Apprentissage du français comme langue étrangère (L3+) par des étudiants indiens

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Targeted apoptosis in ovarian cancer cells through mitochondrial dysfunction in response to Sambucus nigra agglutinin

Ovarian carcinoma (OC) patients encounter the severe challenge of clinical management owing to lack of screening measures, chemoresistance and finally dearth of non-toxic therapeutics. Cancer cells deploy various defense strategies to sustain the tumor microenvironment, among which deregulated apoptosis remains a versatile promoter of cancer progression. Although recent research has focused on identifying agents capable of inducing apoptosis in cancer cells, yet molecules efficiently breaching their survival advantage are yet to be classified. Here we identify lectin, Sambucus nigra agglutinin (SNA) to exhibit selectivity towards identifying OC by virtue of its specific recognition of α-2, 6-linked sialic acids. Superficial binding of SNA to the OC cells confirm the hyper-sialylated status of the disease. Further, SNA activates the signaling pathways of AKT and ERK1/2, which eventually promotes de-phosphorylation of dynamin-related protein-1 (Drp-1). Upon its translocation to the mitochondrial fission loci Drp-1 mediates the central role of switch in the mitochondrial phenotype to attain fragmented morphology. We confirmed mitochondrial outer membrane permeabilization resulting in ROS generation and cytochrome-c release into the cytosol. SNA response resulted in an allied shift of the bioenergetics profile from Warburg phenotype to elevated mitochondrial oxidative phosphorylation, altogether highlighting the involvement of mitochondrial dysfunction in restraining cancer progression. Inability to replenish the SNA-induced energy crunch of the proliferating cancer cells on the event of perturbed respiratory outcome resulted in cell cycle arrest before G2/M phase. Our findings position SNA at a crucial juncture where it proves to be a promising candidate for impeding progression of OC. Altogether we unveil the novel aspect of identifying natural molecules harboring the inherent capability of targeting mitochondrial structural dynamics, to hold the future for developing non-toxic therapeutics for treating OC

Membrane protein glycan markers of epithelial ovarian cancer: discrimination of serious tumours of ovary, peritoneum and tube

Thesis by publication.Includes bibliographical references.Epithelial ovarian cancer is characterised by a low 5-year survival rate of 43% of those diagnosed. The overall prognosis is poor due to many factors such as the lack of reliable and sensitive markers for early stage detection, the heterogeneity of the tumours, rapid metastasis of the disease which extends beyond the ovaries and the different cancer tissue origins (ovary, peritoneum and tube). As these cancers are derived from the epithelial surface, the comprehensive glycosylation of the cell membrane proteins and lipids from cell lines and serous cancer tissues was investigated to unravel cancer-specific biomarkers for improved diagnosis and personalised tumour-specific treatments.In the first phase of this study, N- and O-glycans were enzymatically released by PNGase F and reductive β-elimination, respectively, from extracted membrane glycoproteins derived from 'in vitro'-based cell line models of cancerous and non-cancerous ovarian cells. Released glycan alditols were separated using porous graphitised carbon (PGC) chromatography, analysed using electrospray ionisation mass spectrometry (LC-ESI-MS/MS) and characterised based on negative ion tandem MS fragmentation patterns and LC retention times. Glycan structural features such as bisecting N-acetylglucosamine (GlcNAc) and sialylated N-N-diacetyllactosamine (LacdiNAc)-type structures, together with increased levels of α2-6 sialylation were detected on membrane glycoproteins derived from ovarian cancer cells. The corresponding gene expression and epigenetic regulation of specific glycosyltransferases responsible for the biosynthesis of relevant N-glycan structures were also investigated using qRT-PCR and de-methylation by 5-Aza treatment. Their presence correlated with the corresponding glycosyltransferase gene expression of ST6GAL1, B4GALNT3, and MGAT3, in which MGAT3 was found to be also epigenetically regulated by DNA hypomethylation.The presence of specific glycan structures implicated in the preliminary cell-line models were further verified in membrane proteins of serous cancer tissues from different origins (14 ovarian, 14 peritoneal and 4 tubal). Several statistical analyses such as analysis of variance (ANOVA), principal component analysis (PCA), partial-least-square discrimination analysis (PLSDA) and receiver-operating curve (ROC) were employed to evaluate differences in the expression of glycan structures and to discriminate between all three serous cancers. Bi-antennary sialylated N-glycans with prominent α2-6 sialylation appeared to be the most common feature in all serous cancer tissues, whilst the unique expression of sialylated LacdiNAc N-glycans was detected specifically in serous ovarian-derived cancer tissues and these structures were statistically classified as 'highly accurate' biomarkers of serous ovarian cancer by ROC. The expression of 11 N-glycans, including the LacdiNAc-type glycans contributed significantly (p=0.00186) to the classification accuracy (78.6 %) of serous ovarian and peritoneal cancers.In addition to the structural identification of serous cancer membrane N- and O-glycans, a second analytical platform employing PGC-LC-ESI-MS/MS was also developed to characterise glycans released enzymatically (Endoglycoceramidase II) from PVDF-immobilised glycosphingolipids (GSLs) derived from cancer tissue samples and cell line. The analysis of the glycosylation of ovarian cancer membrane lipids revealed the presence of several isomeric and isobaric structures which were differentiated using specific diagnostic and structural feature ions produced by negative ion mode MS/MS fragmentation. This approach led to the identification of P blood group-related as well as fucosylated/non-fucosylated Type 1 and Type 2 antigens which were not expressed on N- and O-glycans of membrane proteins of serous cancers analysed in this study. These antigens were also implicated in the immune recognition by auto-antibodies found in the plasma and ascites fluid of ovarian cancer patients.The identification and understanding of the regulation of membrane protein and lipid glycan epitopes unique to ovarian cancer could be utilised to distinguish serous ovarian from peritoneum and tubal cancers. More importantly, such structures may facilitate the diagnosis and development of different drug targets to improve survival rates of this malignancy.Mode of access: World wide web1 online resource (xvi, 269 pages) illustrations, chart

A Platform for the structural characterization of glycans enzymatically released from glycosphingolipids extracted from tissue and cells

Rationale: Glycosphingolipids (GSLs) constitute a highly diverse class of glyco-conjugates which are involved in many aspects of cell membrane function and disease. The isolation, detection and structural characterization of the carbohydrate (glycan) component of GSLs are particularly challenging given their structural heterogeneity and thus rely on the development of sensitive, analytical technologies. Methods: Neutral and acidic GSL standards were immobilized onto polyvinylidene difluoride (PVDF) membranes and glycans were enzymatically released using endoglycoceramidase II (EGCase II), separated by porous graphitized carbon (PGC) liquid chromatography and structurally characterized by negative ion mode electrospray ionization tandem mass spectrometry (PGC-LC/ESI-MS/MS). This approach was then employed for GSLs isolated from 100 mg of serous and endometrioid cancer tissue and from cell line (10⁷ cells) samples. Results: Glycans were released from GSL standards comprising of ganglio-, asialo-ganglio- and the relatively resistant globo-series glycans, using as little as 1 mU of enzyme and 2 µg of GSL. The platform of analysis was then applied to GSLs isolated from tissue and cell line samples and the released isomeric and isobaric glycan structures were chromatographically resolved on PGC and characterized by comparison with the MS² fragment ion spectra of the glycan standards and by application of known structural MS² fragment ions. This approach identified several (neo-)lacto-, globo- and ganglio-series glycans and facilitated the discrimination of isomeric structures containing Lewis A, H type 1 and type 2 blood group antigens and sialyl-tetraosylceramides. Conclusion: We describe a relatively simple, detergent-free, enzymatic release of glycans from PVDF-immobilized GSLs, followed by the detailed structural analysis afforded by PGC-LC-ESI-MS/MS, to offer a versatile method for the analysis of tumour and cell-derived GSL-glycans. The method uses the potential of MS²fragmentation in negative ion ESI mode to characterize, in detail, the biologically relevant glycan structures derived from GSLs.17 page(s

Tissue glycomics distinguish tumour sites in women with advanced serous adenocarcinoma

In the era of precision medicine, the tailoring of cancer treatment is increasingly important as we transition from organ-based diagnosis towards a more comprehensive and patient-centric molecular diagnosis. This is particularly the case for high-grade serous adenocarcinomas of the ovary and peritoneum, which are commonly diagnosed at an advanced stage, and collectively treated and managed similarly. We characterized the N- and O-glycome of serous ovarian (OC) and peritoneal cancer (PC) tissues using PGC-LC-ESI-IT-MS/MS profiling and validated the discriminatory glycans and their corresponding glyco-gene expression levels using cell lines and transcriptomic data from 232 patients. Overall, the N- and O-glycan repertoires of both cancer types were found to comprise mostly of α2,6-sialylated glycan structures, with the majority of N-glycans displaying the biantennary mono- and disialylation as well as bisecting-type biantennary glycans. The MS profiling by PGC-LC also revealed several glycan structural isomers that corresponded to LacdiNAc-type (GalNAcβ1-4GlcNAc) motifs that were unique to the serous ovarian cancers and that correlated with elevated gene expression of B4GALNT3 and B4GALNT4 in patients with serous cancer. Statistical evaluation of the discriminatory glycans also revealed 13 N- and 3 O-glycans (P < 0.05) that significantly discriminated tumour-sampling sites, with LacdiNAc-type N-glycans (m/z 1205.02- and m/z 1059.42- ) being associated with ovarian-derived cancer tissue and bisecting GlcNAc-type (m/z 994.92- ) and branched N-glycans (m/z 1294.02- and m/z 1148.42- ) upregulated at the metastatic sites. Hence, we demonstrate for the first time that OC and PC display distinct molecular signatures at both their glycomic and transcriptomic levels. These signatures may have potential utility for the development of accurate diagnosis and personalized treatments

The glycosphingolipid P1 is an ovarian cancer-associated carbohydrate antigen involved in migration

Background The level of plasma-derived naturally circulating anti-glycan antibodies (AGA) to P1 trisaccharide has previously been shown to significantly discriminate between ovarian cancer patients and healthy women. Here we aim to identify the Ig class that causes this discrimination, to identify on cancer cells the corresponding P1 antigen recognised by circulating anti-P1 antibodies and to shed light into the possible function of this glycosphingolipid. Method An independent Australian cohort was assessed for the presence of anti-P1 IgG and IgM class antibodies using suspension array. Monoclonal and human derived anti-glycan antibodies were verified using three independent glycan-based immunoassays and flow cytometry-based inhibition assay. The P1 antigen was detected by LC-MS/MS and flow cytometry. FACS-sorted cell lines were studied on the cellular migration by colorimetric assay and real-time measurement using xCELLigence system. Results Here we show in a second independent cohort (n=155) that the discrimination of cancer patients is mediated by the IgM class of anti-P1 antibodies (P=0.0002). The presence of corresponding antigen P1 and structurally related epitopes in fresh tissue specimens and cultured cancer cells is demonstrated. We further link the antibody and antigen (P1) by showing that human naturally circulating and affinity-purified anti-P1 IgM isolated from patients ascites can bind to naturally expressed P1 on the cell surface of ovarian cancer cells. Cell-sorted IGROV1 was used to obtain two study subpopulations (P1-high, 66.1%; and P1-low, 33.3%) and observed that cells expressing high P1-levels migrate significantly faster than those with low P1-levels. Conclusions This is the first report showing that P1 antigen, known to be expressed on erythrocytes only, is also present on ovarian cancer cells. This suggests that P1 is a novel tumour-associated carbohydrate antigen recognised by the immune system in patients and may have a role in cell migration. The clinical value of our data may be both diagnostic and prognostic; patients with low anti-P1 IgM antibodies present with a more aggressive phenotype and earlier relapse

Cell surface protein glycosylation in cancer

Glycosylation of proteins is one of the most important PTMs, with more than half of all human proteins estimated to be glycosylated. It is widely known that aberrant glycosylation has been implicated in many different diseases due to changes associated with biological function and protein folding. In cancer, there is increasing evidence pertaining to the role of glycosylation in tumour formation and metastasis. Alterations in cell surface glycosylation, particularly terminal motifs, can promote invasive behaviour of tumour cells that ultimately lead to the progression of cancer. While a majority of studies have investigated protein glycosylation changes in cancer cell lines and tumour tissue for individual cancers, the review presented here represents a comprehensive, in-depth overview of literature on the structural changes of glycosylation and their associated synthetic enzymes in five different cancer types originating from the breast, colon, liver, skin and ovary. More importantly, this review focuses on key similarities and differences between these cancers that reflect the importance of structural changes of cell surface N- and O-glycans, such as sialylation, fucosylation, degree of branching and the expression of specific glycosyltransferases for each cancer. It is envisioned that the understanding of these biologically relevant glycan alterations on cellular proteins will facilitate the discovery of novel glycan-based biomarkers which could potentially serve as diagnostic and prognostic indicators of cancer.22 page(s

Specific glycosylation of membrane proteins in epithelial ovarian cancer cell lines : glycan structures reflect gene expression and DNA methylation status

Epithelial ovarian cancer is the fifth most common cause of cancer in women worldwide bearing the highest mortality rate among all gynecological cancers. Cell membrane glycans mediate various cellular processes such as cell signaling and become altered during carcinogenesis. The extent to which glycosylation changes are influenced by aberrant regulation of gene expression is nearly unknown for ovarian cancer and remains crucial in understanding the development and progression of this disease. To address this effect, we analyzed the membrane glycosylation of non-cancerous ovarian surface epithelial (HOSE 6.3 and HOSE 17.1) and serous ovarian cancer cell lines (SKOV 3, IGROV1, A2780, and OVCAR 3), the most common histotype among epithelial ovarian cancers. Nglycans were released from membrane glycoproteins by PNGase F and analyzed using nano-liquid chromatography on porous graphitized carbon and negative-ion electrospray ionization mass spectrometry (ESI-MS). Glycan structures were characterized based on their molecular masses and tandem MS fragmentation patterns. We identified characteristic glycan features that were unique to the ovarian cancer membrane proteins, namely the "bisecting N-acetyl-glucosamine" type N-glycans, increased levels of α 2-6 sialylated N-glycans and "N,N'-diacetyllactosamine" type N-glycans. These N-glycan changes were verified by examining gene transcript levels of the enzymes specific for their synthesis (MGAT3, ST6GAL1, and B4GALNT3) using qRT-PCR. We further evaluated the potential epigenetic influence on MGAT3 expression by treating the cell lines with 5-azacytidine, a DNA methylation inhibitor. For the first time, we provide evidence that MGAT3 expression may be epigenetically regulated by DNA hypomethylation, leading to the synthesis of the unique "bisecting GlcNAc" type N-glycans on the membrane proteins of ovarian cancer cells. Linking the observation of specific N-glycan substructures and their complex association with epigenetic programming of their associated synthetic enzymes in ovarian cancer could potentially be used for the development of novel anti-glycan drug targets and clinical diagnostic tools.20 page(s