Inhibition of cancer cell migration and invasion through suppressing the Wnt1-mediating signal pathway by G-quadruplex structure stabilizers.

WNT1 encodes a multifunctional signaling glycoprotein that is highly expressed in several malignant tumors. Patients with Wnt1-positive cancer are usually related to advanced metastasis. Here, we found that a stretch of G-rich sequences located at the WNT1 promoter region is capable of forming G-quadruplex structures. The addition of G-quadruplex structure stabilizers, BMVC and BMVC4, raises the melting temperature of the oligonucleotide formed by the WNT1 promoter G-rich sequences. Significantly, the expression of WNT1 was repressed by BMVC or BMVC4 in a G-quadruplex-dependent manner, suggesting that they can be used to modulate WNT1 expression. The role of G-quadruplex stabilizers on Wnt1-mediated cancer migration and invasion was further analyzed. The protein levels of ß-catenin, a mediator of the Wnt-mediated signaling pathway, and the downstream targets MMP7 and survivin were down-regulated upon BMVC or BMVC4 treatments. Moreover, the migration and invasion activities of cancer cells were inhibited by BMVC and BMVC4, and the inhibitory effects can be reversed by WNT1-overexpression. Thus the Wnt1 expression and its downstream signaling pathways can be regulated through the G-quadruplex sequences located at its promoter region. These findings provide a novel approach for future drug development to inhibit migration and invasion of cancer cells.

Conformational transition of a hairpin structure to G-quadruplex within the WNT1 gene promoter.

The role of G-quadruplexes (G4s) in biological systems has been widely studied. It is found that they have an important function in gene transcription and regulation. In this work, we have identified two topologies of hairpin and G4 structures formed by a native G-rich sequence (WT22: 5'-GGGCCACCGGGCAGGGGGCGGG-3') from the WNT1 promoter region using nuclear magnetic resonance (NMR) spectroscopy. With the help of site-specific isotope labeling, the topologies of these two structures are unambiguously characterized. Circular dichroism and NMR results are analyzed to determine the kinetics associated with the potassium ion-induced hairpin-to-G4 transition, which is very slow-on the time scale of 4800 s-compared to the previously reported folding kinetics of G4 formation. In addition, the free energies of the unfolding of these two structures are obtained using differential scanning calorimetry. Combining the kinetic and thermodynamic data, we have established the free energy landscape of this two-state folding system. Considering that similar conformational change may exist in other native G-rich sequences, this work highlights an important hairpin to G4 conformational transition which can be used in manipulation of gene regulation or ligand modulation in vivo.

Ions and hydrogen bonding in a hydrophobic environment: CCl(4).

It is generally expected that ions in an aqueous ionic solution in contact with a hydrophobic phase enter the hydrophobic phase accompanied by a hydration shell. This expectation suggests that the ion mole fraction in the hydrophobic phase is less than, or at most, equal to that of water. Both gravimetric and spectroscopic evidence shows that for a model hydrophobic phase, carbon tetrachloride, this is not the case: In contact with a 1 M simple salt solution (sodium or potassium halide), the salt concentration in carbon tetrachloride ranges from 1.4 to nearly 3 times that of water. Infrared spectra of the OH stretch region support a model in which water associates with the cation, primarily as water monomers. Salts containing larger, more polarizable anions can form outer-sphere ion pairs that support water dimers, giving rise to a spectral signature at 3440 cm(-1). In CCl(4), the infrared spectral signature of the normally strongly ionized acid HCl clearly shows the presence of molecular HCl. Additionally, the presence of a Q branch for HCl indicates restricted rotational motion. The spectral and gravimetric data provide compelling evidence for ion clusters in the hydrophobic phase, which is a result that may have implications for hydrophobic matter in both biological and environmental systems.

Unfolding kinetics of human telomeric G-quadruplexes studied by NMR spectroscopy.

Characterization of the unfolding kinetics of G-quadruplexes (G4s) is the key to a better understanding of the biological function of G4s and is important for biomedical research and material design. Of interest is that slight variations of human telomeric sequences can form different types of G4 structures. In general, there is a correlation between unfolding kinetics and thermal stability. Here we examined this correlation by first systematic analysis of the unfolding kinetics of a variety of telomeric G4 structures using the real-time imino proton NMR spectra of DNA hybridization and hydrogen-deuterium exchange (HDX). We then measured the melting temperature (Tm) and determined the Gibbs free energy (ΔG) of these G4 structures using differential scanning calorimetry (DSC). Our results showed that both Tm and ΔG are slightly structure-dependent, except the Tm of the parallel G4 structure is ∼10 °C higher than that of nonparallel G4 structures. The hybridization results showed that the decay times of different imino proton signals for each telomeric G4 structure are quite similar, which are also consistent with the time constant of the central G-tetrad obtained from HDX measurements. It is suggested that global unfolding is the rate-determining step for HDX, and each real-time imino proton NMR measurement can provide the intrinsic unfolding rate constant. The key finding is that the unfolding times of these various G4 structures are quite different and show no correlation between thermal stability and unfolding kinetics. Our results raised an issue that the folding and unfolding kinetics is more relevant for better understanding of biological function of G4 structures.

Rotational structure of water in a hydrophobic environment: carbon tetrachloride.

Infrared spectroscopy has been used to probe the interaction between water and the hydrophobic solvent, carbon tetrachloride. At room temperature, water exists as monomers in carbon tetrachloride, presenting a system for studying the rotational properties of water free of strong hydrogen-bonding. The rotational structure suggests a very anisotropic motion consisting of essentially free rotation about the symmetry axis and highly hindered rotation about the two perpendicular axes of the asymmetric water molecule. The rotational lifetime is significantly shortened relative to gas-phase water. An upper limit of 0.93 ps is deduced from the spectrum. Interaction with carbon tetrachloride also slightly enhances the intensity of the symmetric stretch. The results are compared with results of interactions between water and the cations Li+, Na+, K+, and Cs+. It is concluded that the attractive interaction is between the oxygen of water and the electropositive carbon of carbon tetrachloride.