Pharmacoproteomic Study of the Natural Product Ebenfuran
III in DU-145 Prostate Cancer Cells: The Quantitative and Temporal
Interrogation of Chemically Induced Cell Death at the Protein Level
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Abstract
A naturally
occurring benzofuran derivative, Ebenfuran III (Eb
III), was investigated for its antiproliferative effects using the
DU-145 prostate cell line. Eb III was isolated from <i>Onobrychis
ebenoides</i> of the <i>Leguminosae family</i>, a plant
endemic in Central and Southern Greece. We have previously reported
that Eb III exerts significant cytotoxic effects on certain cancer
cell lines. This effect is thought to occur via the isoprenyl moiety
at the C-5 position of the molecule. The study aim was to gain a deeper
understanding of the pharmacological effect of Eb III on DU-145 cell
death at the translational level using a relative quantitative and
temporal proteomics approach. Proteins extracted from the cell pellets
were subjected to solution phase trypsin proteolysis followed by iTRAQ-labeling.
The labeled tryptic peptide extracts were then fractionated using
strong cation exchange chromatography and the fractions were analyzed
by nanoflow reverse phase ultraperformance liquid chromatography–nanoelectrospray
ionization-tandem mass spectrometry analysis using a hybrid QqTOF
platform. Using this approach, we compared the expression levels of
1360 proteins analyzed at ≤1% global protein false discovery
rate (FDR), commonly present in untreated (control, vehicle only)
and Eb III-treated cells at the different exposure time points. Through
the iterative use of Ingenuity Pathway Analysis with hierarchical
clustering of protein expression patterns, followed by bibliographic
research, the temporal regulation of the Calpain-1, ERK2, PAR-4, RAB-7,
and Bap31 proteins were identified as potential nodes of multipathway
convergence to Eb III induced DU-145 cell death. These proteins were
further verified with Western blot analysis. This gel-free, quantitative
2DLC–MS/MS proteomics method effectively captured novel modulated
proteins in the DU-145 cell line as a response to Eb III treatment.
This approach also provided greater insight to the multifocal and
combinatorial signaling pathways implicated in Eb III-induced cell
death