Synergistic cytotoxic effect of doxorubicin and bortezomib on lung cancer cell lines using 2D and 3D models

• DOX and BOR induces apoptosis and boosts therapeutic efficacy in lung cancer cell lines • 3D cell culture showed a decreased sensitivity to anticancer drugs in compression to 2D cell culture • Gene expression analysis indicated significant connections between BOR treatment and regulation of BCL2 and NFκB1, indicating their roles in apoptosis induction Lung cancer is the most prevalent cause of cancer-related mortality globally. Anti-cancer monotherapy outcomes have proven insufficient to meet the clinical needs for treating lung cancer; therefore, combination therapy has been employed to overcome the limitations of the present therapeutic modalities used in lung cancer. In this study, the anti-proliferative effects of doxorubicin (DOX) and bortezomib (BOR) were investigated separately and in combination using two-dimensional (2D) and three-dimensional (3D) cell culture models on 3 different lung cancer cell lines, H69AR, H69 and A549. Proliferation was assessed using resazurin dye colorimetric method, while quantitative real-time polymerase chain reaction (qRT-PCR) was performed to quantify the expression of genes, including PIK3CA, AKT, PTEN, NFκB1 and BCL-2 . In 3D cultures, drug treatments had significantly higher half maximal inhibitory concentration (IC₅₀) values compared to 2D cultures, indicating that cells are more resistant in this environment. Drugs combination resulted in significant synergistic effects in H69 and A549 cell lines in both 2D and 3D models, whereas, a synergistic effect was only seen in the 3D model of the H69AR cell line, based on combination index (CI) calculations. Moreover, of the genes analyzed, the expression of NFκB1 was significantly downregulated in 2D cultures in all tested cells treated with BOR alone or in combination with DOX. Overall, the study sheds light on the differences in drug responses and gene expression profiles between 2D and 3D culture models under single and combination treatments, emphasizing the increased physiological importance of 3D systems in simulating in vivo tumor behavior.