Integrated In Silico and In Vitro Assessment of the Anticancer Potential of Origanum vulgare L. Essential Oil

Oregano essential oil (OEO) has gained attention for its broad pharmacological activities, such as anti-inflammatory, antimicrobial, and anticancer properties. This study aimed to analyze the phytochemical composition and biological activity of OEO obtained from wild-growing Origanum vulgare L. in Romania. Gas chromatography–mass spectrometry (GC–MS) analysis identified p-cymene (43.98%), γ-terpinene (22.16%), and thymol (11.46%) as major constituents, with notable differences from previously reported chemotypes. Antioxidant activity was assessed using the DPPH, ABTS radical scavenging assay, and TPC. OEO has a moderate antioxidant activity, with IC50 values of 134.67 ± 1.32 µg/mL (DPPH) and 88.15 ± 0.045 Inh% (ABTS) and a TPC of 159.63 mg GAE/g extract. The cytotoxicity of the simple water dispersion of OEO, OEO solubilized with polyethylene glycol 400 (OEO-PEG), and that solubilized with Tween 20 (OEO-Tw) was evaluated on human melanoma (A375) and human colorectal adenocarcinoma (HT-29) cancer cell lines, as well as on the normal human immortalized keratinocytes (HaCaT) cell line. The results demonstrated a significant inhibition of cancer cell viability with no recorded cytotoxic effect on normal cells. The highest inhibition of cell viability was recorded for OEO-PEG 200 µg/mL (7.22% ± 6.51 in A375 cell line and 22.25% ± 10.08 in HT-29 cell line). In cancer cells, OEO and its formulations significantly reduced malondialdehyde (MDA) levels (up to 41.24% in A375 cells and up to 48.58% in HT-29 cells), suggesting potent antioxidant activity. Moreover, treatment with OEO increased caspase 3/7 activation two-fold in treated A375 cells, while high-resolution respirometry studies revealed that OEO induces mitochondrial dysfunction by acting as a potential uncoupling agent. Molecular docking analysis suggested that β-caryophyllene oxide (CPO), a minor constituent of OEO, may act as a potential inhibitor of 3-phosphoinositide-dependent protein kinase-1 (PDPK1), indicating a possible mechanism of anticancer activity. Our findings highlight the potential of OEO as a natural anticancer agent, emphasizing the need for further investigations to elucidate its exact molecular mechanisms and therapeutic applicability.