5-Fluorouracil sensitizes colorectal tumor cells towards double stranded DNA breaks by interfering with homologous recombination repair

Abstract

Malignant tumors of the rectum are treated by neoadjuvant radiochemotherapy. This involves a combination of 5-fluorouracil (5-FU) and double stranded DNA-break (DSB)-inducing radiotherapy. Here we explored how 5-FU cooperates with DSB-induction to achieve sustainable DNA damage in colorectal cancer (CRC) cells. After DSB induction by neocarzinostatin, phosphorylated histone 2AX (γ-H2AX) rapidly accumulated but then largely vanished within a few hours. In contrast, when CRC cells were pre-treated with 5-FU, gammaH2AX remained for at least 24 hours. GFP-reporter assays revealed that 5-FU decreases the efficiency of homologous recombination (HR) repair. However, 5-FU did not prevent the initial steps of HR repair, such as the accumulation of RPA and Rad51 at nuclear foci. Thus, we propose that 5-FU interferes with the continuation of HR repair, e. g. the synthesis of new DNA strands. Two key mediators of HR, Rad51 and BRCA2, were found upregulated in CRC biopsies as compared to normal mucosa. Inhibition of HR by targeting Rad51 enhanced DNA damage upon DSB-inducing treatment, outlining an alternative way of enhancing therapeutic efficacy. Taken together, our results strongly suggest that interfering with HR represents a key mechanism to enhance the efficacy when treating CRC with DNA-damaging therapy.

XRCC2-Deficient Cells are Highly Sensitive to 5-Fluorouracil in Colorectal Cancer

“…Recently, several reports have described 5-FU-induced DNA lesions and their repair in CRC cells [45]. XRCC2 plays an important role in the repair of DSBs based in the HRs and thereby decreases the sensitivity to 5-FU.…”

“…Double-strand breaks (DSBs) are the most deleterious DNA lesions that occur in cells after treatment with chemotherapy and/or radiation [4, 5]. The repair of these DSBs largely determines the outcome of cancer therapy [6, 7].…”

Heterogeneity of colorectal cancer cell positions as a cell viability biometrie

“…Human colorectal cell lines HCT-116, SW-620 and SW-480 were cultured on 75 cm 2 cell culture flask based on the incubation process reported in [11]. In this process, first the chamber slides were coated with matrigel basement membrane matrix with a density of 2 mg/ml and then kept into an incubator at 37 • C for one-hour period to polymerize the matrigel.…”

Quantification of cell apoptosis for in-vitro colorectal cancer cell cultures based on morphological features

“…Human colorectal cancer cells of HCT-116 were cultured on 75 cm 2 cell culture flask using a well-known incubation process described in [9]. To prepare the experimental setup, chamber slides were coated with matrigel basement membrane matrix with a density of 2 mg/ml.…”