High-throughput screen identifies disulfiram as a potential therapeutic for triple-negative breast cancer cells: Interaction with IQ motif-containing factors

Triple-negative breast cancer (TNBC) represents an aggressive subtype, for which radiation and chemotherapy are the only options. Here we describe the identification of disulfiram, an FDA-approved drug used to treat alcoholism, as well as the related compound thiram, as the most potent growth inhibitors following high-throughput screens of 3185 compounds against multiple TNBC cell lines. The average IC₅₀ for disulfiram was ~300 nM. Drug affinity responsive target stability (DARTS) analysis identified IQ motif-containing factors IQGAP1 and MYH9 as direct binding targets of disulfiram. Indeed, knockdown of these factors reduced, though did not completely abolish, cell growth. Combination treatment with 4 different drugs commonly used to treat TNBC revealed that disulfiram synergizes most effectively with doxorubicin to inhibit cell growth of TNBC cells. Disulfiram and doxorubicin cooperated to induce cell death as well as cellular senescence, and targeted the ESA⁺/CD24^-/low/CD44⁺ cancer stem cell population. Our results suggest that disulfiram may be repurposed to treat TNBC in combination with doxorubicin.     

Repeated oral administration of capsaicin increases anxiety-like behaviours with prolonged stress-response in rats

This study was conducted to examine the psycho-emotional effects of repeated oral exposure to capsaicin, the principal active component of chili peppers. Each rat received 1 mL of 0.02% capsaicin into its oral cavity daily, and was subjected to behavioural tests following 10 daily administrations of capsaicin. Stereotypy counts and rostral grooming were significantly increased, and caudal grooming decreased, in capsaicin-treated rats during the ambulatory activity test. In elevated plus maze test, not only the time spent in open arms but also the percent arm entry into open arms was reduced in capsaicin-treated rats compared with control rats. In forced swim test, although swimming duration was decreased, struggling increased in the capsaicin group, immobility duration did not differ between the groups. Repeated oral capsaicin did not affect the basal levels of plasma corticosterone; however, the stress-induced elevation of plasma corticosterone was prolonged in capsaicin treated rats. Oral capsaicin exposure significantly increased c-Fos expression not only in the nucleus tractus of solitarius but also in the paraventricular nucleus. Results suggest that repeated oral exposure to capsaicin increases anxiety-like behaviours in rats, and dysfunction of the hypothalamic-pituitary-adrenal axis may play a role in its pathophysiology.     

Evaluation of a Model for Glycemic Prediction in Critically Ill Surgical Patients

We evaluated a neural network model for prediction of glucose in critically ill trauma and post-operative cardiothoracic surgical patients. A prospective, feasibility trial evaluating a continuous glucose-monitoring device was performed. After institutional review board approval, clinical data from all consenting surgical intensive care unit patients were converted to an electronic format using novel software. This data was utilized to develop and train a neural network model for real-time prediction of serum glucose concentration implementing a prediction horizon of 75 minutes. Glycemic data from 19 patients were used to “train” the neural network model. Subsequent real-time simulated testing was performed in 5 patients to whom the neural network model was naive. Performance of the model was evaluated by calculating the mean absolute difference percent (MAD%), Clarke Error Grid Analysis, and calculation of the percent of hypoglycemic (≤70 mg/dL), normoglycemic (>70 and <150 mg/dL), and hyperglycemic (≥150 mg/dL) values accurately predicted by the model; 9,405 data points were analyzed. The models successfully predicted trends in glucose in the 5 test patients. Clark Error Grid Analysis indicated that 100.0% of predictions were clinically acceptable with 87.3% and 12.7% of predicted values falling within regions A and B of the error grid respectively. Overall model error (MAD%) was 9.0% with respect to actual continuous glucose modeling data. Our model successfully predicted 96.7% and 53.6% of the normo- and hyperglycemic values respectively. No hypoglycemic events occurred in these patients. Use of neural network models for real-time prediction of glucose in the surgical intensive care unit setting offers healthcare providers potentially useful information which could facilitate optimization of glycemic control, patient safety, and improved care. Similar models can be implemented across a wider scale of biomedical variables to offer real-time optimization, training, and adaptation that increase predictive accuracy and performance of therapies.     

Optical coherence tomography to detect acute esophageal radiation‐induced damage in mice: A validation study

Radiation therapy for patients with non‐small‐cell lung cancer is hampered by acute radiation‐induced toxicity in the esophagus. This study aims to validate that optical coherence tomography (OCT), a minimally invasive imaging technique with high resolution (~10 μm), is able to visualize and monitor acute radiation‐induced esophageal damage (ARIED) in mice. We compare our findings with histopathology as the gold standard. Irradiated mice receive a single dose of 40 Gy at proximal and distal spots of the esophagus of 10.0 mm in diameter. We scan mice using OCT at two, three, and seven days post‐irradiation. In OCT analysis, we define ARIED as a presence of distorted esophageal layering, change in backscattering signal properties, or change in the esophageal wall thickness. The average esophageal wall thickness is 0.53 mm larger on OCT when ARIED is present based on histopathology. The overall sensitivity and specificity of OCT to detect ARIED compared to histopathology are 94% and 47%, respectively. However, the overall sensitivity of OCT to assess ARIED is 100% seven days post‐irradiation. We validate the capability of OCT to detect ARIED induced by high doses in mice. Nevertheless, clinical studies are required to assess the potential role of OCT to visualize ARIED in humans.      

Effect of overexpression of a neutral sphingomyelinase on CD95-induced ceramide production and apoptosis

We previously showed that ceramide (Cer) formed during the execution phase of apoptosis is derived from plasma membrane sphingomyelin (SM), most likely by a neutral sphingomyelinase activity (Tepper et al., J. Cell Biol. 150, 2000, 155-164). In this study, we investigated the involvement of a cloned putative human neutral sphingomyelinase (nSMase1) in this process. Site-directed mutagenesis of predicted catalytic residues (Glu(49), Asn(180), and His(272)) to Ala residues abolished the catalytic activity of nSMase1. Jurkat cells were retrovirally transduced with either wildtype or inactive (with all three point mutations) Myc-tagged nSMase1. Cells overexpressing wildtype nSMase1 showed dramatically elevated in vitro nSMase activity. However, nSMase1 gene transduction (wildtype or mutant) did not alter steady-state levels of SM, Cer, or glucosylceramide. Moreover, the Cer response and apoptosis sensitivity to ligation of the CD95/Fas receptor in cells overexpressing wildtype or mutant nSMase1 were identical to vector-transduced cells. We conclude that not nSMase1 but a different, yet to be identified, nSMase accounts for the generation of Cer during the execution phase of death receptor-induced apoptosis.     

A redundant role of the CD3 gamma-immunoreceptor tyrosine-based activation motif in mature T cell function

At least four different CD3 polypeptide chains are contained within the mature TCR complex, each encompassing one (CD3gamma, CD3delta, and CD3epsilon) or three (CD3zeta) immunoreceptor tyrosine-based activation motifs (ITAMs) within their cytoplasmic domains. Why so many ITAMs are required is unresolved: it has been speculated that the different ITAMs function in signal specification, but they may also serve in signal amplification. Because the CD3zeta chains do not contribute unique signaling functions to the TCR, and because the ITAMs of the CD3-gammadeltaepsilon module alone can endow the TCR with normal signaling capacity, it thus becomes important to examine how the CD3gamma-, delta-, and epsilon-ITAMs regulate TCR signaling. We here report on the role of the CD3gamma chain and the CD3gamma-ITAM in peripheral T cell activation and differentiation to effector function. All T cell responses were reduced or abrogated in T cells derived from CD3gamma null-mutant mice, probably because of decreased expression levels of the mature TCR complex lacking CD3gamma. Consistent with this explanation, T cell responses proceed undisturbed in the absence of a functional CD3gamma-ITAM. Loss of integrity of the CD3gamma-ITAM only slightly impaired the regulation of expression of activation markers, suggesting a quantitative contribution of the CD3gamma-ITAM in this process. Nevertheless, the induction of an in vivo T cell response in influenza A virus-infected CD3gamma-ITAM-deficient mice proceeds normally. Therefore, if ITAMs can function in signal specification, it is likely that either the CD3delta and/or the CD3epsilon chains endow the TCR with qualitatively unique signaling functions.     

Genetic evidence of an essential role for cytomegalovirus small capsid protein in viral growth

Many steps in the replication cycle of cytomegalovirus (CMV), like cell entry, capsid assembly, and egress of newly synthesized virions, have not been completely analyzed yet. In order to facilitate these studies, we decided to construct a recombinant CMV that incorporates the green fluorescent protein (GFP) into the nucleocapsid. A comparable herpes simplex virus type 1 (HSV-1) mutant has recently been generated by fusion of the GFP open reading frame (ORF) with the HSV-1 ORF encoding small capsid protein (SCP) VP26 (P. Desai and S. Person, J. Virol. 72:7563-7568, 1998). Recombinant CMV genomes expressing a fusion protein consisting of GFP and the SCP were constructed by the recently established bacterial artificial chromosome mutagenesis procedure. In transfected cells, the SCP-GFP fusion protein localized to distinct foci in the nucleus that may represent sites for capsid assembly (assemblons). However, no viable progeny was reconstituted from these mutant CMV genomes. CMV genomes with deletion of the SCP ORF also did not give rise to infectious virus. Rescue of the mutation by insertion of the SCP gene at an ectopic position in an SCP knockout genome indicates that, in contrast to the HSV-1 SCP, the CMV SCP is essential for viral growth. Expression of the SCP-GFP fusion protein together with the authentic SCP blocked the CMV infection cycle, suggesting that the SCP-GFP fusion protein exerts a dominant-negative effect on the assembly of new virions. The results of this study are discussed with regard to recently published data about the structure of the CMV virion and its differences from the HSV-1 virion.     

Mechanism of action of Drosophila Reaper in mammalian cells: Reaper globally inhibits protein synthesis and induces apoptosis independent of mitochondrial permeability

Drosophila Reaper can bind inhibitor of apoptosis proteins (IAP) and thereby rescue caspases from proteasomal degradation. In insect cells, this is sufficient to induce apoptosis. Reaper can also induce apoptosis in mammalian cells, in which caspases need to be activated, usually via the mitochondrial pathway. Nevertheless, we find that Reaper efficiently induces apoptosis in mammalian cells in the absence of mitochondrial permeabilisation and cytochrome c release. Moreover, this capacity was only marginally affected by deletion of Reaper's amino-terminal IAP-binding motif. Independent of this motif, Reaper could globally suppress protein synthesis. Deletion of 20 amino acids from the carboxy-terminus of Reaper fully abrogated its potential to inhibit protein synthesis and to induce apoptosis in the absence of IAP-binding. Our findings indicate that the newly identified capacity of Reaper to suppress protein translation can operate in mammalian cells and may be key to its pro-apoptotic activity.