Analysis of inhibition of topoisomerase IIalpha and cancer cell proliferation by ingenolEZ

Abstract

We previously reported that many ingenol compounds derived from Euphorbia kansui exhibit topoisomerase inhibitory activity and/or inhibitory activity of cell proliferation. The inhibitory effects of 20-O-(2'E,4'Z-decadienoyl) ingenol and 3-O-(2'E,4'Z-decadienoyl)-ingenol among these compounds on topoisomerase II activity and on the cell proliferative activity and arrest phase of the cell cycle were studied using a mouse breast cancer (MMT) cell line. Although 20-O-ingenolEZ exerted inhibitory effects on both topoisomerase II activity and cell proliferative activity, 3-O-ingenolEZ exerted inhibitory activity on neither. The 20-O-ingenolEZ-induced cell arrest of MMT-cell proliferation led to a cell cycle arrest in the G2/M phase. Topoisomerase II inhibition can be divided into the poison and catalytic inhibitor types. A checkpoint mechanism is activated when cells are treated with these topoisomerase II inhibitors. Poison-type inhibition occurs via induction of the DNA damage checkpoint and the catalytic-type inhibition occurs via induction of the DNA-decatenation checkpoint, suggestive of distinct checkpoint reactions. 20-O-ingenolEZ inhibited topoisomerase IIalpha activity through inhibition of ATPase, and induced DNA-decatenation checkpoint without signaling for phosphorylation of H2AX.

Figure 1

Structures of the diterpene compounds. (a) 20‐O‐(2′E,4′Z‐decadienoyl)ingenol; (b) 3‐O‐(2′E,4′Z‐decadienoyl)‐ingenol.

Figure 2

Topoisomerase II mediated supercoiled pBR322 relaxation. To determine inhibition of relaxation of the supercoils, pBR322 DNA was incubated with topoisomerase IIα. Each reaction mixture (20 μL) contained 30 ng pBR322 plasmid DNA, 0.2 U of human topoisomerase IIα, and 4, 40, or 200 μm 20‐O‐ingenolEZ or 3‐O‐ingenolEZ. After incubation for 1 h at 37°C, the mixture was loaded onto a gel well for agarose gel electrophoresis. Lanes: 1, no enzyme; 2, plus enzyme, 3, plus 20‐O‐ingenolEZ; 4, plus 3‐O‐ingenolEZ; 4 μm of lane 4, no sample. R, relaxed DNA; S, supercoiled DNA.

Figure 3

Kinetoplast DNA (kDNA) decatenation assay. To demonstrate inhibition of decatenation, kDNA was incubated with nuclear protein in mouse breast cancer (MMT) cells. Each mixture (20 μL) contained 0.4 μg of kDNA, nuclear protein, and 40, 100, or 200 μm 20‐O‐ingenolEZ. After incubation for 1 h at 37°C, the mixture was loaded onto a gel well for agarose gel electrophoresis. Lanes: 1, no enzyme; 2, plus enzyme; 3, plus 40 μm 20‐O‐ingenolEZ; 4, plus 100 μm 20‐O‐ingenolEZ; 5, plus 200 μm 20‐O‐ingenolEZ. C, catenated kDNA; D, decatenated kDNA.

Figure 4

Effects on ATPase activity of topoisomerase II by 20‐O‐ingenolEZ. DNA‐dependent ATPase activity was measured in a reaction mixture containing 20 mm Tris, 40 mm NaCl, 4 mm MgAc₂, 0.5 mm EDTA, 5 mm ATP, 200 ng pBR322, and two units of human topoisomerase IIα (pH 7.5) at various 20‐O‐ingenolEZ concentrations. The free phosphate measure was carried out with a malachite green reagent. Relative ATPase activity is shown by normalizing against activity under conditions without inhibitors.

Figure 5

Effects of ingenolEZ on cell proliferative activity. Mouse breast cancer (MMT) cells were cultured in microplates at 37°C for 3 days in the absence or presence of 20, 40, 60, and 100 μm 20‐O‐ingenolEZ or 3‐O‐ingenolEZ. Relative cell growth was determined by MTT assay. The cell growth in untreated MMT cells was set as 100%, and the cell growth of the MMT cells treated with 20, 40, 60, and 100 μm 20‐O‐ingenolEZ or 3‐O‐ingenolEZ was expressed relative to the level in untreated MMT cells (100%). The expressions were assessed in triplicate and the data are shown as means ± SD.

Figure 6

Cell cycle analysis. 3T3‐fibrobrast cells were cultured at 37°C for 24 h with 20 μm 20‐O‐ingenolEZ. Mouse breast cancer (MMT) cells were cultured at 37°C for 12, 24, and 48 h with 20‐O‐ingenolEZ at 10 μg/mL. The cells stained with propidium iodide were subjected to flow‐cytometric analysis (a). (b) Shows the results of (a) with % the cell distributions in each G1, S, and G2/M phase of the cell cycle in untreated cells and at 12, 24, and 48 h after 20‐O‐ingenolEZ treatment.

Figure 7

Influence of 20‐O‐ingenolEZ on phosphorylation of H2AX. For immunoblotting of γ‐H2AX in the mouse breast cancer (MMT) cells, cells were cultured in the presence of 20 μm 20‐O‐ingenolEZ or 0.9 μm adriamycin for 24 h. The nuclear protein fraction (20 μg) was resolved by SDS‐PAGE, followed by Western blot analysis and chemiluminescence detection. γ‐H2AX was detected using the specific antibody against γH2AX. Lane 1, control; lane 2, 20‐O‐ingenolEZ; lane 3, adriamycin.

Figure 8

Effects of 20‐O‐ingenolEZ on mouse breast cancer (MMT) cell apoptosis. MMT cells were treated at 37°C with 0.9 μm adriamycin, 20 μm 20‐O‐ingenol EZ, or control for 24 and 48 h. Apoptosis of the cells was detected by DAPI staining and the arrows in the photograph indicate examples of apoptotic cells after 24 h treatment with adriamycin (a). The percentage of apoptotic MMT cells at different time‐points after adriamycin and 20‐O‐ingenolEZ treatments are shown (b).