Abstract
Annexin V and propidium iodide staining is widely used for determining the cellular death through apoptosis. In the presence of Ca2+ ions, annexin V has a strong binding affinity for phosphatidylserine, a membrane phospholipid that during apoptosis is translocated from the inner side of the cell membrane to its outer side. On the other hand, propidium iodide has ability for DNA binding and it can only enter into necrotic or late apoptotic cells. This chapter describes a commonly used method for detection of apoptosis in a non-small cell lung cancer cell line using annexin V and propidium iodide dye. We describe the detection of different stages of apoptosis in the A549 lung cancer cell line treated with dihydroartemisinin (DHA). This apoptosis detection method can be used to determine the efficacy of different kinds of drugs on cultured cancer cell lines.
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References
Pérez-Garijo A, Fuchs Y, Steller H (2013) Apoptotic cells can induce non-autonomous apoptosis through the TNF pathway. Elife 2:e01004
Deschesnes RG, Huot J, Valerie K et al (2001) Involvement of p38 in apoptosis-associated membrane blebbing and nuclear condensation. Mol Biol Cell 12(6):1569–1582
Willenberg H, Bornstein S, Dumser T et al (1998) Morphological changes in adrenals from victims of suicide in relation to altered apoptosis. Endocr Res 24(3-4):963–967
D’Avila H, Freire-de-Lima CG, Roque NR et al (2011) Host cell lipid bodies triggered by Trypanosoma cruzi infection and enhanced by the uptake of apoptotic cells are associated with prostaglandin E2 generation and increased parasite growth. J Infect Dis 204(6):951–961
Nunez R, Sancho-Martínez S, Novoa J et al (2010) Apoptotic volume decrease as a geometric determinant for cell dismantling into apoptotic bodies. Cell Death Differ 17(11):1665
Eckhart L, Ballaun C, Uthman A et al (2005) Identification and characterization of a novel mammalian caspase with proapoptotic activity. J Biol Chem 280(42):35077–35080
Wei W, Norton DD, Wang X et al (2002) Aβ 17–42 in Alzheimer’s disease activates JNK and caspase-8 leading to neuronal apoptosis. Brain 125(9):2036–2043
Fulda S, Meyer E, Friesen C et al (2001) Cell type specific involvement of death receptor and mitochondrial pathways in drug-induced apoptosis. Oncogene 20(9):1063
Li J, Yang Z, Li Y et al (2016) Cell apoptosis, autophagy and necroptosis in osteosarcoma treatment. Oncotarget 7(28):44763
Chang DW, Xing Z, Pan Y et al (2002) c-FLIPL is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis. EMBO J 21(14):3704–3714
Argun M, Tök L, Uğuz A et al (2014) Melatonin and amfenac modulate calcium entry, apoptosis, and oxidative stress in ARPE-19 cell culture exposed to blue light irradiation (405 nm). Eye 28(6):752
Katoh I, Sato S, Fukunishi N et al (2008) Apaf-1-deficient fog mouse cell apoptosis involves hypo-polarization of the mitochondrial inner membrane, ATP depletion and citrate accumulation. Cell Res 18(12):1210
Caroppi P, Sinibaldi F, Fiorucci L et al (2009) Apoptosis and human diseases: mitochondrion damage and lethal role of released cytochrome C as proapoptotic protein. Curr Med Chem 16(31):4058–4065
Sanchis D, Mayorga M, Ballester M et al (2003) Lack of Apaf-1 expression confers resistance to cytochrome c-driven apoptosis in cardiomyocytes. Cell Death Differ 10(9):977
Schamberger CJ, Gerner C, Cerni C (2005) Caspase-9 plays a marginal role in serum starvation-induced apoptosis. Exp Cell Res 302(1):115–128
Arbab IA, Abdul AB, Sukari MA et al (2013) Dentatin isolated from Clausena excavata induces apoptosis in MCF-7 cells through the intrinsic pathway with involvement of NF-κB signalling and G0/G1 cell cycle arrest: a bioassay-guided approach. J Ethnopharmacol 145(1):343–354
Liu G, Pei F, Yang F et al (2017) Role of autophagy and apoptosis in non-small-cell lung cancer. Int J Mol Sci 18(2):367
Pore MM, Hiltermann TJN, Kruyt FA (2013) Targeting apoptosis pathways in lung cancer. Cancer Lett 332(2):359–368
Crowley LC, Marfell BJ, Scott AP et al (2016) Quantitation of apoptosis and necrosis by annexin V binding, propidium iodide uptake, and flow cytometry. Cold Spring Harb Protoc 2016(11):pdb prot087288
Hingorani R, Deng J, Elia J et al (2011) Detection of apoptosis using the BD Annexin V FITC assay on the BD FACSVerse™ system. BD Biosciences 1:1–12
Kumar R, Singh M, Meena J et al (2019) Hyaluronic acid-dihydroartemisinin conjugate: synthesis, characterization and in vitro evaluation in lung cancer cells. Int J Biol Macromol 133:495–502
Wlodkowic D, Skommer J, Darzynkiewicz Z (2009) Flow cytometry-based apoptosis detection. Apoptosis. Humana Press, New Jersey
Acknowledgments
The authors acknowledge the financial support from National Institute of Immunology for carrying out this research.
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Kumar, R., Saneja, A., Panda, A.K. (2021). An Annexin V-FITC—Propidium Iodide-Based Method for Detecting Apoptosis in a Non-Small Cell Lung Cancer Cell Line. In: Santiago-Cardona, P.G. (eds) Lung Cancer. Methods in Molecular Biology, vol 2279. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1278-1_17
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DOI: https://doi.org/10.1007/978-1-0716-1278-1_17
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