Article

In Silico ERK Pathway Prediction and Experimental Validation of IB-DK143-Induced Apoptosis in MDA-MB-231 Breast Cancer Cells

Abstract

Background/Objectives: IB-DK143, an ibuprofen-linked chalcone derivative previously investigated in cervical cancer cells, has been associated with cytotoxicity, mitochondrial stress, p38 MAPK activation, and a decreasing tendency of ERK1/2 phosphorylation. In the present study, we extended this mechanistic concept to MDA-MB-231 breast cancer cells and investigated whether IB-DK143 induces ERK suppression-associated, caspase-dependent apoptosis.

Methods: To examine the potential interaction of IB-DK143 with ERK2/MAPK1 and its upstream regulator MEK1/MAP2K1, target prediction, molecular docking, and molecular dynamics simulation were performed. To experimentally validate the in silico findings, MDA-MB-231 breast cancer cells were treated with increasing concentrations of IB-DK143. Cytotoxicity was assessed using the WST-1 assay. Apoptosis-associated nuclear morphological changes were evaluated by Hoechst staining, including quantification of apoptosis-positive cells, mean fluorescence intensity, and nuclear area. Caspase-3 changes were analyzed as an apoptosis-related marker, and ERK1/2 expression and/or phosphorylation was evaluated as an early signaling readout of ERK pathway modulation.

Results: In silico analysis identified ERK2/MAPK1 and MEK1/MAP2K1 as candidate ERK pathway-associated targets of IB-DK143. Experimentally, IB-DK143-treated MDA-MB-231 cells showed concentration-dependent cytotoxicity and apoptosis-associated nuclear morphology, including increased apoptosis-positive nuclei, increased nuclear fluorescence intensity, and reduced nuclear area. Caspase-3-related changes were detected, and a decreasing trend in ERK1/2 signaling was also observed. Based on these findings and prior cervical cancer data, an in silico-guided working model was constructed in which IB-DK143 may suppress ERK-mediated survival signaling and promote caspase-dependent apoptosis.

Conclusions: The current findings support the feasibility of a combined computational and experimental study to test whether IB-DK143 induces ERK suppression-associated, caspase-3-dependent apoptosis in MDA-MB-231 breast cancer cells. Further validation using quantitative Western blotting, ERK rescue experiments, kinase activity assays, and final docking/MD simulation metrics is required before causal conclusions can be made.

Keywords

IB-DK143, MDA-MB-231, ERK1/2, MAPK; MEK1, caspase-3, apoptosis, molecular docking, molecular dynamics, breast cancer