## 4 Discussion  
Chemotherapy is one of the most mainstreams methods used for treating OSCC, which is known to kill cancer cells mainly through apoptosis [1; 32]. The burst of poly(APD-ribosyl)ation of nuclear proteins following PARP cleavage catalyzed by caspase-3 is required for apoptosis [33]. By using a human OSCC cell line (SCC4), this study suggested that 5-FU, a cytotoxic chemotherapeutic agent, can induce OSCC cell death through apoptosis followed by caspase-3-mediated PARP cleavage, which is consistent with a previous research  
Figure 3: Refametinib mitigates 5-FU-induced PD-L1 expression in SCC4 cells. (**A–D**) The cells were treated with increasing concentrations of 5-FU (50, 100, 200, and 400 \(\upmu\)M) for 24 h. The (**A**) Western blot analysis and (**B**) relative densitometric graph showing the total level of PD-L1 expression. The (**C**) flow cytometry graph and (**D**) relative fluorescence intensity showing the surface expression of PD-L1. (**E–J**) The cells were pre-incubated with (**E–G**) Refametinib (**R**e; 50 nM) or (**H–J**) U0126 (U0 or U; 50 nM) for 1 h and then treated with increasing concentrations of 5-FU (100, 200, and 400 \(\upmu\)M) for 24 h. The (**E,H**) Western blot analyses showing the total level of PD-L1 expression. The (**F,J**) flow cytometry graph and (**G,J**) relative fluorescence intensity showing the surface expression of PD-L1. * \(p<0.05\), as compared to the control or as compared between the two indicated groups. NS: not significant; Veh: vehicle; 5 + R: 5-FU + Refametinib; 5 + U: 5-FU + U0126; Re: Refametinib; U0 or U: U0126. All experiments were independently performed at least three times. reporting the caspase-dependent chemocytotoxicity of 5-FU in the cultured oral cancer cells [7]. Our results also demonstrated the anti-proliferative effect of 5-FU treatment in SCC4 cell line. Furthermore, the dose-response experiments indicated that a higher drug concentration was correlated with lower cell viability, less colony formation, and higher levels of apoptotic markers. However, it is important to notice a therapeutic tightrope regarding the balance between the maximum cancer cell death with the tolerable toxicity. Therefore, the combination of anticancer drugs may help to achieve a better therapeutic efficacy than the use of a single agent without exceeding the maximum tolerable dose of each. Moreover, drug combinations may provide some benefits to prevent the development of drug resistance [5; 34]. The MAPK pathway comprises different signaling cascades that transduce diverse extracellular signals to the nucleus upon the phosphorylation activation, thereby regulating the expression of a vast array of molecules involved in cell survival, proliferation, apoptosis, invasion, metastasis, and many cellular processes. ERK1/2, a critical component of MAPK pathway, plays a crucial role in promoting cell survival and proliferation [10]. In this research, ERK1/2 phosphorylation was significantly enhanced through 5-FU treatment in SCC4 cell line. A previous study reported the overexpression of ERK1/2 in the biopsy specimens of OSCC, which was associated with the proliferation in OSCC [11]. Thus, ERK1/2 activation through 5-FU may promote cancer cell proliferation, indicating the paradoxically pro-survival effect of 5-FU that counteracts its principal cytotoxic activity and compromises its efficacy. Logically, pharmacologic blockage of the pathway with MEK inhibitors may inhibit 5-FU-induced ERK activation and downstream effects. To elucidate this assumption, a combination of 5-FU with an orally bioavailable selective MEK1/2 inhibitor, Refametinib, was used to treat the SCC4 cell line. Our results found that Refametinib could sensitize SCC4 cells to 5-FU treatment for synergistically inhibiting cell proliferation and promoting apoptosis. Similarly, preclinical evidences have demonstrated that MEK/ERK pathway inhibition increased the sensitivity to 5-FU in different colorectal cancer cell lines and murine colorectal tumor xenograft models [35; 36; 37].