Study of effectiveness of mifepristone for glioma cell line growth suppression.

OBJECTIVE: Glioblastoma multiforme is a malignant primary brain tumour with very limited treatment options. Any addition to existing treatment options which can improve prognosis and life expectancy is useful. In our study, we look at the usefulness of anti-progestogen mifepristone in causing growth suppression of glioma cell lines in the laboratory. METHODS: We cultured five cell lines in the lab and exposed them to mifepristone in different doses for a total of 96 h. Five different doses of mifepristone were used. Progesterone and dexamethasone were also used as growth stimulants. Immunostaining was used to identify progesterone receptors (PRs) in the cell lines. RESULTS: U257/7 and IN1265 showed statistically significant growth suppression (36% and 11%, P = 0.001 and 0.03 respectively), maximal at 96 h. Growth suppression in U257/7 showed a dose response progression except with the lowest dose which was not explicable. The response of IN1265 was seen only with the highest dose of mifepristone. There was no significant growth stimulation with either dexamethasone or progesterone. None of the cell lines showed any significant positivity for PRs. CONCLUSION: We were able to produce enough growth suppression of glioma cell lines using mifepristone. This is in keeping with some of the published results in literature. This raises the possibility of using mifepristone in treating GBMs which have very limited treatment options. This, however, needs further work probably on primary glioma cultures first followed by in vivo studies before it can be used in patients.

A hetero-dimer model for concerted action of vitamin K carboxylase and vitamin K reductase in vitamin K cycle.

Vitamin K carboxylase (VKC) is believed to convert vitamin K, in the vitamin K cycle, to an alkoxide-epoxide form which then reacts with CO(2) and glutamate to generate γ-carboxyglutamic acid (Gla). Subsequently, vitamin K epoxide reductase (VKOR) is thought to convert the alkoxide-epoxide to a hydroquinone form. By recycling vitamin K, the two integral-membrane proteins, VKC and VKOR, maintain vitamin K levels and sustain the blood coagulation cascade. Unfortunately, NMR or X-ray crystal structures of the two proteins have not been characterized. Thus, our understanding of the vitamin K cycle is only partial at the molecular level. In this study, based on prior biochemical experiments on VKC and VKOR, we propose a hetero-dimeric form of VKC and VKOR that may explain the efficient oxidation and reduction of vitamin K during the vitamin K cycle.

A molecular dynamics study of the early stages of amyloid-beta(1-42) oligomerization: the role of lipid membranes.

As research progresses toward understanding the role of the amyloid-beta (Abeta) peptide in Alzheimer's disease, certain aspects of the aggregation process for Abeta are still not clear. In particular, the accepted constitution of toxic aggregates in neurons has shifted toward small oligomers. However, the process of forming these oligomers in cells is also not full clear. Even more interestingly, it has been implied that cell membranes, and, in particular, anionic lipids within those membranes, play a key role in the progression of Abeta aggregation, but the exact nature of the Abeta-membrane interaction in this process is unknown. In this work, we use a thermodynamic cycle and umbrella sampling molecular dynamics to investigate dimerization of the 42-residue Abeta peptide on model zwitterionic dipalmitoylphosphatidylcholine (DPPC) or model anionic dioleoylphosphatidylserine (DOPS) bilayer surfaces. We determined that Abeta dimerization was strongly favored through interactions with the DOPS bilayer. Further, our calculations showed that the DOPS bilayer promoted strong protein-protein interactions within the Abeta dimer, whereas DPPC favored strong protein-lipid interactions. By promoting dimer formation and subsequent dimer release into the solvent, the DOPS bilayer acts as a catalyst in Abeta aggregation.

Interaction between amyloid-beta (1-42) peptide and phospholipid bilayers: a molecular dynamics study.

The amyloid-beta (Abeta) peptide is a key aggregate species in Alzheimer's disease. Although important aspects of Abeta peptide aggregation are understood, the initial stage of aggregation from monomer to oligomer is still not clear. One potential mediator of this early aggregation process is interactions of Abeta with anionic cell membranes. We used unconstrained and umbrella sampling molecular dynamics simulations to investigate interactions between the 42-amino acid Abeta peptide and model bilayers of zwitterionic dipalmitoylphosphatidylcholine (DPPC) lipids and anionic dioleoylphosphatidylserine (DOPS) lipids. Using these methods, we determined that Abeta is attracted to the surface of DPPC and DOPS bilayers over the small length scales used in these simulations. We also found supporting evidence that the charge on both the bilayer surface and the peptide affects the free energy of binding of the peptide to the bilayer surface and the distribution of the peptide on the bilayer surface. Our work demonstrates that interactions between the Abeta peptide and lipid bilayer promotes a peptide distribution on the bilayer surface that is prone to peptide-peptide interactions, which can influence the propensity of Abeta to aggregate into higher-order structures.

A quantum chemical study of the mechanism of action of Vitamin K carboxylase (VKC) III. Intermediates and transition states.

A reaction path including transition states is generated for the Dowd mechanism [P. Dowd, R. Hershlne, S.W. Ham, S. Naganathan. Vitamin K and energy transduction: a base strength amplification mechanism. Science 269 (2005) 1684-1691] of action for Vitamin K carboxylase (VKC) using quantum chemical methods (B3LYP/6-311G**). VKC, an essential enzyme in mammalian systems, catalyzes the conversion of hydroquinone form of Vitamin K to the epoxide form in the presence of oxygen. An intermediate species of the oxidation of Vitamin K, an alkoxide, acts apparently to abstract the gamma hydrogen from specifically located glutamate residues. We are able to follow the Dowd proposed path to generate this alkoxide species. The geometries of the proposed model intermediates and transition states in the mechanism are energy optimized. We find that the most energetic step in the mechanism is the uni-deprotonation of the hydroquinone - once this occurs, there is only a small barrier of 3.5kcal/mol for the interaction of oxygen with the carbon to be attacked - and then the reaction proceeds downhill in free energy to form the critical alkoxide species. The results are consistent with the idea that the enzyme probably acts to facilitate the formation of the epoxide by reducing the energy required to deprotonate the hydroquinone form.

A quantum chemical study of the mechanism of action of Vitamin K epoxide reductase (VKOR) II. Transition states.

A reaction path including transition states is generated for the Silverman mechanism [R.B. Silverman, Chemical model studies for the mechanism of Vitamin K epoxide reductase, J. Am. Chem. Soc. 103 (1981) 5939-5941] of action for Vitamin K epoxide reductase (VKOR) using quantum mechanical methods (B3LYP/6-311G**). VKOR, an essential enzyme in mammalian systems, acts to convert Vitamin K epoxide, formed by Vitamin K carboxylase, to its (initial) quinone form for cellular reuse. This study elaborates on a prior work that focused on the thermodynamics of VKOR [D.W. Deerfield II, C.H. Davis, T. Wymore, D.W. Stafford, L.G. Pedersen, Int. J. Quant. Chem. 106 (2006) 2944-2952]. The geometries of proposed model intermediates and transition states in the mechanism are energy optimized. We find that once a key disulfide bond is broken, the reaction proceeds largely downhill. An important step in the conversion of the epoxide back to the quinone form involves initial protonation of the epoxide oxygen. We find that the source of this proton is likely a free mercapto group rather than a water molecule. The results are consistent with the current view that the widely used drug Warfarin likely acts by blocking binding of Vitamin K at the VKOR active site and thereby effectively blocking the initiating step. These results will be useful for designing more complete QM/MM studies of the enzymatic pathway once three-dimensional structural data is determined and available for VKOR.

Insights into thermophilic archaebacterial membrane stability from simplified models of lipid membranes.

Lipid aggregation into fluid bilayers is an essential process for sustaining life. Simplified models of lipid structure, which allow for long time scales or large length scales not obtainable with all-atom simulations, have recently been developed and show promise for describing lipid dynamics in biological systems. Here, we describe two simplified models, a reduced-lipid model and a bola-lipid model for thermophilic bacterial membranes, developed for use with the rapid discrete molecular dynamics simulation method. In the reduced-lipid model, we represent the lipid chain by a series of three beads interacting through pairwise discrete potentials that model hydrophobic attractions between hydrocarbon tails in implicit solvent. Our phase diagram recapitulates those produced by continuous potential models with similar coarse-grained lipid representations. We also find that phase transition temperatures for our reduced-lipid model are dependent upon the flexibility of the lipid chain, giving an insight into archaebacterial membrane stability and prompting development of a bola-lipid model specific for archaebacteria lipids. With both the reduced-lipid and bola-lipid model, we find that the reduced flexibility inherent in archaebacteria lipids yields more stable bilayers as manifested by increased phase transition temperatures. The results of these studies provide a simulation methodology for lipid molecules in biological systems and show that discrete molecular dynamics is applicable to lipid aggregation and dynamics.

Quantum Chemical Study of the Mechanism of Action of Vitamin K Carboxylase in Solvent.

We investigate the post-translational generation of Gla (γ-carboxy glutamic acid) from Glu (glutamic acid) by vitamin K carboxylase (VKC) in solvent. VKC is thought to convert vitamin K, in the vitamin K cycle, to an alkoxide-epoxide form, which then reacts with CO(2) to generate an essential ingredient in blood coagulation, γ-carboxyglutamic acid (Gla). The generation of Gla from Glu is found to be exergenic (-15 kcal/mol) in aqueous solution with the SM6 method. We also produced the free energy profile for this model biochemical process with other solvent methods (polarizable continuum model, dielectric polarizable continuum model) and different dielectric constants. The biological implications are discussed.

Structure of the amyloid-beta (1-42) monomer absorbed to model phospholipid bilayers: a molecular dynamics study.

The amyloid-beta (Abeta) peptide, the 39 to 43 amino acid peptide that plays a substantial role in Alzheimer's disease, has been shown to interact strongly with lipids both in vitro and in vivo. Abeta-lipid interactions have been proposed as a considerable factor in accelerating Abeta aggregation through the templating role of membranes in aggregation disorders. Previous work has shown that anionic lipids are able to significantly increase Abeta aggregation rate and induce a structural conversion in Abeta from a random coil to a beta-structure that is similar to the monomer structure observed in mature fibrils. However, it is unclear if this structural change occurs with the Abeta monomer because of direct interactions with the lipids or if the structural change results from protein-protein interactions during oligomerization. We use extensive replica exchange molecular dynamics simulations of an Abeta monomer bound to a homogeneous model zwitterionic or anionic lipid bilayer. From these simulations, we do not observe any significant beta-structure formation except for a small, unstable beta-hairpin formed on the anionic dioleylphosphatidylserine bilayer. Further, we see that the Asp23-Lys28 salt bridge that plays a role in beta-hairpin formation is not substantially formed on the bilayer surface and that Lys28 preferentially interacts with lipids when bound to the bilayer. These results suggest that the structural conversion seen in experiments are not due to the ordering of monomeric Abeta on the bilayer surface but are a result of protein-protein interactions enhanced by Abeta binding to the cell membrane.

Quantum chemical study of the mechanism of action of vitamin K carboxylase (VKC). IV. Intermediates and transition states.

We studied proposed steps for the enzymatic formation of gamma-carboxyglutamic acid by density functional theory (DFT) quantum chemistry. Our results for one potentially feasible mechanism show that a vitamin K alkoxide intermediate can abstract a proton from glutamic acid at the gamma-carbon to form a carbanion and vitamin K epoxide. The hydrated carbanion can then react with CO2 to form gamma-carboxyglutamic acid. Computations at the B3LYP/6-311G** level were used to determine the intermediates and transition states for the overall process. The activation free energy for the gas-phase path is 22 kcal/mol, with the rate-limiting step for the reaction being the attack of the carbanion on CO2. Additional solvation studies, however, indicate that the formation of the carbanion step can be competitive with the CO2 attack step in high-dielectric systems. We relate these computations to the entire vitamin K cycle in the blood coagulation cascade, which is essential for viability of vertebrates.