SIRT2 reduces actin polymerization and cell migration through deacetylation and degradation of HSP90.

SIRT2, a member of the class III histone deacetylase family, has been identified as a tumor suppressor, which is associated with various cellular processes including metabolism and proliferation. However, the effects of SIRT2 on cancer cell migration caused by cytoskeletal rearrangement remain uncertain. Here we show that SIRT2 inhibits cell motility by suppressing actin polymerization. SIRT2 regulates actin dynamics through HSP90 destabilization and subsequent repression of LIM kinase (LIMK) 1/cofilin pathway. SIRT2 directly interacts with HSP90 and regulates its acetylation and ubiquitination. In addition, the deacetylase activity of SIRT2 is required for the regulation of actin polymerization and the ubiquitin-mediated proteasomal degradation of HSP90 induced by SIRT2.

Polydiacetylene single-walled carbon nanotubes nano-hybrid for cellular imaging applications.

The functionalization of single-walled carbon nanotubes (SWCNTs) by forming self-assembled supramolecular structure of 10,12-pentacosadiynoic acid (PCDA) on the carbon nanotube wall is reported. PCDA assemblies on SWCNTs (PCDA/SWCNTs) were polymerized by UV irradiation to extensively conjugated polydiacetylene (PDA). PDA/SWCNT was identified by absorption and emission spectroscopy, scanning and transmission electron microscopies (SEM and TEM) and atomic force microscopy (AFM). PDA/SCWNTs showed strong near-infrared (NIR) fluorescence caused by fluorescence resonance energy transfer (FRET) between PDA network and semiconducting SWCNT core. The micro-patterning of biotinylated PDA/SWCNT with FITC-avidin on biotinylated glass surface demonstrated the potential application for a bio-sensing device. Furthermore, the biocompatibility for mammalian cancer cells was tested by viability experiments, which revealed that the PDA/SWCNTs had very low toxicity below 31.3 mg/L in terms of pristine SWCNTs concentration. Also, PDA/SWCNTs inside the cells can be observed by NIR microscopy. This unique modular method of preparation can contribute to diverse functionalities for practical applications in various non-invasive cellular imaging.

Hepatitis B virus X protein promotes epithelial-mesenchymal transition of hepatocellular carcinoma cells by regulating SOCS1.

Hepatocellular carcinoma (HCC), a primary type of liver cancer, is one of the leading causes of cancer related deaths worldwide. HCC patients have poor prognosis due to intrahepatic and extrahepatic metastasis. Hepatitis B virus (HBV) infection is one of the major causes of various liver diseases including HCC. Among HBV gene products, HBV X protein (HBx) plays an important role in the development and metastasis of HCC. However, the mechanism of HCC metastasis induced by HBx has not been elucidated yet. In this study, for the first time, we report that HBx interacts with the suppressor of cytokine signaling 1 (SOCS1) which negatively controls NF-κB by degrading p65, a subunit of NF-κB. NF-κB activates the transcription of factors associated with epithelial-mesenchymal transition (EMT), a crucial cellular process associated with invasiveness and migration of cancer cells. Here, we report that HBx physically binds to SOCS1, subsequently prevents the ubiquitination of p65, activates the transcription of EMT transcription factors and enhance cell migration and invasiveness, suggesting a new mechanism of HBV-associated HCC metastasis. [BMB Reports 2022; 55(5): 220-225].

Pseudomonas aeruginosa eliminates natural killer cells via phagocytosis-induced apoptosis.

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes the relapse of illness in immunocompromised patients, leading to prolonged hospitalization, increased medical expense, and death. In this report, we show that PA invades natural killer (NK) cells and induces phagocytosis-induced cell death (PICD) of lymphocytes. In vivo tumor metastasis was augmented by PA infection, with a significant reduction in NK cell number. Adoptive transfer of NK cells mitigated PA-induced metastasis. Internalization of PA into NK cells was observed by transmission electron microscopy. In addition, PA invaded NK cells via phosphoinositide 3-kinase (PI3K) activation, and the phagocytic event led to caspase 9-dependent apoptosis of NK cells. PA-mediated NK cell apoptosis was dependent on activation of mitogen-activated protein (MAP) kinase and the generation of reactive oxygen species (ROS). These data suggest that the phagocytosis of PA by NK cells is a critical event that affects the relapse of diseases in immunocompromised patients, such as those with cancer, and provides important insights into the interactions between PA and NK cells.

Sonochemical preparation of silica nanorods for gene delivery using single-walled carbon nanotubes as templates.

Silica nanorods were fabricated with single-walled carbon nanotubes (SWCNTs) via ultrasound. The diameter of the resulting SWCNT-silica particles ranged from 60 to 70 nm. The morphology of this composite material was investigated via scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The individual SWCNTs are uniformly coated with silica and formed a unique nanocomposite material. The important role of ultrasound and the mechanism of silica layer formation on SWCNTs were explained via the hydrolysis of the silica source and the adsorption of the siloxane groups on the SWCNT surfaces under ultrasound irradiation. The amino-functionalized silica nanorods were demonstrated as non-viral vectors for gene delivery.

Synthesis of stable silica-dye hybrid nanomaterial as DNA carrier.

A new method is proposed for the fabrication of fluorescence-labeled and amine-modified silica nanoparticles for application as nonviral vectors in gene delivery. Highly monodisperse, stable fluorescent silica nanoparticles were prepared using 2,5-bis(5-tert-butyl-2-benzoxazolyl)thiophene and the water-in-oil microemulsion method. The green-fluorescent-protein gene can be easily combined onto the positively charged surfaces of nanoparticles to form a nanoparticle-DNA complex. The nanoparticle-DNA complex successfully passed through various barriers into the HeLa and HEK 293 K cells. The cytotoxicity of the PEI-coated and BBOT-encapsulated silica nanoparticles on both the HeLa and HEK 293T cell lines was found to be at an acceptable level for use as gene carriers when the particle concentration was below 125 microg/ml. The fluorescence intracellular images confirm the successful delivery of the nanoparticle-DNA complex and gene expression. The present work suggests the potential use of dye-incorporated silica nanoparticles in nonviral gene delivery.

Hepatitis B virus X protein enhances liver cancer cell migration by regulating calmodulin-associated actin polymerization.

Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC), which is a highly aggressive cancer. HBV X protein (HBx), one of four HBV gene products, plays pivotal roles in the development and metastasis of HCC. It has been reported that HBx induces liver cancer cell migration and reorganizes actin cytoskeleton, however the molecular basis for actin cytoskeleton reorganization remains obscure. In this study, we for the first time report that HBx promotes actin polymerization and liver cancer cell migration by regulating calcium modulated protein, calmodulin (CaM). HBx physically interacts with CaM to control the level of phosphorylated cofilin, an actin depolymerizing factor. Mechanistically, HBx interacts with CaM, liberates Hsp90 from its inhibitory partner CaM, and increases the activity of Hsp90, thus activating LIMK1/cofilin pathway. Interestingly, the interaction between HBx and CaM is calcium-dependent and requires the CaM binding motif on HBx. These results indicate that HBx modulates CaM which plays a regulatory role in Hsp90/LIMK1/cofilin pathway of actin reorganization, suggesting a new mechanism of HBV-induced HCC metastasis specifically derived by HBx. [BMB Reports 2021; 54(12): 614-619].

Antiaging and antioxidant effects of topical autophagy activator: A randomized, placebo-controlled, double-blinded study.

BACKGROUND: Recently, potential roles of autophagy in skin homeostasis received many interests. But, little has been reported for the potential antiaging effects of autophagy activator. OBJECTIVE: With the newly synthesized autophagy activator, heptasodium hexacarboxymethyl dipeptide-12 (Aquatide™) in vitro and clinical efficacy of the topical autophagy activator as an antiaging cosmeceutical ingredient was evaluated. METHODS: Antioxidant effect of Aquatide™ was evaluated by radical scavenging assay. In vitro effect was assessed by measuring the cytotoxicity of hydrogen peroxide in cultured normal human epidermal keratinocytes. Clinical evaluation was performed by a randomized, placebo-controlled, double-blinded study. Antioxidant efficacy was observed by measuring the carbonylated proteins in stratum corneum (SC) by fluorescein-5-thiosemicarbazide (FTZ) staining. RESULTS: Radical scavenging effects of Aquatide were observed with the ABTS assay, and significant reduction in hydrogen peroxide-induced cytotoxicity was observed in Aquatide™-treated cells. Autophagy inhibitor treatment abrogated cytoprotective effects of Aquatide™. In a clinical study, statistically significant increase in skin elasticity was observed after 4 and 8 weeks. Quantitative analysis of carbonylated proteins in SC also showed significant reduction in Aquatide™-treated group, which is consistent with the in vitro data. CONCLUSION: These results suggest that autophagy plays important roles in antioxidant system and aging process in skin, and topical autophagy activators can be potential cosmeceutical ingredients for skin antiaging.

HSV-1 ICP27 suppresses NF-kappaB activity by stabilizing IkappaBalpha.

Nuclear factor kappaB (NF-kappaB) is associated with the transcriptional activation of genes encoding chemokines, adhesion molecules, cytokines, and anti-apoptotic proteins, which are key components in immune responses and viral infection. Many viruses modulate NF-kappaB through numerous viral gene products to allow productive infections and immune escape. Here we report that herpes simplex virus-1 infected cell protein 27 (HSV-1 ICP27), an immediate early protein of HSV-1, represses NF-kappaB activity through binding to inhibitor of kappaB (IkappaBalpha), blocking phosphorylation and ubiquitination of IkappaBalpha, and stabilizing IkappaBalpha. These data may explain how NF-kappaB activity is regulated by ICP27 to escape immune responses during the very early period of HSV-1 infection.

Inhibition of NF-kappaB acetylation and its transcriptional activity by Daxx.

We propose a biochemical mechanism by which Daxx modulates NF-kappaB transcriptional activity. Both chromatin immunoprecipitation (ChIP) assay and electrophoretic mobility shift assay (EMSA) have confirmed Daxx-mediated repression of transcriptional competence of NF-kappaB in HeLa cells. Overexpression of Daxx repressed the expression of NF-kappaB-regulated genes such as I kappa B alpha and IL8. Co-immunoprecipitation assay revealed the existence of intermolecular association between endogenous Daxx and p65 subunit of NF-kappaB stimulated by TNFalpha. Here, we suggest that Daxx-mediated repression of NF-kappaB transactivation correlates with the inhibition of p65 acetylation by Daxx. Based on the finding that the Daxx binding N-terminal side of p65 includes the major sites of acetylation mediated by p300/CBP, we further propose that the physical interaction between Daxx and p65 provides a functional framework for the inhibition of p65 acetylation by p300/CBP and subsequent repression of NF-kappaB transcriptional activity.

Hepatitis B virus X protein induces apoptosis by enhancing translocation of Bax to mitochondria.

Hepatitis B virus X protein (HBx) is essential for viral replication and plays an important role in viral pathogenesis. HBx transactivates many viral and cellular genes and participates in cellular signal transduction pathways, proliferation, and apoptosis. In the present study, we report that HBx induces apoptosis by enhancing the translocation of Bax to mitochondria, followed by inducing the loss of mitochondrial membrane potential and release of cytochrome C. In addition, Bcl-2, inhibitor of Bax, rescues the disruption of mitochondrial membrane potential and DNA fragmentation induced by serum starvation in HepG2-X cells expressing HBx. We also found that HBx binds directly to Bax and interferes with the interaction between Bax and 14-3-3epsilon to enhance the translocation of Bax to mitochondria. Taken together, our data suggest that HBx induces apoptosis by interacting with Bax and enhancing its translocation to mitochondria.

14-3-3epsilon inhibits MK5-mediated cell migration by disrupting F-actin polymerization.

The signal pathway by which 14-3-3epsilon inhibits cell migration induced by MAPK-activated protein kinase 5 (MK5) was investigated in cultured HeLa cells. Both in vivo and in vitro analyses have revealed that 14-3-3epsilon interacts with MK5. 14-3-3epsilon bound to MK5 inhibits the phosphorylation of HSP27, a known substrate of MK5. Disturbance of actin cytoskeleton organization by 14-3-3epsilon was shown in transfected cells transiently expressing 14-3-3epsilon as well as established cells stably expressing 14-3-3epsilon. Moreover, overexpression of 14-3-3epsilon resulted in the inhibition of cell migration induced by MK5 overexpression or TNFalpha treatment. Our results suggest that 14-3-3epsilon bound to MK5 inhibits cell migration by inhibiting the phosphorylation of HSP27 whose phosphorylation regulates F-actin polymerization, actin cytoskeleton organization and subsequent actinfilament dynamics.

VBP1 represses cancer metastasis by enhancing HIF-1α degradation induced by pVHL.

von Hippel-Lindau-binding protein 1 (VBP1) physically interacts with pVHL, an E3-ubiquitin ligase, which degrades HIF-1α in an oxygen-dependent manner. HIF-1 is a key regulator of adaptive responses to a lack of oxygen that controls glucose metabolism, angiogenesis, proliferation, invasion, and metastasis. However, the role of VBP1 in pVHL-mediated degradation of HIF-1α is not yet known. In this study, we show that VBP1 enhances the stability of pVHL and facilitates pVHL-mediated ubiquitination of HIF-1α. Furthermore, VBP1 suppresses HIF-1α-induced epithelial-mesenchymal transition in vitro and tumor metastasis in vivo. These findings suggest that VBP1 is a bona fide tumor suppressor protein associated with HIF-1α regulation.

HSV-1 ICP27 induces apoptosis by promoting Bax translocation to mitochondria through interacting with 14-3-3θ.

The subcellular localization of Bax plays a crucial role during apoptosis. In response to apoptotic stimuli, Bax translocates from the cytoplasm to the mitochondria, where it promotes the release of cytochrome c to the cytoplasm. In cells infected with HSV-1, apoptosis is triggered or blocked by diverse mechanisms. In this study, we demonstrate how HSV-1 ICP27 induces apoptosis and modulates mitochondrial membrane potential in HEK 293T cells. We found that ICP27 interacts with 14-3-3θ which sequesters Bax to the cytoplasm. In addition, ICP27 promotes the translocation of Bax to the mitochondria by inhibiting the interaction between 14-3-3θ and Bax. Our findings may provide a novel apoptotic regulatory pathway induced by ICP27 during HSV-1 infection. [BMB Reports 2017; 50(5): 257-262].

HSV-1 ICP27 represses NF-κB activity by regulating Daxx sumoylation.

Herpes simplex virus type 1 ICP27 is a multifunctional protein responsible for viral replication, late gene expression, and reactivation from latency. ICP27 interacts with various cellular proteins, including Daxx. However, the role of interaction between ICP27 and Daxx is largely unknown. Since Daxx is known to repress NF-κB activity, there is a possibility that ICP27 may influence the inhibitory effect of Daxx on NF-κB activity. In this study, we tested whether ICP27 affects the NF-κB activity through its interaction with Daxx. Interestingly, ICP27 enhanced the Daxx-mediated repression of NF-κB activity. In addition, we found that sumoylation of Daxx regulates its interaction with p65. ICP27 binds to Daxx, inhibits Daxx sumoylation, and enhances p65 deacetylation induced by Daxx. Consequently, ICP27 represses the NF-B activity, by elevating the inhibitory effect of Daxx on NF-κB activity through desumoylation of Daxx. [BMB Reports 2017; 50(5): 275-280].

Aquatide Activation of SIRT1 Reduces Cellular Senescence through a SIRT1-FOXO1-Autophagy Axis.

Ultraviolet (UV) irradiation is a relevant environment factor to induce cellular senescence and photoaging. Both autophagy- and silent information regulator T1 (SIRT1)-dependent pathways are critical cellular processes of not only maintaining normal cellular functions, but also protecting cellular senescence in skin exposed to UV irradiation. In the present studies, we investigated whether modulation of autophagy induction using a novel synthetic SIRT1 activator, heptasodium hexacarboxymethyl dipeptide-12 (named as Aquatide), suppresses the UVB irradiation-induced skin aging. Treatment with Aquatide directly activates SIRT1 and stimulates autophagy induction in cultured human dermal fibroblasts. Next, we found that Aquatide-mediated activation of SIRT1 increases autophagy induction via deacetylation of forkhead box class O (FOXO) 1. Finally, UVB irradiation-induced cellular senescence measured by SA-ß-gal staining was significantly decreased in cells treated with Aquatide in parallel to occurring SIRT1 activation-dependent autophagy. Together, Aquatide modulates autophagy through SIRT1 activation, contributing to suppression of skin aging caused by UV irradiation.

Growth hormone-releasing peptide-biotin conjugate stimulates myocytes differentiation through insulin-like growth factor-1 and collagen type I.

Based on the potential beneficial effects of growth hormone releasing peptide (GHRP)-6 on muscle functions, a newly synthesized GHRP-6-biotin conjugate was tested on cultured myoblast cells. Increased expression of myogenic marker proteins was observed in GHRP-6-biotin conjugate-treated cells. Additionally, increased expression levels of insulin-like growth factor-1 and collagen type I were observed. Furthermore, GHRP-6-biotin conjugate-treated cells showed increased metabolic activity, as indicated by increased concentrations of energy metabolites, such as ATP and lactate, and increased enzymatic activity of lactate dehydrogenase and creatine kinase. Finally, binding protein analysis suggested few candidate proteins, including desmin, actin, and zinc finger protein 691 as potential targets for GHRP6-biotin conjugate action. These results suggest that the newly synthesized GHRP-6-biotin conjugate has myogenic stimulating activity through, at least in part, by stimulating collagen type I synthesis and several key proteins. Practical applications of the GHRP-6-biotin conjugate could include improving muscle condition.

Establishment and use of a cell line expressing HSV-1 thymidine kinase to characterize viral thymidine kinase-dependent drug-resistance.

To understand the mechanisms of antiviral drug resistance and to have a system to examine the cytotoxicity of herpes simplex virus type 1 (HSV-1) inhibitors that are thymidine kinase (TK)-dependent, we have constructed a plasmid pFTK1 by inserting a DNA fragment containing the TK gene of HSV-1 strain F into the eukaryotic expression vector pcDNA3.1/His A. TK-deficient 143B cells were transfected with this vector and neomycin-resistant cells were selected. Cell survival in HAT medium and TK activity of the cell lysates were examined to ascertain HSV-1 TK expression. A cell line expressing the viral TK gene, FTK143B (FTK), was established and used for characterization of two laboratory-derived TK-deficient drug-resistant HSV-1 mutants of strain F. The antiviral activities of several drugs, mostly nucleoside analogues, were compared in the Vero, 143B and FTK cell culture systems. We showed that both mutant viruses lost their resistance to acyclovir and to other HSV-1 TK-dependent compounds in FTK cells but not in Vero and 143B cells. Significantly increased cytotoxicity of ganciclovir and (E)-5-(2-bromovinyl)-2'-deoxyuridine was also observed in the FTK cells. This HSV-1 TK gene-transfected cell model is a useful tool to rapidly determine HSV-1 drug resistance at the viral TK level.

Identification of hepatitis C virus core domain inducing suppression of allostimulatory capacity of dendritic cells.

Hepatitis C virus (HCV) is remarkably efficient at establishing chronic infection. One of the reasons for this appears to be the suppression of the accessory cell function of professional antigen presenting cells. In the present study, the immunosuppressive activity of HCV protein was examined on dendritic cells (DCs) generated from mouse bone marrow progenitor cells in vitro. We found that the DCs forced to express HCV protein have defective allostimulatory ability. DCs expressing HCV protein were phenotypically indistinguishable from normal DCs. However, they were unable to produce IL-12 effectively when stimulated with lipopolysaccharide. The functional domain of the HCV protein essential for immunosuppression was determined using a series of NH2-and C-terminal deletion mutants of HCV core protein. We found that amino acid residues residing between the 21st and the 40th residues from the NH2-terminus of HCV core protein are required for immunosuppression. These findings suggest that HCV core protein suppresses the elicitation of protective Th1 responses by the inhibition of IL-12 production by DCs.

Aqueous extract of Tribulus terrestris Linn induces cell growth arrest and apoptosis by down-regulating NF-κB signaling in liver cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: A medicinal herb Tribulus terrestris Linn has been used to treat various diseases including hepatocellular carcinoma. The aim of the present study was to investigate the anticancer activity of Tribulus terrestris Linn (TT) in liver cancer cells. MATERIALS AND METHODS: The antitumor activity of aqueous TT extract was analyzed by testing the cytotoxicity and the effect on clonogenecity in HepG2 cells. Apoptosis and cell cycle arrest induced by TT were dissected by flow cytometry and its inhibitory effect on NF-κB activity was determined by analyzing the expression levels of NF-κB/IκB subunit proteins. The suppression of NF-κB-regulated gene expression by TT was assessed by RT-PCR. RESULTS: TT extract repressed clonogenecity and proliferation, induced apoptosis, and enhanced accumulation in the G0/G1 phase of liver cancer cells. It also turned out that TT extract inhibited NF-κB-dependent reporter gene expression and NF-κB subunit p50 expression, while it enhanced the cellular level of IκBα by inhibiting the phosphorylation and degradation of IκBα. In addition, IKK activity was inhibited in a dose-dependent manner. Furthermore, TT extract suppressed the transcription of genes associated with cell cycle regulation, anti-apoptosis, and invasion. CONCLUSION: These data showed that TT extract blocks proliferation and induces apoptosis in human liver cancer cells through the inhibition of NF-κB signaling. Aqueous TT extract can be used as an anticancer drug for hepatocellular carcinoma patients.