Polyphenolic compounds from Korean Lonicera japonica Thunb. induces apoptosis via AKT and caspase cascade activation in A549 cells

Abstract

Lonicera japonica Thunb. (L. japonica T.) has historically been used in Korean herbal medicine due to its anticancer and protective effects on the respiratory system. In the present study, the polyphenolic compounds in L. japonica T. were investigated using high-performance liquid chromatography coupled with tandem mass spectrometry, and its anticancer effects on A549 non-small-cell lung cancer cells were studied. Polyphenolic compounds potentially inhibit A549 cells in a dose-dependent manner. Flow cytometry and western blot analysis demonstrated that polyphenolic compounds induce apoptosis by regulating the protein expression levels of caspases, poly-(ADP-ribose) polymerase and the B-cell lymphoma-2-associated X-protein/B-cell lymphoma-extra large ratio. Furthermore, polyphenolic compounds inhibited mitochondrial membrane potential activity. Caspase-3 activity was increased in a dose-dependent manner and polyphenolic compounds inhibited the activation of protein kinase B by dephosphorylation. These results suggest that polyphenolic compounds in A549 cells indicate the anticancer activity through the induction of apoptosis.

Keywords: Lonicera japonica Thunb; anticancer; apoptosis; polyphenolic compounds; protein kinase B.

Figure 1.

Characterization of polyphenolic compounds identified from Korean L. japonica T. in A549 cells. (A) HPLC profiles of Korean L. japonica T. at 280 nm: (1) Caffeoylquinic acid dimer; (2) Caffeoylquinic acid; (3) Caffeoylglycerol; (4) 5-p-coumaroylquinic acid; (5) Feruloylquinic acid; (6) Dicaffeoylquinic acid; (7) Dicaffeoylquinic acid; (8) Kaempferol 3-O-glucoside; (9) Kaempferol- O-rutinoside; (10) Dicaffeoylquinic acid; (11) Apigenin-7-O-glucoside; (12) Apigenin rutinoside; (13) Feruoyl caffeoylquinic acid; (14) Trihydroxymethoxyflavone; (15) Kaempferol; (16) Isorhamnetin glucoside; (17) Caffeic acid derivative; and (18) Feruoyl caffeoylquinic acid. (B) Growth inhibition of A549 cells following treatment with various concentrations (0–1,500 µg/ml) of polyphenolic compounds for 24 h. *P<0.05 compared with the control. HPLC, high performance liquid chromatography; L. japonica T., Lonicera japonica Thunb.

Figure 2.

Effect of polyphenolic compounds on the cell cycle distribution in A549 cells. The cells were incubated with various concentrations (0–1,200 µg/ml) of polyphenolic compounds for 24 h and the distribution of the cell cycle was evaluated using FACS analysis. (A) Flow cytometry of cell cycle phase distribution; (B) statistical analysis of cell cycle phase distribution. The data are presented as the mean ± standard deviation of triplicate independent experiments. *P<0.05 compared with the control.

Figure 3.

Induction of apoptosis in A549 cells by flavonoids. The apoptosis ratio was detected by Annexin V-FITC/PI double staining. (A) Flow cytometry analysis, (B) statistical analysis for apoptosis detection, (C) the cells were incubated with flavonoids for 24 h, fixed and stained with Hoechst 33342. The cells were imaged using a Leica DM6000 B microscope (magnification, 400x). The white arrows indicate chromatin condensation. The data are presented as the mean ± standard deviation of triplicate independent experiments. *P<0.05 compared with the control. FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 4.

Polyphenolic compounds induced mitochondrial membrane potential stability. Cells were treated with various concentrations of polyphenolic compounds (0–1,200 µg/ml) for 24 h. (A) The cells were stained with the carbocyanine dye DiOC₆ and flow cytometry was performed to determine the MMP stability. (B) Fluorescence light was detected by flow cytometry and expressed as a bar graph. The data are presented as the mean ± standard deviation of triplicate independent experiments. *P<0.05 compared with the control. MMP, mitochondrial membrane potential.

Figure 5.

Effect of polyphenolic compounds on apoptosis-associated protein (Bcl-xL, BAX, caspases, PARP and AKT) expression levels and caspase-3 activity in A549 cells. The cells were treated with polyphenolic compounds (0, 100, 400, 800 and 1,200 µg/ml) for 24 h. (A) Whole cell lysates were subjected to SDS-PAGE and analyzed Bcl-xl and Bax by western blotting. (B) Densitometry analysis of the effect of flavonoids on the expression levels of apoptosis-associated proteins and caspase-3 activity is depicted. (C) p-AKT was normalized to the respective total AKT, and is presented relative to the value for the untreated control cells. The data are presented as the mean ± standard deviation of triplicate independent experiments. *P<0.05 compared with the control. Bcl-xL, B-cell lymphoma-extra large; BAX, Bcl-2-associated x-protein; PARP, poly-(ADP-ribose) polymerase; AKT, protein kinase B; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; p-AKT, phosphorylated AKT.