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

Presence of bisecting glcnac-modified proteins on ovarian cancer cells associates with elevated expression of the epigenetically regulated MGAT3 gene

Aims: Changes in the glycosylation of membrane proteins are a common feature of malignant transformation including ovarian cancer development. However, the nature of these changes and their underlying molecular mechanisms are poorly understood. Methods: Membrane protein glycan extraction and LC-ESI-MS (liquid chromatography negative-ion electrospray ionization mass spectrometry for glycan characterization), qRT-PCR (gene expression profiling in cell lines), bisulfite sequencing (analysis of DNA methylation) and Western blotting (protein determination). Results: We identified characteristic glycan features that were unique to membrane proteins of ovarian cancer cells such as the exclusive presence of "bisecting N-acetyl-glucosamine (GlcNAc)" type Nglycans. The bisecting GlcNAc on N-glycosylated proteins is the product of MGAT3 (beta-1,4-N-acetylglucosaminyltransferase 3). The expression of both the MGAT3 gene and the corresponding enzyme was elevated in ovarian cancer cell lines (SKOV3, IGROV1, A2780, OVCAR3) as compared to normal ovarian surface epithelial (HOSE) cells. We found that elevated MGAT3 expression in ovarian cancer cells correlated with DNA hypomethylation at the transcription start site (TSS) of the MGAT3 promoter, suggesting an epigenetic regulatory mechanism for MGAT3. This finding was further confirmed by treating HOSE cells with DNA-methyltransferase inhibitor, 5-Aza, which resulted in increased MGAT3 expression, mirrored by a reduction of DNA methylation at the TSS. Conclusion: Elevated MGAT3 expression in ovarian cancer cells is epigenetically regulated by DNA methylation. The presence of specific N-glycan substructures in combination with the epigenetic regulation of their associated enzymes may stimulate the development of novel anti-glycan drug targets and clinical diagnostic tools in ovarian cancer.2 page(s

Epigenetic activation of MGAT3 and corresponding bisecting GlcNAc shortens the survival of cancer patients

Bisecting GlcNAc on N-glycoproteins is described in E-cadherin-, EGF-, Wnt- and integrin- cancer-associated signaling pathways. However, the mechanisms regulating bisecting GlcNAc expression are not clear. Bisecting GlcNAc is attached to N-glycans through beta 1-4 N-acetylglucosaminyl transferase III (MGAT3), which is encoded by two exons flanked by high-density CpG islands. Despite a recently described correlation of MGAT3 and bisecting GlcNAc in ovarian cancer cells, it remains unknown whether DNA methylation is causative for the presence of bisecting GlcNAc. Here, we narrow down the regulatory genomic region and show that reconstitution of MGAT3 expression with 5-Aza coincides with reduced DNA methylation at the MGAT3 transcription start site. The presence of bisecting GlcNAc on released N-glycans was detected by mass spectrometry (LC-ESI-qTOF-MS/MS) in serous ovarian cancer cells upon DNA methyltransferase inhibition. The regulatory impact of DNA methylation on MGAT3 was further evaluated in 18 TCGA cancer types (n = 6118 samples) and the results indicate an improved overall survival in patients with reduced MGAT3 expression, thereby identifying long-term survivors of high-grade serous ovarian cancers (HGSOC). Epigenetic activation of MGAT3 was also confirmed in basal-like breast cancers sharing similar molecular and genetic features with HGSOC. These results provide novel insights into the epigenetic regulation of MGAT3/bisecting GlcNAc and demonstrate the importance of N-glycosylation in cancer progression

P1 glycosphingolipid is an ovarian cancer-associated carbohydrate antigen and enhances cell migration

Aims: We previously showed that levels of plasma-derived naturally circulating anti-glycan antibodies (AGA) to P1 trisaccharide was lower in ovarian cancer patients than healthy women. We investigated which Ig-class of these AGA accounts for this discrimination "cancer vs. healthy", whether P1 is indeed expressed on ovarian cancer cells, and what the biological functions of P1 may be. Methods: Suspension array was employed to assess the presence of anti-P1 IgG- and IgM-class antibodies in the plasma/ascites. Findings were verified using three independent glycan-based immunoassays and flow cytometry. LC-MS/MS and flow cytometry were performed to detect P1 antigen on tissue and cells. FACS-sorting was used to produce two IGROV1 cell subpopulations (P1-low, P1-high) which were employed in transwell-assay and real-time xCELLigence system to assess cell migration. Results: We show (independent cohort, n=155) that it is the IgM-class of anti-P1 antibodies which accounts for the previously observed discrimination "cancer vs. healthy" (p=0.0002). We also demonstrate that P1 antigen is indeed expressed on cells from fresh tissue specimens and on cultured ovarian cancer cells and that this naturally expressed P1antigen is recognized and bound by naturally circulating and affinity purified anti-P1-IgM isolated from patients ascites. IGROV1 cells expressing high levels of P1 (66%) migrated significantly faster than P1-low cells (33%). Conclusion: P1 antigen is for the first time reported to be expressed on ovarian cancer cells and is thus proposed as a novel ovarian cancer-associated carbohydrate antigen with a potential diagnostic and prognostic value. P1 glycosphingolipid may also play a role in cell migration.2 page(s