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.

Upregulation of DJ-1 in Dopaminergic Neurons by a Physically-Modified Saline: Implications for Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder in human and loss-of-functions DJ-1 mutations are associated with a familial form of early onset PD. Functionally, DJ-1 (PARK7), a neuroprotective protein, is known to support mitochondria and protect cells from oxidative stress. Mechanisms and agents by which the level of DJ-1 could be increased in the CNS are poorly described. RNS60 is a bioactive aqueous solution created by exposing normal saline to Taylor-Couette-Poiseuille flow under high oxygen pressure. Recently we have described neuroprotective, immunomodulatory and promyelinogenic properties of RNS60. Here we delineate that RNS60 is also capable of increasing the level of DJ-1 in mouse MN9D neuronal cells and primary dopaminergic neurons, highlighting another new neuroprotective effect of RNS60. While investigating the mechanism we found the presence of cAMP response element (CRE) in DJ-1 gene promoter and stimulation of CREB activation in neuronal cells by RNS60. Accordingly, RNS60 treatment increased the recruitment of CREB to the DJ-1 gene promoter in neuronal cells. Interestingly, RNS60 treatment also induced the enrollment of CREB-binding protein (CBP), but not the other histone acetyl transferase p300, to the promoter of DJ-1 gene. Moreover, knockdown of CREB by siRNA led to the inhibition of RNS60-mediated DJ-1 upregulation, indicating an important role of CREB in DJ-1 upregulation by RNS60. Together, these results indicate that RNS60 upregulates DJ-1 in neuronal cells via CREB-CBP pathway. It may be of benefit for PD and other neurodegenerative disorders.

RNS60, a physically-modified saline, inhibits glial activation, suppresses neuronal apoptosis and protects memory in a mouse model of traumatic brain injury.

Traumatic brain injury (TBI) is a serious health issue that causes long-term neurological disability, particularly in young adults, athletes and war veterans. Despite the use of different medications or surgical procedures, no effective therapy is currently available to halt its pathogenesis. Here, we have undertaken a novel approach to reduce neuroinflammation and improve cognitive, social and locomotor behaviors in a mouse model of TBI. RNS60 is a physiologic saline solution containing oxygen nanobubbles that is generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. Recently we have delineated that RNS60 inhibits the expression of proinflammatory molecules in glial cells via type 1A phosphatidylinositol-3 kinase (PI3K)-mediated upregulation of IκBα. In this study, we found that TBI decreased the level of IκBα and increased the activation of NF-κB in hippocampus and cortex as monitored by the upregulation of p-p65. However, intraperitoneal administration of RNS60 increased and/or restored the level of IκBα and inhibited the activation of NF-κB in hippocampus and cortex of TBI mice. Accordingly, RNS60 treatment decreased the activation of astrocytes and microglia and reduced neuronal apoptosis in the brain of TBI mice. RNS60 treatment also reduced vascular damage, attenuated blood-brain barrier leakage and decreased the size of lesion in the brain of TBI mice. Importantly, RNS60 treated mice showed significant improvements in memory, social behavior and locomotor activities while displaying reduction in depression-like behaviors. These results delineate a novel neuroprotective property of RNS60 and suggest its possible therapeutic use in TBI.

Correction to: Nebulization of RNS60, a Physically-Modified Saline, Attenuates the Adoptive Transfer of Experimental Allergic Encephalomyelitis in Mice: Implications for Multiple Sclerosis Therapy.

The original version of this article unfortunately contained a mistake. The Figure 3, 4, 5 legends have been misplaced. The updated legends along with the figures are corrected with this erratum.

RNS60 exerts therapeutic effects in the SOD1 ALS mouse model through protective glia and peripheral nerve rescue.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects the motor neuromuscular system leading to complete paralysis and premature death. The multifactorial nature of ALS that involves both cell-autonomous and non-cell-autonomous processes contributes to the lack of effective therapies, usually targeted to a single pathogenic mechanism. RNS60, an experimental drug containing oxygenated nanobubbles generated by modified Taylor-Couette-Poiseuille flow with elevated oxygen pressure, has shown anti-inflammatory and neuroprotective properties in different experimental paradigms. Since RNS60 interferes with multiple cellular mechanisms known to be involved in ALS pathology, we evaluated its effect in in vitro and in vivo models of ALS. METHODS: Co-cultures of primary microglia/spinal neurons exposed to LPS and astrocytes/spinal neurons from SOD1G93A mice were used to examine the effect of RNS60 or normal saline (NS) on the selective motor neuron degeneration. Transgenic SOD1G93A mice were treated with RNS60 or NS (300 µl/mouse intraperitoneally every other day) starting at the disease onset and examined for disease progression as well as pathological and biochemical alterations. RESULTS: RNS60 protected motor neurons in in vitro paradigms and slowed the disease progression of C57BL/6-SOD1G93A mice through a significant protection of spinal motor neurons and neuromuscular junctions. This was mediated by the (i) activation of an antioxidant response and generation of an anti-inflammatory environment in the spinal cord; (ii) activation of the PI3K-Akt pro-survival pathway in the spinal cord and sciatic nerves; (iii) reduced demyelination of the sciatic nerves; and (iv) elevation of peripheral CD4+/Foxp3+ T regulatory cell numbers. RNS60 did not show the same effects in 129Sv-SOD1G93A mice, which are unable to activate a protective immune response. CONCLUSION: RNS60 demonstrated significant therapeutic efficacy in C57BL/6-SOD1G93A mice by virtue of its effects on multiple disease mechanisms in motor neurons, glial cells, and peripheral immune cells. These findings, together with the excellent clinical safety profile, make RNS60 a promising candidate for ALS therapy and support further studies to unravel its molecular mechanism of action. In addition, the differences in efficacy of RNS60 in SOD1G93A mice of different strains may be relevant for identifying potential markers to predict efficacy in clinical trials.

Upregulation of Myelin Gene Expression by a Physically-Modified Saline via Phosphatidylinositol 3-Kinase-Mediated Activation of CREB: Implications for Multiple Sclerosis.

An increase in central nervous system (CNS) remyelination and a decrease in CNS inflammation are important steps to halt the progression of multiple sclerosis (MS). RNS60 is a bioactive aqueous solution generated by subjecting normal saline to Taylor-Couette-Poiseuille flow under elevated oxygen pressure. Recently we have demonstrated that RNS60 exhibits anti-inflammatory properties. Here, we describe promyelinating property of RNS60. RNS60, but not normal saline (NS), RNS10.3 (TCP-modified saline without excess oxygen) or PNS60 (saline containing excess oxygen without TCP modification), stimulated the expression of myelin-specific genes and proteins (myelin basic protein, MBP; myelin oligodendrocyte glycoprotein, MOG and proteolipid protein, PLP) in primary mouse oligodendroglia and mixed glial cells. While investigating the mechanisms, we found that RNS60 treatment induced the activation of cAMP response element binding protein (CREB) in oligodendrocytes, ultimately leading to the recruitment of CREB to the promoters of myelin-specific genes. Furthermore, activation of type 1A p110ß/α, but not type 1B p110γ, phosphatidylinositol-3 (PI-3) kinase by RNS60 together with abrogation of RNS60-mediated activation of CREB and upregulation of myelin genes by LY294002 (a specific inhibitor of PI-3 kinase) suggest that RNS60 upregulates the activation of CREB and the expression of myelin-specific molecules in oligodendrocytes via activation of PI3 kinase. These results highlight a novel promyelinating property of RNS60, which may be of benefit for MS and other demyelinating disorders.

Increase in Mitochondrial Biogenesis in Neuronal Cells by RNS60, a Physically-Modified Saline, via Phosphatidylinositol 3-Kinase-Mediated Upregulation of PGC1α.

This study highlights a novel approach to upregulate mitochondrial biogenesis in neuronal cells. 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. 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), increased the expression of Nrf1, Tfam, Mcu, and Tom20 (genes associated with mitochondrial biogenesis) and upregulated mitochondrial biogenesis in MN9D dopaminergic neuronal cells. Similarly RNS60 also increased mitochondrial biogenesis in primary dopaminergic neurons and in the nigra of MPTP-intoxicated mice. However, RNS60 had no effect on lysosomal biogenesis. Interestingly, we found that RNS60 upregulated PGC1α and siRNA knockdown of PGC1α abrogated the ability of RNS60 to increase mitochondrial biogenesis. Furthermore, we delineated that RNS60 increased the transcription of Pgc1a via type IA phosphatidylinositol (PI) 3-kinase-mediated activation of cAMP-response element-binding protein (CREB). Accordingly, knockdown of the PI3K - CREB pathway suppressed RNS60-mediated mitochondrial biogenesis. These results describe a novel property of RNS60 of enhancing mitochondrial biogenesis via PI 3-kinase-CREB-mediated up-regulation of PGC1α, which may be of therapeutic benefit in different neurodegenerative disorders.

Correction: A Physically-Modified Saline Suppresses Neuronal Apoptosis, Attenuates Tau Phosphorylation and Protects Memory in an Animal Model of Alzheimer's Disease.

[This corrects the article DOI: 10.1371/journal.pone.0103606.].

Nebulization of RNS60, a Physically-Modified Saline, Attenuates the Adoptive Transfer of Experimental Allergic Encephalomyelitis in Mice: Implications for Multiple Sclerosis Therapy.

Developing a new and effective therapeutic approach against multiple sclerosis (MS) is always an important area of research. RNS60 is a bioactive aqueous solution generated by subjecting normal saline to Taylor-Couette-Poiseuille flow under elevated oxygen pressure. Recently we have demonstrated that RNS60, administered through intraperitoneal injection, ameliorated clinical symptoms and disease progression of experimental allergic encephalomyelitis (EAE), an animal model of MS. Since the intravenous route is not preferred for treating a chronic condition, we tested if nebulization of RNS60 could attenuate the disease process of adoptively-transferred EAE in mice. Although we could not directly image RNS60 after nebulization, nebulized Alexa680 reached spleen, spinal cord and different parts of the brain. Nebulization of RNS60 starting from the acute phase attenuated clinical symptoms of relapsing-remitting EAE in female SJL/J mice. RNS60 nebulization also inhibited perivascular cuffing, maintained the integrity of blood-brain and blood-spinal cord barriers, suppressed inflammation, normalized the expression of myelin genes, and blocked demyelination in the CNS of EAE mice. On the immunomodulatory front, nebulization of RNS60 to EAE mice led to the enrichment of anti-autoimmune regulatory T cells (Tregs) and suppression of autoimmune Th17 cells. Together, these results suggest that nebulization of RNS60 may be used to control aberrant immune responses in MS and other autoimmune disorders.

Potential Benefit of the Charge-Stabilized Nanostructure Saline RNS60 for Myelin Maintenance and Repair.

Myelin injury in multiple sclerosis (MS) has been attributed both to "outside-in" primary immune mediated and "inside-out" metabolic stress of oligodendrocyte (OL) related mechanisms. Subsequent remyelination is dependent on recruitment and differentiation of oligodendrocyte progenitor cells (OPCs). RNS60 is a physically-modified saline containing charge-stabilized nanobubbles generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. Administration of RNS60 has been shown to reduce the severity of EAE by dampening the immune response and myelin loss. Additionally, RNS60 has been demonstrated to enhance mitochondrial ATP synthesis in neurons. Here, we used post-natal rat derived OLs and OPCs to assess the impact of RNS60 on the response of OLs to metabolic stress in vitro (glucose-nutrient deprivation, referred to as 'NG') and on OPC differentiation capacity. Under the NG condition, our findings indicate that RNS60 decreases caspases 3/7 activation. Respirometric analyses revealed that RNS60 increased spare glycolytic capacity (SGC) under normal culture conditions. However, RNS60 enhanced OL spare respiratory capacity (SRC) when a metabolic stress was present. Furthermore, we show that RNS60 promotes OPC differentiation under physiological conditions. Our findings provide evidence for the potential therapeutic efficacy of RNS60 through the promotion of OL survival and OPC differentiation.

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.

Protection of dopaminergic neurons in a mouse model of Parkinson's disease by a physically-modified saline containing charge-stabilized nanobubbles.

Neuroinflammation underlies the pathogenesis of various neurodegenerative disorders including Parkinson's disease (PD). Despite intense investigations, no effective therapy is available to stop its onset or halt its progression. RNS60 is a novel therapeutic containing charge-stabilized nanobubbles in saline, generated by subjecting normal saline to Taylor-Couette-Poiseuille flow under elevated oxygen pressure. Recently, we have delineated that RNS60 inhibits the expression of proinflammatory molecules in glial cells via type 1A phosphatidylinositol-3 kinase (PI3K)-mediated upregulation of IκBα. In this study, we demonstrate that RNS60 inhibited the expression of proinflammatory molecules in cultured microglial cells stimulated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridium ion (MPP(+)) and in vivo in the nigra of MPTP-intoxicated mice. While investigating the underlying mechanisms, we found that MPTP intoxication rapidly stimulated the activation of type IB PI3K p110γ in the nigra, while suppressing the activation of type IA PI3K p110α/ß. Interestingly, RNS60 treatment suppressed the activation of p110γ PI3K, while inducing the activation of p110α/ß PI3K in the nigra of MPTP-intoxicated mice. Accordingly, RNS60 treatment increased the level of IκBα and inhibited the activation of NF-κB in the SNpc of MPTP-intoxicated mice. These findings paralleled dopaminergic neuronal protection, normalized striatal neurotransmitters, and improved motor functions in MPTP-intoxicated mice. These results strongly suggest a promising therapeutic role of this simple modified saline in PD and other neuroinflammatory disorders.

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.