MLH1 silencing reprograms PI3K/AKT and JAK/STAT signaling to enhance 5-Fluorouracil sensitivity in colorectal cancer cells.

BACKGROUND/AIM: This study aimed to evaluate the anticancer efficacy of 5-Fluorouracil (5-FU) in Caco-2 human colorectal adenocarcinoma cells treated with siRNA-mediated MLH1 gene inhibition, in terms of cell viability, apoptosis, and related gene/protein expression.

MATERIALS AND METHODS: Caco-2 cells were treated with a dose of 10 µM 5-FU. Cell viability was assessed by CVDK-8 analysis, and apoptosis was determined by flow cytometry using Annexin V-FITC/PI staining. Expression levels of MLH1, CDK2, CDK4, CDK5, CDK6, PTEN, EGFR, mTOR, PI3K, AKT3, ERK, PARP1, and GAPDH genes were examined by RT-qPCR analysis. Protein expression levels were analyzed using Western blot for PI3K, p-AKT, AKT1, mTOR, MAPK-p38, Caspase-3, Bcl-2, STAT3, JAK2, MLH1, MDR1, p53, and proteins; β-Actin was used as an internal control.

RESULTS: CVDK-8 analysis showed that application of 10 µM 5-FU significantly reduced cell viability in Caco-2 cells. Flow cytometry results revealed a significant increase in both early- and late-apoptotic cell populations following 5-FU treatment.

DISCUSSION: Gene and protein expression analyses showed significant changes in signaling pathways associated with apoptosis and cell proliferation. In this study, the effects of combining MLH1 gene silencing with 5-FU on colorectal cancer cells were comprehensively evaluated using Cell viability, RT-qPCR, flow cytometry, and Western blot analyses. It was determined that the combined application significantly reduced cell viability and increased apoptosis. Molecular-level findings on gene and protein expression support this effect. The results suggest that MLH1 gene silencing may increase sensitivity to 5-FU, offering a potential combination therapy approach in colorectal cancer.