Ciliated Cells Express a Novel Pattern of Brain-Derived Neurotrophic Factor in Allergic Rhinitis.

Background: Mounting research indicates that brain-derived neurotrophic factor (BDNF), has great potential to increase neuro-hyperresponsiveness and airway resistance in airway allergic disease. The expression level of BDNF has been found to be notably elevated in lung/nasal lavage (NAL) fluid. However, the expression and position of BDNF in ciliated cells with allergic rhinitis remains unclear. Methods: Nasal mucosal cells were collected from patients with allergic rhinitis (AR) and mice which were performed under different allergen challenge time, then observed the expression and position of BDNF located in ciliated cells through the immunofluorescence staining. Nasal mucosa, serum and NAL fluid were collected also. The expression level of BDNF and IL-4/5/13 were detected by RT-PCR. The expressions of BDNF (in serum and NAL fluid), and total-IgE, ovalbumin sIgE (in serum) were detected by ELISA. Results: We found that MFI of BDNF in AR group's ciliated cells was obviously lower than that in the control group, and a negative correlation was discovered between MFI and VAS score. It can be roughly divided into 5 patterns according to its location in the cytoplasm of ciliated cells. In the mouse model, the expressions of BDNF in serum and NAL fluid increased temporarily after allergen stimulation. The MFI of BDNF in ciliated cells displayed an initial increase followed by a subsequent decrease. Conclusion: Our study shows for the first time that, the expression and localization of BNDF were observed in the human nasal ciliated epithelial cells of allergic rhinitis, and the expression of level was less than the control group under the persistent state of allergy. BDNF expression in ciliated cells was transient increased after allergen stimulation and decreased to normal level after 24h in mouse model of allergic rhinitis. This might be the possible source of the transient increase of BNDF in serum and NAL fluid.

Synergistic Effects of Radiotherapy With JNK Inhibitor-Incorporated Nanoparticle in an Intracranial Lewis Lung Carcinoma Mouse Models.

BACKGROUND: Radiosurgery has been recognized as a reasonable treatment for metastatic brain tumors. Increasing the radiosensitivity and synergistic effects are possible ways to improve the therapeutic efficacy of specific regions of tumors. c-Jun-N-terminal kinase (JNK) signaling regulates H2AX phosphorylation to repair radiation-induced DNA breakage. We previously showed that blocking JNK signaling influenced radiosensitivity in vitro and in an in vivo mouse tumor model. Drugs can be incorporated into nanoparticles to produce a slow-release effect. This study assessed JNK radiosensitivity following the slow release of the JNK inhibitor SP600125 from a poly (DL-lactide-co-glycolide) (LGEsese) block copolymer in a brain tumor model. MATERIALS AND METHODS: A LGEsese block copolymer was synthesized to fabricate SP600125-incorporated nanoparticles by nanoprecipitation and dialysis methods. The chemical structure of the LGEsese block copolymer was confirmed by 1H nuclear magnetic resonance (NMR) spectroscopy. The physicochemical and morphological properties were observed by transmission electron microscopy (TEM) imaging and measured with particle size analyzer. The blood-brain barrier (BBB) permeability to the JNK inhibitor was estimated by BBBflammaTM 440-dye-labeled SP600125. The effects of the JNK inhibitor were investigated using SP600125-incorporated nanoparticles and by optical bioluminescence, magnetic resonance imaging (MRI), and a survival assay in a mouse brain tumor model for Lewis lung cancer (LLC)-Fluc cells. DNA damage was estimated by histone γ H2AX expression and apoptosis was assessed by the immunohistochemical examination of cleaved caspase 3. RESULTS: The SP600125-incorporated nanoparticles of the LGEsese block copolymer were spherical and released SP600125 continuously for 24h. The use of BBBflammaTM 440-dye-labeled SP600125 demonstrated the ability of SP600125 to cross the BBB. The blockade of JNK signaling with SP600125-incorporated nanoparticles significantly delayed mouse brain tumor growth and prolonged mouse survival after radiotherapy. γ H2AX, which mediates DNA repair protein, was reduced and the apoptotic protein cleaved-caspase 3 was increased by the combination of radiation and SP600125-incorporated nanoparticles.

Enhancing Radiotherapeutic Effect With Nanoparticle-Mediated Radiosensitizer Delivery Guided By Focused Gamma Rays In Lewis Lung Carcinoma-Bearing Mouse Brain Tumor Models.

BACKGROUND: Targeting radiosensitizer-incorporated nanoparticles to a tumor could allow for less normal tissue toxicity with more efficient drug release, thus improving the efficacy and safety of radiation treatment. The aim of this study was to improve tumor-specific delivery and bioavailability of a nanoparticle-mediated radiosensitizer in mouse brain tumor models. METHODS: A pH-sensitive nanoparticle, chitoPEGAcHIS, was conjugated to recombinant peptide HVGGSSV that could bind to tax-interaction protein 1 (TIP-1) as a radiation-inducible receptor. Then the c-Jun N-terminal kinase (JNK) inhibitor, SP600125 was incorporated into this copolymer to fabricate a HVGGSSV-chitoPEGAcHIS-SP600125 (HVSP-NP) nanoradiosensitizer. In vitro and in vivo radiation treatment were performed using a Gamma Knife unit. The tumor targetability of HVSP-NP was estimated by optical bioluminescence. Synergistic therapeutic effects of radiation treatment and HVSP-NP were investigated in Lewis lung carcinoma (LLC) cell-bearing mouse brain tumor models. RESULTS: The SP600125 JNK inhibitor effectively reduced DNA damage repair to irradiated LLC cells. A pH sensitivity assay indicated that HVSP-NP swelled at acidic pH and increased in diameter, and its release rate gradually increased. Optical bioluminescence assay showed that radiation induced TIP-1 expression in mouse brain tumor and that the nanoradiosensitizer selectively targeted irradiated tumors. Radiation treatment with HVSP-NP induced greater apoptosis and significantly inhibited tumor growth compared to radiation alone. CONCLUSION: As a novel nanoradiosensitizer, HVSP-NP was found to be able to selectively target irradiated tumors and significantly increase tumor growth delay in LLC-bearing mouse brain tumor models. This research shows that delivering a pH-sensitive nanoradiosensitizer to a brain tumor in which TIP-1 is induced by radiation can result in improved radiosensitizer-release in an acidic microenvironment of tumor tissue and in created synergistic effects in radiation treatment.

Genetically-engineered Salmonella typhimurium expressing TIMP-2 as a therapeutic intervention in an orthotopic glioma mouse model.

Glioma is one of the most devastating and refractory cancers. The main factors underlying therapeutic failure include extremely invasive characteristics and lack of effective methods for drug delivery. Attenuated Salmonella strains presented a high concentration of tumor targets in various types of cancer models, suggesting a role as potential vectors for drug delivery. In this study, we genetically engineered an attenuated strain of Salmonella as an anti-invasive vector for the targeted delivery and expression of tissue inhibitor of metalloproteinases 2 (TIMP-2) in an orthotopic nude mouse model of glioma. The bioluminescence signals related to tumor size significantly declined in the TIMP-2-expressing Salmonella (SLpTIMP-2)-treated group compared with the control group. Compared with the control group with a survival rate of an average of 33 days, the SLpTIMP-2 group showed an extended survival rate by nearly 60% and lasted an average period of 53 days with TIMP-2 induction. These results indicated the promising therapeutic potential of S. typhimurium for targeted delivery and secretion of TIMP-2 in glioma.

Sub-bandgap photo-response of non-doped black-silicon fabricated by nanosecond laser irradiation.

Non-doped black silicon (b-Si) is fabricated on the surface layer of a near-intrinsic Si substrate by nanosecond (ns) laser direct writing in an argon (Ar) atmosphere. The non-doped samples exhibit a near-unity sub-bandgap (1100∼2500 nm) absorptance of more than 50%. Amazingly, the resistivity of the ns laser irradiated b-Si layer is about five orders of magnitude lower than that of the unprocessed Si substrate. The carrier density of the b-Si layer is about 1×1018 cm-3, according to the Hall effect measurement. Temperature-dependent Hall effect measurements show that the non-doped b-Si layer exhibits an energy level of 0.026 eV below the conduction band minimum (CBM). At last, Si infrared photodiodes are made based on the difference of carrier concentration between the ns laser-processed b-Si layer and the high-resistivity Si substrate. The responsivity of the b-Si photodiode for 1310 nm is up to 256 mA/W at a 10-V reverse bias, which is much higher than that of the reported pure Si bulk-structure photodiodes.

Corrigendum to "Osteoporosis Recovery by Antrodia camphorata Alcohol Extracts through Bone Regeneration in SAMP8 Mice".

[This corrects the article DOI: 10.1155/2016/2617868.].

PDIA6 regulation of ADAM17 shedding activity and EGFR-mediated migration and invasion of glioblastoma cells.

OBJECTIVE In patients with glioblastoma, local invasion of tumor cells causes recurrence and shortens survival. The goal of this study was to determine whether protein disulfide isomerase (PDI) A6 regulates migration and invasion of glioblastoma cells and the associated factors. METHODS U87MG cells were treated with either PDIA6 or ADAM17 small interfering RNA (siRNA) fragments or with both types of siRNA fragments, and expression was confirmed by reverse transcription-polymerase chain reaction and Western blot. Migration and invasion were assessed using a wound-healing assay, a Matrigel assay, and an organotypic culture system. After the U87MG cells were treated with siRNAs and epidermal growth factor receptor (EGFR) inhibitors, the expression of matrix metalloproteinase-2 (MMP-2), membrane Type 1-matrix metalloproteinase (MT1-MMP), integrin, phosphorylated focal adhesion kinase (pFAK), and phosphorylated EGFR (pEGFR) was detected by Western blotting and zymography. RESULTS U87MG cell migration and invasion increased significantly after inhibition of PDIA6. The MMP-2 activation ratio and ADAM17 activity (as a sheddase of the proligand) increased, and expression of pEGFR, pFAK, integrin α5ß3, and MT1-MMP was induced, compared with control levels. Furthermore, heparin-binding epidermal growth factor (EGFR signaling ligand) was highly expressed in PDIA6-knockdown cells. After siPDIA6-transfected U87MG cells were treated with EGFR signaling inhibitors, expression of pFAK, MMP-2, and MT1-MMP decreased and invasion decreased significantly. Simultaneous double-knockdown of PDIA6 and ADAM17 reduced pEGFR and pFAK expression, compared with control levels. CONCLUSIONS The authors propose that inhibiting PDIA6 could transduce EGFR signaling by activating and inducing ADAM17 during migration and invasion of U87MG glioblastoma cells. The results of this study suggest that PDIA6 is an important component of EGFR-mediated migration and invasion of U87MG cells. This is the first report of the effects of PDIA6 on migration and invasion in glioblastoma.

Metallothinein 1E Enhances Glioma Invasion through Modulation Matrix Metalloproteinases-2 and 9 in U87MG Mouse Brain Tumor Model.

Malignant glioma cells invading surrounding normal brain are inoperable and resistant to radio- and chemotherapy, and eventually lead to tumor regrowth. Identification of genes related to motility is important for understanding the molecular biological behavior of invasive gliomas. According to our previous studies, Metallothionein 1E (MT1E) was identified to enhance migration of human malignant glioma cells. The purpose of this study was to confirm that MT1E could modulate glioma invasion in vivo. Firstly we established 2 cell lines; MTS23, overexpressed by MT1E complementary DNA construct and pV12 as control. The expression of matrix metalloproteinases (MMP)-2, -9 and a disintegrin and metalloproteinase 17 were increased in MTS23 compared with pV12. Furthermore it was confirmed that MT1E could modulate MMPs secretion and translocation of NFkB p50 and B-cell lymphoma-3 through small interfering ribonucleic acid knocked U87MG cells. Then MTS23 and pV12 were injected into intracranial region of 5 week old male nude mouse. After 4 weeks, for brain tissues of these two groups, histological analysis, and immunohistochemical stain of MMP-2, 9 and Nestin were performed. As results, the group injected with MTS23 showed irregular margin and tumor cells infiltrating the surrounding normal brain, while that of pV12 (control) had round and clear margin. And regrowth of tumor cells in MTS23 group was observed in another site apart from tumor cell inoculation. MT1E could enhance tumor proliferation and invasion of malignant glioma through regulation of activation and expression of MMPs.

Enhancement of radiosensitivity by inhibition of c-Jun N-terminal kinase activity in a Lewis lung carcinoma­bearing subcutaneous tumor mouse model.

Stereotactic radiosurgery has been recognized as an effective treatment approach for metastatic brain tumors. By increasing the sensitivity of the tumor to radiation and decreasing the marginal dose, it is possible to improve therapeutic efficacy and decrease side-effects. In radiation-induced cells, c-Jun N-terminal kinase (JNK) signaling mediates the phosphorylation of H2AX, which indicates DNA damage sensitivity and modulates the effect of radiation. Lewis lung cancer (LLC) and breast cancer (4T1) cells were irradiated with a Gamma Knife in cell culture tubes. To evaluate the relationship between radiosensitivity and JNK activity, clonogenic assay was performed. DNA damage response was estimated by γH2AX focus formation assay and apoptosis­related protein levels were assessed by western blotting. The mice were subcutaneously inoculated with LLC cells, and irradiated concomitantly with JNK inhibitor treatment. The effect of the JNK inhibitor was investigated by tumor volumetry and immunohistochemistry. γH2AX expression, which mediates repair of radiation­induced DNA damage, was reduced in the cancer cell group pretreated with the JNK inhibitor. This finding shows that JNK inhibition may increase the radiosensitivity in radiated lung and breast cancer cells. For the in vivo study, irradiated tumor growth was significantly delayed in the JNK inhibitor-treated mouse group. Blockade of JNK signaling decreased γH2AX expression and increased apoptosis in the radiation-induced cancer cells. JNK inhibitor may be useful for enhancing the radiosensitivity of lung and breast cancer cells and improving the treatment efficacy of radiosurgical approaches for metastatic brain tumors.

Osteoporosis Recovery by Antrodia camphorata Alcohol Extracts through Bone Regeneration in SAMP8 Mice.

Antrodia camphorata has previously demonstrated the efficacy in treating cancer and anti-inflammation. In this study, we are the first to evaluate Antrodia camphorata alcohol extract (ACAE) for osteoporosis recovery in vitro with preosteoblast cells (MC3T3-E1) and in vivo with an osteoporosis mouse model established in our previous studies, ovariectomized senescence accelerated mice (OVX-SAMP8). Our results demonstrated that ACAE treatment was slightly cytotoxic to preosteoblast at 25 µg/mL, by which the osteogenic gene expression (RUNX2, OPN, and OCN) was significantly upregulated with an increased ratio of OPG to RANKL, indicating maintenance of the bone matrix through inhibition of osteoclastic pathway. Additionally, evaluation by Alizarin Red S staining showed increased mineralization in ACAE-treated preosteoblasts. For in vivo study, our results indicated that ACAE inhibits bone loss and significantly increases percentage bone volume, trabecular bone number, and bone mineral density in OVX-SAMP8 mice treated with ACAE. Collectively, in vitro and in vivo results showed that ACAE could promote osteogenesis and prevent bone loss and should be considered an evidence-based complementary and alternative medicine for osteoporosis therapy through the maintenance of bone health.

Nogo-A inhibits the migration and invasion of human malignant glioma U87MG cells.

Nogo or reticulon-4 (RTN4), also known as neurite outgrowth inhibitor, is a member of the reticulon family of genes. Nogo-A, one of the three isoforms, is enriched in the central nervous system (CNS). The extracellular domain of Nogo-A, Nogo-66, has neurite growth inhibitory activity that is specific for neurons and is mediated by the Nogo receptor. However, most of its functions are not known yet. We investigated whether Nogo-A modulates the migration and invasion of a glioblastoma cell line, as well as the factors that have an effect on Nogo-A. The expression of Nogo-A was evaluated using western blotting and immunohistochemistry in human brain tumor specimens. U87MG cells were transfected with a sense-Nogo-A cDNA construct (U87-Nogo-A cells expressing Nogo-A) and an empty vector (U87MG-E cells not expressing Nogo-A). The migration and invasion abilities of these cells were investigated using simple scratch and Matrigel invasion assays. Morphologic and cytoskeletal changes were documented by confocal microscopy. The proliferation rate was estimated using doubling time assay. The effects of Nogo-A on Rho activity and phosphorylated cofilin were determined by a Rho activity assay and western blotting. Among primary brain tumors, Nogo-A expression was found in a higher percentage of oligodendrogliomas (90.0%) compared with the percentage in the glioblastomas (68.4%). In addition, the percentage in mixed gliomas was 42.9%, while it was not expressed in pituitary adenomas or schwannomas. The migration and invasion abilities of the U87-Nogo-A cells were decreased compared with the control. In the U87-Nogo-A cell line, Rho activity and phosphorylated cofilin expression were also decreased and morphology became more flat in comparison with the U87MG-E cell line. Nogo-A may inhibit the migration and invasion of human malignant glioma cells via the downregulation of RhoA-cofilin signaling.

Sublethal dose of irradiation enhances invasion of malignant glioma cells through p53-MMP 2 pathway in U87MG mouse brain tumor model.

BACKGROUND: Glioblastoma is a highly lethal neoplasm that frequently recurs locally after radiotherapy, and most of these recurrences originate from near the irradiated target field. In the present study, we identified the effects of radiation on glioma invasion and p53, TIMP-2, and MMP-2 expression through in vitro and in vivo experiments. METHODS: The U87MG (wt p53) and U251 (mt p53) human malignant glioma cell lines were prepared, and the U2OS (wt 53) and Saos2 (del p53) osteosarcoma cell lines were used as p53 positive and negative controls. The four cell lines and p53 knock-downed U87MG cells received radiation (2-6 Gy) and were analyzed for expression of p53 and TIMP-2 by Western blot, and MMP-2 activity was detected by zymography. In addition, the effects of irradiation on directional invasion of malignant glioma were evaluated by implanting nude mice with bioluminescent u87-Fluc in vivo followed by MMP-2, p53, and TIMP-2 immunohisto-chemistry and in situ zymography. RESULTS: MMP-2 activity and p53 expression increased in proportional to the radiation dose in cell lines with wt p53, but not in the cell lines with del or mt p53. TIMP-2 expression did not increase in U87MG cells. MMP-2 activity decreased in p53 knock-downed U87MG cells but increased in the control group. Furthermore, radiation enhanced MMP-2 activity and increased tumor margin invasiveness in vivo. Tumor cells invaded by radiation overexpressed MMP-2 and p53 and revealed high gelatinolytic activity compared with those of non-radiated tumor cells. CONCLUSION: Radiation-induced upregulation of p53 modulated MMP-2 activity, and the imbalance between MMP-2 and TIMP-2 may have an important role in glioblastoma invasion by degrading the extracellular matrix. Bioluminescent "U87-Fluc"was useful for observing tumor formation without sacrifice after implanting tumor cells in the mouse brain. These findings suggest that the radiotherapy involved field for malignant glioma needs to be reconsidered, and that future trials should investigate concurrent pharmacologic therapies that inhibit invasion associated with radiotherapy.