CA-125, the most frequently used biomarker for ovarian cancer detection, cannot provide accurate diagnosis due to its poor specificity as it may also increase in many benign gynecological conditions. Thus, reducing the false-positive outcomes is urgently needed. Decrease in terminal galactosylated N-glycans of serum IgG has been found in various malignancies compared to healthy controls. Here, this alteration of IgG galactosylation was extended to be investigated between ovarian cancer and benign conditions with similar elevated CA-125 levels, in an attempt to effectively distinguish between false-positive subjects and ovarian cancer patients. In the study of 58 patients with elevated CA-125 levels (>35 U/mL), the degree of IgG galactosylation was measured from the relative intensities of IgG digalactosyl (G2), monogalactosyl (G1), and agalactosylated (G0) N-glycans according to the formula G0/(G1+G2·2). This ratio was found significantly higher in the malignant group than in the benign group (0.74 vs 0.34; p<0.0001). ROC analysis demonstrated an improved specificity from 65.2% (by CA-125 test alone) to 84.6%, while maintaining sensitivity at 90% by incorporating quantitative analysis of IgG galactosylation in the current assay. The results suggest that combining quantitative alteration of IgG galactosylation with CA-125 may generate an overall more robust approach for differential diagnosis of ovarian cancer.
Generally, most of ovarian cancer cannot be detected until large scale and remote metastasis occurs, which is the major cause of high mortality in ovarian cancer. Therefore, it is urgent to discover metastasis-related biomarkers for the detection of ovarian cancer in its occult metastasis stage. Altered glycosylation is a universal feature of malignancy and certain types of glycan structures are well-known markers for tumor progressions. Thus, this study aimed to reveal specific changes of N-glycans in the secretome of the metastatic ovarian cancer. We employed a quantitative glycomics approach based on metabolic stable isotope labeling to compare the differential N-glycosylation of secretome between an ovarian cancer cell line SKOV3 and its high metastatic derivative SKOV3-ip. Intriguingly, among total 17 N-glycans identified, the N-glycans with bisecting GlcNAc were all significantly decreased in SKOV3-ip in comparison to SKOV3. This alteration in bisecting GlcNAc glycoforms as well as its corresponding association with ovarian cancer metastatic behavior was further validated at the glycotransferase level with multiple techniques including real-time PCR, western blotting, transwell assay, lectin blotting and immunohistochemistry analysis. This study illustrated metastasis-related N-glycan alterations in ovarian cancer secretome in vitro for the first time, which is a valuable source for biomarker discovery as well. Moreover, N-glycans with bisecting GlcNAc shed light on the detection of ovarian cancer in early peritoneal metastasis stage which may accordingly improve the prognosis of ovarian cancer patients.
A deep insight into the function and kinetics of ATP-binding cassette (ABC) transporters may aid in the development of pharmaceutics that can minimize the particular facet of chemo-resistance. We utilized bioluminescence imaging to monitor the ABC transporter mediated intracellular drug efflux function. We also investigated the potential association between the intracellular bioluminescent pharmacokinetic profiles and the anti-tumor efficacy of the coix seed extract and gemcitabine against pancreatic cancer cells in vitro and in vivo. The bioluminescent pharmacokinetic parameters and pharmacodynamic index (IC50 and TGI) were determined. The expression levels ABCB1 and ABCG2 were assessed. Results showed that coix seed extract could synergistically enhance the anti-cancer efficacy of gemcitabine (p < 0.05). Meanwhile coix seed extract alone or in combination with gemcitabine could significantly increase the AUCluc while decreasing the Kluc (p < 0.01). Western blot and immunohistochemistry assay demonstrated that coix seed extract could significantly mitigate gemcitabine-induced upregulation of ABCB1 and ABCG2 protein. The Pearson correlation analysis demonstrated that the bioluminescent pharmacokinetic parameters and pharmacodynamic index have strong association in vitro and in vivo. In conclusion coix seed extract could augment the efficacy of gemcitabine therapy in pancreatic cancer cells may at least partly due to the alteration of ABC transporter-mediated drug efflux function.
Human LOX-1/OLR 1 plays a key role in atherogenesis and endothelial dysfunction. The N-glycosylation of LOX-1 has been shown to affect its biological functions in vivo and modulate the pathogenesis of atherosclerosis. However, the N-glycosylation pattern of LOX-1 has not been described yet. The present study was aimed at elucidating the N-glycosylation of recombinant human LOX-1 with regard to N-glycan profile and N-glycosylation sites. Here, an approach using nonspecific protease (Pronase E) digestion followed by MALDI-QIT-TOF MS and multistage MS (MS(3)) analysis is explored to obtain site-specific N-glycosylation information of recombinant human LOX-1, in combination with glycan structure confirmation through characterizing released glycans using tandem MS. The results reveal that N-glycans structures as well as their corresponding attached site of LOX-1 can be identified simultaneously by direct MS analysis of glycopeptides from non-specific protease digestion. With this approach, one potential glycosylation site of recombinant human LOX-1 on Asn(139) is readily identified and found to carry heterogeneous complex type N-glycans. In addition, manual annotation of multistage MS data utilizing diagnostic ions, which were found to be particularly useful in defining the structure of glycopeptides and glycans was addressed for proper spectra interpretation. The findings described herein will shed new light on further research of the structure-function relationships of LOX-1 N-glycan.
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