Determination of In Vitro and In Vivo Effects of Taxifolin and Epirubicin on Epithelial-Mesenchymal Transition in Mouse Breast Cancer Cells.

Background: One of the most significant characteristics of cancer is epithelial-mesenchymal transition and research on the relationship between phenolic compounds and anticancer medications and epithelial-mesenchymal transition is widespread. Methods: In order to investigate the potential effects of Taxifolin on enhancing the effectiveness of Epirubicin in treating breast cancer, specifically in 4T1 cells and an allograft BALB/c model, the effects of Taxifolin and Epirubicin, both individually and in combination, were examined. Cell viability assays and cytotoxicity assays in 4T1 cells were performed. In addition, 4T1 cells were implanted into female BALB/c mice to conduct in vivo studies and evaluate the therapeutic efficacy of Taxifolin and Epirubicin alone or in combination. Tumor volumes and histological analysis were also assessed in mice. To further understand the mechanisms involved, we examined the messenger RNA and protein levels of epithelial-mesenchymal transition-related genes, as well as active Caspase-3/7 levels, using quantitative real-time polymerase chain reaction, western blot, and enzyme-linked immunosorbent assays, respectively. Results: In vitro results demonstrated that the coadministration of Taxifolin and Epirubicin reduced cell viability and cytotoxicity in 4T1 cell lines. In vivo, coadministration of Taxifolin and Epirubicin suppressed tumor growth in BALB/c mice with 4T1 breast cancer cells. Additionally, this combination treatment significantly increased the levels of active caspase-3/7 and downregulated the messenger RNA and protein levels of N-cadherin, ß-catenin, vimentin, snail, and slug, but upregulated the E-cadherin gene. It significantly decreased the messenger RNA levels of the Zeb1 and Zeb2 genes. Conclusion: The in vitro and in vivo results of our study indicate that the concurrent use of Epirubicin with Taxifolin has supportive effects on breast cancer treatment.

Ti (IV) attached-phosphonic acid functionalized capillary monolith as a stationary phase for in-syringe-type fast and robust enrichment of phosphopeptides.

In this study, poly(vinylphosphonic acid-co-ethylene dimethacrylate), poly(VPA-co-EDMA) capillary monolith was synthesized as a starting material for obtaining a stationary phase for microscale enrichment of phosphopeptides. The chelation of active phosphonate groups with Ti (IV) ions gave a macroporous monolithic column with a mean pore size of 5.4 µm. The phosphopeptides from different sources were enriched on Ti (IV)-attached poly(VPA-co-EDMA) monolith using a syringe-pump. The monolithic capillary columns exhibited highly sensitive/selective enrichment performance with phosphoprotein concentrations as low as 1.0 fmol/mL. Six different phosphopeptides were detected with high intensity by the treatment of ß-casein digest with the concentration of 1.0 fmol/mL, using Ti (IV)@poly(VPA-co-EDMA) monolith. Highly selective enrichment of phosphopeptides was also successfully carried out even at trace amounts, in a complex mixture of digested proteins (molar ratio of ß-casein to bovine serum albumin, 1:1500) and three phosphopeptides were successfully detected. Four highly intense signals of phosphopeptides in human serum were also observed with high signal-to-noise ratio and a clear background after enrichment with Ti (IV)@poly(VPA-co-EDMA) monolith. It was concluded that the capillary microextraction system enabled fast, efficient and robust enrichment of phosphopeptides from microscale complex samples. The whole enrichment process was completed within 20 min, which was shorter than in the previously reported studies.

Isolation of RNA and beta-NAD by phenylboronic acid functionalized, monodisperse-porous silica microspheres as sorbent in batch and microfluidic boronate affinity systems.

Monodisperse-porous silica microspheres 5.5 µm in size were obtained by a staged shape templated hydrolysis-condensation method, with a bimodal pore-size distribution. 3-aminophenylboronic acid (APBA) was covalently attached onto the silica microspheres with a capacity of 0.476 mmol APBA/g microspheres. The boronate affinity isolation behaviour of ribonucleic acid (RNA) containing cis-diol at 3'-end was investigated by using APBA attached-silica microspheres as the sorbent in batch fashion. A short-chain diol carrying agent, ß-nicotinamide adenine dinucleotide (ß-NAD) was used as a target molecule with stronger affinity for phenylboronic acid ligand. The maximum equilibrium adsorptions for RNA and ß-NAD were determined as 60 and 159 mg/g sorbent, respectively. By using the synthesized sorbent, phosphate buffer at pH 7.0 containing sorbitol was successfuly used as a mild elution medium for obtaining quantitative desorptions with both RNA and ß-NAD. RNA isolations from mammalian and bacterial cells were successfully performed while protecting the structural integrity of RNA via boronate affinity interaction in batch fashion. A microfluidic boronate affinity system including a microcolumn 300 µm in diameter was also constructed using APBA attached-silica microspheres as the stationary phase. The breakthrough curves of microfluidic system were obtained by studying with different feed concentrations of RNA and ß-NAD. Quantitative desorptions and satisfactory isolation yields were obtained with RNA and ß-NAD in the microfluidic system. The proposed system is useful for boronate affinity applications in genomics or proteomics in which valuable cis-diols at low concentrations are recovered from low-volume samples.

Protein A and protein A/G coupled magnetic SiO2 microspheres for affinity purification of immunoglobulin G.

Protein A carrying magnetic, monodisperse SiO2 microspheres [Mag(SiO2)] with bimodal pore size distribution including both mesoporous and macroporous compartments were proposed as an affinity sorbent for IgG purification. Protein A was tightly bound onto the aldehyde functionalized-Mag(SiO2) microspheres. The mesoporous compartment provided high surface area for protein A binding and IgG adsorption while the macropores made easier the intraparticular diffusion of protein A and IgG. The selection of relatively larger microspheres with high saturation magnetization allowed faster magnetic separation of affinity sorbent from the IgG isolation medium, less than 1min. With these properties, the proposed sorbent is an alternative to the common sorbents in the form of core-shell type, magnetic silica nanoparticles with more limited surface area and slower magnetic response. By using protein A attached-Mag(SiO2) microspheres with the concentrations lower than 50mg/mL, IgG isolation from rabbit serum was performed with a purity higher than 95%, with an isolation yield comparable to commercial magnetic resins, and in shorter isolation periods. IgG could be also quantitatively isolated from rabbit serum with the sorbent concentrations higher than 50mg/mL. Successive IgG isolation runs indicated that no significant protein A leaching occurred from the magnetic matrix.

Ti(IV) carrying polydopamine-coated, monodisperse-porous SiO2 microspheres with stable magnetic properties for highly selective enrichment of phosphopeptides.

A marked decrease in the saturation magnetization by the formation of functional shells around the magnetic core is an important disadvantage of magnetic core-shell nanoparticles. Another drawback of Ti(IV)-functionalized immobilized metal affinity chromatography (IMAC) sorbents is the acidic character of the binding medium used for Ti4+ attachment onto composite magnetic nanoparticles, which causes an additional decrease in the saturation magnetization owing to the chemical interaction between the acidic moiety and the magnetic core. An IMAC sorbent in the form of magnetic microspheres with superior and stable magnetic properties with respect to magnetic core-shell nanoparticles was designed for phosphopeptide enrichment. Magnetic, monodisperse-porous silica microspheres (MagSiO2) 6µm in size were synthesized by a new staged-shape template hydrolysis-condensation protocol. A porous-silica shell layer was generated around the microspheres to protect the magnetic core from the acidic medium during Ti4+ attachment (MagSiO2@SiO2). The MagSiO2@SiO2 microspheres were coated with a polydopamine shell (MagSiO2@SiO2@PDA) and Ti4+ was attached onto the composite microspheres (MagSiO2@SiO2@PDA@Ti(IV)). Formation of the PDA layer and Ti4+ attachment did not cause any significant decrease in the saturation magnetization. The platform exhibited excellent performance for phosphopeptide enrichment from the digests of phosphorylated proteins. Selectivity was investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The detection limit for phosphopeptide enrichment by the MagSiO2@SiO2@PDA@Ti(IV) microspheres from the tryptic digests of ß-casein was 50 fmol/mL. Usability of the proposed magnetic sorbent with complex biological samples was demonstrated by successful enrichment of four phosphopeptides from human serum. The proposed sorbent showed stable performance over five repeated uses.

Human genomic DNA isolation from whole blood using a simple microfluidic system with silica- and polymer-based stationary phases.

Monodisperse-porous silica microspheres 5.1µm in size with a bimodal pore-size distribution (including both mesoporous and macroporous compartments) were obtained using a newly developed staged-shape templated hydrolysis and condensation protocol. Synthesized silica microspheres and monodisperse-porous polymer-based microspheres with different functionalities, synthesized by staged-shape template polymerization, were comparatively tested as sorbents for human genomic DNA (hgDNA) isolation in a microfluidic system. Microcolumns with a permeability range of 1.8-8.5×10-13m2 were fabricated by the slurry-packing of silica- or polymer-based microspheres. The monodisperse-porous silica microspheres showed the best performance in hgDNA isolation in an aqueous buffer medium; >2500ng of hgDNA was recovered with an isolation yield of about 50%, using an hgDNA feed concentration of 100ng/µL. Monodisperse-porous silica microspheres were also evaluated as a sorbent for genomic DNA isolation from human whole blood in the microfluidic system; 14ng of hgDNA was obtained from 10µL of whole blood lysate with an isolation yield of 64%. Based on these results, we conclude that monodisperse-porous silica microspheres with a bimodal pore size distribution are a promising sorbent for the isolation of hgDNA in larger amounts and with higher yields compared to the sorbents previously tried in similar microfluidic systems.

Highly selective enrichment of phosphopeptides by titanium (IV) attached monodisperse-porous poly(vinylphosphonic acid-co-ethylene dimethacrylate) microspheres.

A seeded polymerization protocol was developed for the synthesis of monodisperse-porous poly(vinylphosphonic acid-co-ethylene dimethacrylate), [poly(VPA-co-EDMA)] microspheres with superior porous properties. The protocol allowed the direct synthesis of phosphonic acid functionalized porous microspheres with the mean size of ∼4µm and the specific surface area of 420m2g-1 without applying any complicated post-derivatization protocol for the attachment of phosphonic acid group. The phosphonic acid content of poly(VPA-co-EDMA) microspheres was determined as 1.5mmol H2PO3g-1 microspheres. Ti(IV) ions were attached onto the microspheres via metal-chelate complex formation by phosphonate-groups and Ti(IV) carrying monodisperse-porous poly(vinylphosphonic acid-co-ethylene dimethacrylate), [Ti(IV)@poly(VPA-co-EDMA)] microspheres were obtained as a new sorbent for phosphopeptide enrichment via immobilized metal affinity chromatography. The phosphopeptides in the enriched samples were identified by matrix-assited laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Four different phosphopeptides were detected with extremely high intensity by the treatment of ß-casein digest prepared with the concentration of 10 fmol/mL with Ti(IV)@poly(VPA-co-EDMA) microspheres. Highly selective enrichment of phosphopeptides was also successfully carried out even at trace amounts in a complex mixture of digested proteins (molar ratio of ß-casein to BSA, 1:1000) and eight different phosphorylated peptides from BSA digest were successfully identified. Moreover, four highly intense signals of the phosphopeptides in human serum were observed with high S/N ratio and clear background after enrichment by using Ti(IV)@poly(VPA-co-EDMA) microspheres.

A Boronate Affinity-Assisted SERS Tag Equipped with a Sandwich System for Detection of Glycated Hemoglobin in the Hemolysate of Human Erythrocytes.

Phenylboronic acid-functionalized, Ag shell-coated, magnetic, monodisperse polymethacrylate microspheres equipped with a glycoprotein-sensitive sandwich system were proposed as a surface-enhanced Raman scattering (SERS) substrate for quantitative determination of glycated hemoglobin (HbA1c). The magnetization of the SERS tag and the formation of the Ag shell on the magnetic support were achieved using the bifunctional reactivity of newly synthesized polymethacrylate microspheres. The hemolysate of human red blood cells containing both HbA1c and nonglycated hemoglobin was used for determination of HbA1c. The working principle of the proposed SERS tag is based on the immobilization of HbA1c by cyclic boronate ester formation between glycosyl residues of HbA1c and boronic acid groups of magnetic polymethacrylate microspheres and the binding of p-aminothiophenol (PATP)-functionalized Ag nanoparticles (Ag NPs) carrying another boronic acid ligand via cyclic boronate ester formation via unused glycosyl groups of bound HbA1c. Then, in situ formation of a Raman reporter, 4,4'-dimercaptoazobenzene from PATP under 785 nm laser irradiation allowed for the quantification of HbA1c bound onto the magnetic SERS tag, which was proportional to the HbA1c concentration in the hemolysate of human erythrocytes. The sandwich system provided a significant enhancement in the SERS signal intensity due to the plasmon coupling between Ag NPs and Ag shell-coated magnetic microspheres, and low HbA1c concentrations down to 50 ng/mL could be detected. The calibration curve obtained with a high correlation coefficient between the SERS signal intensity and HbA1c level showed the usability of the SERS protocol for the determination of the HbA1c level in any person.