Lupeol- and Cis-Vaccenic Acid-Containing Bioactive Fraction of Mondia whitei Induces Vacuolation-Associated Cytotoxicity Through Autophagy–Lysosome Perturbation and Caspase-Dependent Apoptosis in Human Colorectal Adenocarcinoma Cells

Abstract

Plant-derived natural products are essential and prominent contributors to drug discovery, especially as anticancer agents. Medicinal plants used in alternative therapy are often obscured by poor definitions of the underlying mechanisms behind their bioactivity. In this study, we investigated the cytotoxic and mechanistic effects of an enriched bioactive fraction derived from Mondia whitei. Bioactivity-guided fractionation was performed using C18 solid-phase extraction. The cytotoxic effects were evaluated using the MTT assay, while cellular morphology and mechanistic pathways were examined through microscopy, acridine orange staining, cathepsin-based assay, and immunoblotting of apoptosis- and autophagy-related proteins. Chemical profiling of the fraction was conducted using GC–MS analysis. The enriched fraction exhibited enhanced cytotoxicity at low microgram concentrations. Morphological assessment revealed prominent cytoplasmic vacuolation, while acridine orange staining indicated the accumulation of acidic vesicles. Cathepsin-based assays and immunoblot analysis of LC3-I/II confirmed lysosomal involvement and autophagy perturbation, whereas increased p62 levels suggested disruption of the autophagy–lysosome perturbation. In parallel, activation of intrinsic apoptosis was evidenced by the increased expression of caspase-9 and caspase-3. GC–MS profiling tentatively identified lupeol and cis-vaccenic acid as the major constituents of the fraction. The results from these studies demonstrate that the enriched fraction of Mondia whitei induced vacuolation-associated cytotoxicity through autophagy–lysosome perturbation and caspase-dependent apoptosis, providing mechanistic insight into its anticancer potential.