Dimethyl α-Ketoglutarate Promotes the Synthesis of Collagen and Inhibits Metalloproteinases in HaCaT Cells.

We observed that treatment with dimethyl α-ketoglutarate (DMK) increased the amount of intracellular α-ketoglutarate significantly more than that of α-ketoglutarate in HaCaT cells. DMK also increased the level of intracellular 4-hydroxyproline and promoted the production of collagen in HaCaT cells. In addition, DMK decreased the production of collagenase and elastase and down-regulated the expression of selected matrix metalloproteinases (MMPs), such as MMP-1, MMP-9, MMP-10, and MMP-12, via transcriptional inhibition. The inhibition of MMPs by DMK was mediated by the suppression of the IL-1 signaling cascade, leading to the attenuation of ERK1/2 phosphorylation and AP-1 transactivation. Our study results illustrate that DMK, an alkylated derivative of α-ketoglutarate, increased the level of 4-hydroxyproline, promoted the production of collagen, and inhibited the expression of selected MMPs by affecting the IL-1 cascade and AP-1 transactivation in HaCaT cells. The results suggest that DMK might be useful as an anti-wrinkle ingredient.

Enhanced Specificity in Loop-Mediated Isothermal Amplification with Poly(ethylene glycol)-Engrafted Graphene Oxide for Detection of Viral Genes.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method that allows the simple, quick, and low-cost detection of various viral genes. LAMP assays are susceptible to generating non-specific amplicons, as high concentrations of DNA primers can give rise to primer dimerization and mismatched hybridizations, resulting in false-positive signals. Herein, we reported that poly(ethylene glycol)-engrafted nanosized graphene oxide (PEG-nGO) can significantly enhance the specificity of LAMP, owing to its ability to adsorb single-stranded DNA (ssDNA). By adsorbing surplus ssDNA primers, PEG-nGO minimizes the non-specific annealing of ssDNAs, including erroneous priming and primer dimerization, leading to the enhanced specificity of LAMP. The detection of complementary DNAs transcribed from the hepatitis C virus (HCV) RNA was performed by the PEG-nGO-based LAMP. We observed that the inclusion of PEG-nGO significantly enhances the specificity and sensitivity of the LAMP assay through the augmented difference in fluorescence signals between the target and non-target samples. The PEG-nGO-based LAMP assay greatly facilitates the detection of HCV-positive clinical samples, with superior precision to the conventional quantitative real-time PCR (RT-qPCR). Among the 20 clinical samples tested, all 10 HCV-positive samples are detected as positive in the PEG-nGO-based LAMP, while only 7 samples are detected as HCV-positive in the RT-qPCR. In addition, the PEG-nGO-based LAMP method significantly improves the detection precision for the false-positive decision by 1.75-fold as compared to the LAMP without PEG-nGO. Thus, PEG-nGO can significantly improve the performance of LAMP assays by facilitating the specific amplification of target DNA with a decrease in background signal.

Ultrasensitive Electrical Detection of Hemagglutinin for Point-of-Care Detection of Influenza Virus Based on a CMP-NANA Probe and Top-Down Processed Silicon Nanowire Field-Effect Transistors.

Rather than the internal genome nucleic acids, the biomolecules on the surface of the influenza virus itself should be detected for a more exact and rapid point-of-care yes/no decision for influenza virus-induced infectious diseases. This work demonstrates the ultrasensitive electrical detection of the HA1 domain of hemagglutinin (HA), a representative viral surface protein of the influenza virus, using the top-down complementary metal oxide semiconductor (CMOS) processed silicon nanowire (SiNW) field-effect transistor (FET) configuration. Cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-NANA) was employed as a probe that specifically binds both to the aldehyde self-aligned monolayer on the SiNWs and to HA1 simultaneously. CMP-NANA was serially combined with two kinds of linkers, namely 3-aminopropyltriethoxysilane and glutaraldehyde. The surface functionalization used was verified using the purification of glutathione S-transferase-tagged HA1, contact angle measurement, enzyme-linked immunosorbent assay test, and isoelectric focusing analysis. The proposed functionalized SiNW FET showed high sensitivities of the threshold voltage shift (ΔVT) ~51 mV/pH and the ΔVT = 112 mV (63 mV/decade) with an ultralow detectable range of 1 fM of target protein HA1.

Recovery of TRIM25-Mediated RIG-I Ubiquitination through Suppression of NS1 by RNA Aptamers.

Non-structural protein 1 (NS1) of influenza virus has been shown to inhibit the innate immune response by blocking the induction of interferon (IFN). In this study, we isolated two single-stranded RNA aptamers specific to NS1 with K d values of 1.62 ± 0.30 nM and 1.97 ± 0.27 nM, respectively, using a systematic evolution of ligand by exponential enrichment (SELEX) procedure. The selected aptamers were able to inhibit the interaction of NS1 with tripartite motif-containing protein 25 (TRIM25), and suppression of NS1 enabled retinoic acid inducible gene I (RIG-I) to be ubiquitinated regularly by TRIM25. Additional luciferase reporter assay and quantitative real-time PCR (RT-PCR) experiments demonstrated that suppression of NS1 by the selected aptamers induced IFN production. It is noted that viral replication was also inhibited through IFN induction in the presence of the selected aptamers. These results suggest that the isolated aptamers are strongly expected to be new therapeutic agents against influenza infection.

Shikonin induces mitochondria-mediated apoptosis and attenuates epithelial-mesenchymal transition in cisplatin-resistant human ovarian cancer cells.

Cisplatin-based chemotherapy often results in the development of chemoresistance when used to treat ovarian cancer, which is difficult to overcome. The present study investigated the cytotoxic and anti-migratory effects of shikonin, a naphthoquinone compound, on cisplatin-resistant human ovarian cancer A2780 cells (A2780-CR). Shikonin had a potent dose-dependent cytotoxic effect on A2780-CR cells, with 9 µM shikonin treatment reducing A2780-CR cell viability by 50%, validate using an MTT assay. Shikonin induced apoptosis, as evidenced by the increased number of apoptotic bodies, following staining with Hoechst 33342, and terminal deoxynucleotidyl cell transferase dUTP nick end labeling-positive cells following treatment. Flow cytometry and fluorescent microscope imaging, following JC-1 staining, revealed that shikonin increased mitochondrial membrane depolarization. Also it altered the levels of apoptosis-associated proteins, leading to diminished expression of B cell lymphoma-2 (Bcl-2), enhanced expression of Bcl-associated X, and cleavage of caspase-9 and -3, as revealed using western blot analysis. Shikonin activated mitogen-activated protein kinases; while treatment with specific inhibitors of these kinases attenuated the decline in cell viability induced by shikonin treatment. In addition, the cell migration assay and western blot analysis indicated that shikonin decreased the migratory capacity of A2780-CR cells via the upregulation of epithelial-cadherin and downregulation of neural-cadherin. Taken together, the results of the present study indicated that shikonin induces mitochondria-mediated apoptosis and attenuates the epithelial-mesenchymal transition in A2780-CR cells.

A Novel Chemical Compound for Inhibition of SARS Coronavirus Helicase.

We have discovered a novel chemical compound, (E)-3-(furan-2-yl)-N-(4-sulfamoylphenyl) acrylamide, that suppresses the enzymatic activities of SARS coronavirus helicase. To determine the inhibitory effect, ATP hydrolysis and double-stranded DNA unwinding assays were performed in the presence of various concentrations of the compound. Through these assays, we obtained IC50 values of 2.09 ± 0.30 µM (ATP hydrolysis) and 13.2 ± 0.9 µM (DNA unwinding), respectively. Moreover, we found that the compound did not have any significant cytotoxicity when 40 µM of it was used. Our results showed that the compound might be useful to be developed as an inhibitor against SARS coronavirus.

An Aptamer-Based Electrochemical Sensor That Can Distinguish Influenza Virus Subtype H1 from H5.

The surface protein hemagglutinin (HA) mediates the attachment of influenza virus to host cells containing sialic acid and thus facilitates viral infection. Therefore, HA is considered as a good target for the development of diagnostic tools for influenza virus. Previously, we reported the isolation of single-stranded aptamers that can distinguish influenza subtype H1 from H5. In this study, we describe a method for the selective electrical detection of H1 using the isolated aptamer as a molecular probe. After immobilization of the aptamer on Si wafer, enzyme-linked immunosorbent assay (ELISA) and field emission scanning electron microscopy (FE-SEM) showed that the immobilized aptamer bound specifically to the H1 subtype but not to the H5 subtype. Assessment by cyclic voltammetry (CV) also demonstrated that the immobilized aptamer on the indium thin oxide-coated surface was specifically bound to the H1 subtype only, which was consistent with the ELISA and FE-SEM results. Further measurement of CV using various amounts of H1 subtype provided the detection limit of the immobilized aptamer, which showed that a nanomolar scale of target protein was sufficient to produce the signal. These results indicated that the selected aptamer can be an effective probe for distinguishing the subtypes of influenza viruses by monitoring current changes.

Luteolin induces apoptotic cell death via antioxidant activity in human colon cancer cells.

The present study determined whether luteolin induces HT-29 colon cancer cell death through an antioxidant effect such as the activation of antioxidant enzymes. Luteolin decreased cell viability in human colon cancer cells (HT-29), whereas it had no effect on normal colon cells (FHC). Luteolin induced apoptosis by activating the mitochondria-mediated caspase pathway in HT-29 cells. Luteolin caused loss of the mitochondrial membrane action potential, increased mitochondrial Ca2+ level, upregulated Bax, downregulated Bcl-2, induced the release of cytochrome c from mitochondria to the cytosol, and increased the levels of the active forms of caspase-9 and caspase-3. Luteolin-induced apoptosis was accompanied by the activation of intracellular and mitochondrial reactive oxygen species scavenging through the activation of antioxidant enzymes, such as superoxide dismutase and catalase in HT-29 cells. Luteolin increased the level of reduced glutathione (GSH) and the expression of GSH synthetase, which catalyzes the second step of GSH biosynthesis. The apoptotic effect of luteolin was mediated by the activation of the mitogen-activated protein kinase signaling pathway. The present results indicate that luteolin induces apoptosis by promoting antioxidant activity and activating MAPK signaling in human colon cancer cells.

A Benzylideneacetophenone Derivative Induces Apoptosis of Radiation-Resistant Human Breast Cancer Cells via Oxidative Stress.

Benzylideneacetophenone derivative (1E)-1-(4-hydroxy-3-methoxyphenyl) hept-1-en-3-one (JC3) elicited cytotoxic effects on MDA-MB 231 human breast cancer cells-radiation resistant cells (MDA-MB 231-RR), in a dose-dependent manner, with an IC50 value of 6 µM JC3. JC3-mediated apoptosis was confirmed by increase in sub-G1 cell population. JC3 disrupted the mitochondrial membrane potential, and reduced expression of anti-apoptotic B cell lymphoma-2 protein, whereas it increased expression of pro-apoptotic Bcl-2-associated X protein, leading to the cleavage of caspase-9, caspase-3 and poly (ADP-ribose) polymerase. In addition, JC3 activated mitogen-activated protein kinases, and specific inhibitors of these kinases abrogated the JC3-induced increase in apoptotic bodies. JC3 increased the level of intracellular reactive oxygen species and enhanced oxidative macromolecular damage via lipid peroxidation, protein carbonylation, and DNA strand breakage. Considering these findings, JC3 is an effective therapy against radiation-resistant human breast cancer cells.

Endoplasmic reticulum stress induces 5-fluorouracil resistance in human colon cancer cells.

Colon cancer can be treated with 5-fluorouracil (5-FU), but 5-FU resistance frequently occurs. We determined whether 5-FU resistance arises as a result of endoplasmic reticulum (ER) stress. 5-FU-resistant SNUC5 colon cancer cells (SNUC5/FUR cells) expressed higher levels of ER stress-related proteins than drug-sensitive SNUC5 cells. SNUC5/FUR cells also exhibited more intense ER staining and higher level of mitochondrial Ca(2+) overload. SNUC5/FUR cells transfected with siRNA against GRP78, ATF6, ERK, or AKT were more sensitive to 5-FU than siControl RNA-transfected cells. These results suggested that 5-FU resistance was associated with ER stress in colon cancer.

Induction of Endoplasmic Reticulum Stress via Reactive Oxygen Species Mediated by Luteolin in Melanoma Cells.

BACKGROUND: This study aimed to investigate whether luteolin, a flavonoid, induces apoptosis in human melanoma cells via endoplasmic reticulum (ER) stress. MATERIALS AND METHODS: To investigate the effects of luteolin in human melanoma cells, the anti-proliferation, apoptosis, ER stress induction and reactive oxygen species (ROS) generation were evaluated using MTT, Hoechst 33342, ER-tracker Blue White DPX and DCF-DA staining assays, respectively. RESULTS: Luteolin inhibited cell proliferation and increased apoptotic body formation. Luteolin induced ER stress, as shown by ER staining and mitochondrial Ca(2+) overloading. Luteolin increased expression of the ER stress-related proteins; protein kinase RNA-like ER kinase, phospho eukaryotic translation initiation factor 2α, activating transcription factor (ATF) 6, CCAAT/enhancer-binding protein-homologous protein (CHOP), and cleaved caspase 12. Furthermore, luteolin increased the level of intracellular ROS, leading to ROS-mediated apoptosis and ER stress. However, N-acetyl cysteine, a ROS scavenger, decreased ROS levels, apoptosis, and ER stress induced by luteolin treatment. In addition, knockdown of CHOP and ATF6 by small-interfering RNA inhibited luteolin-induced cell death. CONCLUSION: Luteolin induces apoptosis by ER stress via increasing ROS levels.

Identification of a resveratrol tetramer as a potent inhibitor of hepatitis C virus helicase.

BACKGROUND AND PURPOSE: Hepatitis C virus (HCV) infection is responsible for various chronic inflammatory liver diseases. Here, we have identified a naturally occurring compound with anti-HCV activity and have elucidated its mode of antiviral action. EXPERIMENTAL APPROACH: Luciferase reporter and real-time RT-PCR assays were used to measure HCV replication. Western blot, fluorescence-labelled HCV replicons and infectious clones were employed to quantitate expression levels of viral proteins. Resistant HCV mutant mapping, in vitro NS3 protease, helicase, NS5B polymerase and drug affinity responsive target stability assays were also used to study the antiviral mechanism. KEY RESULTS: A resveratrol tetramer, vitisin B from grapevine root extract showed high potency against HCV replication (EC50 = 6 nM) with relatively low cytotoxicity (EC50 >10 µM). Combined treatment of vitisin B with an NS5B polymerase inhibitor (sofosbuvir) exhibited a synergistic or at least additive antiviral activity. Analysis of a number of vitisin B-resistant HCV variants suggested an NS3 helicase as its potential target. We confirmed a direct binding between vitisin B and a purified NS3 helicase in vitro. Vitisin B was a potent inhibitor of a HCV NS3 helicase (IC50 = 3 nM). In vivo, Finally, we observed a preferred tissue distribution of vitisin B in the liver after i.p. injection in rats, at clinically attainable concentrations. Conclusion and Implications Vitisin B is one of the most potent HCV helicase inhibitors identified so far. Vitisin B is thus a prime candidate to be developed as the first HCV drug derived from natural products.

Generation of Reactive Oxygen Species and Endoplasmic Reticulum Stress by Dictyopteris undulata Extract Leads to Apoptosis in Human Melanoma Cells.

In this study, we evaluated the hypothesis that a marine brown algae, Dictyopteris undulata ethanol extract (DUE), provokes apoptosis in a human melanoma cell line, A2058, via reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. DUE inhibited A2058 cell proliferation and increased apoptotic body formation, as indicated by the presence of fragmented nuclei and the activation of caspase-3. Moreover, DUE-treated cells showed elevated ER staining, mitochondrial calcium cation (Ca2+) overloading, augmented levels of ER stress-related and cell death modulatory proteins, including RNA-dependent protein kinase-related ER kinase, phospho-inositol-requiring enzyme 1α, phospho-eukaryotic translation initiation factor 2α, and CCAAT/enhancer-binding protein-homologous protein, as well as increased intracellular ROS levels. However, the antioxidant N-acetyl cysteine reversed the elevated ROS levels, decreased apoptosis, and mitigated ER stress in A2058 cells following DUE treatment. These findings suggest that DUE treatment triggers apoptosis in human melanoma cells through a mechanism involving ER stress and ROS.

Identification of a Novel Small Molecule Inhibitor Against SARS Coronavirus Helicase.

A new chemical inhibitor against severe acute respiratory syndrome (SARS) coronavirus helicase, 7-ethyl-8-mercapto-3-methyl-3,7-dihydro-1H-purine-2,6-dione, was identified. We investigated the inhibitory effect of the compound by conducting colorimetry-based ATP hydrolysis assay and fluorescence resonance energy transfer-based double-stranded DNA unwinding assay. The compound suppressed both ATP hydrolysis and double-stranded DNA unwinding activities of helicase with IC50 values of 8.66 ± 0.26 µM and 41.6 ± 2.3 µM, respectively. Moreover, we observed that the compound did not show cytotoxicity up to 80 µMconcentration. Our results suggest that the compound might serve as a SARS coronavirus inhibitor.

Isolation of single-stranded DNA aptamers that distinguish influenza virus hemagglutinin subtype H1 from H5.

Surface protein hemagglutinin (HA) mediates the binding of influenza virus to host cell receptors containing sialic acid, facilitating the entry of the virus into host cells. Therefore, the HA protein is regarded as a suitable target for the development of influenza virus detection devices. In this study, we isolated single-stranded DNA (ssDNA) aptamers binding to the HA1 subunit of subtype H1 (H1-HA1), but not to the HA1 subunit of subtype H5 (H5-HA1), using a counter-systematic evolution of ligands by exponential enrichment (counter-SELEX) procedure. Enzyme-linked immunosorbent assay and surface plasmon resonance studies showed that the selected aptamers bind tightly to H1-HA1 with dissociation constants in the nanomolar range. Western blot analysis demonstrated that the aptamers were binding to H1-HA1 in a concentration-dependent manner, yet were not binding to H5-HA1. Interestingly, the selected aptamers contained G-rich sequences in the central random nucleotides region. Further biophysical analysis showed that the G-rich sequences formed a G-quadruplex structure, which is a distinctive structure compared to the starting ssDNA library. Using flow cytometry analysis, we found that the aptamers did not bind to the receptor-binding site of H1-HA1. These results indicate that the selected aptamers that distinguish H1-HA1 from H5-HA1 can be developed as unique probes for the detection of the H1 subtype of influenza virus.

Dual effects of duplex RNA harboring 5'-terminal triphosphate on gene silencing and RIG-I mediated innate immune response.

Duplex RNA harboring the 5'-terminal triphosphate RNA is hypothesized to not only execute selective gene silencing via RNA interference, but also induce type I interferon (IFN) through activation of the retinoic acid inducible gene I (RIG-I). We evaluated gene silencing efficacy of the shRNA containing 5'-triphosphate (3p-shRNA) targeting the hepatitis C virus (HCV) RNA genome in hepatic cells. Gene silencing efficacy of the 3p-shRNA was diminished due to the presence of the 5'-triphosphate moiety in shRNA, whereas the shRNA counterpart without 5'-triphosphate (HO-shRNA) showed a strong antiviral activity without significant induction of type I IFN in the cells. 3p-shRNA was observed to be a better activator of the RIG-I signaling than the HO-shRNA with an elevated induction of type I IFN in cells that express RIG-I. Taken together, we suggest that competition for the duplex RNA bearing 5'-triphosphate between RIG-I and RNA interference factors may compromise efficacy of selective gene silencing.

Dictyopteris undulata Extract Induces Apoptosis via Induction of Endoplasmic Reticulum Stress in Human Colon Cancer Cells.

BACKGROUND: Induction of endoplasmic reticulum (ER) stress-mediated apoptosis in cancer cells represents an alternative approach for cancer therapy. The objective of this study was to elucidate whether ethanol extract of the marine brown alga Dictyopteris undulata can induce apoptosis, via ER stress, in human colon adenocarcinoma cells. METHODS: Anti-proliferative activity was evaluated by the colony forming assay. ER stress response was evaluated using flow cytometry and confocal imaging after Rhod2 and ER tracker staining. The expression of ER stress-related proteins was assessed by Western blotting. RESULTS: D. undulata extract (DUE) inhibited colony forming ability in SW480 cells. Furthermore, DUE induced characteristic signs of ER stress: mitochondrial Ca(2+) overloading, ER staining, expression of ER stress-related proteins, phosphorylation of RNA-dependent protein kinase-like ER kinase and inositol requiring enzyme 1, cleavage of activating transcription factor 6, and induction of the pro-apoptotic factors, CCAAT/enhancer-binding protein-homologous protein (CHOP) and caspase-12. Moreover, down-regulation of CHOP by siCHOP RNA attenuated DUE-induced apoptosis. CONCLUSIONS: The ER stress response plays an important role in DUE-induced apoptosis in human colon cancer cells.

Ursolic acid and its natural derivative corosolic acid suppress the proliferation of APC-mutated colon cancer cells through promotion of ß-catenin degradation.

Ursolic acid (UA) and corosolic acid (CA), naturally occurring pentacyclic triterpene acids, exhibit antiproliferative activities against various cancer cells, but a clear chemopreventive mechanism of these triterpenoids in colon cancer cells remains to be answered. Here we used a cell-based reporter system for detection of ß-catenin response transcription (CRT) to identify UA as an antagonist of the Wnt/ß-catenin pathway. UA promoted the degradation of intracellular ß-catenin that was accompanied by its N-terminal phosphorylation at Ser33/37/Thr41 residues, marking it for proteasomal degradation. Consistently, UA down-regulated the intracellular ß-catenin level in colon cancer cells with inactivating mutations of adenomatous polyposis coli (APC). In addition, UA repressed the expression of ß-catenin/T-cell factor (TCF)-dependent genes, thereby inhibiting cell proliferation in colon cancer cells. The functional group analysis revealed that the major structural requirements for UA-mediated ß-catenin degradation are a carboxyl group at position 17 and a methyl group at position 19. Notably, CA (2α-hydroxyursolic acid) was also found to decrease the level of intracellular ß-catenin and to suppress the growth of APC-mutated colon cancer cells. Our findings suggest that UA and CA exert their anticancer activities against colon cancer cells by promoting the N-terminal phosphorylation and subsequent proteasomal degradation of ß-catenin.

Single-stranded DNA aptamer that specifically binds to the influenza virus NS1 protein suppresses interferon antagonism.

Non-structural protein 1 (NS1) of the influenza A virus (IAV) inhibits the host's innate immune response by suppressing the induction of interferons (IFNs). Therefore, blocking NS1 activity can be a potential strategy in the development of antiviral agents against IAV infection. In the present study, we selected a single-stranded DNA aptamer specific to the IAV NS1 protein after 15 cycles of systematic evolution of ligands by exponential enrichment (SELEX) procedure and examined the ability of the selected aptamer to inhibit the function of NS1. The selected aptamer binds to NS1 with a Kd of 18.91±3.95nM and RNA binding domain of NS1 is determined to be critical for the aptamer binding. The aptamer has a G-rich sequence in the random sequence region and forms a G-quadruplex structure. The localization of the aptamer bound to NS1 in cells was determined by confocal images, and flow cytometry analysis further demonstrated that the selected aptamer binds specifically to NS1. In addition, luciferase reporter gene assay, quantitative RT-PCR, and enzyme-linked immunosorbent assay (ELISA) experiments demonstrated that the selected aptamer had the ability to induce IFN-ß by suppressing the function of NS1. Importantly, we also found that the selected aptamer was able to inhibit the viral replication without affecting cell viability. These results indicate that the selected ssDNA aptamer has strong potential to be further developed as a therapeutic agent against IAV.