Mechanistic Insights into Vernonia calvoana-Induced Apoptosis in Ovarian Cancer Cells via the Intrinsic Pathway

Abstract

Vernonia calvoana (VC), a commonly used medicinal plant in West Africa, has been shown by our research team to inhibit the proliferation of OVCAR-3 ovarian cancer cells through mechanisms involving oxidative stress, DNA damage, and S-phase cell cycle arrest. The objective of the current study was to elucidate the intrinsic apoptotic mechanisms triggered by VC fraction seven (VCF7). OVCAR-3 cells were treated with VCF7 (0, 8, 16, and 32 μg/mL) for a duration of 48 h. Apoptosis was assessed using Annexin V/Propidium Iodide (PI) staining followed by flow cytometry analysis. Mitochondrial membrane potential (ΔΨm) was assessed through JC-1 staining and confocal microscopy, while chromatin condensation was analyzed using DAPI staining. DNA fragmentation was examined by agarose gel electrophoresis. Caspase 3 activity was measured using flow cytometry. Protein expression levels of p53, Bcl-2, cytochrome c, caspase-9, and caspase-3 were determined by Western blot analysis, and mRNA expression levels of p53 and Bcl-2 were evaluated using qRT-PCR. VCF7 induced apoptosis in a concentration-dependent manner. Analysis using Annexin V/PI indicated an increase in apoptotic cell populations from 10.5% to 30%, along with a rise in necrotic cells from 7% to 50% across treatment concentrations. A modest, concentration-associated decrease in mitochondrial membrane potential was recorded (0.96-, 0.88-, and 0.85-fold at 8, 16, and 32 μg/mL, respectively; p < 0.05). DAPI staining validated the concentration-dependent chromatin condensation and nuclear fragmentation. The analysis of DNA fragmentation showed progressive internucleosomal degradation, appearing as a smear pattern with distinct fragments at elevated concentrations, indicative of concurrent apoptotic and necrotic cell death. The activation of caspase-3 reached a peak of 28% at 16 μg/mL. Western blot analysis indicated an upregulation of p53, a downregulation of Bcl-2, an increase in total cytochrome c protein levels, and an increased expression of caspase-9 and caspase-3 in a concentration-dependent manner. These findings were corroborated at the transcriptional level by qRT-PCR, which showed increased p53 mRNA and decreased Bcl-2 mRNA expression. Taken together, these results underscore the potential of VCF7 as a promising plant-derived anticancer agent and support the need for further preclinical and clinical studies in ovarian cancer.