Baicalin Targets HSP70/90 to Regulate PKR/PI3K/AKT/eNOS Signaling Pathways.

Baicalin is a major active ingredient of traditional Chinese medicine Scutellaria baicalensis, and has been shown to have antiviral, anti-inflammatory, and antitumor activities. However, the protein targets of baicalin have remained unclear. Herein, a chemical proteomics strategy was developed by combining baicalin-functionalized magnetic nanoparticles (BCL-N3@MNPs) and quantitative mass spectrometry to identify the target proteins of baicalin. Bioinformatics analysis with the use of Gene Ontology, STRING and Ingenuity Pathway Analysis, was performed to annotate the biological functions and the associated signaling pathways of the baicalin targeting proteins. Fourteen proteins in human embryonic kidney cells were identified to interact with baicalin with various binding affinities. Bioinformatics analysis revealed these proteins are mainly ATP-binding and/or ATPase activity proteins, such as CKB, HSP86, HSP70-1, HSP90, ATPSF1ß and ACTG1, and highly associated with the regulation of the role of PKR in interferon induction and the antiviral response signaling pathway (P = 10-6), PI3K/AKT signaling pathway (P = 10-5) and eNOS signaling pathway (P = 10-4). The results show that baicalin exerts multiply pharmacological functions, such as antiviral, anti-inflammatory, antitumor, and antioxidant functions, through regulating the PKR and PI3K/AKT/eNOS signaling pathways by targeting ATP-binding and ATPase activity proteins. These findings provide a fundamental insight into further studies on the mechanism of action of baicalin.

Computational and in vitro experimental analyses of the anti-COVID-19 potential of Mortaparib and MortaparibPlus.

Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has become a global health emergency. Although new vaccines have been generated and being implicated, discovery and application of novel preventive and control measures are warranted. We aimed to identify compounds that may possess the potential to either block the entry of virus to host cells or attenuate its replication upon infection. Using host cell surface receptor expression (angiotensin-converting enzyme 2 (ACE2) and Transmembrane protease serine 2 (TMPRSS2)) analysis as an assay, we earlier screened several synthetic and natural compounds and identified candidates that showed ability to down-regulate their expression. Here, we report experimental and computational analyses of two small molecules, Mortaparib and MortaparibPlus that were initially identified as dual novel inhibitors of mortalin and PARP-1, for their activity against SARS-CoV-2. In silico analyses showed that MortaparibPlus, but not Mortaparib, stably binds into the catalytic pocket of TMPRSS2. In vitro analysis of control and treated cells revealed that MortaparibPlus caused down-regulation of ACE2 and TMPRSS2; Mortaparib did not show any effect. Furthermore, computational analysis on SARS-CoV-2 main protease (Mpro) that also predicted the inhibitory activity of MortaparibPlus. However, cell-based antiviral drug screening assay showed 30-60% viral inhibition in cells treated with non-toxic doses of either MortaparibPlus or Mortaparib. The data suggest that these two closely related compounds possess multimodal anti-COVID-19 activities. Whereas MortaparibPlus works through direct interactions/effects on the host cell surface receptors (ACE2 and TMPRSS2) and the virus protein (Mpro), Mortaparib involves independent mechanisms, elucidation of which warrants further studies.

A standardized extract of Asparagus officinalis stem improves HSP70-mediated redox balance and cell functions in bovine cumulus-granulosa cells.

Heat shock (HS) protein 70 (HSP70), a well-known HS-induced protein, acts as an intracellular chaperone to protect cells against stress conditions. Although HS induces HSP70 expression to confer stress resistance to cells, HS causes cell toxicity by increasing reactive oxygen species (ROS) levels. Recently, a standardized extract of Asparagus officinalis stem (EAS), produced from the byproduct of asparagus, has been shown to induce HSP70 expression without HS and regulate cellular redox balance in pheochromocytoma cells. However, the effects of EAS on reproductive cell function remain unknown. Here, we investigated the effect of EAS on HSP70 induction and oxidative redox balance in cultured bovine cumulus-granulosa (CG) cells. EAS significantly increased HSP70 expression; however, no effect was observed on HSP27 and HSP90 under non-HS conditions. EAS decreased ROS generation and DNA damage and increased glutathione (GSH) synthesis under both non-HS and HS conditions. Moreover, EAS synergistically increased HSP70 and HSF1 expression and increased progesterone levels in CG cells. Treatment with an HSP70 inhibitor significantly decreased GSH level, increased ROS level, and decreased HSF1, Nrf2, and Keap1 expression in the presence of EAS. Furthermore, EAS significantly increased progesterone synthesis. Thus, EAS improves HSP70-mediated redox balance and cell function in bovine CG cells.

HSP70 Inhibition Leads to the Activation of Proteasomal System under Mild Hyperthermia Conditions in Young and Senescent Fibroblasts.

Aging has been characterized with the accumulation of oxidized proteins, as a consequence of progressive decline in proteostasis capacity. Among others, proteasomal system is an efficient protein turnover complex to avoid aggregation of oxidized proteins. Heat shock protein 70 (HSP70) is another critical player that is involved in some key processes including the correct folding of misfolded proteins and targeting aggregated proteins to the proteasome for rapid degradation. The aim of this study was to determine the role of proteasomal system and heat shock proteins to maintain proteome balance during replicative senescence in mild hyperthermia conditions. Our results demonstrated that HSP40/70 machinery is induced by mild hyperthermia conditions independent from senescence conditions. Since HSP70 is largely responsible for the rapidly inducible cell protection following hyperthermia, the activation of "heat shock response" resulted in the elevation of HSP40/70 expressions as well as the proteasome activity. Interestingly, when HSP70 expression was inhibited, increased proteasomal activation was shown to be responsive to mild hyperthermia. Since HSP70 is involved in various stress-related pathways such as oxidative and endoplasmic reticulum stress, depletion of HSP70 expression may induce proteasomal degradation to maintain proteome balance of the cell. Thus, our data suggests that in mild heat stress conditions, molecular chaperone HSP70 plays an important role to avoid protein oxidation and aggregation; however, activities of proteasomal system are induced when HSP70 expression is depleted.

Rapid and simple screening of the apoptotic compounds based on Hsp70 inhibition using luciferase as an intracellular reporter.

HSP70 is a powerful antiapoptotic protein that can block the extrinsic and intrinsic pathways of apoptosis. The present study describes a rapid, sensitive, and inexpensive system using luciferase as a reporter for the functional analysis of apoptotic compounds. For this approach, the co-transformation of Escherichia coli cells was performed with two expression vectors containing Hsp70 and firefly luciferase. It was found that the luciferase inactivated by heat treatment (40-46 °C for 10 min) was approximately reactivated at room temperature and regained 70% of its initial activity before heat inactivation after 60 min. The results show that the reactivation of thermally inactivated luciferase was inhibited in living cells by treatment with VER-155008 and pifitrin-µ as Hsp70 inhibitors, with half-maximal inhibitory concentration of 124 and 384 µM, respectively. The sensitivity of this method for detecting VER-155008 and pifitrin-µ was about 8 and 25 µM, respectively. Also, this reporter system showed no response to doxorubicin and dactinomycin, which bind to DNA, and we used these anticancer compounds as control compounds. Therefore, for the first time, a rapid and simple real-time system using luciferase as a reporter is introduced for the screening of apoptosis-inducing compounds based on suppression of Hsp70 in E. coli cells.

Cell line-specific efficacy of thermoradiotherapy in human and canine cancer cells in vitro.

OBJECTIVE: Aims were to investigate sensitivity of various human and canine cancer cell lines to hyperthermia and the influence of particular treatment conditions, and to analyze the DNA-damage response and mode of cell death in cell line radiosensitized by hyperthermia. Additionally, we were interested in the involvement of HSP70 in radiosensitization. METHODS: Radiosensitization by hyperthermia was determined in a panel of human and canine cancer cell lines using clonogenic cell survival assay, as well as levels of heat shock proteins (HSPs) using immunoblotting. The influence of the hyperthermia-radiotherapy time gap, different temperatures and the order of treatments on clonogenicity of hyperthermia-sensitive A549 cells was investigated. Additionally, DNA damage and cell death were assessed by Comet assay and an apoptosis/necrosis assay. Further we induced transient knockdown in A549 cells to test HSP70's involvement in radiosensitization. RESULTS: Out of eight cell lines tested, only two (A549 and Abrams) showed significant decrease in clonogenic cell survival when pre-treated with hyperthermia at 42°C. Strong induction of HSP70 upon thermoradiotherapy (HT-RT) treatment was found in all cell lines. Transient knockdown of HSP70 in A549 cells did not result in decrease of clonogenic cell survival in response to HT-RT. CONCLUSION: Tumor cell-type, temperature and order of treatment play an important role in radiosensitization by hyperthermia. However, hyperthermia has limited potency to radiosensitize canine cancer cells grown in a 2D cell culture setting presented here. DNA damage and apoptosis/necrosis did not increase upon combined treatment and cytosolic levels of HSP70 appear not to play critical role in the radiosensitization of A549 cells.

Functionality-Independent DNA Encoding of Complex Natural Products.

DNA encoded chemical libraries (DELs) link the powers of genetics and chemical synthesis via combinatorial optimization. Through combinatorial chemistry, DELs can grow to the unprecedented size of billions to trillions. To take full advantage of the DEL approach, linking the power of genetics directly to chemical structures would offer even greater diversity in a finite chemical world. Natural products have evolved an incredible structural diversity along with their biological evolution. Herein, we used traditional Chinese medicines (TCMs) as examples in a late-stage modification toolbox approach to annotate these complex organic compounds with amplifiable DNA barcodes, which could be easily incorporated into a DEL. The method of end-products labeling also generates a cluster of isomers with a single DNA tag at different sites. These isomers provide an additional spatial diversity for multiple accessible pockets of targeted proteins. Notably, a novel PARP1 inhibitor from TCM has been identified from the natural products enriched DEL (nDEL).

HSP-70-Mediated Hyperbaric Oxygen Reduces Brain and Pulmonary Edema and Cognitive Deficits in Rats in a Simulated High-Altitude Exposure.

High-mountain sickness is characterized by brain and pulmonary edema and cognitive deficits. The definition can be fulfilled by a rat model of high-altitude exposure (HAE) used in the present study. This study aimed to investigate the protective effect of hyperbaric oxygen therapy (HBO2T) and to determine the underlying mechanisms. Rats were subjected to an HAE (9.7% O2 at 0.47 absolute atmosphere of 6,000 m for 3 days). Immediately after termination of HAE, rats were treated with HBO2T (100% O2 at 2.0 absolute atmosphere for 1 hour per day for 5 consecutive days) or non-HBO2T (21% O2 at 1.0 absolute atmosphere for 1 hour per day for 5 consecutive days). As compared to non-HAE+non-HBO2T controls, the HAE+non-HBO2T rats exhibited brain edema and resulted in cognitive deficits, reduced food and water consumption, body weight loss, increased cerebral inflammation and oxidative stress, and pulmonary edema. HBO2T increased expression of both hippocampus and lung heat shock protein (HSP-70) and also reversed the HAE-induced brain and pulmonary edema, cognitive deficits, reduced food and water consumption, body weight loss, and brain inflammation and oxidative stress. Decreasing the overexpression of HSP-70 in both hippocampus and lung tissues with HSP-70 antibodies significantly attenuated the beneficial effects exerted by HBO2T in HAE rats. Our data provide in vivo evidence that HBO2T works on a remodeling of brain/lung to exert a protective effect against simulated high-mountain sickness via enhancing HSP-70 expression in HAE rats.

Curcumin supplementation increases survival and lifespan in Drosophila under heat stress conditions.

Harsh climate induces physiological stress thus compromising organismal survival. Our previous studies demonstrated that curcumin (CUR) supplementation increased survival of turtle under heat stress (HS). Here, we span this work to investigate the survival and lifespan of HS Drosophila fed a diet supplemented with CUR. For this purpose, female and male flies were fed basal diet (N) and CUR diet (0.2 mg/g), and exposed to three conditions: 25°C and 29°C continuously, and 34 °C for 2 h at days 1, 4, and 7, then kept at 25 °C. Lifespan analysis showed that, compared to N-25 °C flies, the mean lifespans of N-29 °C and N-34 °C flies were decreased significantly by 8.5-15.7% in males, and 3.7-7.9% in females. Conversely, in the CUR-supplemented diet, mean lifespans of C-29 °C and C-34 °C flies were significantly extended by 8.7-16.4% in males, and by 8.9-12.8% in females, compared to that of temperature-matched flies fed basal diets. The MDA levels of C-34 °C flies were significantly lower than those of N-34 °C flies, indicating CUR reduced oxidative stress caused by HS. Furthermore, CUR palliated the increased oxidative stress caused by HS, by increasing the expression of SOD1, CAT, and PHGPx and decreasing the expression of Hsp70 and Hsp83. Our results indicated that CUR supplementation increases the survival rate of Drosophila by enhancing thermal tolerance. © 2018 BioFactors, 44(6):577-587, 2018.

Grape seed proanthocyanidin reverses pulmonary vascular remodeling in monocrotaline-induced pulmonary arterial hypertension by down-regulating HSP70.

Heat shock protein 70 (HSP70) is a molecular chaperone which has a low content in cytoplasm under normal physiological conditions. A higher intracytoplasmic HSP70 level can be observed in pulmonary arterial smooth muscle cell (PASMC) in pulmonary arterial hypertension (PAH), and this up-regulation can promote pho-IκBα expression, which is an NF-κB signaling pathway inhibitor. NF-κB signaling pathway up-regulation can promote PASMC proliferation and pulmonary vascular remodeling in PAH, resulting in elevation of pulmonary pressure and the subsequent right heart failure caused by right ventricular hypertrophy. Grape seed proanthocyanidin (GSP) is effective in vascular protection and several tumor treatments, and its effect on PAH treatment remains to be elucidated. In this study, we made observations and contrasts in monocrotaline(MCT) -induced PAH rats, and found decrease in mPAP, PVR and RVHI after GSP administration. Our study also proved GSP's effect on down-regulating the intracytoplasmic HSP70 content both in cellular and animal levels. The results indicate a possible mechanism of GSP reversing pulmonary vascular remodeling by down-regulating HSP70, and this change may influence pho-IκBα expression. Therefore, inhibition of NF-κB signaling pathway caused by GSP can lead to inhibition of PASMC proliferation in PAH.

High-throughput screen for inhibitors of protein-protein interactions in a reconstituted heat shock protein 70 (Hsp70) complex.

Protein-protein interactions (PPIs) are an important category of putative drug targets. Improvements in high-throughput screening (HTS) have significantly accelerated the discovery of inhibitors for some categories of PPIs. However, methods suitable for screening multiprotein complexes (e.g. those composed of three or more different components) have been slower to emerge. Here, we explored an approach that uses reconstituted multiprotein complexes (RMPCs). As a model system, we chose heat shock protein 70 (Hsp70), which is an ATP-dependent molecular chaperone that interacts with co-chaperones, including DnaJA2 and BAG2. The PPIs between Hsp70 and its co-chaperones stimulate nucleotide cycling. Thus, to re-create this ternary protein system, we combined purified human Hsp70 with DnaJA2 and BAG2 and then screened 100,000 diverse compounds for those that inhibited co-chaperone-stimulated ATPase activity. This HTS campaign yielded two compounds with promising inhibitory activity. Interestingly, one inhibited the PPI between Hsp70 and DnaJA2, whereas the other seemed to inhibit the Hsp70-BAG2 complex. Using secondary assays, we found that both compounds inhibited the PPIs through binding to allosteric sites on Hsp70, but neither affected Hsp70's intrinsic ATPase activity. Our RMPC approach expands the toolbox of biochemical HTS methods available for studying difficult-to-target PPIs in multiprotein complexes. The results may also provide a starting point for new chemical probes of the Hsp70 system.

Structure-Based Drug Designing and Simulation Studies for Finding Novel Inhibitors of Heat Shock Protein (HSP70) as Suppressors for Psoriasis.

Psoriasis is a chronic immune-mediated inflammatory skin disorder. Heat shock proteins (HSPs) have been witnessed as a potential drug target for inhibition of psoriatic cell differentiation. The expression level of HSP is increased when the cells get exposed to elevated temperature, oxidative stress and nutritional deficiencies and thus plays major role in psoriatic progression pathway. Immunoreactivity intensity distribution index scores for HSP70 expression is significantly higher in psoriatic patients compared to normal. In the present work, the 3D structure of human Hsp70 has been taken. Inhibition of HSP70 can control the severity of psoriasis up to many folds; thus, virtual screening was performed against lead-like, drug-like and some natural product of ZINC database. The screened ligands were further introduced to ADMET prediction and simulations to see the drug proficiency and likeness property. The molecular dynamic of system was found stable during simulation trajectory and not much of significant changes occurred in the conformation of the protein-ligand complex. Thus, present study in all probability might prove useful for future design of new derivatives with higher potency and specificity.

Pharmacophore modeling, virtual screening and molecular docking of ATPase inhibitors of HSP70.

Heat shock protein 70 is an effective anticancer target as it influences many signaling pathways. Hence the study investigated the important pharmacophore feature required for ATPase inhibitors of HSP70 by generating a ligand based pharmacophore model followed by virtual based screening and subsequent validation by molecular docking in Discovery studio V4.0. The most extrapolative pharmacophore model (hypotheses 8) consisted of four hydrogen bond acceptors. Further validation by external test set prediction identified 200 hits from Mini Maybridge, Drug Diverse, SCPDB compounds and Phytochemicals. Consequently, the screened compounds were refined by rule of five, ADMET and molecular docking to retain the best competitive hits. Finally Phytochemical compounds Muricatetrocin B, Diacetylphiladelphicalactone C, Eleutheroside B and 5-(3-{[1-(benzylsulfonyl)piperidin-4-yl]amino}phenyl)- 4-bromo-3-(carboxymethoxy)thiophene-2-carboxylic acid were obtained as leads to inhibit the ATPase activity of HSP70 in our findings and thus can be proposed for further in vitro and in vivo evaluation.

Extracts Obtained from Pterocarpus angolensis DC and Ziziphus mucronata Exhibit Antiplasmodial Activity and Inhibit Heat Shock Protein 70 (Hsp70) Function.

Malaria parasites are increasingly becoming resistant to currently used antimalarial therapies, therefore there is an urgent need to expand the arsenal of alternative antimalarial drugs. In addition, it is also important to identify novel antimalarial drug targets. In the current study, extracts of two plants, Pterocarpus angolensis and Ziziphus mucronata were obtained and their antimalarial functions were investigated. Furthermore, we explored the capability of the extracts to inhibit Plasmodium falciparum heat shock protein 70 (Hsp70) function. Heat shock protein 70 (Hsp70) are molecular chaperones whose function is to facilitate protein folding. Plasmodium falciparum the main agent of malaria, expresses two cytosol-localized Hsp70s: PfHsp70-1 and PfHsp70-z. The PfHsp70-z has been reported to be essential for parasite survival, while inhibition of PfHsp70-1 function leads to parasite death. Hence both PfHsp70-1 and PfHsp70-z are potential antimalarial drug targets. Extracts of P. angolensis and Z. mucronata inhibited the basal ATPase and chaperone functions of the two parasite Hsp70s. Furthermore, fractions of P. angolensis and Z. mucronata inhibited P. falciparum 3D7 parasite growth in vitro. The extracts obtained in the current study exhibited antiplasmodial activity as they killed P. falciparum parasites maintained in vitro. In addition, the findings further suggest that some of the compounds in P. angolensis and Z. mucronata may target parasite Hsp70 function.

Vitamin A Oral Supplementation Induces Oxidative Stress and Suppresses IL-10 and HSP70 in Skeletal Muscle of Trained Rats.

Exercise training intensity is the major variant that influences the relationship between exercise, redox balance, and immune response. Supplement intake is a common practice for oxidative stress prevention; the effects of vitamin A (VA) on exercise training are not yet described, even though this molecule exhibits antioxidant properties. We investigated the role of VA supplementation on redox and immune responses of adult Wistar rats subjected to swimming training. Animals were divided into four groups: sedentary, sedentary + VA, exercise training, and exercise training + VA. Over eight weeks, animals were submitted to intense swimming 5 times/week and a VA daily intake of 450 retinol equivalents/day. VA impaired the total serum antioxidant capacity acquired by exercise, with no change in interleukin-1ß and tumor necrosis factor-α levels. In skeletal muscle, VA caused lipid peroxidation and protein damage without differences in antioxidant enzyme activities; however, Western blot analysis showed that expression of superoxide dismutase-1 was downregulated, and upregulation of superoxide dismutase-2 induced by exercise was blunted by VA. Furthermore, VA supplementation decreased anti-inflammatory interleukin-10 and heat shock protein 70 expression, important factors for positive exercise adaptations and tissue damage prevention. Our data showed that VA supplementation did not confer any antioxidative and/or protective effects, attenuating exercise-acquired benefits in the skeletal muscle.

HSP70 Inhibition Synergistically Enhances the Effects of Magnetic Fluid Hyperthermia in Ovarian Cancer.

Hyperthermia has been investigated as a potential treatment for cancer. However, specificity in hyperthermia application remains a significant challenge. Magnetic fluid hyperthermia (MFH) may be an alternative to surpass such a challenge, but implications of MFH at the cellular level are not well understood. Therefore, the present work focused on the examination of gene expression after MFH treatment and using such information to identify target genes that when inhibited could produce an enhanced therapeutic outcome after MFH. Genomic analyzes were performed using ovarian cancer cells exposed to MFH for 30 minutes at 43°C, which revealed that heat shock protein (HSP) genes, including HSPA6, were upregulated. HSPA6 encodes the Hsp70, and its expression was confirmed by PCR in HeyA8 and A2780cp20 ovarian cancer cells. Two strategies were investigated to inhibit Hsp70-related genes, siRNA and Hsp70 protein function inhibition by 2-phenylethyenesulfonamide (PES). Both strategies resulted in decreased cell viability following exposure to MFH. Combination index was calculated for PES treatment reporting a synergistic effect. In vivo efficacy experiments with HSPA6 siRNA and MFH were performed using the A2780cp20 and HeyA8 ovarian cancer mouse models. A significantly reduction in tumor growth rate was observed with combination therapy. PES and MFH efficacy were also evaluated in the HeyA8 intraperitoneal tumor model, and resulted in robust antitumor effects. This work demonstrated that HSP70 inhibition combination with MFH generate a synergistic effect and could be a promising target to enhance MFH therapeutic outcomes in ovarian cancer. Mol Cancer Ther; 16(5); 966-76. ©2017 AACR.

An in vitro model to consider the effect of 2 mM glutamine and KNK437 on endotoxin-stimulated release of heat shock protein 70 and inflammatory mediators.

OBJECTIVE: Glutamine has been shown to promote the release of heat shock protein 70 (HSP70) both within experimental in vitro models of sepsis and in adults with septic shock. This study aimed to investigate the effects of 2 mM glutamine and an inhibitor of HSP70 (KNK437) on the release of HSP70 and inflammatory mediators in healthy adult volunteers. METHODS: An in vitro whole blood endotoxin stimulation assay was used. RESULTS: The addition of 2 mM glutamine significantly increased HSP70 levels over time (P < 0.05). HSP70 release had a positive correlation at 4 h with IL-1 ß (r = 0.51, P = 0.03) and an inverse correlation with TNF-α (r = -0.56, P = 0.02) and IL-8 levels (r = -0.52, P = 0.03), and there were no significant correlations between HSP70 and IL6 or IL-10 or glutamine. Glutamine supplementation significantly (P < 0.05) attenuated the release of IL-10 at 4 h and IL-8 at 24 h, compared with conditions without glutamine. In endotoxin-stimulated blood there were no significant differences in the release of IL-6, TNF-α, and IL-1 ß with glutamine supplementation at 4 and 24 h. However, glutamine supplementation (2 mM) appeared to attenuate the release of inflammatory mediators (IL-1 ß, IL-6, TNF-α), although this effect was not statistically significant. The addition of KNK437, a HSP70 inhibitor, significantly diminished HSP70 release, which resulted in lower levels of inflammatory mediators (P < 0.05). CONCLUSION: Glutamine supplementation promotes HSP70 release in an experimental model of sepsis. After the addition of KNK437, the effects of glutamine on HSP70 and inflammatory mediator release appear to be lost, suggesting that HSP70 in part orchestrates the inflammatory mediator response to sepsis. The clinical implications require further investigation.

Blocking Hsp70 enhances the efficiency of amphotericin B treatment against resistant Aspergillus terreus strains.

The polyene antifungal amphotericin B (AmB) is widely used to treat life-threatening fungal infections. Even though AmB resistance is exceptionally rare in fungi, most Aspergillus terreus isolates exhibit an intrinsic resistance against the drug in vivo and in vitro. Heat shock proteins perform a fundamental protective role against a multitude of stress responses, thereby maintaining protein homeostasis in the organism. In this study, we elucidated the role of heat shock protein 70 (Hsp70) family members and compared resistant and susceptible A. terreus clinical isolates. The upregulation of cytoplasmic Hsp70 members at the transcriptional as well as translational levels was significantly higher with AmB treatment than without AmB treatment, particularly in resistant A. terreus isolates, thereby indicating a role of Hsp70 proteins in the AmB response. We found that Hsp70 inhibitors considerably increased the susceptibility of resistant A. terreus isolates to AmB but exerted little impact on susceptible isolates. Also, in in vivo experiments, using the Galleria mellonella infection model, cotreatment of resistant A. terreus strains with AmB and the Hsp70 inhibitor pifithrin-µ resulted in significantly improved survival compared with that achieved with AmB alone. Our results point to an important mechanism of regulation of AmB resistance by Hsp70 family members in A. terreus and suggest novel drug targets for the treatment of infections caused by resistant fungal isolates.

Quercetin affects Hsp70/IRE1α mediated protection from death induced by endoplasmic reticulum stress.

Relative to their normal counterparts, tumor cells generally exhibit a greater "stress phenotype" and express heat shock proteins (Hsp) that represent candidate targets for anticancer therapy. Here we investigated the role of Hsp70 in survival induced by endoplasmic reticulum (ER) stressors in human leukemia U937 cells. Quercetin, a major dietary flavonoid, or specific silencing affected the expression level of Hsp70 and did not allow the upregulation of inositol-requiring kinase 1α (IRE1α), the prototype ER stress sensor regulating the unfolded protein response (UPR), that protects the cells against the stress of misfolded proteins in the ER. The reduction of Hsp70 prevented the upregulation of immunoglobulin heavy-chain binding protein (BiP), but not of CCAAT/enhancer-binding protein-homologous protein (CHOP), and induced apoptosis. Also specific silencing of IRE1α or inhibition of its endoribonuclease activity by 4µ8c hampered the upregulation of BiP, but not of CHOP, and induced apoptosis. These results suggest that drugs affecting the Hsp70-IRE1α axis, like quercetin, or affecting directly IRE1α may represent an effective adjuvant antileukemia therapy.

Photothermal therapeutic response of cancer cells to aptamer-gold nanoparticle-hybridized graphene oxide under NIR illumination.

The objective of this study was to synthesize a nanocomposite, aptamer-gold nanoparticle-hybridized graphene oxide (Apt-AuNP-GO), to facilitate targeted treatment of tumor cells by near-infrared (NIR) light-activatable photothermal therapy. We also investigated whether Apt-AuNP-GO with NIR illumination modulates heat shock proteins (HSPs) expression leading to therapeutic response in human breast cancer cells. These findings can provide strategies for improving the photothermal therapy efficacy of cancer. The self-assembled Apt-AuNP-GO nanocomposite could selectively target MUC1-positive human breast cancer cells (MCF-7) due to the specific interaction between the MUC1-binding-aptamer and the MUC1 (type I transmembrane mucin glycoprotein) on cell membrane. In addition, Apt-AuNP-GO has a high light-to-heat conversion capability for photoabsorption of NIR light, and it is able to exert therapeutic effects on MCF-7 cells at an ultralow concentration without inducing adverse effects in healthy cells. The Apt-AuNP-GO nanocomposites combine the advantages of GOs, AuNPs, and Apts, possess specific targeting capability, excellent biocompatibility, and tumor cell destruction ability, suggesting great potential for application in the photothermal therapy of breast cancer. Under NIR illumination, Apt-AuNP-GO induced transient increase in HSP70 expression, which decreased thereafter. This phenomenon may cause irreversible damage to Apt-AuNP-GO-treated MCF-7 cell under NIR illumination. We also demonstrated that the combination therapy of heat and HSP70 inhibitor could synergistically generate marked tumoricidal effects against breast cancer. These results suggest that the degree and duration of HSP70 protein expression are correlated with therapeutic effects against breast cancer for Apt-AuNP-GO-assisted photothermal therapy. We believe that such a nanocomposite can be readily extended to the construction of HSP70 inhibitors-loaded Apt-AuNP-GO, which could deliver both heat and HSP70 inhibitors to tumorigenic regions for the chemo-photothermal therapy.