Yoon Mi Lee

Sirtuin 1 Modulates Cellular Responses to Hypoxia by Deacetylating Hypoxia-Inducible Factor 1α

To survive in hypoxic environments, organisms must be able to cope with redox imbalance and oxygen deficiency. The SIRT1 deacetylase and the HIF-1alpha transcription factor act as redox and oxygen sensors, respectively. Here, we found that SIRT1 binds to HIF-1alpha and deacetylates it at Lys674, which is acetylated by PCAF. By doing so, SIRT1 inactivated HIF-1alpha by blocking p300 recruitment and consequently repressed HIF-1 target genes. During hypoxia, SIRT1 was downregulated due to decreased NAD(+) levels, which allowed the acetylation and activation of HIF-1alpha. Conversely, when the redox change was attenuated by blocking glycolysis, SIRT1 was upregulated, leading to the deacetylation and inactivation of HIF-1alpha even in hypoxia. In addition, we confirmed the SIRT1-HIF-1alpha interaction in hypoxic mouse tissues and observed in vivo that SIRT1 has negative effects on tumor growth and angiogenesis. Our results suggest that crosstalk between oxygen- and redox-responsive signal transducers occurs through the SIRT1-HIF-1alpha interaction.

Nutlin-3, an Hdm2 antagonist, inhibits tumor adaptation to hypoxia by stimulating the FIH-mediated inactivation of HIF-1α

The interplay among hypoxia-inducible factor 1-alpha (HIF-1alpha), p53 and human orthologue of murine double minute 2 (Hdm2) has been introduced as a key event in tumor promotion and angiogenesis. Recently, nutlin-3, a small-molecule antagonist of Hdm2, was demonstrated to inhibit the HIF-1-mediated vascular endothelial growth factor production and tumor angiogenesis. Yet, the mechanism by which nutlin-3 inhibits HIF-1 is an open question. We here addressed the mode-of-action of nutlin-3 with respect to the HIF-1alpha-p53-Hdm2 interplay. The effect of nutlin-3 on HIF-1alpha function was examined by reporter analyses, immunoprecipitation and immunoblotting. Nutlin-3 downregulated HIF-1alpha, which occurred p53-dependently but von Hippel-Lindau-independently. On the contrary, nutlin-3 blunted the hypoxic induction of vascular endothelial growth factor by inactivating HIF-1 even in p53-null cells. The C-terminal transactivation domain (CAD) of HIF-1alpha was inactivated by nutlin-3, and furthermore, the factor-inhibiting hypoxia-inducible factor (FIH) hydroxylation of Asn803 was required for the nutlin-3 action. In terms of protein interactions, Hdm2 competed with FIH in CAD binding and inhibited the Asn803 hydroxylation both in vivo and in vitro, which facilitated p300 recruitment. Moreover, nutlin-3 reinforced the FIH binding and Ans803 hydroxylation by inhibiting Hdm2. In conclusion, Hdm2 functionally activates HIF-1 by inhibiting the FIH interaction with CAD, and the Hdm2 inhibition by nutlin-3 results in HIF-1 inactivation and vascular endothelial growth factor suppression. The interplays among HIF-1alpha, Hdm2, FIH and p300 could be potential targets for treating tumors overexpressing HIF-1alpha.

Silver-Coated Silica Beads Applicable as Core Materials of Dual-Tagging Sensors Operating via SERS and MEF

We have developed dual-tagging sensors, operating via both surface-enhanced Raman scattering (SERS) and metal-enhanced fluorescence (MEF), composed of silver-coated silica beads onto which were deposited SERS markers and dye-grafted polyelectrolytes, for multiplex immunoassays. Initially, a very simple electroless-plating method was applied to prepare Ag-coated silica beads. The Raman markers were then assembled onto the Ag-coated silica beads, after which they were brought to stabilization by the layer-by-layer deposition of anionic and cationic polyelectrolytes including a dye-grafted polyelectrolyte. In the final stage, the dual-tagging sensors were assembled onto them with specific antibodies (antihuman-IgG or antirabbit-IgG) to detect target antigens (human-IgG or rabbit-IgG). The MEF signal was used as an immediate indicator of molecular recognition, while the SERS signals were subsequently used as the signature of specific molecular interactions. For this reason, these materials should find wide application, especially in the areas of biological sensing and recognition that rely heavily on optical and spectroscopic properties.

Rhodamine B isothiocyanate-modified Ag nanoaggregates on dielectric beads: A novel surface-enhanced Raman scattering and fluorescent imaging material

Rhodamine B isothiocyanate (RhBITC) is a prototype dye molecule that is widely used as a fluorescent tag in a variety of biological applications. We report in this work that once RhBITC is adsorbed onto Ag on silica or polystyrene beads, it exhibits not only a strong surface-enhanced Raman scattering (SERS) signal but also a measurable amount of fluorescence. The RhBITC-modified Ag-deposited silica or polystyrene beads disperse well in ethanol, and they are also readily coated in water with polyelectrolytes for their further derivatization with biological molecules of interest that can bind to target molecules. The application prospects of these materials are thus expected to be very high especially in the areas of biological sensing and recognition that rely heavily on optical and spectroscopic properties. For instance, on the basis of the nature of the SERS peaks of RhBITC, those Ag-deposited silica or polystyrene beads were confirmed, after attaching biotin groups over RhBITC, to selectively recognize streptavidin molecules down to concentrations of 10(-13)M based on a signal-to-noise ratio of 3. The biotin-streptavidin interaction was also confirmed from the photoluminescence of RhBITC.

Silver salts of aromatic thiols applicable as core materials of molecular sensors operating via SERS and fluorescence

We have developed dual-tagging sensors, operating via both Raman and fluorescence spectroscopy, composed of silver aromatic thiolates (AgSRs) modified with fluorescent organic dye for multiplex immunoassays. Owing to the photo-induced production of SERS-active Ag nanoparticles, AgSRs exhibit the surface-enhanced Raman scattering (SERS) spectra of corresponding thiols. The fluorescence dye-modified AgSRs were accordingly fabricated using dye-grafted polyelectrolytes during layer-by-layer deposition of cationic and anionic polyelectrolytes onto AgSRs. In the final stage, the tagging sensors assembled with either specific biotin group or specific antibodies (anti-h-IgG or anti-r-IgG) were employed to detect either streptavidin molecules or target antigens (h-IgG or r-IgG), respectively. Since numerous AgSRs can be used as the core materials, multiple bioassays are expected to be accomplishable using the present methodology. The fluorescence signal may be used as an immediate indicator of molecular recognition, while the SERS signals can be used subsequently as the signature of specific molecular interactions.

Facile fabrication of large area nanostructures for efficient surface-enhanced Raman scattering

Large area SERS (surface-enhanced Raman scattering)-active films with closely packed nanoembossed Ag arrays were reproducibly fabricated by the replication method of polymer surfaces with nano-size grooved Al masters. Three different sizes (60, 105 and 420 nm) of nanoembossed polymer surface were produced from three different replication masters obtained by controlled anodization conditions of Al, which could be further coated with silver in various thicknesses by an ion sputtering technique. Nanoembossed Ag films with an emboss size of 420 nm, uniformly formed over all the areas of 1 × 1 cm2, exhibit very even SERS activity and the enhancement factor estimated using benzenethiol as a prototype adsorbate reaches as large as 6.6 × 105. Many replicas of polyethylene and polystyrene could be produced from one replication master by this simple replication method with high reproducibility. Almost the same SERS intensities were observed in ten different spots of each replica and twenty different replicas from one replication master.

Adsorption characteristics of Au nanoparticles onto poly(4-vinylpyridine) surface revealed by QCM, AFM, UV/vis, and Raman scattering spectroscopy

In this work, we report that the adsorption and aggregation processes of Au nanoparticles on a polymer surface can be monitored by means of surface-enhanced Raman scattering (SERS) spectroscopy. Specifically, we were able to analyze the adsorption process of citrate-stabilized Au nanoparticles onto a film of poly(4-vinylpyridine) (P4VP) by taking a series of SERS spectra, during the self-assembly of Au nanoparticles onto the polymer film. In order to better analyze the SERS spectra, we separately conducted quartz crystal microbalance (QCM), UV/vis spectroscopy, and atomic force microscope (AFM) measurements. The adsorption kinetics revealed by QCM under the in situ conditions was in fair agreement with that determined by the ex situ AFM measurement. The number of Au nanoparticles adsorbed on P4VP increased almost linearly with time: 265 Au nanoparticles per 1microm(2) were adsorbed on the P4VP film after 6h of immersion. The SERS signal measured in the ex situ condition showed a more rapid increase than that of QCM; however, its increasing pattern was quite similar to that of UV/vis absorbance at longer wavelengths, suggesting that Au nanoparticles actually became agglomerated on P4VP.

Metal-Enhanced Fluorescence of Rhodamine B Isothiocyanate from Micrometer-Sized Silver Powders

We report here the use of micrometer-sized silver (microAg) powders to enhance the fluorescence emission of rhodamine B isothiocyanate (RhBITC). Our findings clearly show that the RhBITC displays up to a 36-fold increase in fluorescence emission intensity when RhBITC-labeled poly(allylamine hydrochloride) (RhBITC-PAH) is assembled onto a polyelectrolyte layer (about 30 nm thick) above microAg, when compared to that measured for RhBITC-PAH assembled directly onto a glass slide. A similar experiment conducted using a vacuum-evaporated Ag film displayed only a 5-fold increase. The enhanced fluorescence observed for microAg powders is obviously due to their ability to concentrate the electric fields. This suggests that metal-enhanced fluorescence (MEF) can potentially be employed to increase the sensitivity and detection limit of various bioassays that employ RhBITC as a fluorescence label. Since microAg powders are also an efficient substrate for surface-enhanced Raman scattering (SERS), molecular sensors operating via both SERS and MEF may then be able to be fabricated using microAg powders.