Chromenopyrimidinone Controls Stemness and Malignancy by suppressing CD133 Expression in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a highly malignant human cancer that has increasing mortality rates worldwide. Because CD133+ cells control tumor maintenance and progression, compounds that target CD133+ cancer cells could be effective in combating HCC. We found that the administration of chromenopyrimidinone (CPO) significantly decreased spheroid formation and the number of CD133+ cells in mixed HCC cell populations. CPO not only significantly inhibited cell proliferation in HCC cells exhibiting different CD133 expression levels, but also effectively induced apoptosis and increased the expression of LC3-II in HCC cells. CPO also exhibits in vivo therapeutic efficiency in HCC. Specifically, CPO suppressed the expression of CD133 by altering the subcellular localization of CD133 from the membrane to lysosomes in CD133+ HCC cells. Moreover, CPO treatment induced point mutations in the ADRB1, APOB, EGR2, and UBE2C genes and inhibited the expression of these proteins in HCC and the expression of UBE2C is particularly controlled by CD133 expression among those four proteins in HCC. Our results suggested that CPO may suppress stemness and malignancies in vivo and in vitro by decreasing CD133 and UBE2C expression in CD133+ HCC. Our study provides evidence that CPO could act as a novel therapeutic agent for the effective treatment of CD133+ HCC.

Optimization of formulation for enhanced intranasal delivery of insulin with translationally controlled tumor protein-derived protein transduction domain.

Intranasal delivery of insulin is an alternative approach to treat diabetes, as it enables higher patient compliance than conventional therapy with subcutaneously injected insulin. However, the use of intranasal delivery of insulin is limited for insulin's hydrophilicity and vulnerability to enzymatic degradation. This limitation makes optimization of formulation intranasal insulin for commercial purpose indispensable. This study evaluated bioavailability (BA) of various formulations of insulin intranasally delivered with protein transduction domain (PTD) derived from translationally controlled tumor protein. The therapeutic efficacy of newly formulated intranasal insulin + PTD was compared in vivo studies with normal and alloxan-induced diabetic rats, to those of free insulin and subcutaneously injected insulin. BA of insulin in two new formulations was, respectively, 60.71% and 45.81% of subcutaneously injected insulin, while the BA of free insulin was only 3.34%. Histological analysis of tissues, lactate dehydrogenase activity in nasal fluid, and biochemical analysis of sera revealed no detectable topical or systemic toxicity in rats and mice. Furthermore, stability analysis of newly formulated insulin + PTD to determine the optimal conditions for storage revealed that when stored at 4 °C, the delivery capacity of insulin was maintained up to 7 d. These results suggest that the new formulations of intranasal insulin are suitable for use in diabetes therapy and are easier to administer.

Photoinduced Charge Transfer in Single-Molecule p-n Junctions.

We measured photoinduced charge separation in isolated individual C60-tethered 2,5-dithienylpyrrole triad (C60 triad) molecules with submolecular resolution using a custom-built laser-assisted scanning tunneling microscope. Laser illumination was introduced evanescently into the tunneling junction through total internal reflection, and the changes in tunneling current and electronic spectra caused by photoexcitation were measured and spatially resolved. Photoinduced charge separation was not detected for all C60 triad molecules, indicating that the conformations of the molecules may affect the excitation probability, lifetime, and/or charge distribution. A photoinduced signal was not observed for dodecanethiol molecules in the surrounding matrix or for control molecules without C60 moieties, as neither absorbs incident photons at this energy. This spectroscopic imaging technique has the potential to elucidate detailed photoinduced carrier dynamics, which are inaccessible via ensemble-scale (i.e., averaging) measurements, which can be used to direct the rational design and optimization of molecular p-n junctions and assemblies for energy harvesting.

Modified translationally controlled tumor protein-derived protein transduction domain enhances nasal delivery of exendin-4 as shown with insulin.

Protein transduction domains (PTDs) have been shown to promote the delivery of therapeutic proteins or peptides into the living cells. In a previous study, we showed that the double mutant of TCTP-PTD 13, TCTP-PTD 13M2, was more effective in the delivery of insulin than the wild-type TCTP-PTD 13. In this study, we applied this approach to the nasal delivery of a different peptide, exendin-4, using as carriers, several modified TCTP-PTDs, such as TCTP-PTD 13M1, 13M2, and 13M3. Nasal co-administration of TCTP-PTD 13M2 with exendin-4 showed the highest exendin-4 uptake among the three analogs in normal rats, and also decreased blood glucose levels by 43.3% compared with that of exendin-4 alone and by 18.6% compared with that of exendin-4 plus TCTP-PTD 13 in diabetic mice. We also designed an additional covalently linked conjugate of TCTP-PTD 13M2 and exendin-4 and evaluated its hypoglycemic effect after subcutaneous or intranasal delivery. Subcutaneous administration of exendin-4 that its C-terminus is covalently linked to TCTP-PTD 13M2 showed hypoglycemic effect of 42.2% compared to that in untreated group, whereas intranasal delivery was not successful in diabetic mice. We conclude that a simple mixing TCTP-PTD 13M2 with peptide/protein drugs can be potentially a generally applicable approach for intranasal delivery into animals.

Molar uprighting by a nickel-titanium spring based on a setup model.

Molar uprighting is an important adjunctive treatment in orthodontics: repositioning the tilted molar eliminates the potentially pathologic condition and simplifies the ultimate restorative procedure. Although various methods for molar uprighting have been applied successfully, they still have some limitations and disadvantages in tooth movement. This article reports on a new clinical technique for molar uprighting with nickel-titanium springs based on a setup model. Two patients treated with this system are shown.

Exchange reactions between alkanethiolates and alkaneselenols on Au{111}.

When alkanethiolate self-assembled monolayers on Au{111} are exchanged with alkaneselenols from solution, replacement of thiolates by selenols is rapid and complete, and is well described by perimeter-dependent island growth kinetics. The monolayer structures change as selenolate coverage increases, from being epitaxial and consistent with the initial thiolate structure to being characteristic of selenolate monolayer structures. At room temperature and at positive sample bias in scanning tunneling microscopy, the selenolate-gold attachment is labile, and molecules exchange positions with neighboring thiolates. The scanning tunneling microscope probe can be used to induce these place-exchange reactions.

Interaction of translationally controlled tumor protein with Apaf-1 is involved in the development of chemoresistance in HeLa cells.

BACKGROUND: Translationally controlled tumor protein (TCTP), alternatively called fortilin, is believed to be involved in the development of the chemoresistance of tumor cells against anticancer drugs such as etoposide, taxol, and oxaliplatin, the underlying mechanisms of which still remain elusive. METHODS: Cell death analysis of TCTP-overexpressing HeLa cells was performed following etoposide treatment to assess the mitochondria-dependent apoptosis. Apoptotic pathway was analyzed through measuring the cleavage of epidermal growth factor receptor (EGFR) and phospholipase C-γ (PLC-γ), caspase activation, mitochondrial membrane perturbation, and cytochrome c release by flow cytometry and western blotting. To clarify the role of TCTP in the inhibition of apoptosome, in vitro apoptosome reconstitution and immunoprecipitation was used. Pull-down assay and silver staining using the variants of Apaf-1 protein was applied to identify the domain that is responsible for its interaction with TCTP. RESULTS: In the present study, we confirmed that adenoviral overexpression of TCTP protects HeLa cells from cell death induced by cytotoxic drugs such as taxol and etoposide. TCTP antagonized the mitochondria-dependent apoptotic pathway following etoposide treatment, including mitochondrial membrane damage and resultant cytochrome c release, activation of caspase-9, and -3, and eventually, the cleavage of EGFR and PLC-γ. More importantly, TCTP interacts with the caspase recruitment domain (CARD) of Apaf-1 and is incorporated into the heptameric Apaf-1 complex, and that C-terminal cleaved TCTP specifically associates with Apaf-1 of apoptosome in apoptosome-forming condition thereby inhibiting the amplification of caspase cascade. CONCLUSIONS: TCTP protects the cancer cells from etoposide-induced cell death by inhibiting the mitochondria-mediated apoptotic pathway. Interaction of TCTP with Apaf-1 in apoptosome is involved in the molecular mechanism of TCTP-induced chemoresistance. These findings suggest that TCTP may serve as a therapeutic target for chemoresistance in cancer treatment.

Effects of retraction force and anchorage reinforcement on occlusal force: a model study.

OBJECTIVES: The aim of this study was to investigate the effects of retraction force and anchorage reinforcement with orthodontic mini implants on occlusal force. MATERIALS AND METHODS: A strain gauge was attached to the palatal surface of the maxillary right first molar on an en masse retraction model. RESULTS: Occlusal forces were measured from this model, according to different retraction forces that were generated by elastic chain, under varied compressive forces (simulating masticatory forces). This retraction experiment was then performed again, after using anchorage reinforcement with an orthodontic mini implant. Occlusal force decreased as retraction force increased. The decrease showed a significant difference above 150g of retraction force (P < 0.05) and was more definite under compressive force higher than 150 N (P < 0.001). After anchorage reinforcement with the orthodontic mini implant, however, occlusal force did not significantly decrease with increasing retraction force. Significant differences in occlusal force were noted between the conditions with and without anchorage reinforcement when the applied retraction force was greater than 200g. CONCLUSION: Occlusal force tends to decrease during retraction, and this decrease can be prevented by anchorage reinforcement with orthodontic mini implants. LIMITATIONS: Further investigation on the actual masticatory process in humans is required for more clear clinical implication.

Human Noxin is an anti-apoptotic protein in response to DNA damage of A549 non-small cell lung carcinoma.

Human Noxin (hNoxin, C11Orf82), a homolog of mouse noxin, is highly expressed in colorectal and lung cancer tissues. hNoxin contains a DNA-binding C-domain in RPA1, which mediates DNA metabolic processes, such as DNA replication and DNA repair. Expression of hNoxin is associated with S phase in cancer cells and in normal cells. Expression of hNoxin was induced by ultraviolet (UV) irradiation. Knockdown of hNoxin caused growth inhibition of colorectal and lung cancer cells. The comet assay and western blot analysis revealed that hNoxin knockdown induced apoptosis through activation of p38 mitogen-activated protein kinase (MAPK)/p53 in non-small cell lung carcinoma A549 cells. Furthermore, simultaneous hNoxin knockdown and treatment with DNA-damaging agents, such as camptothecin (CPT) and UV irradiation, enhanced apoptosis, whereas Trichostatin A (TSA) did not. However, transient overexpression of hNoxin rescued cells from DNA damage-induced apoptosis but did not block apoptosis in the absence of DNA damage. These results suggest that hNoxin may be associated with inhibition of apoptosis in response to DNA damage. An adenovirus expressing a short hairpin RNA against hNoxin transcripts significantly suppressed the growth of A549 tumor xenografts, indicating that hNoxin knockdown has in vivo anti-tumor efficacy. Thus, hNoxin is a DNA damage-induced anti-apoptotic protein and potential therapeutic target in cancer.

Photoresponsive molecules in well-defined nanoscale environments.

Stimuli-responsive molecules are key building blocks of functional molecular materials and devices. These molecules can operate in a range of environments. A molecule's local environment will dictate its conformation, reactivity, and function; by controlling the local environment we can ultimately develop interfaces of individual molecules with the macroscopic environment. By isolating molecules in well-defined environments, we are able to obtain both accurate measurements and precise control. We exploit defect sites in self-assembled monolayers (SAMs) to direct the functional molecules into precise locations, providing a basis for the measurements and engineering of functional molecular systems. The structure and functional moieties of the SAM can be tuned to control not only the intermolecular interactions but also molecule-substrate interactions, resulting in extraction or control of desired molecular functions. Herein, we report our progress toward the assembly and measurements of photoresponsive molecules and their precise assemblies in SAM matrices.

High-fidelity chemical patterning on oxide-free germanium.

Oxide-free germanium can be chemically patterned directly with self-assembled monolayers of n-alkanethiols via submerged microcontact printing. Native germanium dioxide is water soluble; immersion activates the germanium surface for self-assembly by stripping the oxide. Water additionally provides an effective diffusion barrier that prevents undesired ink transport. Patterns are stable with respect to molecular exchange by carboxyl-functionalized thiols.

The expression of damage-regulated autophagy modulator 2 (DRAM2) contributes to autophagy induction.

Autophagy is a membrane trafficking process involved in intracellular degradation and recycling in eukaryotic cells. DRAM2 (damage-regulated autophagy modulator 2) is a homologue of DRAM that regulates p53-mediated cell death. As its name implies, DRAM expression induces autophagy in a p53-dependent manner; however, the role of DRAM2 in autophagy is not clear. In this study, we report that DRAM2 expression contributes to autophagy induction. Overexpression of DRAM2 induces cytoplasmic GFP-LC3 punctuates, and increases the level of endogenous LC3-II. Moreover, the silencing of endogenous DRAM2 interferes with starvation-induced autophagy. Thus, we propose that DRAM2 as well as DRAM are involved in autophagy.

Directing substrate morphology via self-assembly: ligand-mediated scission of gallium-indium microspheres to the nanoscale.

We have developed a facile method for the construction of liquid-phase eutectic gallium-indium (EGaIn) alloy nanoparticles. Particle formation is directed by molecular self-assembly and assisted by sonication. As the bulk liquid alloy is ultrasonically dispersed, fast thiolate self-assembly at the EGaIn interface protects the material against oxidation. The choice of self-assembled monolayer ligand directs the ultimate size reduction in the material; strongly interacting molecules induce surface strain and assist particle cleavage to the nanoscale. Transmission electron microscopy images and diffraction analyses reveal that the nanoscale particles are in an amorphous or liquid phase, with no observed faceting. The particles exhibit strong absorption in the ultraviolet (∼200 nm), consistent with the gallium surface plasmon resonance, but dependent on the nature of the particle ligand shell.

Dynamic double lattice of 1-adamantaneselenolate self-assembled monolayers on Au{111}.

We report a complex, dynamic double lattice for 1-adamantaneselenolate monolayers on Au{111}. Two lattices coexist, revealing two different binding modes for selenols on gold: molecules at bridge sites have lower conductance than molecules at three-fold hollow sites. The monolayer is dynamic, with molecules switching reversibly between the two site-dependent conductance states. Monolayer dynamics enable adsorbed molecules to reorganize according to the underlying gold electronic structure over long distances, which facilitates emergence of the self-organized rows of dimers. The low-conductance molecules assume a (7 × 7) all-bridge configuration, similar to the analogous 1-adamantanethiolate monolayers on Au{111}. The high-conductance molecules self-organize upon mild annealing into distinctive rows of dimers with long-range order, described by a (6√5 × 6√5)R15° unit cell.

Creating favorable geometries for directing organic photoreactions in alkanethiolate monolayers.

The products of photoreactions of conjugated organic molecules may be allowed by selection rules but not observed in solution reactions because of unfavorable reaction geometries. We have used defect sites in self-assembled alkanethiolate monolayers on gold surfaces to direct geometrically unfavorable photochemical reactions between individual organic molecules. High conductivity and stochastic switching of anthracene-terminated phenylethynylthiolates within alkanethiolate monolayers, as well as in situ photochemical transformations, have been observed and distinguished with the scanning tunneling microscope (STM). Ultraviolet light absorbed during imaging increases the apparent heights of excited molecules in STM images, a direct manifestation of probing electronically excited states.

Design and evaluation of variants of the protein transduction domain originated from translationally controlled tumor protein.

Protein transduction domains (PTDs) have been successfully employed to deliver therapeutic cargos both in vitro and in vivo because of their cellular penetrating ability. We previously reported that a 10-amino acid peptide (MIIYRDLISH) derived from the NH(2)-terminus of human translationally controlled tumor protein (TCTP) functions as a PTD. TCTP-PTD is quite different from other well-known PTDs in its hydrophobic composition and structural character, and the sequence requirements for transduction remain unknown. To identify the role of each residue, we compared the cellular uptake of various deletion mutants and Ala substituents of TCTP-PTD. The results showed that the amino terminal residues and the hydrophobic nature of the peptide, with a minimal length of nine residues, were necessary for transduction. Based on the elucidated sequence requirements, we designed and evaluated variants to improve the efficiency and solubility through sequential modification of TCTP-PTD. During the optimization process, we also delineated the contribution of residues and the advantageous composition of sequences for cellular uptake.

A protein transduction domain located at the NH2-terminus of human translationally controlled tumor protein for delivery of active molecules to cells.

Protein transduction domains (PTDs) are small peptides, able to penetrate biological membranes and deliver various types of cargo both in vitro and in vivo. Because use of PTDs originating from viral origins resulted in undesired effects, PTDs originating from non-viral origins are needed. Here, we report that a 10-amino acid peptide (MIIYRDLISH) derived from the NH(2)-terminus of human translationally controlled tumor protein (TCTP) functions as a PTD. This peptide was internalized through lipid raft-dependent endocytosis and partial macropinocytosis, and did not enter lysosome and nucleus. Beta-galactosidase fused to TCTP-PTD, when injected into mice, was efficiently delivered to liver, kidney, spleen, heart, and lungs of the animals. Preincubation of TCTP-PTD with adenovirus increased adenoviral mediated-gene expression in cells and also improved immune response to intranasally administered adenovirus expressing the triple repeat of G glycoprotein of respiratory syncytial virus (RSV), rAd/3×G. These findings suggest that TCTP-PTD might overcome the limitations of polycation-mediated transduction and serve as an efficient vehicle for drug delivery.

OIP5 is a highly expressed potential therapeutic target for colorectal and gastric cancers.

Previously, we reported that overexpression of Opa (Neisseria gonorrhoeae opacity-associated)-interacting protein 5 (OIP5) caused multi-septa formation and growth defects, both of which are considered cancer-related phenotypes. To evaluate OIP5 as a possible cancer therapeutic target, we examined its expression level in 66 colorectal cancer patients. OIP5 was upregulated about 3.7-fold in tumors and over 2-fold in 58 out of 66 colorectal cancer patients. Knockdown of OIP5 expression by small interfering RNA specific to OIP5 (siOIP5) resulted in growth inhibition of colorectal and gastric cancer cell lines. Growth inhibition of SNU638 by siOIP5 caused an increase in sub-G1 DNA content, as measured by flow cytometry, as well as an apoptotic gene expression profile. These results indicate that knockdown of OIP5 may induce apoptosis in cancer cells. Therefore, we suggest that OIP5 might be a potential cancer therapeutic target, although the mechanisms of OIP5-induced carcinogenesis should be elucidated.

Roles of ERK, PI3 kinase, and PLC-gamma pathways induced by overexpression of translationally controlled tumor protein in HeLa cells.

We reported previously that translationally controlled tumor protein (TCTP) is a cytoplasmic repressor of Na,K-ATPase in HeLa cells. In the current study, we showed that TCTP overexpression using adenovirus as vehicle, induced partial inhibition of Na,K-ATPase; phosphorylation of EGFR tyrosine residues 845, 992,1068, and 1148; activation of Ras/Raf/ERK pathway; activation of PI3K/Akt pathway; and phosphorylation of PLC-gamma in HeLa cells. Specific inhibition of PI3K/Akt pathway in contrast to the inhibition of ERK,significantly decreased TCTP overexpression-induced survival signal. Inhibition of PLC-gamma pathway significantly decreased TCTP overexpression-induced cell migration but inhibition of ERK had less effect. These results suggest that TCTP plays a key physiological role in cell survival through Akt pathway and migration through PLC-gamma pathway.