ABSTRACT
Roxithromycin (RXM) is a semi-synthetic fourteen-membered macrolide antibiotic that shows anti-angiogenic activity in solid tumors. In the present study, we conducted biopanning of T7 phage-displayed peptides either on a 96-well formatted microplate, a flow injection-type quartz-crystal microbalance (QCM) biosensor, or a cuvette-type QCM. RXM-selected peptides of different sequence, length and number were obtained from each mode of screening. Subsequent bioinformatics analysis of the RXM-selected peptides consistently gave positive scores for the extracellular domain (E458-T596) of angiomotin (Amot), indicating that this may comprise a binding region for RXM. Bead pull down assay and QCM analysis confirmed that RXM directly interacts with Amot via the screen-guided region, which also corresponds to the binding site for the endogenous anti-angiogenic inhibitor angiostatin (Anst). Thus, multimodal biopanning of T7PD revealed that RXM binds to the extracellular domain on Amot as a common binding site with Anst, leading to inhibition of angiogenesis-dependent tumor growth and metastasis. These data might explain the molecular basis underlying the mechanism of action for the anti-angiogenic activity of RXM.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Bacteriophage T7/chemistry , Membrane Proteins/antagonists & inhibitors , Peptides/chemistry , Roxithromycin/pharmacology , Angiogenesis Inhibitors/chemical synthesis , Angiogenesis Inhibitors/chemistry , Angiomotins , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Humans , Intercellular Signaling Peptides and Proteins/metabolism , MCF-7 Cells , Membrane Proteins/metabolism , Microfilament Proteins , Molecular Structure , Peptide Library , Quartz Crystal Microbalance Techniques , Roxithromycin/chemical synthesis , Roxithromycin/chemistry , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
By utilizing a quartz crystal microbalance with dissipation (QCM-D), we directly observed the self-oscillating behavior of a polymer chain induced by the Belousov-Zhabotinsky (BZ) reaction. We succeeded in measuring self-oscillations of the resonance frequency (Δf) and dissipation (ΔD), which originate in the self-oscillating behavior of the polymer chain on a gold surface induced by the BZ reaction. We synthesized a self-oscillating polymer chain with Ru as the catalyst of the BZ reaction and a chemical adsorption site, so as to directly observe its periodic swelling and collapse on the gold surface. Distinct self-oscillation of ΔD synchronized with the self-oscillation Δf was observed. The period of the Δf self-oscillation was about 400 s, and the induction time was about 6.5 h. In QCM-D measurements, we found that the peaks of Δf and ΔD oscillations did not coincide in time because the state of the Gaussian chain did not coincide with the maximum value of Δf. Moreover, examination of the relationship between Δf and ΔD revealed that their oscillatory waveforms were identical in frequency but differed in phase and amplitude.
ABSTRACT
In this study, we proposed and demonstrated a novel simultaneous analysis system of biosensing by combining a semiconductor-based field effect transistor (FET) with quartz crystal microbalance with a dissipation (QCM-D) monitoring system. Using the combined system, the changes of not only mass and viscoelasticity but also electrical charge for interaction of charged dextran molecules with substrate, recognition of glucose with low molecular weight, and programmed cell death, apoptosis, were simultaneously and quantitatively monitored in a label-free and real-time manner. The combined system will give more detailed information of biomolecule/substrate interface for development of new biomaterial.
