ATR-FTIR spectroscopy combined with chemometrics reveals molecular alterations and anticancer effects of Nigella sativa extract in human colon cancer cells

Colorectal cancer remains the second leading cause of cancer-related mortality worldwide, with current therapeutic approaches often demonstrating limited efficacy. Nigella sativa L. (NS) seeds, historically valued for their medicinal properties, exhibit promising anticancer potential. This study investigates the molecular effects of NS methanolic extract on CaCo-2 human colon cancer cells, focusing on cellular composition, dynamics, and segregation patterns. Unsupervised chemometric analyses, including principal component and hierarchical cluster analyses, demonstrated a complete separation between control and NS-treated cells, indicating significant molecular divergence, further validated by supervised classification methods. Spectral analysis revealed reductions in unsaturated lipids, proteins, glucose, and DNA levels, along with a shortening of fatty acid acyl chain length. In contrast, saturated lipid and triglyceride content increased, accompanied by enhanced membrane fluidity and lipid disorder, indicating substantial alterations in cellular lipid dynamics and acyl chain flexibility. Furthermore, oxidative stress markers were elevated, as evidenced by increased protein carbonylation, while protein phosphorylation levels declined. NS treatment also induced protein conformational changes, notably an increase in aggregated β-sheet structures, suggesting protein denaturation. These biochemical modifications were strongly associated with NS-enhanced reactive oxygen species (ROS) levels. Overall, this study elucidates the molecular mechanisms underlying the anticancer effects of NS, supporting its potential as an adjunctive therapeutic strategy for colorectal cancer.