O-GlcNAcylation is a dynamic PTM of nuclear and cytoplasmic proteins, regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase, which catalyze the addition and removal of O-GlcNAc, respectively. This modification is associated with glucose metabolism, which plays important roles in many diseases including cancer. Although emerging evidence reveals that some tumor-associated proteins are O-GlcNAc modified, the total O-GlcNAcylation in cancer is still largely unexplored. Here, we demonstrate that O-GlcNAcylation was increased in primary breast malignant tumors, not in benign tumors and that this augmentation was associated with increased expression of OGT level. Using 2D O-GlcNAc immnoblotting and LC-MS/MS analysis, we successfully identified 29 proteins, with seven being uniquely O-GlcNAcylated or associated with O-GlcNAcylation in cancer. Of these identified proteins, some were related to the Warburg effect, including metabolic enzymes, proteins involved in stress responses and biosynthesis. In addition, proteins associated with RNA metabolism, gene expression, and cytoskeleton were highly O-GlcNAcylated or associated with O-GlcNAcylation. Moreover, OGT knockdown showed that decreasing O-GlcNAcylation was related to inhibition of the anchorage-independent growth in vitro. These data indicate that aberrant protein O-GlcNAcylation is associated with breast cancer. Abnormal modification of these O-GlcNAc-modified proteins might be one of the vital malignant characteristics of cancer.

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BackgroundZearalenone (ZEA) is a phytoestrogen from Fusarium species. The aims of the study was to identify mode of human leukemic cell death induced by ZEA and the mechanisms involved.MethodsCell cytotoxicity of ZEA on human leukemic HL-60, U937 and peripheral blood mononuclear cells (PBMCs) was performed by using 3-(4,5-dimethyl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Reactive oxygen species production, cell cycle analysis and mitochondrial transmembrane potential reduction was determined by employing 2',7'-dichlorofluorescein diacetate, propidium iodide and 3,3'-dihexyloxacarbocyanine iodide and flow cytometry, respectively. Caspase-3 and -8 activities were detected by using fluorogenic Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC) and Ile-Glu-Thr-Asp-7-amino-4-methylcoumarin (IETD-AMC) substrates, respectively. Protein expression of cytochrome c, Bax, Bcl-2 and Bcl-xL was performed by Western blot. The expression of proteins was assessed by two-dimensional polyacrylamide gel-electrophoresis (PAGE) coupled with LC-MS2 analysis and real-time reverse transcription polymerase chain reaction (RT-PCR) approach.ResultsZEA was cytotoxic to U937 > HL-60 > PBMCs and caused subdiploid peaks and G1 arrest in both cell lines. Apoptosis of human leukemic HL-60 and U937 cell apoptosis induced by ZEA was via an activation of mitochondrial release of cytochrome c through mitochondrial transmembrane potential reduction, activation of caspase-3 and -8, production of reactive oxygen species and induction of endoplasmic reticulum stress. Bax was up regulated in a time-dependent manner and there was down regulation of Bcl-xL expression. Two-dimensional PAGE coupled with LC-MS2 analysis showed that ZEA treatment of HL-60 cells produced differences in the levels of 22 membrane proteins such as apoptosis inducing factor and the ER stress proteins including endoplasmic reticulum protein 29 (ERp29), 78 kDa glucose-regulated protein, heat shock protein 90 and calreticulin, whereas only ERp29 mRNA transcript increased.ConclusionZEA induced human leukemic cell apoptosis via endoplasmic stress and mitochondrial pathway.

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