Alterations in glycopeptides associated with herceptin treatment of human breast carcinoma mcf-7 and T-lymphoblastoid cells

The therapeutic humanized monoclonal antibody IgG1 known as Herceptin^® has shown remarkable antitumor effects. Although this type of therapy has increased the cancer-free survival of patients, not all tumors respond to this treatment and cancers often develop resistance to the antibody. Despite the fact that Herceptin function has been extensively studied, the precise mechanism underlying its antitumor activity still remains incompletely defined. We previously demonstrated on human breast MCF-7 carcinoma and T-lymphoblastoid CEM cells that monoclonal antibody in combination with Lipoplex consisting of Lipofectamine mixed with plasmid DNA showed a more profound effect on cancer cell viability than antibody alone. The analyses of N-glycans isolated from cancer cells showed dramatic differences in profiles when cells were exposed to Herceptin. Moreover, the investigation of glycosylated peptides from the same cancer cell models after treatment revealed further alterations in the post-translational modifications. Tandem mass spectra obtained from the samples treated confirmed the presence of a series of glycopeptides bearing characteristic oligosaccharides as described in IgG1. However some of them differed by mass differences that corresponded to peptide backbones not described previously and more of them were detected from Herceptin treated samples than from cells transfected with Heceptin/Lipoplex. The results indicate that the presence of Lipoplex prevents antibody transformation and elongates its proper function. The better understanding of the multipart changes described in the glycoconjugates could provide new insights into the mechanism by which antibody induces regression in cancers.Glycosylation of proteins is a ubiquitous type of post-translational modification in living systems. Variations in oligosaccharide structures are associated with many normal and pathological events such as cellular growth, host-pathogen interaction, differentiation, migration, cell trafficking, or tumor invasion (1, 2). Targeted glycosylation research has become important in the area of developing novel therapeutic approaches (3–5). The structures of asparagine-linked oligosaccharides in the conserved C_H2 region of the constant Fc domain of human immunoglobulin-γ (IgG1) have been shown to affect the pharmacokinetics, antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (6, 7). In the last decade, many recombinant antibody molecules have been licensed for the treatment of a variety of cancers and chronic diseases (8). Herceptin, also known as Trastuzumab, marketed by Genentech Inc. is one example of therapeutic IgG1 antibody. It is produced from mammalian cell culture using Chinese hamster ovary cells (9). The main oligosaccharide forms found in this polypeptide chain in the Fc domain at asparagine 297 are biantennary core-fucosylated complex type structures with variable terminal galactosylation (zero, one, or two galactose residues) on their nonreducing termini (10, 11). This humanized monoclonal antibody is known to effectively target breast cancer cells overexpresing the human epidermal growth factor receptor HER2/neu (12). HER2 is a cell membrane surface-bound receptor tyrosine kinase and is normally involved in the signal transduction pathways leading to cell growth and differentiation. It can be found overexpressed in a variety tumors'' cells of epithelial origin and hematological malignancies, including acute lymphoblastic leukemia (13). When antibody binds to defective HER2 protein, this protein no longer causes cells to reproduce uncontrollably. This increases the survival of people with cancer. However, cancers usually develop resistance to trastuzumab. Unfortunately, only 25–30% of patients with HER2/neu positive breast cancer respond to this antibody (14–17). Therefore search for the potential biomarkers that could predict the efficacy of clinical outcomes is needed. More precise investigation on cellular and molecular level might provide many exciting insights in understanding of mechanism resistance cancer cells to the antibody, so that antibody-based therapies can be optimized more individually (18).We recently demonstrated how the carbohydrate moieties of two cancer cell models were affected during treatment with antibody (19). The detailed glycans profiles studied by means of mass spectrometry (MS) from the two most common cancer cell lines—human breast MCF-7 carcinoma and T-lymphoblastoid CEM cells before and after treatment with Herceptin showed significant differences. Dominant high-mannose structures analyzed in both original cancer cells were suppressed after treatment and instead, complex bi- and triantennary glycans were the major structures found in the treated samples. Their ratio or occurrence varied with conditions and time of exposure of the cancer cells to the antibody. The results provided very good evidence for involvement of glycosylation during treatment. In this regard, continuous work presented here on this subject has been aimed to the MS investigation of glycosylated peptides generated by proteolytic digestions of the cancer cells before and after exposure to Herceptin or Herceptin/Lipoplex. Direct analysis of glycopeptides by tandem MS has been shown as one of the most sensitive and fast methods for a site-specific characterization of glycosylation. It can provide information on glycan composition, glycan attachment site with determination of peptide sequence (20–28), and may offer more specific biomarkers to monitor changes in the post-translational modification at the onset, during cancer progression or during treatment.