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. 2013 Mar 26;110(13):5052-7.
doi: 10.1073/pnas.1202653110. Epub 2013 Mar 11.

Cod Glycopeptide With Picomolar Affinity to galectin-3 Suppresses T-cell Apoptosis and Prostate Cancer Metastasis

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Cod Glycopeptide With Picomolar Affinity to galectin-3 Suppresses T-cell Apoptosis and Prostate Cancer Metastasis

Prasun Guha et al. Proc Natl Acad Sci U S A. .
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Abstract

Cancer metastasis and immune suppression are critical issues in cancer therapy. Here, we show that a β-galactoside-binding lectin [galectin-3 (gal3)] that recognizes the Thomsen-Friedenreich disaccharide (TFD, Galβ1,3GalNAc) present on the surface of most cancer cells is involved in promoting angiogenesis, tumor-endothelial cell adhesion, and metastasis of prostate cancer cells, as well as evading immune surveillance through killing of activated T cells. To block gal3-mediated interactions, we purified a glycopeptide from cod (designated TFD100) that binds gal3 with picomolar affinity. TFD100 blocks gal3-mediated angiogenesis, tumor-endothelial cell interactions, and metastasis of prostate cancer cells in mice at nanomolar levels. Moreover, apoptosis of activated T cells induced by either recombinant gal3 or prostate cancer patient serum-associated gal3 was inhibited at nanomolar concentration of TFD100. Because the gal3-TFD interaction is a key factor driving metastasis in most epithelial cancers, this high-affinity TFD100 should be a promising antimetastatic agent for the treatment of various cancers, including prostate adenocarcinoma.

Conflict of interest statement

Conflict of interest statement: The US Patent entitled “Methods of use for a natural Thomsen-Friedenreich disaccharide compound” has been filed in March 2012.

Figures

Fig. 1.
Fig. 1.
Purification and characterization of TFD100. (A) Separation of affinity-purified TFD-containing glycopeptides on a Superdex 75 10/300 GL column calibrated with BSA (dimer 132 kDa and monomer 66 kDa), chymotripsinogen (25 kDa), ribonuclease A (14 kDa), and aprotinin (6.5 kDa). (B) SDS/PAGE under reducing condition on 4–12% Bis-Tris gels followed by silver staining: (i) Crude AFGP (10 μg); (ii) TFD100 (5 μg). (C) Inhibition of gal3 binding to asialofetuin. Inhibition of gal3 binding to asialofetuin by various concentrations of compounds. (D and E) Surface plasmon resonance assay on Biacore. (D) TFD (Galβ1,3GalNAc) was measured from 31 μM to 1,000 μM, and the dissociation constant (KD) was determined to be 6.36e−4 M or 636 μM. (E) TFD100 was measured from 0.312 nM to 10 nM, and the KD was determined to be 9.677e−11 M or 97 pM. The full kinetic analysis results are as follows: ka = 3.105e+6 (1/ms); kd = 3.004e−4 (1/s); KD = 9.677e−11 M; Rmax = 31 (resonance units; RU); Tc = 2.26e+18; χ2 = 0.112 (RU2); U value = 2.
Fig. 2.
Fig. 2.
In vitro angiogenesis. (A) Angiogenesis of HUVECs in the absence or presence of external gal3 and inhibition of angiogenesis with 3.5 nM TFD100 or 50 μM lactose. (B) Quantitation of angiogenesis as measured by number of capillary tube branch point. (C) Quantitation of capillary tube formation by HUVECs after treating with various siRNAs in the presence or absence of TFD100. The data in B and C are shown as the means ± SD from three determinations. ***P < 0.001; **P < 0.01; *P < 0.05; ANOVA.
Fig. 3.
Fig. 3.
In vivo angiogenesis. A mixture of matrigel and VEGF in the absence or presence of gal3 and TFD100 was administered in each mouse (strain C57BL/6 black) under skin at the abdomen. After a week, mice were euthanized and matrigel plugs were removed (A) and stained with hematoxylin and eosin (B), and anti-CD31 antibodies (C). Arrows show blood vessels. (D) Quantitation of microvessels. The data are shown as the means ± SD from three determinations. **P < 0.01; *P < 0.05; ANOVA.
Fig. 4.
Fig. 4.
PC3–HUVEC interactions. (A) Quantitation of bound PC3 cells on HUVECs pretreated with various reagents. (B) Inhibition of PC3 cell adhesion to activated and nonactivated HUVECs with TFD100 (3.5 nM). (C and D) Quantitation of bound PC3 cells pretreated with various reagents on HUVECs. (E) Both PC3 cells and HUVECs were pretreated with TFD100 (3.5 nM), and siRNA and PC3 cell adhesion to HUVECs was performed. All data are shown as the means ± SD from three determinations. ***P < 0.001; ###P < 0.001; **P < 0.01; *P < 0.05; ANOVA. In A, C, and E, data showing significance are against untreated PC3–HUVEC control interactions.
Fig. 5.
Fig. 5.
Apoptosis of nonactivated CD8+ (AD) and activated (EG) CD8+CD25+ T cells. (A) Gating of nonactivated pmel T cells. (B) pmel T cells alone. (C) pmel T cells with gal3 (15 μM). (D) Quantitation showing percent of nonactivated T cells. (E) Gating of pmel activated CD8+CD25+ T cells (shown in red square). (F) Representative cytogram showing apoptosis of pmel activated CD8+CD25+ T cells with gal3 (5 μM). (G) Representative cytogram showing apoptosis of pmel activated CD8+CD25+ T cells with gal3 plus TFD100 (3.5 nM). (H) Quantitation showing percent of activated CD8+CD25+ T cells. (I) Overlay cytograms showing expression of gal3 on B16 melanoma cell surface (red, unstained cells, mean fluorescence unit 214; green, with gal3 Ab, mfu 2832; and blue, with preimmune IgG, mfu 1005. (J) B16 cell-associated apoptosis of pmel activated CD8+CD25+ T cells. Quantitation of B16-mediated apoptosis of pmel activated CD8+CD25+ T cells in the presence of 3.5 nM TFD100 or gal3 siRNA. (K) Apoptosis of activated CD8+CD25+ T cells with gal3-containing or gal3-depleted sera from normal and PCa patients as assessed by annexin V binding. All data are shown as the means ± SD from three determinations. ***P < 0.001; **P < 0.01; *P < 0.05; ANOVA.
Fig. 6.
Fig. 6.
Cancer metastasis induced by PC3 cells expressing a luciferase reporter (PC3-Luc) cells and its inhibition with TFD100. (A) Representative images showing glowscale of luciferase-expressing PC3 cells (wild type or gal3 knockout) injected into the tail vein of nude mice. (B) Western blot showing expression of gal3, gal4, and gal9 in wild-type and gal3−/− (knockout) PC3-Luc cells. (C) Lung photon flux from untreated and TFD100-treated mice. NS, not significant. (D) Lungs of PBS or TFD100-treated mice stained with India ink. White arrow in the PC3-Luc+PBS injected mouse lung indicates tumor. (E) Representative diagrams showing ki67 stain of lung sections from untreated or TFD100-treated mice.

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