Coşkun Orhaner, Mehmet Cudi Tuncer, İlhan Özdemir
Rosmarinic acid (RA), a naturally occurring polyphenolic compound, has attracted increasing attention because of its potential anticancer activity and capacity to modulate oxidative stress-associated signaling pathways. In the present study, the cytotoxic, apoptotic, and antiproliferative effects of RA, alone or in combination with gemcitabine (Gem), were investigated in OVCAR3 ovarian cancer cells and HaCaT keratinocytes using integrated two-dimensional and three-dimensional (3D) experimental models. Cell viability assays demonstrated dose- and time-dependent growth inhibition following RA and Gem treatment, while combination index (CI) analysis revealed synergistic cytotoxic activity in OVCAR3 cells. Flow cytometric analyses showed that combined treatment markedly increased apoptotic cell populations and altered cell cycle progression through enhanced S-phase and G2/M accumulation. Intracellular reactive oxygen species (ROS) levels were significantly elevated following combination treatment, and N-acetyl-L-cysteine (NAC) pretreatment partially attenuated both ROS accumulation and cytotoxicity, indicating a functional contribution of oxidative stress to the observed antitumor response. RT-qPCR analyses demonstrated increased expression of proapoptotic genes (BAX, CASP3, and CASP9) together with suppression of BCL2, MKI67, and CDK4 expression, while immunocytochemical analyses supported enhanced caspase-3 activation at the protein level. In 3D OVCAR3 tumor spheroids, the RA + Gem combination significantly reduced spheroid viability, disrupted spheroid architecture, and increased dead-cell accumulation compared with single-agent treatments. Collectively, these findings suggest that RA may enhance the anticancer activity of Gem in ovarian cancer cells through mechanisms associated with oxidative stress, apoptosis, and proliferation-related signaling pathways under both monolayer and 3D culture conditions.