title: Overexpression of 4EBP1, p70S6K, Akt1 or Akt2 differentially promotes Coxsackievirus B3-induced apoptosis in HeLa cells.
abstract: Our previous studies have shown that the inhibition of phosphatidylinositol 3-kinase (PI3K) or mTOR complex 1 can obviously promote the Coxsackievirus B3 (CVB3)-induced apoptosis of HeLa cells by regulating the expression of proapoptotic factors. To further illustrate it, Homo sapiens eIF4E-binding protein 1 (4EBP1), p70S6 kinase (p70S6K), Akt1 and Akt2 were transfected to HeLa cells, respectively. And then, we established the stable transfected cell lines. Next, after CVB3 infection, apoptosis in different groups was determined by flow cytometry; the expressions of Bim, Bax, caspase-9 and caspase-3 were examined by real-time fluorescence quantitative PCR and western blot analysis; the expression of CVB3 mRNA and viral capsid protein VP1 were also analyzed by real-time fluorescence quantitative PCR, western blot analysis and immunofluorescence, respectively. At the meantime, CVB3 replication was observed by transmission electron microscope. We found that CVB3-induced cytopathic effect and apoptosis in transfected groups were more obvious than that in controls. Unexpectedly, apoptosis rate in Akt1 group was higher than others at the early stage after viral infection and decreased with the viral-infected time increasing, which was opposite to other groups. Compared with controls, the expression of CVB3 mRNA was increased at 3, 6, 12 and 24 h postinfection (p. i.) in all groups. At the meantime, VP1 expression in 4EBP1 group was higher than control during the process of infection, while the expressions in the other groups were change dynamically. Moreover, overexpression of 4EBP1 did not affect the mRNA expressions of Bim, Bax, caspase-9 and caspase-3; while protein expressions of Bim and Bax were decreased, the self-cleavages of caspase-9 and caspase-3 were stimulated. Meanwhile, overexpression of p70S6K blocked the CVB3-induced Bim, Bax and caspase-9 expressions but promoted the self-cleavage of caspase-9. In the Akt1 group, it is noteworthy that the expressions of Bim protein were higher than controls at 3 and 6 h p. i. but lower at 24 h p. i., and the expression of Bax protein were higher at 6 and 24 h p. i., while their mRNA expressions were all decreased. Furthermore, overexpression of Akt1 stimulated the procaspase-9 and procaspase-3 expression but blocked their self-cleavages. Overexpression of Akt2, however, had little effect on Bim, Bax and caspase-3, while prevented caspase-9 from self-cleavage at the late stage of CVB3 infection. As stated above, our results demonstrated that overexpression of 4EBP1, p70S6K, Akt1 or Akt2 could promote the CVB3-induced apoptosis in diverse degree via different mediating ways in viral replication and proapoptotic factors in BcL-2 and caspase families. As 4EBP1, p70S6K and Akt are the important substrates of PI3K and mammalian target of rapamycin (mTOR), we further illustrated the role of PI3K/Akt/mTOR signaling pathway in the process of CVB3-induced apoptosis.
authors.affiliation: Department of Pediatrics, The Third Xiangya Hospital, Central South University, Changsha, China.
authors.forename: X
authors.initials: X
authors.lastname: Li
authors.forename: Z
authors.initials: Z
authors.lastname: Li
authors.forename: W
authors.initials: W
authors.lastname: Zhou
authors.forename: X
authors.initials: X
authors.lastname: Xing
authors.forename: L
authors.initials: L
authors.lastname: Huang
authors.forename: L
authors.initials: L
authors.lastname: Tian
authors.forename: J
authors.initials: J
authors.lastname: Chen
authors.forename: C
authors.initials: C
authors.lastname: Chen
authors.forename: X
authors.initials: X
authors.lastname: Ma
authors.forename: Z
authors.initials: Z
authors.lastname: Yang
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D000072224:Bcl-2-Like Protein 11; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D053453:Caspase 9 / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins; D003588:Cytopathogenic Effect, Viral; D004770:Enterovirus / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D015967:Gene Expression Regulation, Viral; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008954:Models, Biological; D010750:Phosphoproteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism*; D014162:Transfection; D014764:Viral Proteins / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; D018797:Cell Cycle Proteins; C087000:EIF4EBP1 protein, human; D008565:Membrane Proteins; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D014764:Viral Proteins; D051028:bcl-2-Associated X Protein; C494918:AKT1 protein, human; C494921:AKT2 protein, human; D051057:Proto-Oncogene Proteins c-akt; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D053148:Caspase 3; D053453:Caspase 9
references.citation: Sci Signal. 2012 Mar 27;5(217):ra25
references.pmid: 22457331
references.citation: Nature. 2006 May 25;441(7092):424-30
references.pmid: 16724053
references.citation: PLoS One. 2013 Apr 26;8(4):e60730
references.pmid: 23637762
references.citation: Toxicol Appl Pharmacol. 2013 Mar 15;267(3):228-37
references.pmid: 23352504
references.citation: J Virol. 2011 Jan;85(1):296-304
references.pmid: 20980509
references.citation: J Biol Chem. 2010 Jun 4;285(23):17673-80
references.pmid: 20356836
references.citation: Mol Cancer Ther. 2011 Aug;10(8):1440-9
references.pmid: 21632463
references.citation: Cell. 2007 Jun 29;129(7):1337-49
references.pmid: 17604722
references.citation: J Virol. 2006 Sep;80(17):8422-38
references.pmid: 16912293
references.citation: Cell Microbiol. 2012 Jan;14(1):133-46
references.pmid: 21981022
references.citation: J Biol Chem. 2012 Jun 22;287(26):21796-805
references.pmid: 22556409
references.citation: Cell Death Dis. 2012 Nov 15;3:e421
references.pmid: 23152057
references.citation: Gynecol Oncol. 2013 Feb;128(2):383-90
references.pmid: 23107612
references.citation: Hepatology. 2009 Jul;50(1):216-29
references.pmid: 19437488
references.citation: J Thromb Haemost. 2013 Jan;11(1):149-60
references.pmid: 23140172
references.citation: PLoS One. 2013;8(4):e60685
references.pmid: 23577147
references.citation: Nucleic Acids Res. 2012 Jan;40(2):761-74
references.pmid: 21917858
references.citation: Toxicon. 2013 Aug;70:142-52
references.pmid: 23643883
references.citation: Mol Biol Cell. 2012 Sep;23(17):3357-69
references.pmid: 22809628
references.citation: Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2008 Jul;33(7):612-7
references.pmid: 18667775
references.citation: PLoS One. 2012;7(4):e35413
references.pmid: 22523592
references.citation: Cell Signal. 2013 Aug;25(8):1711-9
references.pmid: 23603750
references.citation: Cancer Biol Ther. 2007 May;6(5):755-62
references.pmid: 17426444
references.citation: J Biol Chem. 2013 Jan 4;288(1):368-81
references.pmid: 23152504
references.citation: J Biol Chem. 2009 Feb 13;284(7):4679-88
references.pmid: 19064993
references.citation: J Biol Chem. 2008 Dec 19;283(51):35375-82
references.pmid: 18952604
references.citation: Adv Cancer Res. 2005;94:29-86
references.pmid: 16095999
references.citation: Oncogene. 2005 Nov 14;24(50):7435-42
references.pmid: 16288290
references.citation: PLoS One. 2012;7(6):e37782
references.pmid: 22719850
references.citation: J Infect Dis. 2013 Aug 1;208(3):528-38
references.pmid: 23613163
references.citation: Science. 2007 Feb 9;315(5813):856-9
references.pmid: 17289999
references.citation: J Virol. 2004 Oct;78(20):11030-9
references.pmid: 15452223
references.citation: Cell. 2007 Jan 12;128(1):25-8
references.pmid: 17218252
references.citation: Proc Natl Acad Sci U S A. 2001 Sep 25;98(20):10983-5
references.pmid: 11572954
references.citation: PLoS One. 2011;6(11):e27509
references.pmid: 22110663
references.citation: Cell Death Differ. 2013 Feb;20(2):343-52
references.pmid: 23197294
references.citation: PLoS One. 2012;7(6):e39586
references.pmid: 22761832
references.citation: J Virol. 2004 Apr;78(8):4289-98
references.pmid: 15047842
references.citation: J Biol Chem. 2003 Mar 7;278(10):8091-8
references.pmid: 12506111
references.citation: Cell Metab. 2012 Jun 6;15(6):861-72
references.pmid: 22632897
references.citation: Biochem J. 2007 Jul 1;405(1):11-9
references.pmid: 17437405
references.citation: Cell Death Dis. 2012 May 17;3:e309
references.pmid: 22592316
references.citation: Circ Res. 2006 Aug 18;99(4):354-61
references.pmid: 16840719
references.citation: DNA Cell Biol. 2013 Jul;32(7):359-70
references.pmid: 23705975
references.citation: J Virol. 2006 Feb;80(4):2019-33
references.pmid: 16439558
references.citation: Trends Biochem Sci. 2003 Mar;28(3):130-6
references.pmid: 12633992
references.citation: Cell Death Differ. 2013 Aug;20(8):1043-54
references.pmid: 23645209
references.citation: Cell Microbiol. 2007 Oct;9(10):2358-71
references.pmid: 17848167
references.citation: PLoS One. 2012;7(3):e33266
references.pmid: 22432009