Oridonin induces apoptosis and senescence by increasing hydrogen peroxide and glutathione depletion in colorectal cancer cells

Abstract

We recently demonstrated that oridonin could induce apoptosis and senescence of colon cancer cells in vitro and in vivo. However, the underlying mechanism remains unknown. In this study, the involvement of reactive oxygen species in oridonin-induced cell death and senescence was investigated in colon adenocarcinoma-derived SW1116 cells. Oridonin increased intracellular hydrogen peroxide levels and reduced the glutathione content in a dose-dependent manner. N-acetylcysteine, a reactive oxygen species scavenger, not only blocked the oridonin-induced increase in hydrogen peroxide and glutathione depletion, but also blocked apoptosis and senescence induced by oridonin, as evidenced by the decrease in Annexin V and senescence-associated β-galactosidase- positive cells and the inhibition of oridonin-induced upregulation of p53 and p16 and downregulation of c-Myc. Moreover, exogenous catalase could inhibit the increase in hydrogen peroxide and apoptosis induced by oridonin, but not the glutathione depletion and senescence. Furthermore, thioredoxin reductase (TrxR) activity was reduced by oridonin in vitro and in cells, which may cause the increase in hydrogen peroxide. In conclusion, the increase in hydrogen peroxide and glutathione depletion account for oridonin-induced apoptosis and senescence in colorectal cancer cells, and TrxR inhibition is involved in this process. Given the importance of TrxR as a novel cancer target in colon cancer, oridonin would be a promising clinical candidate. The mechanism of oridonin-induced inhibition of TrxR warrants further investigation.

Figure 1

Effects of oridonin (0, 6.25, 12.5, 25, 50 or 100 μm) on intracellular hydrogen peroxide, superoxide anion and glutathione production in SW1116 cells. SW1116 cells were treated with the indicated concentrations of oridonin for 2 h. Intracellular levels of (A) hydrogen peroxide, (B) superoxide anions, and (C) glutathione production were determined by Flow Cytometry. The graphs show the mean fluorescence levels of (A) DCF, (B) DHE and (C) CMF. Each experiment was repeated three times. ^*P<0.05 when compared with the control group from three independent experiments.

Figure 2

Effects of a ROS scavenger on intracellular ROS and GSH production, oridonin-induced apoptosis and senescence in oridonin-treated SW1116 cells. SW1116 cells were treated with the ROS scavenger NAC and/or oridonin for 2 h. Intracellular (A) hydrogen peroxide, (B) superoxide anion and (C) GSH levels were determined by FACS. The graphs show the mean fluorescence levels of (A) DCF, (B) DHE, and (C) CMF. (D–E) SW1116 cells were treated with NAC and/or oridonin for 12 or 48 h. (D) Annexin-positive cells were measured by flow cytometry. (E) Senescent cells were quantified by senescence-associated β-galactosidase activity analysis (x100). Each experiment was repeated three times. ^*P<0.05 when compared with the cells untreated or treated with oridonin plus NAC from three independent experiments.

Figure 3

Effects of N-acetylcysteine on oridonin-induced apoptotic and senescence-related proteins in SW1116 cells. SW1116 cells were treated with NAC and/or oridonin for (A) 12 h (100 μM) and (B) 48 h (50 μM). Aliquots of protein extracts (40 μg) were immunoblotted with the indicated antibodies.

Figure 4

Effects of exogenous catalase on intracellular ROS production, glutathione depletion, oridonin-induced apoptosis and senescence in oridonin-treated SW1116 cells. SW1116 cells were treated with catalase and/or oridonin for 2 h. Intracellular (A) hydrogen peroxide, (B) superoxide anion, and (C) GSH levels were determined by FACS, respectively. The graphs show the mean fluorescence levels of (A) DCF, (B) DHE and (C) CMF, respectively. Cells were treated with catalase and/or oridonin (50 μM for 48 h or 100 μM for 12 h). (D) Annexin V-positive cells were measured with FACS. (E) Senescent cells were determined by senescence-associated β-galactosidase activity analysis (x100) . ^*P<0.05 compared with the cells treated with oridonin alone.

Figure 5

Inhibitory activity of oridonin on TrxR. (A) Recombinant rat TrxR (0.05 units) was incubated with various concentrations of oridonin for 1 h, and TrxR activity was measured by the dithionitrobenzene reduction assay. Error bars represent the standard deviations of duplicate experiments. (B) SW1116 cells were treated with different concentrations of oridonin for 2 h, and TrxR activity was determined. Error bars represent the standard deviations of duplicate experiments.