The heparan sulfate mimetic PG545 interferes with Wnt/β-catenin signaling and significantly suppresses pancreatic tumorigenesis alone and in combination with gemcitabine

The heparan sulfate mimetic PG545 has been shown to exert anti-angiogenic and anti-metastatic activity in vitro and in vivo cancer models. Although much of this activity has been attributed to inhibition of heparanase and heparan sulfate-binding growth factors, it was hypothesized that PG545 may additionally disrupt Wnt signaling, an important pathway underlying the malignancy of pancreatic cancer. We show that PG545, by directly interacting with Wnt3a and Wnt7a, inhibits Wnt/β-catenin signaling leading to inhibition of proliferation in pancreatic tumor cell lines. Additionally, we demonstrate for the first time that the combination of PG545 with gemcitabine has strong synergistic effects on viability, motility and apoptosis induction in several pancreatic cell lines. In an orthotopic xenograft mouse model, combination of PG545 with gemcitabine efficiently inhibited tumor growth and metastasis compared to single treatment alone. Also, PG545 treatment alone decreased the levels of β-catenin and its downstream targets, cyclin D1, MMP-7 and VEGF which is consistent with our in vitro data. Collectively, our findings suggest that PG545 exerts anti-tumor activity by disrupting Wnt/β-catenin signaling and combination with gemcitabine should be considered as a novel therapeutic strategy for pancreatic cancer treatment.


Animal groups and tumor monitoring using animal imaging system
Tumor volumes were measured weekly by the bioluminescence IVIS Imaging System 200 using a cryogenically cooled imaging system coupled to a data acquisition computer running Living Image software (Xenogen Corp., Alameda, CA). Before imaging, animals were anesthetized in an acrylic chamber with 2.5% isoflurane/air mixture and injected i.p. with 40 mg/mL d-luciferin potassium salt in PBS at a dose of 150 mg/kg body weight. After 10 min of incubation with luciferin, mice were placed in a right lateral decubitus position and a digital grayscale animal image was acquired followed by acquisition and overlay of a pseudo color image. Signal intensity was quantified as the sum of all detected photons within the region of interest per second. Mice were imaged on days 0, 7, 14, 21, 24, and 31 of treatment. Treatment was continued for 4 weeks and all mice were sacrificed 1 week later. Primary tumors in the pancreas were excised and the final tumor volume (V) was measured as V = 2 / 3πr 3 , where r is the mean of the three dimensions (length, width, and depth). Half of the tumor tissue was formalin fixed and paraffin embedded for immunohistochemistry and routine H&E staining. The other half was snap frozen in liquid nitrogen and stored at −80°C for immunoblotting.

Tumor sections for Immunohistochemistry
Tumor sections were cooled on bench top for 30 min, treated with 3% hydrogen peroxide in methanol for 10 min, and blocked with 6% horse serum for 30min at room temperature. Sections were then incubated with the primary antibody at 4°C overnight in humidity chamber. Sections were washed in PBST and incubated with secondary antibody (biotinylated goat anti-rabbit (1:150, Vector Laboratories, Burlingame, CA, USA) or biotinylated rabbit anti-rat IgG (1:150, Abcam, Boston, MA, USA) for 40min in humidity chamber. After further washes, the antibodies were detected with the Vector ABC complex/horseradish peroxidase (HRP) kit (Vector Laboratories, Burlingame, CA, USA), and color developed with 3,3′-diaminobenzidine tetrahydrochloride. For semiquantitation, ten photomicrographs (200×) were taken with a CCD camera, avoiding gross necrotic areas.

Figure S1: Effects of PG545 on Wnt/β-catenin signaling in pancreatic cancer cells.
(A) Various pancreatic cancer cells were treated with PG545 (20 μM) for 6h. (B) AsPC-1 cells were treated with different concentration of Wnt3a (25, 50 ng/ml) for 6 h in the presence of PG545 (10 ng/ml). β-catenin expression were determined by Western blotting. (C) The inhibition of AsPC-1 and Panc-1 cell proliferation by PG545 was measured using the BrdU assay after 24 h exposed to PG545 and Wnt3a (50 ng/ml).

Figure S2: PG545 blocks Wnt7a binding to cells.
(A) AsPC-1 cells were incubated with or withoutWnt3a (50 ng/ml), PG545 (20 µM) and the proteasome inhibitor MG132 (5 µM) for 6 h. Cell lysates were subjected to Western blotting. (B) Blocking effect of PG545 on the binding of recombinant Wnt7a to Panc-1 cells as assessed by immunofluorescence. Panc-1 cells were incubated with PG545 (20 μM) and/or recombinant His-tagged Wnt7a (200 ng/mL) for 6 h. Staining procedures were described in Materials and Methods. Scale bars, 5 µm. (C) AsPC-1 cells were incubated with indicated concentrations of PG545 with or without recombinant His-tagged Wnt7a (100 ng/mL) for 2 h at 4°C and then flow cytometric analysis was conducted. (D) Flow cytometric analysis for detection of FITC positive cells was used to observe the inhibition by PG545 of Wnt7a binding to Panc-1 cells. Cells were incubated with or without PG545 (20 μM) and/or recombinant His-tagged Wnt3a (100 ng/mL) for 2 h at 4°C. Y-axis represents the % of median FITC intensity indicating binding of Wnt ligands to cell surface.

Figure S3: Combination effect of PG545 and gemcitabine on apoptosis induction in gemcitabine resistant pancreatic cancer cells.
AsPC-1, Panc-1 and AsPC-1/GR cells were treated with indicated concentrations of PG545 and gemcitabine for 48 hr. After staining the cells with annexin V-FITC and PI, the apoptotic cells were analyzed by a dot-plot using a flow cytometer. The numbers in each plot indicate the percentage of apoptotic cells, i.e. those staining positively for annexin V alone and both annexin V and PI. Gem: gemcitabine.  AsPC-1 cells were scratched and then treated with PG545 and/or gemcitabine for 48 h. The number of cells that had migrated into the scratched area was expressed as a percentage of the scratch (wound) area. Data represent means ± S.D. from three independent experiments. AsPC-1 cells were treated with indicated concentrations of PG545 and gemcitabine for 48h. Western blot analysis was performed using antibodies against caspase 8 and β-actin. Treatment details given in Materials and Methods. Group1 was given PBS (100 μL, twice weekly by i.p.), group2 was given PG545 (200 mg/kg, twice weekly by i.p. injection), group3 was given gemcitabine (25 mg/kg, twice weekly by i.p. injection) and group4 was given PG545 (10 mg/kg) and gemcitabine (12.5 mg/kg) twice weekly by i.p. injection.