Nutr Res Pract.  2018 Apr;12(2):129-134. 10.4162/nrp.2018.12.2.129.

Reactive oxygen species-dependent apoptosis induction by water extract of Citrus unshiu peel in MDA-MB-231 human breast carcinoma cells

• Kim MY
• Choi EO
• HwangBo H
• Kwon DH
• Ahn KI
• Kim HJ
• Ji SY
• Hong SH
• Jeong JW
• Kim GY
• Park C
• Choi YH

• KMID: 2407809
• DOI: http://doi.org/10.4162/nrp.2018.12.2.129

Abstract

BACKGROUND/OBJECTIVES
Although several recent studies have reported the anti-cancer effects of extracts or components of Citrus unshiu peel, which has been used for various purposes in traditional medicine, the molecular mechanisms for their effects remain unclear. In the present study, the anti-cancer activity of a water-soluble extract of C. unshiu peel (WECU) in MDA-MB-231 human breast carcinoma cells at the level of apoptosis induction was investigated.
MATERIALS/METHODS
Cytotoxicity was evaluated using the MTT assay. Apoptosis was detected using DAPI staining and flow cytometry analyses. Mitochondrial membrane potential, reactive oxygen species (ROS) assay, caspase activity and Western blotting were used to confirm the basis of apoptosis.
RESULTS
The results indicated that WECU-induced apoptosis was related to the activation of caspase-8, and -9, representative initiator caspases of extrinsic and intrinsic apoptosis pathways, respectively, and caspase-3 accompanied by proteolytic degradation of poly(ADP-ribose) polymerase and down-regulation of the inhibitors of apoptosis protein family members. WECU also increased the pro-apoptotic BAX to anti-apoptotic BCL-2 ratio, loss of mitochondrial membrane potential and cytochrome c release from mitochondria to cytoplasm. Furthermore, WECU provoked the generation of ROS, but the reduction of cell viability and induction of apoptosis by WECU were prevented when ROS production was blocked by antioxidant N-acetyl cysteine.
CONCLUSIONS
These results suggest that WECU suppressed proliferation of MDA-MB-231 cells by activating extrinsic and intrinsic apoptosis pathways in a ROS-dependent manner.

Keyword

Citrus unshiu; apoptosis; caspase; cytotoxicity; ROS

MeSH Terms

Apoptosis*
Blotting, Western
Breast Neoplasms*
Breast*
Caspase 3
Caspase 8
Caspases, Initiator
Cell Survival
Citrus*
Cysteine
Cytochromes c
Cytoplasm
Down-Regulation
Flow Cytometry
Humans*
Medicine, Traditional
Membrane Potential, Mitochondrial
Mitochondria
Oxygen*
Poly(ADP-ribose) Polymerases
Reactive Oxygen Species
Water*
Caspase 3
Caspase 8
Caspases, Initiator
Cysteine
Cytochromes c
Oxygen
Poly(ADP-ribose) Polymerases
Reactive Oxygen Species
Water

Figure

• 

  Fig. 1 Inhibition of cell viability and induction of apoptosis by WECU in MDA-MB-231 cells. MDA-MB-231 cells were treated with various concentrations of WECU for 72 h. (A) The cell viability was measured by an MTT assay. The data were expressed as the means ± SD of three independent experiments (* P < 0.05 vs. untreated control). (B) The morphological changes of MDA-MB-231 cells treated with WECU in various concentrations were observed under an inverted microscope (magnification, ×200). (C) The cells were stained with DAPI, and then the nuclei were photographed with a fluorescence microscope using a blue filter (magnification, ×400). (D) The percentages of annexin V-FITC positive cells in the top (PI negative) and bottom (PI positive) right quadrant were indicated. Each point represents the means of two independent experiments.

• 

  Fig. 2 Activation of caspases and inhibition of IAP family proteins expression by WECU in MDA-MB-231 cells. MDA-MB-231 were treated with the indicated concentrations of WECU for 72 h. (A) The equal amounts of cellular proteins were probed with the indicated antibodies, and the proteins were visualized using an ECL detection system. Actin was used as an internal control. (B) The activities of the caspases were evaluated using caspases assay kits. The data were expressed as the means ± SD of three independent experiments (* P < 0.05 vs. untreated control).

• 

  Fig. 3 Effects of WECU on the levels of BCL-2 family proteins in MDA-MB-231 cells. After 72 h incubation with the indicated concentrations of WECU, the cellular proteins were probed with anti-BAX, anti-BCL-2 and anti-BID antibodies. Equal protein loading was confirmed by analysis of actin in the protein extracts.

• 

  Fig. 4 Effects of WECU on the MMP values and cytochrome c expression in MDA-MB-231 cells. A and B) After 72 h incubation with the indicated concentrations of WECU, the changes in MMP were analyzed on a flow cytometer. (A) An example of representative results according to each treatment concentration is presented. (B) The data were expressed as the means ± SD of three independent experiments (* P < 0.05 vs. untreated control). (C and D) Cells cultured under the same conditions were lysed, and cytosolic (C) and mitochondrial (D) proteins were probed with an anti-cytochrome c antibody. Equal protein loading was confirmed by analysis of actin and cytochrome oxidase subunit VI (COX VI) in each protein extract.

• 

  Fig. 5 Induction of ROS generation by WECU in MDA-MB-231 cells. (A) MDA-MB-231 cells were either treated with 1.5 mg/mL WECU for the indicated times or pre-treated with NAC (10 mM) for 1 h before WECU treatment. ROS generation was measured by a flow cytometer. The data are the means of the two different experiments. (B) The cells were either treated with 1.5 mgL WECU for 1 h or pre-treated with NAC (10 mM) for 1 h before WECU treatment and then stained with DCF-DA. Images were obtained using a fluorescence microscope (Original magnifications: 200×). The images presented here are captured from one experiment and are representative of at least three independent experiments.

• 

  Fig. 6 Induction of ROS-dependent apoptosis by WECU in MDA-MB-231 cells. MDA-MB-231 cells were pre-treated with or without 10 mM NAC for 1 h, prior to 1.5 mg/mL WECU treatment. (A) After 72 h incubation, the cells were stained with DAPI, and then the nuclei were photographed (magnification, ×400). (B) The percentages of annexin V-FITC positive cells cultured under the same conditions in the top (PI negative) and bottom (PI positive) right quadrant were indicated. Each point represents the mean of two independent experiments. (C) Cell viability was analyzed using MTT assay. The data are expressed as means ± SD of three independent experiments (* P < 0.05 vs. untreated control; # P < 0.05 vs. WECU-treated cells).

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