Characterization of a human colorectal cancer cell line resistant to 5-fluorouracil in an attempt to reverse the mechanisms of resistance

Abstract

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers locally and worldwide and among the leading causes of cancer-related deaths. Colorectal carcinogenesis follows a well defined adenoma-carcinoma sequence characterized by a series of mutations affecting key regulators such as p53, ras, and mismatch repair genes. Although standard therapies for CRC, including surgery, radiotherapy, and chemotherapy, are effective for some cases; however, the overall survival rate for CRC patients is still relatively low. Studies have focused on developing therapeutic approaches that might prolong CRC patients survival. The chemotherapeutic drug, 5-Fluorouracil (5FU), has long been used as the treatment of choice for CRC. However, patients develop resistance to this drug, stressing the need to study the mechanism underlying 5-FU resistance. In the present study, we used a human resistant colorectal cancer cell line resistant to 5- FU (HCT116-5FUR) and we compared the effects of 5-FU on HCT116 and HCT116-5FUR cell growth. We were able to characterize the inhibition of proliferation by 5-FU only in the HCT116 cells using MTT and the trypan blue dye exclusion assays. Propidium iodide DNA staining followed by flow cytometry analysis demonstrated that 5-FU failed to induce accumulation of the treated cells in the presumably apoptotic sub-G1 region in HCT116- 5FUR cells as it did in the parental counterparts. On a molecular level, 5-FU increased the expression of the tumor suppressor p53 in HCT116 cells only but not in the 5-FU-resistant ones. The treatment with 5-FU did reveal a slight difference in the baseline expression of the anti-apoptotic Bcl-2 in both cell lines. Two key enzymes involved in 5-FU metabolism and mechanism of action, dihydropyrimidine dehydrogenase and thymidylate synthase, did not seem to be involved in the resistance mechanism of HCT116-5FUR cell line. . Our study highlights a new mechanism of resistance developed by colorectal cancer cells against 5-FU. This mechanism does not involve TS or DPD enzymes as it is commonly established but the apoptotic regulators p53 and bcl-2. Further investigation is needed to elaborate the underlying mechanism of resistance.