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.

Glycine Attenuates Lipopolysaccharide-Induced Acute Lung Injury by Regulating NLRP3 Inflammasome and NRF2 Signaling.

Glycine supplementation has been reported to alleviate lipopolysaccharide (LPS)-induced lung injury in mice. However, the underlying mechanisms responsible for this beneficial effect remain unknown. In the present study, male C57BL/6 mice were treated with aerosolized glycine (1000 mg in 5 mL of 0.9% saline) or vehicle (0.9% saline) once daily for 7 continuous days, and then were exposed to aerosolized LPS (5 mg in 5 mL of 0.9% saline) for 30 min to induce lung injury. Sera and lung tissues were collected 24 h post LPS challenge. Results showed that glycine pretreatment attenuated LPS-induced decreases of mucin at both protein and mRNA levels, reduced LPS-triggered upregulation of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interferons, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukins. Further study showed that glycine-reduced LPS challenge resulted in the upregulation of nuclear factor κB (NF-κB), nucleotide binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome. In addition, LPS exposure led to the downregulation of NRF2 and downstream targets, which were significantly improved by glycine administration in the lung tissues. Our findings indicated that glycine pretreatment prevented LPS-induced lung injury by regulating both NLRP3 inflammasome and NRF2 signaling.

Modulatory effects of curcumin on heat shock proteins in cancer: A promising therapeutic approach.

Cancer metastasis represents a multistep process, including alteration of cell adhesion/motility in the microenvironment and sustained angiogenesis, which is essential for supporting cancer growth in tissues that are distant from the primary tumor. There is growing evidence suggesting that heat shock proteins (HSPs) (also known as heat stress proteins), which constitute a family of stress-inducible proteins, may be involved in the pathogenesis of cancer. Curcumin (diferuloylmethane) is a potent anti-inflammatory, antioxidant, antimicrobial, and antitumor agent. Curcumin has been shown to regulate different members of HSPs including HSP27, HSP40, HSP60, HSP70, and HSP90 in cancer. Here, we present extent findings suggesting that curcumin may act as a potential therapeutic agent for the treatment of cancer through its regulation of HSPs.

Maternal high-fat diet induces metabolic stress response disorders in offspring hypothalamus.

Maternal obesity has been shown to increase the risk of obesity and related disorders in the offspring, which has been partially attributed to changes of appetite regulators in the offspring hypothalamus. On the other hand, endoplasmic reticulum (ER) stress and autophagy have been implicated in hypothalamic neuropeptide dysregulation, thus may also play important roles in such transgenerational effect. In this study, we show that offspring born to high-fat diet-fed dams showed significantly increased body weight and glucose intolerance, adiposity and plasma triglyceride level at weaning. Hypothalamic mRNA level of the orexigenic neuropeptide Y (NPY) was increased, while the levels of the anorexigenic pro-opiomelanocortin (POMC), NPY1 receptor (NPY1R) and melanocortin-4 receptor (MC4R) were significantly downregulated. In association, the expression of unfolded protein response (UPR) markers including glucose-regulated protein (GRP)94 and endoplasmic reticulum DNA J domain-containing protein (Erdj)4 was reduced. By contrast, protein levels of autophagy-related genes Atg5 and Atg7, as well as mitophagy marker Parkin, were slightly increased. The administration of 4-phenyl butyrate (PBA), a chemical chaperone of protein folding and UPR activator, in the offspring from postnatal day 4 significantly reduced their body weight, fat deposition, which were in association with increased activating transcription factor (ATF)4, immunoglobulin-binding protein (BiP) and Erdj4 mRNA as well as reduced Parkin, PTEN-induced putative kinase (PINK)1 and dynamin-related protein (Drp)1 protein expression levels. These results suggest that hypothalamic ER stress and mitophagy are among the regulatory factors of offspring metabolic changes due to maternal obesity.

A Systems Biology Approach Using Transcriptomic Data Reveals Genes and Pathways in Porcine Skeletal Muscle Affected by Dietary Lysine.

Nine crossbred finishing barrows (body weight 94.4 ± 6.7 kg) randomly assigned to three dietary treatments were used to investigate the effects of dietary lysine on muscle growth related metabolic and signaling pathways. Muscle samples were collected from the longissimus dorsi of individual pigs after feeding the lysine-deficient (4.30 g/kg), lysine-adequate (7.10 g/kg), or lysine-excess (9.80 g/kg) diet for five weeks, and the total RNA was extracted afterwards. Affymetrix Porcine Gene 1.0 ST Array was used to quantify the expression levels of 19,211 genes. Statistical ANOVA analysis of the microarray data showed that 674 transcripts were differentially expressed (at p ≤ 0.05 level); 60 out of 131 transcripts (at p ≤ 0.01 level) were annotated in the NetAffx database. Ingenuity pathway analysis showed that dietary lysine deficiency may lead to: (1) increased muscle protein degradation via the ubiquitination pathway as indicated by the up-regulated DNAJA1, HSP90AB1 and UBE2B mRNA; (2) reduced muscle protein synthesis via the up-regulated RND3 and ZIC1 mRNA; (3) increased serine and glycine synthesis via the up-regulated PHGDH and PSPH mRNA; and (4) increased lipid accumulation via the up-regulated ME1, SCD, and CIDEC mRNA. Dietary lysine excess may lead to: (1) decreased muscle protein degradation via the down-regulated DNAJA1, HSP90AA1, HSPH1, and UBE2D3 mRNA; and (2) reduced lipid biosynthesis via the down-regulated CFD and ME1 mRNA. Collectively, dietary lysine may function as a signaling molecule to regulate protein turnover and lipid metabolism in the skeletal muscle of finishing pigs.

ArHsp40, a type 1 J-domain protein, is developmentally regulated and stress inducible in post-diapause Artemia franciscana.

Upon diapause termination and exposure to favorable environmental conditions, cysts of the crustacean Artemia franciscana reinitiate development, a process dependent on the resumption of metabolic activity and the maintenance of protein homeostasis. The objective of the work described herein was to characterize molecular chaperones during post-diapause growth of A. franciscana. An Hsp40 complementary DNA (cDNA) termed ArHsp40 was cloned and shown to encode a protein with an amino-terminal J-domain containing a conserved histidine, proline, and aspartic acid (HPD) motif. Following the J-domain was a Gly/Phe (G/F) rich domain, a zinc-binding domain which contained a modified CXXCXGXG motif, and the carboxyl-terminal substrate binding region, all characteristics of type I Hsp40. Multiple alignment and protein modeling showed that ArHsp40 is comparable to Hsp40s from other eukaryotes and likely to be functionally similar. qRT-PCR revealed that during post-diapause development, ArHsp40 messenger RNA (mRNA) varied slightly until the E2/E3 stage and decreased significantly upon hatching. The immunoprobing of Western blots demonstrated that ArHsp40 was also relatively constant until E2/E3 and then declined dramatically. The drop in ArHsp40 when metabolism and protein synthesis were increasing was unexpected and demonstrated developmental regulation. The reduction in ArHsp40 at such an active life history stage indicates, as one possibility, that A. franciscana possesses additional Hsp40s, one or more of which replaces ArHsp40 as development progresses. Increased synthesis upon heat shock established that in addition to being developmentally regulated, ArHsp40 is stress inducible and, because it is found in mature cysts, ArHsp40 has the potential to contribute to stress tolerance during diapause.

Flavokawains A and B from kava (Piper methysticum) activate heat shock and antioxidant responses and protect against hydrogen peroxide-induced cell death in HepG2 hepatocytes.

Context Flavokawains are secondary metabolites from the kava plant (Piper methysticum Forst. f., Piperaceae) that have anticancer properties and demonstrated oral efficacy in murine cancer models. However, flavokawains also have suspected roles in rare cases of kava-induced hepatotoxicity. Objective To compare the toxicity flavokawains A and B (FKA, FKB) and monitor the resulting transcriptional responses and cellular adaptation in the human hepatocyte cell line, HepG2. Materials and methods HepG2 were treated with 2-100 µM FKA or FKB for 24-48 h. Cellular viability was measured with calcein-AM and changes in signalling and gene expression were monitored by luciferase reporter assay, real-time PCR and Western blot of both total and nuclear protein extracts. To test for subsequent resistance to oxidative stress, cells were pretreated with 50 µM FKA, 10 µM FKB or 10 µM sulphoraphane (SFN) for 24 h, followed by 0.4-2.8 mM H2O2 for 48 h, and then viability was assessed. Results FKA (≤100 µM) was not toxic to HepG2, whereas FKB caused significant cell death (IC50=23.2 ± 0.8 µM). Both flavokawains activated Nrf2, increasing HMOX1 and GCLC expression and enhancing total glutathione levels over 2-fold (p < 0.05). FKA and FKB also activated HSF1, increasing HSPA1A and DNAJA4 expression. Also, flavokawain pretreatment mitigated cell death after a subsequent challenge with H2O2, with FKA being more effective than FKB, and similar to SFN. Conclusions Flavokawains promote an adaptive cellular response that protects hepatocytes against oxidative stress. We propose that FKA has potential as a chemopreventative or chemotherapeutic agent.

Caenorhabditis elegans dnj-14, the orthologue of the DNAJC5 gene mutated in adult onset neuronal ceroid lipofuscinosis, provides a new platform for neuroprotective drug screening and identifies a SIR-2.1-independent action of resveratrol.

Adult onset neuronal lipofuscinosis (ANCL) is a human neurodegenerative disorder characterized by progressive neuronal dysfunction and premature death. Recently, the mutations that cause ANCL were mapped to the DNAJC5 gene, which encodes cysteine string protein alpha. We show here that mutating dnj-14, the Caenorhabditis elegans orthologue of DNAJC5, results in shortened lifespan and a small impairment of locomotion and neurotransmission. Mutant dnj-14 worms also exhibited age-dependent neurodegeneration of sensory neurons, which was preceded by severe progressive chemosensory defects. A focussed chemical screen revealed that resveratrol could ameliorate dnj-14 mutant phenotypes, an effect mimicked by the cAMP phosphodiesterase inhibitor, rolipram. In contrast to other worm neurodegeneration models, activation of the Sirtuin, SIR-2.1, was not required, as sir-2.1; dnj-14 double mutants showed full lifespan rescue by resveratrol. The Sirtuin-independent neuroprotective action of resveratrol revealed here suggests potential therapeutic applications for ANCL and possibly other human neurodegenerative diseases.

Chaperones are necessary for the expression of catalytically active potato apyrases in prokaryotic cells.

NTPDases (nucleoside triphosphate diphosphohydrolases) (also called in plants apyrases) hydrolyze nucleoside 5'-tri- and/or diphosphate bonds producing nucleosides di or monophosphate and inorganic phosphate. For years, studies have been carried out to use both plant and animal enzymes for medicine. Therefore, there is a need to develop an efficient method for the quick production of large amounts of homogeneous proteins with high catalytic activity. Expression of proteins in prokaryotic cells is the most common way for the protein production. The aim of our study was to develop a method of expression of potato apyrase (StAPY4, 5, and 6) genes in bacterial cells under conditions that allowed the production of catalytically active form of these enzymes. Apyrase 4 and 6 were overexpressed in BL21-CodonPlus (DE3) bacteria strain but they were accumulated in inclusion bodies, regardless of the culture conditions and induction method. Co-expression of potato apyrases with molecular chaperones allowed the expression of catalytically active apyrase 5. However, its high nucleotidase activity could be toxic for bacteria and is therefore synthesized in small amounts in cells. Our studies show that each protein requires other conditions for maturation and even small differences in amino acid sequence can essentially affect protein folding regardless of presence of chaperones.

Inhibition of polo-like kinase 1 promotes hyperthermia sensitivity via inactivation of heat shock transcription factor 1 in human retinoblastoma cells.

PURPOSE: Hyperthermia (HT) has been recognized as an effective focal treatment in retinoblastoma. However, one of the problems with HT therapy is that cells acquire acquisition. The purpose of this study was to evaluate whether the inhibition of polo-like kinase 1 (PLK1) would promote HT sensitivity in human retinoblastoma cells. METHODS: We examined the effects of PLK1 knockdown by small interfering RNA (siRNA) or by the inhibition of PLK1 activity with PLK1 inhibitor (BI-2536) on the sensitivity to HT (44°C, 1 hour) in human retinoblastoma Y79 and WERI-Rb-1 cells by evaluating apoptosis and cell proliferation using flow cytometry, Western blotting, real-time quantitative polymerase chain reaction, and WST-8 assay. Furthermore, we investigated the effects of activating heat shock transcription factor 1 (HSF1) through a combination of PLK1 knockdown and HT using Western blotting and immunocytochemistry. RESULTS: The combination of PLK1 inhibition and HT enhanced sensitivity to HT synergistically. Furthermore, PLK1 knockdown inhibited HT-induced phosphorylation of HSF1, the nuclear translocation of HSF1 from the cytoplasm, and nuclear granule formation of HSF1. Heat shock transcription factor 1, inactivated by the silencing of PLK1, reduced the expression of heat shock proteins (HSPs), such as HSP70 and HSP40, as well as the expression of Bcl-2-associated athanogene 3 (BAG3). CONCLUSIONS: Polo-like kinase 1 inhibition may attenuate the thermoresistance of HT through the inactivation of HSF1 concomitant with reductions in HSPs and BAG3. The combination of PLK1 inhibition and HT may become an option for HT therapy in patients with retinoblastoma.

Inhibitors of difficult protein-protein interactions identified by high-throughput screening of multiprotein complexes.

Protein-protein interactions (PPIs) are important in all aspects of cellular function, and there is interest in finding inhibitors of these contacts. However, PPIs with weak affinities and/or large interfaces have traditionally been more resistant to the discovery of inhibitors, partly because it is more challenging to develop high-throughput screening (HTS) methods that permit direct measurements of these physical interactions. Here, we explored whether the functional consequences of a weak PPI might be used as a surrogate for binding. As a model, we used the bacterial ATPase DnaK and its partners DnaJ and GrpE. Both DnaJ and GrpE bind DnaK and catalytically accelerate its ATP cycling, so we used stimulated nucleotide turnover to indirectly report on these PPIs. In pilot screens, we identified compounds that block activation of DnaK by either DnaJ or GrpE. Interestingly, at least one of these molecules blocked binding of DnaK to DnaJ, while another compound disrupted allostery between DnaK and GrpE without altering the physical interaction. These findings suggest that the activity of a reconstituted multiprotein complex might be used in some cases to identify allosteric inhibitors of challenging PPIs.

The HLJ1-targeting drug screening identified Chinese herb andrographolide that can suppress tumour growth and invasion in non-small-cell lung cancer.

HLJ1 is a novel tumour suppressor and is a potential druggable target for non-small-cell lung cancer (NSCLC). In this report, using a promoter-containing enhancer region as the HLJ1-targeting drug-screening platform, we identified several herbal compounds from a Chinese herbal bank with the capacity to enhance HLJ1 promoter activity and suppress tumour growth and invasion of NSCLC. Among the herbal drugs identified, the andrographolide (from Andrographis paniculata [Burm. f.] Nees.) most significantly induced HLJ1 expression and suppressed tumorigenesis both in vitro and in vivo. The andrographolide upregulates HLJ1 via JunB activation, which modulates AP-2α binding at the MMP-2 promoter and represses the expression of MMP-2. In addition, silencing of HLJ1 partially reverses the inhibition of cancer-cell invasion by andrographolide. Microarray transcriptomic analysis was performed to comprehensively depict the andrographolide-regulated signalling pathways. We showed that andrographolide can affect 939 genes (analysis of variance, false discovery rate < 0.05) that are dominantly involved in the cell cycle, apoptosis and adhesion-related biological signalling, including mitogen-activated protein kinase, focal adhesion and tight junction pathways, indicating the diverse effects of andrographolide on anticancer invasion and proliferation. In conclusion, the HLJ1-targeting drug-screening platform is useful for screening of novel anticancer compounds. Using this platform, we identified andrographolide is a promising new anticancer agent that could suppress tumour growth and invasion in NSCLC.

Novel inhibitors of heat shock protein Hsp70-mediated luciferase refolding that bind to DnaJ.

Inhibitors of both heat shock proteins Hsp90 and Hsp70 have been identified in assays measuring luciferase refolding containing rabbit reticulocyte lysate or purified chaperone components. Here, we report the discovery of a series of phenoxy-N-arylacetamides that disrupt Hsp70-mediated luciferase refolding by binding to DnaJ, the bacterial homolog of human Hsp40. Inhibitor characterization experiments demonstrated negative cooperativity with respect to DnaJ and luciferase concentration, but varying the concentration of ATP had no effect on potency. Thermal shift analysis suggested a direct interaction with DnaJ, but not with Hsp70. These compounds may be useful tools for studying DnaJ/Hsp40 in various cellular processes.

Co-chaperone HSJ1a dually regulates the proteasomal degradation of ataxin-3.

Homo sapiens J domain protein (HSJ1) is a J-domain containing co-chaperone that is known to stimulate ATPase activity of HSP70 chaperone, while it also harbors two ubiquitin (Ub)-interacting motifs (UIMs) that may bind with ubiquitinated substrates and potentially function in protein degradation. We studied the effects of HSJ1a on the protein levels of both normal and the disease--related polyQ-expanded forms of ataxin-3 (Atx3) in cells. The results demonstrate that the N-terminal J-domain and the C-terminal UIM domain of HSJ1a exert opposite functions in regulating the protein level of cellular overexpressed Atx3. This dual regulation is dependent on the binding of the J-domain with HSP70, and the UIM domain with polyUb chains. The J-domain down-regulates the protein level of Atx3 through HSP70 mediated proteasomal degradation, while the UIM domain may alleviate this process via maintaining the ubiquitinated Atx3. We propose that co-chaperone HSJ1a orchestrates the balance of substrates in stressed cells in a Yin-Yang manner.

Chemical screens against a reconstituted multiprotein complex: myricetin blocks DnaJ regulation of DnaK through an allosteric mechanism.

DnaK is a molecular chaperone responsible for multiple aspects of bacterial proteostasis. The intrinsically slow ATPase activity of DnaK is stimulated by its co-chaperone, DnaJ, and these proteins often work in concert. To identify inhibitors we screened plant-derived extracts against a reconstituted mixture of DnaK and DnaJ. This approach resulted in the identification of flavonoids, including myricetin, which inhibited activity by up to 75%. Interestingly, myricetin prevented DnaJ-mediated stimulation of ATPase activity, with minimal impact on either DnaK's intrinsic turnover rate or its stimulation by another co-chaperone, GrpE. Using NMR, we found that myricetin binds DnaK at an unanticipated site between the IB and IIB subdomains and that it allosterically blocked binding of DnaK to DnaJ. Together, these results highlight a "gray box" screening approach, which might facilitate the identification of inhibitors of other protein-protein interactions.

Hepatitis B virus protein up-regulated HLJ1 expression via the transcription factor YY1 in human hepatocarcinoma cells.

The hepatitis B virus (HBV) protein plays a major role in hepatocellular carcinoma (HCC) development. However, its contribution to tumor invasion and metastasis has not been established so far. HLJ1 was recently cloned and classified as a member of the heat shock protein 40 family (Hsp40/DnaJ) which is abundantly expressed in HBV-related tumors, might be involved in tumor progression. In this study, the role of HBV in activation of HLJ1 was investigated. In HepG2 cells with transit or stable expression of HBV, HLJ1 expression was activated by HBV. The activity assay of HLJ1 promoter revealed that HBV up-regulated HLJ1 expression through the transcription factor YY1 sites within the HLJ1 promoter. YY1 expression was significantly up-regulated by HBV in a concentration-dependent manner. Knockdown of YY1 expression could partially reduce the HBV-induced HLJ1 activation which indicated that YY1 would be involved in HBV-induced HLJ1 expression. In conclusion, our data showed that HBV could promote HLJ1 expression by up-regulating the transcription factor YY1, and this provided a new insight of the mechanism of HBV induction in tumor metastasis.

A cell-based screen for inhibitors of protein folding and degradation.

Cancer cells are exposed to external and internal stresses by virtue of their unrestrained growth, hostile microenvironment, and increased mutation rate. These stresses impose a burden on protein folding and degradation pathways and suggest a route for therapeutic intervention in cancer. Proteasome and Hsp90 inhibitors are in clinical trials and a 20S proteasome inhibitor, Velcade, is an approved drug. Other points of intervention in the folding and degradation pathway may therefore be of interest. We describe a simple screen for inhibitors of protein synthesis, folding, and proteasomal degradation pathways in this paper. The molecular chaperone-dependent client v-Src was fused to firefly luciferase and expressed in HCT-116 colorectal tumor cells. Both luciferase and protein tyrosine kinase activity were preserved in cells expressing this fusion construct. Exposing these cells to the Hsp90 inhibitor geldanamycin caused a rapid reduction of luciferase and kinase activities and depletion of detergent-soluble v-Src::luciferase fusion protein. Hsp70 knockdown reduced v-Src::luciferase activity and, when combined with geldanamycin, caused a buildup of v-Src::luciferase and ubiquitinated proteins in a detergent-insoluble fraction. Proteasome inhibitors also decreased luciferase activity and caused a buildup of phosphotyrosine-containing proteins in a detergent-insoluble fraction. Protein synthesis inhibitors also reduced luciferase activity, but had less of an effect on phosphotyrosine levels. In contrast, certain histone deacetylase inhibitors increased luciferase and phosphotyrosine activity. A mass screen led to the identification of Hsp90 inhibitors, ubiquitin pathway inhibitors, inhibitors of Hsp70/Hsp40-mediated refolding, and protein synthesis inhibitors. The largest group of compounds identified in the screen increased luciferase activity, and some of these increase v-Src levels and activity. When used in conjunction with appropriate secondary assays, this screen is a powerful cell-based tool for studying compounds that affect protein synthesis, folding, and degradation.

Hsp70 expression and induction as a readout for detection of immune modulatory components in food.

Stress proteins such as heat shock proteins (Hsps) are up-regulated in cells in response to various forms of stress, like thermal and oxidative stress and inflammation. Hsps prevent cellular damage and increase immunoregulation by the activation of anti-inflammatory T-cells. Decreased capacity for stress-induced Hsp expression is associated with immune disorders. Thus, therapeutic boosting Hsp expression might restore or enhance cellular stress resistance and immunoregulation. Especially food- or herb-derived phytonutrients may be attractive compounds to restore optimal Hsp expression in response to stress. In the present study, we explored three readout systems to monitor Hsp70 expression in a manner relevant for the immune system and evaluated novel Hsp co-inducers. First, intracellular staining and analysis by flow cytometry was used to detect stress and/or dietary compound induced Hsp70 expression in multiple rodent cell types efficiently. This system was used to screen a panel of food-derived extracts with potent anti-oxidant capacity. This strategy yielded the identity of several new enhancers of stress-induced Hsp70 expression, among them carvacrol, found in thyme and oregano. Second, CD4(+) T-cell hybridomas were generated that specifically recognized an immunodominant Hsp70 peptide. These hybridomas were used to show that carvacrol enhanced Hsp70 levels increased T-cell activation. Third, we generated a DNAJB1-luc-O23 reporter cell line to show that carvacrol increased the transcriptional activation of a heat shock promoter in the presence of arsenite. These assay systems are generally applicable to identify compounds that affect the Hsp level in cells of the immune system.

Mesothelioma cells escape heat stress by upregulating Hsp40/Hsp70 expression via mitogen-activated protein kinases.

Therapy with hyperthermal chemotherapy in pleural diffuse malignant mesothelioma had limited benefits for patients. Here we investigated the effect of heat stress on heat shock proteins (HSP), which rescue tumour cells from apoptosis. In human mesothelioma and mesothelial cells heat stress (39-42 degrees C) induced the phosphorylation of two mitogen activated kinases (MAPK) Erk1/2 and p38, and increased Hsp40, and Hsp70 expression. Mesothelioma cells expressed more Hsp40 and were less sensitive to heat stress compared to mesothelial cells. Inhibition of Erk1/2 MAPK by PD98059 or by Erk1 siRNA down-regulated heat stress-induced Hsp40 and Hsp70 expression and reduced mesothelioma cell survival. Inhibition of p38MAPK by SB203580 or siRNA reduced Hsp40, but not Hsp70, expression and also increased mesothelioma cell death. Thus hyperthermia combined with suppression of p38 MAPK or Hsp40 may represent a novel approach to improve mesothelioma therapy.

Large isoform of MRJ (DNAJB6) reduces malignant activity of breast cancer.

INTRODUCTION: Mammalian relative of DnaJ (MRJ [DNAJB6]), a novel member of the human DnaJ family, has two isoforms. The smaller isoform, MRJ(S), is studied mainly for its possible role in Huntington's disease. There are no reports of any biologic activity of the longer isoform, MRJ(L). We investigated whether this molecule plays any role in breast cancer. Our studies were prompted by interesting observations we made regarding the expression of MRJ in breast cancer cell lines and breast cancer tissue microarrays, as described below. METHODS: Expression of MRJ(L) from several breast cancer cell lines was evaluated using real-time PCR. Relative levels of the small and large isoforms in breast cancer cell lines were studied using Western blot analysis. A breast cancer progression tissue microarray was probed using anti-MRJ antibody. MRJ(L) was ectopically expressed in two breast cancer cell lines. These cell lines were evaluated for their in vitro correlates of tumor aggressiveness, such as invasion, migration, and anchorage independence. The cell lines were also evaluated for in vivo tumor growth and metastasis. The secreted proteome of the MRJ(L) expressors was analyzed to elucidate the biochemical changes brought about by re-expression of MRJ(L). RESULTS: We found that MRJ(L) is expressed at a significantly lower level in aggressive breast cancer cell lines compared with normal breast. Furthermore, in clinical cases of breast cancer expression of MRJ is lost as the grade of infiltrating ductal carcinoma advances. Importantly, MRJ staining is lost in those cases that also had lymph node metastasis. We report that MRJ(L) is a protein with a functional nuclear localization sequence. Expression of MRJ(L) via an exogenous promoter in breast cancer cell line MDA-MB-231 and in MDA-MB-435 (a cell line that metastasizes from the mammary fat pad) decreases their migration and invasion, reduces their motility, and significantly reduces orthotopic tumor growth in nude mice. Moreover, the secreted proteome of the MRJ(L)-expressing cells exhibited reduced levels of tumor progression and metastasis promoting secreted proteins, such as SPP1 (osteopontin), AZGP1 (zinc binding alpha2-glycoprotein 1), SPARC (osteonectin), NPM1 (nucleophosmin) and VGF (VGF nerve growth factor inducible). On the other hand, levels of the secreted metastasis-suppressor KiSS1 (melanoma metastasis suppressor) were increased in the secreted proteome of the MRJ(L)-expressing cells. We confirmed by quantitative RT-PCR analysis that the secreted profile reflected altered transcription of the respective genes. CONCLUSION: Collectively, our data indicate an important role for a totally uncharacterized isoform of DNAJB6 in breast cancer. We show that MRJ(L) is a nuclear protein that is lost in breast cancer, that regulates several key players in tumor formation and metastasis, and that is functionally able to retard tumor growth.