Evaluation of autophagy induction and inhibition in the Huh7.5 cell line through flow cytometry
Archives of Medical Laboratory Sciences,
Vol. 3 No. 4 (2017),
26 September 2018
https://doi.org/10.22037/amls.v3i4.19821
Abstract
Background: Autophagy is a physiologic process in which double membrane vesicles engulf damaged proteins and organelles for delivering them to lysosomein order to degrade and recycle them via lysosomal digestion. Beclin1 is one of the basic proteins involved in the initial step of autophagosome formation. In the current study, the effect of exogenous Beclin1 to induce autophagy and the effect of 3MA to inhibit of autophagy was assessed in Huh7.5 cells as an in vitro models of hepatocellular carcinoma.
Material and methods: The Recombinant pcDNA-Beclin1was transfected into Huh7.5 cells. Also, the cell treated with 3MA. Next, the autophagy induction and inhibition was conducted via LC3 staining as a main autophagy marker using flow cytometry.
Results: The result of this study suggest that the over expression of exogenous Beclin1 in Huh7.5 cells elevated the autophagosome formation as shown by intracellular autophagosomal marker LC3-II staining for about 32.32 % and 3MA decreased it up to2% in compared with control cells in which the stained LC3-II was12.08.
Conclusion: Recombinant beclin1 may be used as a potential autophagy inducer agent and 3-methyl-Adenin inhibits autophagy formation in Huh7.5 cell. The staining autophagy formation marker LC3-II with specific antibody is a reliable method to measure autophagy activation via flow cytometry.
- Autophagy
- Flow cytometry
- LC3-II
How to Cite
References
Kudchodkar SB, Levine B. Viruses and autophagy. Reviews in medical virology. 2009;19(6):359-78.
Sir D, Tian Y, Chen W-l, Ann DK, Yen T-SB, Ou J-hJ. The early autophagic pathway is activated by hepatitis B virus and required for viral DNA replication. Proceedings of the National Academy of Sciences. 2010;107(9):4383-8.
Tanida I, Ueno T, Kominami E. LC3 and Autophagy. Autophagosome and Phagosome. 2008:77-88.
Geng J, Klionsky DJ. The Atg8 and Atg12 ubiquitin‐like conjugation systems in macroautophagy. EMBO reports. 2008;9(9):859-64.
Lorin S, Hamaï A, Mehrpour M, Codogno P, editors. Autophagy regulation and its role in cancer. Seminars in cancer biology; 2013: Elsevier.
Llovet JM, Bruix J. Molecular targeted therapies in hepatocellular carcinoma. Hepatology. 2008;48(4):1312-27.
Kang M, Jeong CW, Ku JH, Kwak C, Kim HH. Inhibition of autophagy potentiates atorvastatin-induced apoptotic cell death in human bladder cancer cells in vitro. International journal of molecular sciences. 2014;15(5):8106-21.
Sun K, Guo X, Zhao Q. Paradoxical role of autophagy in the dysplastic and tumor-forming stages of hepatocarcinoma development in rats. Cell death & disease. 2013;4(2):e501.
Zhang M, Su L, Xiao Z, Liu X, Liu X. Methyl jasmonate induces apoptosis and pro-apoptotic autophagy via the ROS pathway in human non-small cell lung cancer. American journal of cancer research. 2016;6(2):187.
Bartenschlager R, Pietschmann T. Efficient hepatitis C virus cell culture system: what a difference the host cell makes. Proceedings of the National Academy of Sciences of the United States of America. 2005;102(28):9739-40.
Marino G, Niso-Santano M, Baehrecke EH, Kroemer G. Self-consumption: the interplay of autophagy and apoptosis. Nature reviews Molecular cell biology. 2014;15(2):81.
Blight KJ, McKeating JA, Marcotrigiano J, Rice CM. Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture. Journal of virology. 2003;77(5):3181-90.
Taji F, Kouchesfahani HM, Sheikholeslami F, Romani B, Baesi K, Vahabpour R, et al. Autophagy induction reduces telomerase activity in HeLa cells. Mechanisms of ageing and development. 2017;163:40-5.
Eng C, Abraham R. The autophagy conundrum in cancer: influence of tumorigenic metabolic reprogramming. Oncogene. 2011;30(47):4687-96.
Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. The EMBO journal. 2000;19(21):5720-8.
Feizi N, Mehrbod P, Romani B, Soleimanjahi H, Bamdad T, Feizi A, et al. Autophagy induction regulates influenza virus replication in a time-dependent manner. Journal of Medical Microbiology. 2017;66(4):536-41.
Singh P, Godbole M, Rao G, Annarao S, Mitra K, Roy R, et al. Inhibition of autophagy stimulate molecular iodine-induced apoptosis in hormone independent breast tumors. Biochemical and biophysical research communications. 2011;415(1):181-6.
Palmeira-dos-Santos C, Pereira GJ, Barbosa CM, Jurkiewicz A, Smaili SS, Bincoletto C. Comparative study of autophagy inhibition by 3MA and CQ on Cytarabine-induced death of leukaemia cells. Journal of cancer research and clinical oncology. 2014;140(6):909-20.
Mizushima N, Yoshimori T, Levine B. Methods in mammalian autophagy research. Cell. 2010;140(3):313-26.
Mizushima N, Yamamoto A, Hatano M, Kobayashi Y, Kabeya Y, Suzuki K, et al. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. The Journal of cell biology. 2001;152(4):657-68.
- Abstract Viewed: 244 times
- PDF Downloaded: 117 times