ACT001 modulates the NF-κB/MnSOD/ROS axis by targeting IKKß to inhibit glioblastoma cell growth.

Glioblastomas are high-grade brain tumors with poor prognoses, and new therapeutic approaches for these tumors are critically needed. This study revealed the underlying mechanisms of a new orphan drug, ACT001, that is currently in clinical trials for the treatment of advanced glioblastoma in Australia and China. ACT001 significantly suppressed glioma cell proliferation and induced apoptosis and cell cycle arrest in vitro, as determined by Cell Counting Kit-8 assays and flow cytometry. In addition, U-118 MG cells with high expression of p-IKKß were sensitive to ACT001. Changes in the oxidative stress pathway in U-118 MG cells were detected with the isobaric tags for relative and absolute quantitation (iTRAQ) method. We further verified that ACT001 elevated the levels of reactive oxygen species (ROS) by regulating NF-κB-targeted MnSOD. ACT001 markedly inhibited NF-κB activation by directly binding IKKß and inhibiting its phosphorylation. Overexpression of IKKß markedly attenuated the changes in MnSOD and NOX1, indicating that ACT001 increased the levels of ROS by reducing the protein expression of p-IKKß. Furthermore, ACT001 reduced cyclin B1/CDC2 expression and triggered G2/M phase arrest by increasing ROS production. ACT001 also upregulated the expression of Bax and Bim and induced apoptosis in a ROS-dependent manner. ACT001 effectively suppressed the growth of U-118 MG tumors in BALB/c nude mice and GL-261-luciferase tumors in C57BL/6 J mice. Finally, ACT001 downregulated the expression of p-p65, MnSOD, cyclin B1, CDC2, and Ki67 in U-118 MG tumor tissues. Patients with activated NF-κB signaling should thus be given priority for enrollment in future phase II clinical trials. KEY MESSAGES: ACT001 directly bind to IKKß and inhibited its phosphorylation. The inhibition of p-IKKß induced the generation of ROS. ACT001 promoted the generation of ROS by regulating MnSOD expression to induce G2/M phase arrest.

Probing Micro-Newton Forces on Solid/Liquid/Gas Interfaces Using Transmission Phase Shift.

Many of the nature and life systems are driven by capillary interactions on solid/liquid/gas interfaces. Here, we present a profilometry technique called transmission phase shift for visualizing the liquid/gas interfaces in three dimensions with high resolution. Using this approach, we probe the change in tiny forces with particle radius at a solid/liquid/gas interface. We provide the first direct evidence that in the issues of floating versus sinking at small-scale, Archimedes' principle should be generalized to include the crucial role of surface tension and reveal the dominant regimes of floating particles based on the Bond number. Remarkably, the measured forces are in the range of micro-Newtons, suggesting that this terse methodology may guide the future design of a liquid microbalance and will be a universal tool for investigating capillarity and interface issues.

Aluminum hydroxide colloid vaccine encapsulated in yeast shells with enhanced humoral and cellular immune responses.

Aluminum salt (Alum) is one of the most important immune adjuvants approved for use in humans, however it is not suitable for vaccination against various chronic infectious diseases and cancers for not being able to induce cell-mediated (Th1) immunity. Here, we encapsulated an Alum colloid inside ß-glucan particles (GPs), which are a type of natural particles derived from the yeast glucan shells, to prepare hybrid GP-Alum (GP-Al) adjuvant particles with a very uniform size of 2-4 µm. These hybrid particles can be used to load antigen proteins through a simple mixing procedure, and can be highly specifically targeted to antigen-presenting cells (APCs) and strongly activate dendritic cells (DCs) maturation and cytokine secretion. In an animal model, they elicit a strong Th1-biased immune response and extremely high antibody titer, and cause marked prophylactic and therapeutic effects against tumors. As Alum has been proven to be a safe adjuvant to induce strong humoral responses and ß-glucans are safe for human use, this very uniform hybrid Alum particulate system could have important application as a vaccine carrier to stimulate humoral and cellular immune responses at the same time.

A novel antigen delivery system induces strong humoral and CTL immune responses.

New strategies with the ability to enhance both the humoral and cellular immune responses remain a priority for the development of future therapeutic cancer vaccines. In this study, we took advantage of ß-glucan particles (GPs) derived from Saccharomyces cerevisiae baker's yeast and a novel reverse micro-emulsion method to prepare an antigen-loaded GP carrier system for dendritic cell (DC) specific antigen delivery, followed by careful evaluation of the immune functions of the prepared particles in initiating both the humoral and cellular immune responses through in vitro and in vivo experiments. The prepared particles greatly promoted DC activation and cytokine production and cross presented the antigen to CD8 cells, inducing very strong OVA specific humoral and cellular immune responses. Treatment with these particles significantly prevented the growth of implanted EG7-OVA tumors in a prophylactic and pre-established tumor model. These results suggest that our strategy may be able to be utilized as a promising platform for cancer immunotherapy.

IL-4 induces a suppressive IL-10-producing CD8+ T cell population via a Cdkn2a-dependent mechanism.

CD8(+) T cells play an important role in immune regulation and effective immune responses against tumor cells, viral infection, and intracellular pathogens. In this report, using tiger or 10BiT mice, we defined a population of IL-10-producing CD8(+) T cells that were induced by IL-4. These IL-10(+)CD8(+) T cells possessed a strong inhibitory effect on the CD4(+) T cell proliferation in an IL-10-dependent and cell contact-dependent fashion. In comparison with IL-10(-)CD8(+) T cells, IL-10(+)CD8(+) T cells expressed an array of Th2-like cytokines (IL-4, IL-5), perforin, and granzymes, as well as the cell cycle regulatory protein Cdkn2a. Interestingly, knockdown of cdkn2a using siRNA reduced IL-4-induced IL-10 production significantly. Furthermore, CD8(+) T cells from Cdkn2a(-/-) mice produced a significantly lower amount of IL-10, and the effect was limited to CD8(+) T cells but not observed in CD4(+) T cells and APCs. Finally, IL-10(+)CD8(+) T cells played a protective role in the TNBS-induced murine colitis model, indicating a critical role of this population of CD8(+) T cells in regulatory immune responses. Taken together, we have defined a population of IL-10-producing CD8(+) Tregs induced by IL-4 and mediated by Cdkn2a.

Donut-shaped fingerprint in homologous polypeptide relationships--a topological feature related to pathogenic structural changes in conformational disease.

Features of homologous relationship of proteins can provide us a general picture of protein universe, assist protein design and analysis, and further our comprehension of the evolution of organisms. Here we carried out a study of the evolution of protein molecules by investigating homologous relationships among residue segments. The motive was to identify detailed topological features of homologous relationships for short residue segments in the whole protein universe. Based on the data of a large number of non-redundant proteins, the universe of non-membrane polypeptide was analyzed by considering both residue mutations and structural conservation. By connecting homologous segments with edges, we obtained a homologous relationship network of the whole universe of short residue segments, which we named the graph of polypeptide relationships (GPR). Since the network is extremely complicated for topological transitions, to obtain an in-depth understanding, only subgraphs composed of vital nodes of the GPR were analyzed. Such analysis of vital subgraphs of the GPR revealed a donut-shaped fingerprint. Utilization of this topological feature revealed the switch sites (where the beginning of exposure of previously hidden "hot spots" of fibril-forming happens, in consequence a further opportunity for protein aggregation is provided; 188-202) of the conformational conversion of the normal alpha-helix-rich prion protein PrP(C) to the beta-sheet-rich PrP(Sc) that is thought to be responsible for a group of fatal neurodegenerative diseases, transmissible spongiform encephalopathies. Efforts in analyzing other proteins related to various conformational diseases are also introduced.

Biological application of multi-component nanowires in hybrid devices powered by F1-ATPase motors.

In this paper, construction of hybrid device by integrating nanowires with F(1)-ATPase motors is described. The nickel nanowires and multi-segment nanowires, including gold and nickel, were fabricated by electrochemical deposition in nanoporous templates. The nickel nanowires functionalized by biotinylated peptide can be assembled directly onto F(1)-ATPase motors to act as the propellers. If the multi-component nanowires, including gold and nickel, were selectively functionalized by the thiol group modified ssDNA and the synthetic peptide, respectively, the biotinylated F(1)-ATPase motors can be attached to the biotinylated peptide on nickel segment of the nanowires. Then, the multi-component nanowires can also be used as the propellers, and one may observe the rotations of the multi-component nanowires driven by F(1)-ATPase motors. Therefore, introduction of multiple segments along the length of a nanowire can lead to a variety of multiple chemical functionalities, which can be selectively bound to cells and special biomolecules. This method provides an insight for the construction of other hybrid devices with its controlling arrangement of different biomolecule on designed nanometer scale structures.