Asparagine-linked protein glycosylation is a hallmark for glycoprotein structure and function. Its impairment by tunicamycin [a competitive inhibitor of N-acetylglucosaminyl 1-phosphate transferase (GPT)] has been known to inhibit neo-vascularization (i.e., angiogenesis) in humanized breast tumor due to an induction of ER stress-mediated unfolded protein response (UPR). The studies presented here demonstrate that (i) tunicamycin (i) 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.

Angiognesis (proliferation and differentiation of capillary endothelial cells) are essential for normal growth and development as well as for solid tumor progression and metastasis. Asparagine‐linked (N‐linked) glycoproteins play an important role in angiogenesis; “hybrid” but not “complex” type glycans are required for capillary tube formation. Our objective is to understand the dynamic relationship between protein N‐glycosylation and angiogenesis. Using a protein N‐glycosylation inhibitor tunicamycin, we establish that angiogenesis inhibition in humanized breast tumor is due to ER stress‐mediated unfolded protein response (upr). For further evaluation, the proteome was analyzed. The Raman Spectroscopy focused on bands that are characteristics of proteins and originate from C=O stretching vibrations of mono‐substituted amides. In tunicamycin‐treated cells the area under these bands are decreased: 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 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 undergo further degradation. Supported in part by grants from Susan G. Komen for the Cure BCTR0600582 (DKB) and NIH/NIMHD 8G12MD007583 (KB).