Immunohistochemical study of epidermal growth-factor and epidermal growth-factor receptor in breast-carcinoma

We examined the relationship between epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) with clinicopathologic variables and silver-stained nuclear organizer region (Ag-NOR) counts in 93 patients with breast cancer. EGFR expression was significantly associated with axillary lymph node metastases, whereas EGF expression was not significantly associated with any of the clinicopathologic variables. Ag-NOR counts were not significantly different among groups of tumors categorized by EGF and EGFR expression, but a significant correlation was observed between clinical stage and synchronous expression of EGF and EGFR. However, EGF and EGFR expression did not appear to be independent prognostic factors as determined by multivariate analysis in which axillary lymph node metastases were included. We therefore conclude that EGF and EGFR expression may play a role in tumor progression rather than in tumor proliferation, but their expression was not useful in predicting the prognosis of patients with breast cancer.     

Hepatocyte growth factor activation inhibitors (HAI-1 and HAI-2) regulate HGF-induced invasion of human breast cancer cells

Hepatocyte growth factor (HGF) plays a plethora of roles in cancer metastasis and tumour growth. The interaction between tumour cells and their surrounding stromal environment is a crucial factor regulating tumour invasion and metastasis. Stromal fibroblasts are the main source of HGF in the body, and release HGF as an inactive precursor (pro-HGF). HGF activator (HGFA), matriptase, urokinase-type plasminogen activator and hepsin are the main factors responsible for converting pro-HGF into active HGF. HAI-1 and HAI-2 are 2 novel Kunitz-type serine protease inhibitors that regulate HGF activity through inhibition of HGFA, matriptase and hepsin action. Recent studies demonstrate that HAI-1 and HAI-2 may also potently inhibit a number of other pro-metastatic serine proteases and therefore have direct bearing on the spread of tumours. Our study examined the potential of these HAI's to suppress the influence of HGF and regulate cancer metastasis. We generated a retroviral expression system that induced HAI expression in a human fibroblast cell line. Forced expression of either HAI-1 or HAI-2 in these fibroblasts resulted in a dramatic decrease in the production of bioactive hepatocyte growth factor (HGF). This reduction in HGF activity subsequently suppressed HGF's metastatic influence on breast cancer cells. To further assess the anti-cancer properties of HAI-1 and HAI-2 we generated recombinant HAI proteins. These recombinant HAI proteins possessed the ability to potently quench HGF activity. We also demonstrate that these recombinant HAI's suppressed fibroblast-mediated breast cancer invasion. An additional ribozyme transgenes study revealed that elimination of HAI-1 and HAI-2 expression, in an MDA-MB-231 breast cancer cell line, significantly enhanced the migratory, proliferative and invasive nature of these breast cancer cells. Overall, our data demonstrates the important roles of HAI-1 and HAI-2 in cancer metastasis, and reveals that these serine protease inhibitors display strong therapeutic potential.     

Establishment and characterization of pthrp-producing human pancreatic-cancer cell-line

Human pancreatic ductal cell carcinoma cell line, which can secrete parathyroid hormone-related peptide (PTHrP), designated as KP 4, and its daughter cell lines with different PTHrP-secreting activities, termed KP 4-1 and KP 4-2, have been established in tissue culture. KP 4 cells were able to form tumors in nude mice. The absolute production rate of PTHrP in KP 4-1 was 5 to 10 times higher than that in KP 4-2. Similarly, the level of PTHrP mRNA in KP 4-1 was significantly higher than that in KP 4-2. KP 4-2 cells exhibited more rapid growth than KP 4 and KP 4-1 in vitro. Our established cell lines should provide a useful system to study the regulation of PTHrP production and its pathophysiological roles.     

Expression of uPA and its receptor by both neoplastic and stromal cells during xenograft invasion

Recent studies have shown that molecules involved in generation and regulation of extracellular proteolytic activity are often expressed by non-malignant stromal cells during human cancer invasion. We have studied the expression of the urokinase-type plasminogen activator and the urokinase-type plasminogen activator cell-surface receptor in xenografts of human MDA-MB-231 mammary carcinoma cells growing invasively in nude mice. Northern analysis showed the presence of both human and mouse urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor mRNA in tumor extracts. By in situ hybridization, mRNA for human urokinase-type plasminogen activator and its receptor was detected in virtually all the cancer cells, while mouse urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor mRNA was expressed by tumor-infiltrating fibroblast-like and macrophage-like cells. In invasive areas the cells expressing the 2 murine mRNAs were either the same or located immediately adjacent to each other. This model system has several advantages for studies of the mechanism by which cancer cells induce or recruit stromal cells to produce molecules involved in proteolysis. © 1994 Wiley-Liss, Inc.     

Inhibition of vascular endothelial growth-factor induced cell-growth by an angiogenesis inhibitor agm-1470 in capillary endothelial-cells

It is known that an anti-angiogenic compound AGM-1470 inhibits cells in vitro and in vivo, on mitogen-induced cell growth in capillary endothelial cells. In monolayer cultures, 1 ng/ml AGM-1470 completely inhibited both basic fibroblast growth factor (bFGF) induced cell growth and vascular endothelial growth factor (VEGF) induced cell growth in a cytostatic manner. IC50 was 85 pg/ml and 55 pg/ml for bFGF and VEGF induced cell growth, respectively. Moreover, in collagen gels, AGM-1470 suppressed the colony formation induced by bFGF and by VEGF in a dose dependent manner. Few colonies appeared 25 days after co-culuture with I ng/ml AGM-1470 and either 1 ng/ml bFGF or 5 ng/ml VEGF. It is suggested that a potent; inhibition of growth signals of more than one growth factor for endothelial cells might be involved in anti-angiogenic activity of AGM-1470.     

肝细胞生长因子及其受体与肝细胞癌

肝细胞生长因子(HGF)是正常肝细胞增生的强促有丝分裂原.近年研究发现,HGF同样影响肝细胞癌的生物学行为,其受体(c-Met原癌基因编码蛋白)也与原发性肝癌关系密切,因此可能成为原发性肝癌新的诊断标志物和治疗靶点.     

肝细胞生长因子及其受体在多发性骨髓瘤中的研究进展

 肝细胞生长因子（HGF）是一个多功能的细胞因子,作用于细胞表面的跨膜受体c-Met发挥生物学作用。许多肿瘤的生长、侵袭和转移都与HGF-c-Met信号转导通路异常有关。HGF及其受体c-Met与多发性骨髓瘤（MM）的发生、转移、侵袭及预后关系密切     

Immunohistochemical localization of epidermal growth-factor and its receptor in normal, premalignant and malignant oral-mucosa

An important growth factor involved in epithelial carcinogenesis is the epidermal growth factor (EGF). The present study analyze the expression pattern of EGF and its receptor (EGFR) in different stages of tumour progression in oral mucosa (normal epithelium, non-dysplastic and dysplastic leukoplakias and carcinomas). Alterations in expression pattern of EGFR was not significant in the various tissues from normal mucosa to malignancy. In all these stages EGFR positivity was confined to the immature basal or basaloid cells. EGF however showed marked alterations in expression in different stages of tumour progression in oral mucosa. Most of the malignant cells were positive for EGF antibodies while, only a few lower layers of cells were stained in normal mucosa and in dysplastic leukoplakia lesions, more number of cells expressed EGF than normal tissue. The present study thus shows the autocrine role of the EGF and EGFR and the possibility of using EGF as a marker for tumour progression in oral mucosa.     

Biologic effect of human hepatocyte growth-factor on human gastric-carcinoma cell-lines

Toxicity of polyphenols against rat 3Y1 fibroblasts and the cells transformed by human adenovirus (Ad12-3Y1), its EIA gene (EIA-3Y1), or simian virus 40 (SV-3Y1) was examined. Among the diphenol compounds examined, pyrocatechol (o-diphenol) and hydroquinone (p-diphenol) showed selective toxicity against Ad12-3Y1 and EIA-3Y1 cells, while resorcinol (m-diphenol) showed a much weaker non-specific toxicity against these cells. Another o-diphenol (dopamine) and triphenols (gallic acid and pyrogallol) were less toxic but showed selective toxicity. At lower concentrations where they were not toxic, all polyphenols attenuated toxicity of phosphatidylcholine against EIA-3Y1 cells. Among antioxidants examined, ascorbic acid reduced the toxicity of pyrocatechol, but alpha-tocopherol and butyrated hydroxytoluene did not. Oxidation of pyrocatechol was not enhanced in the presence of 3Y1 or EIA-3Y1 cells and their homogenates. These results suggest that the selective toxicity of polyphenols against Ad12-3Y1 and E1A-3Y1 cells is not related to their oxidation velocity but other factors such as the activity of active oxygen-scavenging enzymes.     

Expression and tyrosine phosphorylation of phosphatidyl-inositol-3 kinase in human gastric-cancer cells - its correlation with cell-growth

To reveal the signaling pathway leading to oncogenecity of human cancer cells, we examined the expression and tyrosine-phosphorylation of phosphatidylinositol (PI)-3 kinase in cancer cell lines. Of the 14 cell lines examined, two poorly differentiated human gastric cancer cell lines, NUGC-4 and MKN-45, which were previously found to have aberrant elevation of tyrosine phosphorylation showed elevated levels of PI-3 kinase 85-kDa subunit expression. In these cells, tyrosine-phosphorylation and overall activity of PI-3 kinase were apparently elevated, compared with normal human fibroblasts and another well differentiated gastric cancer cell line, MKN-28. Treatment of these cells with tyrosine kinase inhibitor, genistein, strongly suppressed the PI-3 kinase activity. Furthermore, wortmannin, a potent inhibitor of PI-3 kinase, strongly suppressed the growth of these gastric cancer cells. These results suggest that the growth signaling via tyrosine phosphorylation is required for the activation of PI-3 kinase in NUGC4 and MKN-45, and that this activation plays an important role in oncogenic growth of these cells. However, these two cell lines showed different responses of PI-3 kinase to acid-treatment and tyrosine kinase inhibitors. In MKN-45, activation of PI-3 kinase appeared to be constitutive, and could be relevant to the oncogenic nature of the cell line.     

Epidermal growth-factor and transforming growth factor-alpha in human meningiomas

Recently we have shown that human meningiomas overexpress epidermal growth factor receptor (EGFR) (Torp SH et al APMIS 100: 797-802, 1992). We therefore wanted to examine these tumours for the expression of the EGFR ligands epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). Normal human meningeal tissues were used as controls. Immunohistochemistry (IH) and radioimmunoassay (RIA) demonstrated the presence of EGF and/or TGF-alpha. immunoreactivity in sixteen of nineteen meningiomas. By means of RIA detectable amount of TGF-alpha was also recorded in normal leptomeninges. Discrepancies between IH and RIA were noted and are discussed. Our findings suggest that EGFR/EGF/TGF-alpha play a role in growth regulatory mechanisms in human meningiomas.     

Type-II insulin-like growth-factor receptor in conditioned medium from HT-29 human colon carcinoma cell line.

The HT-29 human colon cancer cell line has previously been shown to secrete high amounts of insulin-like growth factor II (IGF-II). The recent demonstration that soluble IGF-II/mannose 6-phosphate receptor was present in fetal serum prompted us to search for a release of type-II IGF receptor by these human colonic carcinoma cells. Serum-free conditioned medium from the HT-29 cell line was gel filtered on Sephadex G-200. There was significant binding of [125I]IGF-II to the void volume fractions in addition to binding to the 40-kDa IGF-binding protein (IGF-BP) fractions. Competitive binding studies using [125I]IGF-II and the void volume pool showed a pattern typical of the type-II receptor. It exhibited a high affinity for IGF-II (KD = 0.4 nM), but had a low affinity for IGF-I (KD = 6.8 nM), and no detectable affinity for insulin. Additional evidence was provided by affinity cross-linking of [125I]IGF-II to the same high-molecular-weight material which demonstrated a major specific band at 250 kDa after reduction of disulfide bonds. In contrast, the type-I IGF receptor was undetectable. The extracellular type-II IGF receptor was not a significant carrier for IGF-II since virtually all IGF-II secreted by HT-29 cells was associated with IGF-BP. The presence of a soluble IGF-II/mannose 6-phosphate receptor in the culture medium from colonic cancer cells suggests that it may play an important role in tumor pathogenesis.     

Treatment with luteinizing-hormone-releasing hormone antagonist sb-75 decreases levels of epidermal growth-factor receptor and its messenger-RNA in ov-1063 human epithelial ovarian-cancer xenografts in nude-mice

The aim of this study was to investigate the effect of administration of LH-RH antagonist SB-75 and agonist [D-Trp(6)]LH-RH on receptors for epidermal growth factor (EGF) in OV-1063 human epithelial ovarian cancer. Female athymic nude mice bearing xenografts of OV-1063 human epithelial ovarian cancer were treated for 3 weeks with the modern LH-releasing hormone (LH-RH) antagonist [Ac-DNal(2)(1), D-Phe(4Cl)(2), D-Pal(3)(3), D-Cit(6), D-Ala(10)] LH-RH (SB-75, Cetrorelix), the agonist [D-Trp(6)]LH-RH, or bombesin/gastrin-releasing peptide antagonist RC-3095. SB-75 and [D-Trp(6)] LH-RH were injected s.c. at doses of 100 mu g/day, and RC-3095 was injected at a dose of 40 mu g/day. Tumor growth, as measured by percentage change in tumor volume, was significantly inhibited by the treatment with SB-75, but not by [D-Trp(6)] LH-RH or RC-3095. Treatment with SB-75 greatly decreased the levels of mRNA for EGF receptor and reduced the number of EGF binding sites on tumor membranes. Effects of SB-75 on EGF receptors might be related to inhibition of tumor growth. Our findings support the view that LH-RH antagonists such as SB-75 could be considered for possible hormonal therapy of epithelial ovarian cancer.     

Alteration of the C-met oncogene locus in human head and neck-carcinoma

The c-met proto-oncogene encodes the receptor for the hepatocyte growth factor/scatter factor (HGF/SF) which induces eel proliferation and motility. We have analysed the genetic alteration involving the c-met locus on chromosome 7q31 using the pMet H polymorphic probe, in tumor and normal DNA from 87 patients with head and neck squamous cell carcinomas (HNSCC). We report the observation of loss of heterozygosity (LOH) at this locus in 23% of informative cases, contrasting with the previously reported 40% LOH detected in breast cancer. Further, gain of genetic material was also observed in 13% of the HNSCCs. The alterations of c-met gene were not significantly associated with standard pronostic features including tumor size and lymph node status. Involvement of the c-met locus in allelic imbalance, either loss or gain of genetic material, is relatively consistent with complex karyotype patterns detected in head and neck squamous cell carcinomas through previous cytogenetic studies.     

RNAi-mediated downregulation of urokinase plasminogen activator and its receptor in human meningioma cells inhibits tumor invasion and growth

In recent years, RNA interference (RNAi) has emerged as an effective method to target specific genes for silencing. Several groups are actively exploring the use of small interfering RNA (siRNA) for therapeutic applications to treat cancer. Our previous studies have demonstrated the inhibition of various proteases, including serine proteases, cysteine proteases and matrix metalloproteases, via RNA interference (RNAi) in gliomas. Similar to gliomas, malignant meningiomas also exhibit elevated protease levels in comparison to normal brain and benign meningiomas. Here, we used siRNA to simultaneously target urokinase plasminogen activator (uPA) and its receptor, uPAR. A human CMV promoter-driven mammalian expression vector (pU2) was used to produce hairpin double-stranded RNA (hp RNA) to target uPA and uPAR. As determined by Western blotting and fibrin zymography, pU2 effectively inhibited uPAR protein levels and uPA enzymatic activity in meningioma cells (IOMM-Lee). In vitro studies (Matrigel invasion and spheroid migration) revealed reduced meningioma cell invasion and migration. Intratumoral injections of the plasmid vector expressing siRNA for uPA and uPAR resulted in regression of pre-established, subcutaneous tumors in mice. In addition, in vivo studies of mice injected with pU2-transfected meningioma cells revealed inhibition of intracranial tumor formation. These findings suggest that siRNA can be used as a potent and specific therapeutic tool for the treatment of malignant meningiomas in humans.     

The value of epidermal growth-factor receptor (egfr) as a tumor-marker for human oral premalignant and malignant lesions

A previous study demonstrated increased epidermal growth factor receptor (EGFR) in oral dysplasia while another showed decreased EGFR in oral dysplasia. The present study examined immunohistochemical expression of EGFR in 33 dysplastic oral lesions as well as in 9 normal oral mucosa specimens, 12 hyperplastic oral lesions and 10 oral squamous cell carcinomas (SCCs). There were no significant differences in EGFR staining either in intensity or in the epithelial layers stained among the normal oral epithelium, hyperplastic and dysplastic lesions. In addition, no significant difference was noted between keratinized and non-keratinized specimens and among lesions from different sites. Oral SCCs demonstrated significantly stronger staining than the normal oral mucosa, hyperplastic and dysplastic lesions (p=0.0011). At this time, the conflicting data on the EGFR expression in oral dysplastic lesions indicate that this receptor is not a good marker for oral dysplasia. Because most of the available data (including our results) show that the majority of oral SCC overexpress EGFR, this receptor may be useful in the diagnosis and treatment of some oral cancers.     

趋化因子及其受体在肿瘤侵袭转移中的作用

近年来陆续发现了许多新的趋化因子和受体，研究发现趋化因子及其受体参与机体的多种生理和病理过程，并在肿瘤的侵袭转移中通过不同机制发挥着重要作用。本文对其进行简要综述。