*4.11. Statistical Analysis*

Data are presented as mean ± SD. Analysis of variance (ANOVA) with Tukey's Honest Significance post hoc test was carried out to test for significance using SPSS® for Windows, Version 17.0.0. *p* < 0.05 was used as the cut-off value for significance.

#### **5. Conclusions**

In conclusion, while combining terrein with gemcitabine did not improve gemcitabine's resistance fraction, it did however improve its cytotoxic effect against HCT-116 and SW620 cells. The current work focused on HCT-116 as it was the cell line of interest due to the opposite effect of the combination treatment (GCB + Terr) on the cells when treated under normoxic versus hypoxic conditions. Expression of certain genes was affected due to the variable treatment action, more specifically BCL2, Beclin-1, CDK4, and AKT1. This urged us to further investigate the metabolic profile of the HCT-116 cell line after treatment with terrein, gemcitabine, and their combination, and promising results supported our findings. A difference between the metabolites found under each oxygen condition was found, and this could explain the synergistic effect in normoxia versus the antagonistic effect in hypoxia.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/md21050271/s1, Figure S1: The effect of terrein on the cytotoxicity of GCB (A) in normoxia and (B) in hypoxia in HCT-116 cell line. The cells were exposed to serial dilution of terrein (-), GCB (•) or Terr/GCB combination (-) for 72 h. The cell viability was determined using SRB assay; Figure S2: The effect of terrein on the cytotoxicity of GCB (A) in normoxia and (B) in hypoxia in HT-29 cell line. The cells were exposed to serial dilution of terrein (-), GCB (•) or Terr/GCB combination (-) for 72 h. The cell viability was determined using SRB assay; Figure S3: The effect of terrein on the cytotoxicity of GCB (A) in normoxia and (B) in hypoxia in SW620 cell line. The cells were exposed to serial dilution of terrein (-), GCB (•) or Terr/GCB combination (-) for 72 h. The cell viability was determined using SRB assay.

**Author Contributions:** Conceptualization, A.M.A.-A., S.S.E. and N.S.A.; methodology, R.K.A., D.Y.A.S., S.S.E. and A.E.G.; software, R.K.A., D.Y.A.S., N.S.A. and A.E.G.; validation, A.M.A.-A., S.S.E. and N.S.A.; formal analysis, R.K.A. and A.M.A.-A.; investigation, R.K.A., D.Y.A.S., S.S.E. and N.S.A.; resources, A.M.A.-A., S.S.E. and N.S.A.; data curation, R.K.A., D.Y.A.S. and N.S.A.; writing—original draft preparation, R.K.A.; writing—review and editing, A.M.A.-A., S.S.E. and N.S.A.; supervision, A.M.A.-A.; project administration, A.M.A.-A.; funding acquisition, A.M.A.-A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research including the APC was funded by Gulf Medical University, Ajman, UAE, grant number GMU/COP/GR/209-10/004.

**Institutional Review Board Statement:** Ethical review and approval were not applicable for studies not involving humans or animals.

**Data Availability Statement:** All supporting raw data are available upon request.

**Acknowledgments:** The authors would like to thank Thumbay Research Institute for Precision Medicine for providing all the technical support required during the study.

**Conflicts of Interest:** The authors declare no conflict of interest.
