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, 10 (4), 467

β-Carotene-induced Apoptosis Is Mediated With Loss of Ku Proteins in Gastric Cancer AGS Cells

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β-Carotene-induced Apoptosis Is Mediated With Loss of Ku Proteins in Gastric Cancer AGS Cells

Yoona Park et al. Genes Nutr.

Abstract

High dietary intakes and high blood levels of β-carotene are associated with a decreased incidence of various cancers. The anticancer effect of β-carotene is related to its pro-oxidant activity. DNA repair Ku proteins, as a heterodimer of Ku70 and Ku80, play a crucial role in DNA double-strand break repair. Reductions in Ku70/80 contribute to apoptosis. Previously, we showed that reactive oxygen species (ROS) activate caspase-3 which induces degradation of Ku proteins. In the present study, we investigated the mechanism of β-carotene-induced apoptosis of gastric cancer AGS cells by determining cell viability, DNA fragmentation, apoptotic indices (increases in cytochrome c and Bax, decrease in Bcl-2), ROS levels, mitochondrial membrane potential, caspase-3 activity, Ku70/80 levels, and Ku-DNA-binding activity of the cells treated with or without antioxidant N-acetyl cysteine and caspase-3 inhibitor z-DEVED-fmk. As a result, β-carotene induced apoptosis (decrease in cell viability, increases in DNA fragmentation and apoptotic indices) and caspase-3 activation, but decreased Ku70/80 levels and Ku-DNA-binding activity. β-Carotene-induced alterations (increase in caspase-3 activity, decrease in Ku proteins) and apoptosis were inhibited by N-acetyl cysteine and z-DEVED-fmk. Increment of intracellular and mitochondrial ROS levels and loss of mitochondrial membrane potential were suppressed by N-acetyl cysteine, but not by z-DEVED-fmk in β-carotene-treated cells. Therefore, β-carotene-induced increases in ROS and caspase-3 activity may lead to reduction of Ku70/80 levels, which results in apoptosis in gastric cancer cells. Loss of Ku proteins might be the underlying mechanism for β-carotene-induced apoptosis in gastric cancer cells.

Figures

Fig. 1
Fig. 1
β-Carotene induces apoptosis in AGS cells. The cells were cultured with β-carotene (final concentration of 100 μM) for 12 h (a, b) or for various periods (0, 4, 8, 12 h) (c). a Cell viability was determined by counting the number of viable cells. The viability of cells treated without β-carotene (none) was set at 100 %. The results are expressed as the mean ± SEM of four separate experiments. *p < 0.05 versus cells treated without β-carotene (none). b DNA fragmentation was detected by measuring the amount of oligonucleosome-bound DNA in the cell lysate. The relative increase in nucleosomes in the cell lysate, determined at 405 nm, was expressed as an enrichment factor. DNA fragmentation in cells treated without β-carotene (none) was set at 1. The results are expressed as the mean ± SEM of four separate experiments. *p < 0.05 versus cells treated without β-carotene (none). c Apoptotic indices (protein levels of cytochrome c, Bcl-2, and Bax) were assessed with Western blot analysis. Actin served as a loading control
Fig. 2
Fig. 2
β-Carotene induces increases in intracellular ROS levels and caspase-3 activity, and decreases in Ku70/80 levels and Ku-DNA-binding activity in AGS cells. The cells were cultured with β-carotene (final concentration of 100 μM) for 12 h (a, b) or for various periods (0, 4, 8, 12 h) (c, d). a Intracellular ROS levels were determined by measuring DCF fluorescence with a fluorescence multi-well plate reader. The results are expressed as the mean ± SEM of four separate experiments. *p < 0.05 versus cells treated without β-carotene (none). b Caspase-3 activity was determined by measuring the cleavage of the fluorescent peptide substrate DEVDAFC. Caspase-3 activity in cells treated without β-carotene (none) was set at 100 %. The results are expressed as the mean ± SEM of four separate experiments. *p < 0.05 versus cells treated without β-carotene (none). c The levels of Ku70 and Ku80 in total cell lysates were determined with Western blot analysis. Actin served as a loading control. d Ku-DNA-binding activity in nuclear extracts was determined with EMSA
Fig. 3
Fig. 3
Effect of NAC and z-DEVED-fmk on β-carotene-induced apoptosis in AGS cells. The cells were pretreated with NAC (2 mM) or z-DEVED-fmk (5 μM) for 2 h and then cultured with β-carotene (100 μM) for 12 h (ac). a Cell viability was determined by counting the number of viable cells. The viability of cells treated without β-carotene (none) was set at 100 %. The results are expressed as the mean ± SEM of four separate experiments. *p < 0.05 versus cells treated without β-carotene (none); +p < 0.05 versus cells treated with β-carotene alone (control). b DNA fragmentation was detected by measuring the amount of oligonucleosome-bound DNA in the cell lysate. The relative increase in nucleosomes in the cell lysate, determined at 405 nm, was expressed as an enrichment factor. DNA fragmentation in cells treated without β-carotene (none) was set at 1. The results are expressed as the mean ± SEM of four separate experiments. *p < 0.05 versus cells treated without β-carotene (none); +p < 0.05 versus cells treated with β-carotene alone (control). c Apoptotic indices (protein levels of cytochrome c, Bcl-2, and Bax) were assessed with Western blot analysis. Actin served as a loading control
Fig. 4
Fig. 4
Effect of NAC and z-DEVED-fmk on the β-carotene-induced alterations of intracellular ROS levels, caspase-3 activity, Ku70/80 levels, and Ku-DNA-binding activity in AGS cells. The cells were pretreated with NAC (2 mM) or z-DEVED-fmk (5 μM) for 2 h and then cultured with β-carotene (100 μM) for 30 min a and 12 h (bd). a Intracellular ROS levels were assessed by measuring DCF fluorescence. The relative fluorescence intensity of cells treated without β-carotene (none) was set at 100 %. The results are expressed as the mean ± SEM of four separate experiments. *p < 0.05 versus cells treated without β-carotene (none); +p < 0.05 versus cells treated with β-carotene alone (control). b Caspase-3 activity was determined by measuring the cleavage of the fluorescent peptide substrate DEVDAFC. Caspase-3 activity in cells treated without β-carotene (none) was set at 100 %. The results are expressed as the mean ± SEM of four separate experiments. *p < 0.05 versus cells treated without β-carotene (none); +p < 0.05 versus cells treated with β-carotene alone (control). c The levels of Ku70 and Ku80 in total cell lysates were determined with Western blot analysis. Actin served as a loading control. d Ku-DNA-binding activity in nuclear extracts was determined with EMSA. None, cells treated without β-carotene; control, cells treated with β-carotene alone
Fig. 5
Fig. 5
Effect of NAC and z-DEVED-fmk on β-carotene-induced increase in mitochondrial ROS levels, and MMP loss in AGS cells. The cells were pretreated with NAC (2 mM) or z-DEVED-fmk (5 μM) for 2 h and then cultured with β-carotene (100 μM) for 30 min. a Mitochondrial ROS levels were determined by measuring MitoSOX Red signals. Nuclei were stained with DAPI. b MMP was analyzed with flow cytometry. The relative fluorescence intensity of the cells treated without β-carotene (none) was set at 100 %. The results are expressed as the mean ± SEM of four separate experiments. *p < 0.05 versus cells treated without β-carotene; +p < 0.05 versus control cells treated with β-carotene alone
Fig. 6
Fig. 6
Effect of NAC and z-DEVED-fmk on β-carotene-induced decrease in cell surface expression levels of Ku proteins in AGS cells. The cells were pretreated with NAC (2 mM) or z-DEVED-fmk (5 μM) for 2 h and then cultured with β-carotene (100 μM) for 12 h. The expression of Ku70/80 in cell membrane was analyzed by flow cytometry. a Representative histograms of Ku70/80 expression in cell membrane are shown. b Mean fluorescence intensity for Ku70/80 expression in cell membrane was analyzed. The results are expressed as the mean ± SEM of four separate experiments. *p < 0.05 versus corresponding cells treated without β-carotene; +p < 0.05 versus corresponding control cells treated with β-carotene alone

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