P38 MAPK contributes to resistance and invasiveness of HER2- overexpressing breast cancer.

Intrinsic or acquired resistance to the HER2-targeted therapy trastuzumab is a clinical concern in the treatment of patients with HER2-over-expressing metastatic breast cancers. We demonstrate here that multiple models of intrinsic and acquired resistance exhibit increased phosphorylation of p38 MAPK. Kinase inhibition of p38 rescued trastuzumab sensitivity in cells with acquired resistance. In addition, knockdown of p38 increased sensitivity to trastuzumab in an intrinsically resistant cell line. We previously reported that expression of growth differentiation factor 15 (GDF15) is increased in trastuzumab-resistant HER2-overexpressing breast cancer cells. In this study, we found that exogenous GDF15 or stable overexpression of GDF15 stimulated p38 phosphorylation in HER2-positive cells, suggesting a possible mechanism by which p38 is activated in resistant cells.GDF15 stable clones showed significantly increased invasiveness, which was rescued by p38 kinase inhibition, suggesting that p38 plays a role in the pro-invasive phenotype conferred by GDF15. Importantly, immunohistochemical analysis of a breast tumor tissue array indicated a significant (p=0.0053) correlation between HER2 and phosphorylated p38 specifically in GDF15-positive tissues. Our results suggest that p38 signaling drives trastuzumab resistance and invasiveness in HER2-overexpressing breast cancer. Upstream growth factor signals that have previously been implicated in trastuzumab resistance, such as GDF15, may contribute to the increased phosphorylation of p38 found in resistant cells.

In vivo seizure induction and affinity studies of domoic acid and isodomoic acids-D, -E and -F.

Domoic acid and its isomers are produced via algal blooms and are found in high concentrations in shellfish. Here, we assessed the acute seizurogenic potencies of isomers-D, -E and -F and their binding affinities at heterogeneous populations of KA receptors from rat cerebrum. In addition, binding affinities of all six isomers (Iso-A through -F) were assessed at AMPA receptors. Radioligand displacement studies indicated that the seizurogenic potency of Iso-F (E-configuration) closely correlates with its affinities at both KA and AMPA receptors, whereas isomers-D (Z) and -E (E), which exhibit distinctly lower seizurogenic potencies, are quite weak displacers. Previously observed functional potencies for isomers-A, -B and -C (Sawant et al., 2008) correlated with AMPA receptor affinities observed here. Taken together, these findings call into question previous structure-activity rules. Significantly, in our hands, Iso-D was ten-fold less potent than Iso-F. To further explain observed links between structural conformation and functional potency, molecular modeling was employed. Modeling results closely matched the rank order of potency and binding data observed. We further assessed the efficacy of isomers-D, -E and -F as pharmacological preconditioning agents. Acute preconditioning with low-dose Iso-D, -E or -F, before high-dose DA failed to impart behavioural tolerance. This study has shed new light on structural conformations affecting non-NMDA ionotropic glutamate receptor binding and functional potency, and provides a foundation for future work in areas of AMPA and KA receptor modeling.

The use of 2,2,6,6-tetramethyl-4-maleimido-piperidin-1-oxyl in electron paramagnetic resonance spin label studies of drug interactions with erythrocyte membranes.

The electron spin resonance (EPR) spectrum of erythrocyte membranes labeled with 2,2,6,6-tetra-methyl-4-maleimido-piperidin-1-oxyl (MAL-6) indicates both weakly and strongly immobilized labeling sites. Changes in the ratio of the membrane's low field spectral peaks (W/S) have often been used to monitor drug-erythrocyte interactions. We have investigated a number of experimental factors that may influence this ratio even in the absence of drug. Instrumental settings on the EPR spectrometer had no obvious effect. As the weight ratio of label/protein decreased, the W/S ratio increased. Similarly an increase in labeling time and temperature lead to an increase in the ratio. The ratio also increased with time after labeling; this change was most marked in samples kept at 37 degrees C, but was insignificant in samples kept at 4 degrees C. Increasing the viscosity of the sample with low-molecular-weight substances such as sucrose or glycerol, but not with those with high molecular weight such as dextran and PVP, caused a reduction in the ratio. Increasing the pH and changing the buffer concentrations also lead to a small increase in W/S. These results suggest that it is very important that all of these factors be kept constant and at some optimal value if reliable and consistent results are to be obtained using this method to monitor drug-erythrocyte interactions.

ESR measurements on the effects of ethanol on the lipid and protein conformation in biological membranes.

The effects of ethanol on the conformation of proteins or fluidity of lipids was studied in human erythrocyte ghosts and rat brain synaptosomal membranes. A maleimide nitroxide probe (MAL-6) was coupled to membrane protein, or 5-doxylstearic (5NS) was dissolved in the membrane lipid, and the electron spin resonance (ESR) spectra were recorded in the presence of increasing ethanol concentrations and at different temperatures from 4 degrees to 37 degrees. The lipid probe at all temperatures studied showed an increase in fluidity in the membrane lipid when ethanol was added in concentration over 20 mM. The protein probe however required a high concentration of ethanol (200 mM) to produce an increase in the rigidity of the protein conformation of 4 degrees in both erythrocyte and synaptosomal membranes. A decrease in protein rigidity was observed at high ethanol concentrations at 37 degrees only in the erythrocytes. Thus an effect of ethanol on the conformation of membrane protein was observed using MAL-6 only at high ethanol concentrations and depended on the membrane used and the temperature of measurement.