Upregulation Of Hsp70 Research Articles

Pre-induction of Hsp70 expression to protect the grooved carpet shell clam, Ruditapes decussatus, against Micrococcus luteus: A trained immunity strategy

Bacterial pathogens cause disease-associated mortality in Ruditapes decussatus, a model bivalve for aquaculture in Egypt. In this study, it was hypothesized that trained immunity could be achieved to enhance disease resistance against Micrococcus luteus by pre-inducing Hsp70 expression. Clams were challenged via a seawater bath with M. luteus at various concentrations (2.5 × 104, 5.0 × 104, and 7.5 × 104 CFU/ml), gill Hsp70 was quantified using ELISA and infected clams were compared to controls in two experiments (with one 17-day and two 17-day infection periods separated by a 7-day depuration period for recovery, respectively). Infection with M. luteus significantly altered Hsp70 expression compared to the control levels. Downregulation occurred at low concentrations and upregulation at the two higher concentrations. When used in the trained immunity trial, Hsp70 upregulation occurred in infected clams during the first infection and persisted during the 7-day recovery. Re-infection led to the increase of Hsp70 levels, which reached maximum values, and enhanced survival. The results confirmed that Hsp70 is involved in the immune response and disease resistance by protecting R. decussatus against re-infection with M. luteus. These results suggested the use of trained immunity as disease control strategy.

Membrane-anchored heat-shock protein 70 (Hsp70) in cancer.

Hsp70 is a highly conserved and inducible heat shock protein that belongs to the HSP70 family of molecular chaperones and plays a central role in protein homeostasis. The main function of Hsp70 is to protect cells from physiological, pathological and environmental insults, as it assists an ATP-dependent manner the process of protein folding. Since Hsp70 provides critical cell survival functions, cancer cells are assumed to rely on this chaperone. Strong evidence suggests that Hsp70 is upregulated in different type of cancers and is involved in tumor growth, invasion, migration and resistance to anti-cancer therapy. Interestingly, this Hsp70 upregulation induces Hsp70 re-location into plasma membrane. In this review, the role of Hsp70 in cancer will be discussed focusing particularly on the extracellular membrane-bound Hsp70. The mechanism by which Hsp70 is translocated to plasma membrane of tumor cells and the recent discoveries of drugs targeting this Hsp70 in cancer therapy will be also highlighted.

English

Gentamicin (GM) is an aminoglycoside that has harmful effects on the male germ cells and sperm quality. N-3 polyunsaturated fatty acids (n-3 PUFA) are natural antioxidants that influence cell signaling and inflammation. Heme-oxygenase-1 (HO-1) and heat shock proteins (HSP) aid in cellular protection against cellular insults. This study aimed to explore the potential alleviating influences of treatment with n-3 PUFA on GM-induced testicular damage. Thirty-two albino male rats were divided into four equal groups. (1) The control group received normal saline, (2) the n-3 PUFA group received 100 mg/kg body weight/day n-3 PUFA daily for 4 weeks, (3) the GM group received 100 mg/kg/day GM intraperitoneally for 10 consecutive days, and (4) the GM + n-3 PUFA group received intraperitoneal GM for ten days followed by treatment with n-3 PUFA for 4 weeks. Significant reductions in sperm motility, viability, serum testosterone, total testicular protein, and germinal epithelium height were observed in the GM-treated group, with upregulation of the oxidative stress markers, HO-1 mRNA, and HSP70, and downregulation of proliferating cell nuclear antigen (PCNA). We also observed cellular disorganization, vacuolation, tubular distortion, and a significantly higher percentage of collagen. Ultra-structurally, most of the spermatogenic cells were electron dense and degenerated with rarefied cytoplasm. Treatment with n-3 PUFA resulted in a significant increase in sperm motility, viability, serum testosterone, and in the germinal epithelium height. Upregulation of HO-1 mRNA, HSP70, and PCNA expression and a significant reduction in the oxidative stress index were also observed. The findings confirm the potential ameliorative role of and imply novel mechanisms by which n-3 PUFA protects against GM-induced testicular injury. Key words: Polyunsaturated fatty acids, gentamicin, oxidative stress, testis, male infertility. &nbsp

Suppression of HSP70 inhibits the development of acute lymphoblastic leukemia via TAK1/Egr-1

Acute lymphoblastic leukemia (ALL), usually treated with chemotherapy, has limited therapeutic effects and high toxicity. Upregulation of HSP70 induces tumor development, however, the molecular mechanism of HSP70 in ALL remains unclear. In our research, we aimed to investigate the role of HSP70 in ALL, specifically the molecular mechanisms underlying cell apoptosis and proliferation. We found that HSP70 expression in leukomonocytes from ALL patients was increased compared with the control group. HSP70 expression in NALM-6 and BE-13 was also up-regulated contrast with AHH-1. Inhibition of HSP70 significantly promoted cell apoptosis and suppressed cell proliferation in ALL cell lines. Suppression of HSP70 decreased TAK1 and increased Egr-1 protein expression. Further experiments indicated that overexpression of TAK1 ameliorated the effect of HSP70 inhibition on Egr-1 protein expression, cell apoptosis and proliferation. In order to determine whether the effect of HSP70 inhibition on apoptosis and proliferation of ALL cell lines could be achieved via regulation of Egr-1, we performed a loss-of-function experiment for Egr-1. Egr-1 suppression was found to reverse the effect of HSP70 inhibition on cell apoptosis and proliferation in ALL. Taken together, our results suggest that HSP70 inhibition upregulates Egr-1 via TAK1, inducing apoptosis and restricting proliferation in ALL cells.

Arsenic-induced immunomodulatory effects disorient the survival-death interface by stabilizing the Hsp90/Beclin1 interaction

Ground water arsenic contamination is a global menace. Since arsenic may affect the immune system, leading to immunesuppression, we investigated the effects of acute arsenic exposure on the thymus and spleen using Swiss albino mice, exposed to 5 ppm, 15 ppm and 300 ppm of sodium arsenite for 7 d. Effects on cytokine balance and cell survivability were subsequently analyzed. Our data showed that arsenic treatment induced debilitating alterations in the tissue architecture of thymus and spleen. A dose-dependent decrease in the ratio of CD4+-CD8+ T-cells was observed along with a pro-inflammatory response and redox imbalance. In addition, pioneering evidences established the ability of arsenic to induce an up regulation of Hsp90, eventually resulting in stabilization of its client protein Beclin-1, an important autophagy-initiating factor. This association initiated the autophagic process, confirmed by co-immunoprecipitation assay, acridine orange staining and Western blot, indicating the effort of cells trying to survive at lower doses. However, increased arsenic assault led to apoptotic cell death in the lymphoid organs, possibly by increased ROS generation. There are several instances of autophagy and apoptosis taking place either simultaneously or sequentially due to oxidative stress. Since arsenic is a potent environmental stress factor, exposure to arsenic led to a dose-dependent increase in both autophagy and apoptosis in the thymus and spleen, and cell death could therefore possibly be induced by autophagy. Therefore, exposure to arsenic leads to serious effects on the immune physiology in mice, which may further have dire consequences on the health of exposed animals.

Epileptogenesis-Associated Alterations of Heat Shock Protein 70 in a Rat Post-Status Epilepticus Model

Temporal lobe epilepsy is triggered by an initial insult, such as status epilepticus, that initiates the process of epilepsy development. Heat shock protein 70 (Hsp70) is a ubiquitously expressed molecular chaperone, involved in the inflammatory response that is upregulated after status epilepticus. Hsp70 has been described as an endogenous intracellular ligand of Toll-like receptor 4. It is released from damaged or necrotic tissue and by activated immune cells after an inflammatory event. So far, the time course and the pattern of epileptogenesis-associated alterations in Hsp70 expression have not been described in detail.Thus, we investigated immunohistochemical expression of Hsp70 in hippocampus, parahippocampal cortex, parietal cortex, amygdala, and thalamus following status epilepticus in a rat model of temporal lobe epilepsy. The impact of status epilepticus on Hsp70 expression varied during different phases of epileptogenesis, displaying a stronger effect in the early post-insult phase, a milder and more localized effect in the latency phase and no relevant effect in the chronic phase.Cellular-level characterization revealed that Hsp70 colocalized with the neuronal marker NeuN and with Toll-like receptor 4. No colocalization with the astrocytic marker GFAP or the microglia marker Iba1 was found.The intense neuronal Hsp70 upregulation during the early post-insult phase might contribute to the onset of excessive inflammation triggering molecular and cellular reorganization and generation of a hyperexcitable epileptic network. Therefore, development of multi-targeting strategies aiming at prevention of epileptogenesis should consider Hsp70 modulation in the early days following an epileptogenic insult.

HSP70 induces liver X receptor pathway activation and cholesterol reduction in vitro and in vivo

ObjectiveHeat Shock Proteins (HSPs) maintain cellular homeostasis under stress. HSP70 represents a major stress-inducible family member and has been identified as a druggable target in inherited cholesterol-sphingolipid storage diseases. We investigated if HSP70 modulates cholesterol accumulation in more common conditions related to atherogenesis. MethodsWe studied the effects of recombinant HSP70 in cholesterol-laden primary macrophages from human blood donors and pharmacological HSP70 upregulation in high-cholesterol diet fed zebrafish. ResultsRecombinant HSP70 facilitated cholesterol removal from primary human macrophage foam cells. RNA sequencing revealed that HSP70 induced a robust transcriptional re-programming, including upregulation of key targets of liver X receptors (LXR), master regulators of whole-body cholesterol removal. Mechanistically, HSP70 interacted with the macrophage LXRalpha promoter, increased LXRalpha and its target mRNAs, and led to elevated levels of key proteins facilitating cholesterol efflux, including ATP-binding cassette transporters A1 and G1. Pharmacological augmentation of endogenous HSP70 in high-cholesterol diet fed zebrafish activated LXR and its target mRNAs and reduced cholesterol storage at the whole organism level. ConclusionThese data demonstrate that HSP70 exerts a cholesterol lowering effect in primary human cells and animals and uncover a nuclear action of HSP70 in mediating cross-talk between HSP and LXR transcriptional regulation.

Modulated electro-hyperthermia induced p53 driven apoptosis and cell cycle arrest additively support doxorubicin chemotherapy of colorectal cancer in vitro.

ObjectiveModulated electro‐hyperthermia (mEHT), a noninvasive complementary treatment of human chemo‐ and radiotherapy, can generate selective ~42°C heat in cancer due to elevated glycolysis (Warburg‐effect) and electric conductivity in malignant tissues. Here we tested the molecular background of mEHT and its combination with doxorubicin chemotherapy using an in vitro model.MethodsC26 mouse colorectal adenocarcinoma cultures were mEHT treated at 42°C for 2 × 60 minutes (with 120 minutes interruption) either alone or in combination with 1 µmol/L doxorubicin (mEHT + Dox). Cell stress response, apoptosis, and cell cycle regulation related markers were detected using qPCR and immunocytochemistry supported with resazurin cell viability assay, cell death analysis using flow‐cytometry and clonogenic assay.ResultCell‐stress by mEHT alone was indicated by the significant upregulation and release of hsp70 and calreticulin proteins 3 hours posttreatment. Between 3 and 9 hours after treatment significantly reduced anti‐apoptotic XIAP, BCL‐2, and BCL‐XL and elevated pro‐apoptotic BAX and PUMA, as well as the cyclin dependent kinase inhibitor p21waf1 mRNA levels were detected. After 24 hours, major elevation and nuclear translocation of phospho‐p53(Ser15) protein levels and reduced phospho‐Akt(Ser473) levels were accompanied by a significant caspase‐3‐mediated programmed cell death response. While mEHT dominantly induced apoptosis, Dox administration primarily led to tumor cell necrosis, and both significantly reduced the number of tumor progenitor colonies 10 days post‐treatment. Furthermore, mEHT promoted the uptake of Dox by tumor cells and the combined treatment additively reduced tumor cell viability and augmented cell death near to synergy.ConclusionIn C26 colorectal adenocarcinoma mEHT‐induced irreversible cell stress can activate both caspase‐dependent apoptosis and p21waf1 mediated growth arrest pathways, likely to be driven by the upregulated nuclear p53 protein. Elevated phospho‐p53(Ser15) might contribute to p53 escape from mdm2 control, which was further supported by reduced phospho‐Akt(Ser473) protein levels. In combinations, mEHT could promote the uptake and significantly potentiate the cytotoxic effect of doxorubicin.

Phase I trial of the combination of the heat shock protein-90 inhibitor onalespib (AT13387) and the cyclin-dependent kinase inhibitor AT7519M in patients with advanced solid tumors.

2619 Background: The 90kDa heat shock protein (HSP90) participates in the folding, stabilization, activation, and proteolytic turnover of aberrant proteins that contribute to the growth and survival of cancer cells. HSP90 inhibition leads to degradation of these aberrant proteins through the ubiquitin-proteosome pathway, allowing for simultaneous targeting of multiple pathways. Inhibition of HSP90 alone stimulates a compensatory upregulation of HSP70. The transcriptional induction of HSP70 has been linked to the activity of CDK9. Combined inhibition of onalespib-mediated HSP90 inhibition and AT7519-mediated CDK9 inhibition has demonstrated synergistic anti-tumor activity in preclinical models at NOAEL doses, justifying a Phase 1 study. Methods: We conducted an open-label phase 1 study following a 3+3 trial design. Patients received a 1-week lead-in of onalespib alone (C0), followed by onalespib/AT7519M on days 1,4,8, and 11 of a 21-day cycle. Pharmacokinetic samples were obtained on C0D1 after onalespib alone, and on C1D1 and D11 with the combination. HSP70 protein levels were analyzed in PBMC and plasma samples collected at baseline, after onalespib alone, and after the combination, in order to demonstrate AT7519-mediated suppression of HSP70 expression. Patients enrolled to the expansion phase underwent optional paired tumor biopsies for assessment of proof-of-mechanism demonstration of modulation of client proteins. Results: Twenty-eight patients have been treated, 10 of whom were enrolled to the expansion cohort with optional tumor biopsies. The MTD is DL2: onalespib 80 mg/m2 IV + AT7519M 21 mg/m2 IV on days 1,4,8, and 11 of a 21-day cycle. At DL3, DLTs included Grade 3 troponin elevation and mucositis. Drug-related adverse events occurring in ≥ 30% of patients include diarrhea, fatigue, mucositis, nausea, and vomiting, consistent with known toxicities of these agents. Two patients with colorectal and endometrial cancer, respectively, remained on study for 10 cycles with SD as the best response. Modulation of HSP70 were demonstrated in patient plasma samples. Conclusions: The combination of onalespib and AT7519 is tolerable, although the doses of both agents were below the monotherapy MTDs. Prolonged disease stabilizations were observed. Pharmacokinetic and pharmacodynamic analyses are ongoing, including assessment of HSP70 expression in plasma and tumor. Clinical trial information: NCT02503709.

Effects of GLP-1 Receptor Activation on a Pentylenetetrazole-Kindling Rat Model.

Objectives: To study the possible anti-seizure and neuroprotective effect of glucagon like peptide 1 (GLP1) analogue (liraglutide) in a pentylenetetrazole (PTZ) induced kindled rat model and its underlying mechanisms. Methods: Thirty Sprague Dawley rats were allocated into 3 equal groups; i) Normal group: normal rats received normal saline, ii) PTZ (kindling) group: received PTZ (50 mg/Kg intraperitoneally (i.p.)) every other day for 2 weeks and iii) PTZ + GLP1 group: same as the PTZ group but rats received liraglutide (75 µg/kg i.p. daily) for 2 weeks before PTZ injection. Seizure severity score, seizure latency and duration were assessed. Also, the expression of caspase-3 (apoptotic marker) and β-catenin (Wnt pathway) by western blotting, markers of oxidative stress (GSH, CAT and MDA) by biochemical assay and the expression of LC3 (marker of autophagy) and heat shock protein 70 (Hsp70) by immunostaining were assessed in hippocampal regions of brain tissues. Results: PTZ caused a significant increase in Racine score and seizure duration with a significant decrease in seizure latency. These effects were associated with a significant increase in MDA, β-catenin, caspase-3, Hsp70 and LC3 in brain tissues (p < 0.05). Meanwhile, liraglutide treatment caused significant attenuation in PTZ-induced seizures, which were associated with significant improvement in markers of oxidative stress, reduction in LC3, caspase-3 and β-catenin and marked increase in Hsp70 in hippocampal regions (p < 0.05). Conclusion: Activation of GLP1R might have anticonvulsant and neuroprotective effects against PTZ-induced epilepsy. These effects could be due to suppression of oxidative stress, apoptosis and autophagy and upregulation of Hsp70.

Cytoprotective effects of taxifolin against cadmium-induced apoptosis in human keratinocytes.

Cadmium (Cd) is a heavy metal widely used in industry, and the skin is an important target of this metal. Taxifolin (Tax), a natural source of bioflavonoids found in various conifers, exerts multiple biologic effects on skin cells. However, the mechanisms by which Tax protects keratinocytes against Cd are currently unclear. We investigated the cytoprotective effects of Tax against Cd-induced apoptosis in the human HaCaT keratinocyte. The water-soluble tetrazolium salt (WST-1) assay and Annexin V/propidium iodide double-staining assay results showed that Cd-induced cell death was lower in cells treated with Tax (0-100 μM) than in cells treated with Cd alone. Additionally, a reduction of Cd-induced DNA fragmentation by Tax was shown by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling assay. The levels of reactive oxygen species were also lower in Cd/Tax-treated cells than in Cd-treated cells. We employed a two-dimensional electrophoresis-based proteomic analysis to identify treatment-related alterations in protein expression. Tax downregulated cathepsin B and D and upregulated hsp27, cyclophilin A, and peroxiredowin-1. Western blotting confirmed the downregulation of cathepsin B and D and the upregulation of hsp27. The cytoprotective effects of Tax against Cd-induced apoptosis were also characterized by the changes in the activity of caspase 3, -7, poly ADP-ribose polymerase, the cellular proliferation-related ERK1/2, and AKT. Furthermore, the levels of cell cycle-related proteins, such as SP1 and p21, decreased, whereas p53 level increased. We concluded that Tax reduced Cd cytotoxicity and Cd-induced apoptosis by inhibiting the apoptotic pathway.

Abscisic acid mediation of drought priming-enhanced heat tolerance in tall fescue (Festuca arundinacea) and Arabidopsis.

Abscisic acid (ABA) may play roles in mediating cross stress tolerance in plants. The objectives of this study were to investigate the priming effects of drought and ABA on heat tolerance and to determine how ABA may be involved in enhanced heat tolerance by drought. Focusing on the transcriptional level, two independent experiments were conducted, using a perennial grass species, tall fescue (Festuca arundinacea) and Arabidopsis. In experiment 1, tall fescue plants were exposed to mild drought by withholding irrigation for 8 days (drought priming) and foliar sprayed with ABA or an ABA-synthesis inhibitor (fluridone). After that they were subsequently subjected to heat stress (38/33°C day/night) for 25 days in growth chambers. In experiment 2, Arabidopsis Columbia ecotype (wild-type) and ABA-deficient mutant (aba3-1, CS157) were pre-treated with drought priming and then exposed to heat stress (45/40°C) for 3 days. The physiological analysis demonstrated that both drought priming and foliar application of ABA-enhanced heat tolerance in tall fescue, while drought priming had no significant effects on heat tolerance in ABA-deficient Arabidopsis plants. Application of fluridone to tall fescue and ABA-deficient mutants of Arabidopsis exhibited diminished or attenuated positive effects of drought priming on heat tolerance. ABA mediation of acquired heat tolerance by drought priming was associated with the upregulation of CDPK3, MPK3, DREB2A, AREB3, MYB2, MYC4, HsfA2, HSP18, and HSP70. Our study revealed the roles of ABA in drought priming-enhanced heat tolerance, which may involve transcriptional regulation for stress signaling, ABA responses and heat protection.

HSF1 phosphorylation by cyclosporin A confers hyperthermia sensitivity through suppression of HSP expression

Heat shock factor 1 (HSF1) is a transcription factor essential for tumorigenesis, and targeting HSF1 may be effective in combined therapeutics for cervical cancer. Cyclosporin A (CsA) is an immunosuppressant that has revolutionized organ transplantation. However, the roles and regulatory mechanisms by which CsA modulates HSP expression remain largely unknown. In this study, we found that CsA pretreatment prevented induction of HSPs during heat shock by enhancing the phosphorylation of Ser303 and Ser307 on HSF1 and thus inhibiting its transcriptional activity. Suppression of ERK1/2, GSK3β and CK2 activities attenuated CsA-induced down-regulation of HSP expression and up-regulation of HSF1 phosphorylation. CsA interfered with HSF1-SSBP1 complex formation and HSF1 nuclear translocation and recruitment to the HSP70 promoter. CsA clearly caused HeLa cell death during proteotoxic stress through reduced expression of HSPs. These results indicate that CsA suppresses HSP induction during heat shock by regulating the phosphorylation and nuclear translocation of HSF1. Our results provide a conceptual framework for the development of novel therapeutic strategies for cervical cancer through application of CsA during hyperthermia or chemotherapy.

Evidence for different thermal ecotypes in range centre and trailing edge kelp populations

Determining and predicting species' responses to climate change is a fundamental goal of contemporary ecology. When interpreting responses to warming species are often treated as a single physiological unit with a single species-wide thermal niche. This assumes that trailing edge populations are most vulnerable to warming, as it is here where a species' thermal niche will be exceeded first. Local adaptation can, however, result in narrower thermal tolerance limits for local populations, so that similar relative increases in temperature can exceed local niches throughout a species range. We used a combination of common garden temperature heat-shock experiments (8–32 °C) and population genetics (microsatellites) to identify thermal ecotypes of northeast Atlantic range centre and trailing edge populations of the habitat-forming kelp, Laminaria digitata. Using upregulation of hsp70 as an indicator of thermal stress, we found that trailing edge populations were better equipped to tolerate acute temperature shocks. This pattern was consistent across seasons, indicating that between-population variability is fixed. High genetic structuring was also observed, with range centre and trailing edge populations representing highly distinct clusters with little gene flow between regions. Taken together, this suggests the presence of distinct thermal ecotypes for L. digitata, which may mean responses to future warming are more complex than linear range contractions.

Cardiovascular Effects of PCB 126 (3,3',4,4',5-Pentachlorobiphenyl) in Zebrafish Embryos and Impact of Co-Exposure to Redox Modulating Chemicals.

The developing cardiovascular system of zebrafish is a sensitive target for many environmental pollutants, including dioxin-like compounds and pesticides. Some polychlorinated biphenyls (PCBs) can compromise the cardiovascular endothelial function by activating oxidative stress-sensitive signaling pathways. Therefore, we exposed zebrafish embryos to PCB126 or to several redox-modulating chemicals to study their ability to modulate the dysmorphogenesis produced by PCB126. PCB126 produced a concentration-dependent induction of pericardial edema and circulatory failure, and a concentration-dependent reduction of cardiac output and body length at 80 hours post fertilization (hpf). Among several modulators tested, the effects of PCB126 could be both positively and negatively modulated by different compounds; co-treatment with α-tocopherol (vitamin E liposoluble) prevented the adverse effects of PCB126 in pericardial edema, whereas co-treatment with sodium nitroprusside (a vasodilator compound) significantly worsened PCB126 effects. Gene expression analysis showed an up-regulation of cyp1a, hsp70, and gstp1, indicative of PCB126 interaction with the aryl hydrocarbon receptor (AhR), while the transcription of antioxidant genes (sod1, sod2; cat and gpx1a) was not affected. Further studies are necessary to understand the role of oxidative stress in the developmental toxicity of low concentrations of PCB126 (25 nM). Our results give insights into the use of zebrafish embryos for exploring mechanisms underlying the oxidative potential of environmental pollutants.

The dynamic adaptation of primary human endothelial cells to simulated microgravity.

Culture of human endothelial cells for 10 d in real microgravity onboard the International Space Station modulated more than 1000 genes, some of which are involved in stress response. On Earth, 24 h after exposure to simulated microgravity, endothelial cells up-regulate heat shock protein (HSP) 70. To capture a broad view of endothelial stress response to gravitational unloading, we cultured primary human endothelial cells for 4 and 10 d in the rotating wall vessel, a U.S. National Aeronautics and Space Administration-developed surrogate system for benchtop microgravity research on Earth. We highlight the crucial role of the early increase of HSP70 because its silencing markedly impairs cell survival. Once HSP70 up-regulation fades away after 4 d of simulated microgravity, a complex and articulated increase of various stress proteins (sirtuin 2, paraoxonase 2, superoxide dismutase 2, p21, HSP27, and phosphorylated HSP27, all endowed with cytoprotective properties) occurs and counterbalances the up-regulation of the pro-oxidant thioredoxin interacting protein (TXNIP). Interestingly, TXNIP was the most overexpressed transcript in endothelial cells after spaceflight. We conclude that HSP70 up-regulation sustains the initial adaptive response of endothelial cells to mechanical unloading and drives them toward the acquisition of a novel phenotype that maintains cell viability and function through the sequential involvement of different stress proteins.-Cazzaniga, A., Locatelli, L., Castiglioni, S., Maier, J. A. M. The dynamic adaptation of primary human endothelial cells to simulated microgravity.

Abstract B186: Tumor cell-derived proteases contribute to antigen processing and enhance cross-presentation

Abstract Tumor lysates (TLs) offer a source of both undefined tumor antigens and endogenous adjuvants, called danger-associated molecular patterns (DAMPs), to induce antitumor immune responses. TLs can therefore serve clinical applications for immunotherapy, including vaccination and as a platform for the expansion for tumor-specific T-cells for use in adoptive cell therapy. TL-based approaches offer the advantage of inducing antitumor immunity without the need to identify specific tumor antigens, a major challenge limiting the clinical application of immunotherapies for solid tumors. Immunogenic cell death (ICD) in tumor cells has proven crucial for the generation of robust antitumor immune responses, suggesting that the induction of ICD may improve the efficacy of TL-based immunotherapies. Here, we examined the immunogenicity of TLs prepared from tumor cells exposed to increasing levels of heat (37°C, 42°C and 56°C), using the MC38-OVA/OT-I system. Molecular correlates of ICD were assessed during the course of heat-treatment and in TL preparations. Notably, lethal heat-treatment (56°C) induced primary necrosis with concomitant caspase activation, calreticulin exposure, HMGB1 release and HSP upregulation. All TLs promoted maturation of bone marrow-derived dendritic cells (BMDC), as evidenced by CD80 and CD86 upregulation, and elicited protective antitumor immunity in vaccinated mice. However, only TLs prepared from lethal heat-treated tumor cells promoted cross-presentation in BMDC to elicit antigen-specific T-cell activation ex vivo. Marked proteolytic activity was observed in lethal heat-treated tumor cells, which were capable of processing antigen and binding resultant peptides. Interestingly, inhibition of broad protease activity in lethal heat-treated TLs markedly reduced cross-presentation. Selective inhibition of calpains, but not caspases, cathepsins or the proteasome, reduced antigen processing by heat-killed tumor cells.Together, this demonstrates that tumor-derived proteases can contribute to antigen processing and enhance cross-presentation. Our data suggest that in addition to DAMP release, antigenic processing by proteases in dying tumor cells can influence the immunogenicity of cell death. Citation Format: Nicholas J. Shields, Katie A. Young, Christopher Jackson, Sarah L. Young. Tumor cell-derived proteases contribute to antigen processing and enhance cross-presentation [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B186.

De novo transcriptome assembly and gene expression profile of thermally challenged green abalone (Haliotis fulgens: Gastropoda) under acute hypoxia and hypercapnia

Transcriptional regulation constitutes a rapid response of marine organisms facing stressful environmental conditions, such as the concomitant exposure to warming, ocean acidification and hypoxia under climate change. In previous studies, we investigated whole-organism physiological patterns and cellular metabolism in gill and muscle of the marine gastropod Haliotis fulgens in response to increasing temperature (18 °C to 32 °C at +3 °C per day) under hypoxia (50% air saturation), hypercapnia (1000 μatm pCO2) and both factors combined. Here, we report investigations of the molecular responses of H. fulgens to temperature and identify mechanisms concomitantly affected by hypoxia and hypercapnia. A de novo transcriptome assembly with subsequent quantitative PCR and correlation network analysis of genes involved in the molecular response were used to unravel the correlations between gene expression patterns under the different experimental conditions. The correlation networks identified a shift from the expression of genes involved in energy metabolism (down-regulated) to the up-regulation of Hsp70 during warming under all experimental conditions in gill and muscle, indicating a strong up-regulation of damage prevention and repair systems at sustained cellular energy production. However, a higher capacity for anaerobic succinate production was evicted in gill, matching with observations from our previous studies indicating succinate accumulation in gill but not in muscle. Additionally, warming under hypoxia and hypercapnia kept mRNA levels of citrate synthase in both tissues unchanged following a similar pattern as muscle enzyme capacity from a previous study, suggesting an emphasis on maintaining rather than down-regulating mitochondrial activity.

Abstract WP349: L-Glutamine Induces Heat Shock Protein 70 Expression &amp; Attenuates Brain Ischemic Injury in Mice

Introduction: Oxidative stress plays an important role in ischemic stroke pathogenesis. L-glutamine (L-GLN) was shown to have antioxidant activity and was approved by the FDA in July 2017 for the treatment of sickle cell disease. In this study, we aim to investigate the effect of L-GLN in attenuating ischemic brain injury using a mouse middle cerebral artery occlusion (tMCAO) model. Methods: Ninety-minute tMCAO was used to induce a focal cerebral ischemic injury. Animals (n=135) were divided into four groups: (1) Sham, (2) Saline, (3) L-GLN, and (4) L-GLN plus HSP70 ATPase activity inhibitor Apoptozole (Az). Treatments were delivered intraperitoneally once daily for the first three days after ischemic surgery. Neurobehavioral tests were performed before tMCAO and at 1, 3, 7, and 14 days after tMCAO using the modified neurological severity score (mNSS), elevated body swing test (EBST), and rotarod test. Infarction volume was assessed by cresyl violet staining of brain sections. The expression levels of heat shock proteins and its signaling pathway related proteins were evaluated by Western Blot and real time-PCR. The level of oxidative stress was evaluated by malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) assays. Astrocyte and neuron cell cultures and oxygen glucose deprivation (OGD) model was used to further examine the molecular pathways involved in the protective effects by L-GLN. Results: L-GLN reduced brain infarct volume and promoted neurobehavioral recovery in mice suffering from ischemic injury. Such beneficial effect was abolished by the coadministration of AZ. L-GLN induced the up-regulation of HSP70, reduced oxidative stress level, and reduced the expression of IL-1beta and IL-6 in ischemic mice brain. In L-GLN group, SOD and GSH-PX activities are significantly increased compared to the Saline group. Nrf2 was upregulated in the L-GLN group while co-treatment with AZ abolished this upregulation. Cell culture experiments revealed that the conditioned media from astrocytes cultured with the presents of L-GLN attenuated the apoptosis of neurons after OGD. Conclusion: L-GLN attenuates ischemic brain injury in part through HSP70 related pathways and holds potential in post-stroke treatment.

Nuclear Magnetic Coupled Fast Radio Burst Induced Upregulation of Hsp70 in Regeneration of Human Chondrocytes - A Sham Control Study

Manipulating the cell’s inherent vulnerabilities to induce intrinsic changes and re-engineer diseased tissues to drive regeneration is a domain creating significant therapeutic impact through translational medicine. Articular cartilage injuries are recurrently experienced due to trauma, mechanical stress and age-related deterioration. Prevailing mend methods have failed to yield reliable or lasting results. Lack of suitable models that mimic the cellular and extra cellular matrix properties of hyaline cartilage has been one of the reasons for this deficiency. We investigated and observed, joint tissue obtained during total knee replacement surgeries from 7 discrete patients presenting with osteoarthritis and the commonly associated symptoms of inflammation and pain. We used modulated “Nuclear Magnetic Coupled Fast Radio Burst or simply Fast Radio Bursts”, a new modality that seems to trigger the cellular signaling required by the articular cartilage tissue, to mend and regenerate by upregulation of Heat Shock Protein 70. Fast Radio Bursts are high energy, short electromagnetic bursts, in which both electric and magnetic components of the electromagnetic signals are “circularly” polarized. Fast Radio Bursts are produced when a radio signal is traveling through a powerful instantaneous magnetic field on its path to the target. In this Sham controlled study, up-regulation of Hsp 70 protein, to establish its role in an in vitro model was designed to expose 2-Dimensional and 3-Dimensional cultures to Fast Radio Bursts, and compared to a Sham Control, under identical conditions but without exposing Sham Control culture to fast radio bursts. The 2D and 3D reconstructed cartilage tissues were then assessed in both groups. Experiments were conducted to characterize the 2D and 3D cultures to confirm total collagen, fibrillar collagen and proteoglycans, additionally; immunofluorescence and cell viability assay was performed to identify specific bio markers like Collagen 1, Collagen II, Aggrecan, Cell surface Adhesion factor, Hsp70 and cell viability. It could be established in this study that 2D cultures grown in newly defined media and exposed to Fast Radio Burst signals, when compared to 2D cultures grown as Sham culture showed more chondrocyte specific markers and viable matrix properties. In 3D cultures grown similarly also showed better deep layer properties compared to the 3D cultures grown in sham culture. Thus modulated Fast Radio Bursts exposure could play a significant role in specific protein up/down regulation in tissue regeneration.