Dorota Bartusik

N-Glycomic Changes in Human Breast Carcinoma MCF-7 and T-Lymphoblastoid Cells After Treatment with Herceptin and Herceptin/Lipoplex

The humanized monoclonal antibody IgG1 in combination with chemotherapy has been demonstrated to enhance survival benefit in cancer treatment. Despite positive outcomes, some cancer cells develop multidrug resistance. Numerous mechanisms in cancers can be involved in the process of treatment therapy and most of them are not still well understood. To address how the carbohydrate moieties of cells are affected during treatment, the glycan profiles from the two most common cancer cell lines - human breast MCF-7 carcinoma and T-lymphoblastoid CEM cells - were studied here and compared with profiles after treatment with Herceptin alone or in combination with Lipofectamine mixed with plasmid DNA to form Lipoplex. N-Glycans were released from total cells by digestion with PNGaseF and analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). In summary, both original cell lines showed a dominant occurrence of high-mannose glycans. After treatment, these structures were suppressed and biantennary core-fucosylated glycans originating from IgG1 were the major carbohydrate products identified in cells. The high incidence of additional fucosylated or nonfucosylated galactosylated oligosaccharides, which were not detected in original cells or Herceptin, varied with conditions and time of exposure of cells to the antibody. The results presented in this study provide strong evidence for a role of glycosylation during antibody treatment.

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.

Combined treatment of human MCF-7 breast carcinoma with antibody, cationic lipid and hyaluronic acid using ex vivo assays.

The effective targeting of malignant cell surface antigens is essential in cancer therapy. Resistance to treatment and rapid invasion of cancer cells are the main causes of cancer mortality. Despite intense research efforts, treatments often have demonstrated insufficient outcomes in clinical applications. The aim of the present study was to determine whether combined administration of monoclonal antibody (Herceptin, trastuzumab) and anti-HER-2 (clone CB11) with hyaluronic acid (HA) and lipoplex (containing lipofectamine (LipA) and plasmid DNA) can produce a synergistic reaction to increase the therapeutic effect of monoclonal antibodies. To assess the treatment response, we cultured a 3-D MCF-7 cell line overexpressing HER-2 and CD44 receptors. The high density 3-D cell aggregation in the hollow fiber bioreactor (HFB) used for the cell culture was monitored with the use of proton magnetic resonance imaging ((1)H MRI). In addition, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used in combination with HPLC (high performance liquid chromatography) to evaluate structural changes in the proteins contained in treated cells. The study showed that incorporation of antibodies into targeted lipoplex results in more efficient delivery of the complex to tumor cells. The viability of cells decreased mostly due to cellular uptake of lipoplex and binding of the antibodies to the cellular surface receptor. The data also demonstrate that HA could be used to enhance treatment efficacy of trastuzumab and anti-HER-2 (clone CB11) in breast cancer cell cultures.

Application of 19F magnetic resonance to study the efficacy of fluorine labeled drugs in the three-dimensional cultured breast cancer cells.

The cellular monitoring of tumor response to treatments is important for drug discovery and drug development in cancer therapy. We studied efficacy of Herceptin, a common breast cancer drug conjugated with a fluorine organic compound, perfluoro-15-crown-5-ether (PFCE) which easily forms biocompatible emulsions. Three new pharmaceutical forms of Herceptin, Herceptin/PFCE, Herceptin/PFCE/Lipoplex and Herceptin/PFCE/HydraLink were synthesized for the ex vivo study of their efficacy in breast cancer treatment. The emulsions were administered to 10(9)cells mL(-1) of HER-2 positive human adenocarcinoma (MCF-7) cells and the same amount of human mammary epithelial cells (HMEC) cultured in three-dimensional (3D) geometry using hollow fiber bioreactor (HFB) device. Following drugs administration ex vivo, fluorine-19 magnetic resonance imaging ((19)F MRI) was applied for cells imaging to measure their viability and to study drug efficacy over 72h. To ensure optimum drug tracking, HydraLink was used to provide stable binding affinity of emulsified Herceptin to receptor while cationic lipid (Lipofectamine) was used to enhance lipophilicity of the emulsions. After 72h of treatment with Herceptin, Herceptin/PFCE, Herceptin/PFCE/Lipoplex and Herceptin/PFCE/HydraLink the viability of cells was 54+/-2%, 49+/-3%, 43+/-5% and 42+/-1%, respectively, as compared with control 93+/-2%. The efficacy (EC(50)) of Herceptin conjugated with emulsions was found to be 970+/-13 microg mL(-1) for Herceptin/PFCE, 645+/-11 microg mL(-1) for Herceptin/PFCE/Lipoplex, 678+/-7 microg mL(-1) for Herceptin/PFCE/HydraLink and 1000+/-3 microg mL(-1) for Herceptin. The results show that fluorine emulsions improved the efficacy of Herceptin and (19)F signal intensity changes validated drug efficiency. The significant correlations between duration of treatments and MCF-7 cells viability were observed. While we studied breast cancer cells, the fluorine emulsions could be applied for treatment of other cancer cells overexpressing HER-2.

Detection of (19)F-labeled biopharmaceuticals in cell cultures with magnetic resonance.

Magnetic resonance (MR) studies of the therapeutic efficacy of fluorinated drugs have recently become possible due to improvements in detection including the application of very strong magnetic fields up to 9.4Tesla (T). These advances allow tracking, identification, and quantification of (19)F-labeled biopharmaceuticals using (19)F MR imaging ((19)F MRI) and spectroscopy ((19)F MRS). Both techniques are noninvasive, are nondestructive, and enable serial measurements. They also allow for controlled and systematic studies of cellular metabolism in cancerous tissue in vivo (small animals and humans) and in vitro (body fluids, cells culture, tissue extracts and isolated tissues). Here we provide an overview of the (19)F MRI and (19)F MRS techniques used for tracking (19)F labeled anticancer chemotherapeutics and antibodies which allow quantification of drug uptake in cancer cells in vitro.

Derivatives of thiocolchicine and its applications to CEM cells treatment using 19F magnetic resonance ex vivo.

It was shown, that cultured ex vivo human T-Lymphoblastoid (CEM) cells respond to synthesized thiocolchicine and fluorine thiocolchicine derivatives. The preparation of derivatives with substitution at C-3 and C-7 is described. All compounds were used at concentration from 1 nM to 1000 nM. Inhibitory effects of these compounds were examined in the three-dimensional (3-D) culture and cells morphology during treatment was monitored using 9.4 T MRI system. We performed studies of these compounds in CEM cells ex vivo using 1H and 19F Magnetic Resonance Imaging (MRI), 19F Magnetic Resonance Spectroscopy (MRS), High Performance Liquid Chromatography coupled with Ultra Violet (HPLC-UV) and Electron Impact Mass Spectrometry (EIMS). The results of the multi-technique approach are consistent with the fact that the new derivatives are more efficient than colchicine and thiocolchicine ex vivo.

The application of 19F magnetic resonance ex vivo imaging of three-dimensional cultured breast cancer cells to study the effect of δ-tocopherol

The aim of this study was to determine the role of delta-tocopherol in breast cancer cell growth ex vivo. Human gland mammary adenocarcinoma (MCF-7) and human T-lymphoblastoid (CEM) cells were cultured in the presence of delta-tocopherol at various concentrations (0-750 microM) for 5 days. We have grown 3D ex vivo breast cancer cell cultures in the hollow fiber bioreactor (HFBR). (19)F magnetic resonance imaging (MRI) was used to evaluate oxygen concentration in the cell suspension and thus its viability.

Detection of fluorine labeled Herceptin using cellular 19F MRI ex vivo

The aim of this study was to assess the herceptin efficacy in ex vivo cultures of MCF-7 breast carcinoma cells. Herceptin was used with perfluorooctyl bromide (PFOB) and conjugated with lipoplex, containing plasmid DNA and lipofectamine (LipA), to allow fluorine-19 magnetic resonance imaging ((19)F MRI) study. Treatments such as herceptin, herceptin/PFOB and herceptin/PFOB/lipoplex were used for ex vivo targeting of MCF-7 cells cultured in three-dimensional (3D) geometry using hollow fiber bioreactor (HFB) device. The viability of MCF-7 cells after 72h treatments decreased to 54+/-2%, 50+/-3% and 45+/-1% for herceptin, herceptin/PFOB and herceptin/PFOB/Lipoplex, respectively. The EC(50) values were 1000microg/ml, 930microg/ml and 730microg/ml, respectively. The significant correlation between the treatment concentration and efficacy was observed in MCF-7 cell cultures.

19F MRI of 3D CEM cells to study the effects of tocopherols and tocotrienols

Oximetry of the human T-Lymphoblastoid (CEM) cells was measured using (19)F magnetic resonance imaging ((19)F MRI). The cells were treated with the analogues of vitamin E, alpha-, gamma-, delta-tocopherols and corresponding tocotrienols, ex vivo in three-dimensional (3D) cell culture. The study showed that (19)F MRI allows to measure the effect of the analogues due to changes of oxygenation, which were detected using MRI. Hexafluorobenzene was used as a (19)F MRI probe sensitive to oxygen concentrations. After 72h of treatment in HFBR with alpha-, gamma-, delta-tocopherols the oxygen concentration was 19.9+/-0.8%, 19.3+/-1.4%, 16+/-3.5%, respectively. The oxygen concentration in cells treated with alpha-, gamma-, delta-tocotrienols was found to be 14+/-1.5%, 10+/-1.2% and 8.8+/-1.1%, respectively whereas for the control cells it was 22.1+/-1%. The results show that delta-tocopherol and delta-tocotrienol are the most effective treatments in CEM cells among all the tested analogues.

A Review of the Detection of Amino Acids in Tumor Tissue with 1H Magnetic Resonance Spectroscopy in vivo

The biochemical components of tumor tissue are generally different from normal tissue. A variety of techniques, including of magnetic resonance (MR) are used to study metabolic compounds and their associated biochemical pathways in tumor tissue in vivo. This review provides an overview of applications of MR spectroscopy (MRS) to the identification of biochemical characteristics, e.g. amino acids present during tumor growth. MRS is a technology that allows spatial localization and structural studies of amino acids and is gaining in use as a diagnostic tool. This review is intended to provide information about potential applications of MRS to non-invasive probing of the underlying amino acid biochemistry in tumors. Articles discussed in this review are classified into the following categories:hardware for 1H MRS, acquisition methods, software, and 1H MRS of amino acids in tumor tissue in vivo.