Abstract
Resistance to apoptosis represents a major limitation of platinum-based chemotherapy in colorectal carcinoma, frequently arising from impaired p53 signaling and inefficient execution of programmed cell death. In this study, we investigated the anticancer activity of Erica spiculifolia extract (ESE) and its ability to synergistically enhance cisplatin cytotoxicity in HT-29 colorectal carcinoma cells. Cell viability was assessed using the MTT assay, followed by formal combination analysis based on the Chou-Talalay methodology. Combination experiments employed a non-constant ratio regimen in which a fixed ESE concentration (45 µg/mL) was combined with serial cisplatin dilutions (45.0-2.8 µg/mL) to define interaction behavior across multiple effect levels. Quantitative analysis revealed a strong superadditive effect, with Combination Index values well below 1 and markedly elevated Dose Reduction Indices for cisplatin, indicating substantial dose-sparing across effect levels. To elucidate the molecular basis of this synergism, apoptosis-related protein expression was profiled using a membrane-based immunoassay. Combined ESE and cisplatin treatment induced full-scale p53 reactivation, including restoration of phosphorylated p53 isoforms associated with DNA damage-dependent apoptotic signaling. Acridine orange/propidium iodide staining confirmed a pronounced increase in early and late apoptotic/necrotic cells following combination treatment. UHPLC-HRMS analysis identified kaempferol 3-O-glucoside (8830.19 ± 11.01 ng/mg dw) and myricitrin (3074 ± 3.12 ng/mg) as predominant flavonols, followed by naringenin 7-O-glucoside (5958.96 ± 9.98 ng/mg), while chlorogenic, cinnamic, quinic, and gallic acids were the main phenolic acids detected. These constituents may contribute to HT-29 cell sensitization to cisplatin.