Membrane proteins are an important source of potential targets for anticancer drugs or biomarkers for early diagnosis. In this study, we used a modified aqueous two-phase partition system combined with two-dimensional (2D) matrix-assisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry (MS, 2D-MALDI-TOF-TOF-MS/MS) analysis to isolate and identify membrane proteins in PANC-1 pancreatic cancer cells. Using this method, we identified 55 proteins, of which 31 (56.4 %) were membrane proteins, which, according to gene ontology annotation, are associated with various cellular processes including cell signal transduction, differentiation, and apoptosis. Immunohistochemical analysis showed that the expression level of one of the identified mitochondria membrane proteins, prohibitin 1 (PHB1), is correlated with pancreatic carcinoma differentiation; PHB1 is expressed at a higher level in normal pancreatic tissue than in well-differentiated carcinoma tissue. Further studies showed that PHB1 plays a proapoptotic role in human pancreatic cancer cells, which suggests that PHB1 has antitumorigenic properties. In conclusion, we have provided a modified method for isolating and identifying membrane proteins and demonstrated that PHB1 may be a promising biomarker for early diagnosis and therapy of pancreatic (and potentially other) cancers.
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This study was supported by grants from the Technologies R&D Program of Shandong Province, China (2005GG1102003) and the National High Technology Research and Development Program of China (2007AA021004).
Lallet-Daher H, Roudbaraki M, Bavencoffe A, Mariot P, Gackiere F, Bidaux G, et al. Intermediate-conductance Ca2+-activated K+ channels (IKCa1) regulate human prostate cancer cell proliferation through a close control of calcium entry. Oncogene. 2009;28(15):1792–806. doi:10.1038/onc.2009.25.CrossRefPubMedGoogle Scholar
Schindler J, Lewandrowski U, Sickmann A, Friauf E. Aqueous polymer two-phase systems for the proteomic analysis of plasma membranes from minute brain samples. J Proteome Res. 2008;7(1):432–42. doi:10.1021/pr0704736.CrossRefPubMedGoogle Scholar
Morre DM, Morre DJ. Aqueous two-phase partition applied to the isolation of plasma membranes and Golgi apparatus from cultured mammalian cells. J Chromatogr B Biomed Sci Appl. 2000;743(1–2):377–87.CrossRefPubMedGoogle Scholar
Nuell MJ, Stewart DA, Walker L, Friedman V, Wood CM, Owens GA, et al. Prohibitin, an evolutionarily conserved intracellular protein that blocks DNA synthesis in normal fibroblasts and HeLa cells. Mol Cell Biol. 1991;11:1372–81.CrossRefPubMedPubMedCentralGoogle Scholar
Terashima M, Kim KM, Adachi T, Nielsen PJ, Reth M, Kohler G, et al. The IgM antigen receptor of B lymphocytes is associated with prohibitin and a prohibitin-related protein. EMBO J. 1994;13:3782–92.PubMedPubMedCentralGoogle Scholar
Nijtmans LG, de Jong L, Artal Sanz M, Coates PJ, Berden JA, Back JW, et al. Prohibitins act as a membrane-bound chaperone for the stabilization of mitochondrial proteins. EMBO J. 2000;19:2444–51.CrossRefPubMedPubMedCentralGoogle Scholar
Fusaro G, Dasgupta P, Rastogi S, Joshi B, Chellappan S. Prohibitin induces the transcriptional activity of p53 and is exported from the nucleus upon apoptotic signaling. J Biol Chem. 2003;278:47853–61.CrossRefPubMedGoogle Scholar
Berger KH, Yaffe MP. Prohibitin family members interact genetically with mitochondrial inheritance components in Saccharomyces cerevisiae. Mol Cell Biol. 1998;18(7):4043–52.CrossRefPubMedPubMedCentralGoogle Scholar
Coates PJ, Jamieson DJ, Smart K, Prescott AR, Hall PA. The prohibitin family of mitochondrial proteins regulate replicative lifespan. Curr Biol. 1997;7(8):607–10.CrossRefPubMedGoogle Scholar
Coates PJ, Nenutil R, McGregor A, Picksley SM, Crouch DH, Hall PA, et al. Mammalian prohibitin proteins respond to mitochondrial stress and decrease during cellular senescence. Exp Cell Res. 2001;265(2):262–73. doi:10.1006/excr.2001.5166.CrossRefPubMedGoogle Scholar
Dell’Orco RT, McClung JK, Jupe ER, Liu XT. Prohibitin and the senescent phenotype. Exp Gerontol. 1996;31(1–2):245–52.CrossRefPubMedGoogle Scholar
Wu TF, Wu H, Wang YW, Chang TY, Chan SH, Lin YP, et al. Prohibitin in the pathogenesis of transitional cell bladder cancer. Anticancer Res. 2007;27(2):895–900.PubMedGoogle Scholar
Gregory-Bass RC, Olatinwo M, Xu W, Matthews R, Stiles JK, Thomas K, et al. Prohibitin silencing reverses stabilization of mitochondrial integrity and chemoresistance in ovarian cancer cells by increasing their sensitivity to apoptosis. Int J Cancer. 2008;122(9):1923–30. doi:10.1002/ijc.23351.CrossRefPubMedPubMedCentralGoogle Scholar
Ko KS, Tomasi ML, Iglesias-Ara A, French BA, French SW, Ramani K, et al. Liver-specific deletion of prohibitin 1 results in spontaneous liver injury, fibrosis, and hepatocellular carcinoma in mice. Hepatology. 2010;52(6):2096–108. doi:10.1002/hep.23919.CrossRefPubMedPubMedCentralGoogle Scholar
Zhang L, Ji Q, Ni ZH, Sun J. Prohibitin induces apoptosis in BGC823 gastric cancer cells through the mitochondrial pathway. Asian Pac J Cancer Prev APJCP. 2012;13:3803–7.CrossRefPubMedGoogle Scholar
Sato S, Murata A, Orihara T, Shirakawa T, Suenaga K, Kigoshi H, et al. Marine natural product aurilide activates the OPA1-mediated apoptosis by binding to prohibitin. Chem Biol. 2011;18:131–9.CrossRefPubMedGoogle Scholar
Liu Y-H, Peck K, Lin J-Y (2012) Involvement of prohibitin upregulation in abrin-triggered apoptosis. Evid base Compl Alternative Med 2012:605154Google Scholar
Savulescu D, Feng J, Ping YS, Mai O, Boehm U, He B, et al. Gonadotropin-releasing hormone-regulated prohibitin mediates apoptosis of the gonadotrope cells. Mol Endocrinol. 2013;27:1856–70.CrossRefPubMedGoogle Scholar