HY-1 induces G(2)/M cell cycle arrest in human colon cancer cells through the ATR-Chk1-Cdc25C and Weel pathways

Abstract

The novel aroylthiourea analogue of podophyllotoxin HY-1 (4β-[benzoyl-thioureido]-4-deoxypodophyllotoxin) was synthesized in our laboratory with the aim of developing multitargeted DNA topoisomerase II inhibitors. The compound showed significant antiproliferative effects on seven cancer cell lines and induced G2 /M phase arrest in HCT116 cells. Moreover, HY-1 showed a potent inhibitory effect on topoisomerase II-mediated kinetoplast DNA decatenation in a dose-dependent manner. Our results showed that cdc2 phosphorylation and decreased cdc2 kinase acitivity through the ATR-Chk1-Cdc25C and Weel pathways were the central mechanisms for G2 /M phase arrest in human colon cancer cells.

Figure 1

Podophyllotoxin and 4β‐(benzoyl‐thioureido)‐4‐deoxypodophyllotoxin (HY‐1).

Figure 2

(a) Effects of 4β‐(benzoyl‐thioureido)‐4‐deoxypodophyllotoxin (HY‐1) and camptothecin (CPT) on human topoisomerase I‐mediated decatenation activity of pBR322. RLX, relaxed pBR322; SC, supercoiled pBR322. (b) Effects of HY‐1 and etoposide (VP‐16) on human topoisomerase II‐mediated decatenation activity of kinetoplast DNA (kDNA). Minicircles indicate no drug. DNA samples were separated by electrophoresis on agarose gels containing ethidium bromide (1 mg/mL). Gels were photographed under UV light.

Figure 3

(a) Antiproliferation effects of 4β‐(benzoyl‐thioureido)‐4‐deoxypodophyllotoxin (HY‐1) and etoposide (VP‐16) on different human cancer cells by MTT assay. Cells were treated with different concentrations of compounds for 72 h. The IC ₅₀ values were calculated using the mean results of three separate experiments, expressed as the mean ± SD (μM). (b) Antiproliferation effects of HY‐1 and VP‐16 on human colon carcinoma HCT116 cells by BrdU ELISA. All data represent the mean ± SD from three independent experiments. *P < 0.05 versus blank control; **P < 0.01 versus blank control; #P < 0.05 versus positive control; ##P < 0.01 versus positive control (n = 5).

Figure 4

(a) Effects of compound 4β‐(benzoyl‐thioureido)‐4‐deoxypodophyllotoxin (HY‐1) on the cell cycle of human colon carcinoma HCT‐116 cells. Cells were incubated with 0, 1.25, 2.5, 5, or 10 μM HY‐1 for 24 h and stained with propidium iodide (PI). Their DNA content was analyzed by fluorescence flow cytometry. (b) Effect of HY‐1 on G₂/M cell cycle regulator proteins. HCT116 cells treated with 0, 1.25, 2.5, 5, or 10 μM HY‐1 for 24 h. The protein levels of Cdc2, p‐Cdc2, Weel, P‐Cdc25C, Chk1, Chk2, and ATR were assessed by Western blotting.

Figure 5

(a) Morphological study of human colon carcinoma HCT‐116 cells treated with 4β‐(benzoyl‐thioureido)‐4‐deoxypodophyllotoxin (HY‐1) for 72 h, assessed by Hoechst 33342 staining assay. Cells were treated with 0, 0.1, or 0.2 μM HY‐1 for 72 h, then visualized using a fluorescence microscope. The cells became rounded and contained fragmented nuclei, typical morphological features of apoptosis. (b) HY‐1‐induced HCT116 cell apoptosis was detected by annexin V/propidium iodide (PI). Cells were treated with various concentrations of HY‐1 for 72 h, then incubated with annexin V and PI dyes. Apoptotic cells were analyzed by flow cytometry. (c) Determination of the rates of apoptosis induced by HY‐1. All data represent the mean ± SD from three independent experiments.

Figure 6

Schematic explanation of the contribution of cell cycle regulatory proteins to the development of G₂/M phase arrest in human colon carcinoma HCT116 cells treated with 4β‐(benzoyl‐thioureido)‐4‐deoxypodophyllotoxin (HY‐1).