Annona muricata leaves induced apoptosis in A549 cells through mitochondrial-mediated pathway and involvement of NF-κB

Abstract

Background: Annona muricata leaves have been reported to have antiproliferative effects against various cancer cell lines. However, the detailed mechanism has yet to be defined. The current study was designed to evaluate the molecular mechanisms of A. muricata leaves ethyl acetate extract (AMEAE) against lung cancer A549 cells.

Methods: The effect of AMEAE on cell proliferation of different cell lines was analyzed by MTT assay. High content screening (HCS) was applied to investigate the suppression of NF-κB translocation, cell membrane permeability, mitochondrial membrane potential (MMP) and cytochrome c translocation from mitochondria to cytosol. Reactive oxygen species (ROS) formation, lactate dehydrogenase (LDH) release and activation of caspase-3/7, -8 and -9 were measured while treatment. The western blot analysis also carried out to determine the protein expression of cleaved caspase-3 and -9. Flow cytometry analysis was used to determine the cell cycle distribution and phosphatidylserine externalization. Quantitative PCR analysis was performed to measure the gene expression of Bax and Bcl-2 proteins.

Results: Cell viability analysis revealed the selective cytotoxic effect of AMEAE towards lung cancer cells, A549, with an IC50 value of 5.09 ± 0.41 μg/mL after 72 h of treatment. Significant LDH leakage and phosphatidylserine externalization were observed in AMEAE treated cells by fluorescence analysis. Treatment of A549 cells with AMEAE significantly elevated ROS formation, followed by attenuation of MMP via upregulation of Bax and downregulation of Bcl-2, accompanied by cytochrome c release to the cytosol. The incubation of A549 cells with superoxide dismutase and catalase significantly attenuated the cytotoxicity caused by AMEAE, indicating that intracellular ROS plays a pivotal role in cell death. The released cytochrome c triggered the activation of caspase-9 followed by caspase-3. In addition, AMEAE-induced apoptosis was accompanied by cell cycle arrest at G0/G1 phase. Moreover, AMEAE suppressed the induced translocation of NF-κB from cytoplasm to nucleus.

Conclusions: Our data showed for the first time that the ethyl acetate extract of Annona muricata inhibited the proliferation of A549 cells, leading to cell cycle arrest and programmed cell death through activation of the mitochondrial-mediated signaling pathway with the involvement of the NF-kB signalling pathway.

Figure 1

Lactate dehydrogenase (LDH) assay showed the cytotoxicity of AMEAE against A549 cells. The result revealed significant cytotoxicity at concentrations of 5 to 40 μg/mL, in a dose-dependent manner. The data represent the means ± SEM of three independent experiments. *p < 0.05 compared with the untreated group.

Figure 2

Fluorescent micrographs of AO/PI-double-stained A549 cells. (A) Untreated A549 cells after 72 h depict healthy structures. Early apoptosis features such as chromatin condensation and blebbing were observed after (B) 24, (C) 48 and 72 h (D) of treatment with AMEAE. Late apoptosis was observed at the late stage of treatment (magnification: 200×). VC: Viable cells; CC: Chromatin condensation; BL: Blebbing of the cell membrane; LA: Late apoptosis.

Figure 3

Time-dependent induction of early and late apoptosis by AMEAE indicated the percentage of early apoptotic, late apoptotic and necrotic cells after (B) 24, (C) 48 and (D) 72 h of treatment. (A) shows the cells treated with vehicle DMSO. The early and late apoptotic events are depicted in the lower right and upper left quadrant of each panel. (E) Representative bar chart demonstrated significant elevation in a number of early and late apoptotic cells after 48 h of treatment. The data represent the means ± SEM of three independent experiments. *p < 0.05 compared with the untreated group.

Figure 4

Flow cytometry analysis on cell cycle progression in A549 cells was carried out after incubation with AMEAE for (A) 0, (B) 24, (C) 48 and (D) 72 h. (E) Representative bar chart demonstrated significant cell cycle arrest at the G₁ phase. The data represent the means ± SEM of three independent experiments. *p < 0.05 compared with the no-treatment group.

Figure 5

Effect of AMEAE on ROS generation in A549 cells. (A) Representative images of A549 cells treated with medium alone and 10 μg/mL of AMEAE for 24 h and stained with dihydroethidium and Hoechst 33342 dyes for 30 min. (B) Representative bar chart indicated dose-dependent elevation in ROS formation in treated A549 cells. The level of ROS was significantly elevated at 5 to 40 μg/mL concentrations. (C) Pretreatment of A549 cells with Cat and SOD for 1 h prior to the incubation with AMEAE caused a significant elevation in cell viability compared to the treatment alone with AMEAE. The data represent the means ± SEM of three independent experiments. *p < 0.05 compared with the untreated group.

Figure 6

Effect of AMEAE on cell membrane permeability, MMP and cytochrome c release. (A) Representative images of A549 cells treated with medium alone and 10 μg/mL of AMEAE, and stained with MMP, cell membrane permeability, Hoechst 33342 and cytochrome c dyes. AMEAE caused a marked elevation in cytochrome c leakage and cell membrane permeability, and a noteworthy decrease in MMP. Representative bar charts indicating dose-dependent increased (B) cell permeability and reduced (C) MMP in treated A549 cells at 5 to 40 μg/mL concentrations. Meanwhile, (D) cytochrome c was significantly released at 10 to 40 μg/mL concentrations. The data represent the means ± SEM of three independent experiments. *p < 0.05 compared with the untreated group.

Figure 7

Effect of AMEAE of caspases activation. (A) Relative luminescence dose-dependent activation of caspase-8, -9 and -3/7 in A549 cells treated with various concentrations of AMEAE. Caspase-9 and -3/7 were significantly activated at 10 to 40 μg/mL concentrations. Meanwhile, caspase-8 was activated at higher concentrations (20 and 40 μg/mL). (B) A549 cells were treated with the indicated concentrations of AMEAE for 24 h. The cell lysates were evaluated for levels of cleaved caspase-3 and -9. AMEAE induced the up-regulation of cleaved caspase-3 and -9. (C) The quantitative analysis was expressed as a ratio to the expression of β-actin. The data represent the means ± SEM of three independent experiments. *p < 0.05 compared with the untreated group.

Figure 8

Effect of AMEAE on mRNA expression of A549 cells. Downregulation of (A) Bcl-2 and upregulation of (B) Bax at gene expression level induced by AMEAE at 20 and 40 μg/mL concentrations after 24 h assessed by quantitative PCR analysis. The data represent the means ± SEM of three independent experiments. *p < 0.05 compared with the untreated group.

Figure 9

Effect of AMEAE on NF-κB activation. (A) Representative images of the intracellular targets of stained A549 cells that were exposed to AMEAE (10 μg/mL) and then stimulated with TNF-α. (B) Representative bar chart revealed the translocation of NF-κB nuclear in treated A549 cells with AMEAE. The data represent the means ± SEM of three independent experiments. *p < 0.05 compared with the untreated control group.