## 3 Results  
### 5-FU Inhibits Cell Proliferation and Causes Apoptosis in SCC4 Cells  
To evaluate the impact of 5-FU on the SCC4 cell line, various concentrations of 5-FU (50, 100, 200, and 400 \(\upmu\)M) were administered for 24 h. The MTS/PMS assay indicated that the viability of SCC4 cells was significantly reduced through 5-FU treatment at all concentrations tested (\(p\) < 0.05) (Figure 1A). Microscopic observations were consistent with the results of the cell viability assay. As shown in Figure 1B, the control cells had a basic density level, while the 5-FU treatment reduced the number and size of cancer cell clusters. Increasing the concentration of 5-FU appeared to correspond with a progressive decrease in cell density in the SCC4 cells. Additionally, 5-FU treatment inhibited colony formation in a concentration-dependent manner (Figure 1C), indicating an anti-proliferative effect of 5-FU. To further elucidate the anti-cancer mechanism of action of 5-FU, the apoptotic markers were assessed. Western blot analysis revealed an increased expression of both cleaved PARP and caspase-3 in SCC4 cells following 5-FU treatment (Figure 1D). The relative densitometric graphs also demonstrated a significant dose-dependent increase in the cleavage of PARP and caspase-3 induced by 5-FU (\(p\) < 0.05) (Figure 1E). These findings therefore suggest that the major anticancer effects of 5-FU in SCC4 cells are anti-proliferation and pro-apoptosis.  
Figure 1: 5-FU inhibits cell proliferation and causes apoptosis in SCC4 cells. The cells were treated with increasing concentrations of 5-FU (50, 100, 200, and 400 \(\upmu\)M) for 24 h. (**A**) The cell viability wasmeasured using MTS/PMS assay. (**B**) The cell density was observed under a light microscope (10\(\times\)). (**C**) Representative images of colony formation assay and their quantitative analysis. The (**D**) Western blot analysis showing the protein expression of PARP, cleaved PARP, pro-caspase-3, and caspase-3. The (**E**) relative densitometric graphs showing PARP and caspase-3 cleavage. * \(p<\) 0.05, as compared to the control. All experiments were independently performed at least three times.  
Refametinib Sensitizes SCC4 Cells to 5-FU Treatment for Suppressing Cell Proliferation and Promoting Apoptosis  
Figure 2A,B demonstrates that treatment with the lowest tested concentration of 5-FU (50 uM) resulted in increased expression of phospho-ERK1/2 over time, while ERK1/2 expression remained unchanged in SCC4 cells, indicating activation of the oncogenic pathway through ERK1/2. To enhance the effectiveness of 5-FU treatment, we investigated the combination of 5-FU at various concentrations (50, 100, 200, and 400 uM) with Refametinib (50 nM), a selective MEK1/2 inhibitor that is orally bioavailable. Our results showed that the combination treatment improved the anticancer effects of 5-FU on SCC4 cells compared to the vehicle (5-FU plus DMSO). This was evidenced by a significant decrease in cell viability (Figure 2C), an increase in apoptotic markers (Figure 2D), a reduction in cell density (Figure 2E), and a decrease in colony formation (Figure 2F). Additionally, it is known that the mitochondrial membrane potential decreases during apoptosis [29]. As demonstrated in Figure 2G,H, the mitochondrial membrane potential in SCC4 cells was significantly lower for the combination treatment than for the vehicle. Overall, our findings suggest that the combination of 5-FU with Refametinib has a synergistic effect on inhibiting cell proliferation and promoting apoptosis in SCC4 cells.