Enhanced in vitro tumoricidal effects of 5-Fluorouracil, thymoquinone, and active vitamin D 3 triple therapy against colon cancer cells by attenuating the PI3K/AKT/mTOR pathway

This study measured the effects of 5-Fluorouracil (5-FU), calcitriol (VD 3 ), and/or thymoquinone (TQ) single/dual/triple therapies on cell cycle progression, apoptosis, inhibition of the PI3K/AKT/mTOR pathway, and oxidative stress against colorectal cancer (CRC). The HT29, SW480 and SW620 cell lines were treated with 5-FU (50 μM), VD 3 (25 μM), and TQ (75 μM), alone or combined for 12 h, prior to cell cycle/apoptosis analyses. TQ monotherapy had greater anticancer effects to active VD 3 or 5-FU, revealing higher expression of p21/p27/PTEN/BAX/Cyto-C/Casp-3 and increased levels of total glutathione, with inhibitions in CCND1/CCND3/BCL-2 and PI3K/AKT/mTOR molecules, alongside higher rates of apoptosis in HT29, SW480 and SW620 cells ( P  < 0.005 for all markers). Additionally, all combination protocols revealed enhanced modulations of the PI3K/PTEN/Akt/mTOR pathway, higher expression of p21/p27/PTEN/BAX/Cyto-C/Casp-3, and better anti-oxidant effects, than the monotherapies. Although TQ/5-FU and TQ/VD 3 co-therapies were better relative to the VD 3 /5-FU regimen, the best tumoricidal effects were observed with triple therapy in the HT29 and SW480 cell lines, possibly by boosted attenuations of the PI3K/AKT/mTOR oncogenic pathway. In contrast, TQ single treatment was more effective than the triple therapy regimen in metastatic SW620 cells, suggesting that this protocol would be more useful therapeutically in late-stage CRC. In conclusion, this study is the first to demonstrated enhanced anti-tumorigenic effects for VD 3, TQ, and 5-FU triple therapy against CRC cells and could represent the best strategy for treating early stages of malignancy, whereas TQ monotherapy could be a better approach for treating metastatic forms of the disease.