Fig. 4.

Treatment with a demethylating agent restores sensitivity to 5-FU toxicity in a MMR-deficient CRC cell line. (A) The top schematic summarizes the treatment plan and results from the in-vitro experiments. Four CRC cell lines - HCT116 and HCT116 + 2 (both MMR-deficient due to mutation in MLH1), HCT116 + 3 (MMR-proficient following restoration of MLH1 gene with chromosome 3 transfer in HCT116 cells) and SW48 (MMR-deficient due to hypermethylation of MLH1) - were cultivated in growth medium or exposed to 5-AZA for 24 h.⁶⁶ Culture medium was exchanged and cells were allowed to form colonies over a period of 10-12 days. During this time, the cells were continuously exposed to increasing concentrations of 5-FU. Cells were washed, fixed, stained and colonies counted. MLH1 methylation status, its expression (mRNA and protein), and sensitivity to 5-FU sensitivity were monitored in each experiment. (B) A representative graph (in the left panel) depicts the mean ± S.D. of three different experiments for the colony forming assay from CRC cell lines exposed to different doses of 5-FU (1 µM, 2.5 µM, and 5 µM). Cells were allowed to form colonies for 10-12 days. The means and S.D. of three independently performed experiments are shown. As indicated, only HCT116 + 3 cells (MMR proficient cells) are sensitive to 5-FU, while all MMR-deficient cells demonstrate more colonies, indicating resistance to the chemotherapeutic drug. However, when SW48 cells were pretreated with 5 µM 5-Aza for 24 h before 5-FU exposure, MMR activity is restored, and they behave similarly to the MMR-proficient HCT116 + 3 cells (right panel).

MMR, mismatch repair; CRC, colorectal cancers.