*2.4. The Effect of Terr, GCB, and Their Combination on the Cell Cycle Distribution of Colorectal Cell Lines*

In the HCT-116 cells, Terr induced a significant G2/M phase arrest and increased the cell population after 24 h from 27.8 ± 1.1% to 38 ± 1.15%. This effect was reversed by GCB where the cell population in the S phase dropped significantly to 32.61 ± 0.5%. As a result, there was no significant difference between the combination treatment (GCB + Terr) and GCB and Terr alone. On the other hand, after 48 h of exposure, there was no significant difference observed between all treatments in terms of the S phase population (Figure 6A,B).

**Figure 6.** Effect of Terr, GCB, and their combination on the cell cycle distribution after 24 h and 48 h. The cell cycle distribution was determined using DNA cytometry analysis and different cell phases were plotted as the percentage of total events. HCT-116 after 24 h and 48 h (**A**,**B**), HT-29 after 24 h and 48 h (**C**,**D**), and SW620 after 24 h and 48 h (**E**,**F**).

In the HT-29 cells, Terr did not induce significant changes at the G0/G1 phase after 24 h of exposure. However, after further exposure (48 h), Terr induced a significant reduction in the cell population at the G0/G1 phase compared to the control untreated cells from 54.81 ± 0.7% to 42.78 ± 1.61%. Terr induced significant G2/M phase arrest after 24 h (from 22.84 ± 0.57% to 26.5 ± 1.99%) and after 48 h (from 22.42 ± 0.99% to 34.42 ± 0.84%). After 24 h of exposure, the combination treatment (GCB + Terr) induced a decrease in the G0/G1cell population when compared to GCB alone from 67.3 ± 1.81% to 54.32 ± 1.49%, where the combination treatment (GCB + Terr) increased the S phase cell population compared to GCB (from 18.48 ± 0.09% to 31.39 ± 0.92%). Similar results were observed after 48 h of exposure (Figure 6C,D).

In the SW620 cells, GCB increases in the G0/G1 phase cell population after 24 h of exposure from 48.04 ± 1.28% to 77.43 ± 2.57%. This effect was also seen for the combination treatment (GCB + Terr), where the cell population in the G0/G1 phase increased significantly to 52.77% when compared to the control untreated cells. After 48 h of exposure, the combination treatment induced significant G2/M phase arrest and increased the S phase cell population from 18.96 ± 0.86% to 27.88 ± 1.14% when compared to the control untreated cells. It also increased the G0/G1 phase cell population from 53.01 ± 2.72% to 60.16 ± 1.08% compared to GCB alone (Figure 6E,F).

In addition, we examined cell cycle regulatory genes by RT-qPCR (CCND1, CDK4, and MCM7). In HCT-116, only CDK4 was over-expressed due to treatment with terrein

by 6.7-fold under normoxic conditions. CCND1 was under-expressed in response to all treatments under both oxygen and hypoxia conditions. Similar results were observed with the MCM7 gene (Figure 7A,B). In HT-29 and SW620, all genes tested for cell cycle regulation were under-expressed in response to all treatments under both normoxia and hypoxia (Figure 7C–E).

**Figure 7.** Fold change of cell cycle regulatory genes due to treatment with Terr, GCB, and their combination under normoxic and hypoxic conditions. A fold change value below 0.5 indicates under-expression, above 2.0 indicates over-expression, and between 0.5 and 2.0 indicates no change in expression. HCT-116 under normoxia and hypoxia (**A**,**B**), HT-29 under normoxia and hypoxia (**C**,**D**), and SW620 under normoxia and hypoxia (**E**). The data are presented as mean fold change ± SD.
