Cytotoxic Effects of Medicinal Plant Extracts on HeLa Cells via Redox Imbalance, L-Arginine Metabolic Reprogramming,...

Cancer cells undergo metabolic reprogramming to sustain proliferation, resist apoptosis, and adapt to oxidative stress. In the present study, we comparatively evaluated the anticancer effects of four medicinal plant extracts (Inula helenium, Hypericum alpestre, Rumex obtusifolius, and Alchemilla smirnovii) on HeLa cervical cancer cells, with particular emphasis on oxidative stress, l-arginine metabolism, and cellular energy pathways. Cell viability was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, while biochemical colorimetric methods were used to determine nitric oxide production, arginase and nitric oxide synthase activities, malondialdehyde levels, superoxide dismutase and catalase activities, proline content, and key glycolytic metabolites. Nuclear morphology changes consistent with apoptotic features were examined by Hoechst 33258 staining. All tested extracts exhibited cytotoxic effects in HeLa cells in a time- and concentration-dependent manner, which were further enhanced when combined with the metabolic inhibitors l-NAME (Nω-nitro-l-arginine methyl ester) or nor-NOHA (Nω-hydroxy-nor-l-arginine). Inula helenium showed the strongest potentiation in combination treatments. Several extracts significantly decreased arginase activity while increasing nitric oxide synthase activity and nitrite levels, indicating modulation of l-arginine metabolism. Treatments also increased malondialdehyde levels and stimulated antioxidant enzyme activities, consistent with redox imbalance and oxidative stress-mediated cytotoxicity. In addition, Inula helenium and Alchemilla smirnovii reduced intracellular glucose, pyruvate, and lactate concentrations, suggesting partial modulation of glycolytic metabolism. Fluorescence microscopy revealed chromatin condensation, nuclear fragmentation, and apoptotic body formation, particularly after treatment with Hypericum alpestre and Rumex obtusifolius. These findings demonstrate that selected medicinal plant extracts exert cytotoxic effects by simultaneously modulating oxidative stress, amino acid metabolism, and energy pathways in cervical cancer cells. The investigated plants may represent promising sources of bioactive compounds for the development of complementary metabolic anticancer strategies.