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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.

Authors+Show Affiliations

No affiliation info availableBiomolecular Research Group, Biochemistry Program, Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia. habsah@um.edu.my.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

25127718

Citation

Moghadamtousi, Soheil Zorofchian, et al. "Annona Muricata Leaves Induced Apoptosis in A549 Cells Through Mitochondrial-mediated Pathway and Involvement of NF-κB." BMC Complementary and Alternative Medicine, vol. 14, 2014, p. 299.
Moghadamtousi SZ, Kadir HA, Paydar M, et al. Annona muricata leaves induced apoptosis in A549 cells through mitochondrial-mediated pathway and involvement of NF-κB. BMC Complement Altern Med. 2014;14:299.
Moghadamtousi, S. Z., Kadir, H. A., Paydar, M., Rouhollahi, E., & Karimian, H. (2014). Annona muricata leaves induced apoptosis in A549 cells through mitochondrial-mediated pathway and involvement of NF-κB. BMC Complementary and Alternative Medicine, 14, p. 299. doi:10.1186/1472-6882-14-299.
Moghadamtousi SZ, et al. Annona Muricata Leaves Induced Apoptosis in A549 Cells Through Mitochondrial-mediated Pathway and Involvement of NF-κB. BMC Complement Altern Med. 2014 Aug 15;14:299. PubMed PMID: 25127718.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Annona muricata leaves induced apoptosis in A549 cells through mitochondrial-mediated pathway and involvement of NF-κB. AU - Moghadamtousi,Soheil Zorofchian, AU - Kadir,Habsah Abdul, AU - Paydar,Mohammadjavad, AU - Rouhollahi,Elham, AU - Karimian,Hamed, Y1 - 2014/08/15/ PY - 2014/06/19/received PY - 2014/08/05/accepted PY - 2014/8/17/entrez PY - 2014/8/17/pubmed PY - 2014/10/24/medline SP - 299 EP - 299 JF - BMC complementary and alternative medicine JO - BMC Complement Altern Med VL - 14 N2 - 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. SN - 1472-6882 UR - https://www.unboundmedicine.com/medline/citation/25127718/Annona_muricata_leaves_induced_apoptosis_in_A549_cells_through_mitochondrial_mediated_pathway_and_involvement_of_NF_κB_ L2 - https://bmccomplementalternmed.biomedcentral.com/articles/10.1186/1472-6882-14-299 DB - PRIME DP - Unbound Medicine ER -