Flavonoids Isolated from Korea Citrus aurantium L. Induce G2/M Phase Arrest and Apoptosis in Human Gastric Cancer AGS Cells

Abstract

Aim of the Study. Citrus species is used in traditional medicine as medicinal herb in several Asian countries including Korea. Flavonioids became known as various properties, such as anti-oxidants, anti-inflammation and anti-cancer, and so forth. The present study, the anti-cancer effect of flavonioids isolated from Citrus aurantium L. in human gastric cancer AGS cells has been investigated. Materials and Methods. The anti-proliferative activity was assayed using MTT assay. Cell cycle analysis was done using flow cytometry and apoptosis detection was done using by hoechst fluorescent staining and Annexin V-propidium iodide double staining. Western blot was used to detect the expression of protein related with cell cycle and apoptosis. Results. Flavonoids isolated from Citrus aurantium L. have the effect of anti proliferation on AGS cells with IC50 value of 99 μg/mL. Flavonoids inhibited cell cycle progression in the G2/M phase and decrease expression level of cyclin B1, cdc 2, cdc 25c. Flavonoids induced apoptosis through activate caspase and inactivate PARP. Conclusions. Flavonoids isolated from Citrus aurantium L. induced G2/M phase arrest through the modulation of cell cycle related proteins and apoptosis through activation caspase. These finding suggest flavonoids isolated from Citrus aurantium L. were useful agent for the chemoprevention of gastric cancer.

Figure 1

HPLC chromatogram patterns of Korea Citrus aurantium L. at 280 nm. (1) naringin, (2) hesperidin, (3) poncirin, (4) isosiennsetin, (5) hexamethoxyflavone, (6) sineesytin, (7) hexamethoxyflavone, (8) tetramrthnl-o-isoscutellaeein, (9) nobiletin, (10) heptamethoxyflavone, (11) 3-hydoxynobiletin, (12) tangeretin, (13) hydroxypentamethoxyflavone, and (14) hexamethoxyflavone.

Figure 2

Effect of flavonoids on the viability of AGS cells. (a) AGS cells were treated with various concentrations of the flavonoids for 24 h. Cell viability was then determined by an MTT assay. Cell viability is represented as the percentage relative absorbance compared to the controls. (b) Morphology of cells treated with or without flavonoids for 24 h and examined under light microscopy (×400). Each value represents the mean of three experiments.

Figure 3

Flavonoids induce G2/M phage arrest on AGS cells. Flavonoids were treated with the indicated concentrations, and cells were incubated for 24 h. Cell cycle proportions were determined by flow cytometry after staining with propidium iodide. (a) Flow cytometry of cell cycle phase distribution. (b) Statistical analysis of cell cycle phase distribution. Each value represents the mean of three experiments; bars, ±SD. (*P < 0.05, **P < 0.01 versus control group.)

Figure 4

Flavonoids alter the expression of cell-cycle-related proteins cdc 2, cyclin B1, and cdc 25c. The expression of proteins was assessed by immunoblotting. (a) Representative blots are shown. Densitometric analysis of the effect of flavonoids on the expression of cyclin B1 (b), cdc 2 (c), and cdc 25c (d). Each value represents the mean of three experiments; bars, ±SD. (*P < 0.05, **P < 0.01 versus control group.)

Figure 5

Flavonoids induce apoptosis of AGS cells. Flavonoids were added at the indicated concentrations, and cells were incubated for 24 h. (a) Hoechst 33258 staining of AGS cells treated with or without flavonoids for 24 h. Fragmented or condensed nuclei could be observed at 1000x magnification in the flavonoids-treated group as indicated by the arrows. (b) Apoptosis was assessed by Annexin V-PI double staining. (c) Statistical analysis of apoptosis rate. Each value represents the mean of three experiments; bars, ±SD. (*P < 0.05, **P < 0.01 versus control group.)

Figure 6

Flavonoids alter the expression of apoptosis-related proteins caspase-8, -9, -3, and -6, and cleavage of PARP. The expression of proteins was assessed by immunoblotting. (a) Representative blots are shown. Densitometric analysis of the effect of flavonoids on the expression of procaspase-8 (b), -9 (c), -3 (d), and -6 (e), and cleaved PARP (f). (g) Caspase-3 activity was determined using a colorimetric caspase-3 activation kit assay kit following the manufacturer's protocol. Each value represents the mean of three experiments; bars, ±SD. (*P < 0.05, **P < 0.01 versus control group.)

Figure 7

Flavonoids alter the expression of mitochondria-dependent pathway of apoptosis-related proteins Bcl-xL and Bax. The expression of proteins was assessed by immunoblotting. (a) Representative blots are shown. (b) Densitometric analysis of the Bax/Bcl-xL ratio. Each value represents the mean of three experiments; bars, ±SD. (*P < 0.05, **P < 0.01 versus control group.)