Exploring potential additive effects of 5-fluorouracil, thymoquinone, and coenzyme Q10 triple therapy on colon cancer cells in relation to glycolysis and redox status modulation.

Abstract

Background: To investigate the anticancer effects of 5-Fluorouracil (5-FU), thymoquinone (TQ), and/or coenzyme Q10 (CQ10), alone and combined, in HT29, SW480, and SW620 human colorectal cancer (CRC) cell lines.

Methods: Cell cycle progression and apoptosis were assessed by flow cytometry. Gene and protein expression of molecules involved in apoptosis (BLC2, survivin, BAX, Cytochrome-C, and Caspase-3), cell cycle (CCND1, CCND3, p21, and p27), the PI3K/AKT/mTOR/HIF1α oncogenic pathway, and glycolysis (LDHA, PDH, and PDHK1) were also analysed by quantitative RT-PCR and Western blot. Oxidative stress markers (ROS/RNS, MDA, and Protein carbonyl groups) and antioxidants (GSH and CAT) were quantified by ELISA.

Results: All treatments resulted in anticancer effects depicted by cell cycle arrest and apoptosis, with TQ demonstrating greater efficacy than CQ10, both with and without 5-FU. However, 5-FU/TQ/CQ10 triple therapy exhibited the most potent pro-apoptotic activity in all cell lines, portrayed by the lowest levels of oncogenes (CCND1, CCND3, BCL2, and survivin) and the highest upregulation of tumour suppressors (p21, p27, BAX, Cytochrome-C, and Caspase-3). The triple therapy also showed the strongest suppression of the PI3K/AKT/mTOR/HIF1α pathway, with a concurrent increase in its endogenous inhibitors (PTEN and AMPKα) in all cell lines used. Additionally, the triple therapy favoured glucose oxidation by upregulating PDH, while decreasing LDHA and PDHK1 enzymes. The triple therapy also displayed the most significant decline in antioxidant levels and the highest increases in oxidative stress markers.

Conclusions: This study is the first to demonstrate the superior anticancer effects of TQ compared to CQ10, with and without 5-FU, in CRC treatment. Moreover, this is the first report to reveal improved anticancer effects of the 5-FU/TQ/CQ10 triple therapy, potentially through promoting oxidative phosphorylation, attenuating the PI3K/AKT/mTOR/HIF1α pathway, and increasing oxidative stress-induced apoptosis.