*2.1. Gemini-Cur and Cisplatin Have a Synergistic Toxic Effect on MDA-MB-231 Cells*

Breast cancer cells were treated with different concentrations of Gemini-Cur and cisplatin and the combination index (CI) was analyzed by using Chou–Talalay equation method [17]. Our results showed that Gemini-Cur has an inhibitory effect on the viability of MDA-MB-231 cells in a time- and dose-dependent manner with IC50 values of 35.06 and 23.48 μM in 48 and 72 h, respectively (Figure 2A). However, cisplatin lonely suppressed the proliferation of MDA-MB-231 cells with IC50 of 58.32 μM for 48 h. Then, serial doses of cisplatin and Gemini-Cur were employed and concentrations with CI < 1 were selected as proper ratio for further studies. The IC50 of cisplatin and Gemini-Cur was adjusted to 13 μM and 20 μM, respectively (Figure 2C). As shown in Figure 2D, the combination

index is <1, pretending synergism between Gemini-Cur and cisplatin in 20 and 13 μM concentrations, respectively.

**Figure 2.** Effect of Gemini curumin and cisplatin on the viability of MDA-MB-231 cells in vitro. MDA-MB-231 cells were grown and treated with Gemini-Cur (**A**), Cisplatin (**B**) and Gemini-Cur/Cis (**C**,**D**). 15 and 20 μM of Gemini-Cur combined with serial concentrations of cisplatin. Data represent mean ± standard deviation of three independent experiments. CI: Combination index.
