Immobilization of Paclitaxel on Hydroxyapatite for Breast Cancer Investigations.

A simple method for immobilization of the chemotherapy drug paclitaxel (PTX) on hydroxyapatite nanoparticles (n-HAP) using the biopolymer chitosan as a trapping agent is described focusing on applications involving breast cancer cells. n-HAP with two distinct crystallinity profiles were used: with predominant crystallization along the long axis and with a more homogeneous crystallization in all directions. In the first scenario, the interactions between chitosan and both the OH and PO43- groups on the surface of the nanoparticles are favored and lead to a more efficient attachment of the drug. In this case, PTX is found to remain mostly attached to the n-HAP for at least 24 h, while being dispersed in aqueous solution. During this time, the activity of the drug is inhibited as corroborated by in vitro assays with breast cancer cells. With that, the in vitro experiments revealed distinct effects from the drug-loaded nanoparticles on the cells depending on the experimental conditions. In a short term, that is, in 24 h, the cells exhibit higher viability than those challenged with nonloaded materials. Nevertheless, after 72 h, even a small content of PTX in the presence of n-HAP can reduce the cells' viability via stimulation of the apoptotic phenotype and suppression of survival stimuli.

Wortmannin targeting phosphatidylinositol 3-kinase suppresses angiogenic factors in shear-stressed endothelial cells.

Modifications on shear stress-based mechanical forces are associated with pathophysiological susceptibility and their effect on endothelial cells (EC) needs to be better addressed looking for comprehending the cellular and molecular mechanisms. This prompted us to better evaluate the effects of shear stress in human primary venous EC obtained from the umbilical cord, using an in vitro model to mimic the laminar blood flow, reaching an intensity 1-4 Pa. First, our data shows there is a significant up-expression of phosphatidylinositol 3-kinase (PI3K) in shear-stressed cells culminating downstream with an up-phosphorylation of AKT and up-expression of MAPK-ERK, concomitant to a dynamic cytoskeleton rearrangement upon integrin subunits (α4 and ß 3) requirements. Importantly, the results show there is significant involvement of nitric oxide synthase (eNOS), nNOS, and vascular endothelial growth factors receptor 2 (VEGFR2) in shear-stressed EC, while cell cycle-related events seem to being changed. Additionally, although diminution of 5-hydroxymethylcytosine in shear-stressed EC, suggesting a global repression of genes transcription, the promoters of PI3K and eNOS genes were significantly hydroxymethylated corroborating with their respective transcriptional profiles. Finally, to better address, the pivotal role of PI3K in shear-stressed EC we have revisited these biological issues by wortmannin targeting PI3K signaling and the data shows a dependency of PI3K signaling in controlling the expression of VGFR1, VGFR2, VEGF, and eNOS, once these genes were significantly suppressed in the presence of the inhibitor, as well as transcripts from Ki67 and CDK2 genes. Finally, our data still shows a coupling between PI3K and the epigenetic landscape of shear-stressed cells, once wortmannin promotes a significant suppression of ten-11 translocation 1 (TET1), TET2, and TET3 genes, evidencing that PI3K signaling is a necessary upstream pathway to modulate TET-related genes. In this study we determined the major mechanotransduction pathway by which blood flow driven shear stress activates PI3K which plays a pivotal role on guaranteeing endothelial cell phenotype and vascular homeostasis, opening novel perspectives to understand the molecular basis of pathophysiological disorders related with the vascular system.