Discussion
Marine organisms constitute an unexplored resource of potential drugs. They produce a rich source of active and potent novel chemical compounds, i.e. secondary metabolites, potential useful as medical treatments. In the project ‘Marine Fungi’ an interdisciplinary approach is employed to identify novel anti-cancer compounds and characterize their targets, mode-of-action and pharmacokinetic properties. Cancer is known to be tightly correlated to deregulated signaling pathways, thus protein kinases are prominent drug targets (Hiroko et al. 2013). The aim of the project was to elucidate the temporal regulation of cellular signaling of cancer cells in response to exposure to a novel anti-cancer compound.
Cell signaling of cancer cells was investigated using quantitative phoshoproteomics. Cancer cells were cultured and exposed to compound ‘CpdX’ at a concentration of 69.8 µM. The concentration was chosen based on previous results indicating a total growth inhibition after 48 hrs. at this concentration (TGI-48h). Cancer cells were sampled at 0 hr., 1 hr. and 6 hr. after introducing CpdX to the culture media, the experiment included three biological replicates. Proteins were enzymatically digested using trypsin, and peptides were chemically labeled by isobaric tags (iTRAQ). Samples within biological replicates were multiplexed and subsequently fractionated by off-line strong cation exchange chromatography (SCX). Each three-plex replicate generated seven SCX fractions, and phosphopeptides of each of these fractions were enriched by titaniumdioxide (TiO2). Phosphoenriched fractions were analyzed by reversed-phase nLC MS/MS (LTQ Orbitrap Velos) using a 180 min gradient and an iTRAQ optimized MS method (Ow et al. 2009). Protein and peptide identification and localization of phosphorylation was performed using Proteome Discoverer and PhosphoRS 3.0. All low and medium confident peptides were excluded from data analysis.
From each replicate 1989 (Rep1), 1439 (Rep2) and 1990 (Rep3) proteins were identified, with 1311 proteins identified in all three replicates. From each replicate approximately 200 phosphopeptides were identified. Despite the enrichment of phosphopeptides was moderate, several phosphopeptides were found to respond reproducibly to exposure of the compound in the three biological replicates. Proteins involved in cellular tight junction processes showed increasing phosphorylation levels, with no increase in overall protein expression level. The temporal regulation of phosphopeptides showed increasing levels of phosphorylation even after 6 hrs. The phosphoenrichment procedure will be optimized and repeated for more in depth analysis of the phosphoproteomic responses to this novel anti-cancer compound.
Kamilla Sofie Pedersen1, Nadia Taouatas1, Johannes Landskron2, Jan Lorenzen1*
1 Danish Technological Institute, Division of Life Science, Medical Biotechnology, Kongsvang allé 29, DK-8000 Aarhus.
2 University of Oslo, The Biotechnology Centre of Oslo, P.O. Box 1125, Blindern, NO-0317 Oslo.
References:
Hiroko, K-H, Yumi, G and Oyama, M. 2013. Phosphoproteomics-Based Characterization of Cancer Cell Signaling Networks, Oncogenomics and Cancer Proteomics - Novel Approaches in Biomarkers Discovery and Therapeutic Targets in Cancer, Dr. Cesar Lopez (Ed.), InTech, DOI: 10.5772/52915.
Ow, SY, Salim, M, Noirel, J et al. 2009. iTRAQ underestimation in simple and complex mixtures: ”The good, the bad and the ugly”. J. Proteome Res. 8: 5347-55.
