## 3 Discussion  
One of the mechanisms by which DSF plays an antitumor role is that it can combine with metal ions to form complexes [9; 10]. In this study, we further confirmed the Cu-dependent cytotoxic effect of DSF on NPC cells. Some research indicated that DSF/Cu achieved the purpose of cancer treatment by inhibiting the growth of ALDH-positive cancer stem cells [28; 29]. However, our study showed that DSF/Cu induced NPC cell apoptosis and necrosis by an ALDH-independent method. Recently, [PERSON] et al. indicated that DSF/Cu can activate the ROS-JNK pathway in acute myeloid leukemia (AML) cells [14]. Consistent with this study, we found that DSF/Cu increase cellular ROS levels and activate the apoptosis-related MAPK pathway. However, only a ROS scavenger, but not a JNK or p38 inhibitor, can abolish the antitumor activity induced by DSF/Cu, suggesting that ROS played a dominant role in DSF/Cu-induced apoptosis and necrosis of NPC cells. Ferroptosis has been indicated as playing a crucial role in suppressing tumor growth [21]. Activation of the MAPK pathway contributes to ferroptotic cancer cell death [29; 30]. Our observations, for the first time, demonstrated that DSF/Cu was an inducer for both ferroptosis and apoptosis in NPC cells. [PERSON] et al., reported that p53-mediated transcriptional activation of SAT1 is critical for ROS-induced ferroptosis [24]. What is more, SAT1 can increase the expression of ALOX15, a lipoxygenase that catalyzes the peroxidation of arachidonic acid, which leads to an increase in lipid ROS levels on the cell membrane, eventually resulting in ferroptosis [24]. In accord with these studies, our results showed that DSF/Cu can induce the expression of p53 protein, and the mRNA levels of SAT1 as well as ALOX15 were also increased in NPC cells. Moreover, both cellular ROS levels and lipid ROS levels were increased under the DSF/Cu treatment. These data demonstrate that ferroptosis played an important role in DSF/Cu-induced cell death. Although ferroptosis is distinct from other forms of cell death like apoptosis, we have detected that DSF/Cu can obviously promote NPC cell apoptosis; the use of the ROS scavenger NAC did not completely restore the morphology and survival of NPC cells, suggesting that the synergistic activation of multiple signature pathways contributed to the strong DSF/Cu-induced antitumor activity, and lipid ROS-induced ferroptosis played the major role in DSF/Cu-induced destruction. It is well known that cancer-associated fibroblasts (CAFs) are crucial to cancer development, progression, and metastasis. CAFs can not only potentially induce chemoresistance acquisition in tumor cells, but can also directly contribute to the suppression of antitumor T-cell responses [31; 32; 33]. Currently, anticancer therapies are suggested to target both cancer cells and the stromal compartment to improve patient outcomes [34]. In this study, we are excited to find that DSF/Cu can induce apoptosis and inhibit the expression of \(\alpha\)-SMA in CAFs. Our NPC xenograft study clearly indicated that, based on the destruction of both CAFs and tumor cells by DSF/Cu, large solid tumors can be turned into necrotic tumor tissues with a \(\alpha\)-SMA-negative CAFs. Overexpression of \(\alpha\)-SMA-positive CAFs has been reported to predict poor prognosis in NPC [35]. Our study, for the first time, demonstrated that DSF/Cu plays strong antitumor roles by targeting both tumor cells and CAFs. Moreover, the combination therapy of antitumor drugs and DSF/Cu showed an effective improvement in their anti-NPC activity. Previously, two studies indicated that DSF/Cu induces NPC cell apoptosis by increasing chloride channel-3 protein expression, or by inducing ROS production and decreasing NF-KB-p65 expression [36; 37]. These studies, together with our findings, indicate that DSF/Cu induces anti-NPC activity through a joint action of multiple pathways. Surprisingly, we also found that DSF/Cu showed a strong inhibitory effect on nasopharyngeal epithelial cells NP69-SV40T in vitro.