Heat Shock Pathway Research Articles

Beyond the heat shock pathway: Heat stress responses in Drosophila development

Beyond the heat shock pathway: Heat stress responses in Drosophila development

Steroid responsiveness in alcohol-associated hepatitis is linked to glucocorticoid metabolism, mitochondrial repair, and heat shock proteins.

Alcohol-associated hepatitis (AH) is one of the clinical presentations of alcohol-associated liver disease. AH has poor prognosis, and corticosteroids remain the mainstay of drug therapy. However, ~40% of patients do not respond to this treatment, and the mechanisms underlying the altered response to corticosteroids are not understood. The current study aimed to identify changes in hepatic protein expression associated with responsiveness to corticosteroids and prognosis in patients with AH. Patients with AH were enrolled based on the National Institute on Alcohol Abuse and Alcoholism inclusion criteria for acute AH and further confirmed by a diagnostic liver biopsy. Proteomic analysis was conducted on liver samples acquired from patients with AH grouped as nonresponders (AH-NR, n = 7) and responders (AH-R, n = 14) to corticosteroids, and nonalcohol-associated liver disease controls (n = 10). The definition of responders was based on the clinical prognostic model, the Lille Score, where a score < 0.45 classified patients as AH-R and a score > 0.45 as AH-NR. Primary outcomes used to assess steroid response were Lille Score (eg, improved liver function) and survival at 24 weeks. Reduced levels of the glucocorticoid receptor and its transcriptional co-activator, glucocorticoid modulatory element-binding protein 2, were observed in the hepatic proteome of AH-NR versus AH-R. The corticosteroid metabolizing enzyme, 11-beta-hydroxysteroid dehydrogenase 1, was increased in AH-NR versus AH-R along with elevated mitochondrial DNA repair enzymes, while several proteins of the heat shock pathway were reduced. Analysis of differentially expressed proteins in AH-NR who survived 24 weeks relative to AH-NR nonsurvivors revealed several protein expression changes, including increased levels of acute phase proteins, elevated coagulation factors, and reduced mast cell markers. This study identified hepatic proteomic changes that may predict responsiveness to corticosteroids and mortality in patients with AH.

Exploring Potential Molecular Mechanisms of Drug Response in FLT3-ITD Negative AML Patients Treated with Quizartinib Vs Placebo Plus Standard Chemotherapy in the Quiwi Trial

Introduction Wild-type FLT3 AML presents significant therapeutic challenges due to limited treatment options. The QUIWI trial investigated quizartinib (Quiz) combined with standard chemotherapy, showing promising results. To understand drug response and refractoriness, we conducted a correlative analysis, exploring differential expression patterns among distinct patient groups. We reasoned that the molecular fingerprints of good responders to Quiz plus chemotherapy would be different with respect to patients who respond only to standard chemotherapy, helping to elucidate critical molecular mechanisms associated with Quiz response. Our study provides a basis for personalized therapeutic strategies in FLT3-ITD negative AML. Methods We performed RNA sequencing (RNAseq) analysis on a subset of patients from the clinical trial. RNA was extracted using standard methods, followed by assessment of nucleic acid integrity (TapeStation) and quantification (Qubit). Total mRNA sequencing was performed using polyA RNAseq with TruSeq technology. A total of 206 adequate samples from bone marrow and peripheral blood were sequenced, out of which 55 cases were FLT3-ITD and were discarded for this analysis. For this analysis, only samples with a minimum of 20% blasts were considered. The sequences were aligned to the GRCh37 reference genome using the Hisat algorithm. Differential expression was assessed with the DESeq2 algorithm, using Blast proportion to adjust for disparities among the samples. P-values were adjusted for multiple testing using the FDR method. OS was defined as time from start of screening to death. Gene ontology and pathways analysis were performed using the WebGestalt portal. Results In this study, we compared the gene expression profiles of Quiz versus placebo-treated patients categorized as good responders, defined arbitrarily as those who continue alive and have a minimum follow-up of 6 months after diagnosis. Out of 82 eligible patients, we identified 100 (q-value &amp;lt; 0.05) and 172 (q-value &amp;lt; 0.1) differentially expressed genes between the Quiz and Placebo groups. Notably, 132 genes were overexpressed in the Quiz group, while 40 genes showed overexpression in the placebo group. Moreover, we observed a highly significant overexpression of ribosomal genes in the Quiz group (17 overlaps, KEGG pathways q-value &amp;lt; 2.2 x 10 -16), and a significant overexpression of genes related to the attenuation of the heat shock protein transcriptional response, which is involved in protein folding, in the placebo group (Reactome q-value, 0.008; gene ids: HSPA1A, HSPA1B and DNAJB1). Additionally, the Quiz group exhibited a significant overexpression of transmembrane receptor protein tyrosine phosphatase-related genes (Gene Ontology Molecular Function q-value, 0.02; gene ids: PTPRB, PTPRG and PTPRU). Conclusion This correlative analysis of the Quiwi trial revealed critical molecular mechanisms associated with Quiz response. The overexpression of ribosomal and transmembrane receptor protein tyrosine phosphatase-related genes in good responders within the Quiz group, along with the overexpression of the heat shock pathway genes in the placebo group, aligns with existing knowledge about the FLT3 pathway biology and the molecular determinants to tyrosine-kinase inhibitors. These findings offer potential biomarkers for personalized therapeutic approaches to improve clinical outcomes in this challenging AML population.

Differential Regulations of Antioxidant Metabolism and Cold-Responsive Genes in Three Bermudagrass Genotypes under Chilling and Freezing Stress.

As a typical warm-season grass, bermudagrass growth and turf quality begin to decrease when the environmental temperature drops below 20 °C. The current study investigated the differential responses of three bermudagrass genotypes to chilling stress (8/4 °C) for 15 days and then freezing stress (2/-2 °C) for 2 days. The three genotypes exhibited significant variation in chilling and freezing tolerance, and Chuannong-3, common bermudagrass 001, and Tifdwarf were ranked as cold-tolerant, -intermediate, and -sensitive genotypes based on evaluations of chlorophyll content, the photochemical efficiency of photosystem II, oxidative damage, and cell membrane stability, respectively. Chuannong-3 achieved better tolerance through enhancing the antioxidant defense system to stabilize cell membrane and reactive oxygen species homeostasis after being subjected to chilling and freezing stresses. Chuannong-3 also downregulated the ethylene signaling pathway by improving CdCTR1 expression and suppressing the transcript levels of CdEIN3-1 and CdEIN3-2; however, it upregulated the hydrogen sulfide signaling pathway via an increase in CdISCS expression under cold stress. In addition, the molecular basis of cold tolerance could be associated with the mediation of key genes in the heat shock pathway (CdHSFA-2b, CdHSBP-1, CdHSP22, and CdHSP40) and the CdOSMOTIN in Chuannong-3 because the accumulation of stress-defensive proteins, including heat shock proteins and osmotin, plays a positive role in osmoprotection, osmotic adjustment, or the repair of denatured proteins as molecular chaperones under cold stress. The current findings give an insight into the physiological and molecular mechanisms of cold tolerance in the new cultivar Chuannong-3, which provides valuable information for turfgrass breeders and practitioners.

USP14 Positively Modulates Head and Neck Squamous Carcinoma Tumorigenesis and Potentiates Heat Shock Pathway through HSF1 Stabilization.

The ubiquitin-proteasome system is a pivotal intracellular proteolysis process in posttranslational modification. It regulates multiple cellular processes. Deubiquitinating enzymes (DUBs) are a stabilizer in proteins associated with tumor growth and metastasis. However, the link between DUBs and HNSCC remains incompletely understood. In this study, therefore, we identified USP14 as a tumor proliferation enhancer and a substantially hyperactive deubiquitinase in HNSCC samples, implying a poor prognosis prediction. Silencing USP14 in vitro conspicuously inhibited HNSCC cell proliferation and migration. Consistently, defective USP14 in vivo significantly diminished HNSCC tumor growth and lung metastasis compared to the control group. Luciferase assays indicated that HSF1 was downstream from USP14, and an evaluation of the cellular effects of HSF1 overexpression in USP14-dificient mice tumors showed that elevated HSF1 reversed HNSCC growth and metastasis predominantly through the HSF1-HSP pathway. Mechanistically, USP14 encouraged HSF1 expression by deubiquitinating and stabilizing HSF1, which subsequently orchestrated transcriptional activation in HSP60, HSP70, and HSP90, ultimately leading to HNSCC progression and metastasis. Collectively, we uncovered that hyperactive USP14 contributed to HNSCC tumor growth and lung metastasis by reinforcing HSF1-depedent HSP activation, and our findings provided the insight that targeting USP14 could be a promising prognostic and therapeutic strategy for HSNCC.

Investigating the molecular mechanisms behind the transcriptional response in pressure adaptation.

Despite 80% of Earth's microbial biomass existing in high pressure habitats in the Deep Ocean and within the continental crust, the mechanisms that allow for survival and growth in these thermodynamically unfavorable environments are poorly understood. Moreover, pressure resistance in pathogenic bacteria commonly found in food, such as Listeria monocytogenes and Escherichia coli, has emerged as more foods are sterilized by high pressure, rather than high temperature. To understand pressure adaptation, we have begun studying the molecular mechanisms of transcriptional adaptation to pressure. One important pathway in the pressure response is the heat shock pathway. To compare the heat shock response to pressure and temperature, we generated green fluorescent protein (GFP) promoter fusions of the central sigma factor genes’ transcriptional promoters and those of their downstream effectors. Activity of these promoter fusions was evaluated after heat shock and after pressure shock using scanning number and brightness (sN&B) fluorescence microscopy to determine the absolute number of GFP molecules produced in single cells and compared to the absolute number of GFP molecules produced in no shock conditions. Assuming transcriptional bursting at these promoters arises from release of supercoiling tension, we calculated the burst size and burst frequency from the distribution of the number of GFP molecules produced in each cell under each condition. These studies were carried out in the mesophilic E. coli MG1655 and in a moderately piezophilic (pressure-loving) strain adapted from MG1655, AN62. Studying both strains allowed us to quantify how the transcriptional response to changes in hydrostatic pressure occurs in both mesophiles and piezophiles, providing a more holistic model of the pressure response and adaptation phenomena.

From Natural Behavior to Drug Screening: Invertebrates as Models to Study Mechanisms Associated with Alcohol Use Disorders.

Humans consume ethanol-containing beverages, which may cause an uncontrollable or difficult-to-control intake of ethanol-containing liquids and may result in alcohol use disorders. How the transition at the molecular level from "normal" ethanol-associated behaviors to addictive behaviors occurs is still unknown. One problem is that the components contributing to normal ethanol intake and their underlying molecular adaptations, especially in neurons that regulate behavior, are not clear. The fruit fly Drosophila melanogaster and the earthworm Caenorhabditis elegans show behavioral similarities to humans such as signs of intoxication, tolerance, and withdrawal. Underlying the phenotypic similarities, invertebrates and vertebrates share mechanistic similarities. For example in Drosophila melanogaster, the dopaminergic neurotransmitter system regulates the positive reinforcing properties of ethanol and in Caenorhabditis elegans, serotonergic neurons regulate feeding behavior. Since these mechanisms are fundamental molecular mechanisms and are highly conserved, invertebrates are good models for uncovering the basic principles of neuronal adaptation underlying the behavioral response to ethanol. This review will focus on the following aspects that might shed light on the mechanisms underlying normal ethanol-associated behaviors. First, the current status of what is required at the behavioral and cellular level to respond to naturally occurring levels of ethanol is summarized. Low levels of ethanol delay the development and activate compensatory mechanisms that in turn might be beneficial for some aspects of the animal's physiology. Repeated exposure to ethanol however might change brain structures involved in mediating learning and memory processes. The smell of ethanol is already a key component in the environment that is able to elicit behavioral changes and molecular programs. Minimal networks have been identified that regulate normal ethanol consumption. Other environmental factors that influence ethanol-induced behaviors include the diet, dietary supplements, and the microbiome. Second, the molecular mechanisms underlying neuronal adaptation to the cellular stressor ethanol are discussed. Components of the heat shock and oxidative stress pathways regulate adaptive responses to low levels of ethanol and in turn change behavior. The adaptive potential of the brain cells is challenged when the organism encounters additional cellular stressors caused by aging, endosymbionts or environmental toxins or excessive ethanol intake. Finally, to underline the conserved nature of these mechanisms between invertebrates and higher organisms, recent approaches to identify drug targets for ethanol-induced behaviors are provided. Already approved drugs regulate ethanol-induced behaviors and they do so in part by interfering with cellular stress pathways. In addition, invertebrates have been used to identify new compounds targeting molecules involved in the regulation in ethanol withdrawal-like symptoms. This review primarily highlights the advances of the last 5years concerning Drosophila melanogaster, but also provides intriguing examples of Caenorhabditis elegans and Apis mellifera in support.

Elucidating host cell response pathways and repurposing therapeutics for SARS-CoV-2 and other coronaviruses

COVID-19, first reported in late 2019, is an ongoing pandemic that has been causing devastation across the globe. Although there are multiple vaccines that can prevent severe symptoms, effective COVID-19 therapeutics are still of importance. Using our proprietary in silico engine, we screened more than 22,000 unique compounds represented by over half a million gene expression profiles to uncover compounds that can be repurposed for SARS-CoV-2 and other coronaviruses in a timely and cost-efficient manner. We then tested 13 compounds in vitro and found three with potency against SARS-CoV-2 with reasonable cytotoxicity. Bortezomib and homoharringtonine are some of the most promising hits with IC50 of 1.39 μM and 0.16 μM, respectively for SARS-CoV-2. Tanespimycin and homoharringtonine were effective against the common cold coronaviruses. In-depth analysis highlighted proteasome, ribosome, and heat shock pathways as key targets in modulating host responses during viral infection. Further studies of these pathways and compounds have provided novel and impactful insights into SARS-CoV-2 biology and host responses that could be further leveraged for COVID-19 therapeutics development.

Systematic Interrogation of the Temperature Perturbation in the Insulin Signaling Pathway for Optogenetic Stimulation.

The application of NIR to optogenetic systems is in great demand due to its superior properties enabling in vivo deep tissue penetration. Irradiation of NIR to tissue samples or cells rapidly generates heat locally. The resultant elevation in temperature affects cells at the molecular level because of the activation of the heat shock pathway and ROS generation. Nevertheless, few reports have presented detailed comparisons of the effects of the temperature change rate on signaling pathway biomolecules, especially those of rapid heat changes. Aiming at broadening the understanding of temperature sensitivity, we investigated seven insulin signaling pathway biomolecules (INSR, IRS1, Akt, GSK3β, p70S6K, FoxO1, and ERK1/2) in three cell lines (C2C12, HepG2, and Fao) at temperatures between 25 and 45 °C. The results show that, except for INSR, pAkt(T308), and FoxO1, biomolecules are sensitive to rapid temperature changes at temperatures higher than 42 °C, at which they are significantly phosphorylated. At 25 °C, around a 50% reduction in phosphorylation occurred. Moreover, p70S6K is sensitive over time. It dephosphorylates quickly (5 min) and then phosphorylates over time. Our findings extend the temperature range to 45 °C, while providing additional time course information about the signaling pathway biomolecule response necessary to advance NIR optogenetic research.

Most cancers carry a substantial deleterious load due to Hill-Robertson interference.

Cancer genomes exhibit surprisingly weak signatures of negative selection (Martincorena et al., 2017; Weghorn, 2017). This may be because selective pressures are relaxed or because genome-wide linkage prevents deleterious mutations from being removed (Hill-Robertson interference; Hill and Robertson, 1966). By stratifying tumors by their genome-wide mutational burden, we observe negative selection (dN/dS ~ 0.56) in low mutational burden tumors, while remaining cancers exhibit dN/dS ratios ~1. This suggests that most tumors do not remove deleterious passengers. To buffer against deleterious passengers, tumors upregulate heat shock pathways as their mutational burden increases. Finally, evolutionary modeling finds that Hill-Robertson interference alone can reproduce patterns of attenuated selection and estimates the total fitness cost of passengers to be 46% per cell on average. Collectively, our findings suggest that the lack of observed negative selection in most tumors is not due to relaxed selective pressures, but rather the inability of selection to remove deleterious mutations in the presence of genome-wide linkage.

Transcriptome analysis of Epichloë strains in tall fescue in response to drought stress

ABSTRACT Epichloë coenophiala, a systemic fungal symbiont (endophyte) of tall fescue (Lolium arundinaceum), has been documented to confer to this grass better persistence than plants lacking the endophyte, especially under stress conditions such as drought. The response, if any, of the endophyte to imposition of stress on the host plant has not been characterized previously. Therefore, we investigated effects on gene expression by E. coenophiala and a related endophyte when plant-endophyte symbiota were subjected to acute water-deficit stress. Plants harboring different endophyte strains were grown in sand in the greenhouse, then half were deprived of water for 48 h and the other half were watered controls. RNA was isolated from different plant tissues, and mRNA sequencing (RNA-seq) was conducted to identify genes that were differentially expressed comparing stress treatment with control. We compared two different plants harboring the common toxic E. coenophiala strain (CTE) and two non–ergot-alkaloid-producing Epichloë strains in tall fescue pseudostems, and in a second experiment we compared responses of E. coenophiala CTE in plant pseudostem and crown tissues. The endophytes responded to the stress with increased expression of genes involved in oxidative stress response, oxygen radical detoxification, C-compound carbohydrate metabolism, heat shock, and cellular transport pathways. The magnitude of fungal gene responses during stress varied among plant-endophyte symbiota. Responses in pseudostems and crowns involved some common pathways as well as some tissue-specific pathways. The fungal response to water-deficit stress involved gene expression changes in similar pathways that have been documented for plant stress responses, indicating that Epichloë spp. and their host plants either coordinate stress responses or separately activate similar stress response mechanisms that work together for mutual protection.

The combination of hypoxia and high temperature affects heat shock, anaerobic metabolism, and pentose phosphate pathway key components responses in the white shrimp (Litopenaeus vannamei).

Due to global warming, world water bodies have higher temperatures and lower oxygen concentrations that affect aquatic species including the white shrimp Litopenaeus vannamei. This species withstands these conditions, but the information of the physiological responses that allow them to survive are scarce. We analyzed the effects of high temperature, hypoxia, reoxygenation, and the combination of these factors on the relative expression of selected genes: HSF1, Hsp70, p53, TIGAR, HIF-1α, and VEGF1-3 in gills of L. vannamei. Additionally, glucose, lactate, NADP, and NADPH were determined. HSF1 was up-regulated in the high temperature and oxygen stress conditions, but Hsp70 was up-regulated only in reoxygenation at both temperatures. HIF-1α was also up-regulated by reoxygenation in both temperatures. Meanwhile, the VEGF genes were not altered by the stress conditions, since none of them changed expression drastically. p53 relative expression remained stable at the tested stress conditions, which prompts to the maintenance of antioxidant defenses. TIGAR expression was induced in normoxia and hypoxia at high temperature, which induced NADPH content helping to scavenge reactive oxygen species (ROS). Additionally, high temperature caused higher glucose and lactate content in normoxia and hypoxia, indicating carbohydrate mobilization and a switch to anaerobic metabolism. The results showed that HSF1, the anaerobic metabolism and the pentose phosphate pathway (PPP) are crucial for the shrimp response to these abiotic stress conditions and contribute to their survival.

Chitosan (CTS) Alleviates Heat-Induced Leaf Senescence in Creeping Bentgrass by Regulating Chlorophyll Metabolism, Antioxidant Defense, and the Heat Shock Pathway.

Chitosan (CTS) is a deacetylated derivative of chitin that is involved in adaptive response to abiotic stresses. However, the regulatory role of CTS in heat tolerance is still not fully understood in plants, especially in grass species. The aim of this study was to investigate whether the CTS could reduce heat-induced senescence and damage to creeping bentgrass associated with alterations in antioxidant defense, chlorophyll (Chl) metabolism, and the heat shock pathway. Plants were pretreated exogenously with or without CTS (0.1 g L−1) before being exposed to normal (23/18 °C) or high-temperature (38/33 °C) conditions for 15 days. Heat stress induced detrimental effects, including declines in leaf relative water content and photochemical efficiency, but significantly increased reactive oxygen species (ROS) accumulation, membrane lipid peroxidation, and Chl loss in leaves. The exogenous application of CTS significantly alleviated heat-induced damage in creeping bentgrass leaves by ameliorating water balance, ROS scavenging, the maintenance of Chl metabolism, and photosynthesis. Compared to untreated plants under heat stress, CTS-treated creeping bentgrass exhibited a significantly higher transcription level of genes involved in Chl biosynthesis (AsPBGD and AsCHLH), as well as a lower expression level of Chl degradation-related gene (AsPPH) and senescence-associated genes (AsSAG12, AsSAG39, Asl20, and Ash36), thus reducing leaf senescence and enhancing photosynthetic performance under heat stress. In addition, the foliar application of CTS significantly improved antioxidant enzyme activities (SOD, CAT, POD, and APX), thereby effectively reducing heat-induced oxidative damage. Furthermore, heat tolerance regulated by the CTS in creeping bentgrass was also associated with the heat shock pathway, since AsHSFA-6a and AsHSP82 were significantly up-regulated by the CTS during heat stress. The potential mechanisms of CTS-regulated thermotolerance associated with other metabolic pathways still need to be further studied in grass species.

Metabolic and Molecular Subacute Effects of a Single Moderate-Intensity Exercise Bout, Performed in the Fasted State, in Obese Male Rats.

Introduction and objectives: Obesity represents a major global public health problem. Its etiology is multifactorial and includes poor dietary habits, such as hypercaloric and hyperlipidic diets (HFDs), physical inactivity, and genetic factors. Regular exercise is, per se, a tool for the treatment and prevention of obesity, and recent studies suggest that the beneficial effects of exercise can be potentiated by the fasting state, thus potentially promoting additional effects. Despite the significant number of studies showing results that corroborate such hypothesis, very few have evaluated the effects of fasted-state exercise in overweight/obese populations. Therefore, the aim of this study was to evaluate the subacute effects (12 h after conclusion) of a single moderate-intensity exercise bout, performed in either a fed or an 8 h fasted state, on serum profile, substrate-content and heat shock pathway–related muscle protein immunocontent in obese male rats. Methods: Male Wistar rats received a modified high-fat diet for 12 weeks to induce obesity and insulin resistance. The animals were allocated to four groups: fed rest (FER), fed exercise (FEE), fasted rest (FAR) and fasted exercise (FAE). The exercise protocol was a 30 min session on a treadmill, with an intensity of 60% of VO2max. The duration of the fasting period was 8 h prior to the exercise session. After a 12 h recovery, the animals were killed and metabolic parameters of blood, liver, heart, gastrocnemius and soleus muscles were evaluated, as well as SIRT1 and HSP70 immunocontent in the muscles. Results: HFD induced obesity and insulin resistance. Soleus glycogen concentration decreased in the fasted groups and hepatic glycogen decreased in the fed exercise group. The combination of exercise and fasting promoted a decreased concentration of serum total cholesterol and triglycerides. In the heart, combination fasting plus exercise was able to decrease triglycerides to control levels. In the soleus muscle, both fasting and fasting plus exercise were able to decrease triglyceride concentrations. In addition, heat shock protein 70 and sirtuin 1 immunocontent increased after exercise in the gastrocnemius and soleus muscles. Conclusions: An acute bout of moderate intensity aerobic exercise, when realized in fasting, may induce, in obese rats with metabolic dysfunctions, beneficial adaptations to their health, such as better biochemical and molecular adaptations that last for at least 12 h. Considering the fact that overweight/obese populations present an increased risk of cardiovascular events/diseases, significant reductions in such plasma markers of lipid metabolism are an important achievement for these populations.

The Novel Protein HSF1 Stress Pathway Inhibitor Bisamide CCT361814 Demonstrates Pre-Clinical Anti-Tumor Activity in Myeloma

IntroductionMultiple myeloma (MM) is a plasma cell malignancy, characterized by the constant production of immunoglobulins (Ig), making the tumor cells reliant on protein homeostasis for their survival. MM cells are therefore highly susceptible to therapies that target protein homeostasis, such as proteasome inhibitors. Several protein homeostasis mechanisms, when disturbed, converge on the heat shock pathway, a critical survival pathway for tumor cells comprising among other components molecular chaperones, the heat shock proteins (HSPs), and heat shock factor 1 (HSF1) - a master transcription factor and regulator of the heat shock response (HSR). Targeting HSP90 with small-molecule inhibitors has anti-myeloma activity (Stuhmer et al, 2009; Ishii et al, 2011), but leads to compensatory activation of the HSR, which makes inhibition of the HSR itself an attractive target. Recently, we discovered a chemical probe bisamide (CCT251236) that disrupts the HSF1 pathway (Cheeseman MD et al, 2017). This led to the identification of CCT361814 with bioavailability features compatible with clinical use. Here we present first data demonstrating marked antitumor activity by CCT361814 in MM.Methods and ResultsWe evaluated CCT361814 activity in 16 human myeloma cell line (HMCL) models including KMS11, KMS12BM, H929, L363 and RPMI-8226 using MTS cell proliferation assay and found that most HMCL cell lines displayed a GI50 in the nanomolar range (20-300nM, median GI50 147nM) whilst bone marrow stromal HS5 cells had a GI50 of around 700nM suggesting a therapeutic window. Cell lines with the t(11;14) translocation affecting CCND1 such as MOLP8, U266 and KMS12BM appeared to be particularly sensitive to the drug with median GI50 values of 88nM. Western blot analysis showed that CCT361814 caused a concentration-dependent loss of HSP27 and HSP72, as markers of HSF-1 pathway inhibition, and a reduced HSF1 phosphorylation at serine 326. Annexin V/DAPI staining was performed in 8 cell lines and demonstrated consistent induction of apoptosis in all lines, with MM1.S as an example showing a 9-fold increase in apoptosis compared to vehicle control (student's t-test, P=<0.0001). Increased autophagy was observed in L363 and RPMI-8226 cells as assessed by flow cytometry. This was also accompanied by increased cleaved caspase 3 and LC3 I-II conversion as detected by western blotting. Gene expression profiling was performed using Affymetrix U133 plus 2 arrays in L363, RPMI-8226 and KMS11 to observe any differentially expressed genes. Data were normalised using robust multi-array average (RMA) and analysed for differential gene expression using the limma package in R, employing a 2.5-fold change cut-off and significant false discovery rate adjusted p-values (P=<0.05). Overrepresentation enrichment analysis identified several upregulated genes belonging to the nucleic acid binding transcription factor activity molecular function including genes such as CEBP (P=0.02), DDIT3 (P=0.01), ATF3 (P=0.04) in all MM lines studied. DDIT3 and ATF3 along with other UPR markers such as XBP-1 splicing were also assayed by qRT-PCR and showed a significant upregulation in a dose-dependent manner in L363, KMS12BM, KMS11, RPMI-8226 and H929 cells (P=<0.05 for all). The analysis also revealed significantly downregulated genes belonging to chaperone binding group (P=0.024) and unfolded protein binding group (P=0.05) and genes encoding heat shock proteins such as HSPA8 (P=0.00011), HSPA1A (P=0.001) and HSP4L (P=0.03), consistent with functional work showing that CCT361814 affects the heat shock response.ConclusionsOur data shows that CCT361814 affects the heat shock pathway as evidenced by a concentration-dependent loss of HSP27 and HSP72 proteins and also has highly potent anti-myeloma activity in all HMCL models studied. Further functional validation of the gene expression data and assessing the effect of CCT361814 treatment in combination with clinically used drugs is underway. Our discovery of CCT361814 and its potent inhibition of the HSR in protein homeostasis-addicted MM cells makes it an attractive drug to be further evaluated in early phase clinical trials. DisclosuresWorkman:Institute of Cancer Research: Employment. Jones:Institute of Cancer Research: Employment. Kaiser:Celgene: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Other: Travel expenses; Amgen: Consultancy, Honoraria; Takeda: Consultancy; Janssen: Honoraria; Chugai: Consultancy.

Hypoxia and heat stress affect epithelial integrity in a Caco-2/HT-29 co-culture

Hypoxia and hyperthermia, which can be induced by high environmental temperature or strenuous exercise, are two common stressors that affect intestinal epithelial integrity and lead to multiple clinical symptoms. In this study, we developed an in-vitro intestinal monolayer model using two human colonic epithelial cell lines, Caco-2 and HT-29, co-cultured in Transwell inserts, and investigated the effects of heat treatment and/or hypoxia on the epithelial barrier function. The monolayer with a ratio of 9:1 (Caco-2:HT-29) showed high trans-epithelial electrical resistance (TEER), low Lucifer Yellow permeability and high mucin production. Hyperthermia and/or hypoxia exposure (2 h) triggered heat shock and oxidative stress responses. HSP-70 and HSF-1 protein levels were up-regulated by hyperthermia, which were further enhanced when hyperthermia was combined with hypoxia. Increased HIF-1α protein expression and Nrf2 nuclear translocation was only caused by hypoxia. Hyperthermia and/or hypoxia exposure disrupted the established monolayer by increasing paracellular permeability, decreasing ZO-1, claudin-3 and occludin protein/mRNA expression, while enhancing E-cadherin protein expression. Tight junction protein distribution in the monolayer was also modulated by the hyperthermia and/or hypoxia exposure. In addition, transcription levels of mucin genes, MUC-2 and MUC-5AC, were increased after 2 h of hyperthermia and/or hypoxia exposure. In conclusion, this Caco-2/HT-29 cell model is valid and effective for studying detrimental effects of hyperthermia and/or hypoxia on intestinal barrier function and related heat shock and oxidative stress pathways and can be used to investigate possible interventions to reverse hyperthermia and/or hypoxia-induced intestinal epithelial injury.

Transcriptome analysis of cepharanthine against a SARS-CoV-2-related coronavirus.

Antiviral therapies targeting the pandemic coronavirus disease 2019 (COVID-19) are urgently required. We studied an already-approved botanical drug cepharanthine (CEP) in a cell culture model of GX_P2V, a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related virus. RNA-sequencing results showed the virus perturbed the expression of multiple genes including those associated with cellular stress responses such as endoplasmic reticulum (ER) stress and heat shock factor 1 (HSF1)-mediated heat shock response, of which heat shock response-related genes and pathways were at the core. CEP was potent to reverse most dysregulated genes and pathways in infected cells including ER stress/unfolded protein response and HSF1-mediated heat shock response. Additionally, single-cell transcriptomes also confirmed that genes of cellular stress responses and autophagy pathways were enriched in several peripheral blood mononuclear cells populations from COVID-19 patients. In summary, this study uncovered the transcriptome of a SARS-CoV-2-related coronavirus infection model and anti-viral activities of CEP, providing evidence for CEP as a promising therapeutic option for SARS-CoV-2 infection.

SEM and qRT-PCR revealed quercetin inhibits morphogenesis of Aspergillus flavus conidia via modulating calcineurin-Crz1 signalling pathway

ABSTRACT Aspergillus flavus exploits diverse mechanisms to survive during exposure to antifungal agents including morphogenesis. Germination of dormant conidia involves cascades of reactions integrated into the signalling pathway. This study documents the effect of phytochemical-quercetin on A. flavus during germination of conidia using scanning electron microscopy (SEM). Significant inhibition of conidial swelling of A. flavus in comparison to control was observed at 4 and 7 h Quantitative real-time PCR for genes from calcium signalling pathway and heat-shock proteins family showed up-regulation of heat shock (Hsp70 and Hsp90) and calcium signalling pathway genes (calcium-transporting ATPase and calmodulin) in response to quercetin at initial 4 h in comparison to control sample whereas up-regulation of Hsp70, calcineurin and transcription factor Crz1, were observed in both the treated samples. Gene encoding for calcium-kinase, cAMP, Rho-gdp, Plc and Pkc showed a constitutively higher level of expression in quercetin-treated sample in comparison to control at both time points. These data showed a clear response from genes encoding calcineurin-Crz1 signalling pathways and may find its application in the screening of antifungal agents. Abbreviations Hsp: Hear shock protein; MIC: Minimum Inhibitory Concentration; SEM: Scanning Electron Microscopy; qRT-PCR: Quantitative Real-Time Polymerase Chain Reaction

Mebendazole Mediates Its Anti-Leukemic Effects By Proteasomal Degradation of GLI Transcription Factors Via Inhibition of HSP70/90-Chaperone Activity in Acute Myeloid Leukemia in a Preclinical and Clinical Setting

Aberrant activation of hedgehog signaling (HH) is associated with a wide variety of neoplasms, including acute myeloid leukemia (AML). The GLI transcription factors are the main downstream effectors of the HH signaling cascade and play a fundamental role in cancer development, progression and maintenance of leukemic stem cells, which are responsible for therapy failure and tumor relapse due to their resistance to chemotherapy. Moreover, the GLI transcription factors represent central hubs in oncogenic signaling and thus are an attractive therapeutic target for anti-cancer therapy. However non-canonical GLI activation by multiple oncogenic signaling pathways limit the use of SMO inhibitors, while treatment strategies directly targeting the GLI transcription factors are limited. Mebendazole (MBZ) is a commonly used anthelmintic drug with a favorable toxicity profile. MBZ has been shown to exhibit strong anti-tumor effects in different cancer entities, including AML. We treated AML cell lines and primary AML samples with different MBZ concentrations and could show a dose-dependent effect on the proliferation, colony formation and apoptosis in AML cells. In addition, therapeutic synergy between cytarabine and MBZ was demonstrated in OCI-AML3 cells with a combination index &lt;1 and a dose reduction index (DRI) of 2.5- to 23.0-fold. GLI1 and -2 protein levels were clearly reduced in AML cell lines 24h after MBZ exposure, whereas mRNA levels did not decrease. To study the functional activity of GLI transcription factors, we transduced various AML cell lines with a construct where luciferase expression is under the control of the GLI promoter. MBZ strongly inhibited GLI signaling activity in a dose-dependent manner. Furthermore, inhibiting the 26s proteasome with bortezomib partly abolished the effect of MBZ on GLI1 and -2 protein levels and GLI activity, indicating that MBZ promotes their proteasomal degradation. The HSP70/HSP90-based chaperone machinery stabilizes a spectrum of client substrates for protein folding, including transcription factors. Inhibition of either HSP90 or HSP70 with small-molecule inhibitors VER-155008 and PU-H71 resulted in markedly reduction of GLI1 and -2 protein levels in Western Blots. Next, we investigated the impact of MBZ on HSP70/HSP90-mediated refolding of the heat-denatured firefly luciferase. In cell-lines constitutively expressing a firefly luciferase, the luciferase signal was recovered without inhibitor following a heat-shock, but incubation of AML cells with MBZ or specific HSP inhibitors impaired recovery of the signal, suggesting strong inhibition of HSP70/90-mediated luciferase refolding. This indicates that the anti-leukemic activity of MBZ is at least partly mediated through GLI protein destabilization by inhibition of cooperative HSP70/HSP90-chaperone activity. This is further supported by our observation that MBZ activates HSF-1, a major regulator of the heat shock pathway. MBZ may address additional pathways by HSP70/HSP90 inhibition such as FLT3-ITD, MYB or MYC degradation and inactivation of ERK and AKT as shown by us and others. To further evaluate if MBZ is a suitable GLI inhibitor in clinical practice, we transferred these findings into the clinical setting by treating two patients with refractory AML with MBZ monotherapy in an off-label setting. We demonstrated an effective MBZ concentration in the plasma using a modified plasma inhibitory assay (PIA) by incubating an indicator cell lines carrying the GLI luciferase promoter transgene with the patient's plasma. In one patient, a clear continuous decrease in leukemic blasts in peripheral blood was noted. In this patient a reduction in the luciferase activity in the PIA assay and a fast reduction in GLI2 levels in peripheral leukemic blood were detected. Moreover, a healthy volunteer ingested MBZ at a dose of 50 mg/kg divided in two doses at time 0h and 12h. Blood was drawn at 4h and at 24h. PIA results indicated a biological active plasma concentration. Taken together, our results demonstrate that MBZ functions as an effective GLI inhibitor with strong anti-leukemic activity in a clinical setting and encourage for further studies to translate the scientific findings into clinical practice. A clinical study of mebendazole plus low dose cytarabine in patients with refractory AML is planned. Figure Disclosures Stamm: AstraZeneca: Employment. Modemann:Servier, Incyte, Gilead, Jazz Pharmaceuticals, Novartis, Teva, Pfizer, Amgen: Other: Support for meeting attendance ; Servier: Honoraria; Daiichi Sankyo: Research Funding. Fiedler:Amgen, Pfizer, Abbvie: Other: Support in medical writing; Amgen: Research Funding; Amgen, Pfizer, Novartis, Jazz Pharmaceuticals, Ariad/Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen, Jazz Pharmaceuticals, Daiichi Sanchyo Oncology, Servier: Other: Support for meeting attendance. OffLabel Disclosure: Mebendazole is an approved oral broad-spectrum anthelmintic drug for the treatment of worm infections. Data from preclinical studies demonstrated strong anti-tumor effects. We used it as an antineoplastic drug in a compassionate and off-label setting for treatment of two AML patients.

Cyclic nucleotide-gated ion channel 6 mediates thermotolerance in Arabidopsis seedlings by regulating nitric oxide production via cytosolic calcium ions

BackgroundWe previously reported the involvement of nitric oxide (NO) and cyclic nucleotide-gated ion channel 6 (CNGC6) in the responses of plants to heat shock (HS) exposure. To elucidate their relationship with heat tolerance in Arabidopsis thaliana, we examined the effects of HS on several groups of seedlings: wild type, cngc6, and cngc6 complementation and overexpression lines.ResultsAfter HS exposure, the level of NO was lower in cngc6 seedlings than in wild-type seedlings but significantly elevated in the transgenic lines depending on CNGC6 expression level. The treatment of seeds with calcium ions (Ca2+) enhanced the NO level in Arabidopsis seedlings under HS conditions, whereas treatment with EGTA (a Ca2+ chelator) reduced it, implicating that CNGC6 stimulates the accumulation of NO depending on an increase in cytosolic Ca2+ ([Ca2+]cyt). This idea was proved by phenotypic observations and thermotolerance testing of transgenic plants overexpressing NIA2 and NOA1, respectively, in a cngc6 background. Western blotting indicated that CNGC6 stimulated the accumulation of HS proteins via NO.ConclusionThese data indicate that CNGC6 acts upstream of NO in the HS pathway, which improves our insufficient knowledge of the initiation of plant responses to high temerature.