Fast, sensitive, and simple methods for quantitative analysis of disparities in glycan expression between different biological samples are essential for studies of protein glycosylation patterns (glycomics) and the search for disease glycan biomarkers. Relative quantitation of glycans based on stable isotope labeling combined with mass spectrometric detection represents an emerging and promising technique. However, this technique is undermined by the complexity of mass spectra of isotope-labeled glycans caused by the presence of multiple metal ion adduct signals, which result in a decrease of detection sensitivity and an increase of difficulties in data interpretation. Herein we report a simplified quantitative glycomics strategy, which features nonreductive isotopic labeling of reducing glycans with either nondeuterated (d0-) or deuterated (d5-) Girard's reagent P (GP) without salts introduced and simplified mass spectrometric profiles of d0- and d5-GP derivatives of neutral glycans as molecular ions without complex metal ion adducts, allowing rapid and sensitive quantitative comparison between different glycan samples. We have obtained optimized GP-labeling conditions and good quantitation linearity, reproducibility, and accuracy of data by the method. Its excellent applicability was validated by comparatively quantitative analysis of the neutral N-glycans released from bovine and porcine immunoglobulin G as well as of those from mouse and rat sera. Additionally, we have revealed the potential of this strategy for the high-sensitivity analysis of sialylated glycans as GP derivatives, which involves neutralization of the carboxyl group of sialic acid by chemical derivatization.

Trastuzumab is an antibody-based therapy drug targeting HER2-overexpressing tumors. While it has been proven to be very successful initially, most patients eventually develop resistance to trastuzumab. The mechanism of drug resistance is not well understood. Identifying pathways that mediate trastuzumab resistance will improve our understanding of the underlying mechanism and is crucial for the development of therapeutic strategies to overcome resistance.Here we report a quantitative proteomics profiling of a trastuzumab-sensitive (T-S) gastric cancer cell line NCI N87 and a trastuzumab-resistant NCI N87 (T-R) subline generated by low-dose, continuous trastuzumab treatment. By identifying proteins differentially expressed in these two cell lines, we show that multiple pathways including mTOR, Wnt, DNA damage response and metabolic pathways are significantly altered. We further confirm by western blotting that protein levels of multiple components of the mTOR pathway, including mTOR, AKT and RPS6KB1, are increased, whereas AKT1S1 is decreased, suggesting the activation of mTOR pathway. Importantly, treatment of AZD8055, an mTOR inhibitor, leads to the decreased phosphorylation levels of mTOR downstream molecules RPS6KB1 at Thr421/Ser424 and AKT at Ser473. Furthermore, AZD8055 also preferentially reduces viability, and inhibits migration and invasion abilities of the T-R cells. Together, our findings indicate that mTOR pathway is among multiple signaling pathways that mediate trastuzumab resistance in NCI N87 T-R cells, and that mTOR inhibitors may be used to treat trastuzumab resistant, HER2-positive gastric cancer tumors.

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Resistance to trastuzumab, which specifically target HER2-positive breast and gastric cancer, can develop ultimately in cancer patients. However, the underlying mechanisms of resistance in gastric cancer have not been fully elucidated. Here, we established trastuzumab-resistant MKN45 and NCI N87 gastric cancer sublines from their parental cells. The resistant cells exhibited characteristics of epithelial-mesenchymal transition (EMT) and acquired higher migratory and invasive capacities. To exploit the activated pathways and develop new strategies to overcome trastuzumab resistance, we investigated MKN45 and MKN45/R cells via label-free quantitative proteomics, and found pathways that were altered significantly in MKN45/R cells, with the Wnt/β-catenin pathway being the most significant. We further confirmed the activation of this pathway by detecting its key molecules in MKN45/R and NCI N87/R cells via Western blot, in which Wnt3A, FZD6, and CTNNB1 increased, whereas GSK-3β decreased, manifesting the activation of the Wnt/β-catenin pathway. Correspondingly, inhibition of Wnt/β-catenin pathway by ICG-001, a specific Wnt/β-catenin inhibitor, preferentially reduced proliferation and invasion of trastuzumab-resistant cells and reversed EMT. Concurringly, CTNNB1 knockdown in stable cell lines potently sensitized cells to trastuzumab and induced more apoptosis. Taken together, our study demonstrates that the Wnt/β-catenin pathway mediates trastuzumab resistance, and the combination of Wnt/β-catenin inhibitors with trastuzumab may be an effective treatment option.