Radiosensitization of Irinotecan is Attributed to G2/M Phase Arrest, Followed by Enhanced Apoptosis Probably Through the ATM/Chk/Cdc25C/Cdc2 Pathway in P53-mutant Colon Cancer Cells

Abstract

Irinotecan, an analog of camptothecin, the inhibitor of Topoisomerase I, is currently used to treat metastatic colorectal cancer. Camptothecin derivatives were demonstrated to have radiosensitization effects on several kinds of cancer cells. However, few studies have been published in terms of colorectal cancer cell lines. In this study, we try to examine the radiosentization effects of irinotecan on p53 mutant colon cancer lines HT29, SW620 and explore the potential mechanisms. HT29 (radiation-resistant) cells and SW620 (radiation-sensitive) cells were chosen for the following experiments. First, 24h IC50s of irinotecan as a single agent were obtained through drug cytotoxicity tests. Concentrations below 10% IC50 were considered to be combined with radiation. Second, cells were treated with irinotecan (24h) followed by radiation at a dosage gradient, and radiosentization effects of irinotecan were examined through clonogenic assays. Next, cells were divided into four arms: control arm, irinotecan arm, radiation arm and the combined arm. Cell growth curves were obtained through CCK-8 assays. Foci of γH2AX were illustrated through immunofluorescence straining. Cell cycle and apoptosis were examined using Flow Cytometry. Expression of proteins related to DNA damage and repair system and cell cycle were examined through Western Blot. The 24h IC50s of irinotecan were 39.84μg/ml (HT29 95% CI 38.27-41.48) and 96.86μg/ml (SW620 95%CI 89.04-105.4), thus concentrations below 4μg/ml were chosen for the experiments. Clonogenic assays showed that irinotecan had radiosentization effects on HT29 and SW620 cells, and the SERs increased with the increase of concentration (SER= 1.41 for HT29 cells, SER=1.87 for SW620 cells, with irinotecan at 2μg/ml). Compared with the control arm, irinotecan arm and radiation arm, the combined arm had the slowest cell growing rate and the most obvious foci of γH2AX. The combined treatment could result in the most significant G2/M phase arrest and last for more than 24h, followed by the most significant increase of apoptosis. And results of western blot indicated that the expression of proteins related to DNA damage and repair system (Ser1981p-ATM, Ser345p-Chk1, Thr68p-Chk2, γ-H2AX)，cell cycle (Tyr15p-Cdc2, Cyclin B1) increased the most in the combined arm. In addition, we found that the expression of Ser216p-Cdc25C also increased the most in the combined arm, which indicating that irinotecan radiosentized the p53-mutant HT29 and SW620 cells probably through ATM/Chk/Cdc25C/Cdc2 pathway. Irinotecan has radiosensitization effects on HT29 and SW620 cells and the SER increases with the increase of concentration. It attributes to the activation of DNA damage and repair system, leading to significant G2/M phase arrest followed by enhanced apoptosis. And the process is probably through ATM/Chk/Cdc25C/Cdc2 pathway in p53-mutant colon cancer cells.