## 2 Results
### Antitumor Activity of DSF/Cu against NPC In Vivo  
To explore the anti-NPC activity of DSF/Cu in vivo, the anti-tumor activity was evaluated in a mouse 5-8F xenograft model; the schematic outline for in vivo drug treatment is shown in Figure 8A. Although there was no significant difference between the combination of CDDP and DSF/Cu or DSF/Cu alone in vitro (Figure S6), the tumor volume in the DSF/Cu, CDDP and CDDP/DSF/Cu groups was suppressed by 60.1%, 63.3% and 77.7%, respectively, after two weeks administration, compared to control group (Figure 8B,C). In addition, the use of CDDP inevitably led to adverse effects of weight loss in mice (\(p<0.01\)), while there was no significant weight change in the DSF/Cu group compared with the control group (Figure 8D). Figure 6: DSF/Cu induces apoptosis and increases the cellular ROS levels in human skin fibroblasts (HSF). (**A**) HSF were exposed to the indicated concentration of DSF or with 1 uM Cu for 24 h, after which inhibition effects were determined by MTT and LDH assays. (**B**) HSF were exposed to DSF/Cu (1 uM/1 uM) for different lengths of time, after which inhibition effects were determined by MTT and LDH assays. (**C**) Representative images of HSF treated with DSF/Cu (1 uM/1 uM) for the indicated time. Scale bar: 200 uM. (**D**) HSF were cultured with DSF/Cu (1 uM/1 uM), and the \(\alpha\)-SMA and FAP-\(\alpha\) expressions were detected by Western blotting. (**E**) Flow cytometry with Annexin V/PI double staining proved that DSF/Cu could significantly increase Annexin V*/PI+ cells, promoting the apoptosis and necrosis of HSF. Data are shown as means \(\pm\) SD. *** \(p<0.001\) vs. control group, \(n=3\). (**F**) The apoptosis rate in HSF, 5-8F and CNE2 after treatment with DSF/Cu for 4 h and 10 h. Data are shown as means \(\pm\) SD. *** \(p<0.001\), \(n=3\). (**G**) HSF were treated with DSF/Cu (1 uM/1 uM) for 4 h, and cellular ROS production was assessed by flow cytometry using DCFH-DA staining. Quantification of cellular ROS levels is shown as means \(\pm\) SD. *** \(p<0.001\), \(n=3\). In addition, the control group formed normal solid tumors, but the other three groups formed necrotic tumor tissues (Figure 8B). In the DSF/Cu and CDDP groups, only a small number of normally formed tumor cells were found to be present at the tissue margins by hematoxylin and eosin (H&E) assay, and most tumor tissues exhibited a large area of necrotic center (about 80-90%) and obvious nucleus pyknosis (Figure 8E). Furthermore, the histopathological evaluation illustrated that there were more necrotic areas in the tumor tissues of the CDDP/DSF/Cu group (about 95%) than in the CDDP and DSF/Cu alone group, and there were few normal as well as actively dividing tumor cells present, which suggests that CDDP/DSF/Cu synergistically enhanced anti-NPC activity in vivo. IHC for ki67 marker was used to further explore cell proliferation, and the result showed that the number of ki67 positive tumor cells was significantly increased in the control group compared to the other three treatment groups (Figure 8F). Figure 7: The inhibitory effect of DSF/Cu on TGF-\(\beta\)1 activated 3T3 cells was more obvious than that of normal 3T3 cells. (**A**) \(\alpha\)-SMA protein was detected by Western blot in 3T3 cells. TGF-\(\beta\)1 (20 ng/mL) was incubated for 48 h. Data are shown as means \(\pm\) SD. * \(p<0.05\), *** \(p<0.001\), \(n=3\). (**B**) 3T3 cells were exposed to DSF/Cu (1 uM/1 uM) for different lengths of time, after which, inhibition effects were determined by MTT assay. TGF-\(\beta\)1 (20 ng/mL) was pretreated for 48 h. Data are shown as means \(\pm\) SD. * \(p<0.05\), *** \(p<0.001\), \(n=3\). (**C**) Representative images of 3T3 treated with DSF/Cu (1 uM/1 uM) for 24 h. TGF-\(\beta\)1 (20 ng/mL) was pretreated for 48 h. Scale bar: 200 uM. (**D**) The expression of \(\alpha\)-SMA was detected by Western blotting in 3T3 after incubation with DSF/Cu for 12 h. TGF-\(\beta\)1 (20 ng/mL) was pretreated for 48 h. Data are shown as means \(\pm\) SD. *** \(p<0.001\), \(n=3\).