Small molecule 2,3-DCPE induces S phase arrest by activating the ATM/ATR-Chk1-Cdc25A signaling pathway in DLD-1 colon cancer cells

Abstract

In our previous study, it was reported that 2[[3-(2,3-dichlorophenoxy)propyl]amino]ethanol (2,3-DCPE) induces apoptosis and cell cycle arrest. The current study aimed to investigate the molecular mechanism involved in 2,3-DCPE-induced S phase arrest. The results demonstrated that 2,3-DCPE upregulated phosphorylated (p-)H2A histone family member X, a biomarker of DNA damage, in the DLD-1 colon cancer cell line. Western blotting revealed that 2,3-DCPE increased the checkpoint kinase (Chk)1 (Ser317 and Ser345) level and decreased the expression of M-phase inducer phosphatase 1 (Cdc25A) in a time-dependent manner. Subsequently, the results demonstrated that the ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia and Rad3-related (ATR) inhibitors wortmannin and caffeine had no effect on the cell cycle; however, the inhibitors partially abrogated 2,3-DCPE-induced S phase arrest. Flow cytometry assays revealed that caffeine (2 mM) reduced the proportion of S phase cells from 83 to 39.6% and that wortmannin (500 nM) reduced the proportion of S phase cells from 83 to 48.2%. Furthermore, wortmannin and caffeine inhibited the 2,3-DCPE-mediated phosphorylation of Chk1 and the degradation of Cdc25A. However, these ATM/ATR inhibitors had limited effect on 2,3-DCPE-induced apoptosis. Taken together, the data of the current study indicated that 2,3-DCPE caused DNA damage in colon cancer cells and that 2,3-DCPE-induced S phase arrest was associated with the activation of the ATM/ATR-Chk1-Cdc25A pathway.

Keywords: 2,3-DCPE; ATM/ATR pathway; DNA damage; S phase arrest; colorectal cancer.

Figure 1.

Effect of 2,3-DCPE on S phase arrest and p-H2A.X expression in the DLD-1 cell line. Cells were treated with 20 μM 2,3-DCPE for 8, 10, 12, 14, 16 or 18 h. (A) Cell cycle distribution is presented for each experimental condition. (B) p-H2A.X and total H2A.X levels in the cellular extracts were determined by western blotting with specific antibodies. β-actin was used as an internal control. 2,3-DCPE, 2[[3-(2,3-dichlorophenoxy)propyl]amino]ethanol; p-, phosphorylated; H2A.X, H2A histone family member X.

Figure 2.

Expression of DNA damage response-related proteins following 2,3-DEPC treatment. (A) Expression level of p-Chk1 (Ser317 and Ser345) was markedly increased and (B) Cdc25Aexpression was markedly decreased following treatment with 20 µM 2,3-DCPE for 16, 24 and 32 h. 2,3-DCPE, 2[[3-(2,3-dichlorophenoxy)propyl]amino]ethanol; p-, phosphorylated; Chk, checkpoint kinase; Cdc25A, M-phase inducer phosphatase 1.

Figure 3.

Effect of ATM inhibitors on the 2,3-DCPE-induced cell cycle arrest in the S phase. (A) Cell cycle distribution was determined by flow cytometry assays and (B) the quantitative analysis is presented in the bar chart. Data are expressed as the mean ± standard deviation. **P<0.01 vs. the 2,3-DCPE group. (C) Western blotting was performed to investigate the p-Chk1 and Cdc25A expression after cells were treated with 2,3-DCPE and wortmannin or caffeine. 2,3-DCPE, 2[[3-(2,3-dichlorophenoxy)propyl]amino]ethanol; p-, phosphorylated; Chk, checkpoint kinase; ATM, ataxia-telangiectasia mutated; Cdc25A, M-phase inducer phosphatase 1; DMSO, dimethyl sulfoxide.

Figure 4.

Effect of wortmannin and caffeine on apoptosis. Flow cytometry assays were performed to determine the proportion of cell undergoing apoptosis following different treatment conditions. 2,3-DCPE, 2[[3-(2,3-dichlorophenoxy)propyl]amino]ethanol. DMSO, dimethyl sulfoxide.

Figure 5.

The working model of 2,3-DCPE in colon cancer cells. 2,3-DCPE induced DNA damage and activated ATM/ATR. S phase arrest was induced by the subsequent phosphorylation of Chk1 and the degradation of Cdc25A. 2,3-DCPE, 2[[3-(2,3-dichlorophenoxy)propyl]amino]ethanol; Chk, checkpoint kinase; ATM, ataxia-telangiectasia mutated; ART, ataxia-telangiectasia and Rad3-related; Cdc25A, M-phase inducer phosphatase 1.