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Colloidal gelation is used to form processable soft solids from a wide range of functional materials. Although multiple gelation routes are known to create gels of different types, the microscopic processes during gelation that differentiate them remain murky. A fundamental question is how the thermodynamic quench influences the microscopic driving forces of gelation, and determines the threshold or minimal conditions where gels form. We present a method that predicts these conditions on a colloidal phase diagram, and mechanistically connects the quench path of attractive and thermal forces to the emergence of gelled states. Our method employs systematically varied quenches of a colloidal fluid over a range of volume fractions to identify minimal conditions for gel solidification. The method is applied to experimental and simulated systems to test its generality toward attractions with varied shapes. Using structural and rheological characterization, we show that all gels incorporate elements of percolation, phase separation, and glassy arrest, where the quench path sets their interplay and determines the shape of the gelation boundary. We find that the slope of the gelation boundary corresponds to the dominant gelation mechanism, and its location approximately scales with the equilibrium fluid critical point. These results are insensitive to potential shape, suggesting that this interplay of mechanisms is applicable to a wide range of colloidal systems. By resolving regions of the phase diagram where this interplay evolves in time, we elucidate how programmed quenches to the gelled state could be used to effectively tailor gel structure and mechanics.

To overcome the hurdles of immunotherapy, we investigated whether calcipotriol, a synthetic vitamin D analog, could overcome the immune evasion of glioblastoma multiforme (GBM) by modulating immune responses and the immunosuppressive tumor microenvironment. Administration of calcipotriol considerably reduced tumor growth. Both in vivo and in vitro studies revealed that CD8+T and natural killer (NK) cell gene signatures were enriched and activated, producing high levels of IFN-γ and granzyme B. In contrast, regulatory T cells (Treg) were significantly reduced in the calcipotriol-treated group. The expression of CD127, the receptor for thymic stromal lymphopoietin (TSLP), is elevated in CD4+T cells and potentially supports T-cell priming. Depleting CD4+T cells, but not NK or CD8+T cells, completely abrogated the antitumor efficacy of calcipotriol. These data highlight that the calcipotriol/TSLP/CD4+T axis can activate CD8+T and NK cells with a concomitant reduction in the number of Tregs in GBM. Therefore, calcipotriol can be a novel therapeutic modality to overcome the immune resistance of GBM by converting immunologically "cold" tumors into "hot" tumors. DATA AVAILABILITY: Data are available upon reasonable request. The RNA-seq dataset comparing the transcriptomes of control and calcipotriol-treated GL261 tumors is available from the corresponding author upon request., Competing Interests: Conflict of interest statement All authors declare that they have no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Modulating the interaction potential between colloids suspended in a fluid can trigger equilibrium phase transitions as well as the formation of non-equilibrium "arrested states," such as gels and glasses. Faithful representation of such interactions is essential for using simulation to interrogate the microscopic details of non-equilibrium behavior and for extrapolating observations to new regions of phase space that are difficult to explore in experiments. Although the extended law of corresponding states predicts equilibrium phases for systems with short-ranged interactions, it proves inadequate for equilibrium predictions of systems with longer-ranged interactions and for predicting non-equilibrium phenomena in systems with either short- or long-ranged interactions. These shortcomings highlight the need for new approaches to represent and disambiguate interaction potentials that replicate both equilibrium and non-equilibrium phase behavior. In this work, we use experiments and simulations to study a system with long-ranged thermoresponsive colloidal interactions and explore whether a resolution to this challenge can be found in regions of the phase diagram where temporal effects influence material state. We demonstrate that the conditions for non-equilibrium arrest by colloidal gelation are sensitive to both the shape of the interaction potential and the thermal quench rate. We exploit this sensitivity to propose a kinetics-based algorithm to extract distinct arrest conditions for candidate potentials that accurately selects between potentials that differ in shape but share the same predicted equilibrium structure. The algorithm selects the candidate that best matches the non-equilibrium behavior between simulation and experiments. Because non-equilibrium behavior in simulation is encoded entirely by the interparticle potential, the results are agnostic to the particular mechanism(s) by which arrest occurs, and so we expect our method to apply to a range of arrested states, including gels and glasses. Beyond its utility in constructing models, the method reveals that each potential has a quantitatively distinct arrest line, providing insight into how the shape of longer-ranged potentials influences the conditions for colloidal gelation.

X-linked inhibitor of apoptosis-associated factor 1 (XAF1) is a pro-apoptotic tumor suppressor that is frequently inactivated in multiple human cancers. However, its candidacy as a suppressor in the pathogenesis of breast cancer remains undefined. Here, we report that XAF1 acts as a molecular switch in estrogen (E2)-mediated cell-fate decisions favoring apoptosis over cell proliferation. XAF1 promoter hypermethylation is observed predominantly in estrogen receptor α (ERα)-positive versus ERα-negative tumor cells and associated with attenuated apoptotic response to E2. XAF1 is activated by E2 through a G protein-coupled estrogen receptor-mediated non-genomic pathway and induces ERα degradation and apoptosis while it is repressed by ERα for E2 stimulation of cell proliferation. The XAF1-ERα mutual antagonism dictates the outcomes of E2 signaling and its alteration is linked to the development of E2-resistant tumors. Mechanistically, XAF1 destabilizes ERα through the assembly of breast cancer-associated gene 1 (BRCA1)-mediated destruction complex. XAF1 interacts with ERα and BRCA1 via the zinc finger (ZF) domains 5/6 and 4, respectively, and the mutants lacking either of these domains fail to drive ERα ubiquitination and apoptosis. E2-induced regression of XAF1 +/+ tumors is abolished by XAF1 depletion while XAF1 -/- tumors recover E2 response by XAF1 restoration. XAF1 and ERα expression show an inverse correlation in primary breast tumors, and XAF1 expression is associated with the overall survival of patients with ERα-positive but not ERα-negative cancer. Together, this study uncovers an important role for the XAF1-ERα antagonism as a linchpin to govern E2-mediated cell-fate decisions, illuminating the mechanistic consequence of XAF1 alteration in breast tumorigenesis., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: X-linked inhibitor of apoptosis-associated factor 1 (XAF1) is a tumor suppressor that is commonly inactivated in multiple human cancers. However, its role in the pathogenesis and therapeutic response of glioma is poorly characterized., Methods: XAF1 activation by temozolomide (TMZ) and its effect on TMZ cytotoxicity were defined using luciferase reporter, flow cytometry, and immunofluorescence assays. Signaling mechanism was analyzed using genetic and pharmacologic experiments. In vivo studies were performed in mice to validate the role of XAF1 in TMZ therapy., Results: Epigenetic alteration of XAF1 is frequent in cell lines and primary tumors and contributes to cancer cell growth. XAF1 transcription is activated by TMZ via JNK-IRF-1 signaling to promote apoptosis while it is impaired by promoter hypermethylation. In tumor cells expressing high O 6 -methylguanine-DNA methyltransferase (MGMT), XAF1 response to TMZ is debilitated. XAF1 facilitates TMZ-mediated autophagic flux to direct an apoptotic transition of protective autophagy. Mechanistically, XAF1 is translocated into the mitochondria to stimulate reactive oxygen species (ROS) production and ataxia telangiectasia mutated (ATM)-AMP-activated protein kinase (AMPK) signaling. A mutant XAF1 lacking the zinc finger 6 domain fails to localize in the mitochondria and activate ROS-ATM-AMPK signaling and autophagy-mediated apoptosis. XAF1-restored xenograft tumors display a reduced growth rate and enhanced therapeutic response to TMZ, which is accompanied with activation of ATM-AMPK signaling. XAF1 expression is associated with overall survival of TMZ treatment patients, particularly with low MGMT cancer., Conclusions: This study uncovers an important role for the XAF1-ATM-AMPK axis as a linchpin to govern glioma response to TMZ therapy., (© The Author(s) 2022. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Chemical strengthening is considered as the most suitable method for strengthening thin glass sheets used in mobile phones. The spray method of chemical strengthening requires a slurry to be sprayed on the glass sample to be strengthened. This slurry is prepared by mixing various compounds. In this study, the influence of α-Al 2 O 3 particle size in the slurry on the physical properties of the chemically strengthened glass prepared by the spray method was investigated. The compressive stress (CS) was dramatically enhanced as the particle size of Al 2 O 3 in the slurry decreased. The glass sample with 13 nm α-Al 2 O 3 including the KNO 3 -Al 2 O 3 slurry exhibited the highest CS of 905 MPa, and the depth-of-layer (DOL) of this sample was 37 μm. The same slurry composition also achieved the highest bending strength of 640 MPa under different heat treatment conditions. The optimization of the heat treatment conditions, such as temperature and time duration, resulted in the highest value of CS (916 MPa) obtained for the sample heated at 400 °C for 1.5 h, and the maximum DOL (65 μm) was obtained for the sample at 480 °C for 4 h., Competing Interests: The authors declare no competing financial interest.

Aims: Aggressive meningioma remains incurable with neither chemo- nor targeted therapies proven effective, largely due to unidentified genetic alterations and/or aberrant oncogenic pathways driving the disease progression. In this study, we examined the expression and function of Forkhead box M1 (FOXM1) transcription factor during meningioma progression., Methods: Human meningioma samples (n = 101) were collected, followed by Western blotting, quantitative PCR, immunohistochemical and progression-free survival (PFS) analyses. For in vitro assays, FOXM1 was overexpressed or knocked-down in benign (SF4433 and SF4068) or malignant (SF3061 and IOMM-Lee) human meningioma cell lines respectively. For in vivo studies, siomycin A (a FOXM1 inhibitor)-pretreated or control IOMM-Lee cells were implanted subcutaneously in nude mice., Results: FOXM1 expression was increased in higher grades of meningioma and correlated with the mitotic index in the tumour tissue. Moreover, FOXM1 was increased in recurrent meningioma compared with the matched primary lesions. The patients who had higher FOXM1 expression had shorter PFS. In the subsequent in vitro assays, knockdown of FOXM1 in malignant meningioma cell lines resulted in decreased tumour cell proliferation, angiogenesis and invasion, potentially via regulation of β-catenin, cyclin D1, p21, interleukin-8, vascular endothelial growth factor-A, PLAU, and epithelial-to-mesenchymal transition-related genes, whereas overexpression of FOXM1 in benign meningioma cell lines had the opposite effects. Last, suppression of FOXM1 using a pharmacological inhibitor, siomycin A, decreased tumour growth in an in vivo mouse model., Conclusions: Our data demonstrate that FOXM1 is a key transcription factor regulating oncogenic signalling pathways in meningioma progression, and a promising therapeutic target for aggressive meningioma., (© 2019 British Neuropathological Society.)

Despite the presence of aggressive treatment strategies, glioblastoma remains intractable, warranting a novel therapeutic modality. An oral antipsychotic agent, penflurido (PFD), used for schizophrenia treatment, has shown an antitumor effect on various types of cancer cells. As glioma sphere-forming cells (GSCs) are known to mediate drug resistance in glioblastoma, and considering that antipsychotics can easily penetrate the blood-brain barrier, we investigated the antitumor effect of PFD on patient-derived GSCs. Using five GSCs, we found that PFD exerts an antiproliferative effect in a time- and dose-dependent manner. At IC50, spheroid size and second-generation spheroid formation were significantly suppressed. Stemness factors, SOX2 and OCT4, were decreased. PFD treatment reduced cancer cell migration and invasion by reducing the Integrin α6 and uPAR levels and suppression of the expression of epithelial-to-mesenchymal transition (EMT) factors, vimentin and Zeb1. GLI1 was found to be involved in PFD-induced EMT inhibition. Furthermore, combinatorial treatment of PFD with temozolomide (TMZ) significantly suppressed tumor growth and prolonged survival in vivo. Immunostaining revealed decreased expression of GLI1, SOX2, and vimentin in the PFD treatment group but not in the TMZ-only treatment group. Therefore, PFD can be effectively repurposed for the treatment of glioblastoma by combining it with TMZ., Competing Interests: All authors declare no financial conflict of interest.

Antimicrobial agents based on organic materials have limited use owing to their low heat resistance and short lifetimes. Therefore, various studies on antibacterial agents that are based on inorganic material systems are increasingly being performed to supplement them. In this study, Cu@SiO₂ core-shells are fabricated using Cu cores and SiO₂ shells, and are known to have antimicrobial effects. The core-shell was coated with SiO₂ using the sol-gel method. Experiments were conducted using X-ray diffraction and the shaking flask method (KS J 4206) to evaluate the characteristics of the core-shell. In the case of X-ray diffraction, both the Cu core and the core-shell fabricated using the sol-gel process were characterized. Escherichia coli and Staphylococcus aureus were evaluated using the KS J 4206 method for their antibacterial properties. Through this study, it is confirmed that a Cu@SiO₂ core-shell can be fabricated via the sol-gel method, and that Cu with a core-shell structure has antibacterial effects.

V₂O 5 -P₂O 5 -TeO₂, a low-temperature vanadate-based glass sealant, was doped with metal oxides (MO = Ag₂O, BaO, or CuO), which generate Ag, Ba, and Cu ions, respectively, to strengthen the glass structure and improve its water resistance. These ions reduce the number of nonbridging oxygen atoms in the glass structure by forming V-O-M or P-O-M crosslinks in the V₂O 5 -P₂O 5 glass system. Structural analysis using Fourier-transform infrared spectroscopy indicated that the numbers of P-O-P, V═O, and V-O-V bonds decreased with increasing metal oxide content. Thermal property analyses revealed that the glass transition temperatures increased by approximately 2-30 °C and that the coefficients of thermal expansion only varied within approximately ±10×10 -7 K -1 among all the glass samples. The contact angles were measured to quantify the wetting properties of the doped glasses. The contact angle increased from 11 to 36° with increasing metal oxide content at 410 °C. As an indication of the water resistances of the doped glasses, the dissolution rates of the 9 mol% Ag₂O-doped and pure glasses were 0.078 and 0.523 g cm -2 , respectively.

As with any solvent, stabilizing a multivalent element at a given oxidation state in glass depends on the thermodynamic conditions. The effects of temperature on the oxidation-reduction equilibrium have been previously noted with higher temperatures being more conducive to reduced states. Herein, 30CeO₂-70P₂O 5 binary system glasses were prepared. The melting temperature and time dependency on Ce 4+ and Ce 3+ ion concentrations were studied using X-ray photoelectron spectroscopy. Different melting conditions were investigated at temperatures ranging from 1300 °C to 1500 °C for 60 min, and at 1400 °C for durations ranging from 30 min to 90 min. The changes in the catalytic properties of the glasses as a function of Ce 4+ and Ce 3+ ion concentrations were confirmed based on the changes in the decomposition starting temperatures using thermogravimetric analysis. The main changes in the oxidation states according to melting conditions were discussed in terms of the catalytic properties of CeO₂-P₂O 5 glass systems.

We investigate how the aspect ratio of micropillar or microwell arrays patterned on a surface affects the rolling and slipping motion of spheres under flooded conditions at low Reynolds numbers. We study arrays of rigid microstructures with aspect ratios varying over two orders of magnitude for surface coverages ranging from 0.04 to 0.96. We investigate how the surface features (dimensions, surface coverage, and geometry) individually impact the motion of the sphere. We find that increasing microstructure height results in higher rotational velocities on all studied surfaces. We then model the motion of the spheres using two physical parameters: an effective separation and a coefficient of friction between the sphere and the incline. We find that a simple superposition of resistance functions, previously shown to accurately predict the motion of spheres for different surface coverages and geometries, indeed shows good agreement with experimental outcomes for all microstructure heights studied. We also perform separate sliding friction measurements via a force microscope to measure the coefficient of friction between the sphere and incline, under identically flooded conditions. A comparison of the sliding friction measurements at different Hersey numbers suggests that the effect of the microstructure on the coefficient of friction becomes more important as the Hersey number increases.

X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is a proapoptotic tumor suppressor that is frequently inactivated in multiple human cancers. However, the molecular basis for the XAF1-mediated growth inhibition remains largely undefined. Here, we report that XAF1 forms a positive feedback loop with interferon regulatory factor-1 (IRF-1) and functions as a transcriptional coactivator of IRF-1 to suppress tumorigenesis. Under various stressful conditions, XAF1 transcription is activated by IRF-1, and elevated XAF1 stabilizes and activates IRF-1. Mechanistically, XAF1 binds to the multifunctional domain 2 of IRF-1 via the zinc finger domain 6, thereby hindering C-terminus of Hsc70-interacting protein (CHIP) interaction with and ubiquitination of IRF-1. Activation of the IRF-1-XAF1 loop greatly increases stress-induced apoptosis and decreases the invasive capability of tumor cells. Oncogenic Ras and growth factors interfere with the IRF-1-XAF1 interplay via Erk-mediated repression of XAF1 transcription. Furthermore, XAF1 enhances IRF-1-mediated transcription of proapoptotic genes via the XAF1-IRF-1 complex formation on these target promoters. Meanwhile, XAF1 inhibits NF-κB-mediated tumor cell malignancy by reinforcing IRF-1 binding to a subset of coregulated promoters. Expression levels of IRF-1 and XAF1 correlate tightly in both cancer cell lines and primary tumors, and XAF1-induced tumor regression is markedly attenuated in IRF-1-depleted tumors. Collectively, this study identifies a novel mechanism of XAF1-mediated tumor suppression, uncovering XAF1 as a feedback coactivator of IRF-1 under stressful conditions.

In organic photovoltaics (OPVs), determining the energy-level alignment of a donor and an acceptor is particularly important since the interfacial energy gap between the highest occupied molecular orbital (HOMO) level of a donor and the lowest unoccupied molecular orbital (LUMO) level of an acceptor (E-E) gives the theoretical maximum value of the open-circuit voltage (V OC ). To increase the E-E, non-fullerene acceptors, which have a lower electron affinity (EA) than C 60 , are receiving increasing attention. In this study, we investigated the energy-level alignment at the interface of a boron chloride subphthalocyanine (SubPc) donor and a halogenated SubPc (Cl 6 SubPc) acceptor using soft X-ray spectroscopy techniques. The estimated E-E of Cl 6 SubPc/SubPc was 1.95 eV, which was significantly higher than that of 1.51 eV found at the interface of C 60 /SubPc. This increased E-E was the origin of the enhanced V OC in OPVs. Additionally, we studied the molecular orientation of Cl 6 SubPc using angle-dependent X-ray absorption spectroscopy. The highly disordered Cl 6 SubPc molecules result in low carrier mobility, which contributes to the lower short-circuit current density of the Cl 6 SubPc acceptor OPVs than the C 60 acceptor OPVs.

Background: Expression of caveolin-1 (Cav-1) is frequently altered in many human cancers and both tumor suppression and promotion functions of Cav-1 have been suggested based on its expression status. However, it remains unanswered how Cav-1 provokes opposite effects in different cancers or different phases of tumor progression., Methods: To explore the implication of Cav-1 alteration in gastric tumorigenesis, the expression and mutational status of Cav-1 and its effects on tumor cell growth were characterized., Results: A substantial fraction of primary tumors and cell lines displayed abnormally low or high Cav-1 mRNA expression, indicating the bidirectional alteration of Cav-1 in gastric cancers. While allelic imbalance and mutational alterations of the Cav-1 gene were rarely detected, aberrant promoter hyper- or hypo-methylation showed a tight correlation with bidirectional alteration of its expression. Abnormally low and high Cav-1 expression was more frequently observed in early and advanced cancers, respectively, suggesting the oncogenic switch of its function in tumor progression. Cell cycle progression, DNA synthesis, and colony forming ability were markedly decreased by Cav-1 transfection in low-expressing tumor cells but by its depletion in high-expressing cells. Interestingly, Cav-1 exerted opposite effects on MEK-ERK signaling in these two cell types through the reciprocal regulation of the RAF-ERK negative feedback loop. A feedback inhibition of RAF by ERK was stimulated by restoration of Cav-1 expression in low-expressing cells but by it depletion in high-expressing cells. As predicted, the opposite effects of Cav-1 on both tumor cell growth and inhibitory RAF phosphorylation were abolished if ERK is depleted., Conclusion: Bidirectional alteration of Cav-1 is linked to its opposite effects on gastric tumor cell growth, which stem from the reciprocal control on the RAF-ERK negative feedback loop.

XIAP-associated factor 1 (XAF1) is a tumor suppressor that is commonly inactivated in multiple human neoplasms. However, the molecular mechanism underlying its proapoptotic function remains largely undefined. Here, we report that XAF1 induction by heavy metals triggers an apoptotic switch of stress response by destabilizing metallothionein 2A (MT2A). XAF1 directly interacts with MT2A and facilitates its lysosomal degradation, resulting in the elevation of the free intercellular zinc level and subsequent activation of p53 and inactivation of XIAP. Intriguingly, XAF1 is activated as a unique transcription target of metal-regulatory transcription factor-1 (MTF-1) in signaling apoptosis, and its protein is destabilized via the lysosomal pathway by MTF-1-induced MT2A under cytostatic stress conditions, indicating the presence of mutual antagonism between XAF1 and MT2A. The antagonistic interplay between XAF1 and MT2A acts as a key molecular switch in MTF-1-mediated cell-fate decisions and also plays an important role in cell response to various apoptotic and survival factors. Wild-type (WT) XAF1 but not MT2A binding-deficient mutant XAF1 increases the free intracellular zinc level and accelerates WT folding of p53 and degradation of XIAP. Consistently, XAF1 evokes a more drastic apoptotic effect in p53 +/+ versus isogenic p53 -/- cells. Clinically, expression levels of XAF1 and MT2A are inversely correlated in primary colon tumors and multiple cancer cell lines. XAF1-depleted xenograft tumors display an increased growth rate and a decreased apoptotic response to cytotoxic heavy metals with strong MT2A expression. Collectively, this study uncovers an important role for XAF1-MT2A antagonism as a linchpin to govern cell fate under various stressful conditions including heavy metal exposure., Competing Interests: The authors declare no conflict of interest.

Microstructured surfaces, such as those inspired by nature, mediate surface interactions and are actively sought after to control wetting, adhesion, and friction. In particular, the rolling motion of spheres on microstructured surfaces in fluid environments is important for the transport of particles in microfluidic devices or in tribology. Here, we characterize the motion of smooth silicon nitride spheres (diameters 3-5 mm) as they roll down inclined planes decorated with hexagonal arrays of microwells and micropillars. For both types of patterned surfaces, we vary the area fraction of the micropatterned features from 0.04 to 0.96. We measure directly and independently the rotational and translational velocities of the spheres as they roll down planes with inclination angles that vary between 5 and 30°. For a given area fraction, we find that spheres have a higher translational and rotational velocity on surfaces with microwells than on micropillars. We rely on the model of Smart and Leighton [Phys. Fluids A 5, 13 (1993)] to obtain an effective gap width and coefficient of friction for all microstructured surfaces investigated. We find that the coefficient of friction is significantly higher for a surface with micropillars than that for one with microwells, likely due to the presence of interconnected drainage channels that provide additional paths for the fluid flow and favor solid-solid contact on the surface with micropillars. We find that while the effective gap width at a very low solid fraction is equal to the height of the patterned features, the effective separation decreases exponentially as the surface coverage of microstructures increases, with little measured differences between the two geometries. Superposition of resistance functions is used to relate the rapid decrease in the effective gap height with increase in the surface coverage observed in experiments.

RASSF1A is a tumor suppressor implicated in many tumorigenic processes; however, the basis for its tumor suppressor functions are not fully understood. Here we show that RASSF1A is a novel antagonist of protumorigenic RhoA activity. Direct interaction between the C-terminal amino acids (256-277) of RASSF1A and active GTP-RhoA was critical for this antagonism. In addition, interaction between the N-terminal amino acids (69-82) of RASSF1A and the ubiquitin E3 ligase Smad ubiquitination regulatory factor 1 (Smurf1) disrupted GTPase activity by facilitating Smurf1-mediated ubiquitination of GTP-RhoA. We noted that the RhoA-binding domain of RASSF1A displayed high sequence homology with Rho-binding motifs in other RhoA effectors, such as Rhotekin. As predicted on this basis, RASSF1A competed with Rhotekin to bind RhoA and to block its activation. RASSF1A mutants unable to bind RhoA or Smurf1 failed to suppress RhoA-induced tumor cell proliferation, drug resistance, epithelial-mesenchymal transition, migration, invasion, and metastasis. Clinically, expression levels of RASSF1A and RhoA were inversely correlated in many types of primary and metastatic tumors and tumor cell lines. Collectively, our findings showed how RASSF1A may suppress tumorigenesis by intrinsically inhibiting the tumor-promoting activity of RhoA, thereby illuminating the potential mechanistic consequences of RASSF1A inactivation in many cancers. Cancer Res; 76(7); 1847-59. ©2016 AACR., (©2016 American Association for Cancer Research.)

In this study, we verify the relationship between the optical properties and structure of cerium-doped zinc borophosphate glasses that have concurrence of non-bridging oxygen (NBO) and bridging oxygen (BO), Ce3+ and Ce4+, and BO3 structure and BO4 structure. We prepared cerium-doped zinc borophosphate glass with various compositions, given by xCeO2-(100-x)[50ZnO-10B2O3 -40P2O5] (x = 1 mol% to 6 mol%), and analyzed their optical band energy, glass transition temperature, crystallization temperature, density, and molar volume. Some of the techniques used for analysis were Fourier transform infrared (FT-IR) spectroscopy and X-ray photoelectron spectroscopy (XPS). In the investigated glasses, the optical band gap energy decreased from 3.28 eV to 1.73 eV. From these results, we can deduce the changes when transitions occur from BO to NBO, from Ce3+ to Ce4+, and from the BO3 structure to the BO4 structure with increasing CeO2 content using FT-IR and XPS analysis. We also verified the changes in structural and physical properties from quantitative properties such as glass transition temperature, crystallization temperature, density, and molar volume.

We investigated the effect of CeO2 content on the catalytic behavior and chemical properties of the (100 - x)(80P2O5-20Fe2O3)-xCeO2 (x = 0, 4, 8, 12, 16, 20 and 24 wt%) glass system. Using thermogravimetric analysis, we confirmed that the catalytic activity increased until a CeO2 content of 16 wt%, beyond which, it decreased. The reasons for the change in the catalytic properties of the glass samples were determined using Fourier transform infrared (FT-IR) spectroscopy, X-ray photoelectron spectroscopy (XPS), and density analyses. It was confirmed using the FT-IR and XPS-01s spectra that CeO2 acts as a network modifier in iron phosphate glass. When the CeO2 content is above 16 wt%, the number of non-bridging oxygen atoms decreases with increasing CeO2 content. For these reasons, the catalytic properties decrease when the CeO2 content is more than 16 wt%. From the dissolution rate measurements, it can be observed that cerium-iron phosphate has a high water resistance. Also, as we expected, it can be confirmed that the chemical durability is improved with increasing CeO2 content.

Recently, White light emitting diodes (WLEDs) have been studied because of many advantages such as lower energy consumption, fast response, high brightness. Glass frit has been interested in LED packages due to their superior properties such as long-term stability and permeability. To maximize the LED light emission characteristic, the glass frit was required a low firing temperature and high refractive index. We selected the bismuth-based glass due to their low melting and high refractive index. This study was investigated characteristics of glass according to the influence of the glass within Bi2O3 content and this glass characteristic change was studied the effects on the optical properties of LED package structure. The properties changes of the glass frit affect the optical property of the mixed paste. With higher contents of Bi203 glass composition, the transmittance and emission intensity of the mixed paste was increased. These results suggest that the difference in refractive index between the phosphor and glass frit is minimized, the loss of light is minimized.

Bi2Te3 thermoelectric thin films were deposited on the flexible polyimide substrates by RF magnetron co-sputtering of a Bi and a Te targets. The influence of the substrate temperature and RF power on the microstructure, chemical composition, and the thermoelectric properties of the sputtered films was investigated by using scanning electron microscopy, X-ray diffraction, energy dispersive X-ray spectroscopy, and in-plane resistivity/Seebeck coefficient measurement. It was shown that the thermoelectric properties of the films depend sensitively on the Bi/Te chemical composition ratio and the substrate temperature, and the layered structure was clearly observed from the cross section of the (00L)-oriented, nearly stoichiometric Bi2Te3 films when the substrate temperature is higher than 250 °C. As-deposited Bi2Te3 films deposited at 300 °C show the highest power factor of 0.97 mW/K(2)m and the Seebeck coefficient of -193 μV/K at 32 °C, which also have (00L) preferred orientation and the layered structure. The durability of the Bi2Te3 films on polyimide against repeated bending was also tested by monitoring the film resistance, and it was concluded that the Bi2Te3 films are applicable reliably on the curved surfaces with the radius of curvature larger than 5 mm.

X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is a tumor suppressor that is frequently inactivated in many human cancers. However, the molecular mechanism underlying its growth-inhibitory function remains largely unknown. Here, we report that XAF1 forms a positive feedback loop with p53 and acts as a molecular switch in p53-mediated cell-fate decisions favoring apoptosis over cell-cycle arrest. XAF1 binds directly to the N-terminal proline-rich domain of p53 and thus interferes with E3 ubiquitin ligase MDM2 binding and ubiquitination of p53. XAF1 stimulates homeodomain-interacting protein kinase 2 (HIPK2)-mediated Ser-46 phosphorylation of p53 by blocking E3 ubiquitin ligase Siah2 interaction with and ubiquitination of HIPK2. XAF1 also steps up the termination of p53-mediated cell-cycle arrest by activating zinc finger protein 313 (ZNF313), a p21(WAF1)-targeting ubiquitin E3 ligase. XAF1 interacts with p53, Siah2, and ZNF313 through the zinc finger domains 5, 6, and 7, respectively, and truncated XAF1 isoforms preferentially expressed in cancer cells fail to form a feedback loop with p53. Together, this study uncovers a novel role for XAF1 in p53 stress response, adding a new layer of complexity to the mechanisms by which p53 determines cell-fate decisions.

ZNF313 encoding a zinc-binding protein is located at chromosome 20q13.13, which exhibits a frequent genomic amplification in multiple human cancers. However, the biological function of ZNF313 remains largely undefined. Here we report that ZNF313 is an ubiquitin E3 ligase that has a critical role in the regulation of cell cycle progression, differentiation and senescence. In this study, ZNF313 is initially identified as a XIAP-associated factor 1 (XAF1)-interacting protein, which upregulates the stability and proapoptotic effect of XAF1. Intriguingly, we found that ZNF313 activates cell cycle progression and suppresses cellular senescence through the RING domain-mediated degradation of p21(WAF1). ZNF313 ubiquitinates p21(WAF1) and also destabilizes p27(KIP1) and p57(KIP2), three members of the CDK-interacting protein (CIP)/kinase inhibitor protein (KIP) family of cyclin-dependent kinase inhibitors, whereas it does not affect the stability of the inhibitor of CDK (INK4) family members, such as p16(INK4A) and p15(INK4B). ZNF313 expression is tightly controlled during the cell cycle and its elevation at the late G1 phase is crucial for the G1-to-S phase transition. ZNF313 is induced by mitogenic growth factors and its blockade profoundly delays cell cycle progression and accelerates p21(WAF1)-mediated senescence. Both replicative and stress-induced senescence are accompanied with ZNF313 reduction. ZNF313 is downregulated during cellular differentiation process in vitro and in vivo, while it is commonly upregulated in many types of cancer cells. ZNF313 shows both the nuclear and cytoplasmic localization in epithelial cells of normal tissues, but exhibits an intense cytoplasmic distribution in carcinoma cells of tumor tissues. Collectively, ZNF313 is a novel E3 ligase for p21(WAF1), whose alteration might be implicated in the pathogenesis of several human diseases, including cancers.

TGF-β1 plays biphasic functions in prostate tumorigenesis, inhibiting cell growth at early stages but promoting malignant progression at later stages. However, the molecular basis for the oncogenic conversion of TGF-β1 function remains largely undefined. Here, we demonstrate that PPARδ is a direct transcription target of TGF-β1 and plays a critical role in oncogenic redirection of TGF-β1 signaling. Blockade of PPARδ induction enhances tumor cell response to TGF-β1-mediated growth inhibition, while its activation promotes TGF-β1-induced tumor growth, migration and invasion. PPARδ-mediated switch of TGF-β1 function is associated with down- and upregulation of Smad and ERK signaling, respectively, and tightly linked to its function to activate ABCA1 cholesterol transporter followed by caveolin-1 (Cav1) induction. Intriguingly, TGF-β1 activation of the PPARδ-ABCA1-Cav1 pathway facilitates degradation of TGF-β receptors (TβRs) and attenuates Smad but enhances ERK response to TGF-β1. Expression of PPARδ and Cav1 is tightly correlated in both prostate tissues and cell lines and significantly higher in cancer vs. normal tissues. Collectively, our study shows that PPARδ is a transcription target of TGF-β1 and contributes to the oncogenic conversion of TGF-β1 function through activation of the ABCA1-Cav1-TβR signaling axis.

Background: CD81 is a transmembrane protein that serves as a putative receptor for hepatitis C virus. In addition, CD81 has been suggested to be involved in a broad range of other cellular functions. Its putative implication in tumorigenesis has so far, however, remained largely unexplored. To assess the candidacy of CD81 as a tumor suppressor in gastric cancer development, we investigated its expression and function in a series of primary gastric tumors and gastric tumor-derived cell lines., Methods: The expression and concomitant methylation status of the CD81 gene and its effect on tumor development and cellular signaling were evaluated., Results: CD81 mRNA levels were found to be low in 16 of 40 (40 %) primary tumors and 9 of 14 (64.2 %) cell lines, and these low expression levels were found to correlate with the stage and grade of the tumors. Genomic alterations of CD81 were not encountered, whereas its expression could be re-activated in low expressing cells upon 5-aza-dC treatment. Bisulfite DNA sequencing analysis of 10 CpG sites within the 5' proximal region of the CD81 gene promoter revealed that the observed transcriptional silencing was tightly associated with aberrant hypermethylation. Subsequent restoration of CD81 expression induced a G1 cell cycle arrest and apoptosis, whereas siRNA-mediated CD81 down-regulation promoted cell proliferation and attenuated cellular responses to various apoptotic stress stimuli. Also the colony-forming ability of the tumor cells could be inhibited and enhanced through CD81 up- and down-regulation, respectively. CD81 was found to inhibit p38 (but not ERK, JNK and AKT) phosphorylation and its growth suppressive effect could be abolished through p38 up- and down-regulation., Conclusion: From our data we conclude that epigenetic inactivation of CD81 is a common feature of gastric tumors and that this inactivation may render growth and survival advantages to the tumor cells, at least partially through p38 signaling.

Caveolin-1 (CAV1) acts as a growth suppressor in various human malignancies, but its expression is elevated in many advanced cancers, suggesting the oncogenic switch of its role during tumor progression. To understand the molecular basis for the growth-promoting function of CAV1, we characterized its expression status, differential roles for tumor growth, and effect on glucose metabolism in colorectal cancers. Abnormal elevation of CAV1 was detected in a substantial fraction of primary tumors and cell lines and tightly correlated with promoter CpG sites hypomethylation. Depletion of elevated CAV1 led to AMPK activation followed by a p53-dependent G1 cell-cycle arrest and autophagy, suggesting that elevated CAV1 may contribute to ATP generation. Furthermore, CAV1 depletion downregulated glucose uptake, lactate accumulation, and intracellular ATP level, supporting that aerobic glycolysis is enhanced by CAV1. Consistently, CAV1 was shown to stimulate GLUT3 transcription via an HMGA1-binding site within the GLUT3 promoter. HMGA1 was found to interact with and activate the GLUT3 promoter and CAV1 increased the HMGA1 activity by enhancing its nuclear localization. Ectopic expression of HMGA1 increased glucose uptake, whereas its knockdown caused AMPK activation. In addition, GLUT3 expression was strongly induced by cotransfection of CAV1 and HMGA1, and its overexpression was observed predominantly in tumors harboring high levels of CAV1 and HMGA1. Together, these data show that elevated CAV1 upregulates glucose uptake and ATP production through HMGA1-mediated GLUT3 transcription, suggesting that CAV1 may render tumor cells growth advantages by enhancing aerobic glycolysis., (©2012 AACR.)

Background: Hemodialysis (HD) patients with functional iron deficiency often develop resistance to recombinant human erythropoietin (rhEPO). Recent studies have shown that intravenous ascorbic acid (IVAA) administration could override rhEPO resistance in HD patients. This study was undertaken to test the effects of IVAA in HD patients with normoferritinemic functional iron deficiency accompanied by EPO-hyporesponsive anemia., Methods: Fifty-eight HD patients with normoferritinemic anemia (between 100 and 500 μg/L) were included and divided into the control (N=25) and IVAA (N=33) groups. IVAA patients received 500 mg of IVAA with each dialysis session for 3 months and an additional 4-month follow-up after the end of the therapy., Results: Twenty patients had a response to IVAA with a significant increase in hemoglobin level (Hgb>1.0 g/dL) and reduction of weekly rhEPO dosage compared with the control group after 3 months of treatment (P<0.05). Compared with non-responders, transferrin saturation (TSAT) was significantly decreased in the responders group (26±11 vs. 35±14%, P<0.05) on baseline data. There was a significant increase in serum iron and TSAT (baseline vs. 3 months, serum iron 57±22 vs. 108±22 μg/dL, TSAT 26±11 vs. 52±7%, P<0.05) and a decrease in serum ferritin (377±146 vs. 233±145 ng/mL, P<0.05) in the responders group (N=20), but no significant changes in the control and non-responders groups (N=13) at 3-month treatment., Conclusion: IVAA can be a potent and effective adjuvant therapy for HD patients with rhEPO-resistant normoferritinemic anemia. In addition, IVAA can reduce the dosage of rhEPO for anemia correction.

Silicon dioxide (SiO2) thin films have gained considerable attention because of their various industrial applications. For example, SiO2 thin films are used in superhydrophilic self-cleaning surface glass, UV protection films, anti-reflection coatings, and insulating materials. Recently, many processes such as vacuum evaporation, sputtering, chemical vapor deposition, and spin coating have been widely applied to prepare thin films of functionally graded materials. However, these processes suffer from several engineering problems. For example, a special apparatus is required for the deposition of films, and conventional wet processes are not suitable for coating the surfaces of substrates with a large surface area and complex morphology. In this study, we investigated the film morphology and optical properties of SiO2 films prepared by a novel technique, namely, liquid phase deposition (LPD). Images of the SiO2 films were obtained by scanning electron microscopy (SEM) and atomic force microscopy (AFM) in order to study the surface morphology of these films: these images indicate that films deposited with different reaction times were uniform and dense and were composed of pure silica. Optical properties such as refractive index and transmittance were estimated by UV-vis spectroscopy and ellipsometry. SiO2 films with porous structures at the nanometer scale (100-250 nm) were successfully produced by LPD. The deposited film had excellent transmittance in the visible wavelength region.

Purpose: PRKCDBP is a putative tumor suppressor in which alteration has been observed in several human cancers. We investigated expression and function of PRKCDBP in colorectal cells and tissues to explore its candidacy as a suppressor in colorectal tumorigenesis., Experimental Design: Expression and methylation status of PRKCDBP and its effect on tumor growth were evaluated. Transcriptional regulation by NF-κB signaling was defined by luciferase reporter and chromatin immunoprecipitation assays., Results: PRKCDBP expression was hardly detectable in 29 of 80 (36%) primary tumors and 11 of 19 (58%) cell lines, and its alteration correlated with tumor stage and grade. Promoter hypermethylation was commonly found in cancers. PRKCDBP expression induced the G(1) cell-cycle arrest and increased cellular sensitivity to various apoptotic stresses. PRKCDBP was induced by TNFα, and its level correlated with tumor cell sensitivity to TNFα-induced apoptosis. PRKCDBP induction by TNFα was disrupted by blocking NF-κB signaling while it was enhanced by RelA transfection. The PRKCDBP promoter activity was increased in response to TNFα, and this response was abolished by disruption of a κB site in the promoter. PRKCDBP delayed the formation and growth of xenograft tumors and improved tumor response to TNFα-induced apoptosis., Conclusions: PRKCDBP is a proapoptotic tumor suppressor which is commonly altered in colorectal cancer by promoter hypermethylation, and its gene transcription is directly activated by NF-κB in response to TNFα. This suggests that PRKCDBP inactivation may contribute to tumor progression by reducing cellular sensitivity to TNFα and other stresses, particularly under chronic inflammatory microenvironment., (©2011 AACR.)

Biomedical applications of magnetic nanoparticles (MNP), including superparamagnetic nanoparticles, have expanded dramatically in recent years. Systematic and standardized cytotoxicity assessment to ensure the biosafety and biocompatibility of those applications is compulsory. We investigated whether exposure to static magnetic field (SMF) from e.g. magnetic resonance imaging (MRI) could affect the cytotoxicity of superparamagnetic iron oxide (SPIO) nanoparticles using mouse hepatocytes and ferucarbotran, a liver-selective MRI contrast agent as a model system. We show that while the SPIO satisfied the conventional cytotoxicity assessment, clinical doses combined with SMF exposure exerts synergistic adverse effects such as reduced cell viability, apoptosis, and cell cycle aberrations on hepatocytes in vitro and in vivo. Concomitant treatments with the SPIO and SMF generated SPIO aggregates, which demonstrated enhanced cellular uptake, was sufficient to induce the cytotoxicity without further SMF, emphasizing that the SPIO aggregates were the predominant source of the cytotoxicity. Interestingly, the apoptotic effect was dependent on levels of reactive oxygen species (ROS) and SPIO uptake while the reduced cell viability was independent of these factors. Moreover, long-term monitoring showed a significant increase in multinuclear giant cells in the cells concomitantly treated with the SPIO and SMF compared with the control. The results demonstrate that the SPIO produces unidentified cytotoxicity on liver in the presence of SMF and the SPIO aggregates predominantly exert the effect. Since aggregation of MNP in biological milieu in the presence of strong SMF is inevitable, a fundamentally different approach to surface fabrication is essential to increase the biocompatibility of MNP., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Transforming growth factor (TGF)-β1 has biphasic functions in prostate tumorigenesis, having a growth-inhibitory effect in the early stages, but in the late stages promoting tumor angiogenesis and metastasis. We demonstrate here that tumor-producing TGF-β1 induces vascular endothelial growth factor (VEGF) in prostate cancer cells, and hypoxia-inducible factor (HIF)-1α and HIF-2α has opposite functions in TGF-β1 regulation of VEGF expression under non-hypoxic conditions. The promoter response of VEGF to TGF-β1 was upregulated by the transfection of HIF-2α or siHIF-1α but downregulated by HIF-1α and siHIF-2α. Both HIF-1α and HIF-2α were induced by TGF-β1 at mRNA and protein levels, however, their nuclear translocation was differentially regulated by TGF-β1, suggesting its association with their opposite effects. VEGF induction by TGF-β1 occurred in a Smad3-dependent manner, and the Smad-binding element 2 (SBE2, -992 to -986) and hypoxia response element (-975 to -968) in the VEGF promoter were required for the promoter response to TGF-β1. Smad3 cooperated with HIF-2α in TGF-β1 activation of VEGF transcription and Smad3 binding to the SBE2 site was greatly impaired by knockdown of HIF-2α expression. Moreover, the VEGF promoter response to TGF-β1 was synergistically elevated by co-transfection of Smad3 and HIF-2α but attenuated by HIF-1α in a dose-dependent manner. Additionally, TGF-β1 was found to increase the stability of VEGF transcript by facilitating the cytoplasmic translocation of a RNA-stabilizing factor HuR. Collectively, our data show that tumor-producing TGF-β1 induces VEGF at the both transcription and post-transcriptional levels through multiple routes including Smad3, HIF-2α and HuR. This study thus suggests that autocrine TGF-β1 production may contribute to tumor angiogenesis via HIF-2α signaling under non-hypoxic conditions, providing a selective growth advantage for prostate tumor cells., (© 2011 Macmillan Publishers Limited)

Background: NORE1 (RASSF5) is a newly described member of the RASSF family with Ras effector function. NORE1 expression is frequently inactivated by aberrant promoter hypermethylation in many human cancers, suggesting that NORE1 might be a putative tumor suppressor. However, expression and mutation status of NORE1 and its implication in colorectal tumorigenesis has not been evaluated., Methods: Expression, mutation, and methylation status of NORE1A and NORE1B in 10 cancer cell lines and 80 primary tumors were characterized by quantitative PCR, SSCP, and bisulfite DNA sequencing analyses. Effect of NORE1A and NORE1B expression on tumor cell growth was evaluated using cell number counting, flow cytometry, and colony formation assays., Results: Expression of NORE1A and NORE1B transcript was easily detectable in all normal colonic epithelial tissues, but substantially decreased in 7 (70%) and 4 (40%) of 10 cancer cell lines and 31 (38.8%) and 25 (31.3%) of 80 primary carcinoma tissues, respectively. Moreover, 46 (57.6%) and 38 (47.5%) of 80 matched tissue sets exhibited tumor-specific reduction of NORE1A and NORE1B, respectively. Abnormal reduction of NORE1 was more commonly observed in advanced stage and high grade tumors compared to early and low grade tumors. While somatic mutations of the gene were not identified, its expression was re-activated in all low expressor cells after treatment with the demethylating agent 5-aza-dC. Bisulfite DNA sequencing analysis of 31 CpG sites within the promoter region demonstrated that abnormal reduction of NORE1A is tightly associated with promoter CpG sites hypermethylation. Moreover, transient expression and siRNA-mediated knockdown assays revealed that both NORE1A and NORE1B decrease cellular growth and colony forming ability of tumor cells and enhance tumor cell response to apoptotic stress., Conclusion: Our data indicate that epigenetic inactivation of NORE1 due to aberrant promoter hypermethylation is a frequent event in colorectal tumorigenesis and might be implicated in the malignant progression of colorectal tumors.

Objective: To suggest an alternative strategy for deriving histocompatible stems cells without undertaking genetic manipulation., Design: Prospective approach using an animal model., Setting: Stem cell and bioevaluation laboratory, Seoul National University., Animal(s): F1 (C57BL6 X DBA2) and outbred (ICR) mice., Intervention(s): Ovarian stroma cells of less than 40 mum in diameter were subcultured with fibroblast monolayer, and colony-forming cells were characterized., Main Outcome Measure(s): Stemness, genotype, and imprinted gene methylation., Result(s): Two-lines of colony-forming cells were established, which expressed markers specific for embryonic stem cells (ESC) and formed embryoid bodies and teratomas. Complete matching of microsatellite markers with the cell donor strain confirmed their establishment from ovarian tissue, and identification of both homozygotic and heterozygotic chromosomes raised the possibility of their derivation from parthenogenetic oocytes. However, the use of cells smaller than mature oocytes for primary culture, the difference in imprinted gene methylation compared with parthenogenetic ESCs, and failure to establish the ESC-like cells by primary follicle culture collectively suggested the irrelevancy to gametes., Conclusion(s): Coculture of adult ovarian cells with somatic fibroblasts can yield colony-forming cells having ESC-like activity, which may provide an alternative for establishing autologous stem cells from adults that can be obtained without genetic manipulation., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background & Aims: HuR is a RNA-binding factor whose expression is commonly upregulated in some human tumor types. We explored the molecular mechanism underlying HuR elevation and its role in gastric cancer tumorigenesis., Methods: HuR expression and subcellular localization were determined by polymerase chain reaction, immunoblot, and immunohistochemical analyses. Its effect on tumor growth was characterized using flow cytometry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, and soft agar analyses. Luciferase reporter, chromatin immunoprecipitation, and electrophoretic mobility shift assays were used to measure transcriptional activation by nuclear factor kappaB (NF-kappaB) signaling., Results: Compared with normal gastric tissues, HuR was expressed at higher levels in gastric tumors, particularly in advanced versus early tumors; this increase was associated with enhanced cytoplasmic translocation of HuR. HuR overexpression increased proliferation of tumor cells, activating the G(1) to S transition of the cell cycle, DNA synthesis, and anchorage-independent growth. Small interfering RNA-mediated knockdown of HuR expression reduced tumor cell proliferation and response to apoptotic stimuli. No genetic or epigenetic alterations of HuR were observed in gastric tumor cell lines or primary tumors; overexpression depended on phosphatidylinositol 3-kinase/AKT signaling and NF-kappaB activity. AKT activation increased p65/RelA binding to a putative NF-kappaB binding site in the HuR promoter, the stability of HuR target transcripts, and the cytoplasmic import of HuR., Conclusions: HuR is a direct transcription target of NF-kappaB; its activation in gastric cancer cell lines depends on phosphatidylinositol 3-kinase/AKT signaling. HuR activation by this pathway has proliferative and antiapoptotic effects on gastric cancer cells.

Purpose: To explore the effects of power frequency magnetic fields (MF) on cell growth in prostate cancer, DU145, PC3, and LNCaP cells were examined in vitro., Materials and Methods: The cells were exposed to various intensities and durations of 60-Hz sinusoidal MF in combination with various serum concentrations in the media. To analyze MF effects on cell growth, cell counting, trypan blue exclusion assay, Western blot analysis, flow cytometry, enzyme-linked immunosorbent assay (ELISA), semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence microscopy, and spectrofluorometry were used., Results: MF exposure induced significant cell growth inhibition and apoptosis in an intensity- and time-dependent manner, in which cell cycle arrest, cleaved Caspase-3, and reactive oxygen species (ROS) increased. Pretreatment with a Caspase-3 inhibitor or antioxidant, N-acetyl-L-cysteine (NAC), significantly attenuated MF-induced cell growth inhibition and cell death. Media replacement experiments failed to show any notable change in the MF effects., Conclusions: These results demonstrate 60-Hz sinusoidal MF-activated cell growth inhibition of prostate cancer in vitro. Apoptosis together with cell cycle arrest were the dominant causes of the MF-elicited cell growth inhibition, mediated by MF-induced ROS. These results suggest that a possibility of using 60-Hz MF in radiation therapy of prostate cancer could usefully be investigated.

hSRBC is a putative tumor suppressor located at 11p15.4, at which frequent genomic loss has been observed in several human malignancies. To explore the candidacy of hSRBC as a suppressor of gastric tumorigenesis, we analyzed the expression and mutation status of hSRBC in gastric tissues and cell lines. hSRBC transcript was expressed in all normal and benign tumor tissues examined, but undetectable or very low in 73% (11/15) cancer cell lines and 41% (46/111) primary tumors. Loss or reduction of hSRBC expression was tumor-specific and correlated with stage and grade of tumors. While allelic loss or somatic mutations of the gene were infrequent, its expression was restored in tumor cells by 5-aza-2'-deoxycytidine treatment and aberrant hypermethylation of 23 CpG sites in the promoter region showed a tight association with altered expression. Transient or stable expression of hSRBC led to a G(1) cell cycle arrest and apoptosis of tumor cells, and strongly suppresses colony forming ability and xenograft tumor growth. In addition, hSRBC elevated apoptotic sensitivity of tumor cells to genotoxic agents, such as 5-FU, etoposide and ultraviolet. Interestingly, hSRBC increased the protein stability of p53 and expression of p53 target genes, such as p21(Waf1), PUMA and NOXA, while hSRBC-mediated cell cycle arrest and apoptosis were abolished by blockade of p53 function. Our findings suggest that hSRBC is a novel tumor suppressor whose epigenetic inactivation contributes to the malignant progression of gastric tumors, in part, through attenuated p53 response to stresses., ((c) 2007 Wiley-Liss, Inc.)

Background & Aims: X-linked inhibitor of apoptosis protein-associated factor 1 (XAF1) is a candidate tumor suppressor located at the chromosome 17p13 region, but the molecular basis underlying its inactivation in human tumors and growth-inhibiting function has not been well defined. We explored the candidacy of XAF1 as a suppressor in colorectal tumorigenesis., Methods: XAF1 expression was characterized by polymerase chain reaction-based cloning, isoform-specific polymerase chain reaction, ribonuclease protection, and immunoblot assays. Allelic loss of the gene was evaluated by loss of heterozygosity (LOH) assay, and promoter CG dinucleotide (CpG) site methylation was determined using bisulfite sequencing. The effect of XAF1 on tumor growth was examined using flow cytometry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, colony formation, and viability assays., Results: Expression of 5 XAF1 variants including 2 novel transcripts was down-regulated concomitantly in 11 of 20 (55%) cell lines and 26 of 65 (40%) primary tumors. XAF1 reduction was tumor-specific and showed a correlation with advanced stage and high grade of tumor. LOH of the gene was found in 12 of 33 (36%) tumors. Promoter CpG site methylation was observed frequently in both cell lines and tumor tissues including many LOH tumors, suggesting that biallelic inactivation of XAF1 might be common in colorectal cancers. XAF1 expression suppressed tumor cell growth and enhanced cellular response to various apoptotic stimuli, such as 5-fluorouracil, etoposide, H(2)O(2), gamma-irradiation, ultraviolet, and tumor necrosis factor-alpha, whereas knockdown of its expression protected cells from the stresses., Conclusions: Genetic and epigenetic alteration of XAF1 is a common event in colorectal tumorigenesis and contributes to the malignant tumor progression by providing survival advantages for tumor cells under various stress conditions.

XIAP-associated factor 1 (XAF1) is a new candidate tumor suppressor, which has been known to exert proapoptotic effects by interfering with the caspase-inhibiting activity of XIAP. To explore the XAF1's candidacy for a suppressor in urogenital tumorigenesis, we investigated the XAF1 status in a series of cancer cell lines and primary tumors derived from the bladder, kidney and prostate. Expression of XAF1 transcript was undetectable or extremely low in 60% (3/5) of bladder, 66% (10/15) of kidney, and 100% (3/3) prostate cancer cell lines. Abnormal reduction of XAF1 was also found in 33% (18/55) of primary bladder and 40% (8/20) of primary kidney tumors, and showed a correlation with advanced stage and high grade of bladder tumor. Hypermethylation at 14 CpG sites in the 5' proximal region of the XAF1 promoter was highly prevalent in cancers versus adjacent normal or benign tissues and tightly associated with reduced gene expression. XAF1 expression enhanced the apoptotic response of tumor cells to chemotherapeutic agents, such as etoposide or 5-FU. While XAF1 expression did not influence the subcellular distribution or expression of XIAP, it elevated the protein stability of p53 and its target gene expression. Moreover, the apoptosis-sensitizing and growth suppression function of XAF1 was markedly impeded by blockade of p53 function. Collectively, our study demonstrates that epigenetic alteration of XAF1 is frequent in human urogenital cancers and may contribute to the malignant progression of tumors by rendering tumor cells a survival advantage partially through the attenuated p53 response to apoptotic stresses.

A simple high performance liquid chromatographic (HPLC) method was developed for the determination of tolperisone in human plasma. Tolperisone and internal standard (chlorphenesin) were isolated from 1 mL of plasma using 8 mL of dichlormethane. The organic phase was collected and evaporated under nitrogen gas. The residue was then reconstituted with 300 mL aliquot of mobile phase and a 100 mL aliquot was injected onto the C18 reverse-phased column. The mobile phase, 45% methanol containing 1% glacial acetic acid and 0.05% 1-hexanesulfonic acid was run at a flow rate of 1 mL/min. The column effluent was monitored using UV detector at 260 nm. The retention times for tolperisone and the internal standard were approximately 7.1 and 8.4 min, respectively. The standard curve was linear with minimal intra-day and inter-day variability. The quantification limit of tolperisone in human plasma was 10 ng/ mL. The proposed method has been applied to the determination of pharmacokinetic profile of tolperisone in Koreans. The Tmax of tolperisone in Koreans (0.94 +/- 0.42 h) was not significantly differ from that reported in Europeans (0.5-1 h), but the mean half-life in Koreans (1.14 +/- 0.27 h) was shorter than that in Europeans (2.56 +/- 0.2 h). The proposed HPLC method is simple, accurate, reproducible and suitable for pharmacokinetic study of tolperisone.

Mitochondrial dysfunction has been suggested as a causal factor for insulin resistance and diabetes. Previously we have shown a decrease of mitochondrial DNA (mtDNA) content in tissues of diabetic patients. The mitochondrial proteins, which regulate the mitochondrial biogensis, including transcription and replication of mtDNA, are encoded by nuclear DNA. Despite the potential function of the proteins bound to the D-loop region of mtDNA in regulating mtDNA transcription/replication, only a few proteins are known to bind the D-loop region of mtDNA. The functional association of these known proteins with insulin resistance is weak. In this study, we applied proteomic analysis to identify a group of proteins (proteome) that physically bind to D-loop DNA of mtDNA. We amplified D-loop DNA (1.1 kb) by PCR and conjugated the PCR fragments to CNBr-activated sepharose. Mitochondria fractions were isolated by both differential centrifugation and Optiprep-gradient ultracentrifugation. The D-loop DNA binding proteome fractions were enriched via this affinity chromatography and analyzed by SDS-PAGE. The proteins on the gel were transferred onto PVDF membrane and the peptide sequences of each band were subsequently analyzed by capillary reverse-phase liquid chromatography/tandem mass spectrometry (RPLC/MS/MS). We identified many D-loop DNA binding proteins, including mitochondrial transcription factor A (mtTFA, Tfam) and mitochondrial single-stranded DNA binding protein (mtSSBP) which were known to bind to mtDNA. We also report the possibility of novel D-loop binding proteins such as histone family proteins and high-mobility group proteins.

X-linked inhibitor of apoptosis (XIAP) is the most potent member of the IAP family that exerts antiapoptotic effects by interfering with the activities of caspases. Recently, XIAP-associated factor 1 (XAF1) and two mitochondrial proteins, Smac/DIABLO and HtrA2, have been identified to negatively regulate the caspase-inhibiting activity of XIAP. To explore the candidacy of XAF1, Smac/DIABLO, and HtrA2 as a tumor suppressor in gastric tumorigenesis, we investigated the expression and mutation status of the genes in 123 gastric tissues and 15 cancer cell lines. Whereas Smac/DIABLO and HtrA2 transcripts were normally expressed in all cancer specimens we examined, XAF1 transcript was not expressed or present at extremely low levels in 40% (6 of 15) of cancer cell lines and in 23% (20 of 87) of primary carcinomas. Abnormal reduction of XAF1 expression showed a strong correlation with stage and grade of tumors, and a tumor-specific down-regulation of XAF1 was observed in 45% (9 of 20) of matched sets. Unlike XAF1, XIAP expression exhibited no detectable alteration in cancers. Whereas loss of heterozygosity within the XAF1 region or somatic mutations of the gene was not detected, expression of XAF1 transcript was reactivated in all nonexpressor cell lines after 5-aza-2-deoxycytidine treatment. The 5' upstream region of the XAF1 gene encompasses no gastric cell-rich region that rigorously satisfies the formal criteria for CpG islands. However, bisulfite DNA sequencing analysis for 34 CpG sites in the promoter region revealed a strong association between hypermethylation and gene silencing. Moreover, transcriptional silencing of XAF1 was tightly associated with hypermethylation of seven CpGs located in the 5' proximal region (nucleotides -23 to -234). Additionally, loss or abnormal reduction of XAF1 expression was found to inversely correlate with p53 mutations, suggesting that epigenetic inactivation of XAF1 and mutational alteration of p53 might be mutually exclusive events in gastric tumorigenesis. Collectively, our study suggests that epigenetic silencing of XAF1 by aberrant promoter methylation may contribute to the malignant progression of human gastric tumors.

Transforming growth factor (TGF)-beta1 acts as a potent growth inhibitor of prostate epithelial cells, and aberrant function of its receptor type I and II correlates with tumor aggressiveness. However, intracellular and serum TGF-beta1 levels are elevated in prostate cancer patients and further increased in patients with metastatic carcinoma, suggesting the oncogenic switch of TGF-beta1 role in prostate tumorigenesis. Recently, we reported the mitogenic conversion of TGF-beta1 effect by oncogenic Ha-Ras in prostate cancer cells. Here, we show that TGF-beta1 activates interleukin (IL)-6, which has been implicated in the malignant progression of prostate cancers, via multiple signaling pathways including Smad2, nuclear factor-kappaB (NF-kappaB), JNK, and Ras. TGF-beta1-induced IL-6 gene expression was strongly inhibited by DN-Smad2 but not by DN-Smad3 while it was further activated by wild-type Smad2 transfection. IL-6 activation by TGF-beta1 was accompanied by nuclear translocation of NF-kappaB, which was blocked by the p38 inhibitors SB202190 and SB203580 or by IkappaBalphaDeltaN transfection, indicating the crucial role for the p38-NF-kappaB signaling in TGF-beta1 induction of IL-6. TGF-beta1 activated c-Jun phosphorylation, and IL-6 induction by TGF-beta1 was severely impeded by DN-c-Jun and DN-JNK or AP-1 inhibitor curcumin, showing that the JNK-c-Jun-AP-1 signaling plays a pivotal role in TGF-beta1 stimulation of IL-6. It was also found that the Ras-Raf-MEK1 cascade is activated by TGF-beta1 and participates in the TGF-beta1 induction of IL-6 in an AP-1-dependent manner. Cotransfection assays demonstrated that TGF-beta1 stimulation of IL-6 results from the synergistic collaboration of the Smad2, p38-NF-kappaB, JNK-c-Jun-AP-1, or Ras-Raf-MEK1 cascades. In addition, a time course IL-6 decay revealed that mRNA stability of IL-6 is modestly increased by TGF-beta1, indicating that TGF-beta1 also regulates IL-6 at the post-transcriptional level. Intriguingly, IL-6 inactivation restored the sensitivity to TGF-beta1-mediated growth arrest and apoptosis, suggesting that elevated IL-6 in advanced prostate tumors might act as a resistance factor against TGF-beta1. Collectively, our data demonstrate that IL-6 expression is stimulated by tumor-producing TGF-beta1 in human prostate cancer cells through multiple signaling pathways including Smad2, p38, JNK, and Ras, and enhanced expression of IL-6 could contribute to the oncogenic switch of TGF-beta1 role for prostate tumorigenesis, in part by counteracting its growth suppression function.

Mutational alterations of PTEN and PIK3CA, which negatively and positively regulate PI3-kinase activity, respectively, have been observed in many types of human cancer. To explore the implication of PTEN and PIK3CA mutations in gastric tumorigenesis, we characterized the expression and mutation status of the genes in 126 gastric tissues and 15 cell lines. Expression of PTEN transcript was abnormally low in 5 of 15 (33%) cell lines and 20 of 55 (36%) primary carcinomas, whereas 0 of 71 noncancerous tissues including 16 benign tumors showed altered expression. Allelotyping analysis using an intragenic polymorphism (IVS4+109) revealed that 14 of 30 (47%) informative cases carried LOH of the gene, which is closely linked to low expression. The LOH rate was significantly higher in advanced tumors [12 of 19 (63%)] compared to early-stage tumors [2 of 11 (18%)] and more frequent in poorly differentiated tumors [9 of 13 (69%)] than well- or moderately differentiated tumors [5 of 17 (29%)]. Interestingly, however, none of the LOH tumors carried mutational disruption of the remaining allele, suggesting haploinsufficiency of PTEN in gastric tumorigenesis. Methylation studies revealed that PTEN pseudogene, but not PTEN, is methylated in cell lines and primary tumors, indicating that PTEN is not a target of epigenetic silencing in gastric cancers and that the pseudogene should be considered more carefully in methylation analysis of the PTEN promoter. Genomic amplification of PIK3CA was found in 9 of 15 (60%) cell lines and 20 of 55 (36.4%) primary tumors but in no noncancerous tissues. Furthermore, PIK3CA amplification was predominantly detected in tumors with no PTEN alterations, suggesting that mutations of PTEN and PIK3CA are mutually exclusive events in gastric tumorigenesis. Amplification of PIK3CA was strongly associated with increased expression of PIK3CA transcript and elevated levels of phospho-AKT. Collectively, our data reveal that 13 of 15 (87%) gastric cell lines and 31 of 55 (56%) primary carcinomas harbored either amplification of PIK3CA or abnormal reduction of PTEN. Mutually exclusive alterations of PTEN and PIK3CA also suggest that mutations of either gene could activate the PI3-kinase/AKT signaling pathway, which is directly linked to the malignant progression of gastric tumor cells., (Copyright 2003 Wiley-Liss, Inc.)

Homozygous deletion or somatic mutations of mitogen-activated protein kinase kinase 4 (MKK4), a candidate tumour suppressor gene located at 17p11, have been observed in many types of human tumours. To explore the likelihood that MKK4 acts as a suppressor in gastric tumorigenesis, we examined the expression and mutation status of MKK4 in 144 gastric tissues and cell line specimens. Expression of the MKK4 transcript was easily detectable in all normal and benign tumour tissues and none of 102 primary carcinomas and cell lines showed an abnormal reduction in MKK4 expression. Expression levels of MKK4 transcript showed no cancer-specific reduction in 43 matched sets and did not correlate with stage, grade and histopathological types of the tumours. Western blot analysis also revealed that MKK4 protein expression in carcinoma tissues and cell lines is comparable to non-cancerous tissues. A significant loss of heterozygosity (LOH) was detected at telomeric markers of the MKK4, locus. However, no allelic deletion of the MKK4 gene or at the centromeric loci was identified. Moreover, no evidences for somatic mutations leading to amino acid substitutions or frameshifts of MKK4 were identified in the carcinoma tissues and cell lines, whereas a substantial fraction of the same set showed allelic loss or mutations of the TP53 gene located at 17p13, suggesting that LOH at telomeric loci or the TP53 locus might not extend into the MKK4 gene in gastric cancers. In this study, we also report the identification of a highly conserved MKK4 processed pseudogene, which shares 95% homology with the coding region of the functional MKK4 transcript. Collectively, our data demonstrate that genomic deletion or somatic mutation of MKK4 is infrequent in gastric cancers, suggesting that MKK4 might not be a critical target of genetic inactivation in gastric tumorigenesis.

p53R2, a recently identified putative tumor suppressor located at 8q23.1, encodes a protein with striking similarity to a small subunit of ribonucleotide reductase. p53R2 is directly induced by wild-type p53 and involved in the p53 checkpoint for repair of damaged DNA, raising the possibility that mutational inactivation of p53R2 may contribute to the development and progression of human malignancies. To explore the p53R2's candidacy for a suppressor in gastric tumorigenesis, we examined the expression and mutation status of p53R2 in 166 gastric specimens including 90 primary adenocarcinomas and 15 cell lines. In response to genotoxic damages, p53R2 transcription was clearly activated in wild-type but not mutant-type p53-carrying cells while basal expression of p53R2 in undamaged cells showed no association with the mutational status of p53. Host cell reactivation assay revealed that p53R2 enhances DNA repair efficiency and plays a role in the p53-mediated repair of damaged DNA, whereas no significant effect of p53R2 on cell growth and apoptosis was detected in flow cytometry and [(3)H]thymidine incorporation assays. p53R2 transcript was expressed in all normal and tumor tissues and its expression levels were not significantly different between normal and malignant carcinoma tissues. p53R2 expression showed no correlation with stage, grade and histological types of tumors. Moreover, no tumor-specific reduction of p53R2 was detected in 30 matched sets. Mutational analysis of p53R2 in 105 carcinomas including 15 cell lines also failed to detect any evidences for genomic deletion or somatic mutations leading to amino acid substitutions or frameshift whereas 31% (28 of 90) of the same primary tumors showed p53 alterations. Whereas 82% (23 of 28) of the mutant p53-carrying primary tumors expressed abnormally low p21(Waf1), no association of p53R2 expression with the p53 status was recognized, suggesting that basal transcription of p53R2 is regulated through the p53-independent mechanism. Collectively, our study indicates that although p53R2 is induced in a p53-dependent manner and involved the p53-mediated DNA repair in gastric epithelial cells, it is not a critical target of genetic inactivation in gastric tumorigenesis., (Copyright 2002 Wiley-Liss, Inc.)

During tumour progression, cancer cells use diverse mechanisms to escape from apoptosis-inducing stimuli, which may include receptor internalization, inhibition of signal pathways, and regulation of specific sets of genes. Substantial numbers of colon cancer cells have been observed to express Fas/Fas ligand, but are resistant to Fas-mediated apoptosis, suggesting that colonic tumours might develop specific mechanisms to overcome Fas-mediated apoptosis. Recently, cellular FLICE-like inhibitory protein (cFLIP) has been identified as an endogenous inhibitor of Fas- or other receptor-mediated apoptosis and its altered high expression has a suspected association with tumour development or progression. In an effort to investigate the prevalence of cFLIP(L) alterations in colon carcinomas and their possible implications for the progression of colon cancers, cFLIP(L) expression was analysed in adenocarcinomas and adenomatous polyps of colon, with matched normal tissues, at RNA and protein levels, by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. cFLIP(L) transcripts were constitutively expressed in colon cancers and expression levels were significantly higher in carcinomas than in normal tissues (p<0.05). Overexpression of cFLIP(L) protein was found exclusively in carcinoma cells in all matched sets analysed and approximately three-fold induction was detected in cancer cells (p<0.05). The expression of cFLIP(L) protein was not significantly altered in adenomatous polyps compared with normal tissues. Taken together, these results strongly suggest that abnormal overexpression of cFLIP(L) is a frequent event in colon carcinomas and might contribute to in vivo tumour transformation., (Copyright 2001 John Wiley & Sons, Ltd.)

To investigate whether the squamous differentiation of primary hamster tracheal epithelial cell, which is induced by retinoic acid deficiency or chronic PMA treatment, regulates Muc1 expression, we first produced and characterized a monoclonal antibody against hamster tracheal Muc1 mucin using pGEX-Muc1 fusion protein as an antigen and the changes of Muc1 mucin expression was determined by Western blot. Squamous differentiation downregulated the expression of Muc1 mucin from HTSE cells. The decrease in the immunoreactivity of Muc1 mucin was parallel to the decrease in the immunoreactivity of high molecular weight mucin, which is secreted from HTSE cells. The data from the present study implicate a possible role of Muc1 mucin in squamous differentiation of HTSE cells., (Copyright 2000 Academic Press.)

The hepatoprotective effect of DA-9601, a quality-controlled extract of Artemisia asiatica, on liver damage induced by acetaminophen (APAP) and carbon tetrachloride (CCl4) was investigated by means of serum-biochemical, hepatic-biochemical, and histopathological examinations. Doses of DA-9601 (10, 30, or 100 mg/kg) were administered intragastrically to each rat on three consecutive days i.e. 48 h, 24 h and 2 h before a single administration of APAP (640 mg/kg, i.p.) or CCl4 (2 ml/kg, p.o.). Four h and 24 h after hepatotoxin treatment, the animals were sacrificed for evaluation of liver damage. Pretreatment of DA-9601 reduced the elevation of serum ALT, AST, LDH and histopathological changes such as centrilobular necrosis, vacuolar degeneration and inflammatory cell infiltration dose-dependently. DA-9601 also prevented APAP- and CCl4-induced hepatic glutathione (GSH) depletion and CCl4-induced increase of hepatic malondialdehyde (MDA), a parameter of lipid peroxidation, in a dose-dependent manner. These findings suggest that pretreatment with DA-9601 may reduce chemically induced liver injury by complex mechanisms which involve prevention of lipid peroxidation and preservation of hepatic GSH.