*2.5. The Effect of GCB, Terr, and Their Combination on the Colorectal Cell Lines' Growth and Proliferation*

The combination of Terr and GCB was found to show a synergistic effect in normoxia and an antagonistic effect in hypoxia. This was evident when checking the cell growth regulators and cell proliferation regulators in both normoxia and hypoxia conditions. We examined and quantified the fold changes of several genes responsible for cellular growth (AKT1, TGF-B1, HIF1-α, and PRKDC) and cellular proliferation (PCNA and RAD18) using the RT-qPCR technique.

In HCT-116, the AKT1 gene was over-expressed when the cells were treated with terrein in normoxia by 45.8-fold and were under-expressed for other treatments in the same condition. The contrary was evident in hypoxia. TGF-1β showed no change in expression for cells treated with terrein but was under-expressed when the cells were treated with other treatment conditions under normoxia. HIF1-α showed similar results to the ones seen by AKT1 under normoxic conditions. PRKDC showed no change in expression for all treatments except the combination treatment (GCB + Terr) under normoxic conditions. At hypoxia, TGF-1β, HIF1-α, and PRKDC were under-expressed in all treatments (Figures 8A,B and 9A,B).

**Figure 8.** Fold change of genes by Terr, GCB, and their combination under normoxic and hypoxic conditions. A fold change value below 0.5 indicates under-expression, above 2.0 indicates overexpression, 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**,**F**). The data are presented as mean fold change ± SD.

**Figure 9.** *Cont*.

**Figure 9.** Fold change of genes by Terr, GCB, and their combination under normoxic and hypoxic conditions. A fold change value below 0.5 indicates under-expression, above 2.0 indicates overexpression, and between 0.5 and 2.0 indicates no change in expression. HCT-116 at 24 h under normoxia and hypoxia (**A**,**B**), HT-29 at 24 h under normoxia and hypoxia (**C**,**D**), and SW620 at 24 h under normoxia and hypoxia (**E**,**F**). The data are presented as mean fold change ± SD.

On the other hand, the cellular proliferation gene, PCNA, was over-expressed when the cells were treated with terrein in normoxic conditions by 40-fold. However, it was under-expressed for the rest of the treatments. At hypoxia, the combination treatment (GCB + Terr), resulted in no change in the expression of PCNA. Yet, the remaining treatments resulted in the under-expression of the gene. The RAD18 gene was under-expressed in all treatments in both oxygen conditions except for terrein in normoxia where the gene did not change in expression (Figure 8A,B).

In HT-29, AKT1 was only over-expressed 2.62-fold after treatment with GCB. TGF-1β was under-expressed in all treatments under both oxygenation conditions. HIF1-α was under-expressed in all treatments and oxygen conditions, except after treatment with (GCB + Terr) where it showed no change in expression. PRKDC showed no change in expression in normoxia in response to all treatment conditions (Figure 8C,D). PCNA was under-expressed due to hypoxic conditions and no change in expression was observed under normoxia (Figure 9C,D).

In SW620, AKT1 showed no change in gene expression for all treatments under normoxia and hypoxia. However, TGF-1β and HIF1-α were under-expressed in all treatments under normoxia and hypoxia. The PRKDC gene was only expressed under hypoxia conditions for all treatments (Figure 8C,D). PCNA and RAD18 were under-expressed in this cell line for all treatment groups under both oxygenation conditions (Figure 9E,F).
