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Pure Appl. Chem., 2012, Vol. 84, No. 9, pp. 1907-1918

Published online 2012-07-30

Balancing life with glycoconjugates: Monitoring unfolded protein response-mediated anti-angiogenic action of tunicamycin by Raman spectroscopy

Maria O. Longas1*, Ashok Kotapati1, Kilari PVRK Prasad2, Aditi Banerjee3*, Jesus Santiago3, Krishna Baksi4 and Dipak K. Banerjee3,5*

1 Department of Chemistry and Physics, Purdue University Calumet, Hammond, IN 46323-2094, USA
2 Department of Computer and Information Technology, Purdue University Calumet, Hammond, IN 46323-2094, USA
3 Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan, PR 00936-5067, USA
4 Department of Anatomy and Cell Biology, Universidad Central del Caribe, Bayamon, PR 00960-3001, USA
5 Institute of Functional Nanomaterials, University of Puerto Rico, Rio Piedras Campus, San Juan, PR 00931-1907, USA

Abstract: Asparagine-linked protein glycosylation is a hallmark for glycoprotein structure and function. Its impairment by tunicamycin [a competitive inhibitor of N-acetylglucos-aminyl 1-phosphate transferase (GPT)] has been known to inhibit neo-vascularization (i.e., angiogenesis) in humanized breast tumor due to an induction of endoplasmic reticulum (ER) stress-mediated unfolded protein response (UPR). The studies presented here demonstrate that (i) tunicamycin inhibits capillary endothelial cell proliferation in a dose-dependent manner; (ii) treated cells are incapable of forming colonies upon its withdrawal; and (iii) tunicamycin treatment causes nuclear fragmentation. Tunicamycin-induced ER stress-mediated UPR event in these cells was studied with the aid of Raman spectroscopy, in particular, the interpretation of bands at 1672, 1684, and 1694 cm–1, which are characteristics of proteins and originate from C=O stretching vibrations of mono-substituted amides. In tunicamycin-treated cells, these bands decreased in area as follows: at 1672 cm–1 by 41.85 % at 3 h and 55.39 % at 12 h; at 1684 cm–1 by 20.63 % at 3 h and 40.08 % at 12 h; and also at 1994 cm–1 by 33.33 % at 3 h and 32.92 % at 12 h, respectively. Thus, in the presence of tunicamycin, newly synthesized protein chains fail to arrange properly into their final secondary and/or tertiary structures, and the random coils they form had undergone further degradation.