Abstract

5-Fluorouracil (5-FU) is a chemotherapeutic medication commonly used to treat colorectal cancer (CRC); however, the drug-associated adverse effects and toxicity have greatly affected its clinical use. Exploring another therapeutic strategy that lowers the toxicity of 5-FU while having a synergistic effect against CRC is thus a viable option. Diosmetin, a natural flavonoid, has been shown to inhibit the proliferation of many cancer cells, including CRC cells. This study aims to investigate the synergistic effect of diosmetin and 5-FU on HCT116 and HT29 colorectal cancer cells and to explore the apoptotic activity of this combination. The MTT assay was used to assess the viability of cells treated with monotherapy and combination therapy. The combination index (CI) and dose reduction index (DRI) were calculated using the CompuSyn software (version 1.0). The SynergyFinder 2.0 software was used to calculate the synergy score, while the Combenefit software was employed to perform isobologram analysis and synergism determination. The AO/PI double staining technique was used to detect the apoptotic characteristics of cells, whereas the flow cytometry technique was used to investigate the apoptosis induction and cell cycle arrest in cells. The combination of 5-FU and diosmetin showed a synergistic effect in HCT116 cells with a mean CI value of 0.66 ± 0.4, and an additive effect in HT29 cells with a CI value of 1.0 ± 0.2. The DRI of 5-FU in HCT116 cells was three times lower in the combination therapy compared to monotherapy of 5-FU. AO/PI microscopic examination and Annexin V analysis revealed that the combination-treated cells had more apoptotic cells than the monotherapy-treated cells, which was activated mainly through intrinsic apoptosis pathway. HCT116 cell death was confirmed by mitotic arrest in the G2/M phase. Our findings suggest that 5-FU/diosmetin combination exhibits synergistic effect against HCT116 cancer cells, and potentially reduces the unfavorable adverse effect of 5-FU while enhancing the anticancer efficacy by inducing apoptosis and interrupting mitosis. Further research studies are needed to validate the combination’s anti-tumorigenic activities in a xenograft animal model.

Highlights

  • Colorectal cancer (CRC) is the third most prevalent cancer worldwide and the second most deadly cancer

  • HCT116 and HT29 cells were seeded at a density of 5 × 103 cell/well and exposed to a two-fold serial dilution of either 5-FU ranging from 100–0.78 μg/mL (768.8–6.0 μM) or diosmetin ranging from 100–0.78 μg/mL (333.0–2.6 μM)

  • Similar to HCT116 cells, the effect of monotherapy and combination therapy was investigated in HT26 colon cancer cells

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Summary

Introduction

Colorectal cancer (CRC) is the third most prevalent cancer worldwide and the second most deadly cancer. In 2020, GLOBOCAN estimated 1,148,515 new CRC cases and. 576,858 colorectal cancer deaths [1]. There have been substantial advances in the chemotherapeutic agents against CRC, severe adverse effects and toxicity are the major clinical problems. The synergistic effect of existing chemotherapeutic drugs in combination with natural and safe bioactive agents is an effective approach that has been considered in previous studies [3,4,5,6,7]. There are several chemotherapeutic drugs such as doxorubicin, cisplatin, methotrexate, 5-fluorouracil (5-FU), and paclitaxel that are used in combination therapy [8]. The current combination therapies include FOLFOX regimen

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