Plant lectin can target receptors containing sialic acid, exemplified by podoplanin, to inhibit transformed cell growth and migration

Abstract

Cancer is a leading cause of death of men and women worldwide. Tumor cell motility contributes to metastatic invasion that causes the vast majority of cancer deaths. Extracellular receptors modified by α2,3-sialic acids that promote this motility can serve as ideal chemotherapeutic targets. For example, the extracellular domain of the mucin receptor podoplanin (PDPN) is highly O-glycosylated with α2,3-sialic acid linked to galactose. PDPN is activated by endogenous ligands to induce tumor cell motility and metastasis. Dietary lectins that target proteins containing α2,3-sialic acid inhibit tumor cell growth. However, anti-cancer lectins that have been examined thus far target receptors that have not been identified. We report here that a lectin from the seeds of Maackia amurensis (MASL) with affinity for O-linked carbohydrate chains containing sialic acid targets PDPN to inhibit transformed cell growth and motility at nanomolar concentrations. Interestingly, the biological activity of this lectin survives gastrointestinal proteolysis and enters the cardiovascular system to inhibit melanoma cell growth, migration, and tumorigenesis. These studies demonstrate how lectins may be used to help develop dietary agents that target specific receptors to combat malignant cell growth.

Figure 1. Src activation induces Pdpn expression and cell migration.

(a) PDPN, active Src kinase (phosphorylated at Y-416), and β-actin were detected by Western blotting of protein (20 µg per lane) from LA-25 cells (NRK cells containing temperature sensitive v-Src) grown overnight at 33°C (permissive temperature) or 40°C (non-permissive temperature). (b) PDPN, active Src (phosphorylated at Y416), and β-actin were detected by Western blotting of protein (15 µg per lane) from nontransformed cells or Src transformed mouse embryonic cells (MEFs) as indicated. (C) Wound healing migration assays were performed on confluent monolayers of Src transformed or nontransformed cells. Data are shown as the number of cells that migrated into a 300×300 micron area along the center of the wound in 24 hours (mean + SEM, n = 7). Double asterisks indicate p<0.01 compared to untreated Src controls.

Figure 2. MASL associates with PDPN and inhibits migration of Src transformed cells.

(a) Src transformed cells were exposed to MASL conjugated to HiLyte Fluor TR (red), and PDPN was detected by immunofluorescence microscopy (green). Colocalization of lectin and PDPN (yellow) is apparent in merged images, including orthogonal views of the z-axis which is 14 microns thick (bar = 20 microns). (b) Protein from Src transformed cells (750 μg) was precipitated with agarose beads linked to MASL, or control beads, and examined for PDPN by Western blotting. Cell lysate (15 μg/lane) were also examined as indicated. (c) Wound healing migration assays were performed on confluent monolayers of cells treated with concentrations of MASL as indicated. Data are shown as the number of cells that migrated into a 300x300 micron area along the center of the wound in 24 hours (mean + SEM, n = 7). Double asterisks indicate p<0.01 compared to nontransformed cells.

Figure 3. MASL targets PDPN to decrease cell migration and viability.

(a) PDPN and β-actin were detected by Western blotting of protein (15 µg per lane) from nontransformed mouse embryonic cells (MEFs) transfected with cDNA encoding PDPN or empty parental vector (EF4) as indicated. (b) Wound healing migration assays were performed on confluent monolayers treated with concentrations of MASL as indicated. Data are shown as the number of cells that migrated into a 300×300 micron area along the center of the wound in 24 hours (mean + SEM, n = 7). (c) MASL toxicity was evaluated by Trypan blue staining of cells, and shown as the percent of live cells from each well (mean + SEM, n = 4). Single and triple asterisks indicate p<0.05 and p<0.001, respectively, compared to untreated controls.

Figure 4. MASL inhibits melanoma cell motility and viability.

(a) PDPN and β-actin were detected by Western blotting of protein (15 µg per lane) from Melan-a melanocytes and B16 melanoma cells. (b) Wound healing migration assays were performed on confluent MelanA and B16 monolayers. Data are shown as the number of cells that migrated into a 400×500 micron area along the center of the wound in 24 hours (mean + SEM, n = 5). (c) Wound healing migration assays were performed on confluent monolayers treated with MASL as indicated. Data are shown as the number of cells that migrated into a 400×500 micron area along the center of the wound in 24 hours (mean + SEM, n = 5). (d) Melan-a and B16 cells were treated with MASL, and cell viability was evaluated by Alamar blue assay. Data are shown as percent of nontreated cells (mean + SEM, n = 2). (e) Melanoma cell viability and Transwell migration assays were performed on 600,000 cells plated on cell culture inserts containing membranes with an 8 micron pore size in 6-well plates. Cell viability was evaluated by Alamar blue assay and shown as percent of nontreated cells, while Transwell migration was measured after 24hours as the percent of cells found on the underside of the membrane (mean + SEM, n = 2). Double and triple asterisks indicate p<0.01 and p<0.001 compared to corresponding treatments of untreated cells or Melan-a cells in panels d and e, respectively.

Figure 5. MASL targets PDPN to inhibit melanoma cell growth.

(a) PDPN and β-actin were detected by Western blotting of protein (5 µg per lane) from B16 melanoma cells transfected with control siRNA or siRNA targeted against PDPN, as indicated. (b) Wound healing migration assays were performed on confluent B16 monolayers transfected with control siRNA or siRNA targeted against PDPN, as indicated. Data are shown as the number of cells that migrated into a 500×400 micron area along the center of the wound in 24 hours (mean + SEM, n = 4). (c) MASL toxicity was evaluated by Trypan blue staining of cells, and shown as the percent of live cells from each well (mean + SEM, n = 2). Single, double, and triple asterisks indicate p<0.05, p<0.01, and p<0.001, respectively, compared to nontransformed cells, untreated Src transformed cells, or control transfectants as indicated.

Figure 6. Effects of MASL on PARP cleavage.

(top) PARP and β-actin were examined by Western blotting of protein from B16 melanoma cells treated for 24 hours with MASL or 37 µM puromycin as indicated. (bottom) Signal was quantitated by image densitometry (NIH Image) and shown as the percent of cleaved PARP compared to total PARP (mean + SEM, n = 2).

Figure 7. Dietary MASL bioactivity survives gastrointestinal proteolysis to enter the cardiovascular circulatory system and inhibit melanoma cell migration.

Wound healing migration assays were performed on confluent monolayers of B16 melanoma cells treated with serum from mice fed MASL to achieve doses of 1, 100 mg/kg, or 200 mg/kg, or without mouse serum (controls) as indicated. Data are shown as the number of cells that migrated into a 200×300 micron area along the center of the wound in 24 hours (mean + SEM, n = 7). Triple asterisks indicate p<0.001 compared to controls.

Figure 8. Dietary MASL inhibits melanoma growth in vivo.

(a) Mice were fed MASL to achieve a dosage of 0 or 25 mg/kg once weekly (indicated by asterisks) and inoculated subcutaneously with B16 melanoma cells (100,000 cells per mouse on day indicated by arrow). Tumors were measured daily by caliper, and data is shown as cubic cm (mean + SEM, n = 4). (b) Tumors were evaluated for PDPN expression by IHC as indicated (bar  = 50 microns). “bc” indicates blood filled vascular spaces lined by tumor cells. (c) Tumors were examined by hematoxylin and eosin staining (H&E) to visualize vascularization and morphology. (c) Tumor vascularization was quantified as the percent of each field (0.8 mm2) occupied by blood vessels and shown as mean + SEM (n = 7).

Figure 9. Human melanoma cells express PDPN and respond to MASL.

(a) PDPN was detected by Western blotting of protein (15 µg per lane) from a variety of human specimens including normal skin, primary melanoma, melanoma in transit prior to lymph node metastasis, nodal metastasis, lung metastasis, and three melanoma cells lines (HT-144, SK-MEL-2, SK-MEL-5). Detection of MAPK and nonspecific bands by Western blotting and India ink staining of membranes is also shown to verify equal loading of these samples from different patients and cell lines. (b) Wound healing migration assays were performed on confluent HT-144 monolayers treated with MASL as indicated. Data are shown as the number of cells that migrated into a 400×500 micron area along the center of the wound in 24 hours (mean + SEM, n = 5). (d) HT-144 cells were treated with MASL, and cell viability was evaluated by Alamar blue assay. Data are shown as percent of nontreated cells (mean + SEM, n = 2). Single, double, and triple asterisks indicate p<0.05, p<0.01, and p<0.001 compared to untreated cells, respectively.

Figure 10. Analysis of MASL protein subunits.

(a) MASL from Sigma (lane 1) or Sentrimed (lane 2) was analyzed by reducing and nonreducing SDS-PAGE. (b) Coomassie stained bands were excised, extracted and digested with trypsin, reduced and alkylated with iodoacetamide, and sequenced using data dependent acquisition by LC-MS/MS on a Thermo Scientific LTQ Orbitrap XL. All peptides were identical in primary sequence and contained a single cysteine at residue 243 (bold in figure).

Figure 11. Comparison of Pdpn and Vegfr2 (Kdr) mRNA expression in human tissues.

Values from Affymetrix probe sets representing Pdpn and Vegfr2 are shown as presented on BioGPS (http://biogps.gnf.org).