Pukkavadee Netsirisawan1, Daranee Chokchaichamnankit2, Chantragan Srisomsap3, Jisnuson Svasti3, Voraratt Champattanachai4. 1. Applied Biological Sciences Program, Chulabhorn Graduate Institute, Laksi, Bangkok, Thailand. 2. Laboratory of Biochemistry, Chulabhorn Research Institute, Laksi, Bangkok, Thailand. 3. Applied Biological Sciences Program, Chulabhorn Graduate Institute, Laksi, Bangkok, Thailand Laboratory of Biochemistry, Chulabhorn Research Institute, Laksi, Bangkok, Thailand. 4. Applied Biological Sciences Program, Chulabhorn Graduate Institute, Laksi, Bangkok, Thailand Laboratory of Biochemistry, Chulabhorn Research Institute, Laksi, Bangkok, Thailand voraratt@cri.or.th.
Abstract
BACKGROUND: O-GlcNAcylation is a unique intracellular protein modification; however, few extracellular O-GlcNAc-modified proteins have been discovered. We have previously demonstrated that many cellular proteins were aberrant in O-GlcNAcylation in breast cancer tissues. In the present study, therefore, we investigated whether O-GlcNAc-modified proteins were abnormally secreted from breast cancer cells. MATERIALS AND METHODS: Intracellular and extracellular proteins were prepared from cell lysates of breast cancer cells (MCF-7 and MDA-MB-231) and normal breast cells (HMEC) and from their serum-free media (SFM), respectively. O-GlcNAcylation level was examined by immunoblotting. O-GlcNAc-Modified proteins were identified using two-dimensional gel electrophoresis and Liquid Chromatography-tandem Mass Spectrometry. RESULTS: O-GlcNAcylation level was significantly increased in the extracellular compartment of both types of cancer cells compared to normal cells. Interestingly, O-GlcNAc patterns differed between intracellular and extracellular proteins. Proteomic analysis revealed that many O-GlcNAc spots in MCF-7 secretions were abnormally increased in comparison to those in HMEC secretions. Among these, transitional endoplasmic reticulum ATPase (TER ATPase) and heat-shock 70 kDa (HSP70) were confirmed to be O-GlcNAc-modified. The levels of O-GlcNAc-HSP70 and O-GlcNAc-TER ATPase were higher in SFM from MCF-7 cells than in that from HMEC. CONCLUSION: O-GlcNAcomic study of the extracellular compartments reveals aberrant O-GlcNAc-secreted proteins, which may be of interest as potential biomarkers in breast cancer. Copyright
BACKGROUND: O-GlcNAcylation is a unique intracellular protein modification; however, few extracellular O-GlcNAc-modified proteins have been discovered. We have previously demonstrated that many cellular proteins were aberrant in O-GlcNAcylation in breast cancer tissues. In the present study, therefore, we investigated whether O-GlcNAc-modified proteins were abnormally secreted from breast cancer cells. MATERIALS AND METHODS: Intracellular and extracellular proteins were prepared from cell lysates of breast cancer cells (MCF-7 and MDA-MB-231) and normal breast cells (HMEC) and from their serum-free media (SFM), respectively. O-GlcNAcylation level was examined by immunoblotting. O-GlcNAc-Modified proteins were identified using two-dimensional gel electrophoresis and Liquid Chromatography-tandem Mass Spectrometry. RESULTS: O-GlcNAcylation level was significantly increased in the extracellular compartment of both types of cancer cells compared to normal cells. Interestingly, O-GlcNAc patterns differed between intracellular and extracellular proteins. Proteomic analysis revealed that many O-GlcNAc spots in MCF-7 secretions were abnormally increased in comparison to those in HMEC secretions. Among these, transitional endoplasmic reticulum ATPase (TER ATPase) and heat-shock 70 kDa (HSP70) were confirmed to be O-GlcNAc-modified. The levels of O-GlcNAc-HSP70 and O-GlcNAc-TER ATPase were higher in SFM from MCF-7 cells than in that from HMEC. CONCLUSION:O-GlcNAcomic study of the extracellular compartments reveals aberrant O-GlcNAc-secreted proteins, which may be of interest as potential biomarkers in breast cancer. Copyright
Authors: Bethany Mason; Susanne Flach; Felipe R Teixeira; Raquel Manzano Garcia; Oscar M Rueda; Jean E Abraham; Carlos Caldas; Paul A W Edwards; Heike Laman Journal: Cell Mol Life Sci Date: 2019-09-27 Impact factor: 9.261