Heat shock factor 1 inhibition enhances the effects of modulated electro hyperthermia in a triple negative breast cancer mouse model.

Female breast cancer is the most diagnosed cancer worldwide. Triple negative breast cancer (TNBC) is the most aggressive type and there is no existing endocrine or targeted therapy. Modulated electro-hyperthermia (mEHT) is a non-invasive complementary cancer therapy using an electromagnetic field generated by amplitude modulated 13.56 MHz frequency that induces tumor cell destruction. However, we have demonstrated a strong induction of the heat shock response (HSR) by mEHT, which can result in thermotolerance. We hypothesized that inhibition of the heat shock factor 1 (HSF1) can synergize with mEHT and enhance tumor cell-killing. Thus, we either knocked down the HSF1 gene with a CRISPR/Cas9 lentiviral construct or inhibited HSF1 with a specific small molecule inhibitor: KRIBB11 in vivo. Wild type or HSF1-knockdown 4T1 TNBC cells were inoculated into the mammary gland's fat pad of BALB/c mice. Four mEHT treatments were performed every second day and the tumor growth was followed by ultrasound and caliper. KRIBB11 was administrated intraperitoneally at 50 mg/kg daily for 8 days. HSF1 and Hsp70 expression were assessed. HSF1 knockdown sensitized transduced cancer cells to mEHT and reduced tumor growth. HSF1 mRNA expression was significantly reduced in the KO group when compared to the empty vector group, and consequently mEHT-induced Hsp70 mRNA upregulation diminished in the KO group. Immunohistochemistry (IHC) confirmed the inhibition of Hsp70 upregulation in mEHT HSF1-KO group. Demonstrating the translational potential of HSF1 inhibition, combined therapy of mEHT with KRIBB11 significantly reduced tumor mass compared to either monotherapy. Inhibition of Hsp70 upregulation by mEHT was also supported by qPCR and IHC. In conclusion, we suggest that mEHT-therapy combined with HSF1 inhibition can be a possible new strategy of TNBC treatment with great translational potential.

StHsfB5 Promotes Heat Resistance by Directly Regulating the Expression of Hsp Genes in Potato.

With global warming, high temperatures have become a major environmental stress that inhibits plant growth and development. Plants evolve several mechanisms to cope with heat stress accordingly. One of the important mechanisms is the Hsf (heat shock factor)-Hsp (heat shock protein) signaling pathway. Therefore, the plant transcription factor Hsf family plays important roles in response to heat stress. All Hsfs can be divided into three classes (A, B, and C). Usually, class-A Hsfs are transcriptional activators, while class-B Hsfs are transcriptional repressors. In potato, our previous work identified 27 Hsfs in the genome and analyzed HsfA3 and HsfA4C functions that promote potato heat resistance. However, the function of HsfB is still elusive. In this study, the unique B5 member StHsfB5 in potato was obtained, and its characterizations and functions were comprehensively analyzed. A quantitative real-time PCR (qRT-PCR) assay showed that StHsfB5 was highly expressed in root, and its expression was induced by heat treatment and different kinds of phytohormones. The subcellular localization of StHsfB5 was in the nucleus, which is consistent with the characterization of transcription factors. The transgenic lines overexpressing StHsfB5 showed higher heat resistance compared with that of the control nontransgenic lines and inhibitory lines. Experiments on the interaction between protein and DNA indicated that the StHsfB5 protein can directly bind to the promoters of target genes small Hsps (sHsp17.6, sHsp21, and sHsp22.7) and Hsp80, and then induce the expressions of these target genes. All these results showed that StHsfB5 may be a coactivator that promotes potato heat resistance ability by directly inducing the expression of its target genes sHsp17.6, sHsp21, sHsp22.7, and Hsp80.

Nanoparticle-mediated TRPV1 channel blockade amplifies cancer thermo-immunotherapy via heat shock factor 1 modulation.

The survival of malignant tumors is highly dependent on their intrinsic self-defense pathways such as heat shock protein (HSP) during cancer therapy. However, precisely dismantling self-defenses to amplify antitumor potency remains unexplored. Herein, we demonstrate that nanoparticle-mediated transient receptor potential vanilloid member 1 (TRPV1) channel blockade potentiates thermo-immunotherapy via suppressing heat shock factor 1 (HSF1)-mediated dual self-defense pathways. TRPV1 blockade inhibits hyperthermia-induced calcium influx and subsequent nuclear translocation of HSF1, which selectively suppresses stressfully overexpressed HSP70 for enhancing thermotherapeutic efficacy against a variety of primary, metastatic and recurrent tumor models. Particularly, the suppression of HSF1 translocation further restrains the transforming growth factor ß (TGFß) pathway to degrade the tumor stroma, which improves the infiltration of antitumor therapeutics (e.g. anti-PD-L1 antibody) and immune cells into highly fibrotic and immunosuppressive pancreatic cancers. As a result, TRPV1 blockade retrieves thermo-immunotherapy with tumor-eradicable and immune memory effects. The nanoparticle-mediated TRPV1 blockade represents as an effective approach to dismantle self-defenses for potent cancer therapy.

Identification, classification, and expression profile analysis of heat shock transcription factor gene family in Salvia miltiorrhiza.

In response to abiotic stresses, transcription factors are essential. Heat shock transcription factors (HSFs), which control gene expression, serve as essential regulators of plant growth, development, and stress response. As a model medicinal plant, Salvia miltiorrhiza is a crucial component in the treatment of cardiovascular illnesses. But throughout its growth cycle, S.miltiorrhiza is exposed to a series of abiotic challenges, including heat and drought. In this study, 35 HSF genes were identified based on genome sequencing of Salvia miltiorrhiza utilizing bioinformatics techniques. Additionally, 35 genes were classified into three groups by phylogeny and gene structural analysis, comprising 22 HSFA, 11 HSFB, and two HSFC. The distribution and sequence analysis of motif showed that SmHSFs were relatively conservative. In SmHSF genes, analysis of the promoter region revealed the presence of many cis-acting elements linked to stress, hormones, and growth and development, suggesting that these factors have regulatory roles. The majority of SmHSFs were expressed in response to heat and drought stress, according to combined transcriptome and real-time quantitative PCR (qRT-PCR) analyses. In conclusion, this study looked at the SmHSF gene family using genome-wide identification, evolutionary analysis, sequence characterization, and expression analysis. This research serves as a foundation for further investigations into the role of HSF genes and their molecular mechanisms in plant stress responses.

Knockdown of heat shock transcription factor 1 decreases temperature stress tolerance in Bemisia tabaci MED.

The primary function of heat shock transcription factor (HSF) in the heat shock response is to activate the transcription of genes encoding heat shock proteins (HSPs). The phloem-feeding insect Bemisia tabaci (Gennadius) is an important pest of cotton, vegetables and ornamentals that transmits several plant viruses and causes enormous agricultural losses. In this study, the gene encoding HSF (Bthsf1) was characterized in MED B. tabaci. The full-length cDNA encoded a protein of 652 amino acids with an isoelectric point of 5.55. The BtHSF1 deduced amino acid sequence showed strong similarity to HSF in other insects. Expression analyses using quantitative real-time PCR indicated that Bthsf1 was significantly up-regulated in B. tabaci adults and pupae during thermal stress. Although Bthsf1 was induced by both hot and cold stress, the amplitude of expression was greater in the former. Bthsf1 had distinct, significant differences in expression pattern during different duration of high but not low temperature stress. Oral ingestion of dsBthsf1 repressed the expression of Bthsf1 and four heat shock proteins (Bthsp90, Bthsp70-3, Bthsp20 and Bthsp19.5) in MED B. tabaci during hot and cold stress. In conclusion, our results show that Bthsf1 is differentially expressed during high and low temperature stress and regulates the transcription of multiple hsps in MED B. tabaci.

Zinc supplementation prior to heat shock enhances HSP70 synthesis through HSF1 phosphorylation at serine 326 in human peripheral mononuclear cells.

Zinc supplementation prior to heat shock increases HSP70 (heat shock protein 70) expression, which has cytoprotective effects in tissue cells during inflammation. Effects of zinc deficiency in this regard have been discussed controversially. Whether zinc modulates the expression of HSP70 in the human immune system as well and thus affects cell survival during heat stress is so far largely unknown. Therefore, we investigated the effect of alterations in the cellular zinc status on HSP70 expression and on cellular survival in human monocytes and lymphocytes. Three cell lines (Jurkat, THP-1, and Ramos) and enriched primary human monocytes and lymphocytes from young subjects were subjected to zinc deficiency or supplementation and subsequently heat shock at 42 °C. HSP70 mRNA expression was analyzed by real-time PCR, whereas HSP70 protein expression was analyzed by western blotting. In all cells other than Ramos cells, zinc supplementation and deficiency augmented heat shock-induced HSP70 expression. Further experiments in primary monocytes and lymphocytes indicated that this may be explained by the enhanced phosphorylation of HSF1 (Heat shock factor 1) at Ser326, which plays a significant role in HSP70 induction, as observed in zinc deficient and supplemented cells. While zinc supplementation had negligible effects on cell viability, acute zinc deficiency further increased cell death, induced by heat shock. Our results emphasize the importance of an optimal cellular zinc status. Moreover, we present a possible mechanism behind zinc's influence on HSP70 expression in human leukocytes. Our data form the basis for further in vivo and ex vivo studies to investigate how the zinc status may affect cellular damage in transient high temperature situations.

Hyperthermia increases HSP production in human PDMCs by stimulating ROS formation, p38 MAPK and Akt signaling, and increasing HSF1 activity.

BACKGROUND: Human placenta-derived multipotent cells (hPDMCs) are isolated from a source uncomplicated by ethical issues and are ideal for therapeutic applications because of their capacity for multilineage differentiation and proven immunosuppressive properties. It is known that heat shock preconditioning induces the upregulation of heat shock proteins (HSPs), which enhance survival and engraftment of embryonic stem cells (ESCs) during transplantation in live animal models, although whether heat shock preconditioning has the same effects in hPDMCs is unclear. METHODS: The hPDMCs were isolated from placenta of healthy donors. The cells were treated with heat shock (43 °C, 15 min), followed by evaluation of cell viability. Furthermore, the HSPs expression was assessed by Western blot, qPCR. The reactive oxygen species (ROS) production and signal pathway activation were determined by flow cytometry and Western blot, respectively. The regulatory pathways involved in HSPs expression were examined by pretreatment with chemical inhibitors, and siRNAs of MAPK, Akt, and heat shock factor 1 (HSF1), followed by determination of HSPs expression. RESULTS: This study demonstrates that heat shock treatment induced ROS generation and HPSs expression in hPDMCs. Heat shock stimulation also increased p38 MAPK and Akt phosphorylation. These effects were reduced by inhibitors of ROS, p38 MAPK and Akt. Moreover, we found that heat shock treatment enhanced nuclear translocation of the HSF1 in hPDMCs, representing activation of HSF1. Pretreatment of hPDMCs with ROS scavengers, SB203580 and Akt inhibitors also reduced the translocation of HSF1 induced by heat shock. CONCLUSIONS: Our data indicate that heat shock acts via ROS to activate p38 MAPK and Akt signaling, which subsequently activates HSF1, leading to HSP activation and contributing to the protective role of hPDMCs.

Transcription factor YY1 inhibits the expression of THY1 to promote interstitial pulmonary fibrosis by activating the HSF1/miR-214 axis.

Interstitial pulmonary fibrosis (IPF) is a progressive disease of diverse etiology manifesting with proliferation of lung fibroblasts and accumulation of extracellular matrix deposition in pulmonary interstitium. Recent studies show aberrant expression of mRNAs and microRNAs (miRNAs) in human embryonic pulmonary fibroblasts (HEPFs). In this study, we investigated effects of the YY1/HSF1/miR-214/THY1 axis on the functions of HEPFs and IPF. Loss- and gain-of-function tests were conducted to identify roles of YY1, HSF1, miR-214, and THY1 in IPF. As determined by RT-qPCR or western blot assay, silencing YY1 down-regulated HSF1 expression and attenuated the expression of pro-proliferative and fibrosis markers in HEPFs. Meanwhile, viability of HEPFs was impeded by YY1 knockdown. The binding relationship between miR-214 and THY1 was verified using dual-luciferase reporter assay. In HEPFs, down-regulation of HSF1 reduced miR-214 expression to repress proliferation and fibrogenic transformation of HEPFs, while inhibition of miR-214 expression could restrain the fibrogenic transformation property of HEPFs by up-regulating THY1. Subsequently, IPF model in mice was induced by bleomycin treatment. These animal experiments validated the protective effects of YY1 knockdown against IPF-induced lung pathological manifestations, which could be reversed by THY1 knockdown. Our study demonstrates the important involvement of YY1/HSF1/miR-214/THY1 axis in the development of IPF.

Buxus alkaloid compound destabilizes mutant p53 through inhibition of the HSF1 chaperone axis.

BACKGROUND: P53 is the most frequently mutated gene in most tumour types, and the mutant p53 protein accumulates at high levels in tumours to promote tumour development and progression. Thus, targeting mutant p53 for degradation is one of the therapeutic strategies used to manage tumours that depend on mutant p53 for survival. Buxus alkaloids are traditionally used in the treatment of cardiovascular diseases. We found that triterpenoid alkaloids extracted from Buxus sinica found in the Yunnan Province exhibit anticancer activity by depleting mutant p53 levels in colon cancer cells. PURPOSE: To explore the anticancer mechanism of action of the triterpenoid alkaloid KBA01 compound by targeting mutant p53 degradation. STUDY DESIGN AND METHODS: Different mutant p53 cell lines were used to evaluate the anticancer activity of KBA01. MTT assay, colony formation assay and cell cycle analysis were performed to examine the effect of KBA01 on cancer cell proliferation. Western blotting and qPCR were used to investigate effects of depleting mutant p53, and a ubiquitination assay was used to determine mutant p53 ubiquitin levels after cells were treated with the compound. Co-IP and small interfering RNA assays were used to explore the effects of KBA01 on the interaction of Hsp90 with mutant p53. RESULTS: The triterpenoid alkaloid KBA01 can induce G2/M cell cycle arrest and the apoptosis of HT29 colon cancer cells. KBA01 decreases the stability of DNA contact mutant p53 proteins through the proteasomal pathway with minimal effects on p53 mutant protein conformation. Moreover, KBA01 enhances the interaction of mutant p53 with Hsp70, CHIP and MDM2, and knocking down CHIP and MDM2 stabilizes mutant p53 levels in KBA01-treated cells. In addition, KBA01 disrupts the HSF1-mutant p53-Hsp90 complex and releases mutant p53 to enable its MDM2- and CHIP-mediated degradation. CONCLUSION: Our study reveals that KBA01 depletes mutant p53 protein in a chaperone-assisted ubiquitin/proteasome degradation pathway in cancer cells, providing insights into potential strategies to target mutant p53 tumours.

Introduction of Arabidopsis's heat shock factor HsfA1d mitigates adverse effects of heat stress on potato (Solanum tuberosum L.) plant.

Thermal stress induces a wide array of morphological and physiological changes in potato affecting its development and economic yield. Response to thermal stress in plants is mostly regulated by heat shock factors (hsfs). The current study aimed at improving heat tolerance by transforming potato plant with heat shock factor, HsfA1d, using Agrobacterium. Gateway cloning strategy was adopted for isolation of HsfA1d from Arabidopsis thaliana and cloning into plant expression vector. The target gene was introduced into potato by infecting internodal explants with Agrobacterium strain GV3101 carrying pGWB402Ω-HsfA1d construct. Upon exposure to heat stress, the wild-type plants turned yellowish, whereas no phenotypic effect on transgenic plants was observed. Expression of HsfA1d in transgenic plants was increased by 5.8-fold under thermal stress compared to room temperature. Transgenic plants exhibited 6-fold increase in the expression of downstream HSP70 under thermal stress compared to wild-type plants. Both chlorophyll a and b were significantly decreased in wild-type plants while no such decrease was recorded in transgenic plants under thermal stress. Heat stress was found to have no significant effect on carotenoid pigments of both wild-type and transgenic plants. Significantly lower electrolyte leakage from transgenic plants was witnessed compared to wild type upon exposure to thermal stress. Transgenic plants accumulated significantly higher proline content compared to wild-type plants under heat stress. It is concluded that HsfA1d plays a vital role in plant thermotolerance and hence can be effectively used to enhance the resistance of crop plants against heat stress.

Genome-wide investigation of the heat shock transcription factor (Hsf) gene family in Tartary buckwheat (Fagopyrum tataricum).

BACKGROUND: Heat shock transcription factor (Hsfs) is widely found in eukaryotes and prokaryotes. Hsfs can not only help organisms resist high temperature, but also participate in the regulation of plant growth and development (such as involved in the regulation of seed maturity and affects the root length of plants). The Hsf gene was first isolated from yeast and then gradually found in plants and sequenced, such as Arabidopsis thaliana, rice, maize. Tartary buckwheat is a rutin-rich crop, and its nutritional value and medicinal value are receiving more and more attention. However, there are few studies on the Hsf genes in Tartary buckwheat. With the whole genome sequence of Tartary buckwheat, we can effectively study the Hsf gene family in Tartary buckwheat. RESULTS: According to the study, 29 Hsf genes of Tartary buckwheat (FtHsf) were identified and renamed according to location of FtHsf genes on chromosome after removing a redundant gene. Therefore, only 29 FtHsf genes truly had the functional characteristics of the FtHsf family. The 29 FtHsf genes were located on 8 chromosomes of Tartary buckwheat, and we found gene duplication events in the FtHsf gene family, which may promote the expansion of the FtHsf gene family. Then, the motif compositions and the evolutionary relationship of FtHsf proteins and the gene structures, cis-acting elements in the promoter, synteny analysis of FtHsf genes were discussed in detail. What's more, we found that the transcription levels of FtHsf in different tissues and fruit development stages were significantly different by quantitative real-time PCR (qRT-PCR), implied that FtHsf may differ in function. CONCLUSIONS: In this study, only 29 Hsf genes were identified in Tartary buckwheat. Meanwhile, we also classified the FtHsf genes, and studied their structure, evolutionary relationship and the expression pattern. This series of studies has certain reference value for the study of the specific functional characteristics of Tartary buckwheat Hsf genes and to improve the yield and quality of Tartary buckwheat in the future.

Gene expression of heat shoc kproteins/factors (HSP60, HSP70, HSP90, HSF-1, HSF-3) and antioxidant enzyme activities in heat stressed broilers treated with vitamin C.

In broiler chickens, the relationship between dietary supplementation of vitamin C and hepatic, cardiac and renal heat shock proteins (HSP60, HSP70 and HSP90), heat shock factors (HSF-1 and HSF-3) and enzymatic antioxidants requires further investigation. The current study aimed to investigate this relationship at cellular and molecular levels in a 42 days experiment. Two hundred, one-day-old broiler chicks (Ross 308) were allocated into four equal groups. Chicks in the first and third groups were thermo-neutral (TN; 22°C for 24 hours/day) and fed basal diet without or with vitamin C (1g/kg basal diet), respectively. Chicks in the second and fourth groups were heat stressed (HS; 34°C for 8 hours/day) and fed basal diet without or with vitamin C, respectively. Performance parameters were recorded throughout the experiment. Levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione S-transferase (GST), glutathione peroxidase (GPX), Catalase (CAT) and gene expression of heat shock proteins (HSP60, 70 and 90) and heat shock factors (HSF 1 and 3) were analyzed in liver, heart and kidney tissues of the studied groups. Heat stress induced a negative impact on performance parameters, significant reduction in activities of all examined antioxidant enzymes and a significant up-regulation in heat shock proteins and factors genes in all studied tissues. Dietary supplementation of vitamin C corrected these parameters towards the normal control values. Conclusively, dietary supplementation of the examined dose of vitamin C was efficient at ameliorating the detrimental effects of heat stress on liver, heart and kidney tissues of broilers chickens at cellular and molecular levels.

[Double Labeling and Simultaneous Monitoring for Hsp70 and Hsf-1 Kinetics in SCC-25 Cells with a Short-Term Dietary Restriction of Leucine Following Heat Shock].

To develop a quantum-dot-based multiplexed imaging system for the simultaneous monitoring of Hsf- 1/Hsp70 after heat shock, and to evaluate the effects of combined thermotherapy and leucine deprivation therapy on Hsf-1 inactivation. SCC-25 cells were leucine starved for 0, 1, 2, 3 or 4 days following which the cells underwent heat shock at 42°C for 30 min. At 6 h after heat shock, Hsf-1 activation and translocation to the nucleus was observed in cells that were leucine starved for 0, 1 and 2 days, and the synthesis of Hsp70 and Hsf-1 reached their maximum values and had a tendency to gather in the nucleus. However, in cells that were leucine starved for 3 and 4 days, Hsf-1 activity and Hsp70 synthesis level was dramatically decreased. Dietary restriction of leucine for at least three days could result in the inactivation of Hsf-1, leading to a reduction in Hsp70 synthesis. The combination of thermotherapy and short-term leucine deprivation therapy may become effective approach for the treatment of oral tumors.

Transcriptional Regulation of Selenoprotein F by Heat Shock Factor 1 during Selenium Supplementation and Stress Response.

Changes of Selenoprotein F (SELENOF) protein levels have been reported during selenium supplementation, stressful, and pathological conditions. However, the mechanisms of how these external factors regulate SELENOF gene expression are largely unknown. In this study, HEK293T cells were chosen as an in vitro model. The 5'-flanking regions of SELENOF were analyzed for promoter features. Dual-Glo Luciferase assays were used to detect promoter activities. Putative binding sites of Heat Shock Factor 1 (HSF1) were predicted in silico and the associations were further proved by chromatin immunoprecipitation (ChIP) assay. Selenate and tunicamycin (Tm) treatment were used to induce SELENOF up-regulation. The fold changes in SELENOF expression and other relative proteins were analyzed by Q-PCR and western blot. Our results showed that selenate and Tm treatment up-regulated SELENOF at mRNA and protein levels. SELENOF 5'-flanking regions from -818 to -248 were identified as core positive regulatory element regions. Four putative HSF1 binding sites were predicted in regions from -1430 to -248, and six out of seven primers detected positive results in ChIP assay. HSF1 over-expression and heat shock activation increased the promoter activities, and mRNA and protein levels of SELENOF. Over-expression and knockdown of HSF1 showed transcriptional regulation effects on SELENOF during selenate and Tm treatment. In conclusion, HSF1 was discovered as one of the transcription factors that were associated with SELENOF 5'-flanking regions and mediated the up-regulation of SELENOF during selenate and Tm treatment. Our work has provided experimental data for the molecular mechanism of SELENOF gene regulation, as well as uncovered the involvement of HSF1 in selenotranscriptomic for the first time.

Anti-inflammatory effect on genes expression after four days of Qigong training in peripheral mononuclear blood cells in healthy women.

INTRODUCTION: Some studies have shown the influence of Qigong on gene expression in different cells, but there is little data associated with the influence of this kind of therapy on genes expression in pheripheral monocellucar blood cells. OBJECTIVE: The aim of this study was to evaluate changes in the expression of genes associated with cellular stress response in peripheral mononuclear blood cells (PMBC) in healthy women. MATERIAL AND METHODS: The experiment took place at the Japanese Martial Arts Centre "DOJO" in Stara Wies, Poland, conducted over the course of a 4-day qigong training session. To evaluate the genes effect of this training, blood samples were taken before and after the training period. This experiment involved 20 healthy women (aged 56.2±9.01, body height 164.8±6.5 and mass 65.5±8.2). To determine the expression of HSF-1, HSPA1A, NF-kB, IL10 and CCL2 mRNA, 3 ml of venous blood was collected. The blood samples were placed in tubes allowing for separation (BD Vacutainer CPT TM) before and after the 4 days of qigong training. Isolated PMBC were used to determine gene expression using real-time qRT-PCR (quantitative reverse transcription polymerase chain reaction). RESULTS: Significant decreases in NF-kB and CCL2 mRNA and increases in IL10, HSF1 and HSPA1A m-RNA were detected after 4 days of qigong training. The obtained findings suggest that qigong caused a reduction in the inflammatory and intensified anti-inflammatory gene expression, as well as a higher expression of HSF-1 and HSPA1A. CONCLUSIONS: The adaptive response to qigong training was similar to the adaptive response to physical activity and was detected through gene expression in PMBC. Furthermore, this kind of training is especially indicated for women because of their higher susceptibility to psychosocial stress when compared to men.

Protective effects of red grape (Vitis vinifera) juice through restoration of antioxidant defense, endocrine swing and Hsf1, Hsp72 levels in heat stress induced testicular dysregulation of Wister rat.

Ability of red grape juice (RGJ), a known antioxidant, on testis of adult Wister rat to protect from oxidative stress induced damages by heat stress has been investigated in this study. Heat stress was induced maintaining body and testicular temperature at 43°C for 30min/day for 15 days using a hyperthermia induction chamber. Four groups of rats (n=6 per group) comprising of Group-I (control) -kept at 32°C, Group-II -exposed to heat stress alone, Group-III received RGJ (0.8ml/rat/day) alone and Group-IV -exposed to heat stress and received RGJ at same dose. Analysis of blood and testicular tissue exhibited significant reduction in serum testosterone, testicular superoxide dismutase, testicular catalase and testicular glutathione (all p < 0.001); whereas, significant rise in the level of serum corticosteroid, testicular lipid peroxidase and the apoptotic enzyme caspase-3 of testis (all p < 0.001) were observed along with substantial increase in testicular Hsp72 and Hsf-1, and decrease in 17ß-HSD3 were noted in heat stressed rats compared to controls. In Group-IV rats, RGJ administration could restore these parameters to normal levels. The signs of retention were clear in Group-IV rats and found to be significantly different as compared to that of the Group-II rats. In testicular histology of rats exposed to heat stress alone revealed remarkable germ cell degeneration and tubular deformations which were prevented by RGJ treatment (Group-IV). The reduced number of sperm level in Group-II also restored in RGJ treatment (Group-IV). The above results indicate that consumption of RGJ may substantially protect testis from heat stress induce dysfunctions.

Tanshinone-induced ERs suppresses IGFII activation to alleviate Ang II-mediated cardiac hypertrophy.

Cardiomyopathy involves changes in myocardial ultrastructure and cardiac hypertrophy. Angiotensin II (AngII) has previously been shown to stimulate the expression of IGF-2 and IGF-2R in H9c2 cardiomyoblasts and increase of blood pressure, and cardiac hypertrophy. Estrogen receptors (ERs) exert protective effects, such as anti-hypertrophy in cadiomyocytes. Tanshinone IIA (TSN), a main active ingredient from a Chinese medical herb, Salvia miltiorrhiza Bunge (Danshen), was shown to protect cardiomyocytes hypertrophy by different stress signals. We aimed to investigate whether TSN protected H9c2 cardiomyocytes from AngII-induced activation of IGF-2R pathway and hypertrophy by mediating through ERs. AngII resulted in H9c2 cardiomyoblast hypertrophy and increased inflammatory molecular markers. These were down-regulated by TSN via estrogen receptors. AngII resulted in elevation in MAPKs, IGF-2R and hypertrophic protein markers. These, again, were reduced by addition of the phytoestrogen with activation of ERs. Finally, AngII induced phosphorylation of heat shock factor-1 (HSF1) and decreased sirtuin-1 (SIRT1). In addition, AngII also caused an increase in distribution of IGF-2R molecules on cell membrane. In contrast, TSN reduced HSF1 phosphorylation and cell surface IGF-2R while elevating SIRT1 via ERs. TSN was capable of attenuating AngII-induced IGF-2R pathway and hypertrophy through ERs in H9c2 cardiomyoblast cells.

Azadiradione ameliorates polyglutamine expansion disease in Drosophila by potentiating DNA binding activity of heat shock factor 1.

Aggregation of proteins with the expansion of polyglutamine tracts in the brain underlies progressive genetic neurodegenerative diseases (NDs) like Huntington's disease and spinocerebellar ataxias (SCA). An insensitive cellular proteotoxic stress response to non-native protein oligomers is common in such conditions. Indeed, upregulation of heat shock factor 1 (HSF1) function and its target protein chaperone expression has shown promising results in animal models of NDs. Using an HSF1 sensitive cell based reporter screening, we have isolated azadiradione (AZD) from the methanolic extract of seeds of Azadirachta indica, a plant known for its multifarious medicinal properties. We show that AZD ameliorates toxicity due to protein aggregation in cell and fly models of polyglutamine expansion diseases to a great extent. All these effects are correlated with activation of HSF1 function and expression of its target protein chaperone genes. Notably, HSF1 activation by AZD is independent of cellular HSP90 or proteasome function. Furthermore, we show that AZD directly interacts with purified human HSF1 with high specificity, and facilitates binding of HSF1 to its recognition sequence with higher affinity. These unique findings qualify AZD as an ideal lead molecule for consideration for drug development against NDs that affect millions worldwide.

Transient Proteotoxicity of Bacterial Virulence Factor Pyocyanin in Renal Tubular Epithelial Cells Induces ER-Related Vacuolation and Can Be Efficiently Modulated by Iron Chelators.

Persistent infections of biofilm forming bacteria, such as Pseudomonas aeruginosa, are common among human populations, due to the bacterial resistance to antibiotics and other adaptation strategies, including release of cytotoxic virulent factors such as pigment pyocyanin (PCN). Urinary tract infections harbor P. aeruginosa strains characterized by the highest PCN-producing capacity, yet no information is available on PCN cytotoxicity mechanism in kidney. We report here that renal tubular epithelial cell (RTEC) line NRK-52E responds to PCN treatments with paraptosis-like activity features. Specifically, PCN-treated cells experienced dilation of endoplasmic reticulum (ER) and an extensive development of ER-derived vacuoles after about 8 h. This process was accompanied with hyper-activation of proteotoxic stress-inducible transcription factors Nrf2, ATF6, and HSF-1. The cells could be rescued by withdrawal of PCN from the culture media before the vacuoles burst and cells die of non-programmed necrosis after about 24-30 h. The paraptosis-like activity was abrogated by co-treatment of the cells with metal-chelating antioxidants. A microscopic examination of cells co-treated with PCN and agents aiming at a variety of the cellular stress mediators and pathways have identified iron as a single most significant co-factor of the PCN cytotoxicity in the RTECs. Among biologically relevant metal ions, low micromolar Fe2+ specifically mediated anaerobic oxidation of glutathione by PCN, but catechol derivatives and other strong iron complexing agents could inhibit the reaction. Our data suggest that iron chelation could be considered as a supplementary treatment in the PCN-positive infections.

Transcriptional regulation of heat shock proteins and ascorbate peroxidase by CtHsfA2b from African bermudagrass conferring heat tolerance in Arabidopsis.

Heat stress transcription factor A2s (HsfA2s) are key regulators in plant response to high temperature. Our objectives were to isolate an HsfA2 gene (CtHsfA2b) from a warm-season grass species, African bermudagrass (Cynodon transvaalensis Burtt-Davy), and to determine the physiological functions and transcriptional regulation of HsfA2 for improving heat tolerance. Gene expression analysis revealed that CtHsfA2b was heat-inducible and exhibited rapid response to increasing temperature. Ectopic expression of CtHsfA2b improved heat tolerance in Arabidopsis and restored heat-sensitive defects of Arabidopsis hsfa2 mutant, which was demonstrated by higher survival rate and photosynthetic parameters, and lower electrolyte leakage in transgenic plants compared to the WT or hsfa2 mutant. CtHsfA2b transgenic plants showed elevated transcriptional regulation of several downstream genes, including those encoding ascorbate peroxidase (AtApx2) and heat shock proteins [AtHsp18.1-CI, AtHsp22.0-ER, AtHsp25.3-P and AtHsp26.5-P(r), AtHsp70b and AtHsp101-3]. CtHsfA2b was found to bind to the heat shock element (HSE) on the promoter of AtApx2 and enhanced transcriptional activity of AtApx2. These results suggested that CtHsfA2b could play positive roles in heat protection by up-regulating antioxidant defense and chaperoning mechanisms. CtHsfA2b has the potential to be used as a candidate gene to genetically modify cool-season species for improving heat tolerance.