The Antitumor Effect of Gekko Sulfated Glycopeptide by Inhibiting bFGF-Induced Lymphangiogenesis

Abstract

Objective. To study the antilymphangiogenesis effect of Gekko Sulfated Glycopeptide (GSPP) on human lymphatic endothelial cells (hLECs). Methods. MTS was conducted to confirm the antiproliferation effect of GSPP on hLECs; flow cytometry was employed to detect hLECs cycle distribution; the antimigration effect of GSPP on hLECs was investigated by wound healing experiment and transwell experiment; tube formation assay was used to examine its inhibitory effect on the lymphangiogenesis; western blotting was conducted to detect the expression of extracellular signal-regulated kinase1/2 (Erk1/2) and p-Erk1/2 after GSPP and basic fibroblast growth factor (bFGF) treatment. Nude mice models were established to investigate the antitumor effect of GSPP in vivo. Decreased lymphangiogenesis caused by GSPP in vivo was verified by immunohistochemical staining. Results. In vitro, GSPP (10 μg/mL, 100 μg/mL) significantly inhibited bFGF-induced hLECs proliferation, migration, and tube-like structure formation (P < 0.05) and antagonized the phosphorylation activation of Erk1/2 induced by bFGF. In vivo, GSPP treatment (200 mg/kg/d) not only inhibited the growth of colon carcinoma, but also inhibited the tumor lymphangiogenesis. Conclusion. GSPP possesses the antitumor ability by inhibiting bFGF-inducing lymphangiogenesis in vitro and in vivo, which may further inhibit tumor lymphatic metastasis.

Figure 1

GSPP inhibited bFGF-induced proliferation of hLECs and Erk phosphorylation. (a) Morphological characteristics of hLECs in vitro culture hLECs were compressed ovoid, short fusiform, or polygon and formed a single layer with the characteristic of “pebbles.” (b) hLECs growth curves (1 × 10⁵ cells/mL) were incubated with different concentration of GSPP (0, 10, and 100 µg/mL) and/or bFGF (10 ng/mL) for 0, 1, 2, 3, 4, 5, and 6 d, then cell proliferation was quantified by MTS assay, and cell growth curve was made. (c) The changes of Erk and p-Erk protein expression level of hLECs cultured in 6-well plate were incubated with different concentration of GSPP (0, 10, and 100 µg/mL) and/or bFGF (10 ng/mL); Erk and p-Erk protein levels were monitored by western blot analysis of whole-cell lysates. (d) Cell cycle analysis. Left, histogram of cell cycle distribution. Right, statistical analysis of cell cycle percentage. After exposure to GSPP (0, 10, and 100 µg/mL) and/or bFGF (10 ng/mL) for 48 h, cell cycle distribution was determined by propidium iodide labeling. (A) Control, (B) bFGF 10 ng/mL, (C) GSPP 10 µg/mL, (D) GSPP 100 µg/mL, (E) GSPP 10 µg/mL + bFGF 10 ng/mL, and (F) GSPP 100 µg/mL + bFGF 10 ng/mL. Data were presented as mean ± SD of three independent experiments. hLECs, human lymphatic endothelial cells; bFGF, basic fibroblast growth factor; GSPP, Gekko Sulfated Glycopeptide.

Figure 2

GSPP inhibits bFGF-induced migration of hLECs. (a) Wound healing assay. Left, representative images of injury width in wound healing assay (×40). Right, quantification of the migration ratio of hLECs compared to control. hLECs (1 × 10⁵ cells/well) were seeded in 24-well plates and wounds were generated after cell confluence. After hLECs were treated with different concentrations of GSPP (0, 10, and 100 μg/mL) and/or bFGF (10 ng/mL) for 0 h and 6 h, the photos were taken and the injury width was measured. The migration ratio was calculated as the migration width of experiment group/the migration width of control group. (b) Transwell assays. Left, representative images of migrated LECs in transwell assay (×40). Right, quantification of migrated LECs compared to control. hLECs (5 × 10⁴ cells/well) in EBM-2 with different concentrations of GSPP (0, 10, and 100 μg/mL) were added to the upper chamber of the transwell insert. EBM-2 containing bFGF (10 ng/mL) or not was added to the lower chamber to induce cell migration. After 12 h at 37°C, cells on the top surface of the membranes were wiped off with cotton balls, and the cells that migrated on the underside of inserts were fixed with methanol and stained with crystal violet. Five different digital images were taken per well, and the number of migrated cells was counted. (A) Control, (B) bFGF 10 ng/mL, (C) GSPP 10 µg/mL, (D) GSPP 100 µg/mL, (E) GSPP 10 µg/mL + bFGF 10 ng/mL, and (F) GSPP 100 µg/mL + bFGF 10 ng/mL. Data were presented as mean ± SD of three independent experiments; ^□ P < 0.05 versus control group and ^▽ P < 0.05 versus bFGF-single use group.

Figure 3

GSPP inhibits bFGF-induced lymphangiogenesis in vitro. In vitro tube formation assay. hLECs (1.5 × 10⁴ cells/well) were seeded in Matrigel-coated 96-well plates and treated with different concentration of GSPP (0, 10, and 100 μg/mL) and/or bFGF (10 ng/mL) for 4 h and the tube-like structure formation was observed. (a) Representative images of tube formation (×40). (b) Quantification of inhibitory ratios of tube branches. (A) Control, (B) bFGF 10 ng/mL, (C) GSPP 10 µg/mL, (D) GSPP 100 µg/mL, (E) GSPP 10 µg/mL + bFGF 10 ng/mL, and (F) GSPP 100 µg/mL + bFGF 10 ng/mL. Data were presented as mean ± SD of three independent experiments; ^□ P < 0.05 versus control group and ^▽ P < 0.05 versus bFGF-single use group.

Figure 4

GSPP inhibited colon carcinoma HT-29 xenograft growth and lymphangiogenesis in vivo. Male nu/nu nude mice were inoculated subcutaneously with colon carcinoma HT-29 cells. Three days after inoculation, mice were treated with GSPP (20 or 200 mg/kg) or PBS every day for 21 days via intraperitoneal injection. The lengths and widths of tumors were measured individually every 3 days. At the end of the experiment, the implanted tumors were sectioned. (a) Effect of GSPP on tumor volume. Left, image of excised tumors. Right, tumor growth curves. (b) Effect of GSPP on tumor weight. (c) Effect of GSPP on mouse weight. Mice were weighed at the end of the experiment. (d) Tumor lymphatic microvessel density. The implanted tumors were sectioned and stained against LYNE-1 antibody. Tumor lymphatic vessels are shown as LYNE-1 positive (yellow color). Data were presented as mean ± SD of three independent experiments; ^∗ P < 0.05 versus control group.