# Novel Drug Combinations in Lung Cancer: New Potential Synergies Between 5-FU and Repurposed Drugs
## 3 Results
### Synergistic Inhibition of Colony Formation by Resveratrol and 5-FU in A549 Cells  
Based on the initial cell viability assays, Resveratrol significantly enhanced the cytotoxic effects of both 5-FU and Levosimendan in A549 cells. However, the combination of Resveratrol with 5-FU showed a more consistent and pronounced reduction in cell viability compared to its combination with Levosimendan. Given the well-established efficacy of 5-FU as a chemotherapeutic agent, we hypothesized that combining it with Resveratrol would offer a stronger foundation for investigating potential improvements in cancer treatment.  
Figure 12: Cell viability of A549 cells treated with the drugs Levosimendan and Resveratrol alone, in a combination of Levosimendan with Resveratrol, and Resveratrol with IC50 5-FU. Values are expressed as percentages of control and represent means \(\pm\) SD. Each experiment was done three times independently (\(n=3\)); ns: non-significant; * statistically significant vs. drug alone at \(p<0.05\). The different gray bars are used solely to differentiate between data groups and do not represent specific experimental variables.  
Levosimendan alone exhibited limited cytotoxicity in A549 cells. While the addition of Resveratrol enhanced this effect, the synergistic impact was not as significant as that observed with 5-FU. To maximize the potential therapeutic impact and explore the most  
Figure 13: Morphological analysis of Levosimendan and Resveratrol alone and in combination in A549 cells. Cells were treated with vehicle (0.1% DMSO). Results are representative of three independent experiments. Scale bar: 50 μm.  
promising combination, we decided to focus subsequent experiments, including the clonogenic assay, on the Resveratrolrol and 5-FU combination. This approach allowed us to better understand Resveratrol's capacity to enhance the antitumor effects of a well-characterized chemotherapeutic drug, offering valuable insights into potential combination therapies for lung cancer.  
To further investigate Resveratrol's potential in enhancing 5-FU antitumor effects, a clonogenic assay was conducted, focusing on the combination of Resveratrol with the IC\({}_{50}\) concentration of 5-FU. The results demonstrated that Resveratrol significantly suppressed colony formation in A549 cells, particularly when combined with IC\({}_{50}\) 5-FU (Figure 14). Resveratrol alone reduced the number of colonies in a dose-dependent manner, indicating its pronounced effect on long-term cell proliferation.  
Interestingly, the combination of Resveratrol with IC\({}_{50}\) 5-FU resulted in a more substantial reduction in clonogenicity compared to either treatment alone. This highlights Resveratrol's potential to not only enhance the immediate cytotoxic effects of 5-FU, but to also inhibit the long-term survival and proliferation of lung cancer cells. These findings emphasize the importance of selecting effective drug combinations to optimize therapeutic outcomes, suggesting that Resveratrol could be a valuable addition to 5-FU-based treatments in lung cancer therapy.  
Figure 14: Clonogenic assay of human lung cancer cell line treated with IC\({}_{50}\) concentration of 5-FU and Resveratrol, both alone and in combination. (**A**) Image displaying the colonies formed by the human cancer cell line following plating 100 cells and incubation for 14 days. (**B**) Cell colonies were quantified, with errors bar representing the mean \(\pm\) SD from three independent experiments; ns: non-significant; ** statistically significant vs. control at \(p\) < 0.01; *** statistically significant vs. control at \(p\) < 0.001.