Chemical conjugation of aptamer-sphingomyelin nanosystems and their potential as inhibitors of tumour cell proliferation in breast cancer cells.

Breast cancer is a complex and heterogeneous disease with a high mortality rate due to non-specific cytotoxicity, low intratumoral accumulation and drug resistance associated with the ineffectiveness of chemotherapy. In recent years, all efforts have been focused on finding new markers and therapeutic targets, protein kinase MNK1b being a promising candidate. Recently, an aptamer known as apMNK2F showed a highly specific interaction with this protein kinase, leading to a significant reduction in tumour cell proliferation, migration and colony formation. However, as aptamers are unable to penetrate the cell membrane and reach the target, these small biomolecules need to be conjugated to suitable vectors that can transport and protect them inside the cells. In this work, covalent conjugation between biocompatible and non-harmful nanoemulsions of vitamin E and sphingomyelin and the aptamer was performed to facilitate intracellular delivery of the therapeutic aptamer apMNK2F. All strategies employed were based on 2-step bioconjugation and optimized to get the simplest and most reproducible vehicle with the highest association efficiency (about 70% in all cases). The ability of the nanosystems to successfully deliver the conjugated therapeutic aptamer was demonstrated and compared to other commercial transfection agents such as Lipofectamine 2000, leading to an effective decrease of breast cancer cell proliferation in the MDA-MB-231 cell line. The proliferation inhibition of the aptamer nanoconjugates compared to the non-conjugated aptamer provides evidence that the antitumoral capacity derived from kinase interaction is improved in a dose-dependent manner. Furthermore, various experiments including cell migration and colony formation assays, along with apoptosis induction experiments, emphasize the significant antitumoral potential. Overall, the obtained results indicate that the developed formulation could be a promising therapy for the treatment of breast cancer.

Solvent effects on de-excitation channels in the p-coumaric acid methyl ester anion, an analogue of the photoactive yellow protein (PYP) chromophore.

In an attempt to shed light on the environmental effects on the deactivation channels of the PYP chromophore, radiative and non-radiative deactivation mechanisms of the anionic p-coumaric acid methyl ester (pCE-) in the gas phase and water solution are compared at the CASPT2//CASSCF/cc-pVDZ level and, when necessary, at the CASPT2//CASPT2/cc-pVDZ level. We find that the solvent produces dramatic modifications on the free energy profile of the S1 state. Two twisted structures that are minima in the gas phase could not be localized in aqueous solution. Furthermore, the relative stability of minima and conical intersections (CIs) is reverted with respect to the gas phase values, affecting the prevalent de-excitation paths. As a consequence of these changes, three competitive de-excitation channels are open in aqueous solution: the fluorescence emission from a planar minimum on S1, the trans-cis photoisomerization through a CI that involves the rotation of the vinyl double bond and the non-radiative, non-reactive, de-excitation through the CI associated with the rotation of the single bond adjacent to the phenyl group. In the gas phase, the minima are the structures with lower energy, while in solution the CIß structure, characterized by a large charge separation, is strongly stabilized by interactions with water molecules and becomes the structure with the lowest energy on S1. These facts explain the low fluorescence signal of pCE- in aqueous solution and the presence of partial trans-cis photoisomerization in this system.

Analysis of the protein expression changes during taxol-induced apoptosis under translation inhibition conditions.

Taxol is currently used in chemotherapeutic treatments of different types of cancers. In this article, we demonstrate that taxol induces apoptosis and translation down-regulation in human embryonic kidney (HEK293T) cells. Antibody arrays are a promising new tool for the analysis of protein levels changes in cells responding to different stimuli. Using this approach, we have identified changes in the expression of 38 proteins (20 down-regulated and 18 up-regulated), implicated in several cellular processes mainly in apoptosis, cell cycle and signal transduction pathways, and also cytoskeleton proteins. Among them, we have confirmed a considerable decrease in the expression of p14(ARF) and a significant increase in the levels of dystrophin and c-Myc. It is known that c-Myc mRNA has an internal ribosome entry segment (IRES) element in its 5'UTR that could regulate its expression under global protein synthesis inhibition conditions. We demonstrate that after taxol treatment, the c-Myc IRES activity is maintained meanwhile cap-dependent activity is inhibited. In addition, an increase in c-Myc mRNA was also observed after taxol treatment. We conclude that taxol-induced c-Myc expression is regulated at both transcriptional and translational levels, the last of them by a mechanism mediated by IRES.

Solvatochromic Shifts on Absorption and Fluorescence Bands of N,N-Dimethylaniline.

A theoretical study of the absorption and fluorescence UV/vis spectra of N,N-dimethylaniline in different solvents has been performed, using a method combining quantum mechanics, molecular mechanics, and the mean field approximation. The transitions between the three lowest-lying states have been calculated in vacuum as well as in cyclohexane, tetrahydrofuran, and water. The apparent anomalies experimentally found in water (a blue shift in the absorption bands with respect to the trend in other solvents, and an abnormally high red shift for the fluorescence band) are well reproduced and explained in view of the electronic structure of the solute and the solvent distribution around it. Additional calculations were done with a mixture of cyclohexane and tetrahydrofuran as solvent, which displays a nonlinear solvatochromic shift. Results, although not conclusive, are consistent with experiment and provide a possible explanation for the nonlinear behavior in the solvent mixture.