Apoptosis of AGS human gastric adenocarcinoma cells by methanolic extract of Dictamnus

Abstract

Background: The root bark of Dictamnus dasycarpus Turcz has traditionally been used in East Asia to treat skin diseases such as eczema, atopic dermatitis, and psoriasis. However, it has also been reported to exhibit an anti-proliferative effect on cancer cells.

Objective: To investigate the anti-cancer effects of a methanol extract of Dictamnus dasycarpus root bark (MEDD) on AGS cells (a human gastric adenocarcinoma cell-line).

Materials and methods: An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium assay, a caspase activity assay, cell cycle analysis, mitochondrial membrane potential (MMP) measurements, and western blotting were used to investigate the anti-cancer effects of MEDD on AGS cells.

Results: Treatment with MEDD significantly and concentration-dependently inhibited AGS cell growth. MEDD treatment in AGS cells led to increased accumulation of apoptotic sub-G1 phase cells in a concentration-dependent manner. Also, MEDD reduced the expressions of pro-caspase-3, -8 and -9, and increased the active form of caspase-3. Furthermore, subsequent Western blotting revealed elevated levels of poly (ADP-ribose) polymerase protein. MEDD treatment reduced levels of MMP and anti-apoptotic Bcl-2 and Bcl-xL proteins. Pretreatment with SB203580 (a specific inhibitor of p38 mitogen-activated protein kinases), SP600125 (a potent inhibitor of C-Jun N-terminal kinases), or PD98059 (a potent inhibitor of extracellular signal-regulated kinases) did not modify the effects of MEDD treatment. However, pretreatment with LY294002 (a specific inhibitor of Akt) significantly enhanced MEDD-induced cell death.

Conclusion: These results suggest that MEDD-mediated cell death is associated with the intrinsic apoptotic pathway and that inhibition of Akt signaling contributes to apoptosis induction by MEDD.

Keywords: A human gastric adenocarcinoma cell-line; Akt; Dictamnus dasycarpus; LY294002; apoptosis; gastric cancer.

Figure 1

Induction of apoptosis by methanolic extract of Dictamnus dasycarpus (MEDD) Turcz in AGS cells. (a) Cells were treated with the indicated concentrations of MEDD for 24 h. Cell viabilities were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium assay. The significances of difference were determined using the Student's t-test (*P < 0.05 vs. untreated cells) (b) to quantify the degree of apoptosis induced by MEDD, cells grown under the same conditions as (a) were evaluated by flow cytometry for sub-G1 DNA contents (a surrogate of apoptotic DNA degradation). Results are the mean ± standard deviations of two different experiments

Figure 2

Activations of caspases and degradation of poly (ADP-ribose) polymerase protein by methanolic extract of Dictamnus dasycarpus (MEDD) in AGS cells. (a) AGS cells were treated with the indicated concentrations of MEDD for 24 h. Membranes were probed with the indicated antibodies. Actin was used as the internal control. (b) After 24 h incubation with the indicated concentrations of MEDD, cells were lysed, and aliquots were assayed for in vitro caspase-3 and -9 activities using DEVD-p-nitroaniline (pNA) and LEHD-pNA as substrates, respectively. Results are the mean ± standard deviations of three different experiments. *P < 0.05 versus untreated control

Figure 3

Effects of methanolic extract of Dictamnus dasycarpus (MEDD) on the protein levels of Bcl-2 and Bcl-xL and on mitochondrial membrane potential levels in AGS cells. (a) Cells were treated with the indicated concentrations of MEDD for 24 h, collected, and incubated with JC-1 (10 μM) for 20 min at 37°C in the dark. They were then washed once with PBS and subjected to deoxyribonucleic acid flow cytometry. (b) Cell lysates obtained from cells grown under the same conditions as (a) They were separated in SDS-polyacrylamide gels and transferred to nitrocellulose membranes, which were probed with the indicated antibodies. Actin was used as an internal control

Figure 4

Effects of Akt inhibition on methanolic extract of Dictamnus dasycarpus (MEDD)-induced AGS cell death. (a) Cells were pretreated with the indicated mitogen-activated protein kinases inhibitors (SB203580 (20 μM), SP600125 (20 μM), or PD98059 (50 μM)) for 1 h and then treated with MEDD (350 μg/ml) for 24 h. (b) cells were pretreated with the Akt inhibitor (LY294002 (20 μM)) for 1 h and then treated with MEDD (280 μg/ml) for 24 h. Cell viabilities were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium assay. Results are the means ± standard deviations of two different experiments. *P < 0.05 versus untreated cells; ^#P < 0.05 versus MEDD-treated cells; n.s.: Not significant versus MEDD-treated cells

Figure 5

Effects of Akt inhibition on methanolic extract of Dictamnus dasycarpus (MEDD)-induced apoptosis in AGS cells. Cells were pretreated with Akt inhibitor (LY294002 (20 μM)) for 1 h and then treated with MEDD (280 μg/ml) for 24 h. (a) Cells were collected and deoxyribonucleic acid contents were analyzed by flow cytometry. (b) Equal amounts of cell lysates were extracted, separated in SDS-polyacrylamide gels and transferred to nitrocellulose membranes, which were probed with the indicated antibodies. Proteins were visualized using an enhanced chemiluminescence detection system. Actin was used as an internal control