heparitin-sulfate and Colorectal-Neoplasms

Heparan sulfate (HS) is a highly sulfated natural carbohydrate that plays crucial roles in cancer, inflammation, and angiogenesis. Heparanase (HPSE) is the sole HS degrading endoglycosidase that cleaves HS at structure-dependent sites along the polysaccharide chain. Overexpression of HPSE by cancer cells correlates with increased tumor size and enhanced metastasis. Previously we have shown that a tetramer HS mimetic is a potent HPSE inhibitor displaying remarkable anticancer activity in vivo. Building on that work, we report the synthesis and testing of a novel library of single entity trimer glycolipid mimetics that effectively inhibit HPSE at low nanomolar concentrations. A lipophilic arm was introduced to assess whether an improvement of pharmacokinetics and plasma residence time would offset the reduction in charge and multivalency. Preclinical tests in a mouse syngeneic model showed effective tumor growth inhibition by the tetramer but not the trimer glycomimetic.

Topics: Animals; Colorectal Neoplasms; Glycolipids; Heparitin Sulfate; Mice; Neovascularization, Pathologic

Cancer-related cognitive impairment (CRCI) has been frequently reported in colorectal cancer survivors. Heparan sulfate (HS) was gradually considered to be related to cognitive disorders. The effect and potential mechanism of HS on CRCI in colorectal cancer patients were unexplored. In this study, all participants were divided into a cognitive impaired group and a cognitive normal group. The concentrations of oxidative stress factors and HS in serum were detected. Associations among HS, oxidative stress factors and CRCI were evaluated. Participants with cognitive impairment exhibited increased levels of HS, GSH, SOD and MDA, compared to the patients with normal cognitive performance. The independent significant association was found between HS and CRCI after controlling for various covariates. The higher concentrations of HS were related to the decreased cognitive performance among survivors who reported higher levels of GSH (β = 0.080, p = 0.002). Moreover, the nonlinear association between the level of HS and cognitive scores was confirmed using the restricted cubic splines (p < 0.001). These results indicated that the increased concentrations of circulating HS had a nonlinear negative connection with cognitive performance in colorectal cancer survivors, which was moderated by GSH. HS might be a new biomolecule for the identification and management of patients with CRCI.

Topics: Cancer Survivors; Cognitive Dysfunction; Colorectal Neoplasms; Heparitin Sulfate; Humans; Oxidative Stress

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

Coxsackievirus B3 (CVB3), a single-stranded RNA virus of the picornavirus family, has been described as a novel oncolytic virus. However, the CVB3 strain used induced hepatitis and myocarditis in vivo. It was hypothesized that oncolytic activity and safety of CVB3 depends on the virus strain and its specific receptor tropism. Different laboratory strains of CVB3 (Nancy, 31-1-93, and H3), which use the coxsackievirus and adenovirus receptor (CAR), and the strain PD, which uses N- and 6-O-sulfated heparan sulfate (HS) for entry into the cells, were investigated for their potential to lyse tumor cells and for their safety profile. The investigations were carried out in colorectal carcinoma. In vitro investigations showed variable infection efficiency and lysis of colorectal carcinoma cell lines by the CVB3 strains. The most efficient strain was PD, which was the only one that could lyse all investigated colorectal carcinoma cell lines. Lytic activity of CAR-dependent CVB3 did not correlate with CAR expression on cells, whereas there was a clear correlation between lytic activity of PD and its ability to bind to HS at the cell surface of colorectal carcinoma cells. Intratumoral injection of Nancy, 31-1-93, or PD into subcutaneous colorectal DLD1 cell tumors in BALB/c nude mice resulted in strong inhibition of tumor growth. The effect was seen in the injected tumor, as well as in a non-injected, contralateral tumor. However, all animals treated with 31-1-93 and Nancy developed systemic infection and died or were moribund and sacrificed within 8 days post virus injection. In contrast, five of the six animals treated with PD showed no signs of a systemic viral infection, and PD was not detected in any organ. The data demonstrate the potential of PD as a new oncolytic virus and HS-binding of PD as a key feature of oncolytic activity and improved safety.

Topics: Animals; CD55 Antigens; Cell Line, Tumor; Colorectal Neoplasms; Cytotoxicity, Immunologic; Enterovirus B, Human; Heparitin Sulfate; Humans; Mice, Inbred BALB C; Mice, Nude; Oncolytic Viruses; Organ Specificity; Receptors, Virus; Viral Load; Virulence

To explore possible roles of heparanase in cancer-host crosstalk, we examined whether heparanase influences expression of inflammatory chemokines in colorectal cancer cells. Murine colorectal carcinoma cells incubated with heparanase upregulated MCP-1, KC, and RANTES genes and released MCP-1 and KC proteins. Heparanase-dependent production of IL-8 was detected in two human colorectal carcinoma cell lines. Addition of a heparanase inhibitor Heparastatin (SF4) did not influence MCP-1 production, while both latent and mature forms of heparanase augmented MCP-1 release, suggesting that heparanase catalytic activity was dispensable for MCP-1 production. In contrast, addition of heparin to the medium suppressed MCP-1 release in a dose-dependent manner. Similarly, targeted suppression of Ext1 by RNAi significantly suppressed cell surface expression of heparan sulfate and MCP-1 production in colon 26 cells. Taken together, it is concluded that colon 26 cells transduce the heparanase-mediated signal through heparan sulfate binding. We propose a novel function for heparanase independent of its endoglycosidase activity, namely as a stimulant for chemokine production.

Topics: Catalysis; Cell Line, Tumor; Chemokines; Colorectal Neoplasms; Enzyme Activation; Glucuronidase; Heparitin Sulfate; Humans; Inflammasomes

Heparan sulfate (HS) plays a role in the majority of essential hallmarks of cancer, yet its ability to modulate self-renewal, especially of cancer stem cells (CSCs), remains unknown. We have discovered that a non-anticoagulant HS hexasaccharide (HS06) sequence, but not other shorter or longer sequences, selectively inhibited CSC self-renewal and induced apoptosis in colorectal, pancreatic, and breast CSCs suggesting a very general phenomenon. HS06 inhibition of CSCs relied upon early and sustained activation of p38α/β mitogen activated protein kinase (MAPK) but not other MAPKs family members i.e. ERK and JNK. In contrast, polymeric HS induced exactly opposite changes in MAPK activation and failed to inhibit CSCs. In fact, TCF4 signaling, a critical regulator of CSC self-renewal, was inhibited by HS06 in a p38 activation dependent fashion. In conclusion, HS06 selectively inhibits CSCs self-renewal by causing isoform specific activation of p38MAPK to inhibit TCF4 signaling. These observations on chain length-induced specificity carry major mechanistic implications with regard to HS in cancer biology, while also presenting a novel paradigm for developing novel anti-CSC hexasaccharides that prevent cancer relapse.Heparan sulfate (HS) of specific length, i.e., hexasaccharide (HS06), but not longer or shorter sequences, selectively inhibit cancer stem cells (CSCs) through isoform specific activation of p38 mitogen-activated protein kinase. These findings will have major implication for developing chemical probes to decipher complex signaling events that govern cancer stem cells. Additionally, there are direct implications for designing glycosaminoglycan based cancer therapies to selectively target CSCs that escape killing by traditional chemotherapy threatening cancer relapse.

Topics: Breast Neoplasms; Cell Self Renewal; Colorectal Neoplasms; Drug Discovery; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; Heparitin Sulfate; Humans; Neoplastic Stem Cells; p38 Mitogen-Activated Protein Kinases; Pancreatic Neoplasms; Signal Transduction; Transcription Factor 4; Tumor Cells, Cultured

Heparan sulfate endosulfatase-1 and -2 (SULF1 and SULF2) are two important extracellular 6-O-endosulfatases that remove 6-O sulfate groups of N-glucosamine along heparan sulfate (HS) proteoglycan chains often found in the extracellular matrix. The HS sulfation pattern influences signaling events at the cell surface, which are critical for interactions with growth factors and their receptors. SULFs are overexpressed in several types of human tumors, but their role in cancer is still unclear because their molecular mechanism has not been fully explored and understood. To further investigate the functions of these sulfatases in tumorigenesis, stable overexpression models of these genes were generated in the colorectal cancer cells, Caco-2 and HCT-116. Importantly, mimicking overexpression of these sulfatases resulted in increased viability and proliferation, and augmented cell migration. These effects were reverted by shRNA-mediated knockdown of SULF1 or SULF2 and by the addition of unfractionated heparin. Detailed structural analysis of HS from cells overexpressing SULFs showed reduction in the trisulfated disaccharide UA(2S)-GlcNS(6S) and corresponding increase in UA(2S)-GlcNS disaccharide, as well as an unexpected rise in less common disaccharides containing GlcNAc(6S) residues. Moreover, cancer cells transfected with SULFs demonstrated increased Wnt signaling. In summary, SULF1 or SULF2 overexpression contributes to colorectal cancer cell proliferation, migration, and invasion.. This study reveals that sulfatases have oncogenic effects in colon cancer cells, suggesting an important role for these enzymes in cancer progression.

Topics: Caco-2 Cells; Cell Movement; Cell Proliferation; Cell Survival; Colorectal Neoplasms; HCT116 Cells; Heparitin Sulfate; Humans; Sulfatases; Sulfotransferases; Wnt Signaling Pathway

Exostoses like-3 (EXTL3) is a putative tumour suppressor gene but its involvement in colorectal cancer (CRC) is unclear. We have investigated the role of methylation of the EXTL3 promoter as a mechanism for EXTL3 regulation and tested the hypothesis that loss of EXTL3 expression is associated with mucinous differentiation and alteration of glycoprotein expression in CRC cells. The methylation status of the EXTL3 gene promoter was analysed by methylation-specific PCR following bisulphite modification in CRC cell lines and microdissected primary CRC tissues and their corresponding adjacent normal colorectal mucosa. EXTL3 promoter methylation was detected in seven of 11 mucinous CRCs (63.6%) but in none of 26 non-mucinous CRCs examined. EXTL3 promoter methylation was also detected in the normal colonic mucosa of three patients with mucinous CRC but not in the normal colonic mucosa of any patients with non-mucinous CRC. The presence of EXTL3 methylation was significantly associated with the partial loss of HS expression in mucinous CRC lesions. The mucinous CRC cell lines, Colo201 and Colo205, showed EXTL3 promoter methylation and loss of EXTL3 mRNA expression. However 5-aza-2'-deoxycytidine treatment demethylated the EXTL3 gene promoter and restored its mRNA expression. Furthermore, the basal expression of HS in CRC cells was abolished by treatment with EXTL3-siRNA. We conclude that EXTL3 promoter methylation and its related loss of EXTL3 expression are involved in the loss of HS expression in mucinous CRCs.

Topics: Adenocarcinoma, Mucinous; Aged; Biomarkers, Tumor; Colorectal Neoplasms; DNA Methylation; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Heparitin Sulfate; Humans; Male; Middle Aged; N-Acetylglucosaminyltransferases; Polymerase Chain Reaction; Promoter Regions, Genetic; Tumor Suppressor Proteins

Angiogenesis is crucial for the progression of colorectal carcinomas in which the bioavailability of Vascular Endothelial Growth Factor (VEGF) plays a major role. VEGF bioavailability is regulated by proteolytic release or cleavage. In colorectal cancer patients, we observed a significant correlation between circulating VEGF and tumour tissue Matrix Metalloproteinase-9 (MMP-9) levels but not with MMP-2. Therefore, we evaluated the role of MMP-9 in regulating VEGF bioavailability and subsequent angiogenesis in 3-dimensional human cell culture models. MMP-9 treatment released VEGF dose-dependently from HT29 colon carcinoma spheroids, comparable to heparitinase, a known mediator of VEGF release. Conditioned medium from human neutrophils, containing high amounts of active MMP-9, released VEGF comparable to recombinant MMP-9, in contrast to myofibroblast medium. MMP-9 treated spheroids showed decreased extracellular levels of heparan sulphates, required for VEGF binding to the matrix, whereas the levels in the medium were increased. Western blot analysis revealed that VEGF(165) is the major isoform released by MMP-9 treatment. In vitro experiments indicated that MMP-9 is not capable to cleave VEGF(165) into smaller isoforms, like plasmin does. These data suggested that MMP-9 mediates release rather than the cleavage of larger VEGF isoforms. Medium from MMP-9 treated HT29 spheroids induced endothelial cell sprouting in an angiogenesis assay, comparable to the effect of recombinant VEGF(165). Anti-VEGF antibody treatment resulted in a strongly reduced number of sprouts. In conclusion, we have shown that neutrophil-derived MMP-9 is able to release biologically active VEGF(165) from the ECM of colon cancer cells by the cleavage of heparan sulphates.

Topics: Angiogenesis Inhibitors; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Bevacizumab; Cell Hypoxia; Colorectal Neoplasms; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix; Heparitin Sulfate; HT29 Cells; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neovascularization, Pathologic; Neutrophils; Spheroids, Cellular; Vascular Endothelial Growth Factor A

Recently, we reported that the laminin alpha5 synthetic peptide A5G27 (RLVSYNGIIFFLK, residues 2,892-2,904) binds to the CD44 receptor of B16-F10 melanoma cells via the glycosaminoglycans on CD44 and inhibits tumor cell migration, invasion, and angiogenesis in a dominant-negative manner. Here, we have identified the potential mechanism of A5G27 activity using WiDr human colorectal carcinoma cells. WiDr cells bound to the laminin A5G27 peptide via the heparin-like and chondroitin sulfate B glycosaminoglycan side chains of CD44. Cell binding to fibroblast growth factor (FGF2) was blocked by laminin peptide A5G27 but not by either a scrambled version of this peptide or by another laminin peptide known to bind cell surface proteoglycans. FGF2 signaling involving tyrosine phosphorylation was also blocked by laminin peptide A5G27 but was not affected by peptide controls. Finally, we have shown that peptide A5G27 directly blocks FGF2 binding to heparin. Peptide A5G27 has sequence homology to a region on FGF2 that binds heparin and the FGF receptor and is important in FGF2 central cavity formation. We conclude that peptide A5G27 inhibits metastasis and angiogenesis by blocking FGF2 binding to the heparan sulfate side chains of CD44 variant 3, thus decreasing FGF2 bioactivity.

Topics: Amino Acid Sequence; Antibodies; Antibody Specificity; Cell Line, Tumor; Colorectal Neoplasms; Fibroblast Growth Factor 2; Flow Cytometry; Glycoproteins; Heparitin Sulfate; Humans; Hyaluronan Receptors; Laminin; Molecular Sequence Data; Neoplasm Metastasis; Neovascularization, Pathologic; Peptide Fragments; Phosphorylation; Protein Binding

Heparanase (HPSE-1) is involved in the degradation of both cell-surface and extracellular matrix (ECM) heparan sulfate (HS) in normal and neoplastic tissues. Degradation of heparan sulfate proteoglycans (HSPG) in mammalian cells is dependent upon the enzymatic activity of HPSE-1, an endo-beta-d-glucuronidase, which cleaves HS using a specific endoglycosidic hydrolysis rather than an eliminase type of action. Elevated HPSE-1 levels are associated with metastatic cancers, directly implicating HPSE-1 in tumor progression. The mechanism of HPSE-1 action to promote tumor progression may involve multiple substrates because HS is present on both cell-surface and ECM proteoglycans. However, the specific targets of HPSE-1 action are not known. Of particular interest is the relationship between HPSE-1 and HSPG, known for their involvement in tumor progression. Syndecan-1, an HSPG, is ubiquitously expressed at the cell surface, and its role in cancer progression may depend upon its degradation. Conversely, another HSPG, perlecan, is an important component of basement membranes and ECM, which can promote invasive behavior. Down-regulation of perlecan expression suppresses the invasive behavior of neoplastic cells in vitro and inhibits tumor growth and angiogenesis in vivo. In this work we demonstrate the following. 1) HPSE-1 cleaves HS present on the cell surface of metastatic melanoma cells. 2) HPSE-1 specifically degrades HS chains of purified syndecan-1 or perlecan HS. 3) Syndecan-1 does not directly inhibit HPSE-1 enzymatic activity. 4) The presence of exogenous syndecan-1 inhibits HPSE-1-mediated invasive behavior of melanoma cells by in vitro chemoinvasion assays. 5) Inhibition of HPSE-1-induced invasion requires syndecan-1 HS chains. These results demonstrate that cell-surface syndecan-1 and ECM perlecan are degradative targets of HPSE-1, and syndecan-1 regulates HPSE-1 biological activity. This suggest that expression of syndecan-1 on the melanoma cell surface and its degradation by HPSE-1 are important determinants in the control of tumor cell invasion and metastasis.

Topics: Adenocarcinoma; Animals; Cell Membrane; Colorectal Neoplasms; Extracellular Matrix; Glucuronidase; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Hydrogen-Ion Concentration; Melanoma; Membrane Glycoproteins; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Proteoglycans; Recombinant Proteins; Substrate Specificity; Syndecan-1; Syndecans; Tumor Cells, Cultured

Patients with malignancy often present with a variety of coagulation abnormalities which may ultimately lead to recurrent arterial and venous thromboses. Recently the presence of antiphospholipid antibodies in cancer patients has been proposed as one of the potential mechanisms promoting hypercoagulability. Here we report two consecutive patients with localized tumors, one suffering from breast cancer and another presenting with colorectal cancer, who experienced dramatic exacerbation of the antiphospholipid antibody syndrome (APAS) within 4 weeks after surgery. In the first patient who had also received one course of adjuvant chemotherapy, major ischemic stroke and recurrent venous thromboembolism were paralleled by the development of ulcerative livedoid vasculitis and pancytopenia, constituting the diagnosis of systemic lupus erythematosus with secondary APAS. In the second patient, progressive thrombotic occlusion of the superior and inferior vena cava was associated with bilateral pulmonary embolism, acute renal failure, and disabling soft tissue edema. Although not fulfilling the classic criteria of "catastrophic" APAS, the clinical features were life threatening and appeared to be refractory to oral anticoagulation with phenprocoumon. In addition, a diagnosis of Trousseau's syndrome was unlikely due to missing evidence of gross metastatic disease. Besides a suggested treatment strategy comprising high doses of low-molecular-weight heparin, potential pathogenic mechanisms are discussed in consideration of a recently proposed "thrombotic storm," which may cause multiple thromboses after an initial provocation in patients with known hypercoagulability.

Topics: Adult; Antiphospholipid Syndrome; Breast Neoplasms; Chemotherapy, Adjuvant; Chondroitin Sulfates; Colorectal Neoplasms; Dermatan Sulfate; Drug Combinations; Female; Heparin, Low-Molecular-Weight; Heparitin Sulfate; Humans; Lupus Erythematosus, Systemic; Middle Aged; Neoplasms; Stroke; Thromboembolism; Thrombophilia

CD44 variant exon (CD44v) 3 is a heparan sulfate-binding isoform of CD44. The role of CD44v3 in invasion and metastasis associated with heparan sulfate in colon cancer cell lines and cases of colon cancer was examined. Expression of CD44v3 mRNA and protein was observed in five of six human colorectal cancer cell lines. Colo320 and WiDr cells expressed CD44v3 at high levels. Heparan sulfate treatment increased the invasive activity of Colo320 and WiDr cells to rates 14.3 and 12.6 times higher, respectively, than that of untreated cells. However, heparan sulfate treatment did not affect cell growth. Repression of CD44v3 protein production by antisense S-oligodeoxynucleotide treatment reduced the binding affinities and capacities for heparan sulfate by Colo320 and WiDr cells in comparison with that of control cells, and it also reduced the invasiveness of both cell lines to one-fifth that of control cells. In heparan sulfate-treated Colo320 cells, the levels of CD44v3 protein in the Triton X-100-insoluble fraction and moesin-precipitated fraction were increased, suggesting that heparan sulfate treatment facilitates association of CD44 molecules with the cytoskeleton. Immunohistochemical analysis showed CD44v3 to be expressed in 21 of 37 (57%) colorectal cancer cases. Positive CD44v3 expression was associated with more advanced pathological stage and poorer prognosis than negative CD44v3 expression. These data support a role for CD44v3 in invasion and metastasis by colorectal carcinoma cells.

Topics: Antigens, CD; Base Sequence; Cell Division; Colonic Neoplasms; Colorectal Neoplasms; Cytoskeleton; Exons; Gene Expression Regulation, Neoplastic; Genetic Variation; Heparitin Sulfate; Humans; Hyaluronan Receptors; Kinetics; Neoplasm Invasiveness; Oligodeoxyribonucleotides, Antisense; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Rate; Thionucleotides; Time Factors; Tumor Cells, Cultured

Biosynthesis of glycosaminoglycans (GAGs) was studied in morphologically normal colonic mucosa, in peritumoral and tumoral areas, and in colorectal polyps of tumor-bearing patients. After GAG purification, overall biosynthesis was determined: the general trend was a decrease in GAG production in neoplastic colon, lowest GAG synthesis being observed in Dukes' stage C tumors. Separation by ion-exchange chromatography of various GAG species and further characterization revealed the presence of hyaluronic acid (HA) and heparan sulfate (HS) molecules in all specimens studied. Chondroitin-4 sulfate (CS4) was occasionally found in tumor samples. The relative proportion of HA and HS was modified in tumor tissue: i.e. increased HA and decreased HS were observed. Differences in DEAE-chromatographic behavior were obvious in pathological samples as compared to controls, the hydrodynamic form of HA and the charge density of HS being decreased. The latter could be attributed to undersulfatation of HS molecules. Immunocytochemical detection of HS proteoglycan molecules revealed regular and bright labelling at epithelial-stromal interface in control samples. In pathological samples, staining was patchy and discontinuous, showing large areas of basement membrane interruption.

Topics: Adenocarcinoma; Chondroitin Sulfate Proteoglycans; Colon; Colonic Polyps; Colorectal Neoplasms; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Immunohistochemistry; Intestinal Mucosa; Rectum