Domain Of Hepatocyte Growth Factor Research Articles

Computer‐Assisted Identification of Inhibitor with Novel Pharmacophore Targeting First Kringle Domain of Hepatocyte Growth Factor

AbstractHepatocyte growth factor (HGF) participates in multiple biological and pathogenic processes, such as liver regeneration, wound healing, and tumor metastasis. These biological effects are initiated by its binding to Met, so it is anticipated that compounds that can disturb the HGF‐Met association could become novel modulating drugs targeting HGF and its related signaling. In this study, a chemical library composed of 154,118 compounds was screened by in silico structure‐based drug screening (SBDS) techniques to obtain HGF‐targeting compound. X‐ray structural data of the N‐terminal and first kringle domains of HGF (PDBid: 5CS9) were analyzed using the binding energy of known inhibitors and false positive inhibitors. The top five candidate compounds, identified by the DOCK‐GOLD combined screening program, were subjected to cell‐based ELISA assay to confirm experimentally these inhibitory activity on Met activation. The present in silico SBDS study enabled identification of compound which inhibits HGF‐promoted Met phosphorylation (compound 2). The present study also suggested that Trp188/Tyr198 in HGF are essential for compound 2 binding as well as Met activation.

Entamoeba histolytica Trophozoites Interact with the c-Met Receptor at the Surface of Liver Origin Cells through the Gal/GalNAc Amoebic Lectin

Amoebiasis in humans is caused by the protozoan parasite Entamoeba histolytica, which cytotoxic activity has been demonstrated on a wide variety of target cells. The process involves the adherence of the parasite to the cell, and such adherence is mediated by an amoebic surface lectin, known as Gal/GalNAc lectin. It is composed of heavy, intermediate, and light subunits. The carbohydrate recognition domain (CRD) has been identified within a cysteine-rich region in the lectin heavy subunit and has an amino acid sequence identity to the receptor-binding domain of hepatocyte growth factor (HGF). Recombinant CRD has been previously shown to compete with HGF for binding to the c-Met receptor IgG fusion protein. In the present study, we searched for evidence of interaction between the Gal/GalNAc lectin at the surface of trophozoites with the c-Met receptor expressed at the surface of HepG2 in coculture assays. Immunoprecipitation of the coculture lysate indicated interaction of the c-Met with a 60 kDa peptide recognized by antiamoebic lectin antibody. Colocalization of both molecules was detected by fluorescence confocal microscopy. Incubation of HepG2 cells with HGF before coculture with trophozoites prevents the cytotoxic effect caused by the parasites but not their adherence to the cells. Our results point to Gal/GalNAc lectin as a ligand of the c-Met receptor at the surface of HepG2 cells.

Notice of Concern: Kawas LH, Yamamoto BJ, Wright JW, and Harding JW (2011) Mimics of the Dimerization Domain of Hepatocyte Growth Factor Exhibit Anti-Met and Anticancer Activity, J Pharmacol Exp Ther, 339: 509-518; DOI: https://doi.org/10.1124/jpet.111.185694.

The editors wish to express concern about the article by Kawas et al. of possible image manipulation after reviewing information received from several sources. They have shared these concerns with the corresponding author and their institution, Washington State University, and will await the results

Peglated-H1/pHGFK1 nanoparticles enhance anti-tumor effects of sorafenib by inhibition of drug-induced autophagy and stemness in renal cell carcinoma

BackgroundTumor targeting small molecular inhibitors are the most popular treatments for many malignant diseases, including cancer. However, the lower clinical response and drug resistance still limit their clinical efficacies. HGFK1, the first kringle domain of hepatocyte growth factor, has been defined as a potent anti-angiogenic factor. Here, we aimed to develop and identify novel nanoparticles—PH1/pHGFK1 as potential therapeutic agents for the treatment of renal cell carcinoma (RCC).MethodsWe produced a novel cationic polymer—PH1 and investigated the anti-tumor activity of PH1/pHGFK1 nanoparticle alone and its combination therapy with sorafenib in RCC cell line xenografted mice model. Then, we figured out its molecular mechanisms in human RCC cell lines in vitro.ResultsWe firstly demonstrated that intravenous injection of PH1/pHGFK1 nanoparticles significantly inhibited tumor growth and prolonged the survival time of tumor-bearing mice, as well as synergistically enhanced anti-tumor activities of sorafenib. Furthermore, we elucidated that recombinant HGFK1 improved sorafenib-induced cell apoptosis and arrested cell cycle. In addition, HGFK1 could also decrease sorafenib-induced autophagy and stemness via blockading NF-κB signaling pathway in RCC both in vitro and in vivo.ConclusionsHGFK1 could inhibit tumor growth, synergistically enhance anti-tumor activities of sorafenib and reverse its drug resistance evolution in RCC. Our results provide rational basis for clinical application of sorafenib and HGFK1 combination therapy in RCC patients.

Overexpressing kringle 1 domain of hepatocyte growth factor with adeno-associated virus inhibits the pathological retinal neovascularization in an oxygen-induced retinopathy mouse model

Current clinical treatments for ocular neovascularization are characterized by high possibility of damaging healthy tissues and high recurrence rates. It is necessary to develop new treatment methods to control neovascularization with a stable and effective effect. Kringle1 domain of hepatocyte growth factor (HGFK1) has anti-angiogenesis activity. Here, we established oxygen-induced retinopathy (OIR) model to study if using adeno-associated virus (AAV) as a delivery system to overexpression HGFK1 in retinal cells could benefit retinal neovascularization. We show that, overexpressed exogenous gene was mainly expressed in the inner and outer nuclear layer of the retina. Compared with control mice, the mice pretreated with rAAV-HGFK1 at P3 showed relatively normal vascular branches examined by fluorescence fundus angiography. Subsequent H&E staining and immunohistochemical staining of CD31 of the eye tissue sections showed that the mice received rAAV-HGFK1 had a relatively normal distribution of vascular endothelial cells. Additionally, immunohistochemical staining indicated a lower expression of VEGF in the eye tissues of rAAV-HGFK1 treated OIR mice. Further in vitro studies showed that HGFK1 could inhibit the proliferation but promote the apoptosis of bovine retinal microvascular endothelial cells (BRECs) under the presence of VEGF. Moreover, HGFK1 could inhibit VEGF induced ERK activation but promote p38 activation in BRECs. Therefore, we propose that intravitreal injection of rAAV-HGFK1 might be used to improve the retinal neovascularization and HGFK1 may function through regulating VEGF signaling pathway to inhibit neovascularization.

H1/pHGFK1 nanoparticles exert anti-tumoural and radiosensitising effects by inhibition of MET in glioblastoma

Background:The therapeutic resistance to ionising radiation (IR) and anti-angiogenesis mainly impair the prognosis of patients with glioblastoma. The primary and secondary MET aberrant activation is one crucial factor for these resistances. The kringle 1 domain of hepatocyte growth factor (HGFK1), an angiogenic inhibitor, contains a high-affinity binding domain of MET; however, its effects on glioblastoma remain elusive.Methods:We formed the nanoparticles consisting of a folate receptor-targeted nanoparticle-mediated HGFK1 gene (H1/pHGFK1) and studied its anti-tumoural and radiosensitive activities in both subcutaneous and orthotopic human glioma cell-xenografted mouse models. We then elucidated its molecular mechanisms in human glioblastoma cell lines in vitro.Results:We demonstrated for the first time that peritumoural injection of H1/pHGFK1 nanoparticles significantly inhibited tumour growth and prolonged survival in tumour-bearing mice, as well as enhanced the anti-tumoural efficacies of IR in vivo by reducing Ki-67 expression, enhancing TUNEL staining-indicated apoptotic indexes, reducing microvascular intensity and reversing IR-induced MET overexpression in tumour tissues. Furthermore, we showed that HGFK1 suppressed the proliferation and induced cell apoptosis and enhanced sensitivity to IR in glioblastoma cell lines, mainly by suppressing the activities of MET receptor, down-regulating ATM-Chk2 axis but up-regulating Chk1.Conclusions:H1/pHGFK1 exerts anti-tumoural and radiosensitive activities mainly through the inhibition and reversal of IR-induced MET and ATM–Chk2 axis activities in glioblastoma. H1/pHGFK1 nanoparticles are a potential radiosensitiser and angiogenic inhibitor for glioblastoma treatment.

CAR T-cell immunotherapy of MET-expressing malignant mesothelioma

ABSTRACTMesothelioma is an incurable cancer for which effective therapies are required. Aberrant MET expression is prevalent in mesothelioma, although targeting using small molecule-based therapeutics has proven disappointing. Chimeric antigen receptors (CARs) couple the HLA-independent binding of a cell surface target to the delivery of a tailored T-cell activating signal. Here, we evaluated the anti-tumor activity of MET re-targeted CAR T-cells against mesothelioma. Using immunohistochemistry, MET was detected in 67% of malignant pleural mesotheliomas, most frequently of epithelioid or biphasic subtype. The presence of MET did not influence patient survival. Candidate MET-specific CARs were engineered in which a CD28+CD3ζ endodomain was fused to one of 3 peptides derived from the N and K1 domains of hepatocyte growth factor (HGF), which represents the minimum MET binding element present in this growth factor. Using an NIH3T3-based artificial antigen-presenting cell system, we found that all 3 candidate CARs demonstrated high specificity for MET. By contrast, these CARs did not mediate T-cell activation upon engagement of other HGF binding partners, namely CD44v6 or heparan sulfate proteoglycans, including Syndecan-1. NK1-targeted CARs demonstrated broadly similar in vitro potency, indicated by destruction of MET-expressing mesothelioma cell lines, accompanied by cytokine release. In vivo anti-tumor activity was demonstrated following intraperitoneal delivery to mice with an established mesothelioma xenograft. Progressive tumor regression occurred without weight loss or other clinical indicators of toxicity. These data confirm the frequent expression of MET in malignant pleural mesothelioma and demonstrate that this can be targeted effectively and safely using a CAR T-cell immunotherapeutic strategy.

Construction of oncolytic adenovirus carrying kringle 1 domain of hepatocyte growth factor gene and its lethal effect to gastric cancer cells

Objective To construct an double-regulated oncolytic adenovirus carrying kringle 1 domain of hepatocyte growth factor (HGFK1) gene which exerts anti-tumor and anti-angiogenesis effect and observe its lethal effect to gastric cancer cells. Methods Adenoviral E1a and E1b genes were controlled by Human telomerase reverse transcriptase promoter (hTERT) and hypoxia regulatory element (HRE). TH-Ad-HGFK1-EGFP or TH-Ad-EGFP was constructed by inserting HGFK1 gene into the viral genome or not. The adenovirus was divided into three groups: the experimental group A (TH-Ad-HGFK1-EGFP), the experimental group B (TH-Ad-EGFP) and the control group C (Ad-EGFP). Gastric cancer cells SGC7901 and the normal human fibroblasts cell HF were infected by adenovirus, then the proliferation of adenovirus was counted by the half-cell culture infection dose (TCID50) method. Methyl thiazolyl tetrazolium (MTT) method was used to detect the inhibitory effect. 4, 6-Diamidino-2 -Phenylindole, Dihydrochloride (DAPI) method and Terminal -deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) method was used to observe the cell apoptosis. Results TH-Ad-HGFK1-EGFP and TH-Ad-EGFP was confirmed constructed successfully by DNA sequencing and PCR analyses. All the titer of the adenovirus was 2×1010 pfu/ml. The TCID50 results showed the proliferation of group A and group B in SGC7901 cells was 1.47×104 and 1.60×104 fold, which were significantly higher than that in group C. However in HF cells, the fold change of replication of group A and group B was only 110 and 124, which was significantly lower than that in group C. Group A showed potent inhibitory effect on the growth of SC7901 cells, but Group B and group C showed no obviously inhibitory effect. The inhibition ratio of group A (MOI=100, 72 h and 96 h) on SGC7901 cells was (71.34±8.27)%, (74.56±1.78)%, which had significant difference compared with those in group B (23.40±2.29)%, (31.94±6.62) % (P=0.002, P=0.003) or group C (21.18±2.32%, 21.18±2.32%)(P=0.003, P=0.000). The inhibition ratio of group A and group B (MOI=100, 96 h) on HF cells was 18.94±0.88% and 22.84±3.34%, which were significant lower than that in group C [(53.25±3.50)%, P=0.003, P=0.001]. Group A also showed cell apoptosis on SGC7901 cells (18.66±4.04%), which had significant difference in other groups. Conclusion The oncolytic adenovirus TH-Ad-HGFK1-EGFP showed remarkably replication capability, promoted apoptosis and inhibited proliferation of SGC7901. Key words: Gastric cancer; Kringle 1 domain of hepatocyte growth factor; Oncolytic adenovirus; SGC7901

Myogenic induction of adult and pluripotent stem cells using recombinant proteins

Met Activating Genetically Improved Chimeric Factor 1 (Magic-F1) is a human recombinant protein, derived from dimerization of the receptor-binding domain of hepatocyte growth factor. Previous experiments demonstrate that in transgenic mice, the skeletal muscle specific expression of Magic-F1 can induce a constitutive muscular hypertrophy, improving running performance and accelerating muscle regeneration after injury.In order to evaluate the therapeutic potential of Magic-F1, we tested its effect on multipotent and pluripotent stem cells. In murine mesoangioblasts (adult vessel-associated stem cells), the presence of Magic-F1 did not alter their osteogenic, adipogenic or smooth muscle differentiation ability. However, when analyzing their myogenic potential, mesoangioblasts expressing Magic-F1 differentiated spontaneously into myotubes. Finally, Magic-F1 inducible cassette was inserted into a murine embryonic stem cell line by homologous recombination. When embryonic stem cells were subjected to myogenic differentiation, the presence of Magic-F1 resulted in the upregulation of Pax3 and Pax7 that enhanced the myogenic commitment of transgenic pluripotent stem cells.Taken together our results candidate Magic-F1 as a potent myogenic stimulator, able to enhance muscular differentiation from both adult and pluripotent stem cells.

H-RN, a novel antiangiogenic peptide derived from hepatocyte growth factor inhibits inflammation in vitro and in vivo through PI3K/AKT/IKK/NF-κB signal pathway

H-RN, a novel antiangiogenic peptide derived from the kringle 1 domain of hepatocyte growth factor (HGF), consists of the sequence RNPRGEEGGPW (molecular weight: 1254.34Da). Emerging evidence indicates that HGF and the kringle domain exhibit anti-inflammatory effects in inflammatory diseases. In the present study, we assessed the anti-inflammatory effect of H-RN in models of experimental ocular inflammation, including endotoxin-induced uveitis (EIU) and experimental autoimmune uveitis (EAU). The results demonstrated that intravitreal treatment of H-RN concentration-dependently suppressed clinical manifestation, inhibited ocular inflammatory cytokine production and improved histopathologic scores. Moreover, H-RN attenuated the LPS-induced mRNA and protein expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in RAW 264.7 cells and inhibited cell chemotactic migration toward LPS. We also demonstrated that H-RN suppressed TNF-α-induced adhesion molecule expression in HUVECs, including ICAM-1, VCAM-1 and E-selectin, which contributed to its suppressive effect on adherence of U937 cells to endothelial cells. We also demonstrated the possible anti-inflammation mechanism of H-RN. Western blot and immunofluorescence staining analyses revealed that H-RN significantly suppressed LPS-induced phosphorylation of nuclear factor (NF)-κB-p65 at Ser276. Based on examination of upstream pathways, we found that H-RN inhibited PI3K-p85 and AKTSer473 phosphorylation, which may result in the attenuation of LPS-induced IKK complex activation and IκB degradation. Thus, our studies suggest that the 11-amino-acid peptide H-RN exhibits anti-inflammatory effects in vitro and in vivo and may represent a promising candidate for ocular inflammatory diseases.

Targeted Disruption of Heparan Sulfate Interaction with Hepatocyte and Vascular Endothelial Growth Factors Blocks Normal and Oncogenic Signaling

Hepatocyte growth factor (HGF) and vascular endothelial cell growth factor (VEGF) regulate normal development and homeostasis and drive disease progression in many forms of cancer. Both proteins signal by binding to receptor tyrosine kinases and heparan sulfate (HS) proteoglycans on target cell surfaces. Basic residues comprising the primary HS binding sites on HGF and VEGF provide similar surface charge distributions without underlying structural similarity. Combining three acidic amino acid substitutions in these sites in the HGF isoform NK1 or the VEGF isoform VEGF165 transformed each into potent, selective competitive antagonists of their respective normal and oncogenic signaling pathways. Our findings illustrate the importance of HS in growth factor driven cancer progression and reveal an efficient strategy for therapeutic antagonist development.

Mimics of the Dimerization Domain of Hepatocyte Growth Factor Exhibit Anti-Met and Anticancer Activity

The angiotensin IV analog norleual [Nle-Tyr-Leu-ψ-(CH(2)-NH(2))-Leu-His-Pro-Phe] has been shown recently to act as a hepatocyte growth factor (HGF)/Met antagonist capable of blocking the binding of HGF to the Met receptor, inhibiting HGF-dependent activation of Met, and attenuating HGF-dependent cellular activities. In addition, norleual exhibited marked anticancer activity. Homology between norleual and the dimerization domain (hinge region) of HGF led to the hypothesis that norleual acts by interfering with HGF dimerization/multimerization and functions as a dominant-negative hinge region mimic. To test this hypothesis we investigated the ability of norleual to bind to and inhibit the dimerization of HGF. To further evaluate the idea that norleual was acting as a hinge region mimic, we synthesized a hexapeptide representing the HGF hinge sequence and established its capacity to similarly block HGF-dependent activation of Met and HGF-dependent cellular functions. The hinge peptide not only bound with high affinity directly to HGF and blocked its dimerization but it also inhibited HGF-dependent Met activation, suppressed HGF-dependent cellular functions, and exhibited anticancer activity. The major implication of this study is that molecules targeting the dimerization domain of HGF may represent novel and viable anticancer therapeutic agents; the development of such molecules should be feasible using norleual and the hinge peptide as synthetic templates.

Protective effects of HGF-MSP chimer (metron factor-1) on liver ischemia-reperfusion injury in rat model

It has been reported that metron factor-1 (MF-1), an engineered chimerical factor containing selected functional domains of hepatocyte growth factor and macrophage-stimulating protein (HGF-MSP), could prevent apoptosis and have an anti-inflammatory effect. In this study, we investigate the protective effect of MF-1 on liver ischemia-reperfusion (I/R) injury. Overall 30 Sprague Dawley rats were randomly divided into three groups: the I/R model group (n=12), the MF-1 treatment group (n=12), and the sham-operated group (n=6). Liver I/R injury was induced by clamping the blood supply to the left and median lobes of liver by an atraumatic clamp for 90 min, then removing the clamp and allowing reperfusion. Blood samples were obtained on days 1, 2, 3 and 7 to assess liver biochemistry and the histology of liver tissue. Levels of malondialdehyde (MDA), superoxide dismutase (SOD), nitric oxide (NO), endothelial nitric oxide synthase and inducible nitric oxide synthase were measured. In addition, the anti-oxidative effect of MF-1 on hepatocytes was assessed in vitro. MF-1 treatment improved the rat survival rate significantly (P < 0.05). Liver biochemistry and histological changes were significantly ameliorated. MDA increased and SOD and NO decreased in the liver tissue. In vitro, MF-1 protected the human hepatic cell line HL-7702 from damage of oxidative stress. MF-1 could protect the liver from I/R injury, which might involve the reduction of oxygen free radicals and the increase of NO synthesis in an injured liver.

Abstract SY23-03: Oncogenic signal transduction via the hepatocyte growth factor/Met receptor kinase pathway

Abstract SY23-03: Oncogenic signal transduction via the hepatocyte growth factor/Met receptor kinase pathway

Recombinant kringle 5 from plasminogen antagonises hepatocyte growth factor-mediated signalling

The blood protein plasminogen is proteolytically cleaved to produce angiostatin and kringle 5 (K5), both of which are known angiogenesis inhibitors. A common structural element between K5, angiostatin and other endogenous angiogenesis inhibitors is the presence of the kringle protein-interacting domain. Another kringle domain-containing protein, hepatocyte growth factor (HGF), promotes angiogenesis by binding to and stimulating the tyrosine kinase receptor Met. HGF binding to Met is dependent on the kringle domains of HGF. Because both K5 and HGF contain kringle motifs and because these proteins have opposite effects on angiogenesis, we hypothesised that K5 can antagonise HGF-mediated signalling in a Met-dependent manner. We determined that K5 binding to H1299 cells is competed by HGF suggesting that these two proteins bind to the same protein. Purified K5 immunoprecipitates with Met and this interaction is abolished by increasing doses of HGF. Using proliferation, phosphorylation of Met and Akt as markers of HGF activity, we determined that K5 inhibits HGF-mediated signalling. Taken together, these data support a model by which K5 binds to Met and functions as a competitive antagonist of HGF signalling and presents a novel mechanism of action of K5.

Anti-Cancer Approach with NK4: Bivalent Action and Mechanisms

We identified NK4, the N-terminal and four kringle domains of hepatocyte growth factor (HGF), as a specific inhibitor of HGF. NK4 binds to the Met/HGF receptor, but does not activate the Met receptor, thereby competitively inhibiting the HGF-Met pathway. Independent of its inhibition of HGF-Met, NK4 acts as an angiogenesis inhibitor. The angioinhibitory action of NK4 is mediated by perlecan, a multidomain proteoglycan involved in vascular basement membrane assembly. The extracellular binding of NK4 to perlecan inhibits cell-associated assembly of fibronectin, and the impaired fibronectin assembly suppresses integrin-dependent angiogenic responses, i.e., endothelial cell proliferation, migration and tube formation. NK4 or an NK4-like fragment is generated by proteases expressed in inflammatory cells, suggesting regulation of physiological or pathological processes by NK4 or NK4-like fragments. In a variety of cancer models, NK4 exhibited anti-cancer effects due to its bifunctional characteristics, including inhibition of invasion and metastasis, inhibition of angiogenesis-dependent tumor growth, and promotion of survival. Several lines of strategies and different molecules that inhibit the HGF-Met pathway have been developed, including small molecular inhibiters of Met tyrosine kinase. The biological action of NK4 as an angiogenesis inhibitor has definite advantages over other molecules. In addition to the well-acknowledged role of HGF-Met in cancer invasion and metastasis, recent studies indicate that activation of the HGF-Met pathway makes tumor-initiating cells invasive and resistant to chemical and radiation therapy. Treatment with NK4 could offer a new therapeutic option for the inhibition of cancer metastasis and growth, and better outcomes for cancer patients.

RETRACTED ARTICLE: Enhanced suppression of tumor growth using a combination of NK4 plasmid DNA-PEG engrafted cationized dextran complex and ultrasound irradiation

This investigation aims to determine experimentally whether or not ultrasound (US) irradiation is effective in enhancing the in vivo gene expression of NK4 plasmid DNA and suppressing tumor growth. NK4, composed of the NH2-terminal hairpin and subsequent four-kringle domains of hepatocyte growth factor (HGF), acts as an HGF-antagonist and angiogenesis inhibitor. Dextran was cationized by introducing spermine to the hydroxyl groups to allow for polyionic complexation with NK4 plasmid DNA. The cationized dextran was additionally modified with poly(ethylene glycol) (PEG) molecules giving PEG engrafted cationized dextran. Significant suppression of tumor growth was observed when PEG engrafted cationized dextran-NK4 plasmid DNA complexes were intravenously injected into mice carrying a subcutaneous Lewis lung carcinoma tumor mass with subsequent US irradiation when compared with the cationized dextran-NK4 plasmid DNA complex and naked NK4 plasmid DNA with or without US irradiation. We conclude that complexation with PEG-engrafted cationized dextran in combination with US irradiation is a promising way to target the NK4 plasmid DNA to the tumor for gene expression.

Controlled Release Technology Suppresses the Progression of Disseminated Pancreatic Cancer Cells

NK4, composed of the NH2-terminal hairpin and subsequent four-kringle domains of hepatocyte growth factor (HGF), acts as a potent angiogenesis inhibitor. This study is an investigation to evaluate the feasibility of controlled release of NK4 plasmid DNA in suppressing tumor growth. Controlled release by a biodegradable hydrogel enabled the NK4 plasmid DNA to enhance the tumor suppression effects. Biodegradable microspheres of cationized gelatin were prepared for the controlled release of a NK4 plasmid DNA. The cationized gelatin microspheres incorporating NK4 plasmid DNA were subcutaneously injected to tumor-bearing mice to evaluate the suppressive effects on tumor angiogenesis and growth. The cationized gelatin microspheres incorporating NK4 plasmid DNA could release over 28 days. When the cationized gelatin microspheres incorporating NK4 plasmid DNA were injected into the subcutaneous tissue of mice intraperitoneally inoculated with pancreatic cancer cells, their survival time period was prolonged. Tumor growth was suppressed to a significantly greater extent than free NK4 plasmid DNA. The controlled release of NK4 plasmid DNA suppressed angiogenesis and increased cell apoptosis in the tumor tissue, while it enhanced and prolonged the serum level of NK4 protein. We conclude that the controlled release technology was promising to enhance the tumor suppression effects of NK4 plasmid DNA.

Suppression of the progress of disseminated pancreatic cancer cells by NK4 plasmid DNA released from cationized gelatin microspheres.

NK4, composed of the NH2-terminal hairpin and subsequent four-kringle domains of hepatocyte growth factor (HGF), acts as a potent angiogenesis inhibitor. This study is an investigation to evaluate the feasibility of controlled release of NK4 plasmid DNA in suppressing the tumor growth. Controlled release by a biodegradable hydrogel enabled the NK4 plasmid DNA to exert the tumor suppression effects. Biodegradable cationized gelatin microspheres were prepared for the controlled release of an NK4 plasmid DNA. The cationized gelatin microspheres incorporating NK4 plasmid DNA were subcutaneously injected to tumor-bearing mice to evaluate the suppressive effects on tumor angiogenesis and growth. The cationized gelatin microspheres incorporating NK4 plasmid DNA could release over 28 days as a result of microspheres degradation. The injection of cationized gelatin microspheres incorporating NK4 plasmid DNA into the subcutaneous tissue of mice inoculated with pancreatic cancer cells prolonged their survival time period. An increase in the tumor number was suppressed to a significantly greater extent than free NK4 plasmid DNA. The controlled release of NK4 plasmid DNA suppressed angiogenesis and increased the cell apoptosis in the tumor tissue while it enhanced and prolonged the NK4 protein level in the blood circulation. We conclude that the controlled release technology is promising to enhance the tumor suppression effects of NK4 plasmid DNA.

Suppression of tumor metastasis by NK4 plasmid DNA released from cationized gelatin.

NK4, composed of the NH(2)-terminal hairpin and subsequent four-kringle domains of hepatocyte growth factor (HGF), acts as an HGF-antagonist and angiogenesis inhibitor. This study is an investigation to evaluate the feasibility of controlled release formulation of NK4 plasmid DNA in suppressing the tumor growth, and lung metastasis. Biodegradable cationized gelatin microspheres were prepared for the controlled release of an NK4 plasmid DNA. The cationized gelatin microspheres incorporating NK4 plasmid DNA could continuously release plasmid DNA over 28 days as a result of microspheres degradation following the subcutaneous injection. The injection of cationized gelatin microspheres incorporating NK4 plasmid DNA into the subcutaneous tissue significantly prolonged the survival time period of the mice bearing Lewis lung carcinoma tumor. Increases in the tumor volume and the number of lung metastatic nodules of NK4 plasmid DNA release group were suppressed to a significantly greater extent than that of solution-injected group (77.4 and 64.0%, respectively). The number of blood vessels and the apoptosis cells in the tumor tissue were significantly suppressed (80.4%) and increased (127.3%) against free NK4 plasmid DNA-injected group. Thus, the controlled release of NK4 plasmid DNA augmented angiogenesis suppression and apoptosis of tumor cells, which resulted in suppressed tumor growth. We conclude that this controlled release technology is promising to enhance the tumor suppression achieved by gene expression of NK4.