Associação Portuguesa de Investigação em Cancro
Combined effects of glucosamine and anti-cancer drug «bortezomib»
Combined effects of glucosamine and anti-cancer drug «bortezomib»
Ana Sofia Carvalho1,2, Helena Ribeiro1, Paula Voabil1, Deborah Penque2, Ole N. Jensen3, Henrik Molina4 & Rune Matthiesen*1,2
1Proteolysis in Diseases, IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal.
2Laboratory of Proteomics, Human Genetics Department, National Institute of Health Dr Ricardo Jorge, Av. Padre Cruz, 1649- 016 Lisboa, Portugal.
3Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense.
4Proteomics Resource Center, The Rockefeller University, 1230 York Avenue, New York, NY 10065-6399, USA
Global drug profiling study lead to the interesting discovery that glucosamine can protect cancer cells from the effects of the anti-cancer drug “bortezomib”. We investigated the molecular effects of glucosamine supplements, a popular and safe alternative to non-steroidal anti-inflammatory drugs, for decreasing pain, inflammation and maintaining healthy joints. Numerous studies have reported an array of molecular effects after glucosamine treatment. We questioned whether the differences in the effects observed in previous studies were associated with the focus on a specific subproteome or with the use of specific cell lines or tissues. To address this question, global mass spectrometry- and transcription array-based glucosamine drug profiling was performed on malignant cell lines from different stages of lymphocyte development. We combined global label-free MS-based protein quantitation with an open search for modifications to obtain the best possible proteome coverage. Our data were largely consistent with previous studies in a variety of cellular models. We mainly observed glucosamine induced O-GlcNAcylation/O-GalNAcylation (O-HexNAcylation); however, we also observed global and local changes in acetylation, methylation and phosphorylation. For example, our data provides two additional examples of "yin-yang" between phosphorylation and O-HexNAcylation. Furthermore, we mapped novel O-HexNAc sites on GLU2B and calnexin. GLU2B and calnexin are known to be located in the ER (endoplasmic reticulum) and involved in protein folding and quality control. The O-HexNAc sites were regulated by glucosamine treatment and correlated with the up regulation of the ER stress marker GRP78. The occupancy of the O-HexNAc on GLU2B and calnexin sites differed between the cytosolic and nuclear fractions with a higher occupancy in the cytosolic fraction. Based on our data we propose the hypothesis that O-HexNAc either inactivates calnexin and/or targets it to the cytosolic fraction. Further, we hypothesize that O-HexNAcylation induced by glucosamine treatment enhances protein trafficking. This study was conducted in the group of Rune Matthiesen and is in line with his main research interests, to use MS-based proteomics for global drug profiling studies to obtain a better understanding of how drugs work and for patient stratification in therapeutics. The outcome of this study will now be further explored in the Proteostasis cost action network (http://www.cost.eu/domains_actions/bmbs/Actions/BM1307).
Molecular and Cellular Proteomics
