Synergistic Anticancer Effects of DSF and Cu

RKO and Ht29 cell lines were purchased from the American Type Culture Collection. DSF was procured from Macklin. In previous studies, it is demonstrated that the anticancer function of DSF is dependent on the presence of a Cu²⁺ ion (Skrott et al., 2017 (link); Huang et al., 2021 (link)). In our study, we establish that 10 μM Cu (Copper (II) chloride dihydrate, Solarbio) is nontoxic in vitro and in vivo. In vitro cytotoxicity analysis was performed according to manufacturer’s instructions: RKO and Ht29 cells (7000 cells/well) were cultured overnight in 96-well plates; exposed to DSF (0.25 μM), Cu (10 μM), or a combination of both for 24, 48, and 96 h; and then analyzed for cell viability using the Cell Counting Kit-8 (KeyGEN BioTech, Nanjing, China). In parallel, CRC cells were planted into a six-well plate containing 5 ml medium at a density of 500 cells/well and exposed to DSF, Cu, or a combination of both for 96 h. Then, the cells were rinsed with 0.01% PBS, fixed with 4% paraformaldehyde for 15 min, and stained with crystal violet. The number of clones was counted using Image J software.