Snjezana Zaja-Milatovic

Cytokine Receptor CXCR4 Mediates Estrogen-Independent Tumorigenesis, Metastasis, and Resistance to Endocrine Therapy in Human Breast Cancer

Estrogen independence and progression to a metastatic phenotype are hallmarks of therapeutic resistance and mortality in breast cancer patients. Metastasis has been associated with chemokine signaling through the SDF-1-CXCR4 axis. Thus, the development of estrogen independence and endocrine therapy resistance in breast cancer patients may be driven by SDF-1-CXCR4 signaling. Here we report that CXCR4 overexpression is indeed correlated with worse prognosis and decreased patient survival irrespective of the status of the estrogen receptor (ER). Constitutive activation of CXCR4 in poorly metastatic MCF-7 cells led to enhanced tumor growth and metastases that could be reversed by CXCR4 inhibition. CXCR4 overexpression in MCF-7 cells promoted estrogen independence in vivo, whereas exogenous SDF-1 treatment negated the inhibitory effects of treatment with the anti-estrogen ICI 182,780 on CXCR4-mediated tumor growth. The effects of CXCR4 overexpression were correlated with SDF-1-mediated activation of downstream signaling via ERK1/2 and p38 MAPK (mitogen activated protein kinase) and with an enhancement of ER-mediated gene expression. Together, these results show that enhanced CXCR4 signaling is sufficient to drive ER-positive breast cancers to a metastatic and endocrine therapy-resistant phenotype via increased MAPK signaling. Our findings highlight CXCR4 signaling as a rational therapeutic target for the treatment of ER-positive, estrogen-independent breast carcinomas needing improved clinical management.

Protection of DFP-induced oxidative damage and neurodegeneration by antioxidants and NMDA receptor antagonist.

Prophylactic agents acutely administered in response to anticholinesterases intoxication can prevent toxic symptoms, including fasciculations, seizures, convulsions and death. However, anticholinesterases also have long-term unknown pathophysiological effects, making rational prophylaxis/treatment problematic. Increasing evidence suggests that in addition to excessive cholinergic stimulation, organophosphate compounds such as diisopropylphosphorofluoridate (DFP) induce activation of glutamatergic neurons, generation of reactive oxygen (ROS) and nitrogen species (RNS), leading to neurodegeneration. The present study investigated multiple affectors of DFP exposure critical to cerebral oxidative damage and whether antioxidants and NMDA receptor antagonist memantine provide neuroprotection by preventing DFP-induced biochemical and morphometric changes in rat brain. Rats treated acutely with DFP (1.25 mg/kg, s.c.) developed onset of toxicity signs within 7-15 min that progressed to maximal severity of seizures and fasciculations within 60 min. At this time point, DFP caused significant (p<0.01) increases in biomarkers of ROS (F2-isoprostanes, F2-IsoPs; and F4-neuroprostanes, F4-NeuroPs), RNS (citrulline), and declines in high-energy phosphates (HEP) in rat cerebrum. At the same time, quantitative morphometric analysis of pyramidal neurons of the hippocampal CA1 region revealed significant (p<0.01) reductions in dendritic lengths and spine density. When rats were pretreated with the antioxidants N-tert-butyl-alpha-phenylnitrone (PBN, 200 mg/kg, i.p.), or vitamin E (100 mg/kg, i.p./day for 3 days), or memantine (18 mg/kg, i.p.), significant attenuations in DFP-induced increases in F2-IsoPs, F4-NeuroPs, citrulline, and depletion of HEP were noted. Furthermore, attenuation in oxidative damage following antioxidants or memantine pretreatment was accompanied by rescue from dendritic degeneration of pyramidal neurons in the CA1 hippocampal area. These findings closely associated DFP-induced lipid peroxidation with dendritic degeneration of pyramidal neurons in the CA1 hippocampal area and point to possible interventions to limit oxidative injury and dendritic degeneration induced by anticholinesterase neurotoxicity.

Protective effects of antioxidants and anti-inflammatory agents against manganese-induced oxidative damage and neuronal injury

Exposure to excessive manganese (Mn) levels leads to neurotoxicity, referred to as manganism, which resembles Parkinsons disease (PD). Manganism is caused by neuronal injury in both cortical and subcortical regions, particularly in the basal ganglia. The basis for the selective neurotoxicity of Mn is not yet fully understood. However, several studies suggest that oxidative damage and inflammatory processes play prominent roles in the degeneration of dopamine-containing neurons. In the present study, we assessed the effects of Mn on reactive oxygen species (ROS) formation, changes in high-energy phosphates and associated neuronal dysfunctions both in vitro and in vivo. Results from our in vitro study showed a significant (p<0.01) increase in biomarkers of oxidative damage, F(2)-isoprostanes (F(2)-IsoPs), as well as the depletion of ATP in primary rat cortical neurons following exposure to Mn (500 μM) for 2h. These effects were protected when neurons were pretreated for 30 min with 100 of an antioxidant, the hydrophilic vitamin E analog, trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), or an anti-inflammatory agent, indomethacin. Results from our in vivo study confirmed a significant increase in F(2)-IsoPs levels in conjunction with the progressive spine degeneration and dendritic damage of the striatal medium spiny neurons (MSNs) of mice exposed to Mn (100mg/kg, s.c.) 24h. Additionally, pretreatment with vitamin E (100mg/kg, i.p.) or ibuprofen (140 μg/ml in the drinking water for two weeks) attenuated the Mn-induced increase in cerebral F(2)-IsoPs? and protected the MSNs from dendritic atrophy and dendritic spine loss. Our findings suggest that the mediation of oxidative stress/mitochondrial dysfunction and the control of alterations in biomarkers of oxidative injury, neuroinflammation and synaptodendritic degeneration may provide an effective, multi-pronged therapeutic strategy for protecting dysfunctional dopaminergic transmission and slowing of the progression of Mn-induced neurodegenerative processes.

RAF265 inhibits the growth of advanced human melanoma tumors.

Purpose: The purpose of this preclinical study was to determine the effectiveness of RAF265, a multikinase inhibitor, for treatment of human metastatic melanoma and to characterize traits associated with drug response. Experimental Design: Advanced metastatic melanoma tumors from 34 patients were orthotopically implanted to nude mice. Tumors that grew in mice (17 of 34) were evaluated for response to RAF265 (40 mg/kg, every day) over 30 days. The relation between patient characteristics, gene mutation profile, global gene expression profile, and RAF265 effects on tumor growth, mitogen-activated protein/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) phosphorylation, proliferation, and apoptosis markers was evaluated. Results: Nine of the 17 tumors that successfully implanted (53%) were mutant BRAF (BRAFV600E/K), whereas eight of 17 (47%) tumors were BRAF wild type (BRAFWT). Tumor implants from 7 of 17 patients (41%) responded to RAF265 treatment with more than 50% reduction in tumor growth. Five of the 7 (71%) responders were BRAFWT, of which 1 carried c-KITL576P and another N-RASQ61R mutation, while only 2 (29%) of the responding tumors were BRAFV600E/K. Gene expression microarray data from nonimplanted tumors revealed that responders exhibited enriched expression of genes involved in cell growth, proliferation, development, cell signaling, gene expression, and cancer pathways. Although response to RAF265 did not correlate with pERK1/2 reduction, RAF265 responders did exhibit reduced pMEK1, reduced proliferation based upon reduced Ki-67, cyclin D1 and polo-like kinase1 levels, and induction of the apoptosis mediator BCL2-like 11. Conclusions: Orthotopic implants of patient tumors in mice may predict prognosis and treatment response for melanoma patients. A subpopulation of human melanoma tumors responds to RAF265 and can be characterized by gene mutation and gene expression profiles. Clin Cancer Res; 18(8); 2184–98. ©2012 AACR.

Pharmacologic suppression of oxidative damage and dendritic degeneration following kainic acid-induced excitotoxicity in mouse cerebrum.

Intense seizure activity associated with status epilepticus and excitatory amino acid (EAA) imbalance initiates oxidative damage and neuronal injury in CA1 of the ventral hippocampus. We tested the hypothesis that dendritic degeneration of pyramidal neurons in the CA1 hippocampal area resulting from seizure-induced neurotoxicity is modulated by cerebral oxidative damage. Kainic acid (KA, 1 nmol/5 microl) was injected intracerebroventricularly to C57Bl/6 mice. F2-isoprostanes (F2-IsoPs) and F4-neuroprostanes (F4-NeuroPs) were used as surrogate measures of in vivo oxidative stress and biomarkers of lipid peroxidation. Nitric oxide synthase (NOS) activity was quantified by evaluating citrulline level and pyramidal neuron dendrites and spines were evaluated using rapid Golgi stains and a Neurolucida system. KA produced severe seizures in mice immediately after its administration and a significant (p<0.001) increase in F2-IsoPs, F4-NeuroPs and citrulline levels were seen 30 min following treatment. At the same time, hippocampal pyramidal neurons showed significant (p<0.001) reduction in dendritic length and spine density. In contrast, no significant change in neuronal dendrite and spine density or F2-IsoP, F4-NeuroPs and citrulline levels were found in mice pretreated with vitamin E (alpha-tocopherol, 100mg/kg, i.p.) for 3 days, or with N-tert-butyl-alpha-phenylnitrone (PBN, 200mg/kg, i.p.) or ibuprofen (inhibitors of cyclooxygenase, COX, 14 microg/ml of drinking water) for 2 weeks prior to KA treatment. These findings indicate novel interactions among free radical-induced generation of F2-IsoPs and F4-NeuroPs, nitric oxide and dendritic degeneration, closely associate oxidative damage to neuronal membranes with degeneration of the dendritic system, and point to possible interventions to limit severe damage in acute neurological disorders.

Molecular determinants of melanoma malignancy: selecting targets for improved efficacy of chemotherapy

The BRAFV600E mutation is common in human melanoma. This mutation enhances IκB kinase (IKK)/nuclear factor-κB (NF-κB) and extracellular signal-regulated kinase/activator protein signaling cascades. In this study, we evaluated the efficacy of targeting either B-Raf or IKKβ in combination with the DNA alkylating agent temozolomide for treatment of advanced metastatic melanoma. Xenografts of Hs294T human metastatic melanoma cells exhibiting the BRAFV600E mutation were treated with inhibitors of IKKβ (BMS-345541), B-Raf (BAY 54-9085), and/or temozolomide. Drug response was mechanistically analyzed in vitro and in vivo. In this study, we determined that the antitumor activity of all three drugs depends on inhibition of NF-κB. BMS-345541 inhibits IKKβ-mediated phosphorylation of IκBα and thus blocks the nuclear localization of NF-κB, whereas BAY 54-9085 inhibits activation of NF-κB through a mechanism that does not involve stabilization of IκBα. Moreover, BMS-345541, but not BAY 54-9085, activates the death pathways of p53 and c-Jun-NH2-kinase, contributing to the killing of melanoma cells. Temozolomide inhibits both NF-κB and extracellular signal-regulated kinase activity, conferring effective in vivo antitumor activity. Thus, temozolomide, but not BAY 54-9085, has a synergistic in vivo antitumor effect with BMS-345541. We conclude that the efficacy of antimelanoma therapy depends on inhibition of expression of antiapoptotic genes transcriptionally regulated by NF-κB. In contrast, drug targeting of the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway alone in melanoma cells is ineffective for melanoma therapy in cases where NF-κB is not also targeted. [Mol Cancer Ther 2009;8(3):OF636–12]

Morphometric Analysis in Neurodegenerative Disorders

The study of dendritic length and spine density has become standard in the analysis of neuronal abnormalities, since a considerable number of neurological diseases have their foundation in alterations in these structures. One of the best ways to study possible alterations in neuronal morphometry is the use of Golgi impregnation. Introduced more than a century ago, it is still the standard and state‐of‐the‐art technique for visualization of neuronal architecture. We successfully applied the Golgi method to mouse, rat, monkey, and human brain tissues for studying both the normal and abnormal morphology of neurons. We were able to discover subtle morphological alterations in neuronal dendrites and dendritic spines in different brain areas. Although Golgi preparations can be examined by electronic microscopy, we used light microscopy and reconstruction using Neurolucida software to quantitatively explore the relationship between total dendritic length and spine density in different types of neurons. This unit summarizes the methodology used to quantify neuronal abnormalities and discusses the utility of these techniques in different models of neurodegeneration. Curr. Protoc. Toxicol. 43:12.16.1‐12.16.14.

Excitotoxicity, Oxidative Stress, and Neuronal Injury

Publisher Summary This chapter explores mechanisms associated with organophosphates (OP) induced neurotoxicity by probing their effects on oxidative stress and associated dendritic degeneration of pyramidal neurons in the CA1 hippocampal area. It also investigates different pathways to attenuate biomarkers of oxidative damage associated with anticholinesterase exposure and the extent to which such attenuation is accompanied by rescue from neurodegeneration. Exposure to OP nerve agents induces seizures, rapidly progressing to status epilepticus and profound structural brain damage. The progression of events includes initial high cholinergic activity followed by activation of glutamatergic neurons as a result of release of glutamate. Moreover, glutamate stimulates the continuous release of ACh, contributing to further excitatory stimulation, prolongation of the seizures, and excitotoxic neurodegeneration in vulnerable brain areas. The ensuing neuronal damage is thought to result from intense transient influx of calcium leading to mitochondrial functional impairment, cytochrome c inactivation, depletion of ATP, simultaneous formation of free radical species, and oxidative stress. Among promising candidates as antidotes against CNS intoxication by OP nerve agents, memantine (MEM) has been shown to pose both antiexcitotoxic and antiepileptic properties. Memantine is an uncompetitive NMDA receptor antagonist, clinically used for the treatment of Alzheimers disease, Parkinsons disease, and spasticity, in the absence of serious side effects.

Peaceful Use of Disastrous Neurotoxicants

The increasing exposure to environmental neurotoxicants in the last decades caused serious health problems in the world population. Some of the neurotoxic agents are being used in agriculture and household such as insecticides and rodenticides and others are of natural origin like snake and scorpion venoms. Additional group of harmful substances is the chemical warfare agents including nerve and blistering agents that are known for their disastrous effects on neuronal tissues. The present paper presents a combination of epidemiological/clinical and molecular approaches for investigating the effect of certain groups of neurotoxicants on a variety of pathologies. The work of Finkelstein and coworkers describes epidemiological and clinical studies on acute and chronic organophosphate (OP)-induced neurotoxicity in certain populations in Israel. They mainly investigated the neurotoxic effects of low-level long-term exposure to OP in agricultural areas but also dealt with acute exposures as well. A molecular approach to OP mechanism of neuronal injury was described by Milatovic and coworkers. They demonstrated OP-induced oxidative injury in pyramidal neurons in the CA1 hippocampal area and its suppression by antioxidants. Lecht and coworkers described the novel snake venom angioneurins as important mediators of the physiological cross-talk between the cardiovascular and nervous systems. They also showed that under certain conditions these angioneurins may induce pathologies such as tumor development or disruption of the vascular barrier function during envenomation. Additional mechanistic/therapeutic approach was presented by Brodsky, Rosengarten, Proscura, Shapira and Wormser. They developed a novel anti-inflammatory peptide that reduced skin irritation induced by heat and sulfur mustard (SM) stimuli. Since SM causes neuropsychiatric symptoms and alterations in neurological functions this peptide may serve as a potential treatment of neuronal injuries caused by environmental neurotoxicants. These reviews highlight different aspects of neurotoxicity, addressing epidemiology and mechanisms of toxicity; and identifying novel potential therapies.

Biomarkers of oxidative/nitrosative stress and neurotoxicity

Oxidative stress is implicated as one of the major underlying mechanisms in a variety of human diseases. Excess formation of free radicals may overwhelm the capacity of endogenous cellular antioxidant defense mechanisms, cause alterations in normal cell and organ physiology, and activate and/or accelerate disease processes. Reactive radicals derived from molecular oxygen (reactive oxygen species, ROS) and nitrogen (reactive nitrogen species, RNS) readily attack a variety of critical biological molecules, including lipids, DNA, and essential cellular proteins. High content of unsaturated lipids in the brain leads to pronounced lipid peroxidation, a central feature of oxidant injury in the brain. Oxidative stress is also associated with cell response to a variety of toxicants. This chapter analyzes oxidative stress and the cell response to diverse toxicants, and presents evidence of ROS/RNS generation and neurodegeneration in different toxicity models.