Neuroprotective effect of the RNS60 in a mouse model of transient focal cerebral ischemia.

BACKGROUND: Stroke is a major cause of death, disability, and public health problems. Its intervention is limited to early treatment with thrombolytics and/or endovascular clot removal with mechanical thrombectomy without any available subacute or chronic neuroprotective treatments. RNS60 has reduced neuroinflammation and increased neuronal survival in several animal models of neurodegeneration and trauma. The aim here was to evaluate whether RNS60 protects the brain and cognitive function in a mouse stroke model. METHODS: Male C57BL/6J mice were subjected to sham or ischemic stroke surgery using 60-minute transient middle cerebral artery occlusion (tMCAo). In each group, mice received blinded daily administrations of RNS60 or control fluids (PNS60 or normal saline [NS]), beginning 2 hours after surgery over 13 days. Multiple neurobehavioral tests were conducted (Neurological Severity Score [mNSS], Novel Object Recognition [NOR], Active Place Avoidance [APA], and the Conflict Variant of APA [APAc]). On day 14, cortical microvascular perfusion (MVP) was measured, then brains were removed and infarct volume, immunofluorescence of amyloid beta (Aß), neuronal density, microglial activation, and white matter damage/myelination were measured. SPSS was used for analysis (e.g., ANOVA for parametric data; Kruskal Wallis for non-parametric data; with post-hoc analysis). RESULTS: Thirteen days of treatment with RNS60 reduced brain infarction, amyloid pathology, neuronal death, microglial activation, white matter damage, and increased MVP. RNS60 reduced brain pathology and resulted in behavioral improvements in stroke compared to sham surgery mice (increased memory-learning in NOR and APA, improved cognitive flexibility in APAc). CONCLUSION: RNS60-treated mice exhibit significant protection of brain tissue and improved neurobehavioral functioning after tMCAo-stroke. Additional work is required to determine mechanisms, time-window of dosing, and multiple dosing volumes durations to support clinical stroke research.

A physically-modified saline suppresses neuronal apoptosis, attenuates tau phosphorylation and protects memory in an animal model of Alzheimer's disease.

Alzheimer's disease (AD), the leading cause of dementia in the aging population, is characterized by the presence of neuritic plaques, neurofibrillary tangles and extensive neuronal apoptosis. Neuritic plaques are mainly composed of aggregates of amyloid-ß (Aß) protein while neurofibrillary tangles are composed of the hyperphosphorylated tau protein. Despite intense investigations, no effective therapy is currently available to halt the progression of this disease. Here, we have undertaken a novel approach to attenuate apoptosis and tau phosphorylation in cultured neuronal cells and in a transgenic animal model of AD. RNS60 is a 0.9% saline solution containing oxygenated nanobubbles that is generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. In our experiments, fibrillar Aß1-42, but not the reverse peptide Aß42-1, induced apoptosis and cell death in human SHSY5Y neuronal cells. RNS60, but not NS (normal saline), RNS10.3 (TCP-modified saline without excess oxygen) or PNS60 (saline containing excess oxygen without TCP modification), attenuated Aß(1-42)-induced cell death. RNS60 inhibited neuronal cell death via activation of the type 1A phosphatidylinositol-3 (PI-3) kinase-Akt-BAD pathway. Furthermore, RNS60 also decreased Aß(1-42)-induced tau phosphorylation via (PI-3 kinase-Akt)-mediated inhibition of GSK-3ß. Similarly, RNS60 treatment suppressed neuronal apoptosis, attenuated Tau phosphorylation, inhibited glial activation, and reduced the burden of Aß in the hippocampus and protected memory and learning in 5XFAD transgenic mouse model of AD. Therefore, RNS60 may be a promising pharmaceutical candidate in halting or delaying the progression of AD.

Enhancement of morphological plasticity in hippocampal neurons by a physically modified saline via phosphatidylinositol-3 kinase.

Increase of the density of dendritic spines and enhancement of synaptic transmission through ionotropic glutamate receptors are important events, leading to synaptic plasticity and eventually hippocampus-dependent spatial learning and memory formation. Here we have undertaken an innovative approach to upregulate hippocampal plasticity. RNS60 is a 0.9% saline solution containing charge-stabilized nanobubbles that are generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. RNS60, but not NS (normal saline), PNS60 (saline containing a comparable level of oxygen without the TCP modification), or RNS10.3 (TCP-modified normal saline without excess oxygen), stimulated morphological plasticity and synaptic transmission via NMDA- and AMPA-sensitive calcium influx in cultured mouse hippocampal neurons. Using mRNA-based targeted gene array, real-time PCR, immunoblot, and immunofluorescence analyses, we further demonstrate that RNS60 stimulated the expression of many plasticity-associated genes in cultured hippocampal neurons. Activation of type IA, but not type IB, phosphatidylinositol-3 (PI-3) kinase by RNS60 together with abrogation of RNS60-mediated upregulation of plasticity-related proteins (NR2A and GluR1) and increase in spine density, neuronal size, and calcium influx by LY294002, a specific inhibitor of PI-3 kinase, suggest that RNS60 upregulates hippocampal plasticity via activation of PI-3 kinase. Finally, in the 5XFAD transgenic model of Alzheimer's disease (AD), RNS60 treatment upregulated expression of plasticity-related proteins PSD95 and NR2A and increased AMPA- and NMDA-dependent hippocampal calcium influx. These results describe a novel property of RNS60 in stimulating hippocampal plasticity, which may help AD and other dementias.

Suppression of nuclear factor-κB activation and inflammation in microglia by physically modified saline.

Chronic inflammation involving activated microglia and astroglia is becoming a hallmark of many human diseases, including neurodegenerative disorders. Although NF-κB is a multifunctional transcription factor, it is an important target for controlling inflammation as the transcription of many proinflammatory molecules depends on the activation of NF-κB. Here, we have undertaken a novel approach to attenuate NF-κB activation and associated inflammation in activated glial cells. RNS60 is a 0.9% saline solution containing charge-stabilized nanostructures that are generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. RNS60, but not normal saline, RNS10.3 (TCP-modified saline without excess oxygen), and PNS60 (saline containing excess oxygen without TCP modification) were found to inhibit the production of nitric oxide (NO) and the expression of inducible NO synthase in activated microglia. Similarly, RNS60 also inhibited the expression of inducible NO synthase in activated astroglia. Inhibition of NF-κB activation by RNS60 suggests that RNS60 exerts its anti-inflammatory effect through the inhibition of NF-κB. Interestingly, RNS60 induced the activation of type IA phosphatidylinositol (PI) 3-kinase and Akt and rapidly up-regulated IκBα, a specific endogenous inhibitor of NF-κB. Inhibition of PI 3-kinase and Akt by either chemical inhibitors or dominant-negative mutants abrogated the RNS60-mediated up-regulation of IκBα. Furthermore, we demonstrate that RNS60 induced the activation of cAMP-response element-binding protein (CREB) via the PI 3-kinase-Akt pathway and that RNS60 up-regulated IκBα via CREB. These results describe a novel anti-inflammatory property of RNS60 via type IA PI 3-kinase-Akt-CREB-mediated up-regulation of IκBα, which may be of therapeutic benefit in neurodegenerative disorders.

Protection of Tregs, suppression of Th1 and Th17 cells, and amelioration of experimental allergic encephalomyelitis by a physically-modified saline.

In multiple sclerosis (MS) and other autoimmune diseases, the autoreactive T cells overcome the resistance provided by the regulatory T cells (Tregs) due to a decrease in the number of Foxp3-expressing Tregs. Therefore, upregulation and/or maintenance of Tregs during an autoimmune insult may have therapeutic efficacy in autoimmune diseases. Although several immunomodulatory drugs and molecules are available, most present significant side effects over long-term use. Here we have undertaken an innovative approach to upregulate Tregs and achieve immunomodulation. RNS60 is a 0.9% saline solution generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. RNS60, but not NS (normal saline), RNS10.3 (TCP-modified saline without excess oxygen) and PNS60 (saline containing excess oxygen without TCP modification), was found to upregulate Foxp3 and enrich Tregs in MBP-primed T cells. Moreover, RNS60, but not NS, RNS10.3 and PNS60, inhibited the production of nitric oxide (NO) and the expression of iNOS in MBP-primed splenocytes. Incubation of the cells with an NO donor abrogated the RNS60-mediated upregulation of Foxp3. These results suggest that RNS60 boosts Tregs via suppression of NO production. Consistent to the suppressive activity of Tregs towards autoreactive T cells, RNS60, but not NS, RNS10.3, or PNS60, suppressed the differentiation of Th17 and Th1 cells and shifted the balance towards a Th2 response. Finally, RNS60 treatment exhibited immunomodulation and ameliorated adoptive transfer of experimental allergic encephalomyelitis, an animal model of MS, via Tregs. These results describe a novel immunomodulatory property of RNS60 and suggest its exploration for therapeutic intervention in MS and other autoimmune disorders.

Urinary-type plasminogen activator receptor/alpha 3 beta 1 integrin signaling, altered gene expression, and oral tumor progression.

Oral squamous cell carcinoma (OSCC) has 50% 5-year survival rate, highlighting our limited understanding of the molecular events that contribute to disease progression. Microarray analyses of primary oral tumors have identified urinary-type plasminogen activator (uPA) and its receptor (uPAR) as key genes associated with human OSCC progression. The uPAR functions as both a proteinase receptor and an integrin ligand, modifying proteolysis, migration, integrin signaling, and cellular transcription. In the current study, uPAR expression levels were modified in OSCC cells followed by analysis of tumor growth in an in vivo orthotopic xenograft model and by transcriptional profiling. Overexpression of uPAR resulted in more infiltrative and less differentiated tumors, with ill-defined borders, cytologic atypia, and enhanced vascularity. Analysis of serial sections of both murine experimental tumors and microarrayed human OSCC showed a statistically significant association between uPAR and alpha(3) integrin colocalization in areas exhibiting extracellular signal-regulated kinase phosphorylation, suggesting that uPAR/alpha(3) integrin interaction potentiates extracellular signal-regulated kinase signaling in vivo. This is supported by cDNA microarray analysis, which showed differential expression of 148 genes (113 upregulated and 35 downregulated). Validation of gene expression changes in human OSCC using immunohistochemistry and quantitative real-time PCR showed increased growth factors, proteinases/inhibitors, and matrix components in uPAR-overexpressing tumors. Together, these results support a model wherein increased uPAR expression promotes alpha(3)beta(1) integrin association, resulting in increased mitogen-activated protein kinase signaling and transcriptional activation, leading to the formation of more aggressive tongue tumors. This combined approach has efficacy to identify additional biomarkers and/or prognostic indicators associated with aggressive human OSCC.

Multiple kallikrein (KLK 5, 7, 8, and 10) expression in squamous cell carcinoma of the oral cavity.

Oral squamous cell carcinoma (OSCC) represents 3% of all cancer deaths in the U.S. and is ranked one of the top 10 cancers worldwide. The 5-year survival rate has remained at a low 50% for the past several decades, necessitating discovery of novel biomarkers of aggressive disease and therapeutic targets. As overexpression of urinary type plasminogen activator and receptor (uPA/R) in OSCC is associated with malignant progression and poor outcome, cell lines were generated with either overexpression (SCC25-uPAR+) or silencing (SCC25-uPAR-KD) of uPAR. As SCC25-uPAR+ tumors behaved more aggressively both in vitro and in vivo, comparative cDNA microarray analysis was used to identify additional genes that may be associated with aggressive tumors. Four members of the human tissue kallikrein family (KLK 5, 7, 8, and 10) were identified and real-time RT-PCR (qPCR) was used to verify and quantify gene expression. qPCR analysis revealed 2.8-, 5.3-, 4.0-, and 3.5-fold increases in gene expression for KLK5, 7, 8, and 10, respectively, in SCC25-uPAR+ versus SCC25-uPAR-KD. Immunohistochemical analysis demonstrated strong reactivity for KLKs 5, 7, 8 and 10 in both orthotopic murine tumors and human OSCC tissues. Control experiments show lack of reactivity against KLK3 (prostate specific antigen). These results demonstrate that kallikreins 5, 7, 8, and 10 are abundantly expressed in human OSCC and may be implicated in malignant progression.

Polypyrimidine tract-binding protein (PTB) differentially affects malignancy in a cell line-dependent manner.

RNA processing is altered during malignant transformation, and expression of the polypyrimidine tract-binding protein (PTB) is often increased in cancer cells. Although some data support that PTB promotes cancer, the functional contribution of PTB to the malignant phenotype remains to be clarified. Here we report that although PTB levels are generally increased in cancer cell lines from multiple origins and in endometrial adenocarcinoma tumors, there appears to be no correlation between PTB levels and disease severity or metastatic capacity. The three isoforms of PTB increase heterogeneously among different tumor cells. PTB knockdown in transformed cells by small interfering RNA decreases cellular growth in monolayer culture and to a greater extent in semi-solid media without inducing apoptosis. Down-regulation of PTB expression in a normal cell line reduces proliferation even more significantly. Reduction of PTB inhibits the invasive behavior of two cancer cell lines in Matrigel invasion assays but enhances the invasive behavior of another. At the molecular level, PTB in various cell lines differentially affects the alternative splicing pattern of the same substrates, such as caspase 2. Furthermore, overexpression of PTB does not enhance proliferation, anchorage-independent growth, or invasion in immortalized or normal cells. These data demonstrate that PTB is not oncogenic and can either promote or antagonize a malignant trait dependent upon the specific intra-cellular environment.

Synthesis and anticancer activities of 6-amino amonafide derivatives.

Amonafide is a DNA intercalator and topoisomerase II inhibitor in clinical development for the treatment of neoplastic diseases. Amonafide contains a free arylamine, which causes it to be metabolized in humans by N-acetyl transferase-2 (NAT2) into a toxic form. To eliminate the NAT2 acetylation of amonafide while retaining the anticancer properties, we have synthesized nine derivatives that are structurally similar to amonafide that should not be acetylated. Eight derivatives have arylamines at the 6-position (vs. 5-position of amonafide) and one derivative completely lacks the arylamine. The derivative with a free amine in the 6-position and one with a substituted amine in the 6-position are not acetylated, whereas amonafide is extensively acetylated as determined by an NAT2 assay. The biological activities of these compounds were evaluated to determine whether they behaved similarly to amonafide in purified systems and in vitro. We found that three compounds had similar cancer cell-selective growth inhibition to amonafide, while retaining similar subcellular localization, DNA intercalation and topoisomerase II inhibition activities. In addition, these compounds were able to eliminate a marker of metastatic potential, the perinucleolar compartment. These three compounds (named numonafides) might thus allow for better patient management than those treated with amonafide; hence, they should be developed further as potential clinical replacements for amonafide or as novel anticancer drugs.

Engagement of collagen-binding integrins promotes matrix metalloproteinase-9-dependent E-cadherin ectodomain shedding in ovarian carcinoma cells.

Reversible modulation of cell-cell adhesion, cell-matrix adhesion, and proteolytic activity plays a critical role in remodeling of the neoplastic ovarian epithelium during metastasis, implicating cadherins, integrins, and proteinases in i.p. metastatic dissemination of epithelial ovarian carcinoma (EOC). Aberrant epithelial differentiation is an early event in ovarian carcinogenesis; thus, in contrast to most carcinomas that lose E-cadherin expression with progression, E-cadherin is abundant in primary EOC. Metastasizing EOCs engage in integrin-mediated adhesion to submesothelial interstitial collagens and express matrix metalloproteinases (MMP) that facilitate collagen invasion, thereby anchoring secondary lesions in the submesothelial matrix. As metalloproteinases have also been implicated in E-cadherin ectodomain shedding, the current study was undertaken to model the effects of matrix-induced integrin clustering on proteinase-catalyzed E-cadherin ectodomain shedding. Aggregation of collagen-binding integrins induced shedding of an 80-kDa E-cadherin ectodomain [soluble E-cadherin (sE-cad)] in a MMP- and Src kinase-dependent manner, and sE-cad was prevalent in ascites from ovarian cancer patients. Expression of MMP-9 was elevated by integrin aggregation, integrin-mediated ectodomain shedding was inhibited by a MMP-9 function blocking antibody, and incubation of cells with exogenous MMP-9 catalyzed E-cadherin ectodomain shedding. In contrast to other tumors wherein sE-cad is released into the circulation, EOC tumors maintain direct contact with sE-cad-rich ascites at high concentration, and incubation of EOC cells with physiologically relevant concentrations of recombinant sE-cad disrupted adherens junctions. These data support a novel mechanism for posttranslational modification of E-cadherin function via MMP-9 induction initiated by cell-matrix contact and suggest a mechanism for promotion of EOC metastatic dissemination.

Functional relevance of urinary-type plasminogen activator receptor-alpha3beta1 integrin association in proteinase regulatory pathways.

Squamous cell carcinoma of the oral cavity is characterized by persistent, disorganized expression of integrin alpha3beta1 and enhanced production of urinary-type plasminogen activator (uPA) and its receptor (uPAR) relative to normal oral mucosa. Because multivalent aggregation of alpha3beta1 integrin up-regulates uPA and induces a dramatic co-clustering of uPAR, we explored the hypothesis that lateral ligation of alpha3beta1 integrin by uPAR contributes to uPA regulation in oral mucosal cells. To investigate mechanisms by which uPAR/alpha3beta1 binding enhances uPA expression, integrin-dependent signal activation was assessed. Both Src and ERK1/2 were phosphorylated in response to integrin aggregation, and blocking Src kinase activity completely abrogated ERK1/2 activation and uPA induction, whereas inhibition of epidermal growth factor receptor tyrosine kinase activity did not alter uPA expression. Proteinase up-regulation occurred at the transcriptional level and mutation of the AP1 (-1967) site in the uPA promoter blocked the uPAR/integrin-mediated transcriptional activation. Because uPAR is redistributed to clustered alpha3beta1 integrins, the requirement for uPAR/alpha3beta1 interaction in uPA regulation was assessed. Clustering of alpha3beta1 in the presence of a peptide (alpha325) that disrupts uPAR/alpha3beta1 integrin binding prevented uPA induction. Depletion of cell surface uPAR using small interfering RNA also blocked uPA induction following integrin alpha3beta1 clustering. These results were confirmed using a genetic strategy in which alpha3 null epithelial cells reconstituted with wild type alpha3 integrin, but not a mutant alpha3 unable to bind uPAR, induced uPA expression upon integrin clustering, confirming the critical role of uPAR in integrin-regulated proteinase expression. Disruption of uPAR/alpha3beta1 binding using peptide alpha325 or small interfering RNA blocked filopodia formation and matrix invasion, indicating that this interaction stimulates invasive behavior. Together these data support a model wherein matrix-induced clustering ofalpha3beta1 integrin promotes uPAR/alpha3beta1 interaction, thereby potentiating cellular signal transduction pathways culminating in activation of uPA expression and enhanced uPA-dependent invasive behavior.

Loss of adhesion-regulated proteinase production is correlated with invasive activity in oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity. However, the cellular and biochemical factors that underlie locoregional and distant spread of the disease are poorly understood. Invasion of OSCC requires multiple cellular events including dissolution of cell-cell junctions, basement membrane attachment, extracellular matrix proteolysis, and migration. METHODS: We evaluated these properties in vitro using premalignant gingival keratinocytes (ppl26) and two OSCC lines (SCC15 and SCC68). Expression of adhesion molecules integrins and cadherins, cytoplasmic intermediate filaments (IF) vimentin and keratin as well as matrix degrading proteins were evaluated. Moreover, regulation of protease production by adhesion molecules was tested. RESULTS: All cell lines contained comparable levels of the epithelial cell-cell adhesion molecule, E-cadherin. Differential expression of cytoplasmic IF was evident between premalignant pp126 cells and OSCC cell lines. Expression levels of the alpha3beta1 integrin, utilized for attachment to laminin-5 and other matrix proteins, was high in SCC68 cells, moderate in SCC15 cells, and low in ppl26 cells. alpha3beta1 integrin clustering up-regulates expression of urinary-type plasminogen activator (uPA) in ppl26 cells via a mechanism involving ERK activation. Both ppl26 and SCC15 cells were responsive to alpha3beta1 clustering, resulting in enhanced uPA expression. However, basal uPA levels were high in SCC68 cells and integrin clustering did not further stimulate uPA production. ERK was constitutively activated in SCC68 cells and treatment of cells with an inhibitor of ERK activation (PD98059) reduced uPA expression. Consistent with the enhanced proteolytic potential, SCC68 cells readily penetrated Matrigel and invasion was blocked by an anticatalytic uPA antibody. CONCLUSIONS: These data suggest that loss of adhesion-regulated proteinase production may lead to elevated pericellular proteinase activity and coincident alterations in cytoskeletal IF protein expression, thereby contributing to the invasive potential of OSCC.

Proteinase suppression by E-cadherin-mediated cell-cell attachment in premalignant oral keratinocytes.

The expression and activity of epithelial proteinases is under stringent control to prevent aberrant hydrolysis of structural proteins and disruption of tissue architecture. E-cadherin-dependent cell-cell adhesion is also important for maintenance of epithelial structural integrity, and loss of E-cadherin expression has been correlated with enhanced invasive potential in multiple tumor models. To address the hypothesis that there is a functional link between E-cadherin and proteinase expression, we have examined the role of E-cadherin in proteinase regulation. By using a calcium switch protocol to manipulate junction assembly, our data demonstrate that initiation of de novo E-cadherin-mediated adhesive contacts suppresses expression of both relative matrix metalloproteinase-9 levels and net urinary-type plasminogen activator activity. E-cadherin-mediated cell-cell adhesion increases both phosphatidylinositol 3'-kinase (PI3-kinase)-dependent AKT phosphorylation and epidermal growth factor receptor-dependent MAPK/ERK activation. Pharmacologic inhibition of the PI3-kinase pathway, but not the epidermal growth factor receptor/MAPK pathway, prevents E-cadherin-mediated suppression of proteinases and delays junction assembly. Moreover, inhibition of junction assembly with a function-blocking anti-E-cadherin antibody stimulates proteinase-dependent Matrigel invasion. As matrix metalloproteinase-9 and urinary-type plasminogen activator potentiate the invasive activity of oral squamous cell carcinoma, these data suggest E-cadherin-mediated signaling through PI3-kinase can regulate the invasive behavior of cells by modulating proteinase secretion.