RESEARCH ARTICLE Metformin and thymoquinone co-treatment enhance 5-fluorouracil cytotoxicity by suppressing the PI3K/mTOR/ HIF1α pathway and increasing oxidative stress in colon cancer cells Wesam F. Farrash 1 | Akhmed Aslam 1 | Riyad Almaimani 2 | Faisal Minshawi 1 | Hussain Almasmoum 1 | Aiman Alsaegh 1 | Mohammad S. Iqbal 1 | Aisha Tabassum 1 | Mohamed E. Elzubier 2 | Mahmoud Z. El-Readi 2,3 | Amani A. Mahbub 1 | Shakir Idris 1 | Bassem Refaat 1 1 Laboratory Medicine Department, Faculty of Applied Medical Sciences, Umm Al-Qura University, Makkah, Saudi Arabia 2 Department of Biochemistry, Faculty of Medicine, Umm Al-Qura University, Makkah, Saudi Arabia 3 Biochemistry Department, Faculty of Pharmacy, Al-Azhar University, Assuit, Egypt Correspondence Bassem Refaat, Laboratory Medicine Department, Faculty of Applied Medical Sciences, Umm Al-Qura University, Holy Makkah, PO Box 7607, Saudi Arabia. Email: barefaat@uqu.edu.sa; bassem. refaat@yahoo.co.uk Funding information The Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia, Grant/Award Number: IFP22UQU4320045DSR101 Abstract This study investigated the chemotherapeutic effects of 5-fluorouracil (5-FU), metformin (Met), and/or thymoquinone (TQ) single/dual/triple therapies in the HT29, SW480 and SW620 colon cancer (CRC) cell lines. Cell cycle/ apoptosis were measured by flow cytometry. The gene and protein expression of apoptosis (PCNA/survivin/BAX/Cytochrome-C/Caspase-3) and cell cycle (CCND1/CCND3/p21/p27) molecules, the PI3K/mTOR/HIF1α oncogenic pathway, and glycolysis regulatory enzymes were measured by quantitative- PCR and Western blot. Markers of oxidative stress were also measured by col- orimetric assays. Although all treatments induced anti-cancer effects related to cell cycle arrest and apoptosis, the triple therapy showed the highest pro- apoptotic actions that coincided with the lowest expression of CCND1/ CCND3/PCNA/survivin and the maximal increases in p21/p27/BAX/Cyto- chrome-C/Caspase-3 in all cell lines. The triple therapy also revealed the best suppression of the PI3K/mTOR/HIF1α pathway by increasing its endogenous inhibitors (PTEN/AMPKα) in all cell lines. Moreover, the lowest expression of lactate dehydrogenase and pyruvate dehydrogenase kinase-1 with the highest expression of pyruvate dehydrogenase were seen with the triple therapy, which also showed the highest increases in oxidative stress markers (ROS/RNS/ MDA/protein carbonyl groups) alongside the lowest antioxidant levels (GSH/CAT) in all cell lines. In conclusion, this is the first study to reveal enhanced anti-cancer effects for metformin/thymoquinone in CRC that were superior to all monotherapies and the other dual therapies. However, the triple Wesam F. Farrash and Bassem Refaat contributed equally to this work. Received: 10 January 2023 Accepted: 4 March 2023 DOI: 10.1002/biof.1947 BioFactors. 2023;1–18. wileyonlinelibrary.com/journal/biof © 2023 International Union of Biochemistry and Molecular Biology. 1