Inhibits Heat Shock Research Articles

Inhibition of heat shock transcription factor binding by a linear polyamide binding in an unusual 1:1 mode.

Heat shock proteins (HSPs) are known to protect cells from heat, oxidative stress, and the cytotoxic effects of drugs, and thus can enhance cancer cell survival. As a result, HSPs are a newly emerging class of protein targets for chemotherapy. Among the various HSPs, the HSP70 family is the most highly conserved and prevalent. Herein we describe the development of a β-alanine rich linear polyamide that binds the GGA heat shock elements (HSEs) 3 and 4 in the HSP70 promoter in an unusual 1:1 mode and inhibits heat shock transcription factor 1 (HSF1) binding in vitro.

Heat stress activates ER stress signals which suppress the heat shock response, an effect occurring preferentially in the cortex in rats

Although heat stress induces a variety of illnesses, there have been few studies designed to uncover the molecular mechanisms underlining the illnesses. We here demonstrate that heat activates ER stress, which inhibits heat shock responses (HSR) via translational block. In heat-stressed rats, ER stress responses, as represented by eIF2α phosphorylation and XBP1 splicing, occurred mainly in the cortex, where the HSR was substantially inhibited. Heat exposure also activated ER stress signals in primary cortical neurons. Since HSF1 knockdown enhanced heat-induced ER stress and subsequent cell death, HSR inhibition in turn augments ER stress, implying a vicious spiral of both stresses. Taken together, heat-induced ER stress impairs the HSR and enhances cell damage, thereby manifesting its unique effect on heat stress.

Rhein inhibits angiogenesis and the viability of hormone-dependent and -independent cancer cells under normoxic or hypoxic conditions in vitro

Hypoxia is a hallmark of solid tumors, including breast cancer, and the extent of tumor hypoxia is associated with treatment resistance and poor prognosis. Considering the limited treatment of hypoxic tumor cells and hence a poor prognosis of breast cancer, the investigation of natural products as potential chemopreventive anti-angiogenic agents is of paramount interest. Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid), the primary anthraquinone in the roots of Cassia alata L., is a naturally occurring quinone which exhibits a variety of biologic activities including anti-cancer activity. However, the effect of rhein on endothelial or cancer cells under hypoxic conditions has never been delineated. Therefore, the aim of this study was to investigate whether rhein inhibits angiogenesis and the viability of hormone-dependent (MCF-7) or -independent (MDA-MB-435s) breast cancer cells in vitro under normoxic or hypoxic conditions. Rhein inhibited vascular endothelial growth factor (VEGF 165)-stimulated human umbilical vein endothelial cell (HUVEC) tube formation, proliferation and migration under normoxic and hypoxic conditions. In addition, rhein inhibited in vitro angiogenesis by suppressing the activation of phosphatidylinositol 3-kinase (PI3K), phosphorylated-AKT (p-AKT) and phosphorylated extracellular signal-regulated kinase (p-ERK) but showed no inhibitory effects on total AKT or ERK. Rhein dose-dependently inhibited the viability of MCF-7 and MDA-MB-435s breast cancer cells under normoxic or hypoxic conditions, and inhibited cell cycle in both cell lines. Furthermore, Western blotting demonstrated that rhein inhibited heat shock protein 90alpha (Hsp90α) activity to induce degradation of Hsp90 client proteins including nuclear factor-kappa B (NF-κB), COX-2, and HER-2. Rhein also inhibited the expression of hypoxia-inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor (VEGF 165), epidermal growth factor (EGF), and the phosphorylation of inhibitor of NF-κB (I-κB) under normoxic or hypoxic conditions. Taken together, these data indicate that rhein is a promising anti-angiogenic compound for breast cancer cell viability and growth. Therefore, further studies including in vivo and pre-clinical need to be performed.

Terutroban, a thromboxane/prostaglandin endoperoxide receptor antagonist, prevents hypertensive vascular hypertrophy and fibrosis

Thromboxane A(2) and other eicosanoids such as isoprostanes contribute to vascular proliferation and atherosclerosis by binding to the thromboxane/prostaglandin endoperoxide receptors. The effects of terutroban, a thromboxane/prostaglandin endoperoxide receptor antagonist, on aorta remodeling were evaluated in spontaneously hypertensive stroke-prone rats (SHRSPs), a model of severe hypertension, endothelial dysfunction, vascular inflammation, and cerebrovascular diseases. Male SHRSPs were allocated to three groups receiving a standard diet (n = 5) or a high-sodium permissive diet plus vehicle (n = 6) or plus terutroban (30 mg · kg(-1) · day(-1); n = 6). After 6 wk of dietary treatment, all of the animals were injected with bromodeoxyuridine and simultaneously euthanized for aorta collection. The aortic media thickness-to-lumen ratio significantly (P < 0.0001) increased in the salt-loaded rats compared with the rats fed a standard diet, whereas terutroban treatment completely prevented media thickening (P < 0.001). When compared with vehicle, terutroban was also effective in preventing cell proliferation in the media, as indicated by the reduced number of bromodeoxyuridine-positive (P < 0.0001) and proliferating cell nuclear antigen-positive cells (P < 0.0001). Severe fibrosis characterized by a significant accumulation of collagen and fibronectin in the vascular wall was observed in the vehicle-treated rats (P < 0.01) but was completely prevented by terutroban (P < 0.001). The latter also inhibited heat shock protein-47 (P < 0.01) and TGF-1β expression (P < 0.001), which were significantly increased by the high-salt diet. In conclusion, terutroban prevents the development of aorta hyperplasia and has beneficial effects on fibrotic processes by affecting TGF-β and heat shock protein-47 expression in SHRSPs. These findings provide mechanistic data supporting the beneficial effects of terutroban in preventing or retarding atherogenesis.

Gambogic acid inhibits Hsp90 and deregulates TNF-α/NF-κB in HeLa cells

Gambogic acid (GB) is an important anti-cancer drug candidate, but the target protein by which it exerts its anti-cancer effects has not been identified. This study is the first to show that GB inhibits heat shock protein 90 (Hsp90) and down-regulates TNF-α/NF-κB in HeLa cells. The effects of GB on Hsp90 were studied by characterizing its physical interactions with Hsp90 upon binding, the noncompetitive inhibition of Hsp90 ATPase activity, and the degradation of Hsp90 client proteins (i.e., Akt, IKK) in HeLa cells. GB seems to bind to the N-terminal ATP-binding domain of Hsp90. Additionally, GB suppresses the activation of TNF-α/NF-κB and decreases XIAP expression levels and the ratio of Bcl-2/Bax, which in turn induces HeLa cell apoptosis. Thus, GB represents a promising therapeutic agent for cancer; it may also be useful as a probe to increase understanding of the biological functions of Hsp90.

Abstract 2638: Hsp90 inhibitor STA-9090 induces HIF-1α degradation in the hypoxic regions of solid tumors

Abstract BACKGROUND: Solid tumors typically exhibit regions of poor oxygen supply, or hypoxia, due to disorganized tumor microvasculature limiting the efficient circulation of blood. The hypoxia-inducible transcription factor HIF-1α regulates an adaptive response to hypoxia that is characterized by increased glycolytic metabolism, angiogenesis and a propensity to metastasize. HIF-1α expression has also been correlated with resistance to radiation and chemotherapy, as well as poor clinical prognosis. However, inhibiting HIF-1α has proven to be challenging, not only because transcription factors are difficult to directly target, but also because its expression is confined to the most poorly vascularized regions of tumors, which therefore limits efficient distribution of therapeutic agents. An alternative approach to targeting HIF-1α is by inhibiting heat shock protein 90 (Hsp90), a molecular chaperone that regulates the proper folding and stability of HIF-1α. STA-9090 is a highly potent and less toxic, next-generation Hsp90 inhibitor that is structurally unrelated to first-generation agents such as 17-AAG and IPI-504. STA-9090 is currently being evaluated in multiple Phase 1/2 clinical trials in sold tumor and hematologic malignancies. We have examined the ability of STA-9090 to disrupt the hypoxic tumor response mediated by HIF-1α in preclinical models. RESULTS: In vitro in hypoxic NCI-H1975 lung cancer cells, STA-9090 was found to potently down-regulate the expression of HIF-1α and other Hsp90 client proteins. STA-9090 also inhibited the expression of downstream HIF-1α targets. In vivo in the NCI-H1975 xenograft model, a single MTD dose of STA-9090 reduced HIF-1α expression by 6-fold at 24 hr after treatment, despite a 39% decrease in microvascular density and a 2.5-fold increase in tumor hypoxia relative to control tumors. Importantly, this effect on HIF-1α was found to be uniform throughout tumors and independent of distance from microvasculature, indicating that STA-9090 was efficiently distributed to the hypoxic regions of tumors &amp;gt;150 μM from the nearest blood vessels. Similarly, STA-9090 treatment decreased EGFR expression, inhibited cell proliferation and induced apoptosis in tumors independent of vascular distance. CONCLUSIONS: Our results demonstrate that STA-9090 significantly inhibits HIF-1α expression in the hypoxic regions of tumors. In contrast to many other chemotherapeutic agents, STA-9090 is also efficiently distributed into the poorly vascularized, hypoxic tumor tissue where HIF-1α is expressed. Hsp90 inhibition using STA-9090 represents a promising approach to indirectly target HIF-1α and disrupt the hypoxic tumor response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2638.

Abstract 2622: C282Y HFE polymorphism imparts temozolomide and 17-DMAG resistance via p16INK4A

Abstract Cancer cells have a robust requirement for iron. The HFE protein interacts with transferrin receptor and limits the cellular iron uptake. HFE polymorphisms - the most common genetic variants in Caucasians - have been associated with a variety of cancers. Therefore we hypothesized that HFE polymorphisms would impact the phenotype of cancer cells. Using stably transfected human neuroblastoma cell lines and a number of human glioma cell lines we determined the effect of HFE polymorphisms on the therapy resistance. In the HFE variants stably transfected neuroblastoma cells, cells carrying the C282Y allelic variant were significantly more resistant to the chemotherapeutic agent temozolomide (currently the standard of care for brain tumors) than the same cell line carrying wildtype or H63D variant of HFE. The resistance to temozolomide was also found in commercially available human astrocytoma cell lines with the C282Y allele. The mechanism of greater resistance in the cells with the C282Y allele did not appear to involve O6-methylguanine-DNA methyltransferase traditionally associated with temozolomide resistance. In addition to temozolomide, the C282Y allele expressing glioma cells were also resistant to geldanamycin and its derivatives, 17-allylaminogeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG). These ansamycin compounds inhibit heat shock protein 90 (Hsp90) and thus have a different mechanism of action on the cancer cells than temozolomide. To discover the mechanism by which C282Y HFE variant cells were resistant to different therapeutic agents, we performed functional gene arrays in HFE stably transfected neuroblastoma cells. The expression of p16INK4A (cyclin dependent kinase inhibitor 2A) gene was found significantly increased in the C282Y cells compared to the wildtype cells. Consistent with the gene expression, p16INK4A protein expression was elevated in association with the C282Y allele relative to wildtype HFE. C282Y expressing astrocytoma cells also expressed high levels of p16INK4A protein. Therefore, we further determined the role of p16INK4A in drug resistance using p16INK4A siRNA, and found that p16INK4A knockdown sensitized C282Y expressing cells to temozolomide and 17-DMAG. Furthermore, siRNA of HFE decreased p16INK4A expression in C282Y cells. Taking together, these data suggest that C282Y HFE polymorphism impacts the cancer phenotype such as drug resistance, and the mechanism appears to be via increased expression of p16INK4A. Given the frequency of the HFE variants in the general population, these data provide compelling evidence that C282Y HFE polymorphism should be a pharmacogenetic consideration for evaluating treatment efficacy in cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2622.

Potential of Anti- Candida Antibodies in Immunoprophylaxis

The need for new options for the treatment of invasive candidiasis has fuelled the use of antibodies in combination with conventional antifungal therapy. After a long period of time in which antibodies were considered irrelevant in the resistance against invasive candidiasis, it was demonstrated that a number of antibodies or their engineered derivatives directed against Candida albicans cell-wall polysaccharides and glycopeptides, as well as against some protein epitopes, confer protection against invasive candidiasis. This has confirmed this approach as a new strategy for the prophylaxis of invasive candidiasis. Of particular interest is Mycograb, a human recombinant monoclonal antibody that inhibits heat shock protein 90, and has been administrated in combination with lipid-associated amphotericin B to patients with invasive candidiasis, and the fungicidal anti-beta-glucan antibodies induced by the glycoconjugate vaccine composed of a beta-glucan polysaccharide conjugated with the diphtheria toxoid CRM 197. However, despite the promising data obtained in vitro and in animal models, at present there is very little clinical experience on the use of antibodies in Candida immunoprophylaxis.

In VitroActivity of Geldanamycin Derivatives againstSchistosoma japonicumandBrugia malayi

Geldanamycin (GA) is a benzoquinone-containing ansamycin that inhibits heat shock protein 90. GA derivatives are being evaluated as anti-neoplastic agents, but their utility against parasites whose heat shock proteins (Hsps) have homology with human Hsp90 is unknown. The activities of four synthetic GA derivatives were tested in vitro using adult Brugia malayi and Schistosoma japonicum. Two of the derivatives, 17-N-allyl-17-demethoxygeldanamycin (17-AAG) and 17-N-(2-dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG), are currently in human clinical trials as anticancer drugs. Using concentrations considered safe peak plasma concentrations for these two derivatives, all four derivatives were active against both parasites. The less toxic derivative 17-AAG was as effective as GA in killing S. japonicum, and both DMAG and 5′-bromogeldanoxazinone were more active than 17-AAG against B. malayi. This work supports continued evaluation of ansamycin derivatives as broad spectrum antiparasitic agents.

Plasmodial heat shock proteins: targets for chemotherapy

Heat shock proteins act as molecular chaperones, facilitating protein folding in cells of living organisms. Their role is particularly important in parasites because environmental changes associated with their life cycles place a strain on protein homoeostasis. Not surprisingly, some heat shock proteins are essential for the survival of the most virulent malaria parasite, Plasmodium falciparum. This justifies the need for a greater understanding of the specific roles and regulation of malarial heat shock proteins. Furthermore, heat shock proteins play a major role during invasion of the host by the parasite and mediate in malaria pathogenesis. The identification and development of inhibitor compounds of heat shock proteins has recently attracted attention. This is important, given the fact that traditional antimalarial drugs are increasingly failing, as a consequence of parasite increasing drug resistance. Heat shock protein 90 (Hsp90), Hsp70/Hsp40 partnerships and small heat shock proteins are major malaria drug targets. This review examines the structural and functional features of these proteins that render them ideal drug targets and the challenges of targeting these proteins towards malaria drug design. The major antimalarial compounds that have been used to inhibit heat shock proteins include the antibiotic, geldanamycin, deoxyspergualin and pyrimidinones. The proposed mechanisms of action of these molecules and the pathways they inhibit are discussed.

Effects of RNAi on keloid formation by inhibiting heat shock protein

Objective To study the significance of HSP47 gene in keloid formation after in vivo study with RNAi technology and the recombinant HSP4 7 siRNA against heat shock protein 47 to keloid in a nude mice model.Methods We injected RNAi mixture into the keloid of a nude mice model in experimental group and PBS water(0.25 ml)into control group at the 16th days after establishing the models.After interference we observed the specimens and harvested specimens at 7th days for biochemical and pathological analysis.Results The expression of HSP47 mRNA reduced obviously and the collagen content also reduced in the experimental group.The rusults had statistical significance.Conclusion We can suppress the expression of HSP47 gene and then reduce the production of collagen after in vivo interfering experiment with HSP4 7 siRNA in keloid nude mice models using RNAi technique.This study cornfirms the mechanism that HSP47 promotes the keloid formation,which provides a new target to treat keloid. Key words: RNA interference; Heat shock protein 47; Keloid; Interference in vivo

An inhibitory role of p53 via NF-κB element on the cyclin D1 gene under heat shock

Little is known about the mechanisms underlying heat shock-mediated inhibition of cyclin D1 transcription. Here, we report that NF-κB site-mediated cyclin D1 transcription is inhibited by heat shock. The mRNA level of cyclin D1 decreased under heat shock (40–60%). This inhibition of transcription is promoter activity dependent and is mediated by the proximal NF-κB site. However, P65 overexpression did not influence the heat-inducible inhibitory pattern and heat shock did not significantly change the binding activity of p65. P53 can inhibit cyclin D1 promoter activity via an NF-κB site-dependent manner and its binding activity increased after heat shock. Importantly, p53 overexpression can prevent cyclin D1 promoter activation by p65. Therefore, we can deduce that p53 inhibits promoter activity under heat shock. These results reveal that the mechanism of heat shock-mediated inhibition of cyclin D1 transcription involves an NF-κB site. The data presented provide a new insight into the underlying heat shock inhibition of gene transcription.

Age‐related Alterations in PI3‐Kinase/Akt and Heat Shock Protein 90 signaling in Dilation of Rat Soleus Feed Arteries

The purpose of this study was to test the hypothesis that impaired eNOS activation contributes to age‐induced endothelial dysfunction in skeletal muscle feed arteries. Soleus muscle feed arteries (SFA) were isolated from young (4 mo, n=25) and old (24 mo, n=25) Fischer 344 rats and cannulated with two glass micropipettes for examination of vasodilator responses to flow, acetylcholine (ACh) and sodium nitroprusside (SNP) in the absence or presence of Akt Inhibitor (AktI, 5µM) to inhibit Akt, LY‐294002 (25µM) to inhibit PI3K, or Radicicol (RAD, 20µM) to inhibit Heat Shock Protein 90 (HSP90). In the absence of inhibitors, flow‐and ACh‐induced dilations were attenuated in SFA from old rats in comparison to SFA from young rats. Incubation with AktI, did not alter flow‐, ACh‐ or SNP‐induced dilation in young or old SFA. Incubation with LY‐294002 attenuated flow‐ and ACh‐induced dilation in young (not old) SFA. RAD attenuated ACh‐induced dilation in young SFA but augmented ACh‐induced dilation in old SFA. RAD augmented SNP‐induced dilation in both age groups. Collectively, these results suggest that impaired PI3K (not Akt) signaling contributes to the age‐related impairment of endothelial function in SFA and that HSP90 may play a role in SFA smooth muscle contraction. Research supported by AHA 0765043Y, an ASCM Foundation Research Grant, and a Huffines Institute Graduate Student Research Grant.

Heat shock protein 90 inhibition abrogates hepatocellular cancer growth through cdc2-mediated G2/M cell cycle arrest and apoptosis

17-(demethoxy), 17-allylamino geldanamycin (17-AAG) suppresses growth in some cancers by inhibiting Heat shock protein 90 (Hsp90). We examined the effects of 17-AAG-mediated Hsp90 inhibition on human hepatocellular carcinoma (HCC) growth in vitro and in vivo. Human HCC cell lines, Hep3B and HuH7, were exposed to 17-AAG and cell viabilities and apoptosis were determined. Cell cycle profiles were analyzed and the G(2)/M cell cycle checkpoint proteins cdc2 and cyclin B1 were examined. Studies were performed to determine whether 17-AAG-mediated cdc2 decrease was due to altered gene expression, transcription, or protein degradation. The effects of 17-AAG on Hep3B and HuH7 xenograft growth in athymic nude mice were also examined. Hep3B and HuH7 treated with 17-AAG versus untreated controls showed decreased cell viability and increased apoptosis. Cells treated with 17-AAG also showed an increased fraction in G(2)/M phase and an associated decrease in cdc2 through protein degradation rather than through other mechanisms. Hsp90 inhibition by 17-AAG also decreased HCC xenograft growth in association with decreased cdc2 expression. 17-AAG-mediated inhibition of Hsp90 abrogates human HCC cell growth in vitro and in vivo through cdc2 decrease, which in turn induces G(2)/M cell cycle arrest and apoptosis. Hsp90 is a mediator of HCC growth and survival and its inhibition may serve as a potential treatment.

Kava, a Tonic for Relieving the Irrational Development of Natural Preventive Agents

The last two decades have witnessed explosive growth in the study of natural and other cancer chemopreventive agents ([1][1], [2][2]). Extensive preclinical data have been generated for natural agents, and many (such as green tea, curcumin, phenyl isothiocyanate, indole-3-carbinol, silibinin,

Polyamine compound deoxyspergualin inhibits heat shock protein-induced activation of immature dendritic cells

Polyamine compound deoxyspergualin (DSG) is a potent immunosuppressive agent that has been applied clinically for protecting graft rejection and treatment of Wegener's granulomatosis. Though DSG can bind to heat-shock proteins (HSPs) in cells, its mechanism of immunosuppressive action remains unknown. It is widely accepted that extracellular HSPs are capable of stimulating dendritic cells (DC) through cell surface receptors, leading to DC activation and cytokine release. In this study, we examined if DSG analogs could inhibit HSP70-induced DC activation. Bone marrow derived immature mouse DCs and peripheral blood mononuclear cell-derived immature human DCs were generated and incubated with Alexa 488-labeled Hsp70 in the presence of methoxyDSG (Gus-1) that had comparable HSP70-binding affinity to DSG or DSG analog GUS-7, which had much more reduced binding affinity for HSP70. The binding of HSP70 to immature DCs was analyzed by laser microscopy and flow cytometry. HSP70-induced DC activation was assessed by TNF-alpha release by enzyme-linked immunosorbent assay. Binding of Hsp70 to the cell surface of immature DCs was inhibited under the presence of Gus-1, but not under the presence of Gus-7. Immature DCs were activated and released TNF-alpha by the stimulation with HSP70 for 12 hours; however, the HSP70-induced TNF-alpha release was suppressed under the presence of Gus-1, and partially suppressed under the presence of Gus-7. Similar results were observed when immature human DCs were stimulated under the same conditions. Immunosuppressive mechanism of DSG may be explained, at least in part, by the inhibition of extracellular HSP70-DC interaction and HSP70-induced activation of immature DCs.

Oxidative Stress Plays a Critical Role in Inactivating Mutant BRAF by Geldanamycin Derivatives

The geldanamycin derivatives 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) are promising chemotherapeutic drugs that inhibit heat shock protein 90 (HSP90) function. Previous studies have shown that 17-AAG/DMAG treatment induces the degradation of mutant BRAF (V600E) and inhibits the activation of mitogen-activated protein/extracellular signal-regulated kinase 1/2 (MEK1/2). We have found, however, that HSP90 inhibition alone is not sufficient for efficient BRAF(V600E) degradation in some cells. HSP90 inhibitors structurally unrelated to geldanamycin, radicicol and novobiocin, while inducing the degradation of the HSP90 client protein RAF-1 fail to induce BRAF(V600E) degradation or inhibit MEK1/2 activation in HT29 human colon cancer cells. Moreover, after treatment with 17-DMAG, the kinase activity of residual, undegraded BRAF(V600E) was also lost. Incubation of cells with a reactive oxygen species (ROS) scavenger, N-acetyl cysteine, partially restored kinase activity and also partially prevented BRAF(V600E) degradation due to 17-DMAG treatment. Conversely, treatment with the ROS producing drug menadione clearly inhibited MEK1/2 and reduced BRAF(V600E). These results suggest that in addition to direct inhibition of HSP90, the antitumor effect of geldanamycin and its derivatives is also mediated though the production of ROS, which may directly inactivate tumorigenic mutant BRAF(V600E).

Heat shock inhibits caspase-1 activity while also preventing its inflammasome-mediated activation by anthrax lethal toxin

Anthrax lethal toxin (LT) rapidly kills macrophages from certain mouse strains in a mechanism dependent on the breakdown of unknown protein(s) by the proteasome, formation of the Nalp1b (NLRP1b) inflammasome and subsequent activation of caspase-1. We report that heat-shocking LT-sensitive macrophages rapidly protects them against cytolysis by inhibiting caspase-1 activation without upstream effects on LT endocytosis or cleavage of the toxin's known cytosolic substrates (mitogen-activated protein kinases). Heat shock protection against LT occurred through a mechanism independent of de novo protein synthesis, HSP90 activity, p38 activation or proteasome inhibition and was downstream of mitogen-activated protein kinase cleavage and degradation of an unknown substrate by the proteasome. The heat shock inhibition of LT-mediated caspase-1 activation was not specific to the Nalp1b (NLRP1b) inflammasome, as heat shock also inhibited Nalp3 (NLRP3) inflammasome-mediated caspase-1 activation in macrophages. We found that heat shock induced pro-caspase-1 association with a large cellular complex that could prevent its activation. Additionally, while heat-shocking recombinant caspase-1 did not affect its activity in vitro, lysates from heat-shocked cells completely inhibited recombinant active caspase-1 activity. Our results suggest that heat shock inhibition of active caspase-1 can occur independently of an inflammasome platform, through a titratable factor present within intact, functioning heat-shocked cells.

A Phase I Study of 17-Allylamino-17-Demethoxygeldanamycin Combined with Paclitaxel in Patients with Advanced Solid Malignancies

17-Allylamino-17-demethoxygeldanamycin (17-AAG) inhibits heat shock protein 90, promotes degradation of oncoproteins, and exhibits synergy with paclitaxel in vitro. We conducted a phase I study in patients with advanced malignancies to determine the recommended phase II dose of the combination of 17-AAG and paclitaxel. Patients with advanced solid malignancies that were refractory to proven therapy or without any standard treatment were included. 17-AAG (80-225 mg/m2) was given on days 1, 4, 8, 11, 15, and 18 of each 4-week cycle to sequential cohorts of patients. Paclitaxel (80-100 mg/m2) was administered on days 1, 8, and 15. Pharmacokinetic studies were conducted during cycle 1. Twenty-five patients were accrued to five dose levels. The median number of cycles was 2. Chest pain (grade 3), myalgia (grade 3), and fatigue (grade 3) were dose-limiting toxicities at dose level 4 (225 mg/m2 17-AAG and 80 mg/m2 paclitaxel). None of the six patients treated at dose level 3 with 17-AAG (175 mg/m2) and paclitaxel (80 mg/m2) experienced dose-limiting toxicity. Disease stabilization was noted in six patients, but there were no partial or complete responses. The ratio of paclitaxel area under the concentration to time curve when given alone versus in combination with 17-AAG was 0.97 +/- 0.20. The ratio of end-of-infusion concentration of 17-AAG (alone versus in combination with paclitaxel) was 1.14 +/- 0.51. The recommended phase II dose of twice-weekly 17-AAG (175 mg/m2) and weekly paclitaxel (80 mg/m2/wk) was tolerated well. There was no evidence of drug-drug pharmacokinetic interactions.

Radiosensitization of Human Vascular Endothelial Cells Through Hsp90 Inhibition With 17- N-Allilamino-17-Demethoxygeldanamycin

In addition to invasive tumor cells, endothelial cells (ECs) of the tumor vasculature are an important target for anticancer radiotherapy. The purpose of the present work is to investigate how 17-N-allilamino-17-demethoxygeldanamycin (17AAG), known as an anticancer drug inhibiting heat shock protein 90 (Hsp90), modifies radiation responses of human vascular ECs. The ECs cultured from human umbilical veins were exposed to gamma-irradiation, whereas some EC samples were pretreated with growth factors and/or 17AAG. Postirradiation cell death/survival and morphogenesis were assessed by means of terminal deoxynucleotidyl transferase biotin-deoxyuridine triphosphate nick end labeling or annexin V staining and clonogenic and tube-formation assays. The 17AAG-affected expression and phosphorylation of radioresistance-related proteins were probed by means of immunoblotting. Dominant negative or constitutively activated Akt was transiently expressed in ECs to manipulate Akt activity. It was found that nanomolar concentrations of 17AAG sensitize ECs to relatively low doses (2-6 Gy) of gamma-irradiation and abolish the radioprotective effects of vascular endothelial growth factor and basic fibroblast growth factor. The drug-induced radiosensitization of ECs seems to be caused by prevention of Hsp90-dependent phosphorylation (activation) of Akt that results in blocking the radioprotective phosphatidylinositol 3-kinase/Akt pathway. Clinically achievable concentrations of 17AAG can decrease the radioresistance intrinsic to vascular ECs and minimize the radioprotection conferred upon them by tumor-derived growth factors. These findings characterize 17AAG as a promising radiosensitizer for the tumor vasculature.