depletion of desmoglein in HaCaT cells via inhibition of heat shock protein 27 phosphorylation.

BACKGROUND: Glucocorticoids are the first-line treatment for Pemphigus vulgaris (PV), but its serious side effects can be life-threatening for PV patients. Tacrolimus (FK506) has been reported to have an adjuvant treatment effect against PV. However, the mechanism underlying the inhibitory effect of FK506 on PV-IgG-induced acantholysis is unclear. OBJECTIVE: The objective of this study was to explore the effect of FK506 on desmoglein (Dsg) expression and cell adhesion in an immortalized human keratinocyte cell line (HaCaT cells) stimulated with PV sera. METHODS: A cell culture model of PV was established by stimulating HaCaT cells with 5% PV sera with or without FK506 and clobetasol propionate (CP) treatment. The effects of PV sera on intercellular junctions and protein levels of p38 mitogen-activated protein kinase (p38MAPK), heat shock protein 27 (HSP27), and Dsg were assayed using western blot analysis, immunofluorescence staining, and a keratinocyte dissociation assay. RESULTS: PV sera-induced downregulation of Dsg3 was observed in HaCaT cells and was blocked by FK506 and/or CP. Immunofluorescence staining revealed that linear deposits of Dsg3 on the surface of HaCaT cells in the PV sera group disappeared and were replaced by granular and agglomerated fluorescent particles on the cell surface; however, this effect was reversed by FK506 and/or CP treatment. Furthermore, cell dissociation assays showed that FK506 alone or in combination with CP increased cell adhesion in HaCaT cells and ameliorated loss of cell adhesion induced by PV sera. Additionally, FK506 noticeably decreased the PV serum-induced phosphorylation of HSP 27, but had no effect on p38MAPK phosphorylation. CONCLUSION: FK506 reverses PV-IgG induced-Dsg depletion and desmosomal dissociation in HaCaT cells, and this effect may be obtained by inhibiting HSP27 phosphorylation.

Electroacupuncture inhibits PDK1/Akt/HCN4 pathway to improve neurogenic urinary retention in rats. / 电针通过抑制PDK1/Akt/HCN4é€šè·¯æ”¹å–„å¤§é¼ ç¥žç»æºæ€§å°¿æ½´ç•™.

OBJECTIVES: To observe the therapeutic effect of electroacupuncture (EA) on neurogenic urinary retention rats, so as to explore the underlying mechanism of EA in treating neurogenic urinary retention by focusing on 3-phosphoinositide-dependent protein kinase 1 (PDK1)/protein kinase B (Akt)/hyperpolarization activated cyclic nucleotide-gated cation channel 4 (HCN4) pathway. METHODS: Female SD rats were randomly divided into sham operation, model, EA, PDK1 inhibitor, HCN4 blocker and EA + HCN4 blocker groups, with 20 rats in each group. The model of sacral spinal cord injury was established by modified Hassan Shaker spinal cord transection method. EA (2 Hz/15 Hz, 0.5 mA) was applied to "Zhongji" (CV3) and "Zhongliao" (BL33) for 20 min, once daily for 10 days. Rats of the PDK1 inhibitor group received intraperitoneal injection of OSU-03012 (20 mg/kg), and rats of the HCN4 blocker group received intraperitoneal injection of ivabradine (10 mg/kg), both once every other day for 10 days. The urodynamic indexes of rats were detected by multi-channel physiological recorder；muscle strip test was used to detect detrusor excitability；the morphological changes of bladder were observed by HE staining. Immunofluorescence double staining was used to detect the co-expression of HCN4 and C-Kit, a specific marker of interstitial cells of Cajal in bladder. Western blot was used to detect the expression of PDK1/Akt/HCN4 pathway proteins in bladder tissue and heat shock protein 27 (HSP27), a protein related to bladder contraction function. RESULTS: Compared with the sham operation group, the rats in the model group showed urinary dysfunction, decreased leak point pressure, isolated detrusor spontaneous contraction frequency, fluorescence intensity of C-Kit positive cells, HCN4+/C-Kit+ co-expression, HCN4 and p-HSP27/HSP27 protein expression in bladder tissue (P<0.05), and increased maximum bladder capacity and comp-liance, minimum tension during contraction of isolated detrusor, PDK1 and p-Akt/Akt protein expression in bladder tissue (P<0.05). Meanwhile, the above index were all reversed after EA and PDK1 inhibitor intervention (P<0.05). In comparison with the EA group, the rats had severe urinary dysfunction, the urine leakage point pressure, spontaneous contraction frequency, fluorescence intensity of C-Kit positive cells, the co-expression of HCN4+/C-Kit+, and the protein expression of HCN4 and p-HSP27/HSP27 were decreased (P<0.05), the maximum bladder capacity and compliance, the minimum tension during contraction of isolated detrusor, and the protein expression of PDK1 and p-Akt/Akt in bladder tissue were increased (P<0.05) in both HCN4 blocker and EA+HCN4 blocker groups. HE staining showed exfoliated bladder epithelium and disordered layers, vacuolization of bladder wall cells, with infiltration of neutrophils in mucosal and muscular layers in the model group, which were relatively milder in the EA and PDK1 inhibitor groups, but worse in the HCN4 blocker and EA + HCN4 blocker groups. CONCLUSIONS: EA can improve the urinary dysfunction in rats with neurogenic urinary retention, which may be related to its effect in inhibiting the activation of PDK1/Akt pathway, promo-ting HCN4-mediated detrusor excitatory contraction and urinary electrical signal activation.

Impact of vitamin D supplementation on modulating heat-shock proteins in postmenopausal women: a randomized, double-blind, placebo-controlled study.

OBJECTIVE: The aim of this study was to examine the effect of vitamin D (VitD) supplementation on serum heat-shock proteins (HSP) in postmenopausal women (PW). METHODS: In this double-blind, placebo-controlled trial, 160 PW aged 45 to 65 years with amenorrhea 12 months or more were randomized into two groups: 80 PW in VitD group (oral supplementation with 1,000 IU VitD 3 /d) or 80 PW in placebo group. The PW were assessed at baseline and after 10 months of intervention. Plasma concentrations of 25-hydroxyVitD (25[OH]D) were measured by high-performance liquid chromatography. HSP27/pS78/pS82, HSP27/total, HSP60, HSP70/72, and HSP90α levels were assessed in serum using a multiplexed bead immunoassay. RESULTS: HSP27 (pS78/pS82 [ P < 0.035] and total [ P < 0.001]) levels increased in the supplemented group when compared with the control group. There was no effect of VitD supplementation on HSP60, HSP70/72, and HSP90α levels. CONCLUSIONS: Vitamin D supplementation increases serum HSP27 level in PW.

Efficacy of electroacupunture at Zusanli (ST36) on jumping-injured muscle based on transcriptome sequencing and genes analysis.

OBJECTIVE: To reveal the efficacy and possible mechanism of electroacupuncture at Zusanli (ST36) on injured skeletal muscle induced by jumping load. METHODS: In the present study, female Sprague-Dawley rats were randomly divided into four groups with 6 of each, including normal control group (NC), jumping-induced muscle injury model group (JI), JI with electroacupuncture stimulation treatment group (EA), and JI with non-electroacupuncture stimulation group (NEA). Transmission electron microscopy, transcriptome sequencing and analysis, prediction of protein interaction networks, real-time polymerase chain reaction verification, and Western blotting were performed on the gastrocnemius muscle of ipsilateral lower limbs. RESULTS: The structural repair of injured gastrocnemius myofibers following jumping training in EA rats was better than that of NEA rats. A total of 136 genes were differentially expressed in EA rats relative to JI rats, with 55 genes upregulated and 81 genes downregulated. According to results of transcriptome analysis, and prediction of protein mutual interaction by the online STRING database, Heat shock protein beta-7 (Hspb7) and myozenin2 (Myoz2) genes were targeted. Expressions of Hspb7 and Myoz2 mRNAs were increased in EA rats relative to JI rats (0.05). The expression of Hspb7 protein was upregulated in EA rats relative to that in NC, JI, and NEA rats (0.01, 0.05, and0.05, respectively). The expression of Myoz2 protein was upregulated in EA rats relative to that in NC and JI rats (both 0.01respectively). CONCLUSIONS: The present results suggest that electroacupuncture stimulation at Zusanli (ST36) could improve muscle healing following jumping-induced muscle injury, owing to the upregulation of Hspb7 and Myoz2 proteins.

Efficacy of Adenovirus-mediated, miR-199a Nanoparticles on Expression of HSP27, HSP70, and Soluble Glycoprotein in Rats with Heart Failure.

Context: Heart failure (HF) refers to abnormal changes in the function of the body's heart pump under the action of a variety of pathogenic factors. Due to the complex etiology and course of HF, current research on its etiology and pathogenesis hasn't yet reached a clear conclusion. So, there are many manifestations of heart failure in patients, and there are also many changes in the treatment. Objectives: The study intended to evaluate the efficacy of adenovirus-mediated miR-199a nanoparticles (NPs) for heart failure (HF). Design: The research team performed an animal study. Setting: The study took place at Shanghai Pudong Hospital at Fudan University Pudong Medical Center in Shanghai, China. Animals: The animals were 40 healthy, adult, male, Sprague-Dawley (SD) rats. They were specific pathogen-free (SPF) grade SD rats, all weighing about 280 g and aged 7-8 weeks. Intervention: The research team: (1) induced HF using coronary artery ligation and established different HF models and (2) randomly divided the rats into two groups with 20 rats in each group-an experimental group, which received high-dose, microR-199a (miR-199a) NPs, and a control group, which received low-dose miR-199a NPs. The treatments occurred for seven days after the induction of HF. Outcome Measures: At baseline and postintervention, the research team measured the left ventricular ejection fraction (LVEF), left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), diastolic and systolic left ventricular anterior wall (LVAW) thickness, left ventricular posterior wall (LVPW) thickness, and expression of heat shock protein 27 (HSP27), HSP70, soluble glycoprotein 130 (SGP130). The team analyzed and studied the effects of the adenovirus-mediated miR-199a NP on that expression, based on the above indicators. Results: The miR-199a prepared with NPs had good specificity through observation. The expression of HSP27, SGP130 was significantly downregulated in the experimental group as compared to the control group (P < .05) and HSP70 was upregulated in the experimental group as compared to the control group (P < .05). The expression decreased, or increased, with an increase in the cardiac-function classification, with substantial differences between the control and experimental groups. Expression levels of HSP27, HSP70, and SGP in the experimental group were negatively correlated with those of controls and negatively correlated with the left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), and left ventricular ejection fraction (LVEF). Conclusions: NP had good specificity. The miR-199a NP downregulated levels of HSP, which had a certain protective effect against HF and had a high clinical-adoption and promotion value.

Mangiferin prevents hepatocyte epithelial-mesenchymal transition in liver fibrosis via targeting HSP27-mediated JAK2/STAT3 and TGF-ß1/Smad pathway.

Hepatocytes has been confirmed to undergo EMT and can be converted into myofibroblasts during hepatic fibrogenesis. However, the mechanism of hepatocyte EMT regulation in hepatic fibrosis, particularly through HSP27 (human homologue of rodent HSP25), remains unclear. Mangiferin (MAN), a compound extracted from Mangifera indica L, has been reported to attenuate liver injury. This study aimed to investigate the mechanisms underlying HSP27 inhibition and the anti-fibrotic effect of MAN in liver fibrosis. Our results revealed that the expression of HSP27 was remarkably increased in the liver tissues of patients with liver cirrhosis and CCl4 -induced fibrotic rats. However, HSP27 shRNA treatment significantly alleviated fibrosis. Furthermore, MAN was found to inhibit CCl4 - and TGF-ß1-induced liver fibrosis and reduced hepatocyte EMT. More importantly, MAN decreased HSP27 expression to suppress the JAK2/STAT3 pathway, and subsequently blocked TGF-ß1/Smad signaling, which were consistent with its protection against CCl4 -induced EMT and liver fibrosis. Together, these results suggest that HSP27 may play a crucial role in hepatocyte EMT and liver fibrosis by activating JAK2/STAT3 signaling and TGF-ß1/Smad pathway. The suppression of HSP27 expression by MAN may be a novel strategy for attenuating the hepatocyte EMT in liver fibrosis.

Urolithins increased anticancer effects of chemical drugs, ionizing radiation and hyperthermia on human esophageal carcinoma cells in vitro.

Despite progress in diagnosis and treatment of esophageal cancer (EC), it is still considered as a serious malignancy with very poor prognosis. Urolithins are colonic microbiota metabolites with a wide range of pharmacological properties including chemopreventive, anti-inflammatory and anticancer activities. In this study, we hypothesized that urolithins might possess the potential to improve the efficacy of chemical drugs, ionizing radiation (IR) and/or hyperthermia on EC cells. After synthesis of urolithin A (UA), methylurolithin A (mUA) and urolithin B (UB), KYSE30 esophageal cancer cells were treated with urolithins + paclitaxel (PTX), + cisplatin (DDP), + different doses of IR or + heat-shock. Viability of cells was then determined by alamarBlue assay. To further elucidate the effects of UA, we used flow cytometry for investigation of induced apoptosis, and qRT-PCR for evaluating changes in the expression of HSP27, CCND1 and BCL2. Assessment of cell viability demonstrated that mUA increased the toxicity of PTX and DDP (up to 22.4 % and 20 %, respectively) and improved the effects of 6 Gy IR (26.5 %). Our main results achieved after UA treatment were improved toxicity of PTX and 6 Gy IR, beside enhanced effects of hyperthermia (37.3 %), which was confirmed by flow cytometry analysis and downregulation of HSP27, CCND1 and BCL2 expression. Taken together, our findings suggest that UA and mUA could be used as promising agents in combination with therapeutic modalities to improve the clinical outcomes of EC treatment.

Nano-selenium and Macleaya cordata Extracts Improved Immune Function and Reduced Oxidative Damage of Sows and IUGR Piglets After Heat Stress of Sows in Late Gestation.

To investigate the effects of nano-selenium (nano-Se) and Macleaya cordata extracts (MCE) on immune function and oxidative damage of sows and intrauterine growth retardation (IUGR) piglets exposed to heat stress (HS) in large-scale farms, a 2 × 2 factorial design was adopted in this test, and the two factors were nano-Se (0, 0.50 mg/kg) and MCE (0, 500 mg/kg). A total of 80 sows ([Landrace × Yorkshire] × Duroc, parity 2) were used in a 25-day trial from day 90 of gestation to delivery with 20 replications per group and 1 sow per replication. The dietary treatments of sows were as follows: (1) CON group, basic diet (0.30 mg/kg added Se, sodium selenite); (2) Nano-Se group, basic diet (0.00 mg/kg added Se) + 0.50 mg/kg added nano-Se; (3) MCE group, basic diet (0.00 mg/kg added Se) + 500 mg/kg added MCE; and (4) Combined group, basic diet (0.00 mg/kg added Se) + 0.50 mg/kg added nano-Se and 500 mg/kg added MCE. The activities of serum SOD, CAT, and GSH-Px of sows and IUGR piglets were significantly increased in MCE group and combined group, and the MDA content was extremely decreased. There were extreme differences in serum IgG level of sows and IUGR piglets, colostrum, and serum IgM level of IUGR piglets in MCE group and combined group compared with CON group. Maternal combined diets increased greatly the levels of serum IL-10 and IFN-γ of sows and IUGR piglets, and decreased extremely the contents of serum IL-1ß and TNF-α. MCE alone or combination with nano-Se in sow diets decreased greatly mRNA level of Hsp70 and increased mRNA level of Hsp27 in sows and IUGR piglets. In conclusion, nano-Se and/or MCE can be added to sow diets for the amelioration of HS-induced oxidative damage through improving immune function.

Expression of heat shock proteins 27, 60, and 70 in amphetamine and cocaine associated deaths.

INTRODUCTION: The hearts of amphetamine and cocaine users demonstrate essentially the same microscopic features: hypertrophy, interstitial fibrosis, myocyte hypertrophy and intimal and medial hyperplasia. According to Karch (2016), some investigations suggest that amphetamines have properties that make users less likely to experience myocardial infarction than cocaine users. The exposure to amphetamine is associated with the production of heat shock proteins (HSP) whereas cocaine is not. Not all the HSP are present in normal living conditions of cells but their expression is increased when cells are exposed to stress, like heat, anoxemia, and ischemia. It has been known before that increased HSP production is a myocardial response in adaptation to cardiac ischemia and that the production of HSP might influence myocardial resistance to infarction. Furthermore, production of HSP is an explanation of the known ability of amphetamines to cause hyperthermia. The hypothesis of a cytoprotective function of HSP in amphetamine-associated deaths in comparison to cocaine-associated deaths and controls was investigated. MATERIAL AND METHODS: Study group: 39 amphetamine-related fatal cases, 27 cocaine-associated deaths. CONTROL GROUP: 42 cases with other causes of death. Immunohistochemical staining of HSP 27, HSP 60, and HSP 70 in heart, liver, and kidney. RESULTS: 16 out of 39 (41.0%) amphetamine-related fatal cases showed a positive HSP expression, predominantly HSP 70 in myocardial tissue. In cocaine-associated deaths 15 out of 27 (55.5%) cases were positive, also mainly HSP 70. In the kidney in amphetamine-associated deaths 18 out of 39 (46.1%) cases were positive, in cocaine-associated deaths 21 out of 27 (77.7%) cases. The cocaine group showed significantly increased expression for HSP 27 and 70 in the liver and HSP 70 in the kidney compared to the control as well as amphetamine group. Furthermore, the cocaine group showed significantly increased expression for HSP 27 and 70 in the heart compared to the control but not the amphetamine group. CONCLUSION: The hypothesis of Karch that in amphetamine-associated deaths a positive HSP expression has in contrast to cocaine-related deaths a cytoprotective function cannot be verified. Furthermore, cocaine and benzoylecgonine seem to independently lead to an increased expression of HSP 27 both in the liver and in the heart.

The nephroprotective properties of taurine-amikacin treatment in rats are mediated through HSP25 and TLR-4 regulation.

Amikacin (AMK) is one of the most effective aminoglycoside antibiotics. However, nephrotoxicity is a major deleterious and dose-limiting side effect associated with its clinical use especially in high dose AMK-treated patients. The present study assessed the ability of taurine (TAU) to alleviate or prevent AMK-induced nephrotoxicity if co-administrated with AMK focusing on inflammation, apoptosis, and fibrosis. Male Sprague Dawley rats were assigned to six equal groups. Group 1: rats received saline (normal control), group 2: normal rats received 50 mg kg-1 TAU intraperitoneally (i.p.). Groups 3 and 4: received AMK (25 or 50 mg kg-1; i.p.). Groups 5 and 6: received TAU (50 mg kg-1; i.p.) concurrently with AMK (25 or 50 mg kg-1; i.p.) for 3 weeks. AMK-induced nephrotoxicity is evidenced by elevated levels of serum creatinine (CRE), blood urea nitrogen (BUN), and uric acid (UA). Histopathological investigations provoked damaging changes in the renal tissues. Heat shock proteins (HSP)25 and Toll-like receptor-4 (TLR-4) elevated levels were involved in the induction of inflammatory reactions and focal fibrosis. The improved activation of TLR-4 may stimulate monocytes to upgrade Interleukin (IL)-18 production rather than IL-10. TAU proved therapeutic effectiveness against AMK-induced renal toxicity through downregulation of HSP25, TLR-4, caspase-3, and IL-18 with up-regulation of IL-10 levels.

Neuroprotective Effects of Asparagus officinalis Stem Extract in Transgenic Mice Overexpressing Amyloid Precursor Protein.

To mimic Alzheimer's disease, transgenic mice overexpressing the amyloid precursor protein (APP) were used in this study. We hypothesize that the neuroprotective effects of ETAS®50, a standardized extract of Asparagus officinalis stem produced by Amino Up Co., Ltd. (Sapporo, Japan), are linked to the inhibition of the apoptosis cascade through an enhancement of the stress-response proteins: heat shock proteins (HSPs). APP-overexpressing mice (double-transgenic APP and PS1 mouse strains with a 129s6 background), ages 6-8 weeks old, and weighing 20-24 grams were successfully bred in our laboratory. The animals were divided into 5 groups. APP-overexpressing mice and wild-type (WT) mice were pretreated with ETAS®50 powder (50% elemental ETAS and 50% destrin) at 200 mg/kg and 1000 mg/kg body weight. Saline, the vehicle for ETAS®50, was administered in APP-overexpressing mice and WT mice. ETAS®50 and saline were administered by gavage daily for 1 month. Cognitive assessments, using the Morris Water Maze, demonstrated that memory was recovered following ETAS®50 treatment as compared to nontreated APP mice. At euthanization, the brain was removed and HSPs, amyloid ß, tau proteins, and caspase-3 were evaluated through immunofluorescence staining with the appropriate antibodies. Our data indicate that APP mice have cognitive impairment along with elevated amyloid ß, tau proteins, and caspase-3. ETAS®50 restored cognitive function in these transgenic mice, increased both HSP70 and HSP27, and attenuated pathogenic level of amyloid ß, tau proteins, and caspsase-3 leading to neuroprotection. Our results were confirmed with a significant increase in HSP70 gene expression in the hippocampus.

MicroRNA216b mediated downregulation of HSP27/STAT3/AKT signaling is critically involved in lambertianic acid induced apoptosis in human cervical cancers.

Since heat shock protein (HSP27) is a prognostic marker in cervical cancer, in the present study, the apoptotic mechanism of lambertianic acid (LA) was investigated in human cervical cancers in association with HSP27/STAT3/AKT signaling axis. LA exerted significant cytotoxicity, induced sub-G1 population, and increased the cleavage of Poly (ADP-ribose) polymerase (PARP) and cysteine aspartyl-specific protease 3 (caspase3) in HeLa and Caski cancer cells. Consistently, LA downregulated anti-apopotic genes such as B-cell lymphoma 2 (Bcl-2) and inhibitors of apoptosis proteins (c-IAP) in HeLa and Caski cells. Furthermore, LA-inhibited phosphorylation of HSP27, signal transducer, and activator of transcription 3 (STAT3) and Protein kinase B (AKT) through disturbing the binding of HSP27 with STAT3 or AKT in HeLa cells. Notably, LA upregulated the level of miR216b in HeLa and Caski cells. Consistently, miR216b mimic suppressed phosphorylation of HSP27 and reduced the expression of pro-PARP, while miR216b inhibitor reversed the ability of LA to attenuate phosphorylation of AKT, HSP27, and STAT3 and to reduce the expression of pro-PARP in HeLa cells. Overall, our findings suggest that miRNA216b mediated inhibition of HSP27/STAT3/ AKT signaling axis is critically involved in LA-induced apoptosis in cervical cancers.

Effect of high-dose vitamin D supplementation on antibody titers to heat shock protein 27 in adolescent girls.

Background Although vitamin D deficiency is associated with several inflammatory conditions, there have been few studies on the effects of vitamin D supplementation on markers of oxidative stress (OS) and inflammation. The aim of the current study was to evaluate the effects of high-dose vitamin D supplementation on heat shock protein 27 antibody (anti-Hsp27) titers in adolescent girls. Methods Five hundred and fifty adolescent girls received vitamin D3 at a dose of 50,000 IU/week for 9 weeks. Demographic, clinical and biochemical markers including serum fasting blood glucose (FBG), lipid profile and anti-Hsp27 titers as well as hematological parameters including white blood cell (WBC) count and red blood cell (RBC) distribution width (RDW) were determined in all the subjects at baseline and at the end of the study. Results Serum vitamin D significantly increased from 6.4 (4.2-9.6) ng/mL to 35.6 (25.8-47.5) ng/mL (p < 0.001) following the intervention. Furthermore, serum anti-Hsp27 titers were significantly lower after the 9-week vitamin D administration period (0.22 [0.12-0.33] optical density [OD] vs. 0.19 [0.11-0.31] OD; p = 0.002). A significant correlation was found between serum anti-Hsp27 and RDW (r = 0.13, p = 0.037). The reduction in RDW values after intervention was particularly evident in subjects with the greatest increase in serum vitamin D levels. Conclusions High-dose vitamin D supplementation was found to reduce antibody titers to Hsp27. Further randomized placebo-controlled trials are warranted to determine the long-term effect of vitamin D administration on the inflammatory process especially that associated with chronic disease.

Phosphoproteome Analysis Reveals Dynamic Heat Shock Protein 27 Phosphorylation in Tanshinone IIA-Induced Cell Death.

Gastric cancer is one of the most common types of cancer worldwide. Nevertheless, effective therapeutic strategies have not yet been discovered. Several studies have shown that tanshinone IIA (TIIA), which is extracted from the traditional herbal medicine plant Danshen (Salvia miltiorrhiza), has potential activity against many kinds of cancer. Our previous research demonstrated that TIIA can induce cell death in gastric cancer. However, the exact signaling pathway response is still unclear. Post-translational modification (PTM) plays a significant role in a wide range of physiological processes in cancer, via regulation of both signal transduction cascades and many cellular pathways. Here, we integrated multilayer omics-transcriptomics and dynamic phosphoproteomics-to elucidate the regulatory networks triggered by TIIA in gastric cancer. We identified the phosphorylation of heat shock protein 27 (HSP27) at serine 82 in response to TIIA, which caused reactive oxygen species (ROS) production and unfolded protein response (UPR). Moreover, the accumulation of cellular stress increased the expression of heat shock factor 1 (HSF1). In addition, the downstream targets of HSF1, which were involved in heat shock stress and apoptosis, were also activated in TIIA-treated cells. In conclusion, this study performs a multiomic approach to clarify a comprehensive TIIA-responsive network leading to cell death in gastric cancer.

Adverse Effects in Skeletal Muscle Following the Medicinal Use of Nicotiana glauca.

Nicotiana glauca is a cosmopolitan shrub, used in medicine to treat swellings, wounds, sores and cancer. However, its users lack of knowledge of the adverse effects. We seek to evaluate the effects of lipid extracts from N. glauca on myoblasts, identifying the compounds which cause undesirable effects. Myoblasts are important in muscle homeostasis, thus a high death rate of them cause myopathies. We performed an ethanolic extraction from leaves of N. glauca and the extract was successively partitioned with hexane, chloroform and ethyl acetate. The effects of extracts in C2C12 cells were analysed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL), Mitotracker and 4',6-diamidino-2-phenylindole (DAPI) staining, Western blotting, real-time PCR and immunofluorescence assays. Caspase activity was studied. The fraction with the highest apoptotic effects was analysed by chromatography, NMR and GC-MS spectrometry were used to identify the apoptotic agent, after which its biological activity was evaluated. The extracts from N. glauca induced apoptosis in C2C12 cells involving caspase-3/7. We found that the extracts trigger a defence response in muscle through Akt and heat shock protein 27 (HSP27). We identified an apoptotic agent as palmitic acid. These data suggest that the use of N. glauca in hormone replacement therapy, or in other therapies affects skeletal muscle homeostasis, worsening the negative effects of the menopause. Thus, the relevance of this work lies in the fact that it is the first time that a report about the molecular mechanism responsible for the side effects of medicinal use of N. glauca, has been shown. Moreover the compound responsible for these effects has been identified.

Novel compound VB-037 inhibits Aß aggregation and promotes neurite outgrowth through enhancement of HSP27 and reduction of P38 and JNK-mediated inflammation in cell models for Alzheimer's disease.

The pathogenesis of Alzheimer's disease (AD) is involved in the aggregation of misfolded amyloid ß (Aß), which upregulates the activity of acetylcholinesterase (AChE), increases the production of reactive oxygen species (ROS), enhances neuroinflammation, and eventually leads to neuronal death. Therefore, compounds targeting these mechanisms may be candidates for multitarget drugs in AD treatment. We found that two quinoline derivatives, VB-030 and VB-037, markedly reduced Aß aggregation and ROS levels in the thioflavin T biochemical assay and Tet-On Aß-green fluorescent protein (GFP) 293 AD cell model. These compounds further improved neurite outgrowth, reduced AChE activity and upregulated the molecular chaperone heat shock protein family B [small] member 1 (HSP27), whereas knockdown of HSP27 counteracted the compounds' neuroprotective effects on the Tet-On Aß-GFP SH-SY5Y AD neuronal model. Furthermore, VB-037 attenuated lipopolysaccharide (LPS)/interferon (IFN)-γ-induced activation of BV-2 microglial cells. In addition, VB-037 demonstrated its potential to diminish LPS/IFN-γ-induced upregulation of caspase 1 activity, expression of interleukin (IL)-1ß, and active phosphorylation of mitogen-activated protein kinase 14 (P38), mitogen-activated protein kinase 8 (JNK), and Jun proto-oncogene, AP-1 transcription factor subunit (JUN) signalings, as well as improve cell viability in the Tet-On Aß-GFP SH-SY5Y AD neuronal model. Our findings strongly indicate the potential of VB-037 for modifying AD progression by targeting multiple mechanisms, thereby offering a new drug development avenue for AD treatment.

Curcumin Reverses 5-Fluorouracil Resistance by Promoting Human Colon Cancer HCT-8/5-FU Cell Apoptosis and Down-regulating Heat Shock Protein 27 and P-Glycoprotein.

OBJECTIVE: To investigate the potential mechanisms that curcumin reverses 5-fluorouracil (5-FU) multidrug resistance (MDR). METHODS: Cell growth and the inhibitory rate of curcumin (2-25 µg/mL) and/or 5-FU (0.05-1000 µg/mL) on human colon cancer HCT-8 and HCT-8/5-FU (5-FU-resistant cell line) were determined using cell counting kit-8 (CCK-8) assay. Apoptosis and cell cycle after 5-FU and/or curcumin treatment were detected by flow cytometry (FCM) and transmission electron microscopy (TEM). The expression of the multidrug resistance related factors p-glycoprotein (P-gp) and heat shock protein 27 (HSP-27) genes and proteins were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting (WB), respectively. RESULTS: The inhibitory rate of curcumin or 5-FU on HCT-8 and HCT-8/5-FU cells proliferation at exponential phase were in a dosedependent manner, HCT-8 cell line was more sensitive to curcumin or 5-FU when compared the inhibitory rate of HCT-8/5-FU. The 50% inhibitory concentration (IC50) of combination 5-FU and curcumin (4.0 µg/mL) in HCT-8/5-FU was calculated as 179.26 µg/mL, with reversal fold of 1.85. Another IC50 of combination 5-FU and curcumin (5.5 µg/mL) in HCT-8/5-FU was calculated as 89.25 µg/mL, with reversal fold of 3.71. Synergistic effect of 5-FU and curcumin on HCT-8 and HCT-8/5-FU cells were found. The cell cycle analysis performed by FCM showed that HCT-8 and HCT-8/5-FU cells mostly accumulated at G0/G1 phase, which suggested a synergistic effect of curcumin and 5-FU to induce apoptosis. FCM analysis found that the percentage of apoptosis of cells treated with curcumin, 5-FU and their combination were significantly increased compared to the control group (P<0.05), and the percentage of apoptosis of the combination groups were slightly higher than other groups (P<0.05). The mRNA levels of P-gp (0.28±0.02) and HSP-27 (0.28±0.09) in HCT-8/5-FU cells treated with combination drugs were lower than cells treated with 5-FU alone (P-gp, 0.48±0.07, P=0.009; HSP-27, 0.57±0.10, P=0.007). The protein levels of P-gp (0.25±0.06) and HSP-27 (0.09±0.02) in HCT-8/5-FU cells treated with combination drugs were decreased when compared to 5-FU alone (P-gp, 0.46±0.02, P=0.005; HSP-27, 0.43±0.01, P=0.000). CONCLUSIONS: Curcumin can inhibit the proliferation of human colon cancer cells. Curcumin has the ability of reversal effects on the multidrug resistance of human colon cancer cells lines HCT-8/5-FU. Down-regulation of P-gp and HSP-27 may be the mechanism of curcumin reversing the drug resistance of HCT-8/5-FU to 5-FU.

Zhenbao Pill reduces Treg cell proportion in acute spinal cord injury rats by regulating TUG1/miR-214/HSP27 axis.

Background: Acute spinal cord injury (SCI) is one of the weakest pathologies that seriously affect the quality of life of patients. Objective: To study the mechanism of how Zhenbao Pill reduces Treg cell proportion and improves acute SCI. Methods: A rat SCI model was established. Flow cytometry analysis was performed to determine the Treg cell proportion. RNA immunoprecipitation (RIP) and RNA pull-down were applied in confirming taurine up-regulated gene 1 (TUG1) and miR-214 binding. Intrathecal injection of TUG1 siRNA was also conducted to determine the effect of TUG1 in vivoResults: Zhenbao Pill promoted the expression of TUG1 and heat shock protein 27 (HSP27) protein, and reduced the expression of miR-214 and forkhead box protein p3 (Foxp3) as well as Treg cell proportion in a concentration-dependent manner in SCI rats or in vitro cultured CD4+ T cells. Knockdown of TUG1 reversed the high protein expression of HSP27 and the inhibition of Treg cell proportion as well as Foxp3 protein induced by Zhenbao Pill, and miR-214 inhibitor canceled the TUG1 knockdown effect. Further, miR-214 mimic reversed the inhibition of Treg cell proportion and Foxp3 protein expression by Zhenbao Pill, which was abolished by the overexpression of HSP27. The mechanism was validated in animal experiments. Conclusion: Zhenbao Pill regulated TUG1/miR-214/HSP27 signaling pathway to reduce Treg cell proportion and thus relieve acute SCI.

Dependence of HSP27 cellular level on protein kinase CK2 discloses novel therapeutic strategies.

BACKGROUND: HSP27 plays a role in various diseases, including neurodegenerative diseases, ischemia, and atherosclerosis. It is particularly important in the regulation of the development, progression and metastasis of cancer as well as cell apoptosis and drug resistance. However, the absence of an ATP binding domain, that is, instead, present in other HSPs such as HSP90 and HSP70, hampers the development of small molecules as inhibitors of HSP27. METHODS: Knockout cell lines generated by Crispr/Cas9 gene editing tool, specific kinase inhibitors and siRNA transfections were exploited to demonstrate that the expression of HSP27 is dependent on the integrity/activity of protein kinase CK2 holoenzyme. The interaction between these proteins has been confirmed by co-immunoprecipitation, confocal immunofluorescence microscopy, and by density gradient separation of protein complexes. Finally, using a proliferation assay this study demonstrates the potential efficacy of a combinatory therapy of heath shock and CK2 inhibitors in cancer treatment. RESULTS: Our data demonstrate that CK2 is able to regulate HSP27 turnover by affecting the expression of its ubiquitin ligase SMURF2 (Smad ubiquitination regulatory factor 2). Moreover, for the first time we show an increased sensitivity of CK2-inhibited tumour cells to hyperthermia treatment. CONCLUSION: Being HSP27 involved in several pathological conditions, including protein conformational diseases (i.e Cystic Fibrosis) and cancer, the need of drugs to modulate its activity is growing and CK2-targeting could represent a new strategy to reduce cellular HSP27 level. GENERAL SIGNIFICANCE: This study identifies CK2 as a molecular target to control HSP27 cellular expression.

Chemotherapy Sensitizes Therapy-Resistant Cells to Mild Hyperthermia by Suppressing Heat Shock Protein 27 Expression in Triple-Negative Breast Cancer.

Purpose: Triple-negative breast cancer (TNBC) is a clinically aggressive disease with poor prognosis. Conventional chemotherapeutics are generally able to shrink the tumor mass, but often fail to completely eradicate cancer stem-like cells (CSCs) that are responsible for high risk of relapse and frequent metastases. In this study, we examined thermal sensibility of CSCs, developed an approach that enabled concurrent elimination of both the bulk of cancer cells and CSCs, and investigated the underlying mechanism.Experimental Design: We designed a platform consisting of gold nanoparticle-coated porous silicon microparticle (AuPSM) that was also loaded with docetaxel micelles (mDTXs) to enable concurrent killing of the bulk of cancer cells by released mDTX and CSCs by mild hyperthermia upon stimulation of AuPSM with near infrared. In addition, we examined the role of heat shock proteins in sensitizing CSC killing. Finally, we applied mDTX-loaded AuPSM to treat mice with SUM159 and 4T1 orthotopic tumors and evaluated tumor growth and tumor metastasis.Results: MDA-MB-231 and SUM159 TNBC cells treated with mDTX-loaded AuPSM and mild hyperthermia displayed significantly reduced efficiencies in mammosphere formation than those treated with mDTX alone or mild hyperthermia alone. Combination treatment also completely inhibited SUM159 orthotopic tumor growth and 4T1 tumor metastasis. Mechanistically, DTX treatment suppressed expression of heat shock protein 27 in cancer cells including the CSCs, rendering cells sensitive to mild hyperthermia.Conclusions: Our results indicate that chemotherapy sensitizes CSC to mild hyperthermia. We have developed an effective therapeutic approach to eliminate therapy-resistant cells in TNBC. Clin Cancer Res; 24(19); 4900-12. ©2018 AACR.