Inhibition of prostate cancer bone metastasis by synthetic TF antigen mimic/galectin-3 inhibitor lactulose-L-leucine

Abstract

Currently incurable, prostate cancer metastasis has a remarkable ability to spread to the skeleton. Previous studies demonstrated that interactions mediated by the cancer-associated Thomsen-Friedenreich glycoantigen (TF-Ag) and the carbohydrate-binding protein galectin-3 play an important role in several rate-limiting steps of cancer metastasis such as metastatic cell adhesion to bone marrow endothelium, homotypic tumor cell aggregation, and clonogenic survival and growth. This study investigated the ability of a synthetic small-molecular-weight nontoxic carbohydrate-based TF-Ag mimic lactulose-L-leucine (Lac-L-Leu) to inhibit these processes in vitro and, ultimately, prostate cancer bone metastasis in vivo. Using an in vivo mouse model, based on intracardiac injection of human PC-3 prostate carcinoma cells stably expressing luciferase, we investigated the ability of Lac-L-Leu to impede the establishment and growth of bone metastasis. Parallel-flow chamber assay, homotypic aggregation assay, modified Boyden chamber assay, and clonogenic growth assay were used to assess the effects of Lac-L-Leu on tumor cell adhesion to the endothelium, homotypic tumor cell aggregation, transendothelial migration, and clonogenic survival and growth, respectively. We report that daily intraperitoneal administration of Lac-L-Leu resulted in a three-fold (P < .05) decrease in metastatic tumor burden compared with the untreated control. Mechanistically, the effect of Lac-L-Leu, which binds and inhibits galectins by mimicking essential structural features of the TF-Ag, was associated with a dose-dependent inhibition of prostate cancer cell adhesion to bone marrow endothelium, homotypic aggregation, transendothelial migration, and clonogenic growth. We conclude that small-molecular-weight carbohydrate-based compounds targeting β-galactoside-mediated interactions could provide valuable means for controlling and preventing metastatic prostate cancer spread to the skeleton.

Figure 1

Intravital bioluminescent imaging of PC-3Luc metastatic burden and subsequent postmortem histopathologic confirmation of skeletal metastases. (A) An example of the bioluminescent imaging using a CCD IVIS system showing PC-3Luc metastatic tumors to the limbs, jaws, and ribs. Two animals from the control group are shown at week 3 after tumor cell injection. A color scale reflecting photon count in pseudocolored images is shown on the left. (B–D) Histopathologic confirmation and analysis of skeletal metastases. (B) Photomicrograph of PC-3Luc bone metastasis (hematoxylin and eosin, decalcified; original magnification, x10). A tumor mass (T) is present in the marrow cavity, as well as smaller tumor cell clusters (T + arrow) in the cortical bone (B). (C and D) Osteoclast-like cells (OC), commonly associated with bone metastases, are present at the interface between the tumor (T) and the cortical bone (B) (hematoxylin and eosin, decalcified; original magnification, x10 [C] and x40 [D]).

Figure 2

The effect of Lac-l-Leu treatment on PC-3Luc bone metastasis. (A) Temporal dynamics of changes in metastatic tumor burden in individual animals from group 1 (daily Lac-l-Leu treatment commenced 1 week before tumor cell injection), group 2 (daily Lac-l-Leu treatment commenced 1 week after tumor cell injection), and control during the 4 weeks after intracardiac tumor cell inoculation as revealed by biophoton count. (B) Cumulative data showing average metastatic tumor burden in each experimental group at week 4 after tumor cell inoculation. Data are presented as means ± SEM. (C) Representative images of animals from each experimental group at 4 weeks after tumor cell injection.

Figure 3

Inhibition of PC-3Luc and DU-145 metastasis-associated adhesive interactions by Lac-l-Leu. (A) Synthetic TF-Ag mimic Lac-l-Leu, but not Lct-l-Leu, inhibited PC-3Luc (open bars) and DU-145 (closed bars) rolling on (left panel) and stable adhesion (right panel) to HBME-1 human bone barrow endothelial cell monolayers under conditions of physiological flow. (B and C) Synthetic TF-Ag mimic Lac-l-Leu inhibited PC-3Luc (left panel) and DU-145 (right panel) tumor cell homotypic aggregation in a dose-dependent manner (B), whereas its inactive isomer Lct-l-Leu failed to inhibit the formation of prostate cancer cell multicellular aggregates (C). The experiments in A and C were performed using 200 µM (final concentration) of the compounds tested. *P < .05, **P < .001.

Figure 4

Inhibition of prostate cancer cell transendothelial migration and clonogenic growth by synthetic TF-Ag mimic Lac-l-Leu. (A and B) Synthetic TF-Ag mimic Lac-l-Leu, but not its inactive isomer Lct-l-Leu, inhibited PC-3 and DU-145 cell transendothelial migration in vitro in transwell chamber experiments. (C) Dose-dependent inhibition of PC-3Luc (left panel) and DU-145 (right panel) cell clonogenic survival and growth by synthetic glycoamine Lac-l-Leu. *P < .05, **P < .001.

Figure 5

Lac-l-Leu mechanisms of action. (A) Heterotypic adhesive interactions between endothelial and cancer cells are inhibited through Lac-l-Leu binding to Gal-3 on endothelial cell surfaces preventing its interactions with TF-Ag presented on tumor cells. (B) Inhibition of tumor cell homotypic aggregation is achieved by blocking Gal-3 clustered on cancer cell outer membranes. (C) Inhibition of intracellular Gal-3 antiapoptotic function on mitochondrial apoptosis pathway reduces metastatic cell clonogenic survival.