On/off-switchable anti-neoplastic nanoarchitecture.

Throughout the world, there are increasing demands for alternate approaches to advanced cancer therapeutics. Numerous potentially chemotherapeutic compounds are developed every year for clinical trial and some of them are considered as potential drug candidates. Nanotechnology-based approaches have accelerated the discovery process, but the key challenge still remains to develop therapeutically viable and physiologically safe materials suitable for cancer therapy. Here, we report a high turnover, on/off-switchable functionally popping reactive oxygen species (ROS) generator using a smart mesoporous titanium dioxide popcorn (TiO2 Pops) nanoarchitecture. The resulting TiO2 Pops, unlike TiO2 nanoparticles (TiO2 NPs), are exceptionally biocompatible with normal cells. Under identical conditions, TiO2 Pops show very high photocatalytic activity compared to TiO2 NPs. Upon on/off-switchable photo activation, the TiO2 Pops can trigger the generation of high-turnover flash ROS and can deliver their potential anticancer effect by enhancing the intracellular ROS level until it crosses the threshold to open the 'death gate', thus reducing the survival of cancer cells by at least six times in comparison with TiO2 NPs without affecting the normal cells

Phosphorylcholine and KR12-Containing Corneal Implants in HSV-1-Infected Rabbit Corneas

Severe HSV-1 infection can cause blindness due to tissue damage from severe inflammation. Due to the high risk of graft failure in HSV-1-infected individuals, cornea transplantation to restore vision is often contraindicated. We tested the capacity for cell-free biosynthetic implants made from recombinant human collagen type III and 2-methacryloyloxyethyl phosphorylcholine (RHCIII-MPC) to suppress inflammation and promote tissue regeneration in the damaged corneas. To block viral reactivation, we incorporated silica dioxide nanoparticles releasing KR12, the small bioactive core fragment of LL37, an innate cationic host defense peptide produced by corneal cells. KR12 is more reactive and smaller than LL37, so more KR12 molecules can be incorporated into nanoparticles for delivery. Unlike LL37, which was cytotoxic, KR12 was cell-friendly and showed little cytotoxicity at doses that blocked HSV-1 activity in vitro, instead enabling rapid wound closure in cultures of human epithelial cells. Composite implants released KR12 for up to 3 weeks in vitro. The implant was also tested in vivo on HSV-1-infected rabbit corneas where it was grafted by anterior lamellar keratoplasty. Adding KR12 to RHCIII-MPC did not reduce HSV-1 viral loads or the inflammation resulting in neovascularization. Nevertheless, the composite implants reduced viral spread sufficiently to allow stable corneal epithelium, stroma, and nerve regeneration over a 6-month observation period

http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-76543 Modeling of Molecular Interaction between Apoptin, BCR-Abl and CrkL- An Alternative Approach to Conventional Rational Drug Design

In this study we have calculated a 3D structure of apoptin and through modeling and docking approaches, we show its interaction with Bcr-Abl oncoprotein and its downstream signaling components, following which we confirm some of the newly-found interactions by biochemical methods. Bcr-Abl oncoprotein is aberrantly expressed in chronic myelogenous leukaemia (CML). It has several distinct functional domains in addition to the Abl kinase domain. The SH3 and SH2 domains cooperatively play important roles in autoinhibiting its kinase activity. Adapter molecules such as Grb2 and CrkL interact with proline-rich region and activate multiple Bcr-Abl downstream signaling pathways that contribute to growth an

On/off-switchable anti-neoplastic nanoarchitecture

Throughout the world, there are increasing demands for alternate approaches to advanced cancer therapeutics. Numerous potentially chemotherapeutic compounds are developed every year for clinical trial and some of them are considered as potential drug candidates. Nanotechnology-based approaches have accelerated the discovery process, but the key challenge still remains to develop therapeutically viable and physiologically safe materials suitable for cancer therapy. Here, we report a high turnover, on/off-switchable functionally popping reactive oxygen species (ROS) generator using a smart mesoporous titanium dioxide popcorn (TiO2 Pops) nanoarchitecture. The resulting TiO2 Pops, unlike TiO2 nanoparticles (TiO2 NPs), are exceptionally biocompatible with normal cells. Under identical conditions, TiO2 Pops show very high photocatalytic activity compared to TiO2 NPs. Upon on/off-switchable photo activation, the TiO2 Pops can trigger the generation of high-turnover flash ROS and can deliver their potential anticancer effect by enhancing the intracellular ROS level until it crosses the threshold to open the 'death gate', thus reducing the survival of cancer cells by at least six times in comparison with TiO2 NPs without affecting the normal cells

Modeled interactions between apoptin and Bcr-Abl.

<p>(<b>A</b>) Shows the interaction between apoptin and the SH3-domain of Bcr-Abl (solid ribbon view, showing the two terminals of two proteins) obtained by performing virtual docking experiment between apoptin model and the X-ray structure of Bcr-Abl-SH3 domain (PDB: 2ABL). (<b>B</b>) Shows the space filling docking view of the interactions between apoptin (pink) and the SH3-domain of Bcr-Abl (blue), the 13 residues (red) of Bcr-Abl and 13 residues (light blue) of apoptin that are within 2.5 Å to each other; some of the proline-rich (PxxP) SH3-binding residues (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028395#pone-0028395-t002" target="_blank">Table 2</a>) are present and at least five direct hydrogen bonding are possible in between them (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028395#pone-0028395-t003" target="_blank">Table 3</a>). Additional information on apoptin interaction with BcrAbl could be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028395#pone.0028395.s004" target="_blank">Coordinates S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028395#pone.0028395.s005" target="_blank">S5</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028395#pone.0028395.s006" target="_blank">S6</a>.</p

Interaction of apoptin with Abl and Bcr-Abl^p210.

<p>(<b>A</b>) Indirect immunofluorescence showing the nuclear localization of Bcr-Abl. 32D^P210 and 32D^DSMZ cells were transiently transfected with GFP-apoptin (green) and subjected to (immuno)fluorescence staining and detection. Apoptin localization was by GFP and Bcr-Abl was detected by staining with Bcr-Abl^p210 with Cy3 tagged (red) secondary antibody. Nuclei were co-stained with DAPI (4, 6-diamidino-2-phenylindole: blue). Column 1 shows DAPI stained nuclei; columns 2 and 3 show the nuclear localization of Bcr-Abl and apoptin. Column 4 shows the merged image of nuclear co-localized of Bcr-Abl^p210 and GFP-apoptin as small clusters (yellow). Abbreviations: Tx = cells transfected with GFP-apoptin, No Tx = no transfection with GFP-apoptin. (<b>B</b>) To demonstrate apoptin and Bcr-Abl interactions, 5–10 µg of GST-apoptin was used in the ‘pull-down assay’. The interaction was tested either on 500 µg of total cell lysates from Bcr-Abl expressing 32D^p210 cells, or on Bcr-Abl non-expressing 32D^DMSZ cells. Lanes from the left: (1) the pull-down products of Bcr-Abl in 32D^DMSZ extracts (negative control), (2) 32D ^p210 extract (positive control), (3) 32D ^p210 extract treated with glutathione-sepharose beads (beads control), and (4) 32D^p210 extract incubated with GST-Apoptin captured with glutathione-sepharose beads. (<b>C</b>) In order to detect apoptin and Bcr-Abl interaction by co-immunoprecipitation assay, 32D^p210 cells were transiently transfected with GFP-apoptin (3 µg of pEGFP-apoptin plasmid for 2×10⁶ cells per transfection using lipofectamine transfection reagent) and cell lysates were incubated with anti-Bcr-Abl antibody followed by immunoprecipitation by protein G-sepharose beads; washed IP products were tested for the presence of apoptin (GFP-apoptin: 40 kDa) by immunoblot using anti-apoptin antibody. Lanes from the left: 1 - GST-Apoptin (positive control), 2 - GFP-apoptin Co-IP from transfected 32D^p210 cells by anti-Bcr-Abl antibody, 3 - Co-IP supernatant/immunodepleted fraction from transfected 32D^p210 cell lysates, 4 - 32D^p210 transfected with GFP (Co-IP, negative control), and 5 - Co-IP from 32D^p210 cells without transfection (Co-IP, negative control). (<b>D</b>) The Abl SH3 domain in Bcr-Abl^p210 facilitates Bcr-Abl interaction with apoptin. 32D^DSMZ cells were transfected with various Bcr-Abl mutant constructs by lipofectamine using 3–4 µg purified plasmid DNA per 2×10⁶ cells. Specific mutant clones of transfected cells were selected by G418. Pull-down assays were performed 7–10 days following the selection and expressed proteins were detected by immunoblotting. The upper representative immunoblot shows various mutants of Bcr-Abl expressed in various 32D^DSMZ clones. The lower immunoblot shows results of GST-apoptin pull-down assay. The Src-homology domain mutant of Bcr-Abl^p210 and GST-apoptin were used in this ‘pull-down’ (><) experiments using lysates from various Bcr-Abl mutant protein expressing 32D^DSMZ clones. The protein-protein complexes were analyzed for apoptin interaction by immunoblotting with rabbit anti-Bcr antibody. As seen (lane 6–9), the presence of an intact SH3 domain in the Bcr-Abl molecule is essential for its interaction with apoptin. Some degree of Bcr-Abl></p

Visualization of pathways, interacting network, and validation of selected downstream regulators.

<p>(<b>A</b>) Bcr-Abl and its downstream effectors are shown by BioCarta pathways. Global gene expression data of K562 leukemia cells was taken from public database, analyzed, and visualized (GenMAPP). The expression values of signal log base e ratio (SLR) are shown outside the colored boxes. The software generated color codes denote up-regulation (dark-red) and down-regulation (blue-green). (<b>B</b>) Direct and indirect interacting network associated with Bcr-Abl was built utilizing global gene expression data and visualized by IPA. The up-regulated genes are shown in red and down-regulated genes are shown as green. The gene expression values (SLR) are also shown. (<b>C, E, G</b>) Apoptin induced inhibition of Bcr-Abl phosphorylation leads to the down-regulation of downstream regulators, STAT5, Akt, and CrkL respectively. K562 and 32D^p210 cells were treated with 1 µM Tat-apoptin, Tat-GFP (negative control) and 1 µM imatinib (positive control) and cell lysates were prepared by harvesting cells after 16 h. Representative Western blots (divided into upper and lower panels representing the K562 cells and the second panel represents the 32D^p210 cells) show the ratio of the expression levels of phosphorylated and total STAT5 (<b>D</b>), Akt (<b>F</b>), and CrkL (<b>H</b>) respectively. In all the immunoblots, lane 1 from no-treatment control cells, lane 2 is from Tat-GFP treated cells, lane 3 is from Tat-apoptin treated cells respectively for both cell lines. STAT5 phosphorylation was significantly inhibited by apoptin indicating that apoptin induced inhibition of Bcr-Abl phosphorylation decreases the activation of STAT5 through phosphorylation. On the other hand, Akt phosphorylation was higher, indicating that apoptin induced Akt activation, as previously published. For CrkL, apoptin induced inhibition of Bcr-Abl phosphorylation lead to the down-regulation of CrkL resulting in lower phosphorytion indicating that apoptin decreases the activation of CrkL, a down-stream substrate of Bcr-Abl. For quantitation, band intensities were scanned by Image Quant software (version 5.2, Molecular Dynamics®). During quantitation, imatinib expression data was omitted in order to enable greater visualization of the apoptin effect. The quantitation data were normalized to the loading control (eIF4E/β-tubulin) and expressed as a ratio of phosphorylated to the total protein and presented as mean ± SEM of three independent experiments.</p

Sequence alignment and 3D model of Apoptin (aa: 1–121).

<p>(<b>A</b>) A representative sequence alignment between apoptin residues and the residues of one of the templates from a group of templates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028395#pone-0028395-t001" target="_blank">Table1</a>) is shown. (<b>B</b>) Solid ribbon view of full-length (aa: 1–121) of 3D model for apoptin and its amino and carboxyl terminals are shown. (<b>C</b>) Space filling view of apoptin model, showing the potential hydrophobic proline rich interacting area (PKPPSK, aa: 81–86, pink colored region, top right) is shown. (<b>D</b>) Ramachandran plot showing the N-Cα and Cα-C bonds in the apoptin polypeptide chain represented by the torsion angles phi (φ) and psi (ψ); quality of the model was examined by this plot (all atoms are within the allowed regions) and by the G-factors values (the overall value for G-factors is −0.35). (<b>E</b>) Solvent accessible surface area shows the regions of hydrophobic (large cream colored region at the surface) where protein-protein interactions could occur and the hydrophilic regions that are involved in hydrogen bonding, hydrogen bond acceptors (red color) and hydrogen bond donors (blue color). Additional information on apoptin structure could be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028395#pone.0028395.s001" target="_blank">Coordinates S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028395#pone.0028395.s002" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028395#pone.0028395.s003" target="_blank">S3</a>.</p

Sequence alignment and interactions between apoptin and adopter proteins CrkL, Akt1 and STAT5.

<p>Space filling views and similarity in sequences between apoptin and the SH2-domain of the adopter protein CrkL (<b>A</b>), apoptin's SH3-binding domain residues (<b>B</b>), 81 to 86 (PKPPSK), are within the SH2-domain of CrkL (<b>C</b>). In addition, the similarity in sequences between apoptin and the adopter proteins Akt1 (<b>D</b>) and STAT5 (<b>E</b>) suggesting that apoptin might directly interact in the CrkL, Akt1 and STAT5 interacting sites in addition to the SH3-binding domain of Bcr-Abl and could block further propagation of survival and proliferation signaling.</p

Amino acid residues forming hydrogen bond between apoptin and Bcr-Abl.

<p>*Hydrogen bonding forming residues between the Bcr-Abl and the proline rich PxxP region of apoptin are shown in <b>bold</b>.</p