Characterisation of the Helix pomatia agglutinin binding glycoproteins of colorectal cancer cell lines and tissue samples

Colorectal cancer (CRC) is one of the most common malignancies in the US and Western Europe and metastatic dissemination after CRC is a leading cause of mortality. New markers predictive of cancer cell behaviour are actively sought both as targets and as a means of predicting patient prognosis. The lectin from the Roman snail Helix pomatia (HPA) has attracted interest as tool for the detection of metastatic colorectal cancer but the HPA binding partners have remained poorly characterised. We established an in vitro model using human colorectal cancer cell lines ranging from HPA negative, non metastatic, to HPA positive and metastatic. Confocal microscopy was used to assess the HPA binding pattern in the cell lines and the monosaccharides N-acetylgalactosamine, N-acetylglucosamine, and sialic acid were used to inhibit the interaction between the lectin and the cancer cells. A proteomic approach based on cell membrane isolation, pre-fractionation using lectin affinity chromatography, followed by 2-dimensional electrophoresis and MALDI-TOF-MS enabled the identification of the HPA binding proteins in the metastatic cancer cell line HT29. The proteins that eluted in the HPA binding fraction were present either by virtue of their ability to bind directly to HPA or as protein complexes of HPA binding partners and included molecules involved in cell adhesion / migration (integrin a6, integrin aV, annexins) re-modeling (filament proteins including oc tubulin, Ptubulin, cytokeratins, actin) and anti-apoptotic pathways (Hsp-70, Hsp-90, Hsp-96 and TNFR-1). Although many of these proteins have previously been described as altered in cancer, we are not aware of a single reagent like HPA which will concurrently bind all of these molecules

Cellular glycosylation affects Herceptin binding and sensitivity of breast cancer cells to doxorubicin and growth factors

Alterations in protein glycosylation are a key feature of oncogenesis and have been shown to affect cancer cell behaviour perturbing cell adhesion, favouring cell migration and metastasis. This study investigated the effect of N-linked glycosylation on the binding of Herceptin to HER2 protein in breast cancer and on the sensitivity of cancer cells to the chemotherapeutic agent doxorubicin (DXR) and growth factors (EGF and IGF-1). The interaction between Herceptin and recombinant HER2 protein and cancer cell surfaces (on-rate/off-rate) was assessed using a quartz crystal microbalance biosensor revealing an increase in the accessibility of HER2 to Herceptin following deglycosylation of cell membrane proteins (deglycosylated cells Bmax: 6.83 Hz; glycosylated cells Bmax: 7.35 Hz). The sensitivity of cells to DXR and to growth factors was evaluated using an MTT assay. Maintenance of SKBR-3 cells in tunicamycin (an inhibitor of N-linked glycosylation) resulted in an increase in sensitivity to DXR (0.1 µM DXR P<0.001) and a decrease in sensitivity to IGF-1 alone and to IGF-1 supplemented with EGF (P<0.001). This report illustrates the importance of N-linked glycosylation in modulating the response of cancer cells to chemotherapeutic and biological treatments and highlights the potential of glycosylation inhibitors as future combination treatments for breast cancer

Identification of O-Linked Glycoproteins Binding to the Lectin Helix pomatia Agglutinin as Markers of Metastatic Colorectal Cancer

Background Protein glycosylation is an important post-translational modification shown to be altered in all tumour types studied to date. Mucin glycoproteins have been established as important carriers of O-linked glycans but other glycoproteins exhibiting altered glycosylation repertoires have yet to be identified but offer potential as biomarkers for metastatic cancer. Methodology In this study a glycoproteomic approach was used to identify glycoproteins exhibiting alterations in glycosylation in colorectal cancer and to evaluate the changes in O-linked glycosylation in the context of the p53 and KRAS (codon 12/13) mutation status. Affinity purification with the carbohydrate binding protein from Helix pomatia agglutinin (HPA) was coupled to 2-dimensional gel electrophoresis with mass spectrometry to enable the identification of low abundance O-linked glycoproteins from human colorectal cancer specimens. Results Aberrant O-linked glycosylation was observed to be an early event that occurred irrespective of the p53 and KRAS status and correlating with metastatic colorectal cancer. Affinity purification using the lectin HPA followed by proteomic analysis revealed annexin 4, annexin 5 and CLCA1 to be increased in the metastatic colorectal cancer specimens. The results were validated using a further independent set of specimens and this showed a significant association between the staining score for annexin 4 and HPA and the time to metastasis; independently (annexin A4: Chi square 11.45, P = 0.0007; HPA: Chi square 9.065, P = 0.0026) and in combination (annexin 4 and HPA combined: Chi square 13.47; P = 0.0002). Conclusion Glycoproteins showing changes in O-linked glycosylation in metastatic colorectal cancer have been identified. The glycosylation changes were independent of p53 and KRAS status. These proteins offer potential for further exploration as biomarkers and potential targets for metastatic colorectal cancer

Réalisation d'un dispositif de mesure de la vitesse et de l'atténuation d'ondes ultrasonores dans des liquides sous pression

Cet article décrit en détail un dispositif de mesure de la vitesse et du coefficient d'atténuation d'ondes ultrasonores dans des liquides pouvant être portés à des pressions comprises entre 1 et 1 000 bars. La cellule de mesure et les conditions spécifiques de son remplissage (lorsque les échantillons étudiés sont issus de fluides de gisement stockés dans des conditions de pression proches de celles du fond du puits) sont présentées plus particulièrement. Des comparaisons entre les valeurs expérimentales obtenues au moyen de ce dispositif et des valeurs relevées dans la littérature pour quelques corps purs (eau, décane, hexadécane) sont aussi rapportées