Targeting hsp90 family members: A strategy to improve cancer cell death.

A crucial process in biology is the conversion of the genetic information into functional proteins that carry out the genetic program. However, a supplementary step is required to obtain functional proteins: the folding of the newly translated polypeptides into well-defined, three-dimensional conformations. Proteins chaperones are crucial for this final step in the readout of genetic information, which results in the formation of functional proteins. In this review, a special attention will be given to the strategies targeting hsp90 family members in order to increase cancer cell death. We argue that disruption of hsp90 machinery and the further client protein degradation is the main consequence of hsp90 oxidative cleavage taking place at the N-terminal nucleotide-binding site. Moreover, modulation of Grp94 expression will be discussed as a potential therapeutic goal looking for a decrease in cancer relapses.

Hsp90 is cleaved by reactive oxygen species at a highly conserved N-terminal amino acid motif.

Hsp90 is an essential chaperone that is necessary for the folding, stability and activity of numerous proteins. In this study, we demonstrate that free radicals formed during oxidative stress conditions can cleave Hsp90. This cleavage occurs through a Fenton reaction which requires the presence of redox-active iron. As a result of the cleavage, we observed a disruption of the chaperoning function of Hsp90 and the degradation of its client proteins, for example, Bcr-Abl, RIP, c-Raf, NEMO and hTert. Formation of Hsp90 protein radicals on exposure to oxidative stress was confirmed by immuno-spin trapping. Using a proteomic analysis, we determined that the cleavage occurs in a conserved motif of the N-terminal nucleotide binding site, between Ile-126 and Gly-127 in Hsp90ß, and between Ile-131 and Gly-132 in Hsp90α. Given the importance of Hsp90 in diverse biological functions, these findings shed new light on how oxidative stress can affect cellular homeostasis.

Restoring specific lactobacilli levels decreases inflammation and muscle atrophy markers in an acute leukemia mouse model.

The gut microbiota has recently been proposed as a novel component in the regulation of host homeostasis and immunity. We have assessed for the first time the role of the gut microbiota in a mouse model of leukemia (transplantation of BaF3 cells containing ectopic expression of Bcr-Abl), characterized at the final stage by a loss of fat mass, muscle atrophy, anorexia and inflammation. The gut microbial 16S rDNA analysis, using PCR-Denaturating Gradient Gel Electrophoresis and quantitative PCR, reveals a dysbiosis and a selective modulation of Lactobacillus spp. (decrease of L. reuteri and L. johnsonii/gasseri in favor of L. murinus/animalis) in the BaF3 mice compared to the controls. The restoration of Lactobacillus species by oral supplementation with L. reuteri 100-23 and L. gasseri 311476 reduced the expression of atrophy markers (Atrogin-1, MuRF1, LC3, Cathepsin L) in the gastrocnemius and in the tibialis, a phenomenon correlated with a decrease of inflammatory cytokines (interleukin-6, monocyte chemoattractant protein-1, interleukin-4, granulocyte colony-stimulating factor, quantified by multiplex immuno-assay). These positive effects are strain- and/or species-specific since L. acidophilus NCFM supplementation does not impact on muscle atrophy markers and systemic inflammation. Altogether, these results suggest that the gut microbiota could constitute a novel therapeutic target in the management of leukemia-associated inflammation and related disorders in the muscle.

Overexpression of GRP94 in breast cancer cells resistant to oxidative stress promotes high levels of cancer cell proliferation and migration: implications for tumor recurrence.

Targeting the altered redox status of cancer cells is emerging as an interesting approach to potentiate chemotherapy. However, to maximize the effectiveness of this strategy and define the correct chemotherapeutic associations, it is important to understand the biological consequences of chronically exposing cancer cells to reactive oxygen species (ROS). Using an H(2)O(2)-generating system, we selected a ROS-resistant MCF-7 breast cancer cell line, namely Resox cells. By exploring different survival pathways that are usually induced during oxidative stress, we identified a constitutive overexpression of the endoplasmic reticulum chaperone, GRP94, in these cells, whereas levels of its cytoplasmic homolog HSP90, or GRP78, were not modified. This overexpression was not mediated by constitutive unfolded protein response (UPR) activation. The increase in GRP94 is tightly linked to an increase in cell proliferation and migration capacities, as shown by GRP94-silencing experiments. Interestingly, we also observed that GRP94 silencing inhibits migration and proliferation of the highly aggressive MDA-MB-231 cells. By immunohistochemistry, we showed that GRP94 expression was higher in recurrent human breast cancers than in their paired primary neoplasias. Similar to the situation in the Resox cells, this increase was not associated with an increase in UPR activation in recurrent tumors. In conclusion, this study suggests that GRP94 overexpression may be a hallmark of aggressiveness and recurrence in breast cancers.

Catalase overexpression in mammary cancer cells leads to a less aggressive phenotype and an altered response to chemotherapy.

Because reactive oxygen species (ROS) are naturally produced as a consequence of aerobic metabolism, cells have developed a sophisticated set of antioxidant molecules to prevent the toxic accumulation of these species. However, compared with normal cells, malignant cells often exhibit increased levels of intracellular ROS and altered levels of antioxidant molecules. The resulting endogenous oxidative stress favors tumor growth by promoting genetic instability, cell proliferation and angiogenesis. In this context, we assessed the influence of catalase overexpression on the sensitivity of breast cancer cells towards various anticancer treatments. Our data show that catalase overexpression in MCF-7 cells leads to a 7-fold increase in catalase activity but provokes a 40% decrease in the expression of both glutathione peroxidase and peroxiredoxin II. Interestingly, proliferation and migration capacities of MCF-7 cells were impaired by the overexpression of catalase, as compared to parental cells. Regarding their sensitivity to anticancer treatments, we observed that cells overexpressing catalase were more sensitive to paclitaxel, etoposide and arsenic trioxide. However, no effect was observed on the cytotoxic response to ionizing radiations, 5-fluorouracil, cisplatin or doxorubicin. Finally, we observed that catalase overexpression protects cancer cells against the pro-oxidant combination of ascorbate and menadione, suggesting that changes in the expression of antioxidant enzymes could be a mechanism of resistance of cancer cells towards redox-based chemotherapeutic drugs.

Ascorbate/menadione-induced oxidative stress kills cancer cells that express normal or mutated forms of the oncogenic protein Bcr-Abl. An in vitro and in vivo mechanistic study.

Numerous studies suggest that generation of oxidative stress could be useful in cancer treatment. In this study, we evaluated, in vitro and in vivo, the antitumor potential of oxidative stress induced by ascorbate/menadione (asc/men). This combination of a reducing agent (ascorbate) and a redox active quinone (menadione) generates redox cycling leading to formation of reactive oxygen species (ROS). Asc/men was tested in several cell types including K562 cells (a stable human-derived leukemia cell line), freshly isolated leukocytes from patients with chronic myeloid leukemia, BaF3 cells (a murine pro-B cell line) transfected with Bcr-Abl and peripheral blood leukocytes derived from healthy donors. Although these latter cells were resistant to asc/men, survival of all the other cell lines was markedly reduced, including the BaF3 cells expressing either wild-type or mutated Bcr-Abl. In a standard in vivo model of subcutaneous tumor transplantation, asc/men provoked a significant delay in the proliferation of K562 and BaF3 cells expressing the T315I mutated form of Bcr-Abl. No effect of asc/men was observed when these latter cells were injected into blood of mice most probably because of the high antioxidant potential of red blood cells, as shown by in vitro experiments. We postulate that cancer cells are more sensitive to asc/men than healthy cells because of their lack of antioxidant enzymes, mainly catalase. The mechanism underlying this cytotoxicity involves the oxidative cleavage of Hsp90 with a subsequent loss of its chaperone function thus leading to degradation of wild-type and mutated Bcr-Abl protein.

Endoplasmic reticulum calcium release potentiates the ER stress and cell death caused by an oxidative stress in MCF-7 cells.

Increase in cytosolic calcium concentration ([Ca2+](c)), release of endoplasmic reticulum (ER) calcium ([Ca2+](er)) and ER stress have been proposed to be involved in oxidative toxicity. Nevertheless, their relative involvements in the processes leading to cell death are not well defined. In this study, we investigated whether oxidative stress generated during ascorbate-driven menadione redox cycling (Asc/Men) could trigger these three events, and, if so, whether they contributed to Asc/Men cytoxicity in MCF-7 cells. Using microspectrofluorimetry, we demonstrated that Asc/Men-generated oxidative stress was associated with a slow and moderate increase in [Ca2+](c), largely preceding permeation of propidium iodide, and thus cell death. Asc/Men treatment was shown to partially deplete ER calcium stores after 90 min (decrease by 45% compared to control). This event was associated with ER stress activation, as shown by analysis of eIF2 phosphorylation and expression of the molecular chaperone GRP94. Thapsigargin (TG) was then used to study the effect of complete [Ca2+](er) emptying during the oxidative stress generated by Asc/Men. Surprisingly, the combination of TG and Asc/Men increased ER stress to a level considerably higher than that observed for either treatment alone, suggesting that [Ca2+](er) release alone is not sufficient to explain ER stress activation during oxidative stress. Finally, TG-mediated [Ca2+](er) release largely potentiated ER stress, DNA fragmentation and cell death caused by Asc/Men, supporting a role of ER stress in the process of Asc/Men cytotoxicity. Taken together, our results highlight the involvement of ER stress and [Ca2+](er) decrease in the process of oxidative stress-induced cell death in MCF-7 cells.

Menadione reduction by pharmacological doses of ascorbate induces an oxidative stress that kills breast cancer cells.

Oxidative stress generated by ascorbate-driven menadione redox cycling kills MCF7 cells by a concerted mechanism including glycolysis inhibition, loss of calcium homeostasis, DNA damage and changes in mitogen activated protein kinases (MAPK) activities. Cell death is mediated by necrosis rather than apoptosis or macroautophagy. Neither 3-methyladenine nor Z-VAD affects cytotoxicity by ascorbate/menadione (Asc/Men). BAPTA-AM, by restoring cellular capacity to reduce MTT, underlines the role of calcium in the necrotic process. Oxidative stress-mediated cell death is shown by the opposite effects of N-acetylcysteine and 3-aminotriazole. Moreover, oxidative stress induces DNA damage (protein poly-ADP-ribosylation and gamma-H2AX phosphorylation) and inhibits glycolysis. Asc/Men deactivates extracellular signal-regulated kinase (ERK) while activating p38, suggesting an additional mechanism to kill MCF7 cells. Since ascorbate is taken up by cancer cells and, due to their antioxidant enzyme deficiency, oxidative stress should affect cancer cells to a greater extent than normal cells. This differential sensitivity may have clinical applications.

Hsp90 cleavage by an oxidative stress leads to its client proteins degradation and cancer cell death.

The heat shock protein 90 (Hsp90) plays a crucial role in the stability of several proteins that are essential for malignant transformation. Hsp90 is therefore an interesting therapeutic target for cancer therapy. In this paper, we investigated whether an oxidative stress generated during ascorbate-driven menadione redox cycling (ascorbate/menadione), affects Hsp90 leading to the degradation of some critical proteins and cell death. Unlike 17-AAG, which inhibits Hsp90 but enhances Hsp70 levels, ascorbate/menadione-treated cells present an additional Hsp90 protein band of about 70kDa as shown by Western blot analysis, suggesting Hsp90 cleavage. This Hsp90 cleavage seems to be a selective phenomenon since it was observed in a large panel of cancer cell lines but not in non-transformed cells. Antibodies raised against either the N-terminus or the C-terminus domains of Hsp90 suggest that the site of cleavage should be located at its N-terminal part. Furthermore, antibodies raised against either the alpha- or the beta-Hsp90 isoform show that Hsp90beta is cleaved while the alpha isoform is down-regulated. We have further shown that different Hsp90 client proteins like Bcr-Abl (a chimerical protein expressed in K562 leukemia cells), RIP and Akt, were degraded when K562 cells were exposed to an oxidative stress. Both Hsp90 cleavage and Bcr-Abl degradation were observed by incubating K562 cells with another H(2)O(2)-generating system (glucose/glucose oxidase) and by incubating KU812 cells (another leukemia cell line) with ascorbate/menadione. Due to the major role of Hsp90 in stabilizing oncogenic and mutated proteins, these results may have potential clinical applications.

Real time in situ ellipsometric and gravimetric monitoring for electrochemistry experiments.

This work describes a new system using real time spectroscopic ellipsometer with simultaneous electrochemical and electrochemical quartz crystal microbalance (EQCM) measurements. This method is particularly adapted to characterize electrolyte/electrode interfaces during electrochemical and chemical processes in liquid medium. The ellipsometer, based on a rotating compensator Horiba Jobin-Yvon ellipsometer, has been adapted to acquire Psi-Delta spectra every 25 ms on a spectral range fixed from 400 to 800 nm. Measurements with short sampling times are only achievable with a fixed analyzer position (A=45 degrees ). Therefore the ellipsometer calibration is extremely important for high precision measurements and we propose a spectroscopic calibration (i.e., determination of the azimuth of elements according to the wavelength) on the whole spectral range. A homemade EQCM was developed to detect mass variations attached to the electrode. This additional instrument provides further information useful for ellipsometric data modeling of complex electrochemical systems. The EQCM measures frequency variations of piezoelectric quartz crystal oscillator working at 5 MHz. These frequency variations are linked to mass variations of electrode surface with a precision of 20 ng cm(-2) every 160 ms. Data acquisition has been developed in order to simultaneously record spectroscopic ellipsometry, EQCM, and electrochemical measurements by a single computer. Finally the electrodeposition of bismuth telluride film was monitored by this new in situ experimental setup and the density of electroplated layers was extracted from the optical thickness and EQCM mass.

Computerized acoustic assessment of treatment efficacy of nebulized epinephrine and albuterol in RSV bronchiolitis.

AIM: We evaluated the use of computerized quantification of wheezing and crackles compared to a clinical score in assessing the effect of inhaled albuterol or inhaled epinephrine in infants with RSV bronchiolitis. METHODS: Computerized lung sounds analysis with quantification of wheezing and crackles and a clinical score were used during a double blind, randomized, controlled nebulized treatment pilot study. Infants were randomized to receive a single dose of 1 mgr nebulized l-epinephrine or 2.5 mgr nebulized albuterol. Computerized quantification of wheezing and crackles (PulmoTrack) and a clinical score were performed prior to, 10 minutes post and 30 minutes post treatment. Results were analyzed with Student's t-test for independent samples, Mann-Whitney U test and Wilcoxon test. RESULTS: 15 children received albuterol, 12 received epinephrine. The groups were identical at baseline. Satisfactory lung sounds recording and analysis was achieved in all subjects. There was no significant change in objective quantification of wheezes and crackles or in the total clinical scores either within the groups or between the groups. There was also no difference in oxygen saturation and respiratory distress. CONCLUSION: Computerized lung sound analysis is feasible in young infants with RSV bronchiolitis and provides a non-invasive, quantitative measure of wheezing and crackles in these infants.

Measurements and theory of normal tracheal breath sounds.

We studied the mechanisms by which turbulent flow induces tracheal wall vibrations detected as tracheal breath sounds (TRBSs). The effects of flow rate at transitional Reynold's numbers (1300-10,000) and gas density on spectral patterns of TRBSs in eight normal subjects were measured. TRBSs were recorded with a contact sensor during air and heliox breathing at four flow rates (1.0, 1.5, 2.0, and 2.5 l/s). We found that normalized TRBSs were proportional to flow to the 1.89 power during inspiration and to the 1.59 power during expiration irrespective of gas density. The amplitude of TRBSs with heliox was lower than with air by a factor of 0.33 +/- 0.12 and 0.44 +/- 0.16 during inspiration and expiration, respectively. The spectral resonance frequencies were higher during heliox than air breathing by a factor of 1.75 +/- 0.2-approximately the square root of the reciprocal of the air/heliox wave propagation speed ratio. In conclusion, the flow-induced pressure fluctuations inside the trachea, which cause tracheal wall vibrations, were detected as TRBSs consist of two components: (1) a dominant local turbulent eddy component whose amplitude is proportional to the gas density and nonlinearly related to the flow; and (2) a propagating acoustic component with resonances whose frequencies correspond to the length of the upper airway and to the free-field sound speed. Therefore, TRBSs consist primarily of direct turbulent eddy pressure fluctuations that are perceived as sound during auscultation.

Methacholine bronchial provocation measured by spirometry versus wheeze detection in preschool children.

BACKGROUND: Determination of PC20-FEV1 during Methacholine bronchial provocation test (MCT) is considered to be impossible in preschool children, as it requires repetitive spirometry sets. The aim of this study was to assess the feasibility of determining PC20-FEV1 in preschool age children and compares the results to the wheeze detection (PCW) method. METHODS: 55 preschool children (ages 2.8-6.4 years) with recurrent respiratory symptoms were recruited. Baseline spirometry and MCT were performed according to ATS/ERS guidelines and the following parameters were determined at baseline and after each inhalation: spirometry-indices, lung auscultation at tidal breathing, oxygen saturation, respiratory and heart rate. Comparison between PCW and PC20-FEV1 and clinical parameters at these end-points was done by paired Student's t-tests. RESULTS AND DISCUSSION: Thirty-six of 55 children (65.4%) successfully performed spirometry-sets up to the point of PCW. PC20-FEV1 occurred at a mean concentration of 1.70+/-2.01 mg/ml while PCW occurred at a mean concentration of 4.37+/-3.40 mg/ml (p < 0.05). At PCW, all spirometry-parameters were markedly reduced: FVC by 41.3+/-16.4% (mean +/-SD); FEV1 by 44.7+/-14.5%; PEFR by 40.5+/-14.5 and FEF25-75 by 54.7+/-14.4% (P < 0.01 for all parameters). This reduction was accompanied by de-saturation, hyperpnoea, tachycardia and a response to bronchodilators. CONCLUSION: Determination of PC20-FEV1 by spirometry is feasible in many preschool children. PC20-FEV1 often appears at lower provocation dose than PCW. The lower dose may shorten the test and encourage participation. Significant decrease in spirometry indices at PCW suggests that PC20-FEV1 determination may be safer.

Correlation between rapid strip test and the quality of sputum.

STUDY OBJECTIVES: To evaluate the use of a rapid strip test for the quick evaluation of sputum quality. DESIGN: Prospective, double-blind study. Sputum and saliva samples were collected. Sputum quality was assessed by the presence of polymorphonuclear neutrophils (PMNs) and squamous epithelial cells (SECs) per low-power (microscopic) field (LPF) [x10 objective]. Sputum was defined as follows: (1) informative (ie, > 25 PMNs and < 10 SECs per LPF); (2) semi-informative (ie, > 25 PMNs or < 10 SECs per LPF, but not both); or (3) uninformative (ie, < 25 PMNs and > 10 SECs per LPF). The first two levels were considered to be "sputum" and the third one was considered to be "nonsputum." The quality of the sputum was compared to results obtained using a rapid strip test (Combur-Test; Roche Diagnostics; Basel, Switzerland) for specific gravity (SG), pH, leukocyte esterase (LE) activity, and levels of nitrites, protein, glucose, and erythrocytes. A Kruskal-Wallis test was used to compare the three levels of sputum quality and the rapid strip test. A Mann-Whitney test compared sputum and nonsputum to the rapid strip reagents. Pearson correlation and kappa tests were used to assess correlation. Receiver operating characteristic was used to calculate the best cut-point values, and the sensitivity and specificity of these values were calculated. RESULTS: Eighty-two samples were included, with 61 samples from hospitalized patients and 21 samples from healthy volunteers. The best predicator of sputum quality was the SG of the reagent. Using an SG threshold definition of > 1.01, the sensitivity was 86.8% and the specificity was 75.9%. The specificity of protein, glucose >/= +1, and LE levels were relatively low. No relationship was found between the results of the reagent strip test for pH, nitrites, and erythrocytes, and the sputum quality. CONCLUSION: Using an SG threshold definition of > 1.01, the rapid reagent strip test has been shown to be a sensitive test for the evaluation of sputum quality, which can be useful when facilities for sputum cytology are not available.

Adenosine bronchial provocation with computerized wheeze detection in young infants with prolonged cough: correlation with long-term follow-up.

BACKGROUND: Chronic cough in babies is often associated with bronchial hyperreactivity (BHR). The objective documentation of BHR in babies is difficult, and acoustic methods have been described (provocative concentration of a substance causing wheeze) for conducting bronchial provocation tests (BPTs). We conducted a study to evaluate automatic computerized wheeze detection (CWD) in determining BHR in young infants with prolonged cough, and its correlation with the subsequent development of wheezing. METHODS: Infants aged < 24 months with prolonged cough (ie, > 2 months) underwent acoustic BPTs with the response determined by CWD and auscultation by a physician. Telephone interviews with parents were conducted after 1 month and yearly for the next 3 years. RESULTS: A total of 28 infants who were 4 to 24 months old with prolonged cough were included in the study. Twenty of these infants (71.4%) had BHR as determined by a positive acoustic BPT result. In 11 of these 20 tests, the CWD occurred earlier, and in 9 tests it occurred at the same step as auscultation by a physician. Rhonchi or whistles often preceded wheezes. Seventeen of the 20 patients with BHR completed 3 years of follow-up. Of these, 14 had recurrent episodes of wheezing and shortness of breath, and 3 were well. Six of the eight adenosine-negative patients completed 3 years of follow-up and had no symptoms of BHR. CONCLUSIONS: Acoustic BPT is a technically feasible test for the detection of BHR in young infants. CWD provides an earlier detection of wheeze than stethoscope auscultation. In our group of infants, a positive acoustic BPT result had high correlation with symptoms compatible with BHR over the next 3 years.

Measurement of inspiratory flow in children with acute asthma.

Dry-powder inhalers (DPIs) have been proposed for treatment of acute asthma. Different DPIs vary in their inspiratory resistance and have different recommended optimal peak inspiratory flows (PIFs). Reduced PIF during acute asthma may result in inadequate drug delivery to the lungs. Our aim was to measure the inspiratory flow in relation to inspiratory resistance during acute asthma in children presenting to the emergency room. School-age (range, 6-18 years) children were referred to the emergency room for acute asthma. PIF measurements were performed by In-Check Dial trade mark device with simulated airflow resistances equivalent to Turbuhaler, Diskus, and free flow. Percent change in PIF between remission and acute asthma (%Delta) was correlated with percent change in clinical score (CS) and percent change in spirometry in children <9 and >9 years old. Thirty-three children (21 males) participated. PIF with simulated Turbuhaler resistance was significantly lower than with simulated Diskus resistance in both acute and remission states (P < 0.0001). PIF with simulated Turbuhaler resistance increased from 62.1 +/- 15.3 (acute) to 74.4 +/- 16.5 l/min (remission, P < 0.0001), while with Diskus it rose from 72.6 +/- 20.5 to 91.1 +/- 18.9 l/min (P < 0.0001). Turbuhaler %Delta PIF correlated with %Delta FEV(1) (P = 0.01) and with %Delta CS (P = 0.0001). A lesser degree of correlation was observed while using Diskus resistance and in children above 9 years old. During acute asthmatic attacks, PIF is reduced; this reduction is particularly prominent in young children who use a high-resistance device. However, the PIF generated is generally within the values considered compatible with adequate lung deposition with both Diskus and Turbuhaler.

Crouzon syndrome: association with absent pulmonary valve syndrome and severe tracheobronchomalacia.

Airway obstruction is common among patients with craniosynostosis. We describe an infant with a clinical and genetic diagnosis of Crouzon syndrome who presented with respiratory distress and heart murmur in early neonatal life. Cardiac evaluation revealed absent pulmonary valve syndrome. She needed intubation at age 1 month, and repeated trials of extubation failed because of marked respiratory distress, stridor, and severe expiratory obstruction and wheezing. Correction of her cardiac anomaly did not relieve her respiratory distress; only after endobronchial stenting and tracheostomy was it possible to gradually wean her from mechanical ventilation. This case report demonstrates and discusses the different causes of airway obstruction in Crouzon syndrome and the morbidity and mortality that can result from pulmonary involvement in this craniosynostotic syndrome. It also demonstrates the difficult therapeutic challenge created by the combination of cardiopulmonary abnormalities in Crouzon patients.

Abdominal compartment syndrome in children: CT findings.

Abdominal compartment syndrome (ACS) is caused by pathological elevation of intra-abdominal pressure (IAP) leading to multiple organ dysfunction syndrome. Since the condition is highly lethal, early diagnosis is imperative. We evaluated the pre-operative abdominal CT scans of three children with proven ACS to identify signs of elevated IAP. Findings common to these patients included narrowing of the inferior vena cava (IVC), direct renal compression or displacement, bowel wall thickening with enhancement and a rounded appearance of the abdomen. The aim of recognising the CT findings in such cases is to plan emergency surgical decompression. Although these findings are not specific for increased IAP, radiologists should be aware of this life-threatening condition and, in the proper clinical setting, should communicate the presence and significance of these findings to the referring clinician.