GRP78 (an M_r 78 kDa calcium dependent glucose binding protein) located in ER lumen. It functions as ER chaperone and translocates proteins for glycosylation at the asparagine residue present in the sequon Asn-X-Ser/Thr. Paraffin sections from N-glycosylation inhibitor tunicamycin treated ER^-/PR^-/HER2⁺ (double negative) breast tumor in athymic nude mice exhibited reduced N-glycan but increased GRP78 expression. We have evaluated the effect of tunicamycin on cellular localization of GRP78 in metastatic human breast cancer cells MDA-MB-231 (ER^-/PR^-/HER2^-). Tunicamycin inhibited cell proliferation in a time and dose-dependent manner. Non-metastatic estrogen receptor positive (ER⁺) MCF-7 breast cancer cells were also equally effective. GRP78 expression (protein and mRNA) was higher in tunicamycin (1.0 μg/ml) treated MCF-7 and MDA-MB-231 cells. GRP78 is an ER stress marker, so we have followed its intracellular localization using immunofluorescence microscopy after subjecting the cancer cells to various stress conditions. Unfixed cells stained with either FITC-conjugated Concanavalin A (Con A), or Texas-red conjugated wheat germ agglutinin (WGA) exhibited surface expression of N-glycans but not GRP78. GRP78 became detectable only after a brief exposure of cells to ice-cold methanol. Western blotting did not detect GRP78 in conditioned media of cancer cells whereas it did for MMP-1. The conclusion, GRP78 is expressed neither on the outer-leaflet of the (ER^-/PR^-/HER2^-) human breast cancer cells nor it is secreted into the culture media during tunicamycin-induced ER stress. Our study therefore suggests strongly that anti-tumorigenic action of tunicamycin can be modeled to develop next generation cancer therapy, i.e., glycotherapy for treating breast and other sold tumors.

中文翻译：

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衣霉素诱导的乳腺癌细胞内质网应激既不在表面表达GRP78也不将其分泌到培养基中

GRP78（一个M _{- [R} 78 kDa的钙依赖性葡萄糖结合蛋白）位于ER腔。它起内质网伴侣的作用，并在序列Asn-X-Ser / Thr中存在的天冬酰胺残基处使蛋白质进行糖基化转移。从石蜡切片Ñ -glycosylation抑制剂衣霉素处理ER ^- / PR ^- / HER2 ⁺（双阴性）乳房在无胸腺裸小鼠的肿瘤表现出降低Ñ聚糖但增加GRP78表达。我们评价衣霉素对GRP78的细胞定位的影响在转移性的人类乳腺癌细胞MDA-MB-231（ER ^- / PR ^- / HER2 ^-）。衣霉素以时间和剂量依赖性方式抑制细胞增殖。非转移性雌激素受体阳性（ER ⁺）MCF-7乳腺癌细胞也同样有效。在衣霉素（1.0μg/ ml）处理的MCF-7和MDA-MB-231细胞中，GRP78表达（蛋白质和mRNA）更高。GRP78是一种ER应激标记，因此在将癌细胞置于各种应激条件下之后，我们使用免疫荧光显微镜观察了其在细胞内的定位。未固定的细胞用FITC缀合的伴刀豆球蛋白A（Con A）或德克萨斯州红色缀合的小麦胚芽凝集素（WGA）染色后显示N的表面表达-聚糖而不是GRP78。仅在将细胞短暂暴露于冰冷的甲醇中后，GRP78才能被检测到。Western blotting在癌细胞的条件培养基中未检测到GRP78，而在MMP-1中却检测到。结束时，GRP78表达既不在（ER的外小叶^- / PR ^- / HER2 ^- ）人乳腺癌细胞也不是衣霉素诱导的ER应激期间分泌到培养基中。因此，我们的研究有力地表明，衣霉素的抗致瘤作用可以被模型化以开发下一代癌症疗法，即用于治疗乳腺癌和其他肿瘤的糖疗法。