Chrysophanol induces necrosis through the production of ROS and alteration of ATP levels in J5 human liver cancer cells

Abstract

Anthraquinone compounds have been shown to induce apoptosis in different cancer cell types. Effects of chrysophanol, an anthraquinone compound, on cancer cell death have not been well studied. The goal of this study was to examine if chrysophanol had cytotoxic effects and if such effects involved apoptosis or necrosis in J5 human liver cancer cells. Chrysophanol induced necrosis in J5 cells in a dose- and time-dependent manner. Non-apoptotic cell death was induced by chrysophanol in J5 cells and was characterized by caspase independence, delayed externalization of phosphatidylserine and plasma membrane disruption. Blockage of apoptotic induction by a general caspase inhibitor (z-VAD-fmk) failed to protect cells against chrysophanol-induced cell death. The levels of reactive oxygen species production and loss of mitochondrial membrane potential (DeltaPsi(m)) were also determined to assess the effects of chrysophanol. However, reductions in adenosine triphosphate levels and increases in lactate dehydrogenase activity indicated that chrysophanol stimulated necrotic cell death. In summary, human liver cancer cells treated with chrysophanol exhibited a cellular pattern associated with necrosis and not apoptosis.

Figure 1

Chrysophanol affected on cell morphology and percentage of viable cells in J5 human liver cancer cells. Cells were examined and photographed by phase-contrast microscopy (200x) (A) and for percentage of viable cells (B). Cells were cultured with various concentrations of chrysophanol for 24, 48 and 72 h. Each point is mean ± S.D. of three experiments. *p<0.05; **p<0.01; ***p<0.001. Significantly different from the control.

Figure 2

Chrysophanol stimulated the necrotic cell death in J5 cells. Cells were exposed to chrysophanol for indicated time periods and the necrotic cells were determined (A and B) by flow cytometry. Data represents mean ± S.D. of three experiments. *p<0.05; **p<0.01; ***p<0.001. Significantly different from the control.

Figure 3

Chrysophanol-induced non-apoptotic cell death and DNA damage in J5 human liver cancer cells. Cells were incubated with various concentrations of chrysophanol for 24 or 48 h. DNA condensation was determined by DAPI staining (A) (200x); FI: fluorescence intensity (folds of control). DNA damage was detected by Comet assay (B) (200x).

Figure 4

Effects of NAC (ROS scavenger) on chrysophanol-induced the production of ROS and viable cells in J5 cells. Cells were pre-treated with NAC then treated with 120 μM chrysophanol before cells were harvested for ROS (A) and viable cells (B) determinations. Data represent mean ± S.D. of three experiments. *p<0.05; ***p<0.001. Significantly different from the control.

Figure 5

Effects of the z-VAD-fmk on chrysophanol-induced caspase-3 activity, caspase-3 and AIF gene expressions and cell death in J5 cells. Cells were pre-treated with z-VAD-fmk for 3 h then treated with or without 120 μM chrysophanol before cells were harvested for caspase-3 activity and cell death determinations. Caspase-3 activity (A); caspase-3 and AIF mRNA expressions (B); cell viability after z-VAD-fmk treatment (C). Data shown represent the mean ± S.D. of three independent experiments. N.S.= Not significant.

Figure 6

Chrysophanol affected the levels of ATP, LDH activity and cell survival in J5 cells. Cells were at various concentrations with chrysophanol at 0, 10, 25, 50, 75, 100 and 120 μM for 24 h then the total levels of ATP (A) and LDH (B) and Viable cells after treatment with IM-54 (C) were prepared and determined. Data represent mean ± S.D. of three experiments. *p<0.05; **p<0.01; ***p<0.001. Significantly different from the control.

Figure 7

Representative Western blotting showing changes in the levels of associated proteins in necrotic cell death of J5 cells after exposure to chrysophanol. Cells were at various conditions with chrysophanol at 120 μM for 0, 6, 12, 24, 48 and 72 h then the total protein were prepared and determined. The levels of associated proteins expressions (A: Cytochrome c, Bax, AIF, Endo G and Apaf-1; B: Caspase-3, Caspase-8, Caspase-9, Caspase-12; C: SOD (Cu/Zn), SOD (Mn), Catalase and GST) were estimated by Western blotting, as described in Materials and Methods.

Figure 8

Effects of chrysophanol on cytochrome c release from mitochondria in J5 cells. Cells were incubated with chrysophanol at 120 μM for 24 h. Cells were stained with cytochrome c and FITC-labeled secondary antibodies were used (green fluorescence) and the proteins were detected by a confocal laser microscopic system. The mitochondrial was stained by Rhodamine 123 (red fluorescence). Areas of colocalization between cytochrome c, expressions and cytoplasm in the merged panels are yellow. Scale bar, 40 μm.

Figure 9

The schematic proposed signaling pathways of chrysophanol-induced necrotic cell death in J5 human liver cancer cells.