Atypical induction of HIF-1α expression by pericellular Notch1 signaling suffices for the malignancy of glioblastoma multiforme cells.

Contact-based pericellular interactions play important roles in cancer progression via juxtacrine signaling pathways. The present study revealed that hypoxia-inducible factor-1α (HIF-1α), induced even in non-hypoxic conditions by cell-to-cell contact, was a critical cue responsible for the malignant characteristics of glioblastoma multiforme (GBM) cells through Notch1 signaling. Densely cultured GBM cells showed enhanced viability and resistance to temozolomide (TMZ) compared to GBM cells at a low density. Ablating Notch1 signaling by a γ-secretase inhibitor or siRNA transfection resensitized resistant GBM cells to TMZ treatment and decreased their viability under dense culture conditions. The expression of HIF-1α was significantly elevated in highly dense GBM cells even under non-hypoxic conditions. Atypical HIF-1α expression was associated with the Notch1 signaling pathway in both GBM and glioblastoma stem cells (GSC). Proteasomal degradation of HIF-1α was prevented by binding with Notch1 intracellular domain (NICD), which translocated to the nuclei of GBM cells. Silencing Notch1 signaling using a doxycycline-inducible Notch1 RNA-interfering system or treatment with chetomin, a HIF pathway inhibitor, retarded tumor development with a significant anti-cancer effect in a murine U251-xenograft model. Using GBM patient tissue microarray analysis, a significant increase in HIF-1α expression was identified in the group with Notch1 expression compared to the group without Notch1 expression among those with positive HIF-1α expression. Collectively, these findings highlight the critical role of cell-to-cell contact-dependent signaling in GBM progression. They provide a rationale for targeting HIF-1α signaling even in a non-hypoxic microenvironment.

Atomic layer deposition of 1D and 2D nickel nanostructures on graphite.

One-dimensional (1D) nanowires (NWs) and two-dimensional (2D) thin films of Ni were deposited on highly ordered pyrolytic graphite (HOPG) by atomic layer deposition (ALD), using NH3 as a counter reactant. Thermal ALD using NH3 gas forms 1D NWs along step edges, while NH3 plasma enables the deposition of a continuous 2D film over the whole surface. The lateral and vertical growth rates of the Ni NWs are numerically modeled as a function of the number of ALD cycles. Pretreatment with NH3 gas promotes selectivity in deposition by the reduction of oxygenated functionalities on the HOPG surface. On the other hand, NH3 plasma pretreatment generates surface nitrogen species, and results in a morphological change in the basal plane of graphite, leading to active nucleation across the surface during ALD. The effects of surface nitrogen species on the nucleation of ALD Ni were theoretically studied by density functional theory calculations. Our results suggest that the properties of Ni NWs, such as their density and width, and the formation of Ni thin films on carbon surfaces can be controlled by appropriate use of NH3.

Protopanaxatirol type ginsenoside Re promotes cyclic growth of hair follicles via inhibiting transforming growth factor ß signaling cascades.

Ginsenosides, the major bio-active ingredients included in Panax ginseng, have been known for the hair growth activity and used to treat patients who suffer from hair loss; however, the detailed mechanisms of this action are still largely unknown. This study was conducted to investigate the molecular and cellular mechanisms responsible for hair growth promoting effect of ginsenoside Re (GRe) in vitro and in vivo. Different doses of minoxidil and GRe were administered topically to the back regions of nude mice for up to 45 days, and hair shaft length and hair cycles were determined for hair promoting activities. Topical treatment of GRe significantly increased the hair shaft length and hair existent time, which was comparable to the action of minoxidil. We also demonstrated that GRe stimulated hair shaft elongation in the ex vivo cultures of vibrissa hair follicles isolated from C57BL/6 mouse. Systemic transcriptome analysis by next generation sequencing demonstrated that TGF-ß-pathway related genes were selectively down-regulated by treatment of GRe in vivo, and the same treatment suppressed TGF-ß-induced phosphorylation of ERK in HeLa cells. The results clearly indicated that GRe is the effective constituent in the ginseng on hair promotion via selective inhibition of the hair growth phase transition related signaling pathways, TGF-ß signaling cascades.

Combined inhibition of vascular endothelial growth factor receptor signaling with temozolomide enhances cytotoxicity against human glioblastoma cells via downregulation of Neuropilin-1.

Glioblastoma multiforme (GBM) is the most common and aggressive type of primary brain tumor with grave prognosis. Despite the growing understanding of the complex signaling networks responsible for the initiation and progression of GBM, many experimental therapies have fallen short of their treatment goals. In the present study, we investigated the novel molecular mechanisms responsible for synergistic action of temozolomide (TMZ) and anti-VEGF therapy in GBM cells. We tested the combined effects of TMZ and VEGF blockade in four human GBM cell lines: TMZ-sensitive U251-MG and U373-MG cells, and TMZ-resistant CRT-MG and LN215-MG cells, which correlated with MGMT promoter methylation status. Treatment of TMZ along with a sublethal dosage range of SU1498, a chemical inhibitor of the VEGF receptor signaling, induced significant cell death in both TMZ-sensitive and TMZ-resistant GBM cells without changing the status of the MGMT promoter methylation. Treatment with TMZ specifically reduced the expression of NRP-1, a coreceptor of VEGF but not those of VEGF-R1 and VEGF-R2. We further confirmed the key role of NRP-1 by showing that the reduction of NRP-1 by siRNA also increased the SU1498-induced cytotoxicity of LN215-MG. These results collectively indicate that combined treatment of TMZ can sensitize GBM cells to blockade of autocrine VEGF signaling through specific down-regulation of NRP-1, which provide a rationale for further evaluation and a potential clinical trial of combinatorial therapy of TMZ and SU1498 or other VEGF inhibitors for intractable brain tumors.

Critical role of TRIF and MyD88 in Mycobacterium tuberculosis Hsp70-mediated activation of dendritic cells.

As a potent immune regulator, heat shock protein 70 derived from Mycobacterium tuberculosis (Mtb Hsp70) has adjuvant effect and activates immune cells such as macrophages and dendritic cells (DCs). Although Toll-like receptors (TLRs) are known to involve in DCs activation by Mtb Hsp70, there is still a controversy and the underlying mechanism is not well understood. In this study, we examined whether TRIF and MyD88, the core adaptor molecules for TLRs signaling, regulate Mtb Hsp70-induced DCs activation. Although Mtb Hsp70 produced substantial level of cytokines (IL-6, IL-12p40, and TNF-α) in TRIF-deficient DCs in a dose-dependent manner, each level was significantly lower than that in WT cells. The cytokines production was almost abolished in MyD88-deficient DCs. Consistent with cytokine results, Mtb Hsp70-induced activation of NF-κB and MAPKs was also impaired in both TRIF- and MyD88-deficient DCs, as compared with WT cells. Inhibitor assay revealed that NF-κB, ERK, and JNK, but not p38, regulate Mtb Hsp70-induced production of cytokines. In addition, the up-regulation of co-stimulatory molecules and MHC class II was mostly TRIF-dependent in DCs in response Mtb Hsp70, whereas MyD88 was only partially involved. Finally, mixed leukocytes reaction (MLR) assay revealed that both TRIF and MyD88 are critical for DCs ability promoted by Mtb Hsp70 to differentiate naïve T cells into effector T cells of producing IFN-γ. Our findings suggest that both TRIF and MyD88 are essential for the activation and maturation of DCs in response to Mtb Hsp70.

Tumor-conditioned Gr-1(+)CD11b(+) myeloid cells induce angiogenesis through the synergistic action of CCL2 and CXCL16 in vitro.

Gr-1(+)CD11b(+) cells can suppress innate and adaptive immunity, and the functional immunosuppressive characteristics of these cells can be modulated by the tumor microenvironment. Since Gr-1(+)CD11(+) cells are also involved in tumor-associated angiogenesis, we hypothesized that the angiogenic nature of Gr-1(+)CD11b(+) cells could be regulated by the tumor milieu. To address this hypothesis, we imitated a tumor microenvironment by exposing Gr-1(+)CD11b(+) cells isolated from spleen of 4T1 mammary carcinoma-bearing mice to tumor-conditioned medium. Supernatants from tumor-conditioned Gr-1(+)CD11b(+) cells significantly induced capillary-like tube formation and migration of human umbilical vein endothelial cells (HUVECs) compared to naive Gr-1(+)CD11b(+) cells. Incubation of Gr-1(+)CD11b(+) cells with tumor-conditioned medium induced production of pro-angiogenic chemokines CCL2 and CXCL16. Pretreatment with an anti-CCL2 antibody, but not an anti-CXCL16 antibody, suppressed the angiogenic effects of tumor-conditioned Gr-1(+)CD11b(+) cells on HUVECs. Simultaneous neutralization of CCL2 and CXCL16 significantly inhibited tube formation and migration of HUVECs compared to the sole neutralization against CCL2. Supernatants from tumor-conditioned Gr-1(+)CD11b(+) cells induced phosphorylation of ERK1/2 in HUVECs, and inhibition of the ERK pathway blocked angiogenic effects. ERK pathway activity was partially abrogated by neutralization of CCL2 and more suppressed by simultaneous neutralization of CCL2 and CXCL16. These results collectively indicate that CCL2 and CXCL16 chemokines produced by tumor-conditioned Gr-1(+)CD11b(+) myeloid cells synergistically induce angiogenesis in vitro by stimulating the ERK1/2 signaling pathway. Thus, regulation of Gr-1(+)CD11b(+) cells in the tumor microenvironment may contribute to angiogenesis through the secretion of pro-angiogenic chemokines.

Hepatitis C virus infection enhances TNFα-induced cell death via suppression of NF-κB.

UNLABELLED: Hepatitis C virus (HCV) infection results in liver injury and long-term complications, such as liver cirrhosis and hepatocellular carcinoma. Liver injury in HCV infection is believed to be caused by host immune responses, not by viral cytopathic effects. Tumor necrosis factor-alpha (TNF-α) plays a pivotal role in the inflammatory processes of hepatitis C. TNF-α induces cell death that can be ameliorated by nuclear factor kappaB (NF-κB) activation. We investigated the regulation of TNF-α signal transduction in HCV-infected cells and identified HCV proteins responsible for sensitization to TNF-α-induced cell death. We studied the effect of HCV infection on TNF-α signal transduction using an in vitro HCV infection model (JFH-1, genotype 2a) with Huh-7 and Huh-7.5 cells. We found that TNF-α-induced cell death significantly increased in HCV-infected cells. HCV infection diminished TNF-α-induced phosphorylation of IκB kinase (IKK) and inhibitor of NF-κB (IκB), which are upstream regulators of NF-κB activation. HCV infection also inhibited nuclear translocation of NF-κB and expression of NF-κB-dependent anti-apoptotic proteins, such as B-cell lymphoma--extra large (Bcl-xL), X-linked inhibitor of apoptosis protein (XIAP), and the long form of cellular-FLICE inhibitory protein (c-FLIP). Decreased levels of Bcl-xL, XIAP, and c-FLIP messenger RNA and protein were also observed in livers with chronic hepatitis C. Transfection with plasmids encoding each HCV protein revealed that core, nonstructural protein (NS)4B, and NS5B attenuated TNF-α-induced NF-κB activation and enhanced TNF-α-induced cell death. CONCLUSION: HCV infection enhances TNF-α-induced cell death by suppressing NF-κB activation through the action of core, NS4B, and NS5B. This mechanism may contribute to immune-mediated liver injury in HCV infection.

Liver resection for metastatic melanoma: equivalent survival for cutaneous and ocular primaries.

BACKGROUND: The value of surgical resection in patients with hepatic metastases from melanoma is poorly documented in the literature. This study sought to determine the clinicopathologic and surgical factors predictive of outcome for melanoma patients who underwent resection of hepatic metastases. METHODS: Thirty-three patients who underwent liver resection for melanoma metastases were identified from the Melanoma Institute Australia research database. Univariate and multivariate analyses were performed to identity factors predictive of recurrence and survival following liver resection for melanoma metastasis. RESULTS: The actuarial 2- and 5-year survival rates were 59% and 42%, respectively, with a median survival of 29 months (range 1-139). The 5-year survival rates for cutaneous and ocular primary melanoma were 44% and 39%, respectively. Improved post-hepatic metastasectomy survival was observed in patients with microscopically clear resection margins (R0, 44 months; R1/2, 12 months; P = 0.04). Although major hepatic resection was associated with improved survival (major, 70 months; minor, 23 months; P = 0.07), major hepatectomies were performed almost exclusively in patients with isolated liver metastases. CONCLUSIONS: Hepatic resection for metastatic melanoma is associated with improved survival in selected patients with both primary ocular and cutaneous melanoma. Surgical treatment of hepatic melanoma metastases should be considered when complete resection is feasible.

A systematic search for endoplasmic reticulum (ER) membrane-associated RING finger proteins identifies Nixin/ZNRF4 as a regulator of calnexin stability and ER homeostasis.

To identify novel regulators of endoplasmic reticulum (ER)-linked protein degradation and ER function, we determined the entire inventory of membrane-spanning RING finger E3 ubiquitin ligases localized to the ER. We identified 24 ER membrane-anchored ubiquitin ligases and found Nixin/ZNRF4 to be central for the regulation of calnexin turnover. Ectopic expression of wild type Nixin induced a dramatic down-regulation of the ER-localized chaperone calnexin that was prevented by inactivation of the Nixin RING domain. Importantly, Nixin physically interacts with calnexin in a glycosylation-independent manner, induces calnexin ubiquitination, and p97-dependent degradation, indicating an ER-associated degradation-like mechanism of calnexin turnover.

Endoplasmic reticulum-specific BH3-only protein BNIP1 induces mitochondrial fragmentation in a Bcl-2- and Drp1-dependent manner.

Bcl-2/adenovirus E1B 19-kDa interacting protein 1 (BNIP1), which is predominantly localized to the endoplasmic reticulum (ER), is a pro-apoptotic Bcl-2 homology domain 3 (BH3)-only protein. Here, we show that the expression of BNIP1 induced not only a highly interconnected ER network but also mitochondrial fragmentation in a BH3 domain-dependent manner. Functional analysis demonstrated that BNIP1 expression increased dynamin-related protein 1 (Drp1) expression followed by the mitochondrial translocation of Drp1 and subsequent mitochondrial fission. Both BNIP1-induced mitochondrial fission and the translocation of Drp1 were abrogated by Bcl-2 overexpression. These results collectively indicate that ER-specific BNIP1 plays an important role in mitochondrial dynamics by modulating the mitochondrial fission protein Drp1 in a BH3 domain-dependent fashion.

Pirfenidone inhibits transforming growth factor-ß1-induced fibrogenesis by blocking nuclear translocation of Smads in human retinal pigment epithelial cell line ARPE-19.

PURPOSE: Transforming growth factor-ß (TGF-ß) plays a key role in transforming retinal pigment epithelial (RPE) cells into mesenchymal fibroblastic cells, which are implicated in proliferative vitreoretinopathy. Herein, we tested the effect of pirfenidone, a novel antifibrotic agent, on TGF-ß1-mediated fibrogenesis in the human RPE cell line ARPE-19. METHODS: The effect of pirfenidone on the TGF-ß1-induced phenotype in ARPE-19 cells was measured with immunocytochemistry as the change in F-actin. Fibronectin and collagen production was measured with enzyme-linked immunosorbent assay, and cell migration activity was investigated using a scratch assay. Immunoblot analyses of cofilin, sma and mad protein (smad) 2/3, p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and extracellular signal-related kinase expression were conducted to elucidate the cell signaling networks that contribute to the antifibrotic effect of pirfenidone. RESULTS: Treatment with TGF-ß1 induced typical phenotypic changes such as formation of stress fiber running parallel to the long axis of cells and enhanced migration and production of extracellular matrix components such as collagen type I and fibronectin. This fibroblast-like phenotype induced by TGF-ß1 was significantly inhibited by pretreatment with pirfenidone in a dose-dependent manner. We also elucidated the TGF-ß signaling pathways as the target of the inhibitory effect of pirfenidone. Pirfenidone inhibited TGF-ß signaling by preventing nuclear accumulation of active Smad2/3 complexes rather than phosphorylation of Smad2/3. CONCLUSIONS: These results collectively provide a rational background for future evaluation of pirfenidone as a potential antifibrotic agent for treating proliferative vitreoretinopathy and other fibrotic retinal disorders.

Reliable permeability assay system in a microfluidic device mimicking cerebral vasculatures.

Since most of the bioavailable drugs are impermeable through the blood-brain barrier (BBB), development of a rapid and reliable permeability assay system has been a challenge in drug discovery targeting central nervous system (CNS). Here, we designed a microfluidic device to monitor the drug permeability into the CNS. Human umbilical vein endothelial cells (HUVECs) were shortly (2 ~ 3 h) incubated with astrocyte-conditioned medium after being trapped on microholes in the microfluidic device and tested for chip-based permeability measurement of drugs. The measured permeability values were highly correlated with those measured by conventional in vitro methods and the brain uptake index representing the quantity of transported substances across the in vivo BBB of rats. Using the microfluidic device, we could easily monitor the effect of hydrogen peroxide on the trans-endothelial permeability, which are consistent with the finding that the same treatment disrupted the formation of tight junctions between endothelial cells. Considering relatively short period of time needed for endothelial cell culture and ability to monitor the BBB physiology continuously, we propose that this novel system can be used as an invaluable first-line tool for CNS-related drug development.

Manufacturing Therapeutic Exosomes: from Bench to Industry.

Process of manufacturing therapeutics exosome development for commercialization. The development of exosome treatment starts at the bench, and in order to be commercialized, it goes through the manufacturing, characterization, and formulation stages, production under Good Manufacturing Practice (GMP) conditions for clinical use, and close consultation with regulatory authorities. Exosome, a type of nanoparticles also known as small extracellular vesicles are gaining attention as novel therapeutics for various diseases because of their ability to deliver genetic or bioactive molecules to recipient cells. Although many pharmaceutical companies are gradually developing exosome therapeutics, numerous hurdles remain regarding manufacture of clinical-grade exosomes for therapeutic use. In this mini-review, we will discuss the manufacturing challenges of therapeutic exosomes, including cell line development, upstream cell culture, and downstream purification process. In addition, developing proper formulations for exosome storage and, establishing good manufacturing practice facility for producing therapeutic exosomes remains as challenges for developing clinicalgrade exosomes. However, owing to the lack of consensus regarding the guidelines for manufacturing therapeutic exosomes, close communication between regulators and companies is required for the successful development of exosome therapeutics. This review shares the challenges and perspectives regarding the manufacture and quality control of clinical grade exosomes.

SiO2 doped Ge2Sb2Te5 thin films with high thermal efficiency for applications in phase change random access memory.

This study examined the various physical, structural and electrical properties of SiO(2) doped Ge(2)Sb(2)Te(5) (SGST) films for phase change random access memory applications. Interestingly, SGST had a layered structure (LS) resulting from the inhomogeneous distribution of SiO(2) after annealing. The physical parameters able to affect the reset current of phase change memory (I(res)) were predicted from the Joule heating and heat conservation equations. When SiO(2) was doped into GST, thermal conductivity largely decreased by ∼ 55%. The influence of SiO(2)-doping on I(res) was examined using the test phase change memory cell. I(res) was reduced by ∼ 45%. An electro-thermal simulation showed that the reduced thermal conductivity contributes to the improvement of cell efficiency as well as the reduction of I(res), while the increased dynamic resistance contributes only to the latter. The formation and presence of the LS thermal conductivity in the set state test cell after repeated switching was confirmed.

Optic atrophy 3 as a protein of the mitochondrial outer membrane induces mitochondrial fragmentation.

The optic atrophy 3 (OPA3) gene, which has no known homolog or biological function, is mutated in patients with hereditary optic neuropathies. Here, we identified OPA3 as an integral protein of the mitochondrial outer membrane (MOM), with a C-terminus exposed to the cytosol and an N-terminal mitochondrial targeting domain. By quantitative analysis, we demonstrated that overexpression of OPA3 significantly induced mitochondrial fragmentation, whereas OPA3 knockdown resulted in highly elongated mitochondria. Cells with mitochondria fragmented by OPA3 did not undergo spontaneous apoptotic cell death, but were significantly sensitized to staurosporine- and TRAIL-induced apoptosis. In contrast, overexpression of a familial OPA3 mutant (G93S) induced mitochondrial fragmentation and spontaneous apoptosis, suggesting that OPA3 mutations may cause optic atrophy via a gain-of-function mechanism. Together, these results indicate that OPA3, as an integral MOM protein, has a crucial role in mitochondrial fission, and provides a direct link between mitochondrial morphology and optic atrophy.

Single-Setting Superior Vena Cava Biopsy and Stenting Utilizing Cone Beam Computed Tomography as an Additional Tool.

BACKGROUND Malignant disease is a common etiology of superior vena cava syndrome (SVCS). Being a medical emergency, it often requires rapid diagnostic evaluation and therapy. Transcaval biopsy and endovascular stenting in a single-setting has been described, but only in a handful of cases. These cases utilized intra-operative venograms. In this study, we also used intra-operative cone beam computed tomography (CBCT) to increase the safety and efficacy of such single-setting procedures. CASE REPORT From January 2017 to July 2019, there were 5 patients with malignant SVCS who underwent single-setting superior vena cava biopsy and endovascular stenting utilizing intra-operative CBCT as an adjunct. Demographic data, clinical presentation, investigation results, procedural details, and patient outcomes were recorded. CBCT was utilized in all cases to optimize sampling of biopsies, visualize subsequent stent positioning, and for early detection of procedure-related complications. Transcaval biopsy was diagnostic in 4 of the 5 patients. Endovascular stents were deployed successfully in all cases, with post-stenting venogram demonstrating relief of prior obstructed segments. One patient had a complication of an apical pneumothorax, with no associated long-term pneumothorax-related morbidity or mortality. CONCLUSIONS This study demonstrates that single-setting transcaval biopsy and stenting in the context of malignant SVCS is a cost-efficient, safe, and feasible approach. In addition, the additional use of intra-operative CBCT is a useful tool to increase procedure efficacy and safety.

Long-term treatment of farnesyltransferase inhibitor FTI-277 induces neurotoxicity of hippocampal neurons from rat embryo in a ROS-dependent manner.

Despite the well established anti-cancer effect of farnesyltransferase inhibitor FTI-277, the neurotoxic effects of the agent are not yet clearly defined at the molecular and cellular levels. Here, we report the neurotoxic effects of FTI-277 and the involvement of reactive oxygen species (ROS) in FTI-induced neurotoxicity. Although there is no significant effect of FTI-277 for 2 days, long-term treatment of FTI-277 for 4 days induced dramatic reduction in outgrowth, maturation and branching of neuritis and considerable cytoxicity in a dose- and time-dependent manner in primary cultured rat embryo hippocampal neurons. Interestingly, FTI-277 for 4 days dramatically decreased expression of synapsin I, a crucial molecule involved in the neuronal growth and plasticity, and increased a cytotoxic G-protein RhoB of which ectopic expression induced the neurotoxicity in hippocampal neurons. Moreover, treatment with FTI-277 dramatically increased intracellular levels of ROS, which was sustained for 4 days; while blockage of ROS rescued FTI-277-induced neurotoxicity as well as both decrease of synapsin I and increase of RhoB. Taken together, these results provide the molecular insights for the mechanisms which might be of use aiming for avoiding neurotoxic side effects by FTI agent for a drug development for a clinical use.

Imaging features of extramedullary plasmacytoma.

Extramedullary plasmacytomas are rare and can occur at any site. The imaging results are often nonspecific. This report highlights the radiographic, sonographic and cross-sectional imaging features of extramedullary plasmacytomas.

Case report of male breast cancer detected on magnetic resonance imaging.

Male breast cancers (MBC) are rare, accounting for <1% of all breast cancers. The use of magnetic resonance imaging (MRI) for male breast cancer is not generally indicated. We present an unusual case where conventional imaging for a suspected MBC was equivocal and MRI was required to assist in diagnosis.