Nutr Res Pract.  2014 Apr;8(2):132-137.

Induction of apoptosis by a hexane extract of aged black garlic in the human leukemic U937 cells

• Park C
• Park S
• Chung YH
• Kim GY
• Choi YW
• Kim BW
• Choi YH

Abstract

BACKGROUND/OBJECTIVES
In this study, the apoptogenic activity and mechanisms of cell death induced by hexane extract of aged black garlic (HEABG) were investigated in human leukemic U937 cells.
MATERIALS/METHODS
Cytotoxicity was evaluated by MTT (3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide) assay. Apoptosis was detected using 4,6-diamidino-2-phenyllindile (DAPI) staining, agarose gel electrophoresis and flow cytometry. The protein levels were determined by Western blot analysis. Caspase activity was measured using a colorimetric assay.
RESULTS
Exposure to HEABG was found to result in a concentration- and time-dependent growth inhibition by induction of apoptosis, which was associated with an up-regulation of death receptor 4 and Fas legend, and an increase in the ratio of Bax/Bcl-2 protein expression. Apoptosis-inducing concentrations of HEABG induced the activation of caspase-9, an initiator caspase of the mitochodrial mediated intrinsic pathway, and caspase-3, accompanied by proteolytic degradation of poly(ADP-ribose)-polymerase. HEABG also induced apoptosis via a death receptor mediated extrinsic pathway by caspase-8 activation, resulting in the truncation of Bid, and suggesting the existence of cross-talk between the extrinsic and intrinsic pathways. However, pre-treatment of U937 cells with the caspase-3 inhibitor, z-DEVD-fmk, significantly blocked the HEABG-induced apoptosis of these cells, and increased the survival rate of HEABG-treated cells, confirming that HEABG-induced apoptosis is mediated through activation of caspase cascade.
CONCLUSIONS
Based on the overall results, we suggest that HEABG reduces leukemic cell growth by inducing caspase-dependent apoptosis through both intrinsic and extrinsic pathways, implying its potential therapeutic value in the treatment of leukemia.

Keyword

Aged black garlic; U937 cells; apoptosis; caspase

MeSH Terms

Apoptosis*
Blotting, Western
Caspase 3
Caspase 8
Caspase 9
Cell Death
Electrophoresis, Agar Gel
Flow Cytometry
Garlic*
Humans*
Leukemia
Receptors, TNF-Related Apoptosis-Inducing Ligand
Survival Rate
U937 Cells*
Up-Regulation
Caspase 3
Caspase 8
Caspase 9
Receptors, TNF-Related Apoptosis-Inducing Ligand

Figure

• 

  Fig. 1 Effect of HEABG on the viability and morphology of U937 cells. (A and B) Cells were cultured with the indicated concentration of HEABG for 24 h (A) or with 10 µM HEABG for the indicated times (B). Cell viability was measured by MTT assay. Each point is the mean ± SD of three independent experiments. The significance was determined using Student's t-test (*P < 0.05 vs. untreated control). (C) The morphological changes of U937 cells treated with the indicated concentration of HEABG for 24 h were visualized by phase-contrast microscopy (200×).

• 

  Fig. 2 Induction of apoptosis by HEABG in U937 cells. Cells were treated with the indicated concentration of HEABG for 24 h. (A) Cells were fixed, stained with DAPI solution, and examined by fluorescence microscopy (400×). (B) DNA fragmentation was examined by 1.0% agarose gel electrophoresis of genomic DNA, followed by EtBr staining. (C) To quantify the degree of apoptosis induced by HEABG, cells were evaluated using a flow cytometer for sub-G1 DNA content, which represents the cells undergoing apoptotic DNA degradation. Each point represents the mean ± SD of three independent experiments. Significance was determined using Student's t-test (*P < 0.05 vs. untreated control).

• 

  Fig. 3 Effect of HEABG on the expression of apoptosis-associated proteins in U937 cells. Cells were treated with the indicated concentration of HEABG for 24 h. Cell lysates were resolved by SDS-polyacrylamide gels and subjected to Western blotting. The proteins were visualized using specific antibodies and an ECL detection system. Actin was used as an internal control. The expression of the indicated proteins were measured by densitometry and expressed as relative ratio. The results were representatives of two independent experiments.

• 

  Fig. 4 Activation of caspases and the degradation of PARP by HEABG in U937 cells. (A) Cells were treated with the indicated concentrations of HEABG for 24 h. The cells were lysed, and the cellular proteins were then separated by SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The proteins were visualized using the indicated antibodies and an ECL detection system. Actin was used as an internal control. (B) After 24 h of incubation with the indicated concentrations of U937, the cells were lysed, and aliquots (50 µg protein) were assayed for in vitro caspase-3, -8, and -9 activity using DEVD-pNA, IETD-pNA and LEHD-pNA as substrates, respectively, at 37℃ for 1 h. The released fluorescent products were measured. Each point represents the mean ± SD of three independent experiments. Significance was determined using Student's t-test (*P < 0.05 vs. untreated control).

• 

  Fig. 5 Inhibition of PARP degradation by caspase-3 inhibitor in HEABG-treated U937 cells. (A) Cells were incubated with 10 µg/ml HEABG for 24 h after 1 h pretreatment with z-DEVD-fmk (50 µM). Cell lysates were resolved by SDS-polyacrylamide gels and subjected to Western blotting. The proteins were visualized using the anti-caspase-3 and anti-PARP antibodies, and an ECL detection system. (B) Cells grown under the same conditions as (A) were assayed for in vitro caspase-3 activity using DEVD-pNA as substrates. Each point represents the mean ± SD of three independent experiments. Significance was determined using Student's t-test (*P < 0.05 vs. HEABG-treated cells).

• 

  Fig. 6 Caspase-3 inhibitor alleviates HEABG-induced apoptosis in U937 cells. (A) Cells grown under the same conditions as Fig. 5 were fixed and stained with DAPI solution. The stained nuclei were observed under a fluorescent microscope (original magnification 400×). (B) DNA fragmentation was examined by 1.0% agarose gel electrophoresis of genomic DNA, followed by EtBr staining. (C) Cells were evaluated by flow cytometry for sub-G1 DNA content, suggestive of apoptotic cell death. (D) Cell viability was determined by MTT assay. Each point represents the mean ± SD of three independent experiments. The significance was determined using Student's t-test (*P < 0.05 vs. HEABG-treated cells).

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