Targeting adriamycin to tumour cells by means of an affinity ligand; a model system for drug delivery

Actively migrating tumour cells possess the proteolytic enzyme guanidinobenzoatase (GB) in an uninhibited form. This enzyme has been used as a target for the delivery of adriamycin to invasive tumour cells in frozen sections. An adriamycin-agmatine complex has been prepared which act as a competitive inhibitor of GB. Competition experiments have demonstrated that the adriamycin-agmatine complex competes with 9-aminoacridine for the active centre of GB associated with invasive tumour cells, located in the lymph nodes and in squamous cell carcinoma of the oral cavity. The technique described should be generally applicable to the targeting of drugs to cells.     

Evidence for distinct isoenzymic forms of a cell surface protease on normal colonic cells and on carcinoma cells from the same colon.

Both normal and carcinoma cells of the colon possess the cell surface protease guanidinobenzoatase (GB). GB is similar to plasminogen activator. In fixed sections, the insolubilised GB can be located by the fluorescent probe 9-amino acridine (9-AA) which binds to the active centre of GB, causing cells possessing active GB to fluoresce yellow. Both cell types possess cytoplasmic proteins which can be extracted in isotonic saline and were shown to inhibit their respective cell surface GB. The formation of an enzyme-inhibitor complex was demonstrated by the failure of 9-AA to react with the cell surface GB inhibitor complex. The data presented indicate that the colonic carcinoma cells possess an isoenzymic form of GB not recognised by the inhibitors of GB extracted from normal colonic cells.     

Normal epithelial antigens recognized by GB3 and GB5 are diminished in intraductal and lost in infiltrating human breast carcinomas

GB3 and GB5 are mouse monoclonal antibodies raised against human amnion. GB3 detects the epidermal basement membrane, and GB5 reacts with the junctional substances between the epithelial cells. These two antibodies were studied on breast tissues by indirect immunofluorescence. All (n = 8) of the normal mammary glandular basement membranes and epithelia reacted strongly with GB3 and GB5. In the intraductal carcinomas (n = 10), although nine out of ten tissues were reactive, their reactivities were greatly reduced. More strikingly, in the infiltrating carcinomas (n = 15), none of the tumor specimens were recognized by GB3 and only one out of fifteen biopsies was moderately reactive with GB5, indicating that the syntheses of the antigens of GB3 and GB5 were discontinued in the metastatic breast adenocarcinomas. These data suggest that these antigens may contribute to the interaction of the epithelium with the extracellular matrix and the intercellular binding between the epithelial cells; the loss of these antigens may facilitate the wide dissemination of tumor cells.     

银杏内脂B对人骨肉瘤Saos-2细胞的抑制作用研究

目的 探讨银杏内脂B（GB）对骨肉瘤（OS）细胞增殖、凋亡和迁移侵袭的影响以及潜在机制。方法 将人骨肉瘤Saos-2细胞分别在含300、600和1200 μmol/L GB的培养液中培养24、48和72 h,采用CCK-8法检测Saos-2细胞的增殖情况,流式细胞仪检测Saos-2细胞凋亡率,Transwell小室迁移与侵袭实验检测GB对Saos-2细胞迁移和侵袭能力的影响;提取GB处理后Saos-2细胞的mRNA和蛋白,采用实时荧光定量PCR和Western blotting检测Bcl-2、Bax、MMP-2和MMP-9的基因及蛋白表达水平,Western blotting法检测Erk和Akt磷酸化及cleaved-caspase-3水平。结果 GB能明显抑制Saos-2细胞的增殖,且呈时间和浓度依赖性,600和1200 μmol/L GB可诱导Saos-2细胞凋亡,GB对Saos-2细胞的迁移及侵袭的抑制作用呈浓度依赖性;GB可升高Bax mRNA和蛋白水平,但降低Bcl-2、MMP-2和MMP-9的mRNA和蛋白水平;高浓度GB能抑制Erk和Akt的磷酸化且升高cleaved-caspase-3水平。结论 GB具有抑制Saos-2细胞增殖及迁移侵袭和诱导细胞凋亡作用,其机制可能与抑制Erk和Akt磷酸化有关。     

Studies on the interaction and exchange of inhibitors and proteases on the surface of tumour cells in frozen sections.

We have used frozen sections of squamous cell carcinoma as a convenient source of a cell surface protease associated with tumour cells. This protease has been referred to as guanidinobenzoatase (GB) and is now known to be functionally identical to tissue plasminogen activator (t-PA). The use of a fluorescent competitive inhibitor of GB enabled the enzymic status of GB to be determined, i.e. was the enzyme active, latent or removed from our test system. The cell surface GB was then demonstrated to interact with extractable cytoplasmic inhibitors obtained from these sections. We then used a protected form of the GB in the absence of these internal inhibitors; such sections were used to transfer the GB to fibrin fibrils, thus exposing the presumptive receptor on the tumour cell surfaces. Texas red labelled t-PA was then shown to bind to the tumour cells in these pretreated sections from which the GB had previously been removed. We believe that the surface of tumour cells can be used to study the interaction of the naturally occurring inhibitors with GB and also that the cell surface receptors for GB can be used to study the binding of t-PA to cell surfaces.     

A simple fluorescent technique for the location of tumour cells in frozen sections of the head and neck region.

Tumour cells possess cell surface proteases referred to as guanidinobenzoatase (GB) which are closely similar to plasminogen activator. Previous studies have demonstrated different isoenzymic forms of GB on tumour cells and normal cells which can be recognised by cytoplasmic protein inhibitors extracted from frozen sections of appropriate tissues. We now show that normal human serum possesses inhibitors which selectively recognise the isoenzymic forms of GB associated with normal cell surfaces but do not recognise the GB on tumour cell surfaces in frozen sections of tissue obtained from the head and neck regions.     

Participation of OCT3/4 and beta-catenin during dysgenetic gonadal malignant transformation

Gonadoblastoma (GB) is an in situ tumor consisting of a heterogeneous population of mature and immature germ cells, other cells resembling immature Sertoli/granulosa cells, and Leydig/lutein-like cells, may also be present. GB almost exclusively affects a subset of patients with intersex disorders and in 30% of them overgrowth of the germinal component of the tumor is observed and the lesion is term dysgerminoma/seminoma. Several pathways have been proposed to explain the malignant process, and abnormal OCT3/4 expression is the most robust risk factor for malignant transformation. Some authors have suggested that OCT3/4 and beta-catenin might both be involved in the same oncogenic pathway, as both genes are master regulators of cell differentiation and, overexpression of either gene may result in cancer development. The mechanism by which beta-catenin participates in GB transformation is not completely clear and exploration of the E-cadherin pathway did not conclusively show that this pathway participated in the molecular pathogenesis of GB. Here we analyze seven patients with mixed gonadal dysgenesis and GB, in an effort to elucidate the participation of beta-catenin and E-cadherin, as well as OCT3/4, in the oncogenic pathways involved in the transformation of GB into seminoma/dysgerminoma. We conclude that the proliferation of immature germ cells in GB may be due to an interaction between OCT3/4 and accumulated beta-catenin in the nuclei of the immature germ cells.     

The targeting of agmatine-liganded mitomycin C to an enzyme on the surface of tumour cells

Tumour cells possess a cell surface protease referred to as guanidinobenzoatase (GB). We have synthesised a liganded form of mitomycin C which we refer to as MMC*. This MMC* has the fluorescent properties of mitomycin C and the additional ability to be directed to the active centre of GB by the agmatine moiety of the ligand. We have demonstrated the selective delivery of MMC* to the cell surface of tumour cells possessing active GB by fluorescent microscopy and competition experiments with molecules known to bind to the active centre of GB. The MMC* has a high affinity for tumour cell surface GB and we hope it may have potential in the selective delivery of mitomycin C in the chemotherapy of tumours in vivo.     

Reversible dissociation of a tumor-cell surface protease-inhibitor complex

Tumour cell surfaces possess a cell surface protease (GB), which can be recognised by a cytoplasmic inhibitor protein prepared from cultured tumour cells. This enzyme inhibitor complex has been shown to be reversibly dissociated by 10-(4)M sodium dodecyl sulphate. Sections of frozen tumour tissue were used to provide cells with active GB and the successful recognition and inhibition of this GB was followed by fluorescence microscopy employing the competitive inhibitor 9-amino acridine as a fluorescent probe.     

Evidence for cells possessing t-PA like activity in smears obtained from patients with oral squamous cell carcinoma

Squamous cell carcinoma cells possess a cell surface protease referred to as guanidinobenzoatase (GB) which is very similar to the single chain form of tissue type plasminogen activator. This enzyme binds fluorescent probes at its active center and cells possessing GB can be distinguished from those that lack this enzyme by fluorescent microscopic techniques. Normal squamous epithelial cells shed from the surface of the oral cavity lack GB and do not exhibit cell surface fluorescence when pretreated with such fluorescent probes. We have used this knowledge to design a simple technique for the rapid location of squamous cell carcinoma cells in oral smears; our results are presented in the form of colour prints in which the tumour cells can be easily distinguished from other cells by their fluorescence.     

Fluorescent location of tumour cells in fine needle aspirates.

Cells obtained by fine needle aspiration of breast lumps were spread onto microscope slides, defatted with xylene and stained with a fluorescent probe for a cell surface protease. In these aspirates, carcinoma cells possess an active cell surface protease, guanidinobenzoatase (GB), very similar to plasminogen activator (1), which binds the fluorescent probe as a competitive inhibitor. Cells obtained from benign and normal breast lack this active GB and can be distinguished easily from the carcinoma cells by fluorescent microscopy. We use this simple technique to examine breast lumps containing carcinoma cells and to demonstrate the similarity of GB on these carcinoma cells to tissue type plasminogen activator (t-PA).     

A rapid technique for the fluorescent location of pleomorphic adenoma cells in frozen sections of human salivary glands

Both acinar cells and pleomorphic adenoma cells possess a cell surface protease, guanidinobenzoatase (GB) which can be located with a fluorescent probe for the active centre of this enzyme. We have developed a rapid, cheap, fluorescent technique for the differential location of pleomorphic adenoma cells and acinar cells in frozen sections of salivary glands. The fluorescence of other cells (which also possess GB) is selectively quenched, such that only the acinar and pleomorphic adenoma cells are visualised by fluorescent microscopy.     

Boron compounds against human leukemic cells

Two new boron compoumds, dihydroxy(oxybiguanido) boron (iii) hydrochloride monohydrate (HB) and guanidine biboric acid adduct (GB) were used in this study to observe the antitumor effect. Leukemic blast cells isolated from chronic myeloid leukemia (CML) patients showed significant cell growth inhibition within twentyfour hours. IC50 of GB and HB was 2mg/ml. The metabolically active cells were found to be inhibited by drug treatment as assessed by MTT test. Inhibition of 3H Thymidine incorporation also supported the above result. In this study we investigated the molecular mechanisms by which HB and GB induce apoptosis in immature blast cells.     

Growth factor receptors signaling in glioblastoma cells: therapeutic implications

In this study, we investigated the protein expression of platelet-derived growth factor receptor (PDGFR), insulin like growth factor-1 receptor (IGF-1R), phosphatidylinositol 3-kinase (PI3-K) and extracellular signal-regulated kinase (ERK1/2) in five primary glioblastoma (GB), with a view to their possible use as therapeutic targets. Our results demonstrated that appreciable levels of these proteins could be detected in the analysed GB cell lines, except for a low level of PDGFR and ERK1/2 expression in one GB cell line. The small molecule inhibitors towards IGF-1R, PDGFR, PI3-K and ERK1/2 respectively, have only modest or no anti-tumour activity on GB cells and therefore their combination with other therapy modalities was analysed. The interaction between small inhibitors and radiation was mostly additive or sub-additive; synergistic interaction was found in five of forty analysed combinations. Our results showed that GB cells are in general resistant to treatment and illustrate the difficulties in predicting the treatment response in malignant gliomas.     

High expression of the antigen recognized by the monoclonal antibody GB24 on human breast carcinomas: A preventive mechanism of malignant tumor cells against complement attack?

Summary GB24 is a mouse monoclonal antibody raised against a common trophoblast-lymphocyte cross-reactive antigen. GB24 detects the membrane cofactor protein (MCP, CD46), a member of the complement regulatory protein family, which serves as a cofactor for factor 1 mediated cleavage of C3b. This study investigated the reactivity of GB24 on 38 breast carcinomas and 34 normal/benign breast tissues by immunochemistry as well as the reactivity of F2B7-2, an antibody specific to the decay accelerating factor (DAF, CD55) of the complement. GB24 staining was present on both normal tissue and benign lesions, but very strong diffuse reactivity was observed on carcinomas. This reactivity increased with the tumor grade. By contrast, malignant tumor cells lacked DAF expression. F2B7-2 antibody reacted weakly with benign epithelial cells. Results were studied by computer assisted image analysis to accurately define the mean optical densities. The densitometric analysis of MCP positive carcinomas showed a high intensity of the staining. Expression of MCP and DAF on MCF-7 cell lines was analyzed by flow cytometry. MCF-7 cell lines were strongly stained by mAb GB24 only. These data suggest that selectively enhanced expression of the antigen recognized by GB24 is associated with malignant breast disorders. This high expression, which may reflect a protective mechanism by which tumor cells survive complement activation, may prove useful as a marker of malignant transformation.     

Isolation of a new cell population in the glioblastoma microenvironment

Glioblastoma (GB) is a highly infiltrative tumor recurring in 90% of cases within a few centimeters of the resection cavity, even in cases of complete tumor resection and adjuvant chemo/radiotherapy. This observation highlights the importance of understanding this special zone of brain tissue surrounding the tumor. It is becoming clear that the nonneoplastic stromal compartment of most solid cancers plays an active role in tumor proliferation, invasion, and metastasis. Very little information, other than that concerning angiogenesis and immune cells, has been collected for stromal cells from GB. As part of a translational research program, we have isolated a new stromal cell population surrounding GB by computer-guided stereotaxic biopsies and primary culture. We named these cells GB-associated stromal cells (GASCs). GASCs are diploid, do not display the genomic alterations typical of GB cells, and have phenotypic and functional properties in common with the cancer-associated fibroblasts (CAFs) described in the stroma of carcinomas. In particular, GASCs express markers associated with CAFs such as fibroblast surface protein, alpha-smooth muscle actin (α-SMA), and platelet-derived growth factor receptor-beta (PDGFRβ). Furthermore, GASCs have a molecular expression profile different from that of control stromal cells derived from non-GB peripheral brain tissues. GASCs were also found to have tumor-promoting effects on glioma cells in vitro and in vivo. The isolation of GASCs in a tumor of neuroepithelial origin was unexpected, and further studies are required to determine their potential as a target for antiglioma treatment.     

Deoxyhypusine hydroxylase: A novel therapeutic target differentially expressed in short-term vs long-term survivors of glioblastoma

Glioblastoma (GB) is the most aggressive neoplasm of the brain. Poor prognosis is mainly attributed to tumor heterogeneity, invasiveness and drug resistance. Only a small fraction of GB patients survives longer than 24 months from the time of diagnosis (ie, long-term survivors [LTS]). In our study, we aimed to identify molecular markers associated with favorable GB prognosis as a basis to develop therapeutic applications to improve patients' outcome. We have recently assembled a proteogenomic dataset of 87 GB clinical samples of varying survival rates. Following RNA-seq and mass spectrometry (MS)-based proteomics analysis, we identified several differentially expressed genes and proteins, including some known cancer-related pathways and some less established that showed higher expression in short-term (<6 months) survivors (STS) compared to LTS. One such target found was deoxyhypusine hydroxylase (DOHH), which is known to be involved in the biosynthesis of hypusine, an unusual amino acid essential for the function of the eukaryotic translation initiation factor 5A (eIF5A), which promotes tumor growth. We consequently validated DOHH overexpression in STS samples by quantitative polymerase chain reaction (qPCR) and immunohistochemistry. We further showed robust inhibition of proliferation, migration and invasion of GB cells following silencing of DOHH with short hairpin RNA (shRNA) or inhibition of its activity with small molecules, ciclopirox and deferiprone. Moreover, DOHH silencing led to significant inhibition of tumor progression and prolonged survival in GB mouse models. Searching for a potential mechanism by which DOHH promotes tumor aggressiveness, we found that it supports the transition of GB cells to a more invasive phenotype via epithelial-mesenchymal transition (EMT)-related pathways.     

Helianthin induces antiproliferative effect on human glioblastoma cells in vitro

A major focus of brain cancer research today is to translate understanding of glioma biology into advances in treatment, by exploring the potential of target therapy. Here we investigated the ability of three compounds belonging to the chemical class of azo dyes (methyl red, methyl yellow, and helianthin) to inhibit glioblastoma (GB) cell growth in vitro. Our results showed that helianthin induced cytotoxicity in two GB cell cultures, cell lines 18 and 38, whereas methyl red and methyl yellow were not cytotoxic. The effect of helianthin on EGFR, IGF-1R, and their common intracellular signaling via PI3-K and ERK1/2 was also analyzed. Treatment with helianthin down-regulated EGFR and IGF-1R activity in both cell lines. Helianthin treatment blocked ERK1/2 phosphorylation without affecting PI3K activity in cell line 18 and reduced both PI3K and ERK1/2 in GB 38 cell line. The cell death was accompanied by degradation of PARP without affecting BCL2 expression in both GB cell cultures. Because of the genetic heterogeneity of malignant gliomas, we tested the effect of helianthin on other two primary GB lines (11 and 15) and two early-passage GB cultures (BT1GB and BT2GB), to assess the general nature of the anti-tumor effect of the drug in GB cells. We found that helianthin treatment induced cell death in all the GB cell cultures analyzed. To our knowledge, this is the first report indicating that helianthin can reduce GB cell growth.