Downregulation of uPA inhibits migration and PI3k/Akt signaling in glioblastoma cells

The ability of glioma cells to migrate great distances from a primary tumor mass is the primary cause of tumor recurrence. The urokinase-type plasminogen activator (uPA) is a serine protease that can initiate proteolytic cascades, which result in remodeling of extracellular matrix and basement membrane, allowing cells to move across and through these barriers. The binding between uPA and its receptor uPAR also mediates several signaling events that seem to contribute to the evolution of a migratory phenotype. In this study, we determined how the downregulation of uPA affects the signaling pathways leading to cell migration. Stably transfecting human glioblastoma cells with antisense uPA decreased the amount of cell-bound uPA and disrupted actin cytoskeleton formation and cell migration. The phosphatidylinositol 3-kinase (PI3k) and Akt signaling pathway has been suggested to mediate migration in various cancer cells. The antisense-uPA clones also had less phosphorylated PI3k and Akt than control cells, a finding associated with decreased cell migration, G2/M-phase arrest, and decreased clonogenic survival. Decreased activation of PI3k and the antiapoptotic factor Akt was not sufficient to induce apoptosis in the antisense-uPA clones, but staurosporine sensitized them to apoptosis to a greater extent than control cells. These results indicate that PI3k/Akt pathway is involved in the signaling cascade required to induce cell migration and that uPA has a direct role in regulating migration.     

Distribution of respiratory mucin proteins in human nasal mucosa

OBJECTIVES/HYPOTHESIS: The upper respiratory tract is involved in many acute and chronic respiratory tract diseases that present with the symptom of mucus hypersecretion. Mucin genes that encode for the backbone of glycoproteins contribute to the viscoelastic property of airway mucus. We examined the cellular expression and distribution of two major respiratory mucus-forming glycoproteins, MUC5AC and MUC5B, in normal human nasal tissues. METHODS: Immunohistochemical analysis using polyclonal antibodies against the mucins MUC5AC and MUC5B was performed in normal human nasal tissues. RESULTS: An abundant staining of submucosal mucus gland and epithelial goblet cells for MUC5B was found. Immunohistochemical analysis of MUC5AC showed staining of surface epithelium goblet cells, whereas there was no staining of glandular cells. Comparison of the expression to lower airways revealed a similar pattern of expression of both mucins. CONCLUSIONS: The data in the present study demonstrated the localization of the two major respiratory mucin proteins in human nasal mucosa with a similar distribution of expression of MUC5AC and MUC5B in normal upper and lower airways. Mucin protein expression parallels that of mucin messenger RNA expression.     

Activation of caspase-9 with irradiation inhibits invasion and angiogenesis in SNB19 human glioma cells

Glioblastoma multiforme, the most common brain tumor, typically exhibits markedly increased angiogenesis, which is crucial for tumor growth and invasion. Antiangiogenic strategies based on disruption of the tumor microvasculature have proven effective for the treatment of experimental brain tumors. Here, we have overexpressed human caspase-9 by stable transfection in the SNB19 glioblastoma cell line, which normally expresses low levels of caspase-9. Our studies revealed that overexpression of caspase-9 coupled with radiation has a synergistic effect on the inhibition of glioma invasion as demonstrated by Matrigel assay (> 65%). Furthermore, sense caspase stable clones cocultured with fetal rat brain aggregates along with radiation showed complete inhibition as compared to the parental and vector controls. During in vitro angiogenesis, SNB19 cells cocultured with human microvascular endothelial cells (HMEC) showed vascular network formation after 48-72 h. In contrast, these capillary-like structures were inhibited when HMEC cells were cocultured with sense caspase stable SNB19 cells. This effect was further enhanced by radiation (5 Gy). Signaling mechanisms revealed that apoptosis is induced by cleavage of caspase-9 by radiation, loss of mitochondrial membrane potential and activation of caspase-3. These results demonstrate that activation of caspase-9 disrupts glioma cell invasion and angiogenesis in vitro. Hence, overexpression of proapoptotic molecules such as caspase-9 may be an important determinant of the therapeutic effect of radiation in cancer therapy.     

Inhibition of cathepsin B and MMP-9 gene expression in glioblastoma cell line via RNA interference reduces tumor cell invasion, tumor growth and angiogenesis

Extracellular proteases have been shown to cooperatively influence matrix degradation and tumor cell invasion through proteolytic cascades, with individual proteases having distinct roles in tumor growth, invasion, migration and angiogenesis. Matrix metalloproteases (MMP)-9 and cathepsin B have been shown to participate in the processes of tumor growth, vascularization and invasion of gliomas. In the present study, we used a cytomegalovirus promoter-driven DNA template approach to induce hairpin RNA (hpRNA)-triggered RNA interference (RNAi) to block MMP-9 and cathepsin B gene expression with a single construct. Transfection of a plasmid vector-expressing double-stranded RNA (dsRNA) for MMP-9 and cathepsin B significantly inhibited MMP-9 and cathepsin B expression and reduced the invasive behavior of SNB19, glioblastoma cell line in Matrigel and spheroid invasion models. Downregulation of MMP-9 and cathepsin B using RNAi in SNB19 cells reduced cell-cell interaction of human microvascular endothelial cells, resulting in the disruption of capillary network formation in both in vitro and in vivo models. Direct intratumoral injections of plasmid DNA expressing hpRNA for MMP-9 and cathepsin B significantly inhibited established glioma tumor growth and invasion in intracranial tumors in vivo. Further intraperitoneal (i.p.) injections of plasmid DNA expressing hpRNA for MMP-9 and cathepsin B completely regressed pre-established tumors for a long time (4 months) without any indication of these tumor cells. For the first time, these observations demonstrate that the simultaneous RNAi-mediated targeting of MMP-9 and cathepsin B has potential application for the treatment of human gliomas.     

Genital warts among 18- to 59-year-olds in the United States, national health and nutrition examination survey, 1999--2004

BACKGROUND: Genital warts are caused by human papillomavirus (HPV); over 90% are due to HPV types 6 and 11. We determined the percentage of persons who reported having been diagnosed with genital warts in the United States from 1999--2004. METHODS: We collected genital wart diagnosis history and sociodemographic and sexual behavior data from 8849 sexually active men and women aged 18 to 59 years as part of the National Health and Nutrition Examination Survey, 1999--2004. RESULTS: Overall, 5.6% of 18-to 59-year olds reported having ever been diagnosed with genital warts. The percentage was higher in women (7.2%, 95% CI, 6.2%-8.4%) than in men (4%, 95% CI, 3.2%-5.0%). History of genital wart diagnosis peaked among 25- to 34-year-old women (10.4%) and 35- to 44-year-old men (6.0%). Sex, age, race/ethnicity, number of lifetime sex partners, and cocaine/street drug use were associated with genital warts in multivariate analysis. CONCLUSIONS: These are the first national data on the burden of genital warts in the United States. The substantial burden of genital warts could be reduced by a prophylactic HPV vaccine to types 6 and 11.     

uPA/uPAR downregulation inhibits radiation-induced migration, invasion and angiogenesis in IOMM-Lee meningioma cells and decreases tumor growth in vivo

Meningioma is a well-known tumor of the central nervous system, and is treated by surgical resection and/or radiation. Recently, ionizing radiation has been shown to enhance invasiveness of surviving tumor cells, and several proteolytic enzyme molecules, including urokinase plasminogen activator (uPA), seem to be upregulated after radiation. uPA and its receptor (uPAR) have been strongly implicated in tumor invasion, angiogenesis and progression. Hence, the tumor-associated uPA-uPAR system is considered a potential target for cancer therapy. In the present study, we show that radiation increases uPA levels in the IOMM-Lee meningioma cells, and subsequently, increases tumor invasion, migration and angiogenesis in vitro. Studies with signaling molecule inhibitors AG1478, U0126 and SB203580 (specific inhibitors of EGFR, MEK1/2 and p38 respectively) showed inhibition of uPA levels in both basal and irradiated-IOMM-Lee cells. The PI3K inhibitor (LY294002) and the AKT inhibitor (AKT inhibitor IV) also partially decreased uPA expression, whereas SP600125, a JNK inhibitor, did not affect uPA levels in either radiated or non-radiated cells. Further, a bicistronic plasmid construct with small interfering RNA (siRNA) against uPA and its receptor inhibited tumor invasion, migration and angiogenesis in radiation-treated IOMM-Lee cells. In addition, siRNA against uPA and its receptor inhibited subcutaneous tumor growth in athymic nude mice in combination with radiation in a synergistic manner. Thus, the specific targeting of proteases via RNA interference could augment the therapeutic effect of radiation and prevent the adverse effects resulting from tumor cells that receive sublethal doses of radiation within the tumor mass.     

New insights into the competition between antioxidant activities and pro-oxidant risks of rosmarinic acid

Direct and indirect antioxidant activities of rosmarinic acid (RA) based on HOO˙/CH₃OO˙ radical scavenging and Fe(iii)/Fe(ii) ion chelation were theoretically studied using density functional theory at the M05-2X/6-311++G(2df,2p) level of theory. First, four antioxidant mechanisms including hydrogen atom transfer (HAT), radical adduct formation (RAF), proton loss (PL) and single electron transfer (SET) were investigated in water and pentyl ethanoate (PEA) phases. Regarding the free radical scavenging mechanism, HAT plays a decisive role with overall rate coefficients of 1.84 × 10³ M⁻¹ s⁻¹ (HOO˙) and 4.49 × 10³ M⁻¹ s⁻¹ (CH₃OO˙) in water. In contrast to PL, RAF and especially SET processes, the HAT reaction in PEA is slightly more favorable than that in water. Second, the [Fe(iii)(H₂O)₆]³⁺ and [Fe(ii)(H₂O)₆]²⁺ ion chelating processes in an aqueous phase are both favorable and spontaneous especially at the O5, site-1, and site-2 positions with large negative Δ_rG⁰ values and great formation constant K_f. Finally, the pro-oxidant risk of RA⁻ was also considered via the Fe(iii)-to-Fe(ii) complex reduction process, which may initiate Fenton-like reactions forming reactive HO˙ radicals. As a result, RA⁻ does not enhance the reduction process when ascorbate anions are present as reducing agents, whereas the pro-oxidant risk becomes remarkable when superoxide anions are found. The results encourage further attempts to verify the speculation using more powerful research implementations of the antioxidant activities of rosmarinic acid in relationship with its possible pro-oxidant risks.

Direct and indirect antioxidant activities of rosmarinic acid (RA) based on HOO˙/CH₃OO˙ radical scavenging and Fe(iii)/Fe(ii) ion chelation were theoretically studied using density functional theory at the M05-2X/6-311++G(2df,2p) level of theory.     

Helix I of beta-arrestin is involved in postendocytic trafficking but is not required for membrane translocation, receptor binding, and internalization

beta-Arrestins bind to phosphorylated, seven-transmembrane-spanning, G protein-coupled receptors (GPCRs), including the type 1 angiotensin II receptor (AT(1)R), to promote receptor desensitization and internalization. The AT(1) R is a class B GPCR that recruits both beta-arrestin1 and beta-arrestin2, forming stable complexes that cotraffic to deep-core endocytic vesicles. beta-Arrestins contain one amphipathic and potentially amphitropic (membrane-targeting) alpha-helix (helix I) that may promote translocation to the membrane or influence receptor internalization or trafficking. Here, we investigated the trafficking and function of beta-arrestin1 and beta-arrestin2 mutants bearing substitutions in both the hydrophobic and positively charged faces of helix I. The level of expression of these mutants and their cytoplasmic localization (in the absence of receptor activation) was similar to wild-type beta-arrestins. After angiotensin II stimulation, both wild-type and beta-arrestin mutants translocated to the cell membrane, although recruitment was weaker for mutants of the hydrophobic face of helix I. For all beta-arrestin mutants, the formation of deep-core vesicles was less observed compared with wild-type beta-arrestins. Furthermore, helix I conjugated to green fluorescent protein is not membrane-localized, suggesting that helix I, in isolation, is not amphitropic. Bioluminescence resonance energy transfer analysis revealed that both wild-type and beta-arrestin mutants retained a capacity to interact with the AT(1)R, although the interaction with the mutants was less stable. Finally, wild-type and mutant beta-arrestins fully supported receptor internalization in human embryonic kidney cells and mouse embryonic fibroblasts deficient in beta-arrestin1 and -2. Thus, helix I is implicated in postmembrane trafficking but is not strongly amphitropic.     

Drug delivery global summit--evaluating emerging technologies

Two day-long sessions at the Drug delivery global summit, organised by SMi Group Ltd, were devoted to discussion on critical aspects of drug delivery, including advances in drug delivery systems and their applications to new products, with a primary focus on oral systems, but also highlighting recent progress in inhalation, parenteral and transdermal delivery. The event included case studies from big pharma, biotech and drug delivery companies to illustrate emerging delivery technologies and how they can be applied to develop innovative products. The conference created a platform for discussion on a range of topics from scientific issues and challenges to ways of establishing mutually beneficial relationships between technology and pharma companies.