*2.3. Gemini-Cur/Cis Modulates Cell Cycle Distribution in MDA-MB-231 Cells*

Cell cycle distribution was studied by propidium iodide staining in flow cytometry. Our data showed that cell cycle distribution is modulated in all treated cells either in singular or combined forms. As Figure 4 shows, the percentage of SubG1 cells as a hallmark of apoptosis is significantly increased in Gemini-Cur/Cis group compared to curcumin or cisplatin groups (*p* < 0.001). Accordingly, the number of live cells (G1 phase) was decrease in combination treatment when compared to the Gemini-Cue and Cis groups (*p* < 0.001). There was a statistically significant interaction between the effects of Cis and Gemini-Cur on interest in sub G1 (*p* = 0.0001).

**Figure 3.** Morphological observation of apoptosis in Gemini-Cur and cisplatin treated cells by Hoechst staining in 48 h. Control shows a uniform exposure of live cells to stain. The color intensity and the number of cells indicating apoptotic features are significantly increased in treated groups. Arrows indicate apoptotic cells with cell shrinkage, nuclear fragmentation and DNA condensation. Magnification: 200×.

**Figure 4.** Analysis of cell cycle by flow cytometry in cancer cells treated with 13 μM cisplatin and 20 μM Gemini-Cur for 24 & 48 h. (**A**) Histograms for MDA-MB-231 cells. (**B**) The percentage of cells in sub-G1 and G1 phases. The data clearly illustrate an increase in the number of SubG1 cells in combination treatment compared to either void cisplatin or Gemini-Cur. Accordingly, the number of live cells is decrease in Gemini-Cur/Cis group versus Gemini-Cur and Cis groups. Data represent mean ± standard deviation of three independent experiments. (\*\*\* *p* < 0.001).
