PE‑induced apoptosis in SMMC‑7721 cells: Involvement of Erk and Stat signalling pathways

Xue,Li; Li,Ming; Chen,Teng; Sun,Haifeng; Zhu,Jie; Li,Xia; Wu,Feng; Wang,Biao; Li,Juping; Chen,Yanjiong

doi:10.3892/ijmm.2014.1777

PE‑induced apoptosis in SMMC‑7721 cells: Involvement of Erk and Stat signalling pathways

Authors:
- Li Xue
- Ming Li
- Teng Chen
- Haifeng Sun
- Jie Zhu
- Xia Li
- Feng Wu
- Biao Wang
- Juping Li
- Yanjiong Chen
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Affiliations: Forensic Medicine College of Xi'an Jiaotong University, Key Laboratory of the Health Ministry for Forensic Medicine, Key Laboratory of the Ministry of Education for Environment and Genes Related to Diseases, Xi'an, Shaanxi 710061, P.R. China, Department of Cardiovascular Surgery, The First Affiliated Hospital of Medical College of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China, Tumour Hospital of Shaanxi Province, Xi'an, Shaanxi 710061, P.R. China, VIP Internal Medicine Department, Lanzhou University Second Hospital, Lanzhou, Gansu 730030, P.R. China, Graduate Teaching and Experimental Centre, Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi 710061, P.R. China, Department of Immunology and Pathogenic Biology, Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi 710061, P.R. China, School of Public Security, Northwest University of Politics and Law, Xi'an, Shaanxi 710063, P.R. China
Published online on: May 9, 2014 https://doi.org/10.3892/ijmm.2014.1777
Pages: 119-129
Copyright: © Xue et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].

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Abstract

Emerging evidence indicates that the redistribution of phosphatidylethanolamine (PE) across the bilayer of the plasma membrane is an important molecular marker for apoptosis. However, the effect of PE on apoptosis and the underlying mechanism of PE remain unclear. In the current study, MTT and flow cytometric assays were used to examine the effects of PE on apoptosis in SMMC‑7721 cells. The level of mitochondrial membrane potential (ΔΨm) and the expression of Bax, Bcl‑2, caspase‑3, phospho‑Erk and phospho‑Stat1/2 in SMMC‑7721 cells that were exposed to PE were also investigated. The results showed that PE inhibited proliferation, caused G0/G1 phase cell cycle arrest and induced apoptosis in SMMC‑7721 cells in a dose‑dependent manner. Rhodamine 123 staining showed that the treatment of SMMC‑7721 cells with different concentrations of PE for 24 h significantly decreased the level of ΔΨm and exerted dose‑dependent effects. Using immunofluorescence and western blotting, we found that the expression of Bax was upregulated, whereas that of Bcl‑2 was downregulated in PE‑induced apoptotic cells. In addition, these events were accompanied by an increase in caspase‑3 expression in a dose‑dependent manner following PE treatment. PE‑induced apoptosis was accompanied by a decrease in Erk phosphorylation and by the activation of Stat1/2 phosphorylation in SMMC‑7721 cells. In conclusion, the results suggested that PE‑induced apoptosis is involved in upregulating the Bax/Bcl‑2 protein ratio and decreasing the ΔΨm. Moreover, the results showed that the Erk and Stat1/2 signalling pathways may be involved in the process of PE‑induced apoptosis.

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1	Zhao M: Lantibiotics as probes for phosphatidylethanolamine. Amino Acids. 41:1071–1079. 2011. View Article : Google Scholar : PubMed/NCBI
2	Post JA, Bijvelt JJ and Verkleij AJ: Phosphatidylethanolamine and sarcolemmal damage during ischemia or metabolic inhibition of heart myocytes. Am J Physiol. 268:H773–H780. 1995.PubMed/NCBI
3	Zhao M, Li Z and Bugenhagen S: 99mTc-labeled duramycin as a novel phosphatidylethanolamine-binding molecular probe. J Nucl Med. 49:1345–1352. 2008. View Article : Google Scholar : PubMed/NCBI
4	Yao Y, Huang C, Li ZF, et al: Exogenous phosphatidylethanolamine induces apoptosis of human hepatoma HepG2 cells via the bcl-2/Bax pathway. World J Gastroenterol. 15:1751–1758. 2009. View Article : Google Scholar : PubMed/NCBI
5	Keeble JA and Gilmore AP: Apoptosis commitment-translating survival signals into decisions on mitochondria. Cell Res. 17:976–984. 2007. View Article : Google Scholar : PubMed/NCBI
6	Itoh K, Hase H, Kojima H, Saotome K, Nishioka K and Kobata T: Central role of mitochondria and p53 in Fas-mediated apoptosis of rheumatoid synovial fibroblasts. Rheumatology (Oxford). 43:277–285. 2004. View Article : Google Scholar : PubMed/NCBI
7	Suen DF, Norris KL and Youle RJ: Mitochondrial dynamics and apoptosis. Genes Dev. 22:1577–1590. 2008. View Article : Google Scholar : PubMed/NCBI
8	Adams JM and Cory S: The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene. 26:1324–1337. 2007. View Article : Google Scholar : PubMed/NCBI
9	Chipuk JE and Green DR: How do BCL-2 proteins induce mitochondrial outer membrane permeabilization? Trends Cell Biol. 18:157–164. 2008. View Article : Google Scholar : PubMed/NCBI
10	Jiang L, Liu Y, Ma MM, Tang YB, Zhou JG and Guan YY: Mitochondria dependent pathway is involved in the protective effect of bestrophin-3 on hydrogen peroxide-induced apoptosis in basilar artery smooth muscle cells. Apoptosis. 18:556–565. 2013. View Article : Google Scholar
11	Brunelle JK and Letai A: Control of mitochondrial apoptosis by the Bcl-2 family. J Cell Sci. 122:437–441. 2009. View Article : Google Scholar : PubMed/NCBI
12	Fan Y, Chen H, Qiao B, et al: Opposing effects of ERK and p38 MAP kinases on HeLa cell apoptosis induced by dipyrithione. Mol Cells. 23:30–38. 2007.PubMed/NCBI
13	Hetman M, Kanning K, Cavanaugh JE and Xia Z: Neuroprotection by brain-derived neurotrophic factor is mediated by extracellular signal-regulated kinase and phosphatidylinositol 3-kinase. J Biol Chem. 274:22569–22580. 1999. View Article : Google Scholar : PubMed/NCBI
14	Tran SE, Holmstrom TH, Ahonen M, Kahari VM and Eriksson JE: MAPK/ERK overrides the apoptotic signaling from Fas, TNF, and TRAIL receptors. J Biol Chem. 276:16484–16490. 2001. View Article : Google Scholar : PubMed/NCBI
15	Boucher MJ, Morisset J, Vachon PH, Reed JC, Lainé J and Rivard N: MEK/ERK signaling pathway regulates the expression of Bcl-2, Bcl-X(L), and Mcl-1 and promotes survival of human pancreatic cancer cells. J Cell Biochem. 79:355–369. 2000. View Article : Google Scholar : PubMed/NCBI
16	Kovarik P, Mangold M, Ramsauer K, et al: Specificity of signaling by STAT1 depends on SH2 and C-terminal domains that regulate Ser727 phosphorylation, differentially affecting specific target gene expression. EMBO J. 20:91–100. 2001. View Article : Google Scholar
17	Battle TE and Frank DA: The role of STATs in apoptosis. Curr Mol Med. 2:381–392. 2002. View Article : Google Scholar : PubMed/NCBI
18	Wen Z, Zhong Z and Darnell JE Jr: Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell. 82:241–250. 1995. View Article : Google Scholar : PubMed/NCBI
19	Levy DE and Darnell JE Jr: Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol. 3:651–662. 2002. View Article : Google Scholar : PubMed/NCBI
20	Schindler C, Shuai K, Prezioso VR and Darnell JE Jr: Pillars article: interferon-dependent tyrosine phosphorylation of a latent cytoplasmic transcription factor. Science. 257:809–813. 1992. View Article : Google Scholar
21	Townsend PA, Scarabelli TM, Davidson SM, Knight RA, Latchman DS and Stephanou A: STAT-1 interacts with p53 to enhance DNA damage-induced apoptosis. J Biol Chem. 279:5811–5820. 2004. View Article : Google Scholar : PubMed/NCBI
22	Townsend PA, Cragg MS, Davidson SM, et al: STAT-1 facilitates the ATM activated checkpoint pathway following DNA damage. J Cell Sci. 118:1629–1639. 2005. View Article : Google Scholar : PubMed/NCBI
23	Stephanou A, Brar BK, Knight RA and Latchman DS: Opposing actions of STAT-1 and STAT-3 on the Bcl-2 and Bcl-x promoters. Cell Death Differ. 7:329–330. 2000. View Article : Google Scholar : PubMed/NCBI
24	Pérez MJ and Cederbaum AI: Antioxidant and pro-oxidant effects of a manganese porphyrin complex against CYP2E1-dependent toxicity. Free Radic Biol Med. 33:111–127. 2002.PubMed/NCBI
25	Baracca A, Sgarbi G, Solaini G and Lenaz G: Rhodamine 123 as a probe of mitochondrial membrane potential: evaluation of proton flux through F(0) during ATP synthesis. Biochim Biophys Acta. 1606:137–146. 2003. View Article : Google Scholar : PubMed/NCBI
26	Nera MS, Vanderbeek G, Johnson RO, Ruben LN and Clothier RH: Phosphatidylserine expression on apoptotic lymphocytes of Xenopus laevis, the South African clawed toad, as a signal for macrophage recognition. Dev Comp Immunol. 24:641–652. 2000.PubMed/NCBI
27	Murphy KM, Ranganathan V, Farnsworth ML, Kavallaris M and Lock RB: Bcl-2 inhibits Bax translocation from cytosol to mitochondria during drug-induced apoptosis of human tumor cells. Cell Death Differ. 7:102–111. 2000. View Article : Google Scholar : PubMed/NCBI
28	Johnson TL, Lai MB, Lai JC and Bhushan A: Inhibition of cell proliferation and MAP kinase and akt pathways in oral squamous cell carcinoma by genistein and biochanin A. Evid Based Complement Alternat Med. 7:351–358. 2010. View Article : Google Scholar : PubMed/NCBI
29	Hellebrand EE and Varbiro G: Development of mitochondrial permeability transition inhibitory agents: a novel drug target. Drug Discov Ther. 4:54–61. 2010.PubMed/NCBI
30	Tomasello F, Messina A, Lartigue L, et al: Outer membrane VDAC1 controls permeability transition of the inner mitochondrial membrane in cellulo during stress-induced apoptosis. Cell Res. 19:1363–1376. 2009. View Article : Google Scholar
31	Orrenius S: Mitochondrial regulation of apoptotic cell death. Toxicol Lett. 149:19–23. 2004. View Article : Google Scholar : PubMed/NCBI
32	Ling YH, Lin R and Perez-Soler R: Erlotinib induces mitochondrial-mediated apoptosis in human H3255 non-small-cell lung cancer cells with epidermal growth factor receptorL858R mutation through mitochondrial oxidative phosphorylation-dependent activation of BAX and BAK. Mol Pharmacol. 74:793–806. 2008. View Article : Google Scholar
33	Kirkin V, Joos S and Zörnig M: The role of Bcl-2 family members in tumorigenesis. Biochim Biophys Acta. 1644:229–249. 2004. View Article : Google Scholar : PubMed/NCBI
34	Adams JM and Cory S: Life-or-death decisions by the Bcl-2 protein family. Trends Biochem Sci. 26:61–66. 2001. View Article : Google Scholar : PubMed/NCBI
35	Newmeyer DD and Ferguson-Miller S: Mitochondria: releasing power for life and unleashing the machineries of death. Cell. 112:481–490. 2003. View Article : Google Scholar : PubMed/NCBI
36	Tsujimoto Y: Cell death regulation by the Bcl-2 protein family in the mitochondria. J Cell Physiol. 195:158–167. 2003. View Article : Google Scholar : PubMed/NCBI
37	Baek D, Nam J, Koo YD, et al: Bax-induced cell death of Arabidopsis is meditated through reactive oxygen-dependent and -independent processes. Plant Mol Biol. 56:15–27. 2004. View Article : Google Scholar
38	Kondo K, Obitsu S, Ohta S, Matsunami K, Otsuka H and Teshima R: Poly(ADP-ribose) polymerase (PARP)-1-independent apoptosis-inducing factor (AIF) release and cell death are induced by eleostearic acid and blocked by alpha-tocopherol and MEK inhibition. J Biol Chem. 285:13079–13091. 2010. View Article : Google Scholar : PubMed/NCBI
39	Gogvadze V, Norberg E, Orrenius S and Zhivotovsky B: Involvement of Ca²⁺and ROS in alpha-tocopheryl succinate- induced mitochondrial permeabilization. Int J Cancer. 127:1823–1832. 2010.
40	Yip KW and Reed JC: Bcl-2 family proteins and cancer. Oncogene. 27:6398–6406. 2008. View Article : Google Scholar : PubMed/NCBI
41	Kroemer G and Reed JC: Mitochondrial control of cell death. Nat Med. 6:513–519. 2000. View Article : Google Scholar
42	Hengartner MO: The biochemistry of apoptosis. Nature. 407:770–776. 2000. View Article : Google Scholar : PubMed/NCBI
43	Paris C, Bertoglio J and Bréard J: Lysosomal and mitochondrial pathways in miltefosine-induced apoptosis in U937 cells. Apoptosis. 12:1257–1267. 2007. View Article : Google Scholar : PubMed/NCBI
44	Johnson GL and Lapadat R: Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science. 298:1911–1912. 2002. View Article : Google Scholar : PubMed/NCBI
45	Wang X, Martindale JL and Holbrook NJ: Requirement for ERK activation in cisplatin-induced apoptosis. J Biol Chem. 275:39435–39443. 2000. View Article : Google Scholar
46	Bacus SS, Gudkov AV, Lowe M, et al: Taxol-induced apoptosis depends on MAP kinase pathways (ERK and p38) and is independent of p53. Oncogene. 20:147–155. 2001. View Article : Google Scholar : PubMed/NCBI
47	Chang L and Karin M: Mammalian MAP kinase signalling cascades. Nature. 410:37–40. 2001. View Article : Google Scholar : PubMed/NCBI
48	Tamura Y, Simizu S and Osada H: The phosphorylation status and anti-apoptotic activity of Bcl-2 are regulated by ERK and protein phosphatase 2A on the mitochondria. FEBS Lett. 569:249–255. 2004. View Article : Google Scholar : PubMed/NCBI
49	Olson JM and Hallahan AR: p38 MAP kinase: a convergence point in cancer therapy. Trends Mol Med. 10:125–129. 2004. View Article : Google Scholar : PubMed/NCBI
50	Dhanasekaran DN and Reddy EP: JNK signaling in apoptosis. Oncogene. 27:6245–6251. 2008. View Article : Google Scholar
51	Yang J and Stark GR: Roles of unphosphorylated STATs in signaling. Cell Res. 18:443–451. 2008. View Article : Google Scholar : PubMed/NCBI
52	Lee HJ, Oh YK, Rhee M, et al: The role of STAT1/IRF-1 on synergistic ROS production and loss of mitochondrial transmembrane potential during hepatic cell death induced by LPS/d-GalN. J Mol Biol. 369:967–984. 2007. View Article : Google Scholar : PubMed/NCBI
53	Krasilnikov M, Ivanov VN, Dong J and Ronai Z: ERK and PI3K negatively regulate STAT-transcriptional activities in human melanoma cells: implications towards sensitization to apoptosis. Oncogene. 22:4092–4101. 2003. View Article : Google Scholar
54	Xu B, Abdel-Fattah R, Yang L, Crenshaw SA, Black MB and Hinton BT: Testicular lumicrine factors regulate ERK, STAT, and NFKB pathways in the initial segment of the rat epididymis to prevent apoptosis. Biol Reprod. 84:1282–1291. 2011. View Article : Google Scholar : PubMed/NCBI