## 2 Results
### DSF/Cu Induces Both Apoptosis and Necrosis in NPC Cells by an ALDH-Independent Method  
A colony-forming assay was further performed to verify the antiproliferative effect of DSF/Cu in NPC cells. We used 0.2, 0.6 or 1 \(\upmu\)M DSF combined with 1 \(\upmu\)M Cu to treat 5-8F cells for 10 days. The number of colony-forming cells of the 0.2 \(\upmu\)M DSF/Cu group was dramatically decreased compared to the control group (\(p<\) 0.001). What is more, with a high dose of DSF (\(>\)0.6 \(\upmu\)M), 5-8F cells almost stopped growing in vitro (Figure 2A). Next, FACS analysis showed that DSF/Cu (1 \(\upmu\)M/1 \(\upmu\)M) induced both apoptosis and necrosis in NPC cells in a time-dependent manner. The percentage of apoptotic cells is represented in the upper right and lower right quadrants, and the necrotic cells are represented in the upper left and the upper right quadrant. 5-8F and CNE2 cells that were treated with DSF/Cu underwent apoptosis starting at 2 or 4 h and reached a high apoptosis rate (about 50%) and a high necrosis rate (about 61%) after 10 h post-incubation (Figure 2B). Furthermore, Western blot analysis revealed that DSF/Cu induced the expression of cleaved-PARP1 and cleaved-caspase3 in 5-8F and promoted caspase3 and PARP1 cleavage within 6 h (Figure 2C). Figure 1: Disulfiram (DSF) reduces the viability of nasopharyngeal carcinoma cells in a Cu-dependent manner. (**A**) The NPC cell lines 5-8F, CNE2 and the nonmalignant nasopharyngeal epithelial cells NP69-SV40T were exposed to the indicated concentration of DSF or with 1 \(\upmu\)M Cu for 24 h, after which inhibition effects were determined by MTT assay (above) and LDH assay (below). (**B**) 5-8F, CNE2 and NP69-SV40T were exposed to DSF/Cu (1 \(\upmu\)M/1 \(\upmu\)M) for different lengths of time, after which inhibition effects were determined by MTT and LDH assays. (**C**) 5-8F cells were exposed to DSF/Cu (1 \(\upmu\)M/1 \(\upmu\)M) for different lengths of time and were cultured for 12 or 24 h after DSF/Cu withdrawal, after which inhibition effects were determined by MTT assay. DMSO solvent was used as a control. Data are shown as means \(\pm\) SD. *** \(p<\) 0.001 vs. after dosing group, \(n=3\). In addition, qRT-PCR and Western blot analysis showed that the expression of ALDH1A1 was absent, whereas the expression of ALDH2 was strong or moderate in all four NPC cell lines (Figure 3A,B). Moreover, ALDH1A1 but not ALDH2 was detected in NP69, and there was no significant change in ALDH1A1 expression after DSF/Cu treatment (Figure 3C). Next, three specific ALDH2 siRNAs were designed to silence the ALDH2 gene expression, and a scrambled siRNA was used as negative control (NC). As shown in Figure 3D,E, all the three siALDH2 sequences were effective in silencing ALDH2 expression in 5-8F and CNE2 cells. Furthermore, there was no obvious difference in the cell viability between DSF/Cu-treated siALDH2-1 (ALDH2 low) and NC (ALDH2 high) NPC cells (Figure 3F). Finally, the MTT assay showed that DSF/Cu had a similar killing effect on both ALDH1A1\({}^{+}\) A549/CDDP (cisplatin) and ALDH2\({}^{+}\) NPC cells (Figure 3F). These data suggest that DSF/Cu could induce apoptotic and necrotic cell death by an ALDH-independent method. Figure 2: DSF/Cu promotes the apoptosis and necrosis of nasopharyngeal carcinoma cells. (**A**) Representative images and quantification of colony formation assay in 6-well plates. 5-8F cells were incubated for 10 days and the medium containing the drug was replaced once. DMSO solvent containing 1 \(\upmu\)M Cu was used as a control. Data are shown as means \(\pm\) SD. ***\(p<0.001\) vs. control group, \(n=3\). (**B**) Flow cytometry with Annexin V/PI double staining proved that DSF/Cu could significantly increase Annexin V\({}^{+}\)/PI\({}^{+}\) cells, and promote the apoptosis and necrosis of 5-8F and CNE2. Data are shown as means \(\pm\) SD. ***\(p<0.001\) vs. control group, \(n=3\).