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

Abstract

BackgroundAnnona 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.MethodsThe 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.ResultsCell 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.ConclusionsOur 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.

Functional proteomic analysis revels that the ethanol extract of Annona muricata L. induces liver cancer cell apoptosis through endoplasmic reticulum stress pathway

“…Leaf and root extracts have comparable anti-proliferative activities, which are higher than twig extract (Zorofchian Moghadamtousi et al 2014).…”

“…However, leaves are considered to be a better raw material for extraction, as they are more abundant and available. Using 95% hot ethanol as solvent, the resulting leaf extract can inhibit the proliferation of HL-60 cells at an IC 50 of less than 12 μg/ml (Zorofchian Moghadamtousi et al 2014). The values of IC 50 for hexane extract are 11.43 ± 1.87 µg/ml and 8.98 ± 1.24 µg/ml against HT-29 and HCT-116 cells respectively (Moghadamtousi et al 2014).…”

Anti-Inflammatory Effects of The Chloroform Extract of Annona muricata Leaves on Phospholipase A2 and Prostaglandin Synthase Activities

“…All parts of Annona muricata are extensively used as traditional medicines against an array of human ailments and diseases. Previous reports on Annona muricata reveal it possesses anti-oxidant effect Baskar [10] anti-proliferative effect [11,12] and anti-trypanosomal effects [13], anti-bacterial effect [14,15], anti-hepatotoxic activity [16,17], wound healing activity [18]. The antinociceptive and anti-inflammatory effect of the ethanol extract of the plant in animal models has also been reported [19][20][21].…”

The Chemopreventive Effect of Tanacetum Polycephalum Against LA7-Induced Breast Cancer in Rats and the Apoptotic Effect of a Cytotoxic Sesquiterpene Lactone in MCF7 Cells: A Bioassay-Guided Approach

“…Flow cytometric analysis of early and late apoptosis was investigated using Annexin-V-FITC/PI staining [20]. Briefly, after treating the cells with HDZC at IC 50 concentrations for 12, 24 and 48 h, MCF7 cells were harvested and washed twice with PBS.…”