Concentration-Associated DNA Methylation Remodeling of Oncogenic Signaling Networks Following Moringa oleifera Treatment in Metastatic PC3 Prostate Cancer Cells

PC3 prostate cancer (PCa) cells exhibit high metastatic potential, androgen independence, and activation of multiple oncogenic signaling pathways. Although Moringa oleifera has recognized anticancer properties, its effects on DNA methylation in aggressive PCa remain largely unexplored. Here, PC3 cells were treated with Moringa oleifera extract (MO) at 100 or 200 μg/mL and evaluated using functional and molecular analyses alongside genome-wide DNA methylation profiling. Both concentrations reduced AKT phosphorylation and the expression of mesenchymal and invasion-associated markers, whereas increased apoptosis was observed at 200 μg/mL MO. ERK1/2 phosphorylation differed between the two concentrations but was not significantly reduced relative to untreated controls. Methylation profiling revealed concentration-specific pathway-enrichment profiles among genes associated with differentially methylated regions (DMRs). At 100 μg/mL, hypermethylated DMR-associated genes were enriched mainly in small GTPase signaling, focal adhesion, cytoskeletal organization, axon guidance, MAPK, Rap1, and RHO signaling. At 200 μg/mL, enrichment involved cell adhesion, Wnt signaling, RAC1/CDC42 GTPase cycles, and VEGF/VEGFR2-mediated signaling. Hypomethylated DMR-associated genes showed distinct but partly overlapping enrichment profiles. In the direct comparison, DMRs with higher methylation at 100 μg/mL were associated with axon guidance, focal adhesion, actin cytoskeleton regulation, MAPK, and EPH–Ephrin signaling, whereas those with higher methylation at 200 μg/mL were associated mainly with NOTCH and RHOB/RHOC signaling. These findings identify concentration-specific DNA methylation changes in genes belonging to networks relevant to aggressive PCa, accompanied by molecular and cellular responses, without establishing a causal relationship between methylation changes and those responses.