The Synergistic Effect of FU5 and Trifolium pratense Extract on the Expression Levels of KRAS and LKB1 Genes in the LS180 Colorectal Cancer Cell Line

Introduction: Colorectal cancer (CC) is recognized as the third most common cancer in men and the second most common cancer in women. 5-Fluorouracil (FU5) is one of the effective chemotherapeutic agents used for the treatment of CC; however, it is associated with numerous side effects. Trifolium pratense possesses strong antioxidant properties that inhibit cancer cell proliferation and protect cells from damage. Therefore, the present study aimed to investigate the effects of FU5 and T.pratense extract on the expression levels of KRAS and LKB1 genes in the LS180 CC cell line. Methods: T.pratense extract was prepared using the Soxhlet extraction method. After obtaining the LS180 cell line from the Pasteur Institute of Iran, cancer cells were treated with different concentrations of the extract (50–800 µg/mL) and various doses of FU5 (5–80 µg/mL) for 24, 48, and 72 hours. Cell viability was assessed using the MTT assay. The expression levels of KRAS and LKB1 genes were determined by real-time PCR. Results: In the present study, the lowest cell viability was observed following the combined treatment with FU5 (5 µg/mL) and T.pratense extract (50 µg/mL) at 24 hours, reaching 3.17%. The expression level of the LKB1 gene increased by 21.41% in response to the combined treatment compared to the control, while the expression of the KRAS gene decreased approximately 20-fold following treatment with T.pratense extract. Conclusion: In this study, an increase in LKB1 gene expression and a decrease in KRAS gene expression were observed in cells treated with FU5 and T.pratense extract compared to the control group. These changes in LKB1 and KRAS gene expression may be attributed to the activation of AMP-activated protein kinase pathways, induction of apoptosis, or modulation of inflammatory pathways, potentially due to the presence of antioxidant compounds such as isoflavones in T.pratense.