Molecular Basis of Simalikalactone D Sensitivity in Triple-Negative Breast Cancer Cells

<b>Background/Objective:</b> Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer (BC) lacking targeted therapies and characterized by high tumor heterogeneity. In this study, we evaluated the anticancer activity and mechanistic profile of Simalikalactone D (SKD), a quassinoid compound derived from the endemic Puerto Rican tree <i>Simarouba tulae</i>, in three TNBC cell lines, MDA-MB-468, MDA-MB-231, and SUM-149. <b>Methods:</b> MDA-MB-468, MDA-MB-231 and SUM-149 TNBC cells were evaluated for cell viability, proliferation and migration following SKD treatment. Phospho-antibody array, proteomics, and Western blot analyses were used to explore the SKD mechanism of action in MDA-MB-468 and MDA-MB-231 cell lines. Molecular docking was performed to assess SKD's interaction with potential intracellular targets. <b>Results:</b> SKD exerted a concentration-dependent effect on the three cell lines. However, MDA-MB-468 cells exhibited an IC₅₀ of 67 nM, while the IC₅₀ values for MDA-MB-231 and SUM-149 were 422 nM and 598 nM, respectively. In MDA-MB-468 cells, 100 nM of SKD induced apoptosis, evidenced by the activated caspase-3 activity, PARP-1 cleavage and decrease in Bcl-2 and survivin protein levels. Sublethal SKD (25 nM) impaired migration in MDA-MB-231 cells and reduced proliferation and motility in SUM149 cells. A 6 h SKD treatment markedly reduced phosphorylation of apoptosis-related proteins (p53, BAD, DAXX, AKT1, JUN) and Jak/STAT pathway components, indicating early disruption of intracellular signaling prior to phenotypic changes. Proteomic profiling showed distinct pathway alterations in both MDA-MB-468 and MDA-MB-231 cells, with reduced Integrin β1 (ITGB1) levels emerging as a shared effector. This suggests that SKD broadly disrupts cell adhesion and migration independently of apoptosis-driven cell death. Western blot validation confirmed reduced ITGB1 protein levels across all three TNBC cell lines examined. In silico docking confirmed favorable binding affinities of SKD to both EGFR (ΔG = -6.718 kcal/mol) and STAT4 (ΔG = -8.481 kcal/mol). <b>Conclusions:</b> Overall, our findings suggest that SKD is a potent anticancer agent in a subgroup of TNBC cells.