Ovatifolin Purified from Leptocarpha rivularis Induces Cell Death in A375 and A2058 Melanoma Cancer Cells

Skin cancer is increasing worldwide, with melanoma being its most aggressive and lethal form due to its high metastatic potential. Despite therapeutic advances, drug resistance remains a challenge, highlighting the need to explore new anticancer agents. <i>Leptocarpha rivularis</i> is a native plant of Chile, locally called "Palo negro", and is traditionally used in medicine by the Mapuche people. <i>L. rivularis</i> produces bioactive germacrene sesquiterpenoids with cytotoxic, antioxidant, anti-inflammatory and anti-angiogenic properties. This study reports for the first time the isolation of ovatifolin from aerial parts of <i>L. rivularis</i> and its identification by NMR and X-ray diffraction, together with its antiproliferative activity against two melanoma cell lines. The results show that ovatifolin has cytotoxic activity against the cell lines A2058 and A375, with an IC₅₀ of 27.6 (90.2 µM) and 18.4 µg/mL (60.1 µM), respectively, evaluated by live-cell IncuCyte^® analysis. Moreover, ovatifolin arrests colony formation in a clonogenic assay, with an IC₅₀ of 3.26 (10.6 μM) and 3.65 µg/mL (11.9 μM) in these same cell lines. Therefore, ovatifolin increased intracellular ROS and decreased the mitochondrial membrane potential (ΔΨ m). Cell death studies using Annexin V showed that its cytotoxic activity is partially caused by non-specific apoptosis, which was corroborated by the caspase inhibitor Z-VAD with an incomplete recovery of the cell death process.