heparitin-sulfate and Lung-Neoplasms

Shedding of the endothelial glycocalyx layer (EGL) is known to occur during major surgery, but its degradation associated with minimally invasive video-assisted thoracoscopy (VATS) remains unclear. We investigated if serum biomarkers of EGL disruption were elevated during VATS lobectomy, and whether the urinary trypsin inhibitor (UTI) ulinastatin exerted a protective effect during this procedure.. Sixty ASA II-III lung cancer patients undergoing elective VATS lobectomy were divided equally into UTI and control groups. UTI group patients received intravenous UTI during surgery. Serum levels of syndecan-1 and heparan sulfate were examined before (T0) and at the end of surgery (T1). Serum albumin and hemoglobin were measured before surgery (BOD) and on the first postoperative day (POD1).. In control group, syndecan-1 levels were significantly elevated at T1 compared with T0 (3.77 ± 3.15 versus 4.28 ± 3.30,. EGL degradation occurs following VATS lobectomy. UTI can alleviate this shedding, thus helping preserve normal vascular permeability.. This trial is registered with ChiCTR-IOC-17010416 (January 13, 2017).

Topics: Acute Lung Injury; Biomarkers; Capillary Permeability; Cohort Studies; Endothelium; Female; Glycocalyx; Glycoproteins; Heparitin Sulfate; Humans; Lung; Lung Neoplasms; Male; Middle Aged; Syndecan-1; Thoracic Surgery, Video-Assisted; Thoracotomy

This double-blind, randomised, multicentre trial in 513 patients having elective surgery for intra-abdominal or intrathoracic malignancy compared the efficacy and safety of venous thrombosis (VT) prophylaxis using 750 anti-factor Xa units of Orgaran (a mixture of low molecular weight heparinoids) given subcutaneously (sc) twice-daily with that of twice-daily injections of 5,000 units standard heparin. The main study endpoints were the development of postoperative VT detected by 125I-fibrinogen leg scanning, and the onset of clinically significant venous thromboembolism or bleeding. "Intent to treat" analysis showed a statistically non-significant trend towards less VT during Orgaran prophylaxis (10.4%) than after heparin (14.9%) and there was no difference in bleeding complications between the two study groups. Results remained similar if only patients who completed the intended course of therapy ("compliant patients") were analysed. Other trials have shown that Orgaran prevents VT after hip surgery and stroke. We now show it is also safe and effective in patients having major surgery for cancer.

Topics: Aged; Anticoagulants; Chondroitin Sulfates; Dermatan Sulfate; Double-Blind Method; Elective Surgical Procedures; Female; Gastrointestinal Neoplasms; Glycosaminoglycans; Hematologic Tests; Hemorrhage; Heparinoids; Heparitin Sulfate; Humans; Lung Neoplasms; Male; Middle Aged; Postoperative Complications; Pulmonary Embolism; Thrombophlebitis

Lung cancer is one of the most common types of cancer with limited therapeutic options, therefore a detailed understanding of the underlying molecular changes is of utmost importance. In this pilot study, we investigated the proteomic and glycosaminoglycan (GAG) profile of ALK rearranged lung tumor tissue regions based on the morphological classification, mucin and stromal content. Principal component analysis and hierarchical clustering revealed that both the proteomic and GAG-omic profiles are highly dependent on mucin content and to a lesser extent on morphology. We found that differentially expressed proteins between morphologically different tumor types are primarily involved in the regulation of protein synthesis, whereas those between adjacent normal and different tumor regions take part in several other biological processes (e.g. extracellular matrix organization, oxidation-reduction processes, protein folding) as well. The total amount and the sulfation profile of heparan sulfate and chondroitin sulfate showed small differences based on morphology and larger differences based on mucin content of the tumor, while an increase was observed in both the total amount and the average rate of sulfation in tumors compared to adjacent normal regions.

Topics: Adenocarcinoma of Lung; Chondroitin Sulfates; Glycosaminoglycans; Heparitin Sulfate; Humans; Lung Neoplasms; Mucins; Pilot Projects; Proteomics; Receptor Protein-Tyrosine Kinases

In this study, we examined the roles of heparanase and IGFBP-3 in regulating A549 and H1299 non-small-cell lung cancer (NSCLC) survival. We found that H1299 cells, known to be p53-null with no expression of IGFBP-3, had higher heparanase levels and activity and higher levels of heparan sulfate (HS) in the media compared to the media of A549 cells. Inhibiting heparanase activity or its expression using siRNA had no effect on the levels of IGFBP-3 in the media of A549 cells, reduced the levels of soluble HS fragments, and led to decreased interactions between IGFBP-3 and HS in the media. HS competed with HA for binding to IGFBP-3 or IGFBP-3 peptide (

Topics: A549 Cells; Carcinoma, Non-Small-Cell Lung; Cell Survival; Heparitin Sulfate; Humans; Insulin-Like Growth Factor Binding Protein 3; Lung Neoplasms; Peptides; Tumor Suppressor Protein p53

Early lung carcinoma development may be modulated by innate host cellular mechanisms that promote tumor growth and invasion. We recently identified how a loss-of-function mutation in the glycan sulfating enzyme N-deacetylase/N-sulfotransferase-1 (Ndst1; involved in heparan sulfate biosynthesis) targeted to antigen presenting cells (APCs) may augment acquired anti-tumor T cell immune mechanisms. Crossing this mutation (Ndst1f/f CD11cCre+) onto a model of inducible spontaneous Kras mutant lung cancer [CCSP-rtTA; (tetO7) CMV-Kras-G12D] allowed us to examine how the APC mutation affects the formation and growth of early lung carcinoma. We examined early bronchocentric adenoma formation in the model, and the frequency of such events was significantly reduced on the mutant background. This was associated with significant reductions in tumor associated FOXP3+ cellular infiltration and CD163+ M2-type macrophage infiltration. The findings evolved prior to effector CD8+ T cell infiltration into tumors. The impact of this unique glycan under-sulfating mutation on inhibiting early Kras G12D mutant bronchocentric adenoma formation along with a cellular phenotype of inhibited tumor infiltration by cells involved in suppressive T-regulatory cell signaling (FOXP3+ cells) or tumor-permissive M2 macrophage functions (CD163+ cells) provides insight on how glycan targeting may modulate innate cellular mechanisms during early lung tumor development. The findings may also impact the future design of host-centered immunologic anti-tumor therapeutic strategies.

Topics: Adenoma; Animals; CD11c Antigen; CD8-Positive T-Lymphocytes; Heparitin Sulfate; Humans; Lung Neoplasms; Lymphocytes, Tumor-Infiltrating; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Myeloid Cells; Polysaccharides; Proto-Oncogene Proteins p21(ras); Sulfates; Sulfotransferases; T-Lymphocytes, Regulatory

Nanoparticles (NPs), such as liposomes, effectively evade the severe toxicity of unexpected accumulation and passively shuttle drugs into tumor tissues by enhanced permeability and retention. In the case of non-small cell lung cancer and pancreatic ductal adenocarcinoma, cancer-associated fibroblasts promote the aggregation of a gel-like extracellular matrix that forms a physical barrier in the desmoplastic stroma of the tumor. These stroma are composed of protein networks and glycosaminoglycans (GAGs) that greatly compromise tumor-penetrating performance, leading to insufficient extravasation and tissue penetration of NPs. Moreover, the presence of heparan sulfate (HS) and related proteoglycans on the cell surface and tumor extracellular matrix may serve as molecular targets for NP-mediated drug delivery. Here, a GAG-binding peptide (GBP) with high affinity for HS and high cell-penetrating activity was used to develop an HS-targeting delivery system. Specifically, liposomal doxorubicin (L-DOX) was modified by post-insertion with the GBP. We show that the

Topics: A549 Cells; Adenocarcinoma of Lung; Animals; Antibiotics, Antineoplastic; Cell Line, Tumor; Doxorubicin; Drug Delivery Systems; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Liposomes; Lung Neoplasms; Mice; Nanoparticles; NIH 3T3 Cells; Polyethylene Glycols; Tissue Distribution; Tumor Microenvironment; Xenograft Model Antitumor Assays

The members of the slit homolog (SLIT) and roundabout homolog (ROBO) families have emerged as important signaling molecules in tumor metastasis. This study analyzed their role in regulating breast cancer (BC) cell motility and chemotaxis and assessed expression of ROBO1 in brain metastases (BMs) of breast, lung, and colon cancer, and in peritoneal metastases (PMs) of ovarian cancer.. The BC cell line MDA-MB231 was subjected to scratch, motility, and chemotaxis assays using heparin and a purified recombinant N-terminal SLIT2 fragment. Protein expression was assessed in primary tumors and metastases by immunohistochemistry.. Exposure to SLIT2 induced MDA-MB231 cell motility, but no significant chemotaxis without the presence of heparin. ROBO1 was expressed in 4/5 primary BC and in 18/21 BC-derived BM samples; 7/9 BM primary lung cancer samples also stained positive. In contrast, BMs from colorectal cancer were negative for ROBO1. Primary ovarian cancer and ovarian PM showed ROBO1 expression in 0/6 and in only 2/6 samples, respectively, whereas SLIT2 was observed in 1/6 primary cancer and in 6/6 PMs samples.. SLIT2 can induce BC cell motility and chemotaxis, but the latter requires the presence of heparin. BM expression of ROBO1 is a common feature of some, but not all cancer types. SLIT2 expression appears to be a general feature of ovarian cancer-derived PMs.

Topics: Brain Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Female; Heparitin Sulfate; Humans; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Nerve Tissue Proteins; Ovarian Neoplasms; Peritoneal Neoplasms; Receptors, Immunologic; Roundabout Proteins

In this report, we used liquid chromatography-mass spectrometry and Western blotting to analyze the content and structure of glycosaminoglycans, glycolipids and selected proteins to compare differences between patient-matched normal and cancerous lung tissues obtained from lung cancer patients. The cancer tissue samples contained over twice as much chondroitin sulfate (CS)/dermatan sulfate (DS) as did the normal tissue samples, while the amount of heparan sulfate (HS) and hyaluronan (HA) in normal and cancer tissues were not significantly different. In HS, several minor disaccharide components, including NS6S, NS2S and 2S were significantly lower in cancer tissues, while the levels of major disaccharides, TriS, NS and 0S disaccharides were not significantly different in normal and cancer tissues. In regards to CS/DS, the level of 4S disaccharide (the major component of CS-type A and DS) decreased and the level of 6S disaccharide (the major component of CS- type C) increased in cancer tissues. We also compared the content and structure of GAGs in lung tissues from smoking and non-smoking patients. Analysis of the glycolipids showed all lipids present in these lung tissues, with the exception of sphingomyelin were higher in cancer tissues than in normal tissues. Western analysis showed that syndecan 1 and 2 proteoglycans displayed much higher expression in cancer tissue/biopsy samples. This investigation begins to provide an understanding of patho-physiological roles on glycosaminoglycans and glycolipids and might be useful in identifying potential biomarkers in lung cancer.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Squamous Cell; Chondroitin Sulfates; Chromatography, Liquid; Dermatan Sulfate; Disaccharides; Female; Glypicans; Heparitin Sulfate; Humans; Hyaluronic Acid; Lung Neoplasms; Male; Middle Aged; Retrospective Studies; Smoking; Syndecan-1; Tandem Mass Spectrometry

Inflammation and cancer are related pathologies acting synergistically to promote tumor progression. In both, hematogenous metastasis and inflammation, P-selectin participates in interactions involving tumor cells, platelets, leukocytes and endothelium. Heparin has been shown to inhibit P-selectin and as a consequence it blunts metastasis and inflammation. Some heparin analogs obtained from marine invertebrates are P-selectin inhibitors and do not induce bleeding effects. The present work focuses on the P-selectin blocking activity of a unique heparan sulfate (HS) from the bivalve mollusk Nodipecten nodosus. Initially, we showed that the mollusk HS inhibited LS180 colon carcinoma cell adhesion to immobilized P-selectin in a dose-dependent manner. In addition, we demonstrated that this glycan attenuates leukocyte rolling on activated endothelium and inflammatory cell recruitment in thioglycollate-induced peritonitis in mice. Biochemical analysis indicated that the invertebrate glycan also inhibits heparanase, a key player in cell invasion and metastasis. Experimental metastasis of Lewis lung carcinoma cells was drastically attenuated by the mollusk HS through a mechanism involving inhibition of platelet-tumor-cell complex formation in blood vessels. These data suggest that the mollusk HS is a potential alternative to heparin for inhibiting P-selectin-mediated events such as metastasis and inflammatory cell recruitment.

Topics: Animals; Anticoagulants; Antineoplastic Agents; Blood Platelets; Carcinoma, Lewis Lung; Cell Adhesion; Cell Line, Tumor; Drug Screening Assays, Antitumor; Glucuronidase; Heparitin Sulfate; Humans; Inhibitory Concentration 50; Leukocyte Rolling; Lung Neoplasms; Mollusca; Neoplasm Transplantation; P-Selectin

Syndecan-1 is a proteoglycan that acts as co-receptor through its heparan sulfate (HS) chains and plays important roles in cancer. HS chains are highly variable in length and sulfation pattern. This variability is enhanced by the SULF1/2 enzymes, which remove 6-O-sulfates from HS. We used malignant mesothelioma, an aggressive tumor with poor prognosis, as a model and demonstrated that syndecan-1 over-expression down-regulates SULF1 and alters the HS biosynthetic machinery. Biochemical characterization revealed a 2.7-fold reduction in HS content upon syndecan-1 over-expression, but an overall increase in sulfation. Consistent with low SULF1 levels, trisulfated disaccharides increased 2.5-fold. ERK1/2 activity was enhanced 6-fold. Counteracting ERK activation, Akt, WNK1, and c-Jun were inhibited. The net effect of these changes manifested in G1 cell cycle arrest. Studies of pleural effusions showed that SULF1 levels are lower in pleural malignancies compared to benign conditions and inversely correlate with the amounts of syndecan-1, suggesting important roles for syndecan-1 and SULF1 in malignant mesothelioma.

Topics: Biosynthetic Pathways; Cell Cycle; Cell Line, Tumor; Gene Expression; Gene Expression Regulation, Neoplastic; Heparitin Sulfate; Humans; Kaplan-Meier Estimate; Lung Neoplasms; Mesothelioma; Mesothelioma, Malignant; Pleural Effusion, Malignant; Proportional Hazards Models; Signal Transduction; Sulfotransferases; Syndecan-1

CXCL14 is a chemokine family member that is involved in various cellular responses in addition to immune cell activation. Although constitutive CXCL14 expression in normal epithelial cells may help protect against infection by activating immune systems, its expression in cancer cells has raised controversy regarding its possible role in tumorigenesis. However, the underlying mechanisms for this disparity remain unknown. Investigation of cellular CXCL14 binding properties might increase our understanding of the peptide's roles in tumorigenesis. In the present study, we found that CXCL14 binds to various cell types. Interestingly, binding to NCI-H460 cells was prevented by heparan sulfate and N-acetyl neuraminic acid. Next, we examined effect of CXCL14 binding in NCI-H460 and NCI-H23. CXCL14 enhanced proliferation and migration in NCI-H460 but had no effect on NCI-H23. A reporter gene assay with various transcription factor response elements revealed that only nuclear factor-κB (NF-κB) signaling was activated by CXCL14 in NCI-H460 cells, which was blocked by BAPTA-AM, TPCA-1, and brefeldin A. Exogenous expression of some glycoproteins such as syndecan-4, podoplanin, and CD43 in these cells enhanced CXCL14 binding and NF-κB activity. Collectively, these results demonstrate that CXCL14 binding to glycoproteins harboring heparan sulfate proteoglycans and sialic acids leads proliferation and migration of some cancer cells.

Topics: Calcium Signaling; Cell Line, Tumor; Cell Membrane; Cell Movement; Cell Proliferation; Chemokines, CXC; Endocytosis; Glycoproteins; HEK293 Cells; Heparitin Sulfate; Humans; Lung Neoplasms; N-Acetylneuraminic Acid; NF-kappa B; Protein Binding; Receptors, Fc

The epidermal growth factor receptor (EGFR) mutation status is a validated biomarker for the stratification of EGFR-tyrosine kinase inhibitor (EGFR-TKIs) treatment in patients with non-small cell lung cancer (NSCLC); however, its use is limited in patients with wild-type EGFR, and new biomarkers are needed. We hypothesized that the serum concentration of heparan sulfate (HS), which activates oncogenic growth factor receptor signaling through EGFR and non-EGFR signaling pathways, may be a novel glycobiological biomarker for EGFR-TKIs treatment in NSCLC.. The pretreatment serum HS concentrations were determined using enzyme-linked immunosorbent assay in 83 patients with stage IV non-small cell lung adenocarcinoma who received EGFR-TKIs treatment. The relationship between the serum HS concentrations and patient characteristics, tumor response, progression-free survival (PFS), and overall survival (OS) were analyzed.. Patient sex, performance status, smoking history, and EGFR mutation status were associated with tumor response. The serum HS concentrations were significantly higher among patients with progressive disease than among those without progressive disease (p = 0.003). Furthermore, the serum HS concentrations were strongly associated with a poor PFS and OS in a univariate Cox analysis (p = 0.0022 and p = 0.0003, respectively). A stratified multivariate Cox model according to the EGFR mutation status showed that higher HS concentrations were significantly associated with a shorter PFS and OS (p = 0.0012 and p = 0.0003).. We concluded that a high-serum HS concentration was strongly related to a poor treatment outcome of EGFR-TKIs and may be a promising noninvasive and repeatable glycobiological biomarker in cancer treatment.

Topics: Adenocarcinoma; Aged; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; ErbB Receptors; Erlotinib Hydrochloride; Female; Gefitinib; Heparitin Sulfate; Humans; Lung Neoplasms; Male; Mutation; Neoplasm Staging; Protein Kinase Inhibitors; Quinazolines; Retrospective Studies; Treatment Failure

Merlin, the protein product of the neurofibromatosis type 2 gene (NF2) acts as a tumor suppressor in mice and humans. In this study, melanoma B16F10 cells were engineered to overexpress the NF2 gene by establishing stable transductants. A cell line overexpressing Merlin (B16F10-M) was generated. When compared to the parental cells, the B16F10-M cells demonstrated differences in their cell surface organization. The overexpressing strain changed its ability to grow in soft agar as well as its cell motility properties. B16F10-M cells were then examined in the in vivo mouse melanoma tumor growth and tumor metastasis models. While tumor growth was marginally affected, the presence of increased Merlin severely reduced the metastastatic ability of the cells. When isolated using specific enzymes with distinct substrate specificity, the cell surface heparan sulfate glycosaminoglycans (HSGAGs) from the overexpressing B16F10-M cells, inhibited the metastatic properties of the parental B16F10 cells. The results obtained provide a causal link between the reorganization/changes to the cell surface HSGAGs by the overexpression of Merlin and the inhibition of the metastatic activity of the mouse melanoma B16F10 cells in vivo.

Topics: Animals; Blotting, Western; Cell Adhesion; Colony-Forming Units Assay; Gene Expression Regulation, Neoplastic; Heparitin Sulfate; Lung Neoplasms; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neurofibromin 2; Transfection; Tumor Cells, Cultured

Expression of the chemokine receptor CXCR4 allows breast cancer cells to migrate towards specific metastatic target sites which constitutively express CXCL12. In this study, we determined whether this interaction could be disrupted using short-chain length heparin oligosaccharides. Radioligand competition binding assays were performed using a range of heparin oligosaccharides to compete with polymeric heparin or heparan sulphate binding to I(125) CXCL12. Heparin dodecasaccharides were found to be the minimal chain length required to efficiently bind CXCL12 (71% inhibition; P<0.001). These oligosaccharides also significantly inhibited CXCL12-induced migration of CXCR4-expressing LMD MDA-MB 231 breast cancer cells. In addition, heparin dodecasaccharides were found to have less anticoagulant activity than either a smaller quantity of polymeric heparin or a similar amount of the low molecular weight heparin pharmaceutical product, Tinzaparin. When given subcutaneously in a SCID mouse model of human breast cancer, heparin dodecasaccharides had no effect on the number of lung metastases, but did however inhibit (P<0.05) tumour growth (lesion area) compared to control groups. In contrast, polymeric heparin significantly inhibited both the number (P<0.001) and area of metastases, suggesting a differing mechanism for the action of polymeric and heparin-derived oligosaccharides in the inhibition of tumour growth and metastases.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Movement; Chemokine CXCL12; Chemokines, CXC; Female; Gene Expression Regulation, Neoplastic; Heparin, Low-Molecular-Weight; Heparitin Sulfate; Humans; Immunohistochemistry; Iodine Radioisotopes; Lung Neoplasms; Mice; Mice, SCID; Oligosaccharides; Polymers; Radioligand Assay; Receptors, CXCR4; Tinzaparin

Proliferation of fibroblasts (desmoplastic reaction) in the lung adenocarcinomas is an important phenomenon that correlates with metastases and poor prognosis. Because basement membranes are often involved in the desmoplastic areas and many cytokines have binding capacity to basement membrane molecules, we hypothesized that basement membrane modify the paracrine effects between cancer cells and fibroblasts via the fibrogenic cytokines and this hypothesis was experimentally investigated.. The effects of conditioned media derived from ten lung carcinoma cell lines and normal airway epithelial cells on DNA synthesis of fetal lung fibroblasts were determined. We focused on fibroblast growth factor 2 (FGF-2) as the candidate paracrine cytokines and examined their diffusion through an experimental basement membrane matrix model, Matrigel.. All the conditioned media promoted DNA synthesis of fetal lung fibroblasts. Detection by ELISA methods and the neutralizing antibodies suggested that FGF-2 was one of the responsible factors for the growth promotion. Diffusion of FGF-2 across the polycarbonate membrane was suppressed by coating with Matrigel. When FGF-2-secreting A549 cells were covered with Matrigel, FGF-2 was stored in Matrigel and its diffusion into the culture media was significantly reduced. Binding of FGF-2 to Matrigel was completely blocked by a basic protein, protamine sulfate. In the presence of protamine sulfate in Matrigel overlaid on A549 cells, diffusion of FGF-2 increased 7-fold as much as that without overlaid Matrigel.. These results suggest that the basement membrane acts as a barrier to the diffusion and a reservoir of cytokines secreted by cancer cells, and that the subsequent degradation of the basement membrane by cancer cells could release the stored cytokines and promote growth of fibroblasts.

Topics: Adenocarcinoma; Antibodies, Blocking; Basement Membrane; Cell Division; Cell Line, Tumor; Collagen; Culture Media, Conditioned; DNA; DNA Replication; Dose-Response Relationship, Drug; Drug Combinations; Fibroblast Growth Factor 2; Fibroblasts; Heparitin Sulfate; Humans; Interleukin-1; Laminin; Lung; Lung Neoplasms; Paracrine Communication; Protamines; Proteoglycans; Recombinant Proteins

Heparin, a widely used anticoagulant, is known to have anti-metastatic activity, although the mechanism is not fully understood. In the present study, we investigated the mechanism of this anti-metastatic activity using periodate-oxidized and borohydride-reduced heparin with low anticoagulant activity (LAC heparin). The anticoagulant activity of LAC heparin is markedly reduced to almost the control level in terms of prothrombin time in vitro, and no hemorrhagic complication was observed with injection of LAC heparin into mice in vivo. LAC heparin injected intravenously with Lewis lung carcinoma cells or 10 min before tumor cell injection significantly inhibited, to the same extent as intact heparin and in a dose- and time-dependent manner, the lung colonization that develops after intravenous injection (i.v.) of tumor cells. Flow cytometric analysis revealed that Lewis lung carcinoma cells strongly express heparan sulfate on their surface. Both the LAC heparin and intact heparin inhibited the adhesion and invasion of tumor cells to Matrigel-coated dishes in vitro without significant effect on the tumor cell growth. LAC heparin also significantly diminished tumor cell retention in the lung after i.v. of LacZ gene-tagged Lewis lung carcinoma cells. These results suggest that LAC heparin may prevent tumor cells from attachment to the subendothelial matrix of lung capillaries by competitively inhibiting cell surface heparan sulfate functions and suppress lung colonization.

Topics: Animals; Anticoagulants; Borohydrides; Carcinoma, Lewis Lung; Cell Adhesion; Cell Division; Dose-Response Relationship, Drug; Flow Cytometry; Heparin; Heparitin Sulfate; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Periodic Acid; Survival Rate; Time Factors; Tumor Cells, Cultured

Heparanase is an endoglycosidase that degrades heparan sulfate, the main polysaccharide constituent of the extracellular matrix and basement membrane. Expression of the heparanase gene is associated with the invasive, angiogenic, and metastatic potential of diverse malignant tumors and cell lines. We used gene-silencing strategies to evaluate the role of heparanase in malignancy and to explore the therapeutic potential of its specific targeting.. We designed plasmid vectors to express hammerhead ribozymes or small interfering RNAs (siRNAs) directed against the human or mouse heparanase mRNAs. Human breast carcinoma (MDA-MB-435) and mouse lymphoma (Eb) and melanoma (B16-BL6) tumor cell lines, which have naturally high levels of endogenous heparanase or have been genetically engineered to overexpress heparanase, were transfected with anti-heparanase ribozyme or siRNA. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and measurements of enzymatic activity were used to confirm the efficient silencing of heparanase gene expression. Cells transfected with the anti-heparanase ribozyme and siRNA vectors were tested for invasiveness in vitro and metastatic dissemination in animal models of experimental and spontaneous metastasis.. Compared with cells transfected with control constructs, cells transfected with the anti-heparanase ribozyme or siRNA vectors had profoundly reduced invasion and adhesion in vitro, regardless of cell type, and expressed less heparanase. In vivo, tumors produced by cells transfected with the anti-heparanase ribozyme and siRNA vectors were less vascularized and less metastatic than tumors produced by cells transfected with the control vectors. Mice injected with cells transfected with the anti-heparanase ribozyme and siRNA vectors lived longer than mice injected with control cells.. The association of reduced levels of heparanase and altered tumorigenic properties in cells with anti-heparanase ribozyme- or siRNA-mediated gene-silencing vectors suggests that heparanase is important in cancer progression. Heparanase gene silencing has potential use as a target for anticancer drug development.

Topics: Angiogenesis Inducing Agents; Angiogenesis Inhibitors; Animals; Basement Membrane; Disease Progression; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Gene Silencing; Genetic Vectors; Glucuronidase; Growth Substances; Heparitin Sulfate; Humans; Immunohistochemistry; Lung Neoplasms; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Neoplasm Metastasis; Neovascularization, Pathologic; Plasmids; Reverse Transcriptase Polymerase Chain Reaction; RNA, Catalytic; RNA, Neoplasm; RNA, Small Interfering; Transfection

We expressed the full-length CD44v2-10 isoform in SKHep1 cells, a nonmetastatic human hepatocellular carcinoma cell line that does not express any endogenous CD44v isoforms. In SCID mice, expression of CD44v2-10 by SKHep1 cells had no effect on s.c. primary tumor development but caused pulmonary metastases in 41% (7 of 17) of animals compared with control SKHep1 cells (0 of 16; P < 0.01). CD44v2-10 expression by SKHep1 cells resulted in enhanced heparan sulfate (HS) attachment and an enhanced capacity to bind heparin-binding growth factors. Mutation of the v3 domain to prevent HS attachment and growth factor binding abolished the metastatic phenotype, demonstrating that HS modification of CD44v2-10 plays a critical role in the development of metastases in this model. However, in vitro proliferation, motility, and invasion were not altered by CD44v2-10 expression.

Topics: Alternative Splicing; Binding Sites; Carcinoma, Hepatocellular; Cell Adhesion; Cell Division; Cell Movement; Growth Substances; Heparitin Sulfate; Humans; Hyaluronan Receptors; Hyaluronic Acid; Liver Neoplasms; Lung Neoplasms; Neoplasm Metastasis; Protein Isoforms; Tumor Cells, Cultured

Heparin (H), heparan sulfate (HS), and related glycosaminoglycans can inhibit cancer cell invasion, possibly due to their ability to interact with vascular growth factors, adhesion molecules, endoglycosidases, and signaling proteins, in addition to the well-known effects on the clotting system. We evaluated the antitumor activity of a series of semisynthetic sulfaminoheparosan sulfates (SAHSs) with different degree and distribution of sulfates, obtained by chemical modifications of the E. coli K5 polysaccharide, namely type A, B, and C compounds. B16-BL6 melanoma cells (10 5 cells/mouse) were injected intravenously (i.v.) in a lateral tail vein of C57BL6 mice at a dose of 0.5 mg/ mouse together with test compounds. Tumor lung nodules were significantly reduced as compared with controls only by H (95.5 +/- 1.0% inhibition), SAHS-2 (84.2 +/- 5.0% inhibition), and SAHS-4 (91.1 +/- 4.2% inhibition), among compounds tested. SAHS-2 and SAHS-4 are type B compounds, with a sulfate/carboxylate ratio similar to that of H. A typical mammalian HS showed only 54.8% inhibition. Supersulfated low-molecular-weight heparin and heparan sulfate (ssLMWH and ssLMWHS) showed an activity similar to that of unfractionated compounds. H and SAHS-4 inhibited dose dependently B16-BL6 lung colonies, with IC-50 values of 0.05 and 0.1 mg/mouse, respectively. The relationship with ex vivo anticoagulant potency was evaluated by activated partial thromboplastin time (aPTT) on mouse plasma at different time intervals after i.v. injection (0.1 to 0.5 mg/mouse) of the compound. H showed a dose-dependent anticoagulant activity lasting up to 2 hours, whereas SAHS-4 showed a potent anticoagulant effect only at a dose of 0.5 mg/mouse. Accordingly, H but not SAHS-4 consistently inhibited B16-BL6 lung colonies when given 1 hour before tumor cells. SAHS-4 derivatives, with different size and/or affinity depleted of AT binding sites, showed an inhibitory effect on B16-BL6 melanoma similar to that of SAHS-4, suggesting that the greater antitumor effect of H was not due to AT-mediated inhibition of blood clotting. Interactions with other blood inhibitors, such as heparin cofactor II or tissue factor pathway inhibitory protein cannot be ruled out. The better effect of H may be due to persistence in the circulation and/or ability to inhibit tumor neoangiogenesis.

Topics: Animals; Anticoagulants; Antineoplastic Agents; Bacterial Capsules; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Heparin; Heparitin Sulfate; Humans; Lung Neoplasms; Melanoma, Experimental; Mice; Partial Thromboplastin Time; Polysaccharides, Bacterial; Structure-Activity Relationship; Sulfates

Exposure to AG73, a synthetic peptide (LQVQLSIR) from the COOH-terminal region of the laminin alpha1 chain, induces a malignant phenotype in B16F10 melanoma cells. Coinjection of this peptide with the cells results in an increase of lung tumors and also the formation of liver tumors in approximately 50% of the mice (W. H. Kim et al., Int. J. Cancer, 77: 632-639, 1998). Here we have characterized the cell surface receptor and its functional groups on B16F10 cells. Peptide affinity chromatography identified a cell surface protein eluting with 1 M NaCl, which ran in SDS gels as a broad band of M(r) approximately 150,000-200,000. Digestion with heparitinase and chondroitinase produced a core protein of lower molecular weight (M(r) approximately 90,000). Involvement of the glycosaminoglycan (GAG) side chains was demonstrated by inhibition of cell binding to the peptide by heparin, heparan sulfate, and chondroitin sulfate B, but not by chondroitin sulfates A or C, or hyaluronic acid. The IC(50) for heparin was the lowest, followed by heparan sulfate, then chondroitin sulfate B, suggesting that the overall sulfation of the GAG side chain is critical. This was confirmed by inhibition of attachment with chemically modified heparin and heparan sulfate, which also showed that N or O linkages were not important for function. Using sized heparin fragments to inhibit cell binding to the peptide demonstrated that 16-mer is the minimum length required. B16F10 cells form a network when grown on Matrigel, and this is prevented by addition of the AG73 peptide. The GAGs alone did not affect network formation, but heparin, heparan sulfate, and chondroitin sulfate B reversed the inhibitory effect of the peptide, whereas other GAGs were inactive. Furthermore, removal of cell surface GAGs inhibited cell attachment to the peptide. Cells treated with glycosidases and coinjected with the peptide formed liver tumors equal to the control group receiving no peptide, suggesting that the GAGs play an early role in peptide-mediated tumor metastasis. These data indicate that the B16F10 cell receptor for a laminin metastasis-promoting sequence is a heparan sulfate/chondroitin sulfate-containing proteoglycan, and these GAG side chains are functionally important in the cell-peptide interaction.

Topics: Animals; Binding Sites; Cell Adhesion; Chondroitin Sulfates; Glycosaminoglycans; Heparitin Sulfate; Laminin; Liver Neoplasms, Experimental; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Peptide Fragments; Proteoglycans; Receptors, Cell Surface

Heparan sulfate inhibits the proliferation of normal human lung fibroblasts (HFL-1) but not of a human lung carcinoma cell-line (A549). In this study we investigated possible mechanisms and structural requirements by which antiproliferative heparan sulfates exerts its effects on binding, uptake and subcellular localisation. Both HFL-1 and A549 cells were incubated with 125I- or rhodamine-labeled L-iduronate-rich antiproliferative heparan sulfate species as well as L-iduronate-poor inactive ones. The antiproliferative heparan sulfate was bound to the cell surface on both HFL-1 and A549 cells, but to a lesser extent and with less affinity to A549 cells. Both cell types bound the antiproliferative heparan sulfate with one high- and with one low affinity site. The L-iduronate-poor heparan sulfate bound to a lesser extent and with less affinity to both cell types compared to the antiproliferative heparan sulfate. The antiproliferative heparan sulfate accumulated in the cytoplasm of HFL-1 cells after 24 h incubation, but after 72 h it was found evenly distributed in the nucleus. The time-scale for antiproliferative activity correlated with nuclear localization. In contrast, in A549 cells it was only found near the nuclear membrane. The inactive heparan sulfate was taken up in considerably smaller amounts compared to the antiproliferative heparan sulfate and could not be detected in the nucleus of either HFL-1 or A549 cells. Our data suggest that the antiproliferative activity of L-iduronate-rich heparan sulfate on normal fibroblasts may be due to direct effects on nuclear processes, such as gene transcription.

Topics: Animals; Binding Sites; Cattle; Cell Nucleus; Cell Transformation, Neoplastic; Cells, Cultured; Fibroblasts; Growth Inhibitors; Heparitin Sulfate; Humans; Lung; Lung Neoplasms; Microscopy, Confocal; Radioligand Assay; Tumor Cells, Cultured

Heparan sulfate (HS) functions as a co-factor in several signal-transduction systems that affect cellular growth, differentiation, adhesion and motility. HS, therefore, may also play a role in the malignant transformation of cells, tumor growth, cell invasiveness and the formation of tumor metastases. To explore this hypothesis, we analyzed the expression of HS and heparan sulfate proteoglycan (HSPG) in histological sections of human lung-cancer tissues and assayed for the presence of HSPGs in extracts of human lung-cancer cell lines, using a panel of native HS-, delta-HS- and HSPG (syndecan, glypican, CD44 and perlecan) core protein-specific monoclonal antibodies. Compared to normal epithelia, non-small-cell lung carcinomas, particularly poorly differentiated tumors, often expressed reduced amounts of the major cell surface-associated HSPGs (most consistently of syndecan-1). CD44 or CD44-variant proteins, in contrast, were found on all tumor cells, irrespective of their differentiation. Perlecan, a matrix-associated HSPG found in the basement membrane of normal bronchial epithelium, was consistently undetectable in invasive bronchogenic carcinomas. Staining reactions for native HS were consistently reduced in squamous-cell lung carcinomas, in the cells in contact with the stroma and in the less differentiated areas of these tumors. Reactions for delta-HS, however, were not reduced, suggesting a structural change in the HS of these tumor cells. Poorly differentiated adenocarcinomas, in contrast, yielded strong HS and delta-HS reactions. Marked differences in HSPG expression also were observed among various non-small-cell lung carcinoma cell lines. Our results suggest that poorly differentiated lung tumors have markedly altered patterns of HSPG expression, which may contribute to their invasive phenotype.

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Hyaluronan Receptors; Lung Neoplasms; Lymphatic Metastasis; Mediastinum; Neoplasm Proteins; Proteoglycans; Tumor Cells, Cultured

Alkaline phosphatase, a marker of differentiation in the human alveolar adenocarcinoma cell line A549, is inducible by conditioned medium from lung fibroblasts and by cytokines including oncostatin M and interleukin 6, but only in the presence of a glucocorticoid, dexamethasone. Dexamethasone was shown to induce incorporation of [3H]glucosamine into three fractions of medium and cell trypsinate from subconfluent A549 cells, eluting from DEAE ion-exchange chromatography. The first peak did not correspond to any of the unlabelled glycosaminoglycans and was not characterized further. Induction was seen in two other peaks, corresponding to hyaluronic acid and heparan sulphate. Of these, heparan sulphate, eluting as one well-defined peak (referred to as HS1) and another of lower activity and less well defined (HS2), was selected as the most likely to interact with growth factors and cytokines and was isolated from the eluate, concentrated and desalted, and used in alkaline phosphatase induction experiments in place of dexamethasone. HS1 isolated from the medium (HS1m) of subconfluent A549 cells was shown to replace dexamethasone in induction experiments with fibroblast-conditioned medium, oncostatin M and interleukin 6. HS1 from the cell trypsinate and HS2 from the medium and trypsinate were inactive. As the activity of HS1m could be abolished by heparinase and heparitinase but not by chondroitinase ABC, it was concluded that HS1m was a fraction of heparan sulphate involved in the regulation of paracrine growth factor activity in lung fibroblast-conditioned medium, and in the regulation of other growth factors with potential roles in the paracrine control of cell differentiation.

Topics: Adenocarcinoma, Bronchiolo-Alveolar; Dexamethasone; Glucocorticoids; Glucosamine; Growth Substances; Heparitin Sulfate; Humans; Lung Neoplasms; Tumor Cells, Cultured

Structural features of heparin potentially important for heparanase-inhibitory activity were examined by measuring the ability of heparin derivatives to affect the degradation of [3H]acetylated heparan sulphate by tumor cell heparanases. IC50 values were determined using an assay which distinguished degraded from undegraded substrate by precipitation of the latter with cetylpyridinium chloride (CPC). Removal of heparin's 2-O-sulphate and 3-O-sulphate groups enhanced heparanase-inhibitory activity (50%). Removal of its carboxyl groups slightly lowered the activity (18%), while combining the treatments abolished the activity. At least one negative charge on the iduronic acid/idose moiety, therefore, is necessary for heparanase-inhibitory activity. Replacing heparin's N-sulphate groups with N-acetyl groups reduced its activity (37%). Comparing this heparin derivative with 2,3-O-desulphated heparin, the placement of sulphate groups appears important for activity since the two structures have similar nominal linear charge density. In addition, unsubstituted uronic acids are nonessential for inhibition since their modification (periodate-oxidation/borohydride-reduction) enhanced rather than reduced heparanase-inhibitory activity. The most effective heparanase inhibitors (2,3-O-desulphated heparin, and [periodate-oxidized, borohydride-reduced] heparin) were tested in the chick chorioallantoic membrane (CAM) bioassay for anti-angiogenic activity and found to be at least as efficacious as heparin. 2,3-O-desulphated heparin also significantly decreased the tumor growth of a subcutaneous human pancreatic (Ca-Pan-2) adenocarcinoma in nude mice and prolonged the survival times of C57BL/6N mice in a B16-F10 melanoma experimental lung metastasis assay.

Topics: Animals; Anticoagulants; Antineoplastic Agents; Chick Embryo; Chondroitin Sulfates; Enzyme Inhibitors; Female; Glucuronidase; Glycoside Hydrolases; Heparin; Heparitin Sulfate; Humans; Lung Neoplasms; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Nude; Molecular Structure; Neoplasm Metastasis; Neoplasms, Experimental; Neovascularization, Physiologic; Pancreatic Neoplasms

In patients with emphysema the integrity of the extracellular matrix (connective tissue skeleton) is compromised. In this study we analyzed glycosaminoglycans, which are main constituents of this matrix, in urines from patients with chronic obstructive pulmonary disease (COPD)/emphysema. Glycosaminoglycans (GAGs) were purified by anion exchange chromatography and quantified using the 1,9-dimethylmethylene blue assay. Heparan sulfate (HS) was assayed using three different chemical methods: digestion with heparitinase or with nitrous acid and by use of an adapted 1,9-dimethylmethylene blue assay. A specific epitope on the HS molecule, defined by the monoclonal antibody JM403, was determined using an inhibition enzyme immunoassay. In patients with COPD total urinary glycosaminoglycan and HS content were not altered. The JM403 epitope of HS, however, was greatly decreased in patients (0.6 versus 4.1 units/mg creatinine for control subjects, p < 0.0001). A similar pattern was observed when patients with bronchial carcinoma with and without emphysema were compared (0.4 versus 2.4 units/mg creatinine respectively, p < 0.0005). Patients with sarcoidosis did not show a decreased epitope content. These results indicate a structural change or an altered processing of the HS molecule in patients with emphysema. Taking into consideration the importance of HS for the stability of the alveolar extracellular matrix, this change may be associated with the pathogenesis of emphysema.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Bronchogenic; Case-Control Studies; Epitopes; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Lung Diseases, Obstructive; Lung Neoplasms; Male; Middle Aged; Pulmonary Emphysema; Sarcoidosis, Pulmonary

A Lewis lung carcinoma-derived low metastatic clone, P29, with a capacity to induce a fibrotic stromal response of host tissue, exhibits tumorigenesis depending on an interstitial matrix formed by the induced stromal cells. Using this clone, in the present study we isolated and characterized a membrane-intercalated proteoglycan that mediates interaction between the tumor cells and interstitial matrix. The tumor cells were cultured in the presence of [3H]glucosamine and [35S]sulfate or [35S]methionine, and hydrophobic proteoglycans were isolated by chromatography on DEAE-Sephacel and then Octyl-Sepharose CL-4B. Proteoglycans with high affinity to the octylresidue were obtained from the cell layer but not to any significant extent from the medium. By CsCl density gradient centrifugation, they were separated into bottom, middle, and top subfractions, which were shown to consist of homogeneous species with estimated M(r) values of 270,000 (named CPGIIIB), 200,000 (CPGIIIM), and 195,000 (CPGIIIT), respectively, by gel filtration on Sepharose CL-4B. These proteoglycans were intercalated into phosphatidylcholine liposomes, suggesting that they are all membrane-intercalated proteoglycans. Analyses of their glycosaminoglycans with chondroitinase ABC and heparitinase I plus II demonstrated that they all contain heparan sulfate as a major glycosaminoglycan (58-85%) and chondroitin 4-sulfate as a minor one (15-42%). Of these three proteoglycans, only CPGIIIB proteoglycan bound specifically to fibronectin-Sepharose 4B under physiological conditions. Molecular analyses of this proteoglycan by Sepharose CL-4B or SDS-PAGE before and after treatments with glycosaminoglycan degradation enzymes or trifluoromethanesulfonic acid demonstrated that CPGIIIB proteoglycan is a hybrid proteoglycan having heparan sulfate and chondroitin 4-sulfate chains on the same core protein with an M(r) of 40,000. Affinity chromatographies of the CPGIIIB proteoglycan on fibronectin-Sepharose 4B after treatments with these enzymes demonstrated that it bound to fibronectin via its heparan sulfate chains. On the basis of the above results, we propose that the CPGIIIB proteoglycan mediates the interaction between the tumor cells and interstitial matrix.

Topics: Animals; Carbohydrate Conformation; Carcinoma; Clone Cells; Collagen; Fibronectins; Heparin; Heparitin Sulfate; Immune Sera; Immunohistochemistry; Lung Neoplasms; Membrane Glycoproteins; Mice; Protein Binding; Proteoglycans; Stromal Cells; Tumor Cells, Cultured

Topics: Carcinoma, Squamous Cell; Dermatan Sulfate; Diabetes Mellitus, Type 1; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Hyaluronic Acid; Keratan Sulfate; Lung Neoplasms; Male; Middle Aged; Protein Binding

This study addresses the characterization of heparan sulphates of the basement-membrane proteoglycans in tumour formed after the subcutaneous implantation of Lewis-lung-carcinoma-derived different metastatic clones (P29, LM12-3 and LM60-D6 clones with low, medium and high metastatic potentials respectively). Heparan sulphate proteoglycans (125-158 micrograms of hexuronate/g dry weight of tissue) were isolated from chondroitin ABC lyase digests of a proteoglycan fraction obtained after DEAE-Sephacel chromatography of tissue extracts. The proteoglycans were separated into three molecular species by Sepharose CL-4B chromatography followed by CsCl-density-gradient centrifugation: large proteoglycans with an estimated M(r) of 820,000-130,000, which consisted of two components with low (< 1.34 g/ml; PGII-M) and high (> 1.37 g/ml; PGII-B) density, and a small proteoglycan with an M(r) of less than 80,000 (PGIII). Of these, only the PGII-M proteoglycan (34-37 micrograms of hexuronate/g dry weight) reacted with the antiserum against proteoglycan of Engelbreth-Holm-Swarm-tumour basement membrane, and represented, therefore, a basement-membrane proteoglycan. Digestion with heparan sulphate lyases I and II of the heparan sulphates (M(r) 36,000) from the PGII-M proteoglycan of the three tumours resulted in almost complete depolymerization to give six unsaturated disaccharides identified as 2-acetamido-2-deoxy-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyranosyluron ic acid)-D-glucose, 2-acetamido-2-deoxy-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyranosyluron ic acid)-6-O-sulpho-D-glucose, 2-deoxy-2-sulphamino-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyrano syluronic acid)-D-glucose, 2-deoxy-2-sulphamino-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyrano syluronic acid)-6-O-sulpho-D-glucose, 2-deoxy-2-sulphamino-4-O-(4-deoxy-2-O-sulpho-alpha-L-threo-hex-4- enopyranosyluronic acid)-D-glucose and 2-deoxy-2-sulphamino-4-O-(4-deoxy-2-O-sulpho-alpha-L-threo-hex-4- enopyranosyluronic acid)-6-O-sulpho-D-glucose. Comparison of the relative amounts of these disaccharides produced from the three tumour-derived heparan sulphates demonstrated that the degree of sulphation of the heparan sulphates correlated with the degree of morphological organization of the tumour basement membranes; the heparan sulphate from the more highly metastatic tumour with more highly organized basement membrane exhibited a higher degree of overall sulphation along the glycosaminoglycan chains, which was due to an increased cont

Topics: Animals; Basement Membrane; Carbohydrate Sequence; Centrifugation, Density Gradient; Chromatography, Gel; Chromatography, High Pressure Liquid; Disaccharides; Electrophoresis, Gel, Two-Dimensional; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Immunohistochemistry; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Neoplasm Metastasis; Proteoglycans; Tumor Cells, Cultured

We report a case of epithelioid haemangioendothelioma involving both lung and liver. The tumour cells were positive for factor-VIII-related antigen. Immunohistochemical analysis of various basement membrane components in tumour tissue of lung and liver showed striking differences. In the liver tumour there was selective expression of collagen IV, with minimal and focal amounts of laminin and basement membrane-associated heparan sulphate proteoglycan. In the lung tumour nodules, in contrast, all these basement membrane components were present. These patterns of basement membrane expression closely resemble those of normal liver and lung basement membrane respectively. We suggest that this provides evidence that epithelioid haemangioendothelioma arises from local endothelial cell proliferation and that it supports the assumption of a multicentric rather than metastatic origin when multiple tumour deposits are found.

Topics: Aged; Basement Membrane; Chondroitin Sulfate Proteoglycans; Collagen; Female; Hemangioendothelioma; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Immunohistochemistry; Liver Neoplasms; Lung Neoplasms; Membrane Glycoproteins; Neoplasms, Multiple Primary; von Willebrand Factor

Heparan sulfate (HS) enhanced the growth of the highly metastatic (HM) 3LL cell line, but not that of the low metastatic (LM) counterpart, in a dose-dependent way. Heparin, chondroitin sulfate, hyaluronate did not produce this effect. At 4 degrees C both cell lines exhibited high affinity binding sites (Kd 10(-8) M) for exogenous HS. Unlike LM cells, the HM ones lost half of the surface-bound HS during the 24-hour incubation at 37 degrees C. HM cells in the exponential growth phase took up the bound HS at lower rate than the LM counterparts. Both cell lines fragmented the exogenous HS intracellularly, but only the HM cells were able to degrade it at the cell surface. The HM cells contained much more heparin-binding proteins--especially in the cell membrane and nuclear fraction--than the LM ones. These results clearly demonstrate that the interactions of tumor cells with exogenous HS are influenced by the invasive phenotype. We suggest also that there could be a correlation between the surface degradation, the slow uptake and the growth-promoting effect of the exogenous HS.

Topics: Animals; Cattle; Cell Division; Extracellular Space; Glycosaminoglycans; Heparitin Sulfate; Lung Neoplasms; Mice; Neoplasm Metastasis; Phenotype; Protein Binding; Tumor Cells, Cultured

The heparan sulfate proteoglycan/heparin-binding proteins of the human lung carcinoma cell line LX-1 have been identified, partially purified, and characterized. Analysis of the binding of [3H]heparin to membranes isolated from LX-1 cells indicated the presence of two classes of binding sites, with Kd values of approximately 2 x 10(-10) and 4 x 10(-8) M and corresponding Bmax values of 1 x 10(5) and 2 x 10(7) binding sites/cell. Binding was also observed with isolated heparan sulfate chains and with intact heparan sulfate proteoglycan isolated from two different cell types. With each ligand, binding was inhibited by addition of unlabeled heparin. The binding proteins were extracted from LX-1 cell membranes in detergent solution, and two size classes of binding proteins were identified by overlaying transblots of electrophoretically separated proteins with radioactive ligands. These two classes of binding proteins were shown to contain doublets with estimated molecular masses of approximately 16 kDa (HSBP1A and HSBP1B) and approximately 32 kDa (HSBP2A and HSBP2B). The proteins were partially purified by heparin-Sepharose chromatography and shown to bind heparin and heparan sulfate proteoglycan. By amino acid composition, N-terminal amino acid sequence, and reactivity with antibody, HSBP1A was shown to be very similar to histone 2B; HSBP1B may also be related to histone 2A. HSBP2A and HSBP2B, however, did not react with antibodies to the major histones and had compositions different from one another and from HSBP1.

Topics: Amino Acid Sequence; Amino Acids; Blotting, Western; Carrier Proteins; Cell Membrane; Chondroitin Sulfate Proteoglycans; Collodion; Electrophoresis, Polyacrylamide Gel; Heparan Sulfate Proteoglycans; Heparin; Heparitin Sulfate; Humans; Lung Neoplasms; Molecular Sequence Data; Molecular Weight; Tumor Cells, Cultured

The effect of all-trans retinoic acid on metastatic B16 melanoma lung colonization and synthesis and properties of glycosaminoglycans was examined. Injection of tumour cells, pretreated with 10(-6) M-retinoic acid or grown to low density, into the tail vein of syngeneic C57 mice produced significantly fewer pulmonary tumours compared to subconfluent control cells. By cochromatography of glycosaminoglycans isolated from control ([14C]glucosamine-labelled) and 10(-6) M-retinoic acid-treated ([3H]glucosamine-labelled) cells on DEAE ion-exchange columns, differences in elution profiles were detected. Chondroitin sulphates isolated from retinoic acid-treated cells eluted at a lower salt concentration than those from control cells, while retinoic acid-treated cells synthesised heparan sulphates of a higher charge density than heparans from control cultures. These changes were apparent in both medium and trypsin-releasable fractions. Retinoic acid-treated cultures were seeded so that they were of a similar density to control cultures when harvested, as cell density was shown to affect glycosaminoglycan synthesis, the glycosaminoglycans from low-density cultures having similar properties to those isolated from retinoic acid-treated cultures. Retinoic acid treatment also reduced the overall synthesis of glycosaminoglycans while having little effect on the composition or distribution between medium, trypsin-releasable and cell-associated fractions. These observed changes in glycosaminoglycans may, in part, be responsible for retinoic acid-induced inhibition of lung colonization, and reduced adhesion to basement membrane components, which we have previously demonstrated.

Topics: Animals; Cell Adhesion; Chondroitin Sulfates; Glycosaminoglycans; Heparitin Sulfate; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Tretinoin

Differential cell adhesion has been proposed to play a role in organ-specific tumor metastasis. To further explore this hypothesis, we have employed a Lewis lung carcinoma cell line and 2 variants that differ in their ability to metastasize to lung and liver. The three cell lines were tested for their ability to adhere to defined extracellular matrix components that had been previously adsorbed to nylon membranes. Our results demonstrate that the parental cell line adheres preferentially to fibronectin relative to all other adhesion molecules tested. The lung colonizing variant, M27, adheres well to fibronectin and also to type V collagen but adheres poorly to laminin, to types I and VI collagen or to heparan sulfate. In contrast, the liver colonizing H59 cell line was highly adherent to laminin as well as to fibronectin but did not adhere to heparan sulfate or to any of the collagen types tested. These results demonstrate that three related cell lines with differing metastatic specificities have marked differences in their abilities to bind to defined matrix molecules. Such differences may play a role in the preferential localization to specific organ beds in vivo.

Topics: Animals; Carcinoma; Cell Adhesion; Collagen; Extracellular Matrix; Fibronectins; Heparitin Sulfate; Kinetics; Laminin; Liver Neoplasms; Lung Neoplasms; Mice; Neoplasm Metastasis

We studied the relationship between the metastatic potential of murine B16 melanoma cells and their surface expression of heparan sulfate glycosaminoglycan (HS-GAG) by using HepSS-1, a monoclonal antibody specific to HS-GAG. Firstly, among five B16 sublines, those capable of developing lung colonies with high efficiencies, such as B16-F10, had relatively low levels of surface HS-GAG. Secondly, a subline freshly prepared from metastatic lung colonies (F1) displayed a level of surface HS-GAG lower than that of injected B16 cells. Thirdly, in vitro selection of B16 cells with low surface HS-GAG by repeated HepSS-1 staining and cell-sorting resulted in cells with a higher metastatic efficiency than that of the original B16 cells. Collectively, B16 melanoma cells with high metastatic activities seem to have low surface HS-GAG. We also found that there was a positive correlation between the surface level of HS-GAG and the susceptibility to natural killer cells in eight B16 sublines.

Topics: Animals; Glycosaminoglycans; Heparitin Sulfate; Killer Cells, Natural; Lung Neoplasms; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Metastasis

The distribution of basement membrane (BM) markers, type IV collagen, laminin (LM), heparan sulphate proteoglycan (HSP) and fibronectin (FN) has been studied by indirect immunofluorescence using specific antibodies, in tumoural pathology. The disrupted pattern of BM by these markers in severe dysplastic lesions of the breasts, the bronchi and uterine cervix provides evidence for malignancy. In invasive carcinomas, there is generally a loss of these BM components, with FN persisting in the stroma. The loss of these markers in BM is concomitant and superimposable in double staining studies. In embryonic tumours, the presence of BM markers is related to a mesenchymal differentiation of malignant cells with pericellular FN and/or maturation towards organoid structures with BM. In sarcomas, there is a loss of the pericellular BM staining around most transformed muscular and Schwann cells and adipocytes. The persistence of this labelling in some well-differentiated areas can help to diagnose the nature of the sarcoma. The persistence of intercellular filaments of FN corresponds to the mesenchymal and/or sarcomatous nature of undifferentiated anaplastic proliferations.

Topics: Basement Membrane; Breast Neoplasms; Cell Transformation, Neoplastic; Collagen; Female; Fibronectins; Fluorescent Antibody Technique; Heparitin Sulfate; Humans; Laminin; Lung Neoplasms; Male; Neoplasms; Neoplasms, Germ Cell and Embryonal; Sarcoma

Distinction between benign and malignant pleural effusions is often a very difficult problem. Glycosaminoglycans play an important role in cellular proliferation and differentiation. The authors examined glycosaminoglycans in 17 samples, 11 malignant and 7 benign. Separation of glycosaminoglycans has been achieved by centrifugation-addition of trichloracetic acid-centrifugation-addition of cold ethanol. Glycosaminoglycans have been analyzed by electrophoresis on cellulose acetate sheets. 6 malignant samples had higher levels of HA and CS than standard. The authors distinguished benign effusions in two subgroups: inflammatory and cardiac. Among inflammatory, 3 had higher levels of HS and 1 of HA and CS. Further investigations are necessary to elucidate possible different patterns.

Topics: Aged; Electrophoresis, Cellulose Acetate; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Hyaluronic Acid; Lung Neoplasms; Male; Middle Aged; Pleural Effusion

Using a modified papain digestion cetylpyridinium salt precipitation method, glycosaminoglycans were isolated from 21 mesotheliomas, 34 primary lung carcinomas, 12 carcinomas of other sites, and 7 soft tissue sarcomas. Qualitatively, hyaluronic acid (HA) was present in 20 of 21 mesotheliomas, about half of the primary lung adenocarcinomas, and all of the soft tissue sarcomas. On the average, HA constituted 45% of the total glycosaminoglycans in the mesotheliomas and 28% of the total in the lung cancers. Quantitatively, mesotheliomas contained statistically greater amounts (mean value, 0.74 mg/g) of HA than primary lung adenocarcinomas (mean value, 0.08 mg/g), but were not statistically different from soft tissue sarcomas (mean value, 2.01 mg/g) or primary ovarian serous neoplasms (mean value, 0.92 mg/g). The study concludes that, contrary to previous reports, HA is neither the sole nor the predominant glycosaminoglycan in most mesotheliomas, but, given the proper clinical and histologic setting, the finding of sufficiently high levels (greater than 0.4 mg/g dry tissue extract) supports the diagnosis of mesothelioma when the alternative diagnosis is primary adenocarcinoma of lung.

Topics: Adenocarcinoma; Carcinoma; Chondroitin Sulfates; Dermatan Sulfate; Diagnosis, Differential; Electrophoresis, Cellulose Acetate; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Hyaluronic Acid; Lung Neoplasms; Mesothelioma; Ovarian Neoplasms; Sarcoma

Heparan sulfate (HS), a prominent component of vascular endothelial basal lamina, is cleaved into large Mr fragments and solubilized from subendothelial basal lamina-like matrix by metastatic murine B16 melanoma cells. We have examined the degradation products of HS and other purified glycosaminoglycans produced by B16 cells. Glycosaminoglycans 3H-labeled at their reducing termini or metabolically labeled with [35S]sulfate were incubated with B16 cell extracts in the absence or presence of D-saccharic acid 1,4-lactone, a potent exo-beta-glucuronidase inhibitor, and glycosaminoglycan fragments were analyzed by high speed gel permeation chromatography. HS isolated from bovine lung, Engelbreth-Holm-Swarm sarcoma, and subendothelial matrix were degraded into fragments of characteristic Mr, in contrast to hyaluronic acid, chondroitin 6-sulfate, chondroitin 4-sulfate, dermatan sulfate, keratan sulfate, and heparin which were essentially undegraded. Heparin, but not other glycosaminoglycans, inhibited HS degradation. The time dependence of HS degradation into particular Mr fragments indicated that HS was cleaved at specific intrachain sites. In order to determine specific HS cleavage points, HS prereduced with NaBH4 was incubated with a B16 cell extract and HS fragments were separated. The newly formed reducing termini of HS fragments were then reduced with NaB[3H]4, and the fragments hydrolyzed to monosaccharides by trifluoroacetic acid treatment and nitrous acid deamination. Since 3H-reduced terminal monosaccharides from HS fragments were overwhelmingly (greater than 90%) L-gulonic acid, the HS-degrading enzyme responsible is an endoglucuronidase (heparanase).

Topics: Animals; Cell Line; Glucuronidase; Glycosaminoglycans; Glycoside Hydrolases; Heparitin Sulfate; Kinetics; Lung Neoplasms; Melanoma; Mice; Substrate Specificity

The quantitative changes of glycosaminoglycans in tumor tissue of human lung cancers (2 squamous cell carcinomas, 4 adenocarcinomas and 5 small cell carcinomas) were studied. The total amount of glycosaminoglycans in human lung cancer tissues increased 1.4 to 4 times in comparison with that in normal lung tissues. The increase in tissue content of glycosaminoglycans was accompanied by an increase in the chondroitin sulfate level in every histologic type of lung cancer, as well as by a marked increase in hyaluronic acid level in squamous cell carcinomas, and a moderate increase in its level in small cell carcinomas. The concentrations of dermatan sulfate and heparan sulfate in lung cancer tissues did not show any significant changes compared with those in normal lung tissues. The increase in total amount and changes in the composition of glycosaminoglycans in human lung cancer tissue were closely related to the histologic type of the tumor.

Topics: Adenocarcinoma; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Chondroitin Sulfates; Dermatan Sulfate; Electrophoresis, Cellulose Acetate; Glycosaminoglycans; Heparitin Sulfate; Humans; Hyaluronic Acid; Lung Neoplasms

The glycosaminoglycans were prepared by exhaustive Pronase digestion and alkaline treatment of squamous cell carcinoma and adenocarcinoma tissues of human lung, and of tissues taken at a site distant from the tumor as a control. The glycosaminoglycan classes were characterized by chemical enzymic, and electrophoretic methods. The presence of oversulfated chondroitin-and/or dermatan-sulfates which have not up till now been found in lung tissues was also demonstrated in carcinoma and control tissues, their contents being higher in the carcinoma tissues. The levels of whole glycosaminoglycans were markedly increased in carcinoma tissue. The classes of glycosaminoglycans which increased in lung carcinoma tissue were predominatly chondroitin-4-and/or-6-sulfates and hyauronic acid.

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; Chondroitin Sulfates; Glycopeptides; Glycosaminoglycans; Heparitin Sulfate; Hexosamines; Humans; Hyaluronic Acid; Lung; Lung Neoplasms; Sulfates