Reduction of lung metastasis, cell invasion, and adhesion in mouse melanoma by statin-induced blockade of the Rho/Rho-associated coiled-coil-containing protein kinase pathway

<p>Abstract</p> <p>Background</p> <p>Melanomas are highly malignant and have high metastatic potential; hence, there is a need for new therapeutic strategies to prevent cell metastasis. In the present study, we investigated whether statins inhibit tumor cell migration, invasion, adhesion, and metastasis in the B16BL6 mouse melanoma cell line.</p> <p>Methods</p> <p>The cytotoxicity of statins toward the B16BL6 cells were evaluated using a cell viability assay. As an experimental model, B16BL6 cells were intravenously injected into C57BL/6 mice. Cell migration and invasion were assessed using Boyden chamber assays. Cell adhesion analysis was performed using type I collagen-, type IV collagen-, fibronectin-, and laminin-coated plates. The mRNA levels, enzyme activities and protein levels of matrix metalloproteinases (MMPs) were determined using RT-PCR, activity assay kits, and Western blot analysis, respectively; the mRNA and protein levels of vary late antigens (VLAs) were also determined. The effects of statins on signal transduction molecules were determined by western blot analyses.</p> <p>Results</p> <p>We found that statins significantly inhibited lung metastasis, cell migration, invasion, and adhesion at concentrations that did not have cytotoxic effects on B16BL6 cells. Statins also inhibited the mRNA expressions and enzymatic activities of matrix metalloproteinases (MMPs). Moreover, they suppressed the mRNA and protein expressions of integrin α₂, integrin α₄, and integrin α₅and decreased the membrane localization of Rho, and phosphorylated LIM kinase (LIMK) and myosin light chain (MLC).</p> <p>Conclusions</p> <p>The results indicated that statins suppressed the Rho/Rho-associated coiled-coil-containing protein kinase (ROCK) pathways, thereby inhibiting B16BL6 cell migration, invasion, adhesion, and metastasis. Furthermore, they markedly inhibited clinically evident metastasis. Thus, these findings suggest that statins have potential clinical applications for the treatment of tumor cell metastasis.</p

Minimum Free Energy Path of Ligand-Induced Transition in Adenylate Kinase

Large-scale conformational changes in proteins involve barrier-crossing transitions on the complex free energy surfaces of high-dimensional space. Such rare events cannot be efficiently captured by conventional molecular dynamics simulations. Here we show that, by combining the on-the-fly string method and the multi-state Bennett acceptance ratio (MBAR) method, the free energy profile of a conformational transition pathway in Escherichia coli adenylate kinase can be characterized in a high-dimensional space. The minimum free energy paths of the conformational transitions in adenylate kinase were explored by the on-the-fly string method in 20-dimensional space spanned by the 20 largest-amplitude principal modes, and the free energy and various kinds of average physical quantities along the pathways were successfully evaluated by the MBAR method. The influence of ligand binding on the pathways was characterized in terms of rigid-body motions of the lid-shaped ATP-binding domain (LID) and the AMP-binding (AMPbd) domains. It was found that the LID domain was able to partially close without the ligand, while the closure of the AMPbd domain required the ligand binding. The transition state ensemble of the ligand bound form was identified as those structures characterized by highly specific binding of the ligand to the AMPbd domain, and was validated by unrestrained MD simulations. It was also found that complete closure of the LID domain required the dehydration of solvents around the P-loop. These findings suggest that the interplay of the two different types of domain motion is an essential feature in the conformational transition of the enzyme

Influence of Structural Symmetry on Protein Dynamics

<div><p>Structural symmetry in homooligomeric proteins has intrigued many researchers over the past several decades. However, the implication of protein symmetry is still not well understood. In this study, we performed molecular dynamics (MD) simulations of two forms of trp RNA binding attenuation protein (TRAP), the wild-type 11-mer and an engineered 12-mer, having two different levels of circular symmetry. The results of the simulations showed that the inter-subunit fluctuations in the 11-mer TRAP were significantly smaller than the fluctuations in the 12-mer TRAP while the internal fluctuations were larger in the 11-mer than in the 12-mer. These differences in thermal fluctuations were interpreted by normal mode analysis and group theory. For the 12-mer TRAP, the wave nodes of the normal modes existed at the flexible interface between the subunits, while the 11-mer TRAP had its nodes within the subunits. The principal components derived from the MD simulations showed similar mode structures. These results demonstrated that the structural symmetry was an important determinant of protein dynamics in circularly symmetric homooligomeric proteins.</p> </div

Intra-subunit fluctuations of TRAP.

<p>(A) RMS intra-subunit fluctuations of Cα atoms are plotted by residue for 11-mer TRAP (blue) and 12-mer TRAP (red), which are averaged over the subunits. The amplitudes of fluctuations are depicted on the structures: (B) 11-mer TRAP and (C) 12-mer TRAP. The main-chain traces are colored according to the amplitudes of the fluctuations.</p

Normal modes of a ring-shaped object.

<p>Normal modes of a circularly symmetric object are viewed along the symmetry axis in the form of stationary waves on the ring. The individual mode of has wave nodes on the ring. The red curves describe the displacements along the modes. The mode is found only in the 12-mer.</p

Ring and close-packed forms.

<p>(A) A schematic representation of a ring shaped oligomer. Subunits are arranged symmetrically (<i>C_n</i> symmetry) around the rotational axis (axis <i>1</i>). Color gradation indicates the top and bottom of the subunit. (B) Schematic representation of a close-packed oligomer. The oligomer composed of <i>n</i> subunits has <i>n/2</i>-fold rotational symmetry around the axis <i>1</i>, and <i>2</i>-fold rotational symmetry around each of axes <i>2</i>–<i>4</i>. (C) The number of homooligomers (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050011#s4" target="_blank"><i>Materials and Methods</i></a> in detail). (D) The number of ring-shaped oligomers.</p

Correlations of the normal modes.

<p>Correlation function of the displacements of two atoms separated by an angle calculated for the normal modes of (A) 11-mer TRAP and (B) 12-mer TRAP. The vertical broken lines indicate the location of the subunit interfaces. The plots are for the normal modes of the 1st (red), 2nd (green), 3rd (blue), 4th (yellow), 5th (cyan), 6th (magenta), and 7th (black) from top to bottom. The pairs of normal modes, the 1st and 2nd, the 3rd and 4th, and the 6th and 7th, are 2-fold degenerate. The 5th mode is a uniform breathing mode corresponding to the subspace.</p

Decomposition of the subunit fluctuations into intra and external fluctuations.

<p>Intra and external (translational and rotational) subunit fluctuations in the <i>z</i>-axis are shown for the two TRAPs. The internal fluctuation was calculated after the superposition of each subunit onto its average structure, and the translational fluctuation was calculated by the variance of the center of mass of the subunit. The fluctuation of the rotation was estimated by subtracting the internal and translational contributions from the sum of the fluctuations without superimposing the subunit.</p

Crystal structures of the 11-mer and 12-mer TRAP.

<p>(A) Crystal structure of 11-mer TRAP (PDB code: 1C9S). Subunits and bound tryptophans are shown in ribbon and sphere, respectively. (B) Crystal structure of 12-mer TRAP (PDB code: 2EXS). (C) Superimposed structures of subunits A and B of the 11-mer and the 12-mer, shown by main-chain trace and the stick model for some side-chains. Hydrogen bonds between tryptophan and the subunit are indicated with the yellow dashed lines. (D) Hydrophobic pockets of subunit B for the 11-mer (left) and 12-mer TRAP (right). Surfaces are colored according to the hydrophobic contribution calculated by VASCo <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050011#pone.0050011-Steinkellner1" target="_blank">[48]</a>. All the figures were prepared using PyMOL.</p

The largest-amplitude principal modes of TRAP.

<p>Top and side views of the largest-amplitude principal mode for (A) 11-mer TRAP and (B) 12-mer TRAP. The gray arrows indicate the displacements along the mode. The structures of the TRAPs are colored according to the correlation function (see text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050011#pone-0050011-g007" target="_blank">Figure 7</a>).</p