Proteomic identification of fucosylated haptoglobin alpha isoforms in ascitic fluids and its localization in ovarian carcinoma tissues from Mexican patients

Abstract

Background: Ovarian cancer is the most lethal gynecologic disease due to delayed diagnosis, and ascites production is a characteristic of patients in advanced stages. The aim of this study was to perform the proteomic analysis of ascitic fluids of Mexican patients with ovarian carcinoma, in order to detect proteins with a differential expression pattern in the continuing search to identify biomarkers for this disease.

Methods: Samples were collected from 50 patients from the Instituto Nacional de Cancerología of México under informed consent and with approval of the bioethics and scientific committees. After elimination of abundant proteins (Albumin/IgGs) samples were processed for 2D electrophoresis and further protein identification by Mass Spectrometry (MALDI-TOF). Molecules of interest were followed by western blot and lectin binding assays, and their tissue location by histo-immunofluorescence and confocal analysis.

Results and discussion: An area with a differential expression pattern among samples was located in the 2D gels. Identified proteins were 6 alpha 1 isoforms and 1 alpha 2 isoform of Haptoglobin, and 2 isoforms of Transthyretin. While Transthyretin isoforms were constitutively expressed in all samples, clear differences in the expression pattern of Haptoglobin alpha isoforms were found. Moreover, increased levels of fucosylation of Haptoglobin alpha isoforms analyzed in 40 samples by Aleuria aurantia lectin binding by 1D overlay assay showed a positive correlation with advanced stages of the disease. Tissue detection of Haptoglobin and its fucosylated form, by histo-immunofluorescence in biopsies of ovarian cancer, also showed a correlation with ovarian cancer progression. Moreover, results show that fucosylated Haptoglobin is produced by tumor cells.

Conclusions: Increased numbers of highly fucosylated Haptoglobin alpha isoforms in ascitic fluids and the presence of fucosylated Haptoglobin in tumor tissues of ovarian cancer Mexican patients associated with advanced stages of the disease, reinforce the potential of fucosylated Haptoglobin alpha isoforms to be characterized as biomarkers for disease progression.

Figure 1

Detection and selection of a differential expression profile zone. Silver staining pattern of a representative 15% SDS-PAGE of an ascitic fluid sample with more than 250 proteins resolved, after elimination of abundant proteins; an area with a differential expression profile showing nine protein spots, present in 100% of samples, was selected (black square); schematic representation of the differential expression area and a closer view of the resolution of the corresponding spots (a-g).

Figure 2

Phenotypes and genotypes of haptoglobin alpha isoforms in Mexican patients. A). Selected area of the protein profiles obtained from 20 different and independent samples, representative of the total 50 samples collected for this study. Protein samples (50 μg) from ascitic fluids were processed for 2D after HAS/IgG depletion. B). Hp alpha genotypes (1–1, 1–2, 2–2) present in all 50 samples were determined and counted. C). Graph showing the number of protein spots detected by 2D in the selected area for each of the 50 patients according to their clinical stage.

Figure 3

Fucosylation profile of haptoglobin. A). Haptoglobin standard (ST), an ascitic fluid sample non-related with cancer (NR), and 40 ascitic fluid samples from ovarian cancer patients were analyzed for fucosylation of haptoglobin alpha. Protein samples (50 μg) from ascitic fluid free of abundant proteins were processed through a 12.5% SDS-PAGE and transferred to a NCP to incubate with biotinylated-Aleuria aurantia lectin (1:1000), specific for fucose residues; variable levels of fucosylation were detected by a colorimetric system using Alkaline Phosphatase-conjugated Streptavidin (1:2000) (lower left panel) or with Horseradish Peroxidase-conjugated Streptavidin (1:1000) (lower right panel). As loading control, the same samples were incubated with an anti-Hp antibody that recognizes the β, α1 and α2 isoforms of Hp to detect total Hp, and a secondary antibody conjugated with Horseradish Peroxidase (1:1000) (upper right panel) or Alkaline Phosphatase-conjugated antibody (1:1000) (upper left panel). B). Graph showing densitometric values of samples’ fucosylation in arbitrary units, comparing clinical stages IIIC and IV vs. a non-cancer related sample. C). Fucosylation levels (low, medium or high) of samples, according to clinical stages.

Figure 4

Immunolocalization of haptoglobin and its fucosylation state in ovarian carcinoma. A). Detection of total (Hp_T) and fucosylated Hp (Hp_F) in a Hp commercial standard (ST) and a tissue extract from an ovarian tumor developed in Nu/Nu mice (T) by Western blot and lectin binding assays (as described in figure 3). B, C, E). Immunolocalization of Hp, fucosylated Hp and fucosylation. Tissue nuclei were stained with DAPI (blue) to help define the tissues’ structures; Hp was stained with a primary anti-Hp antibody and was developed with a secondary TRITC coupled antibody (red); fucosylation was detected by biotinylated-Aleuria aurantia lectin followed by FITC coupled streptavidin (green). All reagents were used at a 1:100 dilution and the scale bar = 100 μm. B).Histo-immunofluorescence of stromal and epithelial (a) and germinal (b) areas of cancer-free ovarian tissue slices. The analysis of tile scan shows a tridimensional image generated by 25 fields’ caption (left panels); from the tile scan, an area was selected (yellow square) and optical zooms were done twice (10X and 40X). Epithelium (E), stroma (S), and follicles (F). C). Tumor tissue sections from four different patients. Fucosylation of Hp was analyzed in broad zones (5x5) of tissues as a tile-scan analysis. A representative region (yellow box) of each tissue was selected to detect fucosylated Hp in optical zooms. D). Graph showing a semi-quantitative analysis of nuclei, Hp and fucosylation content, considering an average area. This analysis was performed with the Software Zen 2011 (blue edition, Carl Zeiss) determining the mean fluorescence intensity for each molecule of interest given in arbitrary units. E). Immunolocalization of fucosylated Hp in individual cells in an optical zoom from a selected region of a tumor tissue section; the crop section analyzed (yellow box) clearly shows the cytoplasmic distribution of fucosylated Hp with approximately 80% of co-localization found in each selected cell.