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.     

Semaphorins as Mediators of Neuronal Apoptosis

Shrinkage and collapse of the neuritic network are often observed during the process of neuronal apoptosis. However, the molecular and biochemical basis for the axonal damage associated with neuronal cell death is still unclear. We present evidence for the involvement of axon guidance molecules with repulsive cues in neuronal cell death. Using the differential display approach, an up-regulation of collapsin response mediator protein was detected in sympathetic neurons undergoing dopamine-induced apoptosis. A synchronized induction of mRNA of the secreted collapsin-1 and the intracellular collapsin response mediator protein that preceded commitment of neurons to apoptosis was detected. Antibodies directed against a conserved collapsin-derived peptide provided marked and prolonged protection of several neuronal cell types from dopamine-induced apoptosis. Moreover, neuronal apoptosis was inhibited by antibodies against neuropilin-1, a putative component of the semaphorin III/collapsin-1 receptor. Induction of neuronal apoptosis was also caused by exposure of neurons to semaphorin III-alkaline phosphatase secreted from 293EBNA cells. Anti-collapsin-1 antibodies were effective in blocking the semaphorin III-induced death process. We therefore suggest that, before their death, apoptosis-destined neurons may produce and secrete destructive axon guidance molecules that can affect their neighboring cells and thus transfer a "death signal" across specific and susceptible neuronal populations.     

Splitting the apoptosome

Assembly of the apoptosome in response to mitochondrial permeabilization, the hallmark of the intrinsic apoptotic pathway, involves binding of cytochrome c to Apaf1, recruitment and auto-processing of the apical/signaling pro-caspase-9, and coupled activation of downstream/executioner caspases like caspase 3. Evidence now indicates that certain apoptotic cascades can bypass the apoptosome and activate caspase-9 independent of the mitochondria. Recently, we have demonstrated that caspase-9 can be activated in Apaf1-mutant primary myoblasts, but not fibroblasts, in response to stimuli that are known to act via the mitochondria. Thus, apoptosomal activation of caspase-9 seems to represent only one of the routes for its activation; other pathways, some of which are yet to be discovered, can bypass the requirement for Apaf1 and activate caspase-9 in a tissue and context specific manner.     

Anti-semaphorin 3A antibodies rescue retinal ganglion cells from cell death following optic nerve axotomy

Damage to the optic nerve in mammals induces retrograde degeneration and apoptosis of the retinal ganglion cell (RGC) bodies. The mechanisms that mediate the response of the neuronal cells to the axonal injury are still unknown. We have previously shown that semaphorins, axon guidance molecules with repulsive cues, are capable of mediating apoptosis in cultured neuronal cells (Shirvan, A., Ziv, I., Fleminger, G., Shina, R., He, Z., Brudo, I., Melamed, E., and Brazilai, A. (1999) J. Neurochem. 73, 961-971). In this study, we examined the involvement of semaphorins in an in vivo experimental animal model of complete axotomy of the rat optic nerve. We demonstrate that a marked induction of type III semaphorin proteins takes place in ipsilateral retinas at early stages following axotomy, well before any morphological signs of RGC apoptosis can be detected. Time course analysis revealed that a peak of expression occurred after 2-3 days and then declined. A small conserved peptide derived from semaphorin 3A that was previously shown to induce neuronal death in culture was capable of inducing RGC loss upon its intravitreous injection into the rat eye. Moreover, we demonstrate a marked inhibition of RGC loss when axotomized eyes were co-treated by intravitreous injection of function-blocking antibodies against the semaphorin 3A-derived peptide. Marked neuronal protection from degeneration was also observed when the antibodies were applied 24 h post-injury. We therefore suggest that semaphorins are key proteins that modulate the cell fate of axotomized RGC. Neutralization of the semaphorin repulsive function may serve as a promising new approach for treatment of traumatic injury in the adult mammalian central nervous system or of ophthalmologic diseases such as glaucoma and ischemic optic neuropathy that induce apoptotic RGC death.     

Molecular mechanisms of selective dopaminergic neuronal death in Parkinson's disease

Parkinson's disease (PD) is a progressive neurological disease caused by selective degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). Although PD has been heavily researched, the precise etiology of nigral cell loss is still unknown and, consequently, treatment is largely symptomatic rather than preventive. There are conflicting data regarding the mode of dopaminergic cell death in PD and, hence, this remains controversial. Several mutations in specific genes have recently been linked with hereditary forms of PD. Although none of these mutations are seen in idiopathic disease cases, the elucidation of these genetic defects sheds light on the nature of idiopathic PD. It is possible that dopaminergic neurogenesis also contributes to the etiology of idiopathic PD. In addition, intracellular as well as extracellular substances found in the SNc are believed to function as damaging pathogenetic factors. These factors, and the interactions among them, might hold the secret to the underlying causes of the selective death of dopaminergic neurons in PD.     

The Israel National Skin Bank: Quality Assurance and Graft Performance of Stored Tissues

The Israel National Skin Bank (INSB) was founded jointly by the Israel Defense Forces (IDF) Medical Corps and the Ministry of Health in 1986. The prime purpose of the Skin Bank is to treat burn victims incurred at war or during mass casualty incidences. The INSB Protocol is comprised of international skin bank protocols and our previous and present research results. They provide the framework for selecting optimal guidelines for procurement, processing, preservation, storage and evaluation of transplantation performance of viable skin grafts. For evaluation and direct comparison of graft performance of glycerolized or cryopreserved skin stored for long periods, we have applied a mouse recipient model developed by us. This model assesses graft performance before the rejection process takes place. The in vivo design has inherent clinical relevance, which is especially appealing. Cryopreserved skin performed better than glycerolized skin (p > 0.027), but fresh skin performed significantly better than cryopreserved skin (p > 0.003), as analyzed by the Mann–Whitney non-parametric test. Then graft performance of skin specimens were cryopreserved by programmed or stepwise freezing and stored at -80°C or in liquid nitrogen for 1 and 6–10 months was evaluated. The average score of skin preserved by programmed freezing and stored in liquid nitrogen is the highest for both storage periods. This method has a highly significant advantage (p < 0.007) over the others for 6–10 months storage, evaluated by graft adherence. Several interaction factors determine the quality of cryopreserved skin. Highly significant is the interaction factor/'combined effect' of sample variability with the method of cryopreservation or with the storage period. Finally, the results of paired comparison of selected histology criteria of cryopreserved to fresh skin indicated that storage of skin for up to 5 years did not impair significantly its performance compared to fresh skin, whereas, after six years of storage, there was a highly significant (p < 0.001) impairment in skin quality. We offer a simplified in vivo model and analysis for cryopreserved skin graft performance, suggesting that the evaluation procedures, which are issues of great interest in skin banking, may help future skin banks to make informed choices and decisions regarding quality issues.     

Direct Transfer of Viral and Cellular Proteins from Varicella-Zoster Virus-Infected Non-Neuronal Cells to Human Axons

Varicella Zoster Virus (VZV), the alphaherpesvirus that causes varicella upon primary infection and Herpes zoster (shingles) following reactivation in latently infected neurons, is known to be fusogenic. It forms polynuclear syncytia in culture, in varicella skin lesions and in infected fetal human ganglia xenografted to mice. After axonal infection using VZV expressing green fluorescent protein (GFP) in compartmentalized microfluidic cultures there is diffuse filling of axons with GFP as well as punctate fluorescence corresponding to capsids. Use of viruses with fluorescent fusions to VZV proteins reveals that both proteins encoded by VZV genes and those of the infecting cell are transferred in bulk from infecting non-neuronal cells to axons. Similar transfer of protein to axons was observed following cell associated HSV1 infection. Fluorescence recovery after photobleaching (FRAP) experiments provide evidence that this transfer is by diffusion of proteins from the infecting cells into axons. Time-lapse movies and immunocytochemical experiments in co-cultures demonstrate that non-neuronal cells fuse with neuronal somata and proteins from both cell types are present in the syncytia formed. The fusogenic nature of VZV therefore may enable not only conventional entry of virions and capsids into axonal endings in the skin by classical entry mechanisms, but also by cytoplasmic fusion that permits viral protein transfer to neurons in bulk.     

A DJ-1 Based Peptide Attenuates Dopaminergic Degeneration in Mice Models of Parkinson's Disease via Enhancing Nrf2

Drugs currently used for treating Parkinson''s disease patients provide symptomatic relief without altering the neurodegenerative process. Our aim was to examine the possibility of using DJ-1 (PARK7), as a novel therapeutic target for Parkinson''s disease. We designed a short peptide, named ND-13. This peptide consists of a 13 amino acids segment of the DJ-1-protein attached to 7 amino acids derived from TAT, a cell penetrating protein. We examined the effects of ND-13 using in vitro and in vivo experimental models of Parkinson''s disease. We demonstrated that ND-13 protects cultured cells against oxidative and neurotoxic insults, reduced reactive oxygen species accumulation, activated the protective erythroid-2 related factor 2 system and increased cell survival. ND-13 robustly attenuated dopaminergic system dysfunction and in improved the behavioral outcome in the 6-hydroxydopamine mouse model of Parkinson''s disease, both in wild type and in DJ-1 knockout mice. Moreover, ND-13 restored dopamine content in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model. These findings validate DJ-1 as a promising therapeutic target in Parkinson''s disease and identify a novel peptide with clinical potential, which may be significant for a broader range of neurological diseases, possibly with an important impact for the neurosciences.