Cellular mechanisms of hepatocyte growth factor-mediated urokinase plasminogen activator secretion by MAPK signaling in hepatocellular carcinoma

AIMS AND BACKGROUND: The hepatocyte growth factor, its receptor c-Met, and urokinase-type plasminogen mediate various cellular responses on activation, including proliferation, survival, invasion, and metastasis. The regulatory mechanisms for the proliferation and the particular invasive phenotypes of hepatocellular carcinoma are not yet fully understood. In order to clarify the intracellular downstream signal for hepatocyte growth factor/c-Met signaling in tumor progression and metastasis in hepatoma, we determined the effects of a specific MEK1 inhibitor (PD 098059) and a p38 kinase inhibitor (SB 203580) on hepatocyte growth factor-mediated cell proliferation and urokinase-type plasminogen expression in hepatoma cell lines (HepG2 and Hep3B). RESULTS: Hepatocyte growth factor treatment induced the phosphorylation of ERK and p38 kinase in a dose-dependent manner, resulting in an early peak of phosphorylation at 3 to 10 min, which then rapidly decreased to a near basal level. Pretreatment with PD 098059 reduced hepatocyte growth factor-mediated cell proliferation and urokinase-type plasminogen secretion. In contrast, SB 203580 pretreatment enhanced cell proliferation and urokinase-type plasminogen secretion due to induction of ERK phosphorylation. Treatment with PD 098059 and SB 203580 resulted in a decrease in phospho-ERK activity. Stable expression of dominant negative-MEK1 in HepG2 cells showed a decrease in hepatocyte growth factor-mediated urokinase-type plasminogen secretion. CONCLUSIONS: Such results suggest that interaction of an MEK/ERK and a p38 kinase might be critical in intrahepatic invasion and metastasis of human hepatoma cells.     

Intramuscular electroporation of a plasmid encoding human plasminogen kringle 5 induces growth inhibition of Lewis lung carcinoma in mice

Tumor growth and metastasis depend critically on blood vessel formation. Antiangiogenesis, therefore, represents a promising strategy for cancer therapy. The kringle 5 (K5) domain of human plasminogen is a potent angiogenesis inhibitor. To investigate whether intramuscular electroporation (EP) of K5 has antitumor activity in mouse tumor models, we constructed a plasmid encoding K5 (pVAX1-K5). Hela cells transfected with this plasmid produced and secreted K5 that inhibited the migration of human microvascular endothelial cells. Intramuscular EP treatment of pVAX1-K5 inhibited the growth of Lewis lung carcinoma and prolonged the survival time of tumor-bearing mice. Angiogenesis was obviously inhibited, and apoptosis was induced in tumor cells of mice that received intramuscular EP of pVAX1-K5. On the contrary, intramuscular injection of pVAX1-K5 without EP failed to show the same effects. The data indicate that intramuscular EP of plasmid DNA encoding the K5 domain is an effective strategy for the experimental treatment of cancer by expressing K5.     

Role of NF-kappaB signaling in hepatocyte growth factor/scatter factor-mediated cell protection

The cytokine scatter factor/hepatocyte growth factor (HGF/SF) protects epithelial, carcinoma, and other cell types against cytotoxicity and apoptosis induced by DNA-damaging agents such as ionizing radiation and adriamycin (ADR, a topoisomerase IIalpha inhibitor). We investigated the role of nuclear factor kappa B (NF-kappaB) signaling in HGF/SF-mediated protection of human prostate cancer (DU-145) and Madin-Darby canine kidney (MDCK) epithelial cells against ADR. HGF/SF caused the rapid nuclear translocation of the p65 (RelA) subunit of NF-kappaB associated with the transient loss of the inhibitory subunit IkappaB-alpha. Exposure to HGF/SF caused the activation of an NF-kappaB luciferase reporter that was blocked or attenuated by the expression of a mutant 'super-repressor' IkappaB-alpha. Electrophoretic mobility shift assay supershift assays revealed that HGF/SF treatment induced the transient binding of various NF-kappaB family proteins (p65, p50, c-Rel, and RelB) with radiolabeled NF-kappaB-binding oligonucleotides. The HGF/SF-mediated protection of DU-145 and MDCK cells against ADR (demonstrated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays) was abrogated by the IkappaB-alpha super-repressor. The ability of HGF/SF to activate NF-kappaB signaling was dependent on c-Akt --> Pak1 (p21-associated kinase-1) signaling (with Pak1 downstream of c-Akt) and was inhibited by the tumor suppressor PTEN (phosphatase and tensin homolog). Inhibitors of phosphatidylinositol-3'-kinase and Src family kinases significantly inhibited HGF/SF-mediated activation of NF-kappaB, while inhibitors of MEK, protein kinase C, and p70 S6 kinase had a modest effect or no effect on NF-kappaB activity. HGF/SF induced the expression of several known NF-kappaB target genes (cIAP-1 (cellular inhibitor of apoptosis-1), cIAP-2, and TRAF-2 (TNF receptor-associated factor-2)) in an NF-kappaB-dependent manner; HGF/SF blocked the inhibition of expression of these genes by ADR. Experimental manipulation of expression of these genes suggests that they (particularly TRAF-2 and cIAP-2) contribute to the protection against ADR by HGF/SF. These findings suggest that HGF/SF activates NF-kappaB through a c-Akt --> Pak1 signaling pathway that is also dependent on Src, and that NF-kappaB contributes to HGF/SF-mediated protection against ADR.     

The recombinant kringle domain of urokinase plasminogen activator inhibits in vivo malignant glioma growth

In a previous report, the recombinant kringle domain (UK1) of the urokinase type plasminogen activator (uPA) showed antiangiogenic activity. Here, we investigated in vivo antitumor effects of the UK1 of human uPA employing a brain tumor model. The systemic administration of UK1 purified from pichia expression (10 and 50 mg/kg/day intraperitoneally for 25 days) led to suppress the growth of a U87 human glioma xenograft, implanted into the brains of male BALB/cSlc nude mice, by 35% and 80%, respectively. In the immunohistochemical analysis, the tumors treated with UK1 showed decreased vascularity and expression of angiogenesis-related factors including vascular endothelial growth factor (VEGF), angiogenin, alpha-smooth muscle actin, von Willebrand's factor, and CD31 (PECAM-1 [Platelet endothelial cell adhesion molecule-1]), and increased apoptosis. UKl inhibited the in vitro proliferation and tube formation of VEGF-stimulated endothelial cells but not the proliferation of glioma cells. These results suggest that UK1 inhibits the malignant glioma growth by suppression of angiogenesis.     

Combination of human plasminogen kringle 5 with ionizing radiation significantly enhances the efficacy of antitumor effect

Antiangiogenic therapy could destroy tumor vasculature and inhibit tumor growth. It might inhibit tumor growth significantly when used as a single treatment modality and its therapeutic benefit may even be greater when used in combination with established treatment modalities such as radiation therapy (RT). In the present report, we investigated the effect of recombinant human plasminogen kringle 5 domain (rhK5) in combination with ionizing radiation on angiogenesis, tumor growth and survival in a murine Lewis lung carcinoma (LLC) tumor model. Combined treatment using rhK5 and radiotherapy displayed obvious suppressive effect on LLC tumor growth as compared with single treatment with either modality (p < 0.05), and resulted in a more additive effect on tumor growth delay in this model. In addition, combined treatment significantly enhanced the survival of mice and no toxic effect, such as weight loss, was observed. The significant antitumor effect of rhK5 plus radiation was associated with a direct suppression effect on early neoangiogenesis and tumor cell apoptosis. Furthermore, the expression of VEGF and HIF-1alpha in tumor tissue correlated well with decreased vessel density. The results suggest that rhK5 significantly enhances the antitumor activity of RT and could be a potent adjuvant therapeutic approach to improve the efficacy of radiotherapy for lung cancer.     

The kringle 1 domain of hepatocyte growth factor has antiangiogenic and antitumor cell effects on hepatocellular carcinoma

The kringle 1 domain of human hepatocyte growth factor (HGFK1) was previously shown to inhibit bovine aortic endothelial cell proliferation, suggesting that it might be an antiangiogenic molecule. Here, we evaluated the in vivo efficacy of a recombinant adenoassociated virus carrying HGFK1 (rAAV-HGFK1) for the treatment of hepatocellular carcinoma (HCC) in a rat orthotopic HCC model and explored its molecular mechanisms in vitro in both endothelial and tumor cells. We first showed that rAAV-HGFK1 treatment significantly prolonged the survival time of rats transplanted with tumor cells. Treatment with rAAV-HGFK1 inhibited tumor growth, decreased tumor microvessel density, and completely prevented intrahepatic, lung, and peritoneal metastasis in this in vivo model. In vitro, rAAV-HGFK1 exhibited both antiangiogenic and antitumor cell effects, inhibiting the proliferation of both murine microvascular endothelial cells (MEC) and tumor cells, and inducing apoptosis and G(0)-G(1) phase arrest in these cells. To our surprise, rAAV-HGFK1 did not act through the hepatocyte growth factor/hepatocyte growth factor receptor pathway. Instead, it worked mainly through epidermal growth factor (EGF)/epidermal growth factor receptor (EGFR) signaling, with more minor contributions from vascular endothelial growth factor/vascular endothelial growth factor receptor and beta fibroblast growth factor (bFGF)/beta fibroblast growth factor receptor (bFGFR) signaling. In both MECs and tumor cells, rAAV-HGFK1 acted through two pathways downstream of EGFR, namely inhibition of extracellular signal-regulated kinase activation and stimulation of p38 mitogen-activated protein kinase/c-Jun-NH(2)-kinase activation. These results suggest for the first time that HGFK1 exerts both antiangiogenic and antitumor cell activities mainly through EGF/EGFR signaling, and may thus be considered as a novel therapeutic strategy for the treatment of HCC.     

腺相关病毒介导的肝细胞生长因子Kringle 1结构域对人前列腺癌裸鼠骨移植瘤生长的影响

目的:探讨肝细胞生长因子Kringle1结构域(kringle1domain of hepatocyte growth factor,HGFK1)基因对人前列腺癌裸鼠骨移植瘤生长的影响。方法:通过向裸鼠胫骨骨髓腔内注射人前列腺癌PC-3细胞,建立人前列腺癌裸鼠骨移植瘤模型;分别向移植瘤和裸鼠尾静脉内注射PBS、携带有HGFK1基因或增强型绿色萤光蛋白(enhanced green fluorescent protein,EGFP)基因的腺相关病毒载体的病毒上清液rAAV-EGFP和rAAV-HGFK1,观察肿瘤生长、体内转移和裸鼠生存时间,免疫组织化学法检查移植瘤组织中Ki67、E-cadherin和CD31的表达。结果:成功建立了人前列腺癌裸鼠骨移植瘤模型,rAAV-HGFK1组的肿瘤体积和质量明显小于PBS和rAAV-EGFP组(P<0.05),抑瘤率达46.69%;rAAV-HGFK1组裸鼠的中位生存时间比PBS和rAAV-EGFP组明显延长(P<0.05);PBS和AAV-EGFP组腹主动脉旁淋巴结转移发生率高于rAAV-HGFK1组(P<0.05);rAAV-HGFK1组肿瘤组织中Ki67和CD31的表达明显低于PBS和AAV-EGFP组(P<0.05),E-cadherin的表达明显高于PBS和AAV-EGFP组(P<0.05)。结论:HGFK1具有抑制人前列腺癌裸鼠骨移植瘤生长、转移和血管生成的作用,并可延长荷瘤裸鼠的生存时间。     

A novel and effective hepatocyte growth factor kringle 1 domain and p53 cocktail viral gene therapy for the treatment of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide, yet effective therapeutic options for advanced HCC are limited. Kringle 1 domain of HGF (HGFK1) has been demonstrated as a potent anti-tumor molecule and p53 is a well established tumor suppressor. Recently we developed AAV transducing HGFK1 (AAV-HGFK1) as a gene therapy for HCC. Here we investigated the possibility of enhancing the effect of AAV-HGFK1 by combining it with Adv transducing p53 (Adv-p53). In vitro expression experiments suggested a small amount of Adv-p53 could increase the expression of AAV transgenes. AAV-HGFK1+Adv-p53 cocktail strongly inhibited the proliferation of microvascular endothelial cell (MEC) and two HCC cell lines, Hepa1-6 and McA-RH7777. In two orthotopic mice and rat HCC models the cocktail gene therapy also significantly reduced the tumor burdens and prolonged the survival time by inhibiting tumor angiogenesis and inducing tumor cell death. Significantly, tumor metastasis was completely prevented. AAV-HGFK1+Adv-p53 viral cocktail may be a promising cancer therapy for the treatment of HCC.     

The geldanamycins are potent inhibitors of the hepatocyte growth factor/scatter factor-met-urokinase plasminogen activator-plasmin proteolytic network

The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), have been implicated in human tumor development and metastasis. HGF/SF induces the expression of urokinase plasminogen activator (uPA) and the uPA receptor (uPAR), important mediators of cell invasion and metastasis. We have developed a cell-based assay to screen for inhibitors of this signaling system using the induction of endogenous uPA and uPAR and the subsequent conversion of plasminogen to plasmin as the biological end point. Assay validation was established using a neutralizing antiserum to HGF/SF and a uPA inhibitor (B428), as well as inhibitors of the MKK-MAPK1/2 pathway, shown previously to be important in the induction of uPA and uPAR. Using this assay, we found several classes of molecules that exhibited inhibition of HGF/SF-dependent plasmin activation. However, we discovered that certain members of the geldanamycin family of anisamycin antibiotics are potent inhibitors of HGF/SF-mediated plasmin activation, displaying inhibitory properties at femtomolar concentrations and nine orders of magnitude below their growth inhibitory concentrations. At nanomolar concentrations, the geldanamycins down-regulate Met protein expression, inhibit HGF/SF-mediated cell motility and invasion, and also revert the phenotype of both autocrine HGF/SF-Met transformed cells as well as those transformed by Met proteins with activating mutations. Thus, the geldanamycins may have important therapeutic potential for the treatment of cancers in which Met activity contributes to the invasive/metastatic phenotype.     

Evidence of epidermal growth factor receptor expression in uveal melanoma: Inhibition of epidermal growth factor-mediated signalling by Gefitinib and Cetuximab triggered antibody-dependent cellular cytotoxicity

Despite advances in surgery and radiotherapy of uveal melanoma (UM), many patients develop distant metastases that poorly respond to therapy. Improved therapies for the metastatic disease are therefore urgently needed. Expression of the epidermal growth factor receptor (EGFR), a target of kinase inhibitors and humanised antibodies in use for several cancers, had been reported.Forty-eight human UMs were analysed by expression profiling. Signalling was tested in three EGFR expressing UM cell lines by Western blotting using phosphorylation specific antibodies for EGFR and the downstream mediators AKT (v-akt murine thymoma viral oncogene homolog) and extracellular signal-regulated kinase (ERK). Evidence for signalling in tumours was obtained through the application of a UM-specific EGF-signature. The EGFR specific kinase inhibitor, Gefitinib and the humanised monoclonal antibody, Cetuximab, were tested for their effect on EGFR signalling. Natural killer cell mediated antibody-dependent cellular cytotoxicity (ADCC) and tumour necrosis factor α (TNF-α) release was analysed for Cetuximab.Fourteen of 48 UMs and three of 14 cell lines (over-)express EGFR, at least in part due to trisomy of the EGFR locus on chromosome 7p12. EGFR and the downstream mediator, AKT, are phosphorylated upon stimulation with EGF in EGFR expressing cell lines. EGFR over-expressing tumours but not EGFR negative tumours show an activated EGF-signature. Gefitinib inhibits EGFR and AKT phosphorylation and Cetuximab induces EGFR phosphorylation but inhibits signalling to AKT induced with EGF. Cetuximab triggers natural killer (NK) cells to lyse EGFR+ cell lines and to release TNF-α.EGFR appears suited as a novel molecular drug target for therapy of uveal melanoma.     

The E5 gene from human papillomavirus type 16 is an oncogene which enhances growth factor-mediated signal transduction to the nucleus.

Although human papillomavirus type 16 (HPV-16) is believed to be a major etiological agent in the development of cervical cancer, the biological function of several of its early genes remains to be established. In the present study, we have defined some of the biological properties of the E5 gene from HPV-16. Expression of the HPV-16 E5 gene in 3T3-A31 cells induced transformation to anchorage-independent growth (colony formation in soft agar). Addition of epidermal growth factor (EGF) to the soft-agar medium caused the E5-expressing cells to form larger colonies than those formed in the absence of EGF. Parental 3T3-A31 cells did not form colonies in soft agar either in the presence or in the absence of EGF. Analysis of clones expressing high levels of E5 mRNA revealed that these cells also expressed higher levels of c-fos mRNA in response to serum, EGF and platelet-derived growth factor (PDGF) than did the parental 3T3-A31 cells. Cells expressing the E5 gene were also capable of accelerated growth in low serum and were more tumorigenic in nude mice than were control cells. We conclude that the E5 gene from HPV-16 is an oncogene which transforms cells in part through enhancing signal transduction from growth factors to the nucleus.     

Kringle 5 of human plasminogen suppresses hepatocellular carcinoma growth both in grafted and xenografted mice by anti-angiogenic activity

Plasminogen kringle 5 (K5), a proteolytic fragment of plasminogen, is an endogenous angiogenic inhibitor. We have previously shown that K5 inhibits ischemia-induced retinal neovascularization in a rat model. However, its anti-angiogenic potential and application in the treatment of neoplastic diseases have not been well investigated. Our present study was designed to test its effect on the neovascularization and growth of hepatocellular carcinoma, a typical hypervascular tumor. Recombinant human K5 was expressed in E. coli and purified by affinity chromatography. K5 inhibited proliferation and induced apoptosis of primary endothelial cells in dose-dependent manner, but no effect on pericytes from the same origin of endothelial cells, which suggested an endothelial cell-specific inhibition. Moreover, K5 had no effect on the proliferation and apoptosis of mouse HepA and human Bel7402 hepatoma cell lines even in the enhanced concentration range, which suggested K5 having no direct effect on tumor cells. Ventral injection of K5 significantly suppressed the tumor growth in graphed hepatocarcinoma mice model, which was established by injection of mouse HepA hepatoma cells. In xenografted hepatocarcinoma athymic mice model, which mimicked human tumors by injection of human Bel7402 hepatoma cells, K5 significantly suppressed the tumor growth. An average of 68% suppression of primary tumor growth was observed in the K5-treated mice compared with control group. K5 also inhibited intratumoral neovascularization in the two cancer models determined by micro vessel density (MVD) analysis. Injection of K5 significantly induced the cleavage of pro-caspase-3 in tumor tissues of grafted mouse model, which suggested K5 also induced apoptosis of tumor tissues and the decreased intratumoral microvascular density in K5 treated group may correlate with K5-induced endothelial cell apoptosis. These results suggest that tumor growth suppression of K5 depends on its anti-angiogenic activity and K5 could have therapeutic potential in hepatocellular carcinoma.     

Reactive oxygen species regulate urokinase plasminogen activator expression and cell invasion via mitogen-activated protein kinase pathways after treatment with hepatocyte growth factor in stomach cancer cells

Background

Reactive oxygen species (ROS) are closely associated with the intracellular signal cascade, thus strongly implicating involvement in tumor progression. However, the mechanism by which ROS are generated and how ROS target downstream molecules to trigger tumor metastasis is unclear. In this study, we investigated the underlying signal pathways in ROS-induced urokinase plasminogen activator (uPA) expression in the human gastric cancer cells, NUGC-3 and MKN-28.

Methods and Results

Intracellular ROS, as determined using the fluorescent probe, 2''-7'' dichlorofluorescein diacetate, decreased after treatment with hepatocyte growth factor (HGF). We confirmed that Rac-1 regulated ROS production after activation of the AKT pathway with HGF. Exogenously added H₂O₂ promoted the expression of HGF, but not in a dose-dependent manner and also showed negative expression of HGF after co-treatment with H₂O₂ and HGF. Treatment with NAC, an intracellular free radical scavenger, decreased the enhancement of uPA production and tumor invasion in both cells. We clarified the downstream pathways regulated by ROS after treatment with H₂O₂, which showed negative control between FRK and p38 kinase activities for uPA regulation.

Conclusion

HGF regulates Rac-1-induced ROS production through the Akt pathway and ROS regulates uPA production and invasion via MAP kinase, which provides novel insight into the mechanisms underlying the progression of gastric cancer.     

Serum levels of hepatocyte growth factor/scatter factor in patients with liver metastases from breast cancer.

OBJECTIVE: Recent studies have shown that the pleiotropic cytokine hepatocyte growth factor/scatter factor (HGF/SF) and its receptor c-Met play major roles in the malignant progression of numerous tumors. For patients with breast cancer liver metastases, increased serum levels of HGF/SF have been reported. We studied the relationship between the clinical course of the disease and the serum levels of HGF/SF in such patients. METHODS: We examined 51 patients treated for breast cancer liver metastases. Serum concentrations of HGF/SF were measured before each therapy cycle and compared to the corresponding tumor marker levels. RESULTS: Mean serum levels of HGF/SF in patients with liver metastases were increased above the reported reference levels of primary breast cancer patients. Serum levels of HGF/SF were correlated with tumor marker levels in a logarithmic relation (r = 0.47, p < 0.001). In some cases serum concentrations of HGF/SF changed similarly to the course of the corresponding tumor markers. CONCLUSIONS: Serum levels of HGF/SF are associated with the clinical course of metastatic breast cancer patients with liver metastases. Further studies are required to clarify the potential value of the HGF/SF serum concentration as a tumor marker. HGF/SF and its receptor c-Met should be further evaluated as therapeutic targets.     

Neoplastic hepatocyte growth associated with cyclin D1 redistribution from the cytoplasm to the nucleus in mouse hepatocarcinogenesis

Cyclin D1 overexpression is a frequent change in hepatocellular carcinomas (HCCs). Our present study demonstrated that cyclin D1 overexpression with abundant cyclin E, cdk4, cdk2, and p27Kip1 (p27) occurred in neoplastic hepatocytes from the early stage of mouse hepatocarcinogenesis. While cyclin D1 expression was mainly found in the cytoplasm of the tumor cells, it shifted to the nucleus in association with cell proliferation after the animals were subjected to a partial hepatectomy (PH), and then returned once more to the cytoplasm when the cells became quiescent. Inhibition of PI3 kinase (PI3K) by Ly294002 in mouse HCC cells in vitro suppressed the nuclear shift of cyclin D1 as well as cell proliferation, while PI3K activation by PTEN suppression failed to induce nuclear shift of cyclin D1, suggesting that PI3K activation is essential but not sufficient for the cyclin D1 nuclear shift. While MEK-ERK1/2 inhibition by PD98059 and mTOR inhibition by rapamycin affected the cyclin D1 nuclear shift and cell proliferation to a lesser extent, both these inhibitors reduced cyclin D1 levels. Finally, although p27, cdk4 and calmodulin (CaM) were detected in the cyclin D1 immunoprecipitates from both quiescent and proliferating HCC cells, Hsc70 and SSeCKS were detected only in the immunoprecipitate from quiescent cells, and p21Waf1/Cip1 (p21) was detected only in that from proliferating cells, suggesting that the cyclin D1 complex is different in quiescent and proliferating cells. These observations indicate that the nuclear/cytoplasmic localization of cyclin D1 plays an important role in the proliferation/quiescence of neoplastic hepatocytes.