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, 11 (3), e0151466
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Artonin E Induces Apoptosis via Mitochondrial Dysregulation in SKOV-3 Ovarian Cancer Cells

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Artonin E Induces Apoptosis via Mitochondrial Dysregulation in SKOV-3 Ovarian Cancer Cells

Mashitoh Abd Rahman et al. PLoS One.

Abstract

Artonin E is a prenylated flavonoid isolated from the stem bark of Artocarpus elasticus Reinw.(Moraceae). This study aimed to investigate the apoptotic mechanisms induced by artonin E in a metastatic human ovarian cancer cell line SKOV-3 in vitro. MTT assay, clonogenic assay, acridine orange and propidium iodide double staining, cell cycle and annexin V analyses were performed to explore the mode of artonin E-induced cell death at different time points. DNA laddering, activation of caspases-3, -8, and -9, multi-parametric cytotoxicity-3 analysis by high-content screening, measurement of reactive oxygen species generation, and Western blot were employed to study the pathways involved in the apoptosis. MTT results showed that artonin E inhibited the growth of SKOV-3 cells, with IC50 values of 6.5±0.5 μg/mL after 72 h treatment, and showed less toxicity toward a normal human ovarian cell line T1074, with IC50 value of 32.5±0.5 μg/mL. Results showed that artonin E induced apoptosis and cell cycle arrest at the S phase. This compound also promoted the activation of caspases-3, -8, and -9. Further investigation into the depletion of mitochondrial membrane potential and release of cytochrome c revealed that artonin E treatment induced apoptosis via regulation of the expression of pro-survival and pro-apoptotic Bcl-2 family members. The expression levels of survivin and HSP70 proteins were also down regulated in SKOV-3 cells treated with artonin E. We propose that artonin E induced an antiproliferative effect that led to S phase cell cycle arrest and apoptosis through dysregulation of mitochondrial pathways, particularly the pro- and anti-apoptosis signaling pathways.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Chemical structure of artonin E.
Fig 2
Fig 2. Microscopic evaluation of SKOV-3 cells treated with artonin E at different time points (200× magnification).
(A) Untreated cells. (B) Cells after 24 h treatment. (C) Cells after 48 h treatment. (D) Cells after 72 h treatment. IC: Intact cell structure; DC: dissociate cell structure; MB: membrane blebbing; and AB: apoptotic body.
Fig 3
Fig 3. Dose-dependent evaluation of SKOV-3 cells treated with artonin E as measured by clonogenic assay.
Cells were exposed to different concentrations of artonin E for 24 h and then incubated for three weeks to form colonies. The colonies formed were fixed with ethanol and stained with crystal violet.
Fig 4
Fig 4. Morphological analysis of SKOV-3 cells double stained with AO and PI as observed under a flourescent microscope.
The treated cells were exposed to 8 μg/mL of artonin E for 24, 48, and 72 h. (A) Untreated cells. (B) Cells after 24 h treatment. (C) Cells after 48 h treatment. (D) Cells after 72 h treatment. VI: viable cells; BL: blebbing of the cell membranes; LA: lateapoptosis; and SN: secondary necrosis.
Fig 5
Fig 5. Quantitative analysis of double stained (fluorescence dyes AO and PI) untreated and treated SKOV-3 cells with artonin E.
(EA: early apoptosis; LA: late apoptosis; and SN: secondary necrosis). Results are presented as mean±SD of three replicates. * indicates significant difference from the control of each phase (p<0.05).
Fig 6
Fig 6. Flow cytometry analysis of Annexin V-FITC in SKOV-3 cells treated with artonin E (8 μg/mL) in a time-dependent manner.
[A] Control (untreated), [B] 24 h treatment, [C] 48 h treatment, and [D] 72 h treatment. [E] Histogram. Results are presented as mean±SD of three replicates. *p<0.05 indicates significant difference from control.
Fig 7
Fig 7. Flow cytometry analysis of cell cycle phase distribution of SKOV-3 cells treated with artonin E (8 μg/mL) in a time-dependent manner.
[A] Untreated cells, [B] cells after 24 h treatment, [C] cells after 48 h treatment, and [D] cells after 72 h treatment. [E] Graphical analysis represented cell cycle arrest in SKOV-3 cells. Results are represented as mean±SD of three replicates. * indicates significant difference from the control of each phase (p<0.05).
Fig 8
Fig 8. Effects of Artonin E on ROS generation in SKOV-3 cells.
Cells were treated with 8 μg/mL of artonin E for 24, 48, and 72 h. Values are expressed as mean±SD from three independent experiments. Statistical significance was expressed as *p<0.05.
Fig 9
Fig 9. Representative image of SKOV-3 cells in a 96-well black plate and exposed to either medium alone or 8 μg/mL artonin E for 24, 48, and 72 h.
The treated cells showed reduction in cell number, increase in total nuclear intensity, increase in cell membrane permeability, loss of MMP, and increase in cytochrome c release. Images were observed at magnification 20×. (A) Untreated SKOV-3 cells and (B) SKOV-3 cells treated with 8 μg/mL of artonin E. (C) Time-dependent quantitative analysis of SKOV-3 cells treated with artonin E for different apoptosis parameters. Average intensities were observed simultaneously in SKOV-3 cells for total nuclear intensity, cell permeability, MMP, and cytochrome c release. All data were expressed as means±SD. *p<0.05 indicates significant difference from control.
Fig 10
Fig 10
(A) Effect of artonin E (8 μg/mL) on the mitochondrial and cytosolic cytochrome c fractions after 24, 48, and 72 h. β-Actin was used as loading control. (B) Quantitative analysis of SKOV-3 cells treated with artonin E. All data were expressed as mean±SD. Statistical significance was expressed as *p<0.05.
Fig 11
Fig 11. Effect of artonin E on caspases activation.
(A) Relative expression levels of caspases- 3, -8, and -9 in SKOV-3 cells treated with 8 μg/mL of artonin E for 24, 48, and 72 h. Results are represented as mean±SD of three replicates. *p<0.05 indicates significant difference from control. (B) Time-dependent of protein expression levels of caspase-3, -8 and -9 in SKOV-3 cells treated with 8 μg/mL of artonin E. Artonin E induced the up-regulation of caspase-9, -3 and -8. (C) The quantitative analysis is expressed as a ratio to the expression of β-actin. The data are represented as mean±SD of three replicates. *p<0.05 indicates significant difference from control.
Fig 12
Fig 12. Time-dependent electrophoresis gel separation of DNA isolated from SKOV-3 cells.
The cells were treated with artonin E(8 μg/mL). Lane A: untreated cells; lane B: SKOV-3 cells treated with artonin E for 24 h; lane C: cells after 48 h treatment; lane D: cells after 72 h treatment; lane E: positive control (HL-60 cells treated with actinomycin D); and lane F: DNA marker (50 base pairs).
Fig 13
Fig 13. (Top) Effect of artonin E (8 μg/mL) on the apoptosis protein expression after 24, 48, and 72 h. β-Actin was used as loading control.
(Bottom) Quantitative analysis of SKOV-3 cells treated with artonin E. All data were expressed as mean±SD. Statistical significance was expressed as *p<0.05.

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Grant support

The authors wish to thank University of Malaya for providing the research grants under UMRG project (RG077- 12BIO), Institute of Research Management and Monitoring Research Grant (PG162-2014B), and Ministry of Higher Education Malaysia under High Impact Research Grant (UM-MOHE UM.C/625/1/HIR/MOHE/SC/09) for their financial support to carry out this research.
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