Oridonin-induced apoptosis in SW620 human colorectal adenocarcinoma cells

Abstract

Oridonin, a diterpenoid isolated from Rabdosia rubescens (Hemsl.) Hara, inhibited the growth of human tumor cell lines SW620 (colon), MCF-7 (breast) and K562 (bone marrow), and induced significant levels of apoptosis in SW620. Morphological changes indicative of cell apoptosis were observed after the cells were exposed to oridonin for 24 h. Growth inhibition was associated with G1 phase arrest, and with time- and dose-dependent increases in caspase-3 activity. We therefore conclude that oridonin inhibits the proliferation of SW620 cells by induction of apoptosis via the activation of caspase-3. Our data suggest that oridonin may have significant potential as an anti-colorectal adenocarcinoma agent.

Figure 1

Chemical structure of oridonin (C₂₀H₂₈O₆).

Figure 2

Effect of oridonin on the growth of different tissue cell lines. Cells (from left to right: SW620, K562, MCF7) were treated for 72 h with different concentrations of oridonin (2, 5, 10, 25 and 50 μmol/l) dissolved in DMSO. Negative controls were treated with DMSO only and positive controls were treated with 5-fluorouracil (5-Fu) (1 μg/ml). Values are the mean ± SD of triplicate determinations. ^*P<0.001 (Student’s t-test).

Figure 3

Photomicrographs of SW620 cells exposed to oridonin. SW620 cells were treated for 24 h with (A) DMSO (negative control), (B) 10 μmol/l oridonin and (C) 25 μmol/l orodonin. (D) Apoptotic cells following exposure to 10 μmol/l oridonin (magnification, ×400). The arrow shows apoptotic bodies. Cell images were captured using an Olympus CKX41 inverted/phase-contrast microscope (magnification, ×200).

Figure 4

FACS analysis of annexin V (AV) and propidium iodide (PI) binding. SW620 cells were treated for 24 h with (A) DMSO (negative control), (B) 10 μmol/l oridonin, (C) 25 mol/l orodonin and (D) 50 μmol/l orodonin as described in Materials and methods. PI and AV-FITC fluorescence was measured by flow cytometry and analyzed (dot-plots). Viable (AV⁻/PI⁻), early apoptotic (AV⁺/PI⁻), apoptotic (AV⁺/PI⁺) and residual damaged (AV⁻/PI⁺) cells are shown in the respective quadrants.

Figure 5

Inhibition of cell cycle progression in SW620 cells by oridonin. Cell cycle analysis of SW620 cells (A) without oridonin treatment, (B) with exposure to 25 μmol/l oridonin for 24 h and (C) exposure to 50 μmol/l oridonin for 24 h. Cells were fixed with ethanol, stained with propidium iodide, and the cell cycle distribution analyzed by flow cytometry. Data from 10,000 cells were collected for each data file. The percentage of cells in G1, S, G2 phases and apoptotic cells were calculated using Multicycle software (D) and are indicated on the right upper side. ^*Significant difference between controls and oridonin-treated cells.

Figure 6

Effect of oridonin on caspase-3 activity in SW620 cells. SW620 cells were treated with different concentrations of oridonin (2, 5 and 10 μmol/l) and caspase-3 activity was monitored. There are three negative controls: the blank well included an assay buffer only, the oridonin 0 well had DMSO-treated cell extract and the inhibitor well had an inhibitor-treated cell extract added. The positive control was purified caspase-3.