N-terminal and C-terminal heparin-binding domain polypeptides derived from fibronectin reduce adhesion and invasion of liver cancer cells
© Tang et al; licensee BioMed Central Ltd. 2010
Received: 2 December 2009
Accepted: 13 October 2010
Published: 13 October 2010
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.
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.
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.
Polypeptides rhFNHN29 and rhFNHC36 could potentially be applicable to human liver cancer as anti-adhesive and anti-invasive agents.
Invasion and metastasis are important biological characteristics of malignant tumors. Metastatic formation requires specific cell-to-cell and cell-to-extracellular matrix (ECM) interactions mediated by integrins , cadherins , selectins , etc. In particular, integrin-mediated adhesion of tumor cells to ECM proteins and cell surface components is considered a crucial event in metastasis. Accordingly, the prevention of tumor cell adhesion to ECM proteins has been an area of interest as a potential target for therapeutic intervention [4–6].
Fibronectin (FN) is a type of adhesive-attraction glycoprotein. Previous studies have shown that the anchoring of FN to ECM in vitro plays an important role in cancer cell metastasis . Moreover, the study of FN expression on cancer cells has determined that decreased FN expression is closely associated with cancer growth and metastasis [8–10], which illustrates that an increase of FN expression in cancer cells may conversely facilitate the reduction of cancer cell metastasis, implying that FN may have the potential for a significant clinical application. Interestingly, our in vitro intervention experiments showed that free FN could inhibit the adhesion and metastasis of hepatocellular carcinoma cells. It is possible that the exposed cell binding site may not be the same in FN between free-status and anchoring-status. Therefore, the free FN has potential value for a therapeutic application. However, obstacles exist for the clinical application of FN, including deficiency of blood plasma, danger of blood-infection disease, and difficulty in engineering the synthesis of the whole-molecule FN with a molecular weight as large as 420 kDa. Fortunately, FN contains several active sites that serve as scaffoldings for cell anchorage , known as the heparin-binding domains, collagen-binding domain, fibrin-binding domain and cell-binding domain, respectively. These domains are involved in a diverse array of cell functions including adhesion, migration, differentiation, apoptosis, morphous change, haemostasis and reparation of damage, etc. Therefore, the replacement of FN with a polypeptide derived from a FN functional domain is considered to be a more feasible method for cancer treatment. Several reports have highlighted this type of application; for example, it has been found that arginine-glycine-aspartic acid (RGD) integrin-binding motif from FN containing the adhesion recognition signal Arg-Gly-Asp partially inhibit intrahepatic metastasis of murine hepatocellular carcinoma (HCC) . The CH50 polypeptide that contains Cell I and Hep II dual domain fragment of FN has been shown to play a role involving inhibition of tumor growth, invasion and angiogenesis . The FNIII14 peptide also effectively inhibited the adhesion and metastasis of lymphoma cells .
Heparin-binding domains are important molecular structures of FN: one (237 mer) at the N-terminal contains five I-type homologous structures , and another (272 mer) at the C-terminal contains three type III homologous structures . In previous studies we obtained purified recombinant N-terminal and C-terminal heparin-binding domain polypeptides of FN (rhFNHN29 and rhFNHC36) using genetic engineering, further testing their characteristics with heparin-binding activity measurements . However, the function of these fragments concerning cancer therapy was still unknown. In this study we investigated the effects of rhFNHN29 and rhFNHC36 on the adhesion and invasion of highly metastatic human HCC cells (MHCC97H) and analyzed the underlying mechanism.
Reagents, Cell Culture, and Animal Model
The reagents for the production of rhFNHN29 and rhFNHC36 were purchased: restriction enzyme XhoI, EcoRI, BamHI, HindIII, Bgl II and T4 ligase from Promega Co.; vector pPIC9K, pGEM-T, pAo815SM and GS115 yeast cells from Invitrogen Co.; purification column S-100, SP and HiTrap Heparin HP column from GE Co. BSA, type I collagen, type IV collagen and FN were purchased from Sigma Co.; and anti-FN pAb, anti-integrin αv (P2W7), β3 (BV4), β1 (8A2) mAb and anti-p-FAK mAb were purchased from Santa Cruz Biotechnology, Inc. Liver carcinoma cell lines (MHCC97H and MHCC97L) were purchased from Liver Cancer Institute of Fudan University (Shanghai, China) and maintained in DMEM medium supplemented with 10% fetal bovine serum (GIBCO) in a 5.0% CO2 incubator at 37°C . Huh-7, HepG2, BEL7404 and SMMC7721 cells were maintained in our laboratory. Male BALB/c nude mice at the age of 5 to 6 weeks were purchased from the Chinese Academy of Sciences (Shanghai) and were given humane care according to the criteria outlined in the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23 revised 1985). The experimental protocol was approved by the Medical Experimental Animal Care Committee of Fujian Medical University.
Production of rhFNHN29 and rhFNHC36
According to the structural features of yeast expression vector pGEM-T, PCR primers to clone the rhFNHN29 and rhFNHC36 genes were designed and the sequences of these primers are as follows:
5'-ATGCTC ↓ TCGAGAAAAGAGAGGCTGAAGCTAGTCAAAGCAAGCCCGGTTGTTA-3'(rhFNHN29-F), 5'-ACGTAG ↓ AATTCTCCGCTCGATGTGGTCTGCA-3'(rhFNHN29-R), 5'-CGG ↓ GATCCGCTATTCCTGCACCAACTGAC-3'(rhFNHC36-F) and 5'-CCCA ↓ AGCTTCTCGTCTGTCTTTTTCCTTCC-3'(rhFNHC36-R), which contain XhoI/EcoRI and BamHI/HindIII sites (underlined), respectively.
The rhFNHN29 gene was then cloned into the integrated yeast expression vector pPIC9K through the transition of the pGEM-T and pAo815SM vector. The recombinant plasmid of pPIC9K-FNHN29 was confirmed by restriction analysis and sequenced upon transfection of competent DH5a E.coli cells and selection of resistant clones. The pPIC9K-FNHN29 plasmid was extracted, digested by BglII enzyme, and transferred into the GS115 yeast cells. The resistant clone with high expression was cultured for amplification. The fermentation liquid was precipitated using 80% ammonium sulfate, and then the dissolved sediments were purified using an S-100 column and an SP column. The UV280 waveform of the purified liquid was monitored by HPLC. The collected peak waveform sample was detected by SDS-PAGE electrophoresis and western blot analysis for FN antigenicity. The yeast fermentation supernatant of rhFNHN29 was diluted by 5 mM PBS with an adjusted 7.0 pH value and conductivity less than 4.0 and then eluted on a HiTrap Heparin HP column. HPLC was used to monitor the UV280 waveform of the purified liquid. The collected peak waveform sample was measured by mass spectrometry to determine the molecular weight. The construction and identification procedure for the recombinant yeast expression vector of rhFNHC36 was identical to the procedure above.
Semiquantitative RT-PCR Analysis
RNA was isolated from the cell lines with Trizol (Invitrogen, USA) and was then reverse-transcribed. Fragments of integrin α4 (296 bp), α5 (362 bp), αv (236 bp), β1 (407 bp) and β3 (327 bp), and MMP-1 (428 bp), MMP-2 (390 bp), MMP-9 (215 bp) and β-actin (500 bp) cDNA were amplified by PCR. The primer sequences were as follows: integrin α4, sense 5'-CAACACGCTGTTCGGCTAC-3' and antisense (AS) 5'-TATGCCCACAAGTCACGATG-3'; integrin α5, sense 5'-CCGAATTCTGGAGTATGCAC-3' and AS 5'-TGGTGACATAGCCGTAAGTG-3'; integrin αv, sense 5'-GGAGCAATTCGACGAGCACT-3' and AS 5'-GCAGGCGTGAACTGGTTAAG-3'; integrin β1, sense 5'-TGAAGGGCGTGTTGGTAGAC-3' and AS 5'-GCCCTTGAAACTTCGGATCT-3'; integrin β3, sense 5'-CACCAGTAACCTGCGGATTG-3' and AS 5'-GTCTTGGCATCAGTGGTAAAC-3'; MMP-1, sense 5'-CTGAAGGTGATGAAGCAGCC-3' and AS 5'-AGTCCAAGAGAATGGCCGAG-3'; MMP-2, sense 5'-GCGACAAGAAGTATGGCTTC-3' and AS 5'-TGCCAAGGTCAATGTCAGGA-3'; MMP-9, sense 5'-AGTTCCCGGAGTGAGTTGAA-3' and AS 5'-CTCCACTCCTCCCTTTCCTC-3'; β-actin sense, 5'-ATGTCACGCACGATTTCCCGC-3' and AS 5'-GGCATGGGTCAGAAGGATTCC-3'.
Cell Adhesion Assay
Part 1: The MHCC97H cell suspensions (2×105/100 μl) with or without Mn2+ (0.1 mM) were added to 96-well plates coated with either BSA, type I collagen, type IV collagen, or FN at 20 μg/ml. After the above incubation at 37°C for 1 h, cells were fixed with formalin, and separated cells were washed off with PBS . Cells that adhered to the substrate were stained with crystal violet. Dye bound to adhered cells was solubilized with 0.1% SDS, and absorbance at 590 nm was measured. The data represent the mean value of three determinations. Part 2: The MHCC97H cell suspensions (2 × 105/100 μl) containing Mn2+ (0.1 mM) were pretreated with function-blocking mAb to αv (P2W7), β3 (BV4) or β1 (8A2) and control IgG at different concentrations (6, 12, 25 and 50 μg/ml), respectively, and were then seeded in 96-well plates coated with FN (20 μg/ml). The following procedures were identical to Part 1 above. Part 3: The MHCC97H cell suspensions (2 × 105/100 μl) containing Mn2+ (0.1 mM) were pretreated with or without 25 μg/ml mAb to αv (P2W7), β3 (BV4) or β1 (8A2) and control IgG, respectively, and were then seeded in 96-well plates coated with FN (20 μg/ml) or poly-L-Lys (20 μg/ml) in the presence or absence of FN, rhFNHN29 and rhFNHC36 at three different concentrations (50, 100 and 200 μg/ml). The procedure that follows is identical to Part1 above.
Matrigel Invasion Assay
Transwell culture inserts with an 8-mm pore-size PET membrane precoated with 20 μg Matrigel (BD, USA) were transferred to a new 24-well plate. The bottom chambers were coated with 10 μl FN (0.5 mg/ml) and air-dried. The MHCC97H cell suspension (5 × 104/100 μl) containing Mn2+ (0.1 mM) was then treated with or without FN, rhFNHN29 and rhFNHC36 (50, 100 and 200 μg/ml) at 37°C for 1 h, and control IgG, or anti-αv (P2W7), -β3 (BV4) or -β1 (8A2) (25 μg/ml) was then added to the top chambers, respectively. After 48 h incubation, the inserts were dipped into a fixing solution (containing 95% ethanol and 5% acetic acid) for 30 min, and the non-invading cells were removed from the upper surface of the membrane by scrubbing with a cotton swab. The cells on the lower surface of the membrane were stained with H&E. Cells from five different fields were counted under an inverted microscope at 100-fold magnification and the mean was calculated.
Experiments of Lung Metastasis
1 × 107 cells in a 0.2 ml volume of normal saline (NS) were inoculated subcutaneously into the right flank of nude mice. Seven days later, a tumor (approximately 5 mm in diameter) appeared at the cell inoculation site in each nude mouse model. 32 of the tumor-bearing nude mice were divided randomly into 4 groups (n = 8 per group), and then injected with NS (100 μl each time), FN, rhFNHN29 and rhFNHC36 (200 μg in 100 μl NS each time), respectively, at the tumor cell inoculation site for 7 consecutive days. At the end of 6 wk, all of the mice were sacrificed, and the lungs were excised, fixed in formalin, imbedded in paraffin, and cut into 4- μm consecutive sections and stained with H&E. Each metastatic focus in the lung was identified as it appeared at the same site on consecutive sections. Finally, all determined foci were counted to evaluate metastasis histologically [20, 21]. Three pathologists performed the counting independently and in a blinded manner.
The MHCC97H cell (1.2 × 106) suspensions were seeded into 6-well plates in DMEM supplemented with 10% FBS and Mn2+ in the absence or presence of differing concentrations of rhFNHN29 and rhFNHC36. After 24 h, cells were incubated in serum-free DMEM for 24 h. The supernatants were collected after centrifugation and the cells were trypsinized and counted. An assay to measure MMP activity was performed using Gelatin Zymography according to Iwai's report . Briefly, a volume of 20 μl of medium was loaded under non-denaturing conditions into a zymogram gel supplemented with 0.1% gelatin to detect the presence of MMP-2 and -9. Electrophoresis was performed at a constant voltage of 120 V for 90 min. Gels were washed in a re-naturing buffer and incubated in an incubation buffer at 37°C for 24 h, stained with Coomassie brilliant blue R-250 (Sigma), and then de-stained with gel-clear de-stain solution. Areas of gelatinolytic degradation appeared as transparent bands on the blue background. The enzymolysis strip volume [area × (gray stripe-gray background)] is expressed as the mean ± SD of the triplicate experiment for each group.
Western Blot Analysis of Integrin Protein
The MHCC97H cells were cultured as per the process described in "MMP activity". 50 μg samples of cell extract were run on 10% SDS-PAGE gels and electro-blotted to NC membranes, which were then visualized by anti-integrin αv, β3 and β1 mAb, followed by HRP-labeled antimouse IgG and detected by enhanced chemiluminescence (ECL). The relative amount of each protein band was quantified as a ratio to the β-actin band indicated underneath each gel using the densitometric scanning software Quantity One (BIO-RAD). The data are expressed as the mean ± SD of the three independent experiments.
Protein Tyrosine Phosphorylation
MHCC97H cells were incubated in serum-free DMEM for 10 h, then in DMEM with or without phenylarsine oxide (PAO, 20 μM) for 5 min, and further in DMEM with Mn2+ (0.1 mM) in the presence or absence of FN, rhFNHN29 and rhFNHC36 (200 μg/ml) at 37°C for 20 min. Cell nuclear proteins were extracted and the phosphotyrosyl proteins (p125FAK) electroblotted to PVDF membrane were visualized by anti-p-FAK (Tyr 397) after SDS-PAGE, followed by HRP-labeled antirabbit IgG and were detected by ECL.
Electrophoretic Mobility Band-Shift Assay (EMSA)
The MHCC97H cells were cultured as per the process described in "Protein tyrosine phosphorylation". According to EMSA Kit (Viagene, USA) manufacturer instructions, 7 μg of nuclear proteins were extracted and incubated with the biotin-labeled activator protein 1 (AP-1) doublestranded oligonucleotides probe (5'-CGCTTGATGACTCAGCCGGAA-3'/3'-GCGAACTACTGAGTCGGCCTT-5') in a binding buffer for 20 min at room temperature, and the activated AP-1 was then electroblotted to a nylon membrane and visualized after 6.5% PAGE.
Results are shown as the mean ± SD. One-way ANOVA (analysis of variance) followed by Fisher's Least Significant Difference post hoc test or Tamhane's T2 test (if equal variance is not assumed) was used for comparison of data from different groups. Differences with P < 0.05 were considered statistically significant. For the counting of the lung metastatic foci, the Intraclass Correlation Coefficient (ICC) value was used to evaluate the inter-observer variability (reproducibility) by the reliability analysis of SPSS (Statistical Product and Service Solutions).
Purification and Heparin Affinity of rhFNHN29 and rhFNHC36 Polypeptides
Expression Pattern of Integrins and MMPs in Human Liver Cancer Cell Lines
RhFNHN29 and RhFNHC36 Inhibit MHCC7H Cell Adhesion and Migration Induced by Integrin αvβ3
When we performed the cell invasion assay using Transwell chambers, as shown in Figure 4D, the MHCC97H cells migrated across the Matrigel-coated filter into the bottom chamber in response to FN, which was prevented by anti-αv (P2W7) and anti-β3 (BV4) mAb; migration was not prevented by anti-β1 (8A2) mAb, indicating that ligation of αvβ3 induced the invasion of MHCC97H cells. RhFNHC36 inhibited the FN-induced, integrin αvβ3-mediated invasion of MHCC97H cells at a lower dose. RhFNHN29 and FN also affected the invasion of MHCC97H cells to the FN substrate but required higher concentrations.
RhFNHN29 and RhFNHC36 Decrease Experimental Metastasis of MHCC97H Cells
RhFNHN29 and RhFNHC36 Reduce Integrin Expression and MMP Activity
Inhibition of Adhesion and Migration by RhFNHN29 and RhFNHC36 Correlates with Tyrosine Phosphorylation of FAK and Activation of AP-1
As the recurrence and metastasis of HCC remains a formidable problem in clinical practice, many efforts have been made to develop a more efficient treatment to inhibit or prevent tumor metastasis. As the expression level of integrins on HCC cells and in the extracellular matrix is related closely to a cell's ability to migrate [26, 27], attempts to realize "anti-adhesion therapy" have been undertaken using synthetic integrin-binding peptides derived from cell adhesive sites of ECM protein. A number of previous studies have proposed that synthetic peptides which contain the RGD cell adhesion motif common in several ECM proteins, including FN, have the potential to prevent tumor metastasis . However, the application of these short peptides is limited due to high dosage requirement and short half-life. To overcome these difficulties, various chemical modifications of the integrin-binding peptides have been performed in order to enhance integrin binding affinity [29, 30]. A synthetic peptide derived from the 14th type III module weakens the integrin α5β1-mediated adhesion of various cell types to the FN substrate in a reversible manner . Wang  designed a derivative of synthesized peptides containing anti-adhesion peptide β (DLYYLMDLSYSMK), in accordance with the conserved sequence of the integrin α and β unit. Liu  demonstrated that treatment with the gene delivery technique resulting in in vivo expression of a recombinant CBD-HepII bifunctional-domain polypeptide of FN, designated as CH50, inhibited tumor metastasis to the liver. However, the impact of this gene therapy on liver cells themselves has not been investigated.
The present study demonstrated that the polypeptides derived from FN, rhFNHN29 and rhFNHC36, have the ability to reduce by ~30% the number of lung metastases of MHCC97H cells in the nude mice metastasis model. This efficacy might be explained as less degradation with longer fragment and less of an effect on the model organism with non-chemical modification and non-gene expression in cells. The mechanisms underlying the inhibition may be very complex, yet they were identified in this study as follows.
First of all, rhFNHN29 and rhFNHC36 could interfere with the function of integrin αv and β3. As to the αvβ3-mediated interaction, rhFNHN29 and rhFNHC36 inhibited the MHCC97H cell migration toward the FN concentration gradient, as well as adhesion to the FN substrate. Previous studies have confirmed that human recombinant fibronectin polypeptide CH50 has the ability to interfere with the expression and activity of integrin αv and β3, resulting in the inhibition of invasive growth of tumor cells and angiogenesis , and a 22-mer FN peptide, termed FNIII14, has also been shown to inhibit β1 integrin-mediated adhesion without binding to integrins and has exhibited the potential to prevent lymphoma cell metastasis . It is understood that no basement membranes exist under liver sinusoidal endothelial cells, thus liver cancer cells can not have the same adhesion to laminin or collagen as other tumor cells, and so it follows that integrin-mediated HCC metastasis and cell adhesion to FN play a decisive role in the formation of metastases [32–34]. This involves several types of FN receptors, such as α4, α5, αv and β1, β3 integrin subunit, of which integrin αvβ3 is the key element that mediates liver cancer invasion and metastasis due to low expression in normal tissue but high expression in HCC . Therefore, intervention with FN-type peptides is more suitable for liver cancer cells that express α4, α5, αv, β1 or β3 integrin.
The Integrin-FAK-AP-1 signaling pathway plays an important role in cell adhesion, proliferation, migration, differentiation, etc. . Intracellular signaling involves the accumulation of several proteins such as focal adhesion kinase (FAK), which is mainly distributed in the adhesion plaque (FAP) structure. After the extracellular domain of integrin α subunit contacts the ECM, the integrins on the membrane cluster and further activate adhesion kinase (FAK phosphorylation) and other protein kinases [36, 37]. The nuclear target proteins, including AP-1 transcription factor, receive the activated signal and further control gene expression. During the adhesion and invasion process of hepatocellular carcinoma cells, the tyrosine kinase-mediated signaling pathways induced by integrin receptors are of great importance . In this study, it was found that rhFNHN29 and rhFNHC36 inhibit MHCC97H cell adhesion and invasion by blocking the integrin-FAK-AP-1 pathway. Expression of p-FAK was exhibited when a tyrosine phosphatase inhibitor (PAO) was added, suggesting that the interaction between rhFNHN29 or rhFNHN36 and integrin alpha V beta 3 results in the blockage of a series of signals into the nucleus including the AP-1 activation signal. The promoters regulated by AP-1 include TRE of integrins  and MMPs , which contain AP-1 binding sites, and TPA response elements . It was also found that the inhibitory impact of rhFNHC36 on integrin and MMP9 expression exhibited dose-dependence, but impact of rhFNHN29 on integrin expression required a high concentration. The rhFNHC36 polypeptide is more effective than rhFNHN29 due to the following: 1) although both rhFNHN29 and rhFNHC36 contain a heparin-binding domain, rhFNHC36 contains another anti-adhesion site, YTIYVIAL, which was found by Kato . This anti-adhesive site is buried within the type III module structure in plasma FN, however, it is exposed in rhFNHC36. 2) AP-1 transcription factors are leucine zipper proteins that bind to a consensus DNA sequence (5'-TGAG/CTCA-3') as a dimeric complex. Various AP-1 dimers bind to DNA with differing affinities. The dimers of AP-1 bound with MMP9 promoter DNA are greater than that of MMP2 [40, 42], hence regulation of AP-1 due to MMP9 expression is more sensitive than through MMP2 expression. We speculated that through the integrin-FAK-AP-1 pathway the effect of rhFNHC36 on MMP9 activity by AP-1 may be greater than the effect of rhFNHN29 in MHCC97H cells.
In this study our intervention is targeted. Different tumor cells express different integrins and MMPs, which are often associated with a different potential for tumor invasion and metastasis. In previous reports, mRNA expression levels of integrins and MMPs have been analysed using a panel of human tumor cell lines with different degrees of differentiation and potential for tumor formation, tissue invasion, and metastasis [23, 24]. In this study, the mRNA expression of integrins and MMPs that was detected in different liver cancer cell lines and MHCC97H cells was found to feature low levels of integrin α5 mRNA expression and high levels of integrin αv, integrin β1, integrin β3, MMP1, MMP2 and MMP9 mRNA expression. This may partly contribute to its invasion and metastasis, indicating that MHCC97H cells can be used as a model for the study of anti-invasion and anti-metastasis.
In summary, our work provides the first evidence that rhFNHN29 and rhFNHC36 may play an important role in safeguarding against human liver cancer through the reduction of integrin expression level and MMP-9 activity and may shed light on a novel strategy for liver cancer therapy.
We found that both rhFNHN29 and rhFNHC36 polypeptides were able to inhibit the adhesion and invasion of MHCC97H cells, and moreover abrogate the tyrosine phosphorylation of focal adhesion kinase (p-FAK) and activation of activator protein 1 (AP-1), which resulted in the decrease of integrin αv, β3 and β1 expression and the reduction of MMP-9 activity. These findings suggest that rhFNHN29 and rhFNHC36 may play an important role in controlling human liver cancer invasion.
- rhFNHN29 and rhFNHC36:
N-terminal and C-terminal heparin-binding domain peptides of FN
focal adhesion kinase
electrophoretic mobility band-shift assays
This study was funded by Key Project of Hematology Laboratory of Fujian Province (No. 2009J1004) and Program for Key Specialty in "211 Project" of Fujian Province.
- Niu JX, Zhang WJ, Ye LY, Wu LQ, Zhu GJ, Yang ZH, Grau GE, Lou JN: The role of adhesion molecules, alpha v beta 3, alpha v beta 5 and their ligands in the tumor cell and endothelial cell adhesion. Eur J Cancer Prev. 2007, 16 (6): 517-527. 10.1097/CEJ.0b013e3280145c00.View ArticlePubMedGoogle Scholar
- Riou P, Saffroy R, Chenailler C, Franc B, Gentile C, Rubinstein E, Resink T, Debuire B, Piatier-Tonneau D, Lemoine A: Expression of T-cadherin in tumor cells influences invasive potential of human hepatocellular carcinoma. FASEB J. 2006, 20 (13): 2291-2301. 10.1096/fj.06-6085com.View ArticlePubMedGoogle Scholar
- Song G, Ohashi T, Sakamoto N, Sato M: Adhesive force of human hepatoma HepG2 cells to endothelial cells and expression of E-selectin. Mol Cell Biomech. 2006, 3 (2): 61-68.PubMedGoogle Scholar
- Oosterling SJ, van der Bij GJ, Bögels M, ten Raa S, Post JA, Meijer GA, Beelen RH, van Egmond M: Anti-beta1 integrin antibody reduces surgery-induced adhesion of colon carcinoma cells to traumatized peritoneal surfaces. Ann Surg. 2008, 247 (1): 85-94. 10.1097/SLA.0b013e3181588583.View ArticlePubMedGoogle Scholar
- Nobumoto A, Nagahara K, Oomizu S, Katoh S, Nishi N, Takeshita K, Niki T, Tominaga A, Yamauchi A, Hirashima M: Galectin-9 suppresses tumor metastasis by blocking adhesion to endothelium and extracellular matrices. Glycobiology. 2008, 18 (9): 735-744. 10.1093/glycob/cwn062.View ArticlePubMedGoogle Scholar
- Gong W, Liu Y, Huang B, Lei Z, Wu FH, Li D, Feng ZH, Zhang GM: Recombinant CBD-HepII polypeptide of fibronectin inhibits alphavbeta3 signaling and hematogenous metastasis of tumor. Biochem Biophys Res Commun. 2008, 367 (1): 144-149. 10.1016/j.bbrc.2007.12.110.View ArticlePubMedGoogle Scholar
- Han S, Ritzenthaler JD, Sitaraman SV, Roman J: Fibronectin increases matrix metalloproteinase 9 expression through activation of c-Fos via extracellular-regulated kinase and phosphatidylinositol 3-kinase pathways in human lung carcinoma cells. J Biol Chem. 2006, 281 (40): 29614-29624. 10.1074/jbc.M604013200.View ArticlePubMedGoogle Scholar
- Liu W, Cheng S, Asa SL, Ezzat S: The melanoma-associated antigen A3 mediates fibronectin-controlled cancer progression and metastasis. Cancer Res. 2008, 68 (19): 8104-8112. 10.1158/0008-5472.CAN-08-2132.View ArticlePubMedGoogle Scholar
- Urtreger AJ, Werbajh SE, Verrecchia F, Mauviel A, Puricelli LI, Kornblihtt AR, Bal de Kier Joffé ED: Fibronectin is distinctly downregulated in murine mammary adenocarcinoma cells with high metastatic potential. Oncol Rep. 2006, 16 (6): 1403-1410.PubMedGoogle Scholar
- Gulubova M, Vlaykova T: Immunohistochemical assessment of fibronectin and tenascin and their integrin receptors alpha5beta1 and alpha9beta1 in gastric and colorectal cancers with lymph node and liver metastases. Acta Histochem. 2006, 108 (1): 25-35. 10.1016/j.acthis.2005.12.001.View ArticlePubMedGoogle Scholar
- Szaniawska B, Kleczkowska P, Trembacz H, Janik P, Lipkowski AW: A novel fibronectin-related structural motif that modulates cell migration. J Pept Res. 2005, 66 (Suppl 1): 114-119.Google Scholar
- Tsuchiya Y, Sawada S, Tsukada K, Saiki I: A new pseudo-peptide of Arg-Gly-Asp (RGD) inhibits intrahepatic metastasis of orthotopically implanted murine hepatocellular carcinoma. Int J Oncol. 2002, 20 (2): 319-324.PubMedGoogle Scholar
- Liu Y, Huang B, Yuan Y, Gong W, Xiao H, Li D, Yu ZR, Wu FH, Zhang GM, Feng ZH: Inhibition of hepatocarcinoma and tumor metastasis to liver by gene therapy with recombinant CBD-HepII polypeptide of fibronectin. Int J Cancer. 2007, 121 (1): 184-192. 10.1002/ijc.22644.View ArticlePubMedGoogle Scholar
- Kato R, Ishikawa T, Kamiya S, Oguma F, Ueki M, Goto S, Nakamura H, Katayama T, Fukai F: A new type of antimetastatic peptide derived from fibronectin. Clin Cancer Res. 2002, 8 (7): 2455-2462.PubMedGoogle Scholar
- Kishore R, Samuel M, Khan MY, Hand J, Frenz DA, Newman SA: Interaction of the NH2-terminal domain of fibronectin with heparin. Role of the omega-loops of the type I modules. J Biol Chem. 1997, 272 (27): 17078-17085. 10.1074/jbc.272.27.17078.View ArticlePubMedGoogle Scholar
- Garcia-Pardo A, Rostagno A, Frangione B: Primary structure of human pkasma fibronectin. Characterization of a 38 kDa domain containing the C-terminal heparin-binding site (Hep III site) and a region of molecular heterogeneity. Biochem J. 1987, 241 (3): 923-928.View ArticlePubMedPubMed CentralGoogle Scholar
- Zou QL, Chen YZ, Chen XF, Wu Y, Chen P, Huang MJ: Expression of Recombinant Heparin-binding Fibronectin Polypeptide in Insect Cell/Baclovirus System and Its Bioactivities. China Biotechnology. 2008, 28 (6s): 32-38. ChineseGoogle Scholar
- Tian J, Tang ZY, Ye SL, Liu YK, Lin ZY, Chen J, Xue Q: New human hepatocellular carcinoma (HCC) cell line with highly metastatic potential (MHCC97) and its expressions of the factors associated with metastasis. Br J Cancer. 1999, 81 (5): 814-821. 10.1038/sj.bjc.6690769.View ArticlePubMedPubMed CentralGoogle Scholar
- Fukai F, Mashimo M, Akiyama K, Goto T, Tanuma S, Katayama T: Modulation of apoptotic cell death by extracellular matrix proteins and a fibronectin-derived antiadhesive peptide. Exp Cell Res. 1998, 242 (1): 92-99. 10.1006/excr.1998.4076.View ArticlePubMedGoogle Scholar
- Li WC, Ye SL, Sun RX, Liu YK, Tang ZY, Kim Y, Karras JG, Zhang H: Inhibition of growth and metastasis of human hepatocellular carcinoma by antisense oligonucleotide targeting signal transducer and activator of transcription 3. Clin Cancer Res. 2006, 12 (23): 7140-7148. 10.1158/1078-0432.CCR-06-0484.View ArticlePubMedGoogle Scholar
- Wang W, Wu F, Fang F, Tao Y, Yang L: Inhibition of invasion and metastasis of hepatocellular carcinoma cells via targeting RhoC in vitro and in vivo. Clin Cancer Res. 2008, 14 (21): 6804-6812. 10.1158/1078-0432.CCR-07-4820.View ArticlePubMedGoogle Scholar
- Iwai S, Nakanishi-Ueda T, Armstrong D, Oguchi K: Zymographical techniques for detection of matrix metalloproteinases. Methods Mol Biol. 2008, 477: 113-121. full_text.View ArticlePubMedGoogle Scholar
- Sun YX, Fang M, Wang J, Cooper CR, Pienta KJ, Taichman RS: Expression and activation of alpha v beta 3 integrins by SDF-1/CXC12 increases the aggressiveness of prostate cancer cells. Prostate. 2007, 67 (1): 61-73. 10.1002/pros.20500.View ArticlePubMedGoogle Scholar
- Zheng H, Takahashi H, Murai Y, Cui Z, Nomoto K, Niwa H, Tsuneyama K, Takano Y: Expressions of MMP-2, MMP-9 and VEGF are closely linked to growth, invasion, metastasis and angiogenesis of gastric carcinoma. Anticancer Res. 2006, 26 (5A): 3579-3583.PubMedGoogle Scholar
- Garcia-Morales P, Minami Y, Luong E, Klausner RD, Samelson LE: Tyrosine phosphorylation in T cells is regulated by phosphatase activity: studies with phenylarsine oxide. Proc Natl Acad Sci USA. 1990, 87 (23): 9255-9259. 10.1073/pnas.87.23.9255.View ArticlePubMedPubMed CentralGoogle Scholar
- Liu YK, Wu WZ, Wu X, Jiang Y, Zhou XD: Metastasis and recurrence of hepatocellular carcinoma--basic and clinical studies. Shanghai: Shanghai scientific and technological education public house. 2003: 93-104.
- Heyder C, Gloria-Maercker E, Hatzmann W, Niggemann B, Zanker KS, Dittmar T: Role of the beta1-integrin subunit in the adhesion, extravasation and migration of T24 human bladder carcinoma cells. Clin Exp Metastasis. 2005, 22 (2): 99-106. 10.1007/s10585-005-4335-z.View ArticlePubMedGoogle Scholar
- Humphries MJ, Olden K, Yamada KM: A synthetic peptide from fibronectin inhibits experimental metastasis of murine melanoma cells. Science. 1986, 233 (4762): 467-470. 10.1126/science.3726541.View ArticlePubMedGoogle Scholar
- Dechantsreiter MA, Planker E, Mathä B, Lohof E, Hölzemann G, Jonczyk A, Goodman SL, Kessler H: N-Methylated cyclic RGD peptides as highly active and selective alpha(V)beta(3) integrin antagonists. J Med Chem. 1999, 42 (16): 3033-3040. 10.1021/jm970832g.View ArticlePubMedGoogle Scholar
- Peyman A, Wehner V, Knolle J, Stilz HU, Breipohl G, Scheunemann KH, Carniato D, Ruxer JM, Gourvest JF, Gadek TR, Bodary S: RGD mimetics containing a central hydantoin scaffold: alpkha(v)beta3 vs alpha(IIb)beta3 selectivity requirements. Bioorg Med Chem Lett. 2000, 10 (2): 179-182. 10.1016/S0960-894X(99)00661-7.View ArticlePubMedGoogle Scholar
- Wang SM, Zhu J, Pan LF, Liu YK: Inhibitory effect of dimeric beta peptide on the recurrence and metastasis of hepatocellular carcinoma in vitro and in mice. World J Gastroenterol. 2008, 14 (19): 3054-3058. 10.3748/wjg.14.3054.View ArticlePubMedPubMed CentralGoogle Scholar
- Lin KT, Yeh SH, Chen DS, Chen PJ, Jou YS: Epigenetic activation of alpha4, beta2 and beta6 integrins involved in cell migration in trichostatin A-treated Hep3B cells. J Biomed Sci. 2005, 12 (5): 803-813. 10.1007/s11373-005-9005-2.View ArticlePubMedGoogle Scholar
- Fu BH, Wu ZZ, Dong C: Integrin beta1 mediates hepatocellular carcinoma cells chemotaxis to laminin. Hepatobiliary Pancreat Dis Int. 2004, 3 (4): 548-551.PubMedGoogle Scholar
- Wu Y, Zuo J, Ji G, Saiyin H, Liu X, Yin F, Cao N, Wen Y, Li JJ, Yu L: Proapoptotic function of integrin beta(3) in human hepatocellular carcinoma cells. Clin Cancer Res. 2009, 15 (1): 60-69. 10.1158/1078-0432.CCR-08-1028.View ArticlePubMedPubMed CentralGoogle Scholar
- Cowles EA, Brailey LL, Gronowicz GA: Integrin-mediated signaling regulates AP-1 transcription factors and proliferation in osteoblasts. J Biomed Mater Res. 2000, 52 (4): 725-737. 10.1002/1097-4636(20001215)52:4<725::AID-JBM18>3.0.CO;2-O.View ArticlePubMedGoogle Scholar
- Hamamura K, Tsuji M, Ohkawa Y, Nakashima H, Miyazaki S, Urano T, Yamamoto N, Ueda M, Furukawa K, Furukawa K: Focal adhesion kinase as well as p130Cas and paxillin is crucially involved in the enhanced malignant properties under expression of ganglioside GD3 in melanoma cells. Biochim Biophys Acta. 2008, 1780 (3): 513-519.View ArticlePubMedGoogle Scholar
- Van Slambrouck S, Grijelmo C, De Wever O, Bruyneel E, Emami S, Gespach C, Steelant WF: Activation of the FAK-src molecular scaffolds and p130Cas-JNK signaling cascades by alpha1-integrins during colon cancer cell invasion. Int J Oncol. 2007, 31 (6): 1501-1508.PubMedGoogle Scholar
- Liang YL, Lei TW, Wu H, Su JM, Wang LY, Lei QY, Zha XL: S-phase delay in human hepatocellular carcinoma cells induced by overexpression of integrin beta1. World J Gastroenterol. 2003, 9 (8): 1689-1696.View ArticlePubMedPubMed CentralGoogle Scholar
- Corbi AL, Jensen UB, Watt FM: The alpha2 and alpha5 integrin genes:identification of transcription factors that regulate promoter activity in epidermal keratinocytes. FEBS Lett. 2000, 474 (2-3): 201-207. 10.1016/S0014-5793(00)01591-X.View ArticlePubMedGoogle Scholar
- Westermarck J, Kähäri VM: Regulation of matrix metalloproteinase expression in tumor invasion. FASEB J. 1999, 13 (8): 781-792.PubMedGoogle Scholar
- Angel P, Karin M: The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta. 1991, 1072 (2-3): 129-157.PubMedGoogle Scholar
- Stamenkovic I: Matrix metalloproteinases in tumor invasion and metastasis. Semin Cancer Biol. 2000, 10 (6): 415-433. 10.1006/scbi.2000.0379.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/552/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.