chondroitin-sulfates and Breast-Neoplasms

Although immune checkpoint blockade (ICB) holds potential for the treatment of various tumors, a considerable proportion of patients show a limited response to ICB therapy due to the low immunogenicity of a variety of tumors. It has been shown that some chemotherapeutics can turn low-immunogenic tumors into immunogenic phenotypes by inducing a cascade of immune responses. In this paper, we synthesized an injectable micelle-incorporated hydrogel, which was able to sequentially release the chemotherapeutic gemcitabine (GEM) and the hydrophobic indoleamine 2, 3-dioxygenase inhibitor, d-1-methyltryptophan (d-1MT) at tumor sites. The hydrogel was formed via the thiol-ene click reaction between the thiolated chondroitin sulfate and the micelle formed by amphiphilic methacrylated Pluronic F127, in which hydrophobic d-1MT was encapsulated in the core of the F127 micelles and the hydrophilic GEM was dispersed in the hydrogel network. The successive release of chemotherapeutics and immune checkpoint inhibitors at tumor tissues will first promote the infiltration of cytotoxic T lymphocytes and subsequently induce a robust antitumor immune response, ultimately exerting a synergetic therapeutic efficacy. In a 4T1 tumor-bearing mice model, our results showed that the combination of chemotherapy and immunotherapy through the micelle-incorporated hydrogel triggered an effective antitumor immune response and inhibited tumor metastasis to the lung. Our results highlight the potential of the injectable micelle-incorporated hydrogel for the localized chemo-immunotherapy in the treatment of breast tumors.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Chondroitin Sulfates; Delayed-Action Preparations; Deoxycytidine; Enzyme Inhibitors; Female; Gemcitabine; Hydrogels; Immunotherapy; Indoleamine-Pyrrole 2,3,-Dioxygenase; Mice, Inbred BALB C; Micelles; Neoplasm Metastasis; Poloxamer; Tryptophan; Tumor Microenvironment

As a major therapeutic approach for cancer treatment, the effectiveness of chemotherapy is challenged by multidrug resistance (MDR). Herein, we fabricated novel redox-responsive, chondroitin sulfate-based nanoparticles that could simultaneously deliver quercetin (chemosensitizer), chlorin e6 (photosensitizer) and paclitaxel (chemotherapeutic agent) to exert enhanced chemo-photodynamic therapy for overcoming MDR and lung metastasis of breast cancer. In vitro cell study showed that nanoparticles down-regulated the expression of P-glycolprotein (P-gp) on MCF-7/ADR cells and thereby improved the anticancer efficacy of PTX against MCF-7/ADR cells. Moreover, NIR laser irradiation could induce nanoparticles to generate cellular reactive oxygen species (ROS), leading to mitochondrial membrane potential loss, and meanwhile facilitating lysosomal escape of drugs. Importantly, the novel nanoplatform exhibited effective in vivo MDR inhibition and anti-metastasis efficacy through enhanced chemo-photodynamic therapy. Thus, the study suggested that the multifunctional nanoplatform had good application prospect for effective breast cancer therapy.

Topics: Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Breast Neoplasms; Cell Proliferation; Chlorophyllides; Chondroitin Sulfates; Combined Modality Therapy; Doxorubicin; Drug Carriers; Drug Resistance, Neoplasm; Female; Gene Expression; Humans; Infrared Rays; Lasers; Lung Neoplasms; MCF-7 Cells; Nanoparticles; Paclitaxel; Photosensitizing Agents; Porphyrins; Quercetin; Reactive Oxygen Species; Xenograft Model Antitumor Assays

Multidrug resistance (MDR) remains the primary issue leading to the failure of chemotherapy. In this study, a d-α-tocopherol polyethylene 1000 glycol succinate (TPGS) and chondroitin sulfate (CS) dual-modified lipid-albumin nanosystem was constructed for targeted delivery of paclitaxel (PTX) in treating MDR cancer. The obtained nanosystem (TLA/PTX@CS) had an average size of around 176 nm and a negative zeta potential of around -18 mV. TPGS was confirmed to improve the intracellular accumulation of PTX and facilitate the mitochondrial-targeting of lipid-albumin nanosystem. Functionalized with the outer CS shell, TLA/PTX@CS entered MDR breast cancer (MCF-7/MDR) cells via CD44 receptor-mediated endocytosis. CS shell was degraded by concentrated hyaluronidase in the lysosomes, thereby releasing PTX into cytoplasm and inhibiting cell proliferation. In vivo studies revealed that TLA/PTX@CS possessed prolonged blood circulation, resulting in elevated tumor accumulation, excellent antitumor efficacy with a tumor inhibition ratio of 75.3%, and significant survival benefit in MCF-7/MDR tumor-bearing mice. Hence, this TPGS and CS dual-modified lipid-albumin nanosystem provides a promising strategy for targeted delivery of chemotherapeutic drug and reversal of MDR in cancer treatment.

Topics: Albumins; Animals; Breast Neoplasms; Cell Proliferation; Cell Survival; Chondroitin Sulfates; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Female; Humans; Hyaluronan Receptors; Lipids; MCF-7 Cells; Mice; Nanoparticles; Paclitaxel; Vitamin E; Xenograft Model Antitumor Assays

Chondroitin sulfate A-deoxycholic acid-polyethylene glycol-maleimide (CSA-DOCA-PEG-MAL; CDPM) nanostructures were designed for the transient binding of MAL with thiol in blood components and cell membranes, in addition to the CD44 receptor targeting, for the therapy of breast cancer. The spontaneous binding of free thiol groups in plasma proteins and blood cells with the MAL group of CDPM was significantly higher than that of CSA-DOCA-PEG (CDP). Enhanced cellular uptake and the in vitro antiproliferation efficacy of docetaxel (D)-loaded CDPM (CDPM/D) nanoparticles (NPs) in MCF-7 cells indicated dual-targeting effects based on MAL-thiol reactions and CSA-CD44 receptor interactions. Following intravenous injection in rats, reduced clearance and an elevated half-life of the drug was observed in the CDPM/D NPs compared to the CDP/D NPs. Taken together, MAL modification of CDP NPs could be a promising approach not only to enhance tumor targeting and penetration but also to extend the blood circulation time of anticancer drugs.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Chondroitin Sulfates; Drug Carriers; Drug Liberation; Female; Humans; Hyaluronan Receptors; Maleimides; MCF-7 Cells; Nanoparticles; Particle Size; Polyethylene Glycols; Rats

Nanogel based drug delivery systems have been broadly used for cancer treatment. In this research, octadecylamine was grafted to chondroitin sulfate using three different mole ratios (10, 20, and 30) and named CS-ODA1, 2, and 3, respectively. The amide bond formation between chondroitin sulfate and octadecylamine was confirmed by 1H-nuclear magnetic resonance (HNMR) in the CS-ODA3 sample; therefore, further analysis was performed on this sample. Curcumin was loaded at defined Cur/CS-ODA ratios (5, 10 and 15%) and CS-ODA3 with 10% curcumin was selected for further experiments due to more entrapment efficiency (79.56% ± 5.56). In vitro release profile of the curcumin loaded nanogels showed >80% release after 70 h. In addition, the results of MTT analysis on the MCF-7 cell line showed almost no toxicity toward blank nanogels, while curcumin-loaded nanogels induced significant death after 24 h. In the end, analysis of the cell cycle using MCF-7 cells also confirmed the cytotoxicity of curcumin loaded nanogels. This study also showed that the presence of curcumin loaded chondroitin sulfate nanogels could successfully increase cellular uptake in comparison with free curcumin. The synthesized nanogels containing curcumin are expected to be effective for further studies in cancer treatment.

Topics: Breast Neoplasms; Chondroitin Sulfates; Curcumin; Drug Delivery Systems; Female; Humans; MCF-7 Cells; Micelles; Nanogels

Topics: beta Catenin; Breast Neoplasms; Cadherins; Cell Line, Tumor; Cell Movement; Chondroitin Sulfates; Extracellular Matrix; Female; Glycosaminoglycans; Humans; Matrix Metalloproteinase 9; Signal Transduction; Sulfotransferases

Hydrophobically-modified polymers based on chondroitin sulfate with different degree of substitution (DS) of deoxycholic acid (DOCA) were developed for docetaxel delivery. Chondroitin sulfate-deoxycholic acid (CSAD) bioconjugates were synthesized via the linker of adipic dihydrazide by amide bond. They were characterized with spherical shape, mean diameter of around 165.2nm and negative zeta potential (-14.87 to -20.53mV). An increase of DOCA DS reduced size of nanoparticles, while increasing drug loading efficiency. Drug release in vitro showed a triphasic sustained pattern and higher accumulative drug release percentage was observed with increased DS of DOCA on polymer. Self-assemblies with higher DS also had enhanced internalization of nanoparticles and stronger cytotoxicity at the cellular level. The self-assemble nanoparticles demonstrate to be excellent targeting drug delivery systems and the desired therapeutics can be achieved via the alteration of DS.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Proliferation; Chondroitin Sulfates; Deoxycholic Acid; Docetaxel; Drug Carriers; Drug Delivery Systems; Drug Liberation; Female; Humans; Hydrophobic and Hydrophilic Interactions; Nanoparticles; Polymers; Taxoids; Tumor Cells, Cultured

There are lines of evidence demonstrating that NEDD9 (Cas-L, HEF-1) plays a key role in the development, progression, and metastasis of breast cancer cells. We previously reported that NEDD9 plays a critical role for promoting migration and growth of MDA-MB-231. In order to further characterize the mechanisms of NEDD9-mediated cancer migration and growth, stable cells overexpressing NEDD9 were generated using HCC38 as a parental cell line which expresses low level of endogenous NEDD9. Microarray studies demonstrated that core proteins of CD44 and Serglycin were markedly upregulated in HCC38(NEDD9) cells compared to HCC38(Vector) cells, while those of Syndecan-1, Syndecan-2, and Versican were downregulated in HCC38(NEDD9). Importantly, enzymes generating chondroitin sulfate glycosaminoglycans (CS) such as CHST11, CHST15, and CSGALNACT1 were upregulated in HCC38(NEDD9) compared to HCC38(Vector). Immunofluorescence studies using specific antibody, GD3G7, confirmed the enhanced expression of CS-E subunit in HCC38(NEDD9). Immunoprecipitation and western blotting analysis demonstrated that CS-E was attached to CD44 core protein. We demonstrated that removing CS by chondroitinase ABC significantly inhibited anchorage-independent colony formation of HCC38(NEDD9) in methylcellulose. Importantly, the fact that GD3G7 significantly inhibited colony formation of HCC38(NEDD9) cells suggests that CS-E subunit plays a key role in this process. Furthermore, treatment of HCC38(NEDD9) cells with chondroitinase ABC or GD3G7 significantly inhibited mammosphere formation. Exogenous addition of CS-E enhanced colony formation and mammosphere formation of HCC38 parental and HCC38(Vector) cells. These results suggest that NEDD9 regulates the synthesis and expression of tumor associated glycocalyx structures including CS-E, which plays a key role in promoting and regulating breast cancer progression and metastasis and possibly stem cell phenotypes.

Topics: Adaptor Proteins, Signal Transducing; Antibodies, Monoclonal; Antigens; Breast Neoplasms; Cell Movement; Cell Proliferation; Chondroitin ABC Lyase; Chondroitin Sulfates; Down-Regulation; Female; Fluorescent Antibody Technique; Humans; Hyaluronan Receptors; Membrane Glycoproteins; N-Acetylgalactosaminyltransferases; Neoplasm Metastasis; Phosphoproteins; Proteoglycans; Spheroids, Cellular; Sulfotransferases; Syndecan-1; Syndecan-2; Tumor Cells, Cultured; Up-Regulation; Versicans; Vesicular Transport Proteins

The chemokine CCL2 (MCP-1) has been identified as a prominent tumor-promoting factor in breast cancer. The major source for CCL2 is in the tumor cells; thus, identifying the mechanisms regulating CCL2 release by these cells may enable the future design of modalities inhibiting CCL2 secretion and consequently reduce tumorigenicity. Using cells deficient in expression of glycosaminoglycans (GAGs) and short hairpin RNAs reducing heparan sulfate (HS) and chondroitin sulfate (CS) expression, we found that intracellular HS and CS (=GAGs) partly controlled the trafficking of CCL2 from the Golgi toward secretion. Next, we determined the secretion levels of GFP-CCL2-WT and GFP-CCL2-variants mutated in GAG-binding domains and/or in the 40s loop of CCL2 ((45)TIVA(48)). We have identified partial roles for R18+K19, H66, and the (45)TIVA(48) motif in regulating CCL2 secretion. We have also demonstrated that in the absence of R24 or R18+K19+(45)TIVA(48), the secretion of CCL2 by breast tumor cells was almost abolished. Analyses of the intracellular localization of GFP-CCL2-mutants in the Golgi or the endoplasmic reticulum revealed particular intracellular processes in which these CCL2 sequences controlled its intracellular trafficking and secretion. The R24, (45)TIVA(48) and R18+K19+(45)TIVA(48) domains controlled CCL2 secretion also in other cell types. We propose that targeting these chemokine regions may lead to reduced secretion of CCL2 by breast cancer cells (and potentially also by other malignant cells). Such a modality may limit tumor growth and metastasis, presumably without affecting general immune activities (as discussed below).

Topics: Amino Acid Motifs; Amino Acid Sequence; Animals; Breast Neoplasms; Cell Line; Cell Line, Tumor; Chemokine CCL2; Chondroitin Sulfates; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Intracellular Space; Mutation; Protein Binding; Protein Interaction Domains and Motifs; Protein Transport; Recombinant Fusion Proteins

The expression of a specific set of genes controls the different structures of heparan sulfate proteoglycans (HSPGs), which are involved in the growth, invasion and metastatic properties of cancerous cells. The purpose of this study is to increase knowledge of HSPG alterations in breast cancer.. Twenty-three infiltrating ductal adenocarcinomas (IDCs), both metastatic and non-metastatic were studied. A transcriptomic approach to the structure of heparan sulfate (HS) chains was used, employing qPCR to analyze both the expression of the enzymes involved in their biosynthesis and editing, as well as the proteoglycan core proteins. Since some of these proteoglycans can also carry chondroitin sulfate chains, we extended the study to include the genes involved in the biosynthesis of these glycosaminoglycans. Histochemical techniques were also used to analyze tissular expression of particular genes showing significant expression differences, of potential interest.. No significant change in transcription was detected in approximately 70% of analyzed genes. However, 13 demonstrated changes in both tumor types (40% showing more intense deregulation in the metastatic), while 5 genes showed changes only in non-metastatic tumors. Changes were related to 3 core proteins: overexpression of syndecan-1 and underexpression of glypican-3 and perlecan. HS synthesis was affected by lower levels of some 3-O-sulfotransferase transcripts, the expression of NDST4 and, only in non metastatic tumors, higher levels of extracellular sulfatases. Furthermore, the expression of chondroitin sulfate also was considerably affected, involving both the synthesis of the saccharidic chains and sulfations at all locations. However, the pro-metastatic enzyme heparanase did not exhibit significant changes in mRNA expression, although in metastatic tumors it appeared related to increased levels of the most stable form of mRNA. Finally, the expression of heparanase 2, which displays anti-metastatic features, experienced a strong deregulation in all patients analyzed.. IDCs show alterations in the expression of HSPG genes; principally the expression and localization of proteoglycans and the sulfation patterns of glycosaminoglycan chains, depending on the metastatic nature of the tumor. In addition, the anti-proliferative molecule heparanase 2 experiences strong deregulation, thus highlighting it as a potentially interesting diagnostic factor.

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Chondroitin Sulfates; Female; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glucuronidase; Glypicans; Heparan Sulfate Proteoglycans; Humans; Immunohistochemistry; Membrane Proteins; Polymerase Chain Reaction; RNA, Messenger; Sulfotransferases; Syndecan-1

The present investigation was aimed to develop and explore the use of chondroitin sulfate-coupled liposomes (CS-LP) for solid tumor targeting. The liposomes were prepared by cast film method and coupled with chondroitin sulfate. The coupling was confirmed by infrared spectroscopy. They were further characterized for various parameters such as vesicle shape and surface morphology, size and size distribution, zeta potential, entrapment efficiency, and in vitro release pattern. The vesicle size of the uncoupled liposome (256 nm) was found to be less than that of CS-LP (310 nm). In vitro drug release exhibited a release of 44.2% from uncoupled liposomal formulation, compared to 38.3% as observed in coupled formulation at the end of 24 hr. The uptake of the CS-LP and uncoupled liposomes by MDA-MB-231 breast cancer cell lines was visualized using fluorescence microscopy that revealed the dependence of liposomes recognition and higher uptake on the coupling of chondroitin sulfate. Coupling of the liposomes significantly enhanced the tumor uptake of drug, which is reflected in the recovery of a higher percentage of the dose from tumor following administration of CS-LP in comparison to uncoupled liposomes or free drug, suggesting that they can be used as vectors for solid tumor targeting.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Chondroitin Sulfates; Drug Carriers; Drug Delivery Systems; Etoposide; Female; Humans; Liposomes; Mice; Mice, Inbred BALB C; Neoplasms, Experimental; Tissue Distribution

We have previously demonstrated that chondroitin sulfate glycosaminoglycans (CS-GAGs) on breast cancer cells function as P-selectin ligands. This study was performed to identify the carrier proteoglycan (PG) and the sulfotransferase gene involved in synthesis of the surface P-selectin-reactive CS-GAGs in human breast cancer cells with high metastatic capacity, as well as to determine a direct role for CS-GAGs in metastatic spread.. Quantitative real-time PCR (qRT-PCR) and flow cytometry assays were used to detect the expression of genes involved in the sulfation and presentation of chondroitin in several human breast cancer cell lines. Transient transfection of the human breast cancer cell line MDA-MB-231 with the siRNAs for carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) and chondroitin sulfate proteoglycan 4 (CSPG4 ) was used to investigate the involvement of these genes in expression of surface P-selectin ligands. The expression of CSPG4 and CHST11 in 15 primary invasive breast cancer clinical specimens was assessed by qRT-PCR. The role of CS-GAGs in metastasis was tested using the 4T1 murine mammary cell line (10 mice per group).. The CHST11 gene was highly expressed in aggressive breast cancer cells but significantly less so in less aggressive breast cancer cell lines. A positive correlation was observed between the expression levels of CHST11 and P-selectin binding to cells (P < 0.0001). Blocking the expression of CHST11 with siRNA inhibited CS-A expression and P-selectin binding to MDA-MB-231 cells. The carrier proteoglycan CSPG4 was highly expressed on the aggressive breast cancer cell lines and contributed to the P-selectin binding and CS-A expression. In addition, CSPG4 and CHST11 were over-expressed in tumor-containing clinical tissue specimens compared with normal tissues. Enzymatic removal of tumor-cell surface CS-GAGs significantly inhibited lung colonization of the 4T1 murine mammary cell line (P = 0.0002).. Cell surface P-selectin binding depends on CHST11 gene expression. CSPG4 serves as a P-selectin ligand through its CS chain and participates in P-selectin binding to the highly metastatic breast cancer cells. Removal of CS-GAGs greatly reduces metastatic lung colonization by 4T1 cells. The data strongly indicate that CS-GAGs and their biosynthetic pathways are promising targets for the development of anti-metastatic therapies.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Chondroitin Sulfate Proteoglycans; Chondroitin Sulfates; Female; Gene Expression Regulation, Neoplastic; Humans; Ligands; Membrane Proteins; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; P-Selectin; Proteoglycans; RNA Interference; RNA, Small Interfering; Sulfotransferases

Experimental studies have established that the sulfated glycosaminoglycans heparan sulfate and chondroitin sulfate act as co-receptors of cytokines and growth factors that drive the malignant cell phenotype and the remodelling of the surrounding tumor stroma. However, the clinical relevance of these studies remains ill-defined. The present study investigates the significance of chondroitin sulfate expression in malignant cells and the stroma, respectively, of tumors from two independent cohorts of breast cancer patients (cohort I: 144 patients, 130 evaluable samples; cohort II: 498 patients, 469 evaluable samples; ER-positive patients ~86% in both cohorts). Kaplan-Meier analysis and Cox proportional hazards modelling were used to assess the relationship between chondroitin sulfate and recurrence-free and overall survival. High chondroitin sulfate expression in malignant cells was shown to predict shorter recurrence-free survival (P=0.007, cohort I; P=0.024, cohort II) and overall survival (cohort I: P=0.044; cohort II: P<0.001) in both cohorts. In multivariate analysis, high chondroitin sulfate in malignant cells was shown to be an independent, predictive factor of poor overall survival (cohort I: hazard ratio 2.28: 95% confidence interval 1.08-4.81, P=0.031; cohort II: hazard ratio 1.71: 95% confidence interval 1.23-2.38, P=0.001). However, chondroitin sulfate in the stroma showed no correlation with known markers of tumor aggressiveness or with clinical outcome in either cohort. Our data suggest that high chondroitin sulfate expression in malignant cells is associated with an adverse outcome in patients with primary breast cancer, supporting the idea of a functional and potentially targetable role of chondroitin sulfate in tumor disease.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Cell Line, Tumor; Chondroitin Sulfates; Disease Progression; Female; Humans; Kaplan-Meier Estimate; Middle Aged; Neoplasm Staging; Phenotype; Prognosis

Topics: Adult; Breast Neoplasms; Chondroitin Sulfates; Chromatography, High Pressure Liquid; Female; Humans; Lactation; Milk, Human; Neoplasm Invasiveness

The present study investigates prospective of tailored nanoparticles as vectors to provide improved therapeutic efficacy of encapsulated anti-cancer drug 5-FU. Condritin Sulphate (CS) conjugated PLGA nanoparticles were prepared using PEG-bis-amine and adipic dihydrazide as spacers and loaded with an anti-cancer drug 5-FU (CS-PEG-PLGA-FU and CS-ADH-PLGA-FU). The formulations were then compared with non CS-anchored monomethoxy(polyethylene glycol) (MPEG-PLGA-FU) nanoparticles. Nanoparticlulate systems were further characterized by FTIR, NMR, TEM studies and particle size/polydispersity index (PDI) analysis. DSC and XRD were also performed to assess the nature of 5-FU inside the nanoparticles. The nanoparticles prepared using amphiphilic block copolymer CS-PEG-PLGA were able to sustain the release of 5-FU up to 48 h whereas those of CS-ADH-PLGA and MPEG-PLGA released the drug up to 24 h. The CS-PEG-PLGA-FU nanoparticles were found to be least haemolytic when compared to free drug, CS-ADH-PLGA-FU and MPEG-PLGA-FU nanoparticles. Cytotoxicity studies were performed on MCF-7/MDA-MD 231 breast cancer cells. PLGA nanoparticles exhibited more potent cytotoxic effect on MCF-7/MDA-MD 231 cells than free doxorubicin. Further, enhanced cytotoxicity and lower hemolytic potential of CS-PEG-PLGA-FU nanoparticles suggest a potential application in cancer chemotherapy.

Topics: Antimetabolites, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Chondroitin Sulfates; Drug Carriers; Female; Fluorouracil; Humans; Lactic Acid; Molecular Structure; Nanoparticles; Particle Size; Polyethylene Glycols; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; X-Ray Diffraction

We present the case of a 58-year-old lady who developed radionecrosis following irradiation of the chest wall following mastectomy. The ensuing radionecrosis of the soft tissue and bony rib cage on the left side of the chest wall progressed to advanced ischaemia with secondary infection and abscess formation. Initially the patient underwent left-sided chest wall reconstruction using a right-sided pedicled TRAM flap. The flap eventually became ischaemic and necrosed over a period of 3 weeks leaving the pleura and ribs exposed. The patient underwent VAC Therapy in order to promote the formation of granulation tissue. Several weeks later the greater Omentum was harvested as a pedicled flap and transposed into the defect which was then in turn covered with Integra and a split-skin graft. Complete wound closure was obtained with this Integra-omental flap reconstructive technique.

Topics: Breast Neoplasms; Chondroitin Sulfates; Collagen; Female; Humans; Mastectomy; Middle Aged; Necrosis; Omentum; Radiodermatitis; Skin Ulcer; Surgical Flaps; Thoracic Wall; Tissue and Organ Harvesting; Treatment Outcome; Wound Healing

The metastatic breast cancer cell line, 4T1, abundantly expresses the oligosaccharide sialylated Lewis x (sLe(x)). SLe(x) oligosaccharide on tumor cells can be recognized by E- and P-selectin, contributing to tumor metastatic process. We observed that both selectins reacted with this cell line. However, contrary to the E-selectin reactivity, which was sLe(x) dependent, P-selectin reactivity with this cell line was sLe(x)-independent. The sLe(x)-Neg variant of the 4T1 cell line with markedly diminished expression of sLe(x) and lack of sLe(a), provided a unique opportunity to characterize P-selectin ligands and their contribution to metastasis in the absence of overlapping selectin ligands and E-selectin binding. We observed that P-selectin binding was Ca(2+)-independent and sulfation-dependent. We found that P-selectin reacted primarily with cell surface chondroitin sulfate (CS) proteoglycans, which were abundantly and stably expressed on the surface of the 4T1 cell line. P-selectin binding to the 4T1 cells was inhibited by heparin and CS glycosaminoglycans (GAGs). Moreover, Heparin administration significantly inhibited experimental lung metastasis. In addition, the data suggest that surface CS GAG chains were involved in P-selectin mediated adhesion of the 4T1 cells to murine platelets and human umbilical vein endothelial cells. The data suggest that CS GAGs are also the major P-selectin-reactive ligands on the surface of human MDA-MET cells. The results warrant conducting clinical studies on the involvement of cell surface CS chains in breast cancer metastasis and evaluation of various CS types and their biosynthetic pathways as target for development of treatment strategies for antimetastatic therapy of this disease.

Topics: Animals; Breast Neoplasms; Calcium; Cell Line, Tumor; Cell Membrane; Chondroitin Sulfates; Fucosyltransferases; Glycosaminoglycans; Heparin; Humans; Ligands; Lung Neoplasms; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Oligosaccharides; P-Selectin; Proteoglycans; Sialyl Lewis X Antigen; Transfection

The purpose is to determine whether the levels of expression of extracellular matrix components in peritumoral stroma are predictive of disease outcome for women with node-negative breast cancer.. Tumor tissue from 86 patients with node-negative breast cancer was examined by immunohistochemical staining for the expression of versican, chondroitin sulfate (CS), tenascin, and hyaluronan (HA). With the exception of HA, the expression of the extracellular matrix components was measured by video image analysis. Statistical correlation of the immunohistochemical data with clinicopathological characteristics and disease outcome was performed.. All of the extracellular matrix components were present in the peritumoral stroma of the entire study cohort. In contrast, immunoreactivity within the cancer cell was observed in 82% of tumors for HA, 12% for CS, and 4% for tenascin; no immunostaining of cancer cells for versican was observed for any of the tumors. Cox regression and Kaplan-Meier analyses indicated that elevated expression of stromal versican predicted increased risk and rate of relapse in this cohort. Elevated expression of tenascin was predictive of increased risk and rate of death only. Although neither CS nor HA were predictive of disease outcome in this cohort, tumor size was predictive of increased risk and rate of both relapse and survival.. Elevated expression within peritumoral stromal matrix of versican and tenascin was predictive of relapse-free and overall survival, respectively, in women with node-negative breast cancer.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Chondroitin Sulfate Proteoglycans; Chondroitin Sulfates; Cohort Studies; Disease-Free Survival; Extracellular Matrix; Female; Humans; Hyaluronic Acid; Immunohistochemistry; Lectins, C-Type; Lymph Nodes; Middle Aged; Regression Analysis; Risk; Tenascin; Time Factors; Treatment Outcome; Versicans

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

The soluble form of the syndecan-1 heparan sulfate proteoglycan acts as a tumor suppressor molecule that inhibits growth and induces apoptosis of some cancer cell lines in vitro. Analogs of syndecan-1 were produced by carbodiimide (EDAC) conjugation of glycosaminoglycan (GAG) chains to a protein scaffold, thereby generating synthetic proteoglycans that were evaluated for anticancer properties. Surprisingly, when analyzing activities of the controls, we discovered that EDAC modified GAG chains inhibit myeloma cell viability even in the absence of protein. Here, we describe the production and the activities of these novel molecules called neoglycans. The GAG chains heparin and chondroitin sulfate (CS) were exposed to EDAC to generate the neoglycans neoheparin and neoCS, respectively. Heparin and CS in the absence of EDAC modification have no effect or a slight growth promoting effect on cancer and normal cell lines. However, neoheparin and neoCS substantially reduce cell viability by induction of apoptosis of myeloma and breast cancer cells in vitro. NeoCS when injected directly into breast tumors growing in nude mice reduces or abolishes their growth without causing apparent toxicity to the adjacent normal tissue. The neoglycans need not be continuously present in cell cultures because a short pulse exposure is sufficient to reduce cell viability. NeoCS fractions purified by size exclusion chromatography reduce myeloma cell viability, confirming the specificity of neoglycan activity. Collectively, the results of this study demonstrate the anticancer activities of this new class of GAG chain-based molecules and provide the foundation for future development of neoglycans as novel therapeutic agents.

Topics: 3T3 Cells; Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Carbodiimides; Cell Division; Cell Survival; CHO Cells; Chondroitin Sulfates; Cricetinae; Dogs; Drug Screening Assays, Antitumor; Female; Glycosaminoglycans; Heparin; Humans; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Multiple Myeloma; Proteoglycans; Syndecan-1; Syndecans; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The origin of myofibroblasts in stromal reaction has been a subject of controversy. To address this question definitively, we developed techniques for purification and characterization of major stromal cell types. We defined a panel of markers that could, in combination, unequivocally distinguish these cell types by immunocytochemistry, iso-electric focusing, immunoblotting, and two-dimensional gel electrophoresis. We then devised an assay to recapitulate in culture, within two weeks of incubation, critical aspects of the microenvironment in vivo including the typical tissue histology and stromal reaction. When confronted with tumor cells in this assay, fibroblasts readily converted into a graded pattern of myogenic differentiation, strongest in the immediate vicinity of tumor cells. Vascular smooth muscle cells (VSMC), in contrast, did not change appreciably and remained coordinately smooth muscle differentiated. Midcapillary pericytes showed only a slight propensity for myogenic differentiation. Analysis of ten primary tumors implicated converted fibroblasts (10/10), vascular smooth muscle cells (4/10), and pericytes (1/10) in the stromal reaction. Tumor cells were shown to specifically denude the venules both in culture and in vivo, explaining the VSMC phenotype in the stroma. The establishment of this assay and clarification of the origin of these cells pave the way for further analysis of the mechanisms of conversion, and of the consequence of such heterogeneity for diagnosis and treatment.

Topics: Biomarkers; Blood Vessels; Breast; Breast Neoplasms; Cell Separation; Chondroitin Sulfates; Cytoskeletal Proteins; Epithelium; Factor VIII; Fibroblasts; HLA-DQ Antigens; Humans; Immunoblotting; Immunoenzyme Techniques; Immunohistochemistry; Integrins; Keratins; Organoids; Tumor Cells, Cultured

The histological and histochemical findings and the clinical course of 160 cases of breast cancer were compared with the biochemical values of the content and pattern of glycosaminoglycans (GAG) in the tumour tissue. For this purpose the cases were divided into 4 groups of increasing malignancy of the tumours, based on the morphological data and the clinical course after radical mastectomy (4-9 years follow-up). Samples of tumour tissue were examined biochemically-analytically for their total content of glycosaminoglycans and for their mg/g values of hyaluronic acid (H), chondroitin-4- and chondroitin-6-sulphates (CA + CC), dermatan sulphate (CB), heparan sulphate (HpS) and component (K). The statistical processing by means of the method of "discriminant analysis" of the corresponding GAG values in relation to the degree of malignancy displayed by the tumours yielded the following results: 1) the glycosaminoglycan patterns of the mammary carcinoma have characteristic features in common for the large majority of cases; 2) an exception of about 5% of the cases developed carcinomata with a very high (up to 100%) content of component. These cases generally have a more favourable clinical course (group 1); 3) the higher the ratio of hyaluronic acid to chondroitin sulphates the better the prognosis (groups 1 and 2, respectively). A ratio of hyaluronic acid to chondroitin sulphates under 0.5 indicates a poor prognosis (group 3 and 4, respectively); 4) high values of heparan sulphate and small ratios of hyaluronic acid to heparan sulphate also signal an unfavourable prognosis (groups 3 or 4). We consider that the above results are a means of defining the degree of malignancy of the breast tumours.

Topics: Breast Neoplasms; Chondroitin Sulfates; Discriminant Analysis; Female; Follow-Up Studies; Glycosaminoglycans; Heparitin Sulfate; Humans; Hyaluronic Acid; Mastectomy, Radical; Prognosis; Survival Analysis

We report a case of a secretory carcinoma of the breast in a 72-year-old woman with a long history of a left breast mass. Rapid growth and skin invasion of the tumor was noticed three months before a radical mastectomy was performed and a lung metastasis was found one month after the operation. A metastasis to the axillary nodes was noted in 6 of 9 resected specimens and a receptor assay of the estrogen and progesterone proved to be negative. This patient is the oldest case of a secretory carcinoma of the breast reported in Japan.

Topics: Adenocarcinoma; Aged; Breast Neoplasms; Chondroitin Sulfates; Female; Humans; Hyaluronic Acid; Lung Neoplasms; Lymphatic Metastasis; Mastectomy, Radical; Neoplasm Invasiveness; Skin

The proteoglycans secreted by a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal human breast cell line (HBL-100). The physicochemical characteristics of these proteoglycans were established by hexosamine analysis, chemical and enzymatic degradations, and dissociative cesium chloride density gradient centrifugation, and by gel filtration before and after alkaline beta-elimination. Both cell lines secreted approximately 70% of the synthesized proteoglycans, which were composed of 20% heparan sulfate and 80% chondroitin sulfate proteoglycans. The MDA cell line secreted large hydrodynamic size (major) and small hydrodynamic size heparan sulfate proteoglycan. In contrast HBL cells secreted only one species having a hydrodynamic size intermediate to the above two. The chondroitin sulfate proteoglycans from MDA medium were slightly larger than the corresponding polymers from HBL medium. All proteoglycans except the small hydrodynamic size heparan sulfate proteoglycan from MDA medium were of high buoyant density. The proteoglycans of both cell lines contained significant proportions of disulfide-linked lower molecular weight components which were more pronounced in the proteoheparan sulfate polymers, particularly those from MDA medium, than in chondroitin sulfate proteoglycans. The glycosaminoglycans of heparan sulfate proteoglycans from MDA medium were more heterogeneous than those from HBL medium. The glycosaminoglycan chains of large hydrodynamic size heparan sulfate proteoglycans from MDA medium were larger in size than those from HBL medium while small hydrodynamic size heparan sulfate proteoglycans contained shorter glycosaminoglycan chains. In contrast to the glycosaminoglycans derived from chondroitin sulfate proteoglycans of both MDA and HBL medium were comparable in size. The heparan sulfate as well as chondroitin sulfate proteoglycans of both cell lines contained both neutral (di- and tetrasaccharides) and sialylated (tri- to hexasaccharides) O-linked oligosaccharides.

Topics: Adenocarcinoma; Breast; Breast Neoplasms; Carbohydrate Conformation; Cell Line; Centrifugation, Density Gradient; Chondroitin Sulfates; Chromatography, Gel; Disulfides; Epithelium; Female; Galactosamine; Glucosamine; Glycosaminoglycans; Heparitin Sulfate; Humans; Molecular Weight; Oligosaccharides; Proteoglycans

Glycosaminoglycans have been characterized from a normal human breast cell line (HBL-100) and two different cell lines from human breast carcinoma (MDA-MB-231 and MCF-7). The glycosaminoglycans were labeled by exposure of cell cultures to [3H]glucosamine and [35S]sulfate and then isolated from both spent media and cells by pronase digestion and cetylpyridinium chloride fractionation. They were further characterized by (a) hexosamine composition, (b) controlled-pore glass exclusion chromatography, (c) reactivity with specific enzymes (hyaluronidase chondroitinase, heparitinase, and heparinase), (d) nitrous acid degradation, and (e) DEAD-Sephadex chromatography. The results indicate that the HBL-100 line synthesizes mainly hyaluronic acid, most of which is secreted into the medium. Chondroitin sulfate and heparan sulfate are the predominant glycosaminoglycans synthesized by the cancer lines; both are found mainly in the spent medium, but the hyaluronic acid synthesized by the MDA-MB-231 line remains cell associated. The cell-associated heparan sulfate had a molecular weight in excess of 13,000 and may contain linkages susceptible to testicular hyaluronidase. The MCF-7 cells produce significantly lower amounts of glycosaminoglycans than do the other two lines.

Topics: Breast; Breast Neoplasms; Cell Line; Chondroitin Sulfates; Glycosaminoglycans; Heparitin Sulfate; Humans; Hyaluronic Acid; Neoplasms, Experimental

The correlation between the content of individual glycosaminoglycans and the histological patterns are studied on breast tumor tissues. The myxomatous stroma of intracanalicular fibroadenoma contained a large amount of glycosaminoglycans, which were mainly hyaluronic acid. The chondroitin 4- and 6-sulfate level was also high. As the supporting stroma of this tumor became denser and more fibrous, the level of hyaluronic acid content was reduced. In the case of pericanalicular fibroadenoma, glycosaminoglycans were small in amount and the levels of hyaluronic acid and chondroitin sulfate were low, but the ratio of dermatan sulfate content was higher. In the case of gynecomastia, the conent was almost the same as that of pericanalicular fibroadenoma. Scirrhous carcinoma tissues contained a relatively large amount of hyaluronic acid and chondroitin sulfate. No remarkable differences in heparan sulfate content were observed in any one of the breast tumors tested. Dermatan sulfate-chondroitin sulfate copolymers were detected in all the tumors. The presence of dermatan sulfate seemed to have an intimate relation with the fibrogenesis in the interstitial stromal element of the tumor tissues.

Topics: Adenocarcinoma, Scirrhous; Adenofibroma; Adult; Breast Neoplasms; Chondroitin Sulfates; Dermatan Sulfate; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Hyaluronic Acid; Middle Aged; Phyllodes Tumor