RESUMO
Inflammatory Breast Cancer (IBC) is a rare and aggressive type of breast cancer with a poor prognosis. Its management is challenging because of a lack of targeted therapies, increased metastatic potential, and high recurrence rates. Interest in using platinum agents such as carboplatin emerged from data suggesting frequent DNA repair defects in breast cancer. Because studies show that medicinal mushroom Ganoderma lucidum (GLE) sensitizes cancer cells to radiation and other drugs; herein, we aimed to investigate the therapeutic potential of GLE, alone or in combination with carboplatin in breast cancer models. Our studies were focused on the regulation of the DNA Damage Response (DDR) and on cancer cell stemness. Carboplatin and GLE were tested in vitro using the IBC cell line, SUM-149, breast cancer non-IBC cells, MDA-MB-231, and in vivo using IBC xenograft models. Our results show that the GLE/carboplatin combination decreased cell viability, induced cell death by two different mechanisms, and delayed the response to DNA damage. Furthermore, the combination suppressed mammosphere formation and the expression of cancer stemness proteins. In xenograft models, the combination showed significant tumor growth inhibitory effects without systemic toxicity. This study emphasizes the potential of this dual therapy for IBC patients.
RESUMO
Small peptides in solution adopt a specific morphology as they function. It is of fundamental interest to examine the structural properties of these small biomolecules in solution and observe how they transition from one conformation to another and form functional structures. In this study, we have examined the structural properties of a simple dipeptide and a five-residue peptide with the application of far-UV circular dichroism (CD) spectroscopy as a function of temperature, fluorescence anisotropy, and all-atom molecular dynamics simulation. Analysis of the temperature dependent CD spectra shows that the simplest dipeptide N-acetyl-tryptophan-amide (NATA) adopts helical, beta sheet, and random coil conformations. At room temperature, NATA is found to have 5% alpha-helical, 37% beta sheet, and 58% random coil conformations. To our knowledge, this type of structural content in a simplest dipeptide has not been observed earlier. The pentapeptide (WK5) is found to have four major secondary structural elements with 8% 310 helix, 14% poly-L-proline II, 8% beta sheet, and 14% turns. A 56% unordered structural population is also present for WK5. The presence of a significant population of 310 helix in a simple pentapeptide is rarely observed. Fluorescence anisotropy decay (FAD) measurements yielded reorientation times of 45 ps for NATA and 120 ps for WK5. The fluorescence anisotropy decay measurements reveal the size differences between the two peptides, NATA and WK5, with possible contributions from differences in shape, interactions with the environment, and conformational dynamics. All-atom molecular dynamics simulations were used to model the structures and motions of these two systems in solution. The predicted structures sampled by both peptides qualitatively agree with the experimental findings. Kinetic modeling with optimal dimensionality reduction suggests that the slowest dynamic processes in the dipeptide involve sidechain transitions occurring on a 1 ns timescale. The kinetics in the pentapeptide monitors the formation of a distorted helical structure from an extended conformation on a timescale of 10 ns. Modeling of the fluorescence anisotropy decay is found to be in good agreement with the measured data and correlates with the main contributions of the measured reorientation times to individual conformers, which we define as dynamic elements. In NATA, the FAD can be well represented as a sum of contributions from representative conformers. This is not the case in WK5, where our analysis suggests the existence of coupling between conformational dynamics and global tumbling. The current study involving detailed experimental measurements and atomically detailed modeling reveals the existence of specific secondary structural elements and novel dynamical features even in the simplest peptide systems.
