Apoptosis-related gene modulation by Juglans regia leaf extract in breast and colon cancer cells

Juglans regia L. (walnut) leaves are rich in phenolic compounds, flavonoids, and terpenoids known for their antioxidant and pharmacological activities. Although J. regia has been widely used in traditional medicine, its potential anticancer mechanisms on different human cell lines remain to be fully elucidated. This study aimed to investigate the cytotoxic and apoptotic effects of ethanol extracts of J. regia leaves on human breast adenocarcinoma (MDA-MB-231), human colorectal adenocarcinoma (HT-29), and human keratinocyte (HaCaT) cell lines. Dried J. regia leaves collected from Balıkesir, Türkiye, were extracted with ethanol using the Soxhlet method. Cytotoxicity was evaluated by MTT assay in a dose- and time-dependent manner (1000–50 μg/μL concentrations; 24, 48, and 72 h). Gene expression analysis of apoptosis-related Bax, Bcl-2, and cell cycle regulator p27 was performed by quantitative real-time PCR (qRT-PCR). The ethanol extract of J. regia leaves exhibited a significant antiproliferative effect on MDA-MB-231 and HT-29 cells, while it showed no cytotoxicity on HaCaT cells. The effective concentration was determined as 100 μg/μL. qRT-PCR analysis revealed upregulation of pro-apoptotic Bax and p27 genes and downregulation of anti-apoptotic Bcl-2 in MDA-MB-231 and HT-29 cells in a time-dependent manner, suggesting induction of apoptosis and cell cycle arrest. These findings demonstrate that ethanol extracts of J. regia leaves possess potent anticancer and antiproliferative activities, likely mediated through the modulation of apoptotic pathways and regulation of the mitotic phase. J. regia may represent a promising natural source for developing novel anticancer agents.