Use of Glycoproteins-Prostate-Specific Membrane Antigen and Galectin-3 as Primary Tumor Markers and Therapeutic Targets in the Management of Metastatic Prostate Cancer

Abstract

Galectins and prostate specific membrane antigen (PSMA) are glycoproteins that are functionally implicated in prostate cancer (CaP). We undertook this study to analyze the "PSMA-galectin pattern" of the human CaP microenvironment with the overarching goal of selecting novel-molecular targets for prognostic and therapeutic purposes. We examined CaP cells and biopsy samples representing different stages of the disease and found that PSMA, Gal-1, Gal-3, and Gal-8 are the most abundantly expressed glycoproteins. In contrast, other galectins such as Gal-2, 4-7, 9-13, were uniformly expressed at lower levels across all cell lines. However, biopsy samples showed markedly higher expression of PSMA, Gal-1 and Gal-3. Independently PSA and Gleason score at diagnosis correlated with the expression of PSMA, Gal-3. Additionally, the combined index of PSMA and Gal-3 expression positively correlated with Gleason score and was a better predictor of tumor aggressiveness. Together, our results recognize a tightly regulated "PSMA-galectin- pattern" that accompanies disease in CaP and highlight a major role for the combined PSMA and Gal-3 inhibitors along with standard chemotherapy for prostate cancer treatment. Inhibitor combination studies show enzalutamide (ENZ), 2-phosphonomethyl pentanedioic acid (2-PMPA), and GB1107 as highly cytotoxic for LNCaP and LNCaP-KD cells, while Docetaxel (DOC) + GB1107 show greater efficacy in PC-3 cells. Overall, 2-PMPA and GB1107 demonstrate synergistic cytotoxic effects with ENZ and DOC in various CaP cell lines.

Keywords: galectins; glycoproteins; metastasis; prostate cancer; prostate specific membrane antigen; tumor microenvironment.

Figure 1

Expression profile of different galectins and PSMA in prostate cancer cell lines varying in aggressive potential and AR expression status. (A) Transcriptional profile of galectins and PSMA by Q-PCR. Results are expressed as galectin or PSMA mRNA relative to β-Actin. Cell line data are shown according to their stated aggressive potential and AR expression status. Left, minimally aggressive, Androgen receptor⁺, LNCaP cells; middle, moderately aggressive, AR⁺ DU-145 cells; right, the most aggressive AR⁻, PC-3 cells. Data are expressed as mean ± SD of 4 independent experiments. (B) immunocytochemical analysis of galectins and PSMA in prostate cancer cells of varying aggressive potential adhered onto poly-l-lysine–coated glasses (magnification, ×40).

Figure 2

Basal expression of different galectins and PSMA in prostate cancer cell lines of varying aggressive potential by Western blot and flow cytometry analysis. (A) Representative Western blot images (Figure S1) showing differential expression of galectins and PSMA in prostate cancer cells probed by specific antibodies for galectins and PSMA. Cells were lysed and equal amount of lysates (10 µg) were loaded/lane, run on 10% SDS reducing gel electrophoresis, transferred to PVDF membranes for Western blotting for β-actin, galectins, and PSMA as described in the Section 2. Representative Western blots from three separate experiments yielded similar results. (B) Quantitative values of densitometric analysis of scans of Western blots normalized to β-actin and expressed as a percentage of β-Actin expression in each cell line. (C) Representative histograms of our flow cytometry data showing expression of galectin-8 (top panel), galectin-1 (second panel), galectin-3 (third panel), and PSMA (bottom panel) in LNCaP, DU-145, and PC-3 cells respectively. Unstained cells of each cell line serve as a control when overlayed onto the stained population and allows identification of the galectin-8, galectin-1, galectin-3, and PSMA expressing cells. Y-axis shows cell counts and X-axis shows FITC positive cells.

Figure 3

Immunofluorescence image intensity and correlational analysis of β-actin, galectin-1, galectin-3 and PSMA in prostate cancer biopsies of varying Gleason score as quantitated by image J Analysis and numerical data on fluorescence intensity analyzed using GraphPad Prism. (A) Representative immunofluorescence images of different Gleason Score biopsied samples. Immunofluorescence staining was done using specific primary antibody as described under the Section 2. The secondary antibody used had Alexa Fluor 594 (Red) and the nucleus was stained with DAPI (blue) in all cases. The expression levels of each were quantitated based on the intensity of the fluorescent signal analyzed using the computer image analysis image J software (National Institutes of Health, Bethesda, MA, USA). Standard immunofluorescence staining procedures as described under the Section 2 were followed. Imaging was performed with the EVOS^® FL Cell Imaging System and images shown are representative images for proteins probed with Gleason Score 9 (left panel), Gleason score 6 (middle panel) and no Gleason Score (right Panel 3). (B) Correlational analysis of numerical data of staining intensity of (I) PSMA pixel units vs. Gal-3-pixel units, (II) PSMA pixel units vs. Gleason Score, (III) galectin-3 pixel units vs. Gleason score and (IV) PSMA + galectin-3 pixel units vs. Gleason score.

Figure 4

Dose dependent cytotoxicity of Enzalutamide (A), 2-Phosphonomethyl pentanedioic acid (2-PMPA) (B), GB 1107 (C) in CaP cells of varying aggressive potential and of all these drugs on PWR-1E cells (D). Prostate cancer cells of varying aggressive potential LNCaP(low), LNCaP-KD(low), PC-3(high) cells and normal prostate epithelial cells PER-1E cells were treated with indicated concentrations of either enzalutamide, 2-PMPA or GB-1107 for 72 h following which viability of surviving cells was determined by the 3 (4,5 dimethylthiazol 2 Yl) 2,5 diphenyltetrazolium bromide (MTT) assay using a microtiter plate reader (Bio Tek Instruments, Inc., Winooski, VT, USA) at 570 nm. Results are the mean ± SD of four independent experiments done in triplicate. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, NS = not significant.

Figure 5

Combination treatment of prostate cancer cells and normal prostate epithelial cells with different chemotherapy drugs. LNCaP, LNCaP KD, PC-3, and PER-1E cells were treated with indicated doses of ENZ, ENZ+ 2-PMPA, or ENZ+ 2-PMA+GB-1107 for 72 h and were analyzed for viability of surviving cells as determined by the 3 (4,5 dimethylthiazol 2 Yl) 2,5 diphenyltetrazolium bromide (MTT) assay. (A) Combination treatment of all three drugs at reported optimal doses in different prostate cancer cells. (B) Combination treatment of ENZ and 2-PMPA at reported optimal doses and increasing concentration of GB-1107 in different prostate cancer cells, and (C) combination treatment of ENZ and 2-PMPA and GB-1107 at reported optimal doses with or without Docetaxel in normal prostate epithelial cells, PWR-1E. Results are the mean ± SD of four independent experiments done in triplicate. Statistical significance: ** p < 0.01, *** p < 0.001.

Figure 6

Combination treatment of prostate cancer cells with varying aggressive potential with Docetaxel and GB 1107. LNCaP, LNCaP KD, and PC-3 cell with varying aggressive potential were treated with indicated doses of Docetaxel for 72 h, and analyzed for viability of surviving cells as determined by the 3 (4,5 dimethylthiazol 2 Yl) 2,5 diphenyltetrazolium bromide (MTT) assay. (A) Dose effect and IC ₅₀ for Docetaxel, (B) combination of IC₅₀ level of Docetaxel with varying concentration of GB 1107 on viability of PC-3 cells as determination by 3 (4,5 dimethylthiazol 2 Yl) 2,5 diphenyltetrazolium bromide (MTT) assay. Results are the mean ± SD of four individual experiments done in triplicate. Statistical significance: ** p < 0.01, *** p < 0.001.