012 | Anticancer activity of Orobanche crenata leaf extract in human colorectal cancer cell lines

Floriana D’Angeli¹, Alfio Distefano², Laura Orlando², Elisa Gili³, Valentina Di Salvatore⁴, Simone Ronsisvalle⁴, Ivana Roberta Romano², Giuliana Mannino², Debora Lo Furno², Giovanni Giurdanella¹, Giovanni Li Volti², Carlo Genovese¹ | ¹Department of Medicine and Surgery, School of Medicine and Surgery, University of Enna "Kore", Enna, Italy; ²Department of Biomedical and Biotechnological Sciences, University of Catania, Italy; ³Department of Clinical and Experimental Medicine, "Regional Referral Center for Rare Lung Diseases", University, Hospital Policlinico "G. Rodolico-San Marco", University of Catania, Italy; ⁴Department of Health and Drug Sciences, University of Catania, Italy.

Colorectal cancer (CRC) is one of the most prevalent malignancies worldwide and remains a leading cause of cancer-related mortality [1]. Despite significant advances in therapy, conventional treatments such as chemotherapy are often limited by severe side effects and the development of drug resistance [2]. In this context, natural compounds with anticancer properties represent promising alternatives to improve treatment efficacy and tolerability. In our previous studies, we demonstrated the anticancer activity of an acetonic extract of Orobanche crenata leaves against hormone-sensitive breast cancer cells through modulation of reactive oxygen species (ROS)[3]. To improve biological relevance and safety of the extract, in the present study we investigated the effects of the aqueous fraction of O. crenata leaves (OCLAqE) on human colorectal cancer cell lines (Caco-2 and HCT-116), as well as its cytotoxicity on non-cancerous human dermal fibroblast (HDF) cells. Cells were treated with increasing concentrations of OCLAqE (10–160 µg/mL) or cisplatin (0.1–100 µM) for 24, 48, and 72 hours. Cytotoxicity was evaluated using MTT assays. Based on these results, two concentrations of OCLAqE (40 and 80 µg/mL) and Cisplatin (1 and 10 µM) were selected for further analysis. The effects of OCLAqE alone or in combination with a subtoxic dose of Cisplatin (1 µM) were assessed by MTT and Annexin V/Propidium Iodide assays. Intracellular and extracellular ROS levels were measured using 2′,7′-dichlorofluorescein diacetate labeling. The chemical composition of OCLAqE was characterized by UPLC-MS/MS, while bioinformatic analyses identified potential bioactive compounds involved in the modulation of apoptotic pathways. The results demonstrated that OCLAqE significantly reduced cancer cell viability in a dose-dependent manner, with a more pronounced effect observed in Caco-2 cells, while no cytotoxic effects were detected in control HDF cells. Annexin V/PI assays revealed that OCLAqE induced apoptotic cell death in Caco-2 cells, particularly after 72 hours of treatment. Notably, OCLAqE did not modulate intracellular or extracellular ROS levels in either colorectal cancer cell line, suggesting a ROS-independent mechanism of action. UPLC-MS/MS analysis identified several bioactive molecules, and bioinformatic predictions highlighted compounds potentially responsible for apoptosis induction. In conclusion, our data suggest that Orobanche crenata aqueous extract may represent a promising therapeutic adjuvant by promoting apoptosis in human colorectal cancer cell lines through ROS-independent mechanisms.