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.
Highlights
Annona muricata leaves have been reported to have antiproliferative effects against various cancer cell lines
The accumulation of reactive oxygen species (ROS) in cancer cells is a critical factor for the induction of apoptosis by natural products [6,7], since it will result in oxidative DNA damage following by a collapse in mitochondrial membrane potential (MMP) and leakage of cytochrome c, which lead to the activation of the caspase cascade [8]
AMEAE inhibited the proliferation of cancer cells We first examined the cytotoxic effect of three extracts against MCF-7, MDA-MB-231, A549, HepG2 and WRL-68 cell lines by using MTT assay
Summary
Annona muricata leaves have been reported to have antiproliferative effects against various cancer cell lines. The current study was designed to evaluate the molecular mechanisms of A. muricata leaves ethyl acetate extract (AMEAE) against lung cancer A549 cells. Lung cancer as one of the critical causes of cancer death throughout the world has the prevalent complication of apoptosis resistance against different anticancer agents [1]. Thereby, continued research into the development of safe and efficient new anticancer agents against lung cancer cells is urgently necessary for further improvements in cancer therapy. Due to the close correlation between the mechanism of apoptosis and the effect of anticancer agents, extensive research has been done on this mode of cell death [5]. Anticancer agents with the ability to suppress the NF-κB translocation are effectively induce the apoptosis in cancer cells
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