Combined Treatment of 5-Fluorouracil and Delta-Tocotrienol Induce of Apoptosis and Autophagy in Colorectal Cancer Cells

Abstract

Background: 5-fluorouracil (5-FU) has been a first choice chemotherapeutic drug used to treat colorectal cancer. However, its therapeutic outcome often be limited by high-dose toxicities and drug resistance development. Hence, the current research advocates combined application of delta-tocotrienol (δ-T3), as a natural chemosensitizer, with 5-FU as a novel treatment approach for colorectal cancer. Aim: To investigate the combinatorial anticancer effects of δ-T3 and 5-FU on colorectal cancer cell lines. Methods: Cell viability assay was performed to investigate the individual and combined effects of δ-T3 (0.1-100 µM) and 5-FU (0.1-100 µM) on HCT116, HCC2998, Caco-2 and SW48 colorectal cancer cells at 72 h. Subsequently, the synergistic combination were evaluated for the effects on clonogenicity, apoptosis and autophagy. Morphologic assessment was conducted by bright-field and fluorescence microscopy. The protein expression profiles of programmed cell death and survival markers were determined by Western blotting. Results: Combined treatment of subeffective dose of δ-T3 significantly lowered the IC50 of 5-FU in Caco-2 and SW48 colorectal adenocarcinomas for 16-fold and fourfold respectively, signifying a chemosensitising effect. Clonogenic survival assay showed that the combined treatment profoundly hampered the cell survival as compared with the individual single treatments. Apoptosis was induced by the combined treatment as confirmed by flow cytometry. Interestingly, both apoptotic and autophagic morphologies were observed, including nuclear condensation, cell shrinkage, cytoplasmic vacuolation and extension. The presence of autophagy was further confirmed by increase LC3A/B ratio and high volume of acidic vesicular organelles. The combined treatment were found to upregulate prodeath proteins such as Bax, caspase-8, caspase-3, PARP and downregulate prosurvival proteins such as cIAP, XIAP and survivin. Conclusion: The enhanced cell death induced by 5-FU and δ-T3 combined treatment involved both apoptosis and autophagy, suggesting a novel and effective treatment approach for colorectal cancer.