N-terminal and C-terminal heparin-binding domain polypeptides derived from fibronectin reduce adhesion and invasion of liver cancer cells

Abstract

Background: Fibronectin (FN) is known to be a large multifunction glycoprotein with binding sites for many substances, including N-terminal and C-terminal heparin-binding domains. We investigated the effects of highly purified rhFNHN29 and rhFNHC36 polypeptides originally cloned from the two heparin-binding domains on the adhesion and invasion of highly metastatic human hepatocellular carcinoma cells (MHCC97H) and analyzed the underlying mechanism involved.

Methods: The MHCC97H cells that adhered to FN in the presence of various concentrations of rhFNHN29 and rhFNHC36 polypeptides were stained with crystal violet and measured, and the effects of rhFNHN29 and rhFNHC36 on the invasion of the MHCC97H cells were then detected using the Matrigel invasion assay as well as a lung-metastasis mouse model. The expression level of integrins and focal adhesion kinase (FAK) phosphotyrosyl protein was examined by Western blot, and the activity of matrix metalloproteinases (MMPs) and activator protein 1 (AP-1) was analyzed by gelatin zymography and the electrophoretic mobility band-shift assay (EMSA), respectively.

Results: Both of the polypeptides rhFNHN29 and rhFNHC36 inhibited adhesion and invasion of MHCC97H cells; however, rhFNHC36 exhibited inhibition at a lower dose than rhFNHN29. These inhibitory effects were mediated by integrin αvβ3 and reversed by a protein tyrosine phosphatase inhibitor. Polypeptides rhFNHN29 and rhFNHC36 abrogated the tyrosine phosphorylation of focal adhesion kinase (p-FAK) and activation of activator protein 1 (AP-1), resulting in the decrease of integrin αv, β3 and β1 expression as well as the reduction of MMP-9 activity.

Conclusions: Polypeptides rhFNHN29 and rhFNHC36 could potentially be applicable to human liver cancer as anti-adhesive and anti-invasive agents.

Figure 1

Expression of purified rhFNHN29 and rhFNHC36 polypeptides. (A), Schematic structure of FN. FN is made up of a series of repeating homology units of three different types (FNI, FNII and FNIII). Yellow rectangles represent FNI modules, blue rhombus represent FNII modules and red ovals represent specific FNIII modules. RhFNHN29 containing N-terminal heparin-binding domain (HBD) is comprised of the FNI 1-5 modules, and rhFNHC36 containing C-terminal HBD is comprised of the FNIII12-14 modules. (B), Purified rhFNHN29 and rhFNHC36 polypeptides were subjected to SDS-PAGE and visualized by Coomassie Blue staining. (C), The purified rhFNHN29 and rhFNHC36 polypeptides were analysed by Western blot. Immunoreactive fragments of 27.9 kDa and 31.0 kDa indicated their antigenicity.

Figure 2

Heparin affinity analysis of rhFNHN29 and rhFNHC36 polypeptides. (A), The chromatography showed the peak fractions (Arrow) of eluted rhFNHN29 and rhFNHC36 polypeptides on a column (1 ml) of HiTrap Heparin HP at a flow rate of 1.5 ml/min. (B), LC/MS analysis indicated the elution position of molecular weight for purified rhFNHN29 (27.897 kDa) and rhFNHC36 polypeptides (31.051 kDa).

Figure 3

Standard RT-PCR analysis of different integrins or MMP expression in human liver cancer cell lines. Total RNA was isolated from human liver cancer cell lines and subsequently reverse-transcribed and analysed by PCR amplification for expression of integrin α4, α5, αv, β1 and β3 (A), MMP1, MMP2 and MMP9 (B) and, as internal control, β-actin (lower panel). Cell lines are as follows: SMMC7721, BEL7404, HepG2, Huh-7, MHCC97L and MHCC97H.

Figure 4

RhFNHN29 and rhFNHC36 inhibit MHCC97H cell adhesion to FN substrate and migration toward FN. (A), MHCC97H cell suspension (2 × 10⁵/100 μl) with or without Mn²⁺ (0.1 mM) was added to a 96-well plate coated with either BSA, type I collagen (20 μg/ml), type IV collagen (20 μg/ml) or FN (20 μg/ml). (B), Effects of different antibody concentrations (6, 12, 25 and 50 μg/ml) on MHCC97H cell adhesion to FN. (C), MHCC97H cell suspension (2 × 10⁵/100 μl) containing Mn²⁺ (0.1 mM) was pretreated with or without 25 μg/ml control IgG and function-blocking mAb to αv (P2W7), β3 (BV4) or β1 (8A2), respectively, and then seeded to a 96-well plate coated with FN (20 μg/ml) or poly-L-Lys (20 μg/ml) in the presence or absence of FN, rhFNHN29 and rhFNHC36 (50, 100 and 200 μg/ml), respectively. The data represent the mean ± SD of three determinations. (D), A cell invasion assay was carried out in blind-well chambers. MHCC97H cell suspension (5 × 10⁴/100 μl) containing Mn²⁺ (0.1 mM) treated with or without FN, rhFNHN29 and rhFNHC36 (50, 100 and 200 μg/ml), and control IgG, or anti-αv (P2W7), -β3 (BV4) or -β1 (8A2) (25 μg/ml) were incubated at 37°C for 1 h and then added to the top chambers, respectively. After incubation for 48 h, the number of cells migrating across the Matrigel-coated filter into the bottom chamber was counted. The data represent the mean ± SD of three determinations. (E), Compared with the morphous of MHCC97H cells under normal conditions (×200 magnification), representative pictures for migrated MHCC97H cells on the bottom chamber (×100 magnification) in the presence of FN, rhFNHN29 and rhFNHC36 (200 μg/ml) showed a fibroblastic appearance.

Figure 5

Effect of rhFNHN29 and rhFNHC36 on metastatic tumor colonies. Representative pictures of metastasis in lungs of the nude mice 6 wk after MHCC97H cell inoculation and being subjected to intervention by NS, FN, rhFNHN29 and rhFNHC36 (HE staining × 100 →lung metastasis). The group data represent the mean ± SD (n = 8). * denotes a statistically significant difference compared to the control (P < 0.05) and # denotes a statistically significant difference with rhFNHN29 (P < 0.05) by One-Way ANOVA.

Figure 6

Reduction of integrin expression and MMP activity by rhFNHN29 and rhFNHC36. (A-1), Representative immunoblots for integrin αv, β3 and β1 proteins. (A-2), The group data represents the mean ± SD (n = 3). The densitometry data were normalized to β-actin. * denotes a statistically significant difference compared to the control (P < 0.05) and # denotes a statistically significant difference compared to rhFNHN29 at the same concentration (P < 0.05) by One-Way ANOVA. (B-1), Analysis of MMP activity. Supernatants from MHCC97H cells cultured with Mn²⁺ in the absence or presence of different concentrations of rhFNHN29 and rhFNHC36 were performed by Gelatin Zymography. (B-2), The group data of enzymolysis strip volume represent mean ± SD (n = 3). * denotes a statistically significant difference compared to the control (P < 0.05) and # denotes a statistically significant difference compared to rhFNHN29 at the same concentration (P < 0.05) by One-Way ANOVA.

Figure 7

Effects of rhFNHN29 and rhFNHC36 on tyrosine phosphorylation of FAK and activation of AP-1. (A), Effect of PAO on the anti-adhesive activity of rhFNHN29 and rhFNHC36. MHCC97H cell suspension (2 × 10⁵/100 μl) with or without Mn²⁺(0.1 mM), with or without PAO (20 μM) were pretreated with FN, rhFNHN29 and rhFNHC36 (200 μg/ml), respectively, and then seeded in a 96-well plate coated with or without FN (2.5 μg/ml). Adhesion of MHCC97H cells to the FN substrate was assayed under the indicated conditions as described in Figure 2. (B), Immunoblotting of p125^FAK and EMSA of activated AP-1. MHCC97H cells were deprived of serum for 10 h, incubated in DMEM with or without PAO (20 μM) for 5 min, and further cultured with Mn²⁺(0.1 mM) in the presence or absence of FN, rhFNHN29 and rhFNHC36 (200 μg/ml) at 37°C for 20 min. The cytoplasmic protein and the nuclear protein were extracted respectively. The phosphotyrosyl proteins electroblotted to the PVDF membrane were visualized by anti-p-FAK (Tyr 397) after SDS-PAGE, and the activated AP-1 electroblotted to Nylon membrane was visualized by specific HRP-labeled probe after 6.5% PAGE for nuclear protein (F zone indicates non-binded free probe).