Determination of ferroptotic, apoptotic, anti-carcinogenic properties of ferrocene-artemisinin hybrid structures

Cancer remains among the leading causes of death worldwide, necessitating the development of effective and targeted new therapeutic approaches. In this context, hybrid molecules have gained prominence in anticancer drug development in recent years due to their capacity to simultaneously target multiple biological pathways. Ferrocene-containing hybrid structures are particularly noteworthy for their high biological activity and low toxicity. To evaluate the cytotoxic activities of the synthesized ferrocene-artemisinin hybrids, their effects on cell viability were determined using the MTT assay across four different cancer cell lines (HeLa, HCT-116, HepG2, and A549) and a healthy cell line (HUVEC). The EC₅₀ values of these hybrid compounds were determined to be 35.1–46.74 µM for HepG2, 25.2–44.35 µM for A549, 11.02–43.28 µM for HCT-116, and 57.05–88.44 µM for HeLa. Apoptosis analyses performed via flow cytometry revealed that the HepG2 cell line was more sensitive to compounds N141 (14.45%), N154 (16.26%), and N159 (19.55%). In HCT-116 and A549 cells, a moderate increase in apoptotic cells was observed. ROS (Reactive Oxygen Species) levels were significantly increased in the HCT-116 cell line treated with N141 (8.9-fold), N154 (8.8-fold), and N159 (9.12-fold). Similarly, all three compounds were observed to increase ROS levels in the HepG2 and A549 cell lines. Lipid peroxidation assays demonstrated that compound N154 induced a significant increase in lipid peroxidation in HCT-116 (2.3-fold), HepG2 (2.4-fold), and A549 (2.4-fold) cell lines; compounds N141 and N159 exhibited similar results.As a result of qPCR analyses, it was determined that compounds N141, N154, and N159 significantly suppressed the anti-apoptotic gene BCL-2 and the anti-ferroptotic genes SLC7A11, GPX4, and GPX1 across all three selected cell lines. Conversely, these compounds were found to increase the expression of pro-apoptotic genes (BAX, CASP3, CASP8, CASP9) and pro-ferroptotic genes (STEAP3, NOX1, NOX2, TFRC, and ACSL4).In this thesis study, it was established that ferrocene-artemisinin hybrid compounds induce apoptosis and ferroptosis, while also exhibiting anti-migratory and anti-proliferative activities. Therefore, it is of great importance to further detail the biological and pharmacological analysis of these ferrocene-artemisinin hybrid compounds at both in vitro and in vivo levels to more clearly understand their therapeutic potential.