Heat Shock Protein Pathway Research Articles

The protective effects of esculetin against Doxorubicin‐Induced hepatotoxicity in rats: Insights into the modulation of Caspase, FOXOs, and heat shock protein pathways

AbstractDoxorubicin (DOX) is an anthracycline antibiotic widely employed to treat carcinoma. Nevertheless, severe cardiotoxic side effects restrict its clinical use. Esculetin, a natural flavonoid, is found abundantly in plants. This study evaluated the protective effects of esculetin against DOX‐induced hepatotoxicity in rat livers. Forty‐eight rats were randomly divided into six groups with eight rats in each group: control (I), DOX (II), esculetin (III, 50 mg/kg), esculetin (IV, 100 mg/kg), DOX+esculetin 50 (V, DOX+esculetin 50 mg/kg), and DOX+esculetin 100 (VI, DOX+esculetin 100 mg/kg). The administration of esculetin effectively mitigated alterations in the measured biochemical parameters induced by DOX. Gene expression analyses demonstrated that esculetin treatment significantly reduced the DOX‐induced expression of Foxo1, Hspa1a, Hsp4a, Hsp5a, Casp3, and Casp9 while increasing the DOX‐induced expression of Foxo3. These findings suggest that esculetin, with its antioxidant and anti‐inflammatory effects, might be a therapeutic option for protecting against DOX‐induced hepatotoxicity.

Potential Antifungal Targets for Aspergillus sp. from the Calcineurin and Heat Shock Protein Pathways.

Aspergillus species, especially A. fumigatus, and to a lesser extent others (A. flavus, A. niger, A. terreus), although rarely pathogenic to healthy humans, can be very aggressive to immunocompromised patients (they are opportunistic pathogens). Although survival rates for such infections have improved in recent decades following the introduction of azole derivatives, they remain a clinical challenge. The fact that current antifungals act as fungistatic rather than fungicide, that they have limited safety, and that resistance is becoming increasingly common make the need for new, more effective, and safer therapies to become more acute. Over the last decades, knowledge about the molecular biology of A. fumigatus and other Aspergillus species, and particularly of calcineurin, Hsp90, and their signaling pathway proteins, has progressed remarkably. Although calcineurin has attracted much interest, its adverse effects, particularly its immunosuppressive effects, make it less attractive than it might at first appear. The situation is not very different for Hsp90. Other proteins from their signaling pathways, such as protein kinases phosphorylating the four SPRR serine residues, CrzA, rcnA, pmcA-pmcC (particularly pmcC), rfeF, BAR adapter protein(s), the phkB histidine kinase, sskB MAP kinase kinase, zfpA, htfA, ctfA, SwoH (nucleoside diphosphate kinase), CchA, MidA, FKBP12, the K27 lysine position from Hsp90, PkcA, MpkA, RlmA, brlA, abaA, wetA, other heat shock proteins (Hsp70, Hsp40, Hsp12) currently appear promising and deserve further investigation as potential targets for antifungal drug development.

Population Genomics, Transcriptional Response to Heat Shock, and Gut Microbiota of the Hong Kong Oyster Magallana hongkongensis

The Hong Kong oyster Magallana hongkongensis, previously known as Crassostrea hongkongensis, is a true oyster species native to the estuarine-coast of the Pearl River Delta in southern China. The species—with scientific, ecological, cultural, and nutritional importance—has been farmed for hundreds of years. However, there is only limited information on its genetics, stress adaptation mechanisms, and gut microbiota, restricting the sustainable production and use of oyster resources. Here, we present population structure analysis on M. hongkongensis oysters collected from Deep Bay and Lantau Island in Hong Kong, as well as transcriptome analysis on heat shock responses and the gut microbiota profile of M. hongkongensis oysters collected from Deep Bay. Single nucleotide polymorphisms (SNPs), including those on the homeobox genes and heat shock protein genes, were revealed by the whole genome resequencing. Transcriptomes of oysters incubated at 25 °C and 32 °C for 24 h were sequenced which revealed the heat-induced regulation of heat shock protein pathway genes. Furthermore, the gut microbe community was detected by 16S rRNA sequencing which identified Cyanobacteria, Proteobacteria and Spirochaetes as the most abundant phyla. This study reveals the molecular basis for the adaptation of the oyster M. hongkongensis to environmental conditions.

The protective effect of nnano-selenium against cadmium-induced cerebellar injury via the heat shock protein pathway in chicken

Cadmium (Cd) is one of the toxic environmental heavy metals that poses health hazard to animals due to its toxicity. Nano-Selenium (Nano-Se) is a Nano-composite form of Se, which has emerged as a promising therapeutic agent for its protective roles against heavy metals-induced toxicity. Heat shock proteins (HSPs) play a critical role in cellular homeostasis. However, the potential protective effects of Nano-Se against Cd-induced cerebellar toxicity remain to be illustrated. To investigate the toxic effects of Cd on chicken's cerebellum, and the protective effects of Nano-Se against Cd-induced cerebellar toxicity, a total of 80 male chicks were divided into four groups and treated as follows: (A) 0 mg/kg Cd, (B) 1 mg/kg Nano-Se (C) 140 mg/kg Cd + 1 mg/kg Nano-Se (D) 140 mg/kg Cd for 90 days. We tested heat shock protein pathway-related factors including heat shock factors (HSFs) HSF1, HSF2, HSF3 and heat shock proteins (HSPs) HSP10, HSP25, HSP27, HSP40, HSP60, HSP70 and HSP90 expressions. Histopathological results showed that Cd treatment caused degradation of Purkinje cells. In addition, HSFs and HSPs expression decreased significantly in the Cd group. Nano-Se co-treatment with Cd enhanced the expression of HSFs and HSPs. In summary, our findings explicated a potential protective effect of Nano-Se against Cd-induced cerebellar injury in chicken, suggesting that Nano-Se is a promising therapeutic agent for the treatment of Cd toxicity.

Molecular and electrophysiological features of spinocerebellar ataxia type seven in induced pluripotent stem cells.

Spinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disease caused by a polyglutamine repeat expansion in the ATXN7 gene. Patients with this disease suffer from a degeneration of their cerebellar Purkinje neurons and retinal photoreceptors that result in a progressive ataxia and loss of vision. As with many neurodegenerative diseases, studies of pathogenesis have been hindered by a lack of disease-relevant models. To this end, we have generated induced pluripotent stem cells (iPSCs) from a cohort of SCA7 patients in South Africa. First, we differentiated the SCA7 affected iPSCs into neurons which showed evidence of a transcriptional phenotype affecting components of STAGA (ATXN7 and KAT2A) and the heat shock protein pathway (DNAJA1 and HSP70). We then performed electrophysiology on the SCA7 iPSC-derived neurons and found that these cells show features of functional aberrations. Lastly, we were able to differentiate the SCA7 iPSCs into retinal photoreceptors that also showed similar transcriptional aberrations to the SCA7 neurons. Our findings give technical insights on how iPSC-derived neurons and photoreceptors can be derived from SCA7 patients and demonstrate that these cells express molecular and electrophysiological differences that may be indicative of impaired neuronal health. We hope that these findings will contribute towards the ongoing efforts to establish the cell-derived models of neurodegenerative diseases that are needed to develop patient-specific treatments.

240 In vitro and in vivo skin efficacy of CBD extract

Cannabidiol (CBD) has been implicated as beneficial in variety of skin conditions, including acne, atopic dermatitis, psoriasis, scleroderma, and pruritis. CBD mediates its effect through skin’s own endocannabinoid system composed of CB1 and CB2 receptors, but detailed mechanism of action in skin and clinical efficacy potential needs to be established. In this study we investigated the role of CBD on modulation of heat shock protein pathway in human dermal fibroblasts. Cells were treated with various concentration of CBD and amount of HSP-70 induced was evaluated by ELISA. In addition, clinical efficacy was assessed using an in vivo model. Erythema was induced by topical methyl nicotinate (MN), leading to prostaglandin release and vasodilatation of the peripheral blood capillaries of the skin. CBD was shown to significantly stimulate HSP70 protein level in human dermal fibroblast cells as well as reduce erythema in a clinical model of redness.

Cadmium alters heat shock protein pathways in SN56 cholinergic neurons, leading to Aβ and phosphorylated Tau protein generation and cell death

Cadmium, a neurotoxic environmental compound, produces cognitive disorders, although the mechanism remains unknown. Cadmium induces a more pronounced cell death on cholinergic neurons from basal forebrain (BF), mediated, in part, by increase in Aβ and total and phosphorylated Tau protein levels, which may explain cadmium effects on learning and memory processes. Cadmium downregulates the expression of heat shock proteins (HSPs) HSP 90, HSP70 and HSP27, and of HSF1, the master regulator of the HSP pathway. HSPs proteins reduce the production of Aβ and phosphorylated Tau proteins and avoid cell death pathways induction. Thus, we hypothesized that cadmium induced the production of Aβ and Tau proteins by HSP pathway disruption through HSF1 expression alteration, leading to BF cholinergic neurons cell death. Our results show that cadmium downregulates HSF1, leading to HSP90, HSP70 and HSP27 gene expression downregulation in BF SN56 cholinergic neurons. In addition, cadmium induced Aβ and total and phosphorylated Tau proteins generation, mediated partially by HSP90, HSP70 and HSP27 disruption, leading to cell death. These results provide new understanding of the mechanisms contributing to cadmium harmful effects on cholinergic neurons.

FcγRIIIa Signaling Modulates Endosomal TLR Responses in Human CD4+ T Cells.

Recognition of Ab-opsonized pathogens by immune cells triggers both TLR and Fc receptor signaling. Fc receptors endocytose modified nucleic acids bound to Abs and deliver them to endosomes, where they are recognized by nucleic acid-sensing TLRs (NA-TLRs). We show that in CD4+ T cells, NA-TLRs, TLR3, TLR8, and TLR9 are upregulated by FcγRIIIa-pSyk cosignaling and localize with FcγRIIIa on the cell surface. TLR9 accumulates on the cell surface, where it recognizes CpG oligonucleotide 2006. Subcellular location of NA-TLRs is a key determinant in discriminating self versus viral nucleic acid. Hydroxychloroquine used for treating systemic lupus erythematosus and a Syk inhibitor blocked NA-TLR localization with FcγRIIIa. Engaging TLR9 with CpG oligonucleotide contributes to the development of IL17A+ and IL-21+ populations. RNA-sequencing analysis showed upregulation of proinflammatory cytokines, NF-κB signaling, and heat shock protein pathway RNA transcripts. These data suggest a role for FcγRIIIa-pSyk cosignaling in modulating NA-TLR responses in human CD4+ T cells by affecting the amounts and cellular distribution. These events are important for understanding of autoimmune pathology.

Transcriptome analysis of microglia in a mouse model of Rett syndrome: differential expression of genes associated with microglia/macrophage activation and cellular stress

BackgroundRett syndrome (RTT) is a severe, neurodevelopmental disorder primarily affecting girls, characterized by progressive loss of cognitive, social, and motor skills after a relatively brief period of typical development. It is usually due to de novo loss of function mutations in the X-linked gene, MeCP2, which codes for the gene expression and chromatin regulator, methyl-CpG binding protein 2. Although the behavioral phenotype appears to be primarily due to neuronal Mecp2 deficiency in mice, other cell types, including astrocytes and oligodendrocytes, also appear to contribute to some aspects of the RTT phenotype. In addition, microglia may also play a role. However, the effect of Mecp2 deficiency in microglia on RTT pathogenesis is controversial.MethodsIn the current study, we applied whole transcriptome analysis using RNA-seq to gain insight into molecular pathways in microglia that might be dysregulated during the transition, in female mice heterozygous for an Mecp2-null allele (Mecp2+/−; Het), from the pre-phenotypic (5 weeks) to the phenotypic phases (24 weeks).ResultsWe found a significant overlap in differentially expressed genes (DEGs) with genes involved in regulating the extracellular matrix, and those that are activated or inhibited when macrophages and microglia are stimulated towards the M1 and M2 activation states. However, the M1- and M2-associated genes were different in the 5- and 24-week samples. In addition, a substantial decrease in the expression of nine members of the heat shock protein (HSP) family was found in the 5-week samples, but not at 24 weeks.ConclusionsThese findings suggest that microglia from pre-phenotypic and phenotypic female mice are activated in a manner different from controls and that pre-phenotypic female mice may have alterations in their capacity to response to heat stress and other stressors that function through the HSP pathway.

Betulinic acid acetate, an antiproliferative natural product, suppresses client proteins of heat shock protein pathways through a CDC37-binding mechanism

CDC37 has emerged as a promising target in antitumor chemotherapy because of its significant role in oncogenic signaling networks.

Proceedings of the 2015 Annual Meeting of the Fetal Alcohol Spectrum Disorders Study Group.

The 2015 Fetal Alcohol Spectrum Disorders Study Group (FASDSG) meeting was titled "Basic Mechanisms and Translational Implications." Despite decades of basic science and clinical research, our understanding of the mechanisms by which ethanol affects fetal development is still in its infancy. The first keynote presentation focused on the role of heat shock protein pathways in the actions of ethanol in the developing brain. The second keynote presentation addressed the use of magnetoencephalography to characterize brain function in children with FASD. The conference also included talks by representatives from several government agencies, short presentations by junior and senior investigators that showcased the latest in FASD research, and award presentations. An important part of the meeting was the presentation of the 2015 Henry Rosett award to Dr. Michael Charness in honor of his achievements in research on FASD.

Antitumor activity of the combination of an HSP90 inhibitor and a PI3K/mTOR dual inhibitor against cholangiocarcinoma.

The PI3K/Akt/mTOR pathway is overactivated and heat shock protein (HSP) 90 is overexpressed in common cancers. We hypothesized that targeting both pathways can kill intrahepatic cholangiocarcinoma (CCA) cells. HSP90 and PTEN protein expression was evaluated by immunohistochemical staining of samples from 78 patients with intrahepatic CCA. CCA cell lines and a thioacetamide (TAA)-induced CCA animal model were treated with NVP-AUY922 (an HSP90 inhibitor) and NVP-BEZ235 (a PI3K/mTOR inhibitor) alone or in combination. Both HSP90 overexpression and loss of PTEN were poor prognostic factors in patients with intrahepatic CCA. The combination of the HSP90 inhibitor NVP-AUY922 and the PI3K/mTOR inhibitor NVP-BEZ235 was synergistic in inducing cell death in CCA cells. A combination of NVP-AUY922 and NVP-BEZ235 caused tumor regression in CCA rat animal model. This combination not only inhibited the PI3K/Akt/mTOR pathway but also induced ROS, which may exacerbate the vicious cycle of ER stress. Our data suggest simultaneous targeting of the PI3K/mTOR and HSP pathways for CCA treatment.

Alanyl-glutamine and glutamine plus alanine supplements improve skeletal redox status in trained rats: Involvement of heat shock protein pathways

AimsWe hypothesized that oral l-glutamine supplementations could attenuate muscle damage and oxidative stress, mediated by glutathione (GSH) in high-intensity aerobic exercise by increasing the 70-kDa heat shock proteins (HSP70) and heat shock factor 1 (HSF1). Main methodsAdult male Wistar rats were 8-week trained (60-min/day, 5days/week) on a treadmill. During the last 21days, the animals were supplemented with either l-alanyl-l-glutamine dipeptide (1.5g/kg, DIP) or a solution containing the amino acids l-glutamine (1g/kg) and l-alanine (0.67g/kg) in their free form (GLN+ALA) or water (controls). Key findingsPlasma from both DIP- and GLN+ALA-treated animals showed higher l-glutamine concentrations and reduced ammonium, malondialdehyde, myoglobin and creatine kinase activity. In the soleus and gastrocnemius muscle of both supplemented groups, l-glutamine and GSH contents were increased and GSH disulfide (GSSG) to GSH ratio was attenuated (p<0.001). In the soleus muscle, cytosolic and nuclear HSP70 and HSF1 were increased by DIP supplementation. GLN+ALA group exhibited higher HSP70 (only in the nucleus) and HSF1 (cytosol and nucleus). In the gastrocnemius muscle, both supplementations were able to increase cytosolic HSP70 and cytosolic and nuclear HSF1. SignificanceIn trained rats, oral supplementation with DIP or GLN+ALA solution increased the expression of muscle HSP70, favored muscle l-glutamine/GSH status and improved redox defenses, which attenuate markers of muscle damage, thus improving the beneficial effects of high-intensity exercise training.

The Protection of Selenium against Cadmium-Induced Cytotoxicity via the Heat Shock Protein Pathway in Chicken Splenic Lymphocytes

Cadmium (Cd) is a heavy metal that poses a hazard to animal health due to its toxicity. Selenium (Se) is an important nutritional trace element. However, the potential protective effects of Se against Cd-induced toxicity remain to be elucidated. To investigate the cytotoxicity of Cd on bird immunocytes in vitro and the protective effects of Se against exposure to Cd, chicken splenic lymphocytes received Cd (10−6 mol/L), Se (10−7 mol/L), and the mixture of 10−7 mol/L Se and 10−6 mol/L Cd and were incubated for 12 h, 24 h, 36 h, 48 h, respectively. The transcription of heat shock protein (HSP) 27, HSP40, HSP60, HSP70 and HSP90 mRNA was tested by fluorescence quantitative PCR. The results showed that the mRNA expression of HSPs exposed to 10−6 mol/L Cd showed a sustained decrease at 12–48 h exposure. A statistically significant increase in the mRNA expression of HSPs in the case of Se group was observed, as compared to the control group of chicken splenic lymphocytes. Concomitantly, treatment of chicken splenic lymphocytes with Se in combination with Cd enhanced the mRNA expression of HSPs which were reduced by Cd treatment. This indicated that the protective effect of Se against the toxicity of Cd might, at least partially, be attributed to stimulation of the level of HSPs.

Hyperthermia potentiates oncolytic herpes viral killing of pancreatic cancer through a heat shock protein pathway

Oncolytic herpes simplex virus-1 (HSV-1) is designed to specifically infect, replicate in, and lyse cancer cells. This study investigates a novel therapeutic regimen, combining the effects of NV1066 (a recombinant HSV-1) and hyperthermia in the treatment of pancreatic cancer. NV1066 is an attenuated HSV-1 that replicates in cells resistant to apoptosis. Heat shock protein 72 (Hsp72) is a member of a family of proteins that is upregulated after hyperthermic insult, lending cellular protection by inhibiting apoptosis. In these experiments, we test the hypothesis that increased Hsp72 expression in response to hyperthermia enhances anti-apoptotic mechanisms, thereby increasing viral replication and tumor cell kill. Hs 700T pancreatic cancer cells were treated with hyperthermia alone (42 degrees C), NV1066 alone, and combination therapy. Cell survival and viral growth were measured. The effect of siRNA-directed Hsp72 knockdown was also measured. Combining hyperthermia and viral treatment produced a synergistic effect on cell kill. Viral growth increased greater than 6-fold in the presence of hyperthermia (P < .05). Hyperthermia alone showed minimal cytotoxic activity against Hs 700T cells, while NV1066 infection resulted in approximately 50% cell kill. The combination of hyperthermia and viral infection significantly increased cell kill to approximately 80% (P < .01). Hsp72 knockdown attenuated this synergistic effect. Hyperthermia enhances NV1066 replication, thereby potentiating the viral oncolytic response against pancreatic cancer cells. This finding has potential clinical application in the use of heated perfusion or permissive hyperthermia for delivery of oncolytic viral therapies.

Hyperthermia Potentiates Oncolytic Herpes Viral Killing of Pancreatic Cancer through a Heat Shock Protein Pathway

Hyperthermia Potentiates Oncolytic Herpes Viral Killing of Pancreatic Cancer through a Heat Shock Protein Pathway

Alveolar macrophages in allergic asthma: An expression signature characterized by heat shock protein pathways

The implication of alveolar macrophages (AM) in asthma, a T h2 disease, has not been well characterized. Thus, the goal of this study is to better characterize AM phenotype of allergic asthmatic compared with normal subjects using genomic expression analyses. Microarray analyses were performed with AM isolated from bronchoalveolar lavage. Robust multiarray analysis (RMA) normalization and Smyth's moderated t test were used to select differentially expressed genes. Fifty differentially expressed genes were identified. Nineteen have been classified in categories linked to stress or immune responses and among them; nine are part of the heat shock protein (HSP) family. Difference of expression for three ( HSPD1, PRNP, SERPINH1) of the five selected genes were validated using real-time reverse transcription–polymerase chain reaction. Enzyme-linked immunosorbent assay was used to measure the protein level of heat shock protein 60 (HSP60), the protein encoded by HSPD1, and showed difference in AM protein level between allergic asthmatic and control subjects. In summary, this study suggests that HSP gene family, particularly HSP60, is involved in AM functions in a context of allergic asthma. These results also support the involvement of AM immune functions in the development of an allergic asthmatic response.

Targeting Akt and heat shock protein 90 produces synergistic multiple myeloma cell cytotoxicity in the bone marrow microenvironment.

We hypothesized that targeting both Akt and heat shock protein (HSP) 90 would induce cytotoxic activity against multiple myeloma (MM) cells and target the bone marrow (BM) microenvironment to inhibit angiogenesis, osteoclast formation, as well as migration and adhesion of MM cells. MM cell lines were incubated with perifosine (5 and 10 micromol/L) and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG; 50 and 100 nmol/L) alone and in combination. The combination of Akt inhibitor perifosine and HSP90 inhibitor 17-DMAG was synergistic in inducing MM cell cytotoxicity, evidenced by inhibition of DNA synthesis and induction of apoptosis. In addition, perifosine and 17-DMAG almost completely inhibited osteoclast formation: perifosine interfered with both early and late stages of osteoclast progenitor development, whereas 17-DMAG targeted only early stages. We next showed that combined therapy overcomes tumor growth and resistance induced by BM stromal cells and endothelial cells as well as the proliferative effect of exogenous interleukin-6, insulin-like growth factor-I, and vascular endothelial growth factor. Moreover, the combination also induced apoptosis and growth inhibition in endothelial cells and inhibited angiogenesis. Finally, we showed that the two agents prevented migration of MM cells toward stromal-derived factor-1 and vascular endothelial growth factor, which are present in the BM milieu, and also prevented adhesion of MM cells to fibronectin. This study provides the preclinical framework for treatment protocols targeting both the Akt and HSP pathways in MM.

Association of Polymorphisms in TGFB1 and Prostate Cancer Prognosis

Because of the role of TGFB1 in prostate cancer and progression, we hypothesized that polymorphisms of TGFB1 at C-509T may be associated with prostate cancer risk and/or more aggressive tumors. This is a case-control study. Controls consisted of male volunteers 40 years old or older with a normal digital rectal examination and prostate specific antigen 2.5 ng/ml or less. Cases consisted of men with biopsy proven prostate cancer. High grade prostate cancer included all cancers of Gleason sum 7 or greater. Poor prognosis in cases was defined as any stage with Gleason sum 8-10, pT3A (if Gleason sum was greater than 7), pT3B or higher (all Gleason sums), any N1 or higher, any M1 or higher, or any documented PSA recurrence (biochemical failure). Single nucleotide polymorphisms were genotyped using allelic discrimination assays. Logistic regression models were used to estimate the OR with the corresponding 95% CI for individual racial/ethnic groups. Allelic frequency across ethnic/racial groups was compared using Pearson's chi-square test. A total of 653 cases and 1,476 controls were genotyped at C-509T. The TT genotype showed a significant protective effect against high grade prostate cancer (OR 0.482, 95% CI 0.274-0.849). In addition, the TT genotype was associated with a decreased risk of poor prognosis prostate cancer (OR 0.488, 95% CI 0.236-1.009). Limiting analysis to nonHispanic white men showed that the TT genotype had an even more pronounced protective effect for poor prognosis prostate cancer (OR 0.297, 95% CI 0.100-0.887). Finally, there was a significant difference in the distribution of allelic frequency across racial/ethnic groups (p <0.0001). We observed an association between single nucleotide polymorphisms of TGFB1 at C-509T and a decreased risk of aggressive prostate cancer. The TT genotype of TGFB1 at C-509T demonstrates a protective effect against high grade prostate cancer and cases with poor prognosis.

Glutamine Attenuation of Cell Death and Inducible Nitric Oxide Synthase Expression Following Inflammatory Cytokine‐Induced Injury Is Dependent on Heat Shock Factor‐1 Expression

Glutamine (GLN) has been shown to improve outcome after experimental and clinical models of critical illness. Enhanced expression of heat shock protein (HSP) has been hypothesized to be responsible for this protection. The heat shock response has been shown to inhibit inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production. This study tested the hypothesis that GLN-mediated activation of the HSP pathway is responsible for improved survival and attenuation of iNOS expression after an inflammatory cytokine-induced injury. Heat shock factor-1 (HSF-1) wild-type and knockout mouse embryonic fibroblasts (HSF-1+/+ and HSF-1-/-) were used in all experiments. Cells were treated with 0 mmol/L or 8 mmol/L GLN and cytomix (tumor necrosis factor-alpha, lipopolysaccharide, and interferon-gamma) in a concurrent treatment model once they had reached confluence. Cell viability was assayed with MTS/PMS mixture. Apoptosis and necrosis were assayed via immunohistochemistry. iNOS and HSP-70 expression were detected via Western blotting. NO production was measured using the Griess reagent. GLN treatment significantly attenuated inflammatory cytokine-induced cell death and apoptosis in HSF-1+/+ cells vs 0 mmol/L GLN treatment; however, GLN's cellular protection was lost in HSF-1-/- cells. GLN supplementation attenuated cytomix-induced iNOS expression and NO production only in HSF-1+/+ cells. Further, GLN induced HSP-70 expression only in HSF-1+/+ cells. This is the first demonstration that GLN-mediated cellular protection after inflammatory cytokine injury is due to HSF-1 expression and cellular capacity to activate an HSP response.