Diosgenin enhances 5-fluorouracil sensitivity in drug-resistant colorectal cancer cells via inhibition of ATR/Chk1-mediated DNA damage response.

BACKGROUND AND OBJECTIVE: Drug resistance to 5-fluorouracil (5-FU) is one of the major challenges in the treatment of colorectal cancer, which is largely associated with the activation of DNA damage response (DDR) pathways, especially the ATR-CHK1 axis, and increased activity of drug efflux pumps. The aim of this study was to investigate the effect of diosgenin, a natural anticancer compound, alone and in combination with an ATR inhibitor (VE-822), on sensitizing 5-FU-resistant colorectal cancer cells and to elucidate the molecular mechanisms involved in this process. METHODS: In this in vitro study, human colorectal cancer cell line Caco-2 and a 5-FU-resistant line (Caco-2/5-FU) were used. Cell viability and proliferation were assessed by MTT and trypan blue assays, and the ability of long-term cell proliferation was examined by colony formation assay. The expression of genes associated with drug resistance (MDR1/P-gp, ABCG2, ABCC1, YB-1), DNA damage response pathway (ATR, CHK1, H2AX) and apoptosis (Bax, Bcl-2, Caspase-3, PARP-1) was measured by qRT-PCR. The level of DNA damage was assessed by examining the γ-H2AX protein with Western blot. The function of the P-gp pump was measured by the rhodamine 123 accumulation assay and the level of apoptosis was measured by the DNA fragment detection ELISA kit. The cells were treated with diosgenin, VE-822, 5-FU and their double and triple combinations. RESULTS: The results showed that the IC50 of 5-FU in resistant Caco-2/5-FU cells was significantly higher than in sensitive cells, indicating the development of a drug resistance phenotype. Diosgenin alone was able to significantly reduce the viability of both sensitive and resistant cells, although resistant cells showed a lower response. Inhibition of ATR with VE-822 significantly reduced cell viability, and the combination of diosgenin with VE-822 in the presence of 5-FU produced a stronger combined effect and significantly reduced the IC50. Trypan blue and colony formation assays showed a severe reduction in cell survival and long-term proliferation in the combination treatment. Molecularly, the combination treatment significantly inhibited the expression and function of P-gp and reduced the expression of MDR1, ABCG2, ABCC1 and YB-1 genes. The expression of ATR and CHK1, as well as γ-H2AX protein expression levels was also significantly reduced. The Bax/Bcl-2 ratio was increased, and the expression of Caspase-3 and PARP-1 was significantly increased. ELISA results also confirmed a significant increase in apoptosis, especially in the triple treatment of diosgenin+VE-822+5-FU. CONCLUSION: The findings of this study suggest that diosgenin may contribute to reducing 5-FU resistance by affecting ATR/CHK1-associated DNA damage response mechanisms, weakening DNA repair mechanisms, and suppressing drug efflux pumps. The combination of diosgenin with an ATR inhibitor may have a strong combined effect in inducing DNA damage, inhibiting drug resistance, and promoting apoptosis-associated molecular changes. This combination approach may represent a potential chemosensitizing approach for further investigation in colorectal cancer; however, additional validation in diverse models and in vivo studies is required.