chondroitin-sulfates and Melanoma

Topics: Ammonium Chloride; Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Biological Transport; Chondroitin; Chondroitin Sulfates; Clinical Trials as Topic; Epitopes; Gangliosides; Humans; Killer Cells, Natural; Melanoma; Melanoma-Specific Antigens; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasm Proteins; Proteoglycans; Rats

Topics: Ammonium Chloride; Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Biological Transport; Chondroitin; Chondroitin Sulfates; Clinical Trials as Topic; Epitopes; Gangliosides; Humans; Killer Cells, Natural; Melanoma; Melanoma-Specific Antigens; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasm Proteins; Proteoglycans; Rats

In the syngeneic, subcutaneous B16F10 mouse model of malignant melanoma, treatment with exogenous ARSB markedly reduced tumor size and extended survival. In vivo experiments showed that local treatment with exogenous N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) led to reduced tumor growth over time (p < 0.0001) and improved the probability of survival up to 21 days (p = 0.0391). Tumor tissue from the treated mice had lower chondroitin 4-sulfate (C4S) content and lower sulfotransferase activity. The free galectin-3 declined, and the SHP2 activity increased, due to altered binding with chondroitin 4-sulfate. These changes induced effects on transcription, which were mediated by Sp1, phospho-ERK1/2, and phospho-p38 MAPK. Reduced mRNA expression of chondroitin sulfate proteoglycan 4 (CSPG4), carbohydrate sulfotransferase 15 (N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase), and matrix metalloproteinases 2 and 9 resulted. Experiments in the human melanoma cell line A375 demonstrated similar responses to exogenous ARSB as in the tumors, and inverse effects followed ARSB siRNA. ARSB, which removes the 4-sulfate group at the non-reducing end of C4S, acts as a tumor suppressor, and treatment with exogenous ARSB impacts on vital cell signaling and reduces the expression of critical genes associated with melanoma progression.

Topics: Animals; Chondroitin Sulfates; Humans; Melanoma; Melanoma, Cutaneous Malignant; Mice; N-Acetylgalactosamine-4-Sulfatase; Signal Transduction; Skin Neoplasms

Malaria infected erythrocytes utilize the parasite protein VAR2CSA to bind to a unique presentation of chondroitin sulfate (CS) for their placenta specific tropism. Interestingly, many cancers express a similar form of CS, thereby termed oncofetal CS (ofCS). The distinctive tropism of malaria infected erythrocytes and the identification of oncofetal CS, therefore, represent potentially potent tools for cancer targeting. Here we describe an intriguing drug delivery platform that effectively mimics infected erythrocytes and their specificity for ofCS. We used a lipid catcher-tag conjugation system for the functionalization of erythrocyte membrane-coated drug carriers with recombinant VAR2CSA (rVAR2). We show that these malaria mimicking erythrocyte nanoparticles (MMENPs) loaded with docetaxel (DTX) specifically target and kill melanoma cells

Topics: Antigens, Protozoan; Biomimetics; Chondroitin Sulfates; Erythrocytes; Humans; Malaria; Malaria, Falciparum; Melanoma; Plasmodium falciparum

Neovascularization is crucial to the occurrence and progression of tumors, and the development of antiangiogenic drugs has essential theoretical value and clinical significance. However, antiangiogenesis therapy alone cannot meet the needs of tumor therapy. Meanwhile, polysaccharides are ideal drug carriers with promising applications in drug modification and delivery. In this research, we developed a novel redox and acid sensitive nanodrug (CDDP-CS-Cys-EA, CCEA) composed of chondroitin sulfate (CS), antiangiogenic peptide (endostatin2-alft1, EA) and chemotherapeutic drug (cisplatin, CDDP). CCEA exhibited redox and acid responsiveness, better blood hemocompatibility (hemolysis rate < 5 %), the ability to target tumors (CD44-mediated endocytosis), and strong antiangiogenesis and antitumor characteristics in vitro. Moreover, CCEA showed excellent antitumor activity and low toxicity in B16 xenograft mice. It also has been confirmed that CCEA induced tumor cell apoptosis through promoting the expression of Bax, suppressing the expression of Bcl-2, decreasing mitochondrial membrane potential, releasing cytochrome C (Cyto C), and enhancing the activities of Caspase 9 and Caspase 3. The results of this paper provided a theoretical basis and insight for the development of antitumor drugs.

Topics: Animals; Apoptosis; Chondroitin Sulfates; Cisplatin; Humans; Hyaluronan Receptors; Immunotherapy; Melanoma; Mice; Nanoparticles

The application of gold nanorods (GNRs) in photothermal therapy is a promising avenue for cancer treatment. The aim of this study was to develop a GNR-based targeted photothermal therapy for melanoma.. We utilized the electrostatic interaction between cationic GNRs and an anionic polymer chondroitin sulfate A (CSA), which has an affinity for binding to melanoma cells, to construct an anionic binary GNR-CSA complex (GNR-CS) at an optimal theoretical charge ratio of the trimethylammonium groups of GNR: carboxyl and sulfate groups of CSA = 1:2.5. The cytotoxicity to normal cells and erythrocyte agglutination activity of GNR-CS were evaluated. After the cellular uptake of GNR-CS by melanoma cells (B16-F10) was investigated, the photothermal performance of GNR-CS against B16-F10 cells was evaluated in vitro.. The particle size and zeta potential of GNR-CS were approximately 35 nm and -20 mV, respectively. GNR-CS showed little cytotoxicity to normal cells and low erythrocyte agglutination activity, indicating good biocompatibility. Compared with negatively-charged GNR, GNR-CS was highly taken up by B16-F10 cells even if it was negatively charged. Cellular uptake was significantly suppressed upon treatment with excess CSA, suggesting the involvement of a CSA-specific uptake pathway. Furthermore, irradiation of the GNR-CS solution with near-infrared (NIR) light increased its temperature in light-intensity and GNR-concentration dependent manners. GNR-CS exhibited significant and GNR-dose dependent cytotoxicity in melanoma cells in combination with NIR light irradiation.. GNRs coated with CSA have the potential as a medicine in targeted photothermal therapy for melanoma.

Topics: Cell Line, Tumor; Chondroitin Sulfates; Gold; Humans; Melanoma; Nanotubes; Photochemotherapy; Photosensitizing Agents; Phototherapy; Photothermal Therapy

Topics: Aged, 80 and over; Chondroitin Sulfates; Collagen; Foot Diseases; Humans; Male; Melanoma; Plastic Surgery Procedures; Surgical Flaps; Treatment Outcome

Despite extensive use of Integra in burn reconstruction, little has been published regarding its utility in complex hand wounds from nonburn trauma or cancer resection. This study aimed to review outcomes following Integra use for hand reconstruction following cancer resection or nonburn trauma with exposed bone, joints, and/or tendons.. Retrospective review was performed of patients undergoing hand reconstruction with Integra for exposed bones, joints, or tendons over a 6-year period at a single institution.. Fourteen patients underwent hand reconstruction using Integra, 8 following cancer resection and 6 following acute nonburn trauma. The mean defect size was 19 cm. Integra is an effective method to treat complex hand wounds with exposed bone, joints, and/or tendons. This technique can be used in the office, lessens the need for local or free flap coverage, and provides an excellent aesthetic outcome. Integra should be considered a viable option in hand reconstruction algorithm.

Topics: Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Chondroitin Sulfates; Collagen; Female; Hand; Hand Injuries; Humans; Male; Melanoma; Middle Aged; Negative-Pressure Wound Therapy; Patient Satisfaction; Retrospective Studies; Skin Neoplasms; Surgical Flaps

In this report, redox/enzyme responsive chondroitin sulfate-ss-deoxycholic acid (CSCD) conjugates were synthesized using cystamine as the linkage which could self-assemble to form self-assembled nanoparticles (175.6 + 5.2 nm) in the aqueous environment. Docetaxel (DTX) was loaded in nanoparticles with desired loading efficiency for the inhibition of tumor growth and metastasis of melanoma. Interestingly, nanoparticles were demonstrated to respond to hyaluronidase-1 (Hyal-1) which could degrade chondroitin sulfate (CS) backbones. In this case, we designed dual-sensitive nanoparticles with enhanced drug release pattern under the presence of glutathione (GSH)/Hyal-1. Compared with Taxotere®, CSCD nanoparticles significantly improved the DTX distribution in tumors and lungs with about 4.4-fold higher area-under-the-curve (AUC) value. In situ tumor volume and pulmonary metastatic formation were reduced upon the administration of DTX-loaded CSCD nanoparticles via DTX-induced apoptosis and decreased metastasis-promotion protein expression. With only minor cytotoxicity, CSCD nanoparticles could be promising nano-drug delivery systems for successful management of melanoma.

Topics: Animals; Apoptosis; Cell Line, Tumor; Chondroitin Sulfates; Docetaxel; Drug Carriers; Drug Delivery Systems; Melanoma; Mice; Nanoparticles; Oxidation-Reduction; Rats; Rats, Wistar; Taxoids

In this issue of Molecular Cell, Lin et al. (2018) report that chondroitin-4-sulfate, which is found in a common supplement meant to alleviate degenerative joint disorders, promotes the growth of BRAF V600E mutant melanoma. This study not only has implications for patient care but also sheds light on a novel mechanism for regulating phosphoinositide 3-kinase signaling.

Topics: Cell Line, Tumor; Chondroitin Sulfates; Dietary Supplements; Humans; Melanoma; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins B-raf; Sulfates

Dietary supplements such as vitamins and minerals are widely used in the hope of improving health but may have unidentified risks and side effects. In particular, a pathogenic link between dietary supplements and specific oncogenes remains unknown. Here we report that chondroitin-4-sulfate (CHSA), a natural glycosaminoglycan approved as a dietary supplement used for osteoarthritis, selectively promotes the tumor growth potential of BRAF V600E-expressing human melanoma cells in patient- and cell line-derived xenograft mice and confers resistance to BRAF inhibitors. Mechanistically, chondroitin sulfate glucuronyltransferase (CSGlcA-T) signals through its product CHSA to enhance casein kinase 2 (CK2)-PTEN binding and consequent phosphorylation and inhibition of PTEN, which requires CHSA chains and is essential to sustain AKT activation in BRAF V600E-expressing melanoma cells. However, this CHSA-dependent PTEN inhibition is dispensable in cancer cells expressing mutant NRAS or PI3KCA, which directly activate the PI3K-AKT pathway. These results suggest that dietary supplements may exhibit oncogene-dependent pro-tumor effects.

Topics: Animals; Antinematodal Agents; Carcinogens; Casein Kinase II; Cell Proliferation; Cell Transformation, Neoplastic; Chondroitin Sulfates; Dietary Supplements; Drug Resistance, Neoplasm; Female; GTP Phosphohydrolases; HEK293 Cells; HT29 Cells; Humans; Melanoma; Membrane Proteins; Mice; Mice, Inbred NOD; Mice, Nude; Mice, Transgenic; Mutation; NIH 3T3 Cells; Nuclear Proteins; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; PTEN Phosphohydrolase; Signal Transduction; Skin Neoplasms; Transcription Factors; Xenograft Model Antitumor Assays

Sonodynamic therapy (SDT) has been proposed as a new modality for cancer management through low-intensity ultrasound induced activation of sonosensitizers. Here, we designed a novel redox/enzyme/ultrasound responsive chondroitin sulfate-chlorin e6-lipoic acid nanoplatform loading docetaxel, combining SDT and chemotherapy, for antiproliferation and antimetastasis of melanoma. The reversibly crosslinked and self-assembled nanoparticles possessed monodispersive size distribution, stability in physical conditions, while showing increased uptake with rapid drug release in simulated tumor microenvironment (reductive potentials and degradative hyaluronidase-1). With synthesized ultrasound sensitive polymer backbones, SDT induced the generation of cellular reactive oxygen species and mitochondrial damage, exerting the apoptotic effect through the release of cytochrome C, the expression of cleaved caspase-9 followed by the functional cleaved caspase-3. Chemo-sonodynamic therapy not only inhibited tumor growth and metastasis with reduced metastatic protein expression, but also caused immune response via the release of tumor-associated antigens. It was initially demonstrated that SDT could induce the tumor cell death, therefore having potentials to recruit cytotoxic lymphocytes into tumor sites. Notably, the nanoplatforms exhibited good in vivo stability and blood compatibility, indicating the safety and efficiency in drug delivery. Our work thus presents a convenient approach to fabricate intelligent multifunctional nanoparticles and paves a path for effective cancer therapies.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Chlorophyllides; Chondroitin Sulfates; Combined Modality Therapy; Docetaxel; Melanoma; Mice, Inbred C57BL; Nanoparticles; Porphyrins; Thioctic Acid; Ultrasonic Therapy

The purpose of this paper is to highlight the risk of early malignant transformation in infants with giant congenital melanocytic nevi (GN) and demonstrate the potential for earlier intervention with aggressive surgery. We describe the case of a child born with a GN who developed a metastatic melanoma early in life, despite early commencement of resection of the nevus. This is contrasted against a second case of a child in which a more radical management was conducted. Despite early commencement of serial resection of the GN, the first child in this series died of metastatic melanoma prior to complete excision of the nevus. With the second child, radical excision combined with the use of Integra™ and negative pressure wound therapy allowed total removal of the GN within the first 6 months of life.

Topics: Cell Transformation, Neoplastic; Chondroitin Sulfates; Collagen; Fatal Outcome; Female; Humans; Infant; Melanoma; Negative-Pressure Wound Therapy; Nevus, Pigmented; Skin Neoplasms; Time-to-Treatment

Dermal regeneration templates may be used in the reconstruction of large defects after the excision of cutaneous malignancies. We describe the successful use of Integra

Topics: Aged, 80 and over; Carcinoma, Squamous Cell; Chondroitin Sulfates; Collagen; Dermatologic Surgical Procedures; Head and Neck Neoplasms; Humans; Male; Melanoma; Neoplasms, Multiple Primary; Plastic Surgery Procedures; Regeneration; Scalp; Skin Neoplasms; Skin Physiological Phenomena

Arylsulfatase B (ARSB; N-acetylgalactosamine 4-sulfatase) is reduced in several malignancies, but levels in melanoma have not been investigated previously. Experiments were performed in melanoma cell lines to determine ARSB activity and impact on melanoma invasiveness. ARSB activity was reduced ~50% in melanoma cells compared to normal melanocytes. Silencing ARSB significantly increased the mRNA expression of chondroitin sulfate proteoglycan(CSPG)4 and pro-matrix metalloproteinase(MMP)-2, known mediators of melanoma progression. Also, invasiveness and MMP activity increased when ARSB was reduced, and recombinant ARSB inhibited invasiveness and MMP activity. Since the only known function of ARSB is to remove 4-sulfate groups from the N-acetylgalactosamine 4-sulfate residue at the non-reducing end of chondroitin 4-sulfate (C4S) or dermatan sulfate, experiments were performed to determine the transcriptional mechanisms by which expression of CSPG4 and MMP2 increased. Promoter activation of CSPG4 was mediated by reduced binding of galectin-3 to C4S when ARSB activity declined. In contrast, increased pro-MMP2 expression was mediated by increased binding of the non-receptor tyrosine phosphatase SHP2 to C4S. Increased phospho-ERK1,2 resulted from SHP2 inhibition. Combined effects of increased C4S, CSPG4, and MMP2 increased the invasiveness of the melanoma cells, and therapy with recombinant ARSB may inhibit melanoma progression.

Topics: Blood Proteins; Cell Line, Tumor; Chondroitin Sulfate Proteoglycans; Chondroitin Sulfates; Galectin 3; Galectins; Humans; Matrix Metalloproteinase 2; Melanoma; Membrane Proteins; N-Acetylgalactosamine-4-Sulfatase; Neoplasm Invasiveness; Promoter Regions, Genetic; Protein Tyrosine Phosphatase, Non-Receptor Type 11

Topics: Chondroitin Sulfates; Collagen; Humans; Male; Melanoma; Middle Aged; Mohs Surgery; Neoplasm Metastasis; Regeneration; Skin Transplantation

Cationic antimicrobial peptides (CAPs) with antitumor activity have potential for use as novel antitumor agents because of their lower risk for induction of resistance. Of these peptides, magainin II (MG2) exhibited cytotoxicity in tumor cells only at high concentrations, likely due to the inefficiency of MG2 in cell membrane binding and cell entry. Conjugation to a cell-penetrating peptide (CPP) might enhance the cytotoxicity of MG2 in tumor cells. Here, we constructed a fusion peptide MG2A by conjugating MG2 to the N-terminus of the CPP penetratin (Antp). It was found that the fusion peptide MG2A is more potent than unconjugated MG2 at tumor cell killing. The IC50s of MG2A for the tumor cells tested were at least 30 times lower than the IC50s of unconjugated MG2. These data indicate that conjugation to Antp significantly enhanced the cytotoxicity of MG2 in tumor cells. Moreover, the IC50s of MG2A for tumor cells are within 2 to 3 μM, which are about three to five times lower than the IC50 for normal cells. Furthermore, chondroitin sulfate (CS) was found to be overexpressed on the surface of the tested tumor cells, and the cytotoxicity of MG2A could be inhibited by the addition of exogenous CS. These results suggest that binding of Antp to CS on tumor cells might be one important cause for the selective cytotoxicity of MG2A in tumor cells. Taken together, conjugation of MG2 to Antp can significantly enhance its antitumor activity, and the fusion of CAP to Antp might be an alternative for cancer-targeted therapy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Carrier Proteins; Caspases; Cell Survival; Cell-Penetrating Peptides; Central Nervous System Neoplasms; Chlorocebus aethiops; Chondroitin Sulfates; Drug Carriers; Glioma; HeLa Cells; Humans; Inhibitory Concentration 50; Kidney Tubules, Proximal; Lung Neoplasms; Magainins; Melanoma; Molecular Targeted Therapy; Neoplasms; Rats; Skin Neoplasms; Vero Cells; Xenopus Proteins

Scalp melanoma is aggressive and has a proclivity for regional metastasis. We hypothesize that subperiosteal scalp melanoma resection reduces in-transit/satellite recurrence, when compared with subgaleal resection.. We identified patients with intermediate to deep, primary scalp melanoma referred to head/neck surgery over an 8-year period. Patients were compared based on scalp resection depth, including subperiosteal (resection to the level of calvarium) and subgaleal (resection including skin, subcutaneous tissue, and galea). The dependent variables were in-transit/satellite recurrence and time to in-transit/satellite recurrence.. Among 48 identified patients, the in-transit/satellite recurrence rate was 16.7%. Subgaleal resection patients had higher in-transit/satellite recurrence rates than subperiosteal resection patients (24.0% vs. 8.7%, P=0.155). Among node-negative patients, subgaleal resection had significantly higher in-transit/satellite metastasis rates when compared with subperiosteal resection (26.3% vs. 0%, P=0.047).. For node-negative, primary scalp melanoma, subperiosteal resection significantly decreases in-transit/satellite recurrence when compared with subgaleal resection. Given our small sample size, further studies are necessary to confirm these results.

Topics: Aged; Chondroitin Sulfates; Collagen; Dermatologic Surgical Procedures; Female; Follow-Up Studies; Head and Neck Neoplasms; Humans; Lymphatic Metastasis; Male; Melanoma; Neoplasm Recurrence, Local; Periosteum; Registries; Retrospective Studies; Scalp; Skin Neoplasms; Time Factors; Treatment Outcome

Scalp reconstruction after wide tumor excision is particularly challenging. Free tissue transfers, local flaps, or skin grafts can be used but present some disadvantages especially with old patients with local advanced cancers, systemic diseases and in patients with a prior history of recurring scalp skin cancers in which the risk of burying a recurring tumor with a flap is likely. The Authors expose their early experience with Integra dermal regeneration template for scalp reconstruction after scalp tumor excision.. Eight patients with primary or secondary scalp tumor underwent a first surgical procedure under local anaesthesia for tumor removal and Integra positioning followed by a second operation performed three weeks later to reconstruct the defect by removing the superficial silicon layer of Integra and by covering the defect with a split thickness skin graft. The average surface area of the defect was 143.27 cm(2). The average operating time was 30.4 minutes for the first operation and 45.6 minutes for the second operation. In six cases Integra was grafted as a classic full-thickness skin graft. In the remaining two cases the Integra template was meshed. The artificial derma was attached to the edge of the wound by either sutures or staples.. There was a full graft take on all cases. The mean follow-up was 24 months. In two cases we were able to detect early tumor recurrence two months after the operation. Satisfactory cosmetic and functional results were obtained in all patients.. In the scalp defect reconstructions after tumor excision, Integra allows to obtain a thicker and more durable coverage than skin graft on the skull, allowing to detect a tumor recurrence earlier than a flap reconstruction with no risk of burying an eventual underlying residual tumor. These operations are performed under local anaesthesia and are therefore suitable for elderly patients.

Topics: Aged, 80 and over; Carcinoma, Squamous Cell; Chondroitin Sulfates; Collagen; Humans; Melanoma; Plastic Surgery Procedures; Sarcoma; Scalp; Skin Neoplasms; Skin, Artificial; Wounds and Injuries

We present the case of a patient with a 1.2mm Breslow thickness subungual melanoma of the thumb who refused the standard treatment of amputation. He underwent a 1cm wide local excision of the lesion down to bone and the defect was covered with Integra and later grafted. The evidence for increasingly conservative treatment of subungual melanoma is growing. He remains disease free and is left with a fully functional thumb and a good cosmetic result.

Topics: Adult; Chondroitin Sulfates; Collagen; Humans; Male; Melanoma; Plastic Surgery Procedures; Skin Neoplasms; Thumb

Integra® has already established its role in acute burn injuries and scar management. It can also be used to cover non-vascularised wounds such as exposed bone resulting from trauma or tumour resection. The aim of this series was to review all cases that underwent Integra® reconstruction following cancer excision. In particular we were interested in the use of Integra for day-case and local anaesthetic procedures in cases where excision was required down to bone or tendon.. All patients who had Integra reconstruction over a three-year period were prospectively followed. A total of 14 cases were identified for inclusion into the series. In each case patient factors such peripheral vascular disease, age and patient choice meant that traditional methods of reconstruction were not possible. As a result a staged Integra® reconstruction was performed.. The 14 cases comprised 11 (78%) males and 3 (22%) females with the majority being diagnosed with Squamous cell carcinomas, 3 (40%) or Malignant Melanomas, 3 (20%). The most common operative sites were digital (5) and scalp (6) in 72% of the cases. The average graft take was 87%. There were 4 early, 4 delayed and 3 late complications in a total of 8 patients mostly resulting in a delay in healing. In 6/14 patients (43%) there were no complications.. Tumour excision and wide local excision may leave patients with defects requiring complex reconstructive surgery. The options available are often compounded by various patient factors. In complex cases we have found the use of Integra® to be a safe and viable alternative to traditional methods of wound closure.

Topics: Carcinoma, Squamous Cell; Chondroitin Sulfates; Collagen; Female; Fingers; Head and Neck Neoplasms; Humans; Male; Melanoma; Plastic Surgery Procedures; Retrospective Studies; Scalp; Skin Neoplasms; Skin, Artificial

Basic fibroblast growth factor (FGF-2) and its respective tyrosine kinase receptors, form an autocrine loop that affects human melanoma growth and metastasis. The aim of the present study was to examine the possible participation of various glycosaminoglycans, i.e. chondroitin sulfate, dermatan sulfate and heparin on basal and FGF-2-induced growth of WM9 and M5 human metastatic melanoma cells. Exogenous glycosaminoglycans mildly inhibited WM9 cell's proliferation, which was abolished by FGF-2. Treatment with the specific inhibitor of the glycosaminoglycan sulfation, sodium chlorate, demonstrated that endogenous glycosaminoglycan/proteoglycan production is required for both basal and stimulated by FGF-2 proliferation of these cells. Heparin capably restored their growth, and unexpectedly exogenous chondroitin sulfate to WM9 and both chondroitin sulfate and dermatan sulfate to M5 cells allowed FGF-2 mitogenic stimulation. Furthermore, in WM9 cells the degradation of membrane-bound chondroitin/dermatan sulfate stimulates basal growth and even enhances FGF-2 stimulation. The specific tyrosine kinase inhibitor, genistein completely blocked the effects of FGF-2 and glycosaminoglycans on melanoma proliferation whereas the use of the neutralizing antibody for FGF-2 showed that the mitogenic effect of chondroitin sulfate involves the interaction of FGF-2 with its receptors. Both the amounts of chondroitin/dermatan/heparan sulfate and their sulfation levels differed between the cell lines and were distinctly modulated by FGF-2. In this study, we show that chondroitin/dermatan sulfate-containing proteoglycans, likely in cooperation with heparan sulfate, participate in metastatic melanoma cell FGF-2-induced mitogenic response, which represents a novel finding and establishes the central role of sulfated glycosaminoglycans on melanoma growth.

Topics: Autocrine Communication; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Chondroitin Sulfates; Fibroblast Growth Factors; Glycosaminoglycans; Heparitin Sulfate; Humans; Melanoma; Neoplasm Metastasis; Protein-Tyrosine Kinases; Proteoglycans

We previously reported that CS (chondroitin sulfate) GAG (glycosaminoglycan), expressed on MCSP (melanoma-specific CS proteoglycan), is important for regulating MT3-MMP [membrane-type 3 MMP (matrix metalloproteinase)]-mediated human melanoma invasion and gelatinolytic activity in vitro. In the present study, we sought to determine if CS can directly enhance MT3-MMP-mediated activation of pro-MMP-2. Co-immunoprecipitation studies suggest that MCSP forms a complex with MT3-MMP and MMP-2 on melanoma cell surface. When melanoma cells were treated with betaDX (p-nitro-beta-D-xylopyranoside) to inhibit coupling of CS on the core protein, both active form and proform of MMP-2 were no longer co-immunoprecipitated with either MCSP or MT3-MMP, suggesting a model in which CS directly binds to MMP-2 and presents the gelatinase to MT3-MMP to be activated. By using recombinant proteins, we determined that MT3-MMP directly activates pro-MMP-2 and that this activation requires the interaction of the C-terminal domain of pro-MMP-2 with MT3-MMP. Activation of pro-MMP-2 by suboptimal concentrations of MT3-MMP is also significantly enhanced in the presence of excess C4S (chondroitin 4-sulfate), whereas C6S (chondroitin 6-sulfate) or low-molecular-mass hyaluronan was ineffective. Affinity chromatography studies using CS isolated from aggrecan indicate that the catalytic domain of MT3-MMP and the C-terminal domain of MMP-2 directly bind to the GAG. Thus the direct binding of pro-MMP-2 with CS through the C-domain would present the catalytic domain of pro-MMP-2 to MT3-MMP, which facilitates the generation of the active form of MMP-2. These results suggest that C4S, which is expressed on tumour cell surface, can function to bind to pro-MMP-2 and facilitate its activation by MT3-MMP-expressing tumour cells to enhance invasion and metastasis.

Topics: Chondroitin Sulfate Proteoglycans; Chondroitin Sulfates; Enzyme Precursors; Gelatinases; Humans; Matrix Metalloproteinase 16; Matrix Metalloproteinase Inhibitors; Melanoma; Membrane Proteins; Metalloendopeptidases; Protein Structure, Tertiary; Tissue Inhibitor of Metalloproteinase-2; Tumor Cells, Cultured

Topics: Administration, Cutaneous; Biopsy; Chondroitin Sulfates; Collagen; Granuloma, Pyogenic; Humans; Male; Melanoma; Middle Aged; Postoperative Care; Silver Nitrate; Skin Care; Skin Diseases; Skin Neoplasms; Skin Transplantation; Wound Healing

Artificial dermis has been used successfully for coverage of full-thickness wounds with a well-vascularized surgical bed. However, the use of artificial dermis in the reconstruction of partial- and full-thickness scalp defects has not been well documented.. Seven patients (six men and one woman; mean age, 70 +/- 14 years) with partial-thickness (three patients) and full-thickness (four patients) soft-tissue defects of the scalp (mean defect area, 97 +/- 58 cm) following resection of recurrent malignant tumors and/or previous failed reconstructions underwent staged scalp reconstruction with a bilaminate skin substitute (Integra). After adequate debridement of scalp wounds, including burring the outer table of the calvaria down to bleeding bone for full-thickness defects, Integra was scored and applied unexpanded. A split-thickness skin graft (0.011 +/- 0.0 inch in thickness) was placed on the operative site at postoperative day 36 +/- 15 after removal of the silicone layer of the artificial dermis. Two patients required repeated applications of artificial dermis to compensate for contour deficits before skin grafting.. Clinically, all reconstructed areas showed well-vascularized neodermis before skin grafting. There was a 100 percent take of the skin grafts, with no infections or other complications noted. All reconstructive procedures were performed in less than 3 hours of combined operative time, with the last stage performed on an outpatient basis.. Artificial dermis can be used successfully for reconstruction of complex scalp defects following oncologic resection, offering minimal donor-site morbidity, expedient operative time, and when needed, temporary quality closure until final pathologic results are known. Integra skin may offer another option for definitive management of extensive full-thickness scalp defects.

Topics: Aged; Aged, 80 and over; Chondroitin Sulfates; Collagen; Debridement; Female; Glioblastoma; Humans; Hutchinson's Melanotic Freckle; Male; Melanoma; Middle Aged; Retreatment; Retrospective Studies; Scalp; Skin Neoplasms; Skin Transplantation; Skin, Artificial

Chondroitin sulfate (CS) belongs to the group of glycosaminoglycans (GAGs), which are linear polysaccharides, located in the extracellular matrix and on the cell surface. To study the structure and distribution of CS in human skin and skin disorders, we have selected antibodies using phage display technique against CS. Four unique human anti-CS single-chain antibodies were selected: IO3D9, IO3H10, IO3H12, and IO4C2. We determined their amino acid sequence and evaluated their CS reactivity using ELISA and immunohistochemistry. Antibodies were reactive with CS, but not with other GAGs except for IO4C2, which was also reactive with heparin. Antibody IO3D9 showed a strong reactivity with highly sulfated CS (CSE). All antibodies displayed a different staining pattern in rat kidney, indicating the recognition of unique CS epitopes. In normal skin, the papillary dermis but not the reticular dermis was strongly stained. Antibody IO3H12 also stained basal keratinocytes. We applied these antibodies to study CS expression and localization in melanoma and psoriasis. A strong immunoreactivity with the extracellular matrix of melanoma metastases could be observed for all four antibodies, while in atypical nevi a less extensive reactivity with only the papillary dermis was observed. In psoriatic lesions, CS could be observed in the papillary dermis and in the reticular dermis, whereas the specific location in the papillary dermis found in normal skin was completely lost. In conclusion, human phage-display-derived anti-CS antibodies have been selected, characterized, and applied to detect CS alterations in skin conditions. Altered CS composition was detected in melanoma and psoriasis.

Topics: Antibodies; Chondroitin Sulfates; Epitopes; Humans; Melanoma; Psoriasis; Sensitivity and Specificity; Skin

Topics: Adult; Aged; Biocompatible Materials; Carcinoma, Basal Cell; Chondroitin Sulfates; Collagen; Female; Humans; Male; Melanoma; Middle Aged; Nose; Nose Neoplasms; Skin Transplantation; Skin, Artificial; Wound Healing

Giant congenital melanocytic nevi represent a surgical challenge, particularly in cases in which the size of the nevus exceeds certain extend and malignant transformations have to be considered.. To discuss through case report considerable surgical options when extensive giant congenital melanocytic nevi with malignant transformation are encountered.. We present an unusual case of a giant congenital melanocytic nevi of the entire back of a 44-year-old patient. To achieve radical resection with direct appropriate wound closure and acceptable outcome, the integument of the entire back was excised and covered with Integra, followed by split-thickness skin grafting after stable integration of the matrix.. The approach resulted in a complete excision of the tumor and acceptable cosmetic and excellent biomechanical outcome.. The introduced practice demonstrates a useful alternative to established methods, particularly if tumor excision in large areas and subsequent wound closure might be achieved in one procedure.

Topics: Adult; Antineoplastic Agents; Axilla; Back; Biocompatible Materials; Cell Transformation, Neoplastic; Chondroitin Sulfates; Collagen; Female; Groin; Humans; Interferon-alpha; Lymph Node Excision; Melanoma; Neoplasm Staging; Nevus, Pigmented; Plastic Surgery Procedures; Skin Neoplasms; Skin Transplantation; Skin, Artificial

The detection of circulating melanoma cells has been the subject of numerous investigations in recent years. We developed a cellular approach to identifying circulating melanoma cells in peripheral blood using immunomagnetic cell sorting. The examination covered 205 blood samples from 155 melanoma patients and 30 samples from healthy persons and nonmelanoma patients. After density gradient centrifugation, the interphase was incubated with the 9.2.27 antibody. Positive cells were labeled with magnetic microbeads and enriched by immunomagnetic cell sorting. Cells were stained using an alkaline phosphatase-anti-alkaline phosphatase assay and examined by light microscopy. In spiking experiments, melanoma cells seeded at a concentration of one melanoma cell per milliliter of whole blood could be detected reliably. Circulating melanoma cells were not found in 30 controls, nor were 9.2.27-positive cells found in 41 patients with primary malignant melanoma. In patients with regional lymph node metastases and disseminated disease, circulating 9.2.27-positive cells could be detected in 3 of 29 patients (10%) and 13 of 85 patients (15%) examined, respectively. We conclude that immunomagnetic cell sorting is a promising method with high sensitivity and specificity. The method is not suitable for early detection of metastases but is a valuable tool for further investigating the biological characteristics of circulating melanoma cells.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Alkaline Phosphatase; Biomarkers, Tumor; Cell Separation; Chondroitin Sulfates; Female; Humans; Immunomagnetic Separation; Leukocytes; Male; Melanoma; Middle Aged; Neoplasm Staging; Neoplastic Cells, Circulating; Skin Neoplasms; Tumor Cells, Cultured

In the current study, two specific glycosaminoglycan lyases, chondroitinase AC and chondroitinase B, were utilized to examine the roles of chondroitin sulfates and dermatan sulfate in tumor metastasis and angiogenesis. Melanoma cells (SK-MEL) or endothelial cells were treated with either medium or chondroitinase enzyme. Chondroitinase AC inhibited melanoma invasion and proliferation as well as endothelial proliferation and angiogenesis. Apoptosis of melanoma and endothelial cells, as measured by the activity of caspase-3, was also increased by chondroitinase AC, but not by chondroitinase B. Chondroitinase B inhibited endothelial and melanoma proliferation and invasion, but to a lesser extent than chondroitinase AC. Neither chondroitinase had a detectable effect on gelatinase secretion by melanoma cells. These results indicate that both chondroitin and dermatan sulfates regulate many cellular activities related to metastasis.

Topics: Animals; Apoptosis; Cattle; Cell Division; Cell Movement; Cells, Cultured; Chondroitin Sulfates; Chondroitinases and Chondroitin Lyases; Dermatan Sulfate; Dose-Response Relationship, Drug; Endothelium, Vascular; Gelatinases; Humans; Melanoma; Neoplasm Metastasis; Neovascularization, Pathologic; Neovascularization, Physiologic; Tumor Cells, Cultured

Even with novel chemotherapeutic agents and external beam radiation therapy, the prognosis of neoplastic meningitis secondary to malignant melanoma is still dismal. The authors report a case study of a 46-year-old white female who presented with progressive hearing loss, severe headaches, nausea, vomiting, and a rapid decline in neurologic status. She was referred to Duke University Medical Center after conventional chemotherapy for malignant melanoma failed. She was enrolled in a Phase I trial of (131)I-labeled monoclonal antibody Mel-14 F(ab')(2) fragment administered intrathecally. Within a year after her treatment, she recovered, having a normal neurologic exam except for residual bilateral hearing loss. The authors discuss dosimetry, preclinical, and clinical studies conducted with Mel-14 F(ab')(2) and introduce a potentially promising therapy option in the treatment of neoplastic meningitis in patients with malignant melanoma. Currently, the patient remains neurologically normal except for a mild bilateral hearing loss more than 4 years after treatment and has no radiographic evidence of neoplastic meningitis.

Topics: Antibodies, Monoclonal; Chondroitin Sulfates; Female; Humans; Iodine Radioisotopes; L-Selectin; Melanoma; Meningitis; Middle Aged; Radiopharmaceuticals

We developed a cellular approach to the identification of circulating melanoma cells in peripheral blood using immunomagnetic cell sorting. One hundred seventy-eight blood samples from 129 melanoma patients and 30 samples from healthy persons and nonmelanoma patients were examined. After density gradient centrifugation the interphase was incubated with the mAb 9.2.27. Positive cells were labeled with magnetic microbeads and enriched by immunomagnetic cell sorting. Cells were stained using an alkaline phosphatase-antialkaline phosphatase assay and examined by light microscopy. In spiking experiments, melanoma cells seeded at a concentration of one melanoma cell per ml whole blood could be detected reliably with the assay. Circulating melanoma cells were not found in 30 controls examined, nor were 9.2.27-positive cells found in 41 patients with primary malignant melanoma. In patients with regional lymph node metastases and in patients with disseminated disease, circulating 9.2.27-positive cells could be detected in 3 out of 22 patients (13.6%) and 10 out of 66 patients (15.2%) examined. We present a sensitive and specific immunocytological approach to detect circulating melanoma cells in peripheral blood. The method is not suitable for early detection of metastases but is a valuable tool for further investigating biological characteristics of circulating melanoma cells.

Topics: Antibodies; Biomarkers, Tumor; Chondroitin Sulfates; Humans; Immunomagnetic Separation; Melanoma; Neoplasm Staging; Neoplastic Cells, Circulating; Sensitivity and Specificity; Skin Neoplasms; Tumor Cells, Cultured

Haptokinetic cell migration across surfaces is mediated by adhesion receptors including beta1 integrins and CD44 providing adhesion to extracellular matrix (ECM) ligands such as collagen and hyaluronan (HA), respectively. Little is known, however, about how such different receptor systems synergize for cell migration through three-dimensionally (3-D) interconnected ECM ligands. In highly motile human MV3 melanoma cells, both beta1 integrins and CD44 are abundantly expressed, support migration across collagen and HA, respectively, and are deposited upon migration, whereas only beta1 integrins but not CD44 redistribute to focal adhesions. In 3-D collagen lattices in the presence or absence of HA and cross-linking chondroitin sulfate, MV3 cell migration and associated functions such as polarization and matrix reorganization were blocked by anti-beta1 and anti-alpha2 integrin mAbs, whereas mAbs blocking CD44, alpha3, alpha5, alpha6, or alphav integrins showed no effect. With use of highly sensitive time-lapse videomicroscopy and computer-assisted cell tracking techniques, promigratory functions of CD44 were excluded. 1) Addition of HA did not increase the migratory cell population or its migration velocity, 2) blocking of the HA-binding Hermes-1 epitope did not affect migration, and 3) impaired migration after blocking or activation of beta1 integrins was not restored via CD44. Because alpha2beta1-mediated migration was neither synergized nor replaced by CD44-HA interactions, we conclude that the biophysical properties of 3-D multicomponent ECM impose more restricted molecular functions of adhesion receptors, thereby differing from haptokinetic migration across surfaces.

Topics: Antibodies, Monoclonal; Cell Movement; Chondroitin Sulfates; Collagen; Extracellular Matrix; Fluorescent Antibody Technique; Humans; Hyaluronan Receptors; Hyaluronic Acid; Image Processing, Computer-Assisted; Integrins; Melanoma; Membrane Glycoproteins; Membrane Proteins; Microscopy, Video; Platelet Glycoprotein GPIb-IX Complex; Receptors, Collagen; Tumor Cells, Cultured

We have previously reported that alpha4beta1 (but not alpha5beta1) integrin-mediated melanoma cell adhesion is inhibited by removal of cell surface chondroitin sulfate glycosaminoglycan (CSGAG), suggesting that melanoma chondroitin sulfate proteoglycan plays a role in modulating the adhesive function of alpha4beta1 integrin. In the current study, we demonstrated that alpha4beta1 integrin binds to CSGAG. We have identified a peptide from within alpha4 integrin termed SG1 (KKEKDIMKKTI) that binds to cell surface melanoma chondroitin sulfate proteoglycan, indicating that SG1 represents a CSGAG binding site within the alpha4 integrin subunit. Soluble SG1 inhibits alpha4beta1 integrin-mediated human melanoma cell adhesion to CS1. Polyclonal antibody generated against the peptide inhibits melanoma cell adhesion to CS1, and the inhibition is reversed by Mn2+ and an activating monoclonal antibody anti-beta1 (8A2). Additionally, pretreatment of cells with anti-SG1 IgG inhibits the expression of the monoclonal antibody 15/7 epitope in the presence of soluble CS1 peptide, suggesting that anti-SG1 IgG prevents ligand binding by alpha4beta1 integrin. These results demonstrate that alpha4beta1 integrin interacts directly with CSGAG through SG1 site, and that this site can affect the ligand binding properties of the integrin.

Topics: Antibodies; Binding Sites; Cell Adhesion; Chondroitin Sulfates; Humans; Integrin alpha4beta1; Integrins; Manganese; Melanoma; Peptide Fragments; Protein Binding; Proteoglycans; Receptors, Lymphocyte Homing; Tumor Cells, Cultured

We previously reported that cultured mammalian cells incubated with 4-methylumbelliferyl (MU) or p -nitrophenyl (pNP) beta-xyloside synthesize an alpha-GalNAc-terminated pentasaccharide resembling the glycosaminoglycan-core protein linkage region. Here we show that human melanoma M21 cells and human neuroblastoma cells incubated with Xylbeta-MU/pNP also make an alpha-GalNAc-terminated heptasaccharide containing one chondroitin disaccharide repeat. High performance liquid chromatography and matrix-assisted laser desorption ionization mass spectrometry analysis of intact or glycosidase-digested xyloside showed the structure as: GalNAcalphaGlcAbeta1,3GalNAcbeta1,4GlcAbeta1,3Galbe ta1,3Galbeta1, 4Xylbeta-MU/pNP. The alpha-GalNAc-terminated xylosides can account for approximately 10% of the total Xylbeta-MU/pNP products ( approximately 1.5 nmol/h/mg). These results show that GalNAcalphaGlcAbeta-modification is relatively abundant, but not unique to the GAG-linkage tetrasaccharide. alpha-GalNAc addition to the GlcA residue does not appear to be an extension of general phase II detoxification of xenobiotics that involve glucuronidation, since M21 cells incubated with MU synthesize only 0.3 pmol GlcAbeta-MU/h/mg protein, and undetectable amount of GalNAcalphaGlcAbeta-MU (<40 fmol/h/mg). Further, subcellular fractionation shows that the alpha- N- acetylgalactosaminyltransferase activity colocalizes in the Golgi with other glycosyl transferases and not in the ER, where xenobiotic detoxification glucuronosyltransferases are found. Although GalNAcalphaGlcAbeta-terminal modification has not been detected on naturally occurring GAG chains, the substantial amount of alpha-GalNAc transferase activity suggests that the alpha-GalNAc transferase could utilize other GlcA-containing glycoconjugates as acceptors.

Topics: Acetylgalactosamine; Carbohydrate Sequence; Chondroitin Sulfates; Chromatography, Affinity; Chromatography, High Pressure Liquid; Glycosides; Humans; Melanoma; Molecular Sequence Data; Neuroblastoma; Oligosaccharides; Spectrometry, Mass, Fast Atom Bombardment; Tumor Cells, Cultured

The association between cytoadherence of Plasmodium falciparum-infected erythrocytes and the severity of malaria has been evaluated. In this study, we investigate adherence to C32 melanoma cells, CD36, intracellular adhesion molecule-1 (ICAM-1), thrombospondin (TSP), E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and chondroitin sulfate A (CSA) of 36 P. falciparum isolates from patients suffering from acute falciparum malaria. Adherence to purified adhesion molecules varied greatly among different parasite isolates. All isolates but one adhered to CD36, but none bound to E-selectin and VCAM-1 beyond control levels. Some P. falciparum isolates adhered to ICAM-1 and to CSA, a newly identified receptor for adherence. There was no correlation between in vitro binding to any one receptor and the patients' conditions. In addition, we investigated the characteristics of adherence to CSA and to C32 melanoma cells. Infected erythrocytes continued to adhere after trypsin digestion and soluble CSA inhibited adherence to C32 melanoma cells in a dose-dependent manner. The results imply a role for CSA in the natural infection of P. falciparum.

Topics: Adolescent; Adult; Animals; CD36 Antigens; Cell Adhesion; Cell Adhesion Molecules; Chondroitin Sulfates; Erythrocytes; Female; Humans; Malaria, Falciparum; Male; Melanoma; Plasmodium falciparum; Trypsin; Tumor Cells, Cultured

Previous data have indicated that the proteoglycan (PG) pattern is different on tumor cells with different liver metastatic potential. We selected "conventional" glycosaminoglycan (GAG) biosynthesis inhibitors, beta-D-xyloside (BX), 2-deoxy-D-glucose (2-DG), ethane-l-hydroxy-l,l-diphosphonate (ETDP) and the newly discovered 5-hexyl-2-deoxyuridine (HUdR), to modulate PGs on highly metastatic/liver-specific 3LL-HH murine carcinoma and HT168 human melanoma cells and to influence their liver colonization potential. These compounds all induced remarkable changes in GAG biosynthesis, but to varying degrees: glucosamine labelling was affected mainly by 2-DG, and HUdR and sulphation by BX and HUdR. Furthermore, the ratio of heparan sulphate/chondroitin sulphate (HS/CS) of PGs was increased by ETDP and decreased after treatment by HUdR. In addition to changes in PG metabolism, tumor-cell proliferation and adhesion to fibronectin were affected; BX and 2-DG stimulated cell proliferation and adhesion, while HUdR inhibited both proliferation and adhesion. Most interestingly, HUdR, the most effective inhibitor of HS/HSPG, depressed the formation of liver colonies, while ETDP, the most effective inhibitor of CS/CSPG, stimulated the appearance of liver colonies. These observations indicated that, at least in these experimental systems, tumor cells with a high HS/CS ratio are more likely to form liver metastases; consequently, anti-HS agents could also be anti-metastatic.

Topics: Animals; Antimetabolites; Antimetabolites, Antineoplastic; Chondroitin Sulfates; Deoxyglucose; Deoxyuridine; Etidronic Acid; Glycosaminoglycans; Glycosides; Heparitin Sulfate; Humans; Liver Neoplasms; Melanoma; Mice; Tumor Cells, Cultured

Adherence of Plasmodium falciparum-infected erythrocytes to cerebral postcapillary venular endothelium is believed to be a critical step in the development of cerebral malaria. Some of the possible receptors mediating adherence have been identified, but the process of adherence in vivo is poorly understood. We investigated the role of carbohydrate ligands in adherence, and we identified chondroitin sulfate (CS) as a specific receptor for P. falciparum-infected erythrocytes. Parasitized cells bound to Chinese hamster ovary (CHO) cells and C32 melanoma cells in a chondroitin sulfate-dependent manner, whereas glycosylation mutants lacking chondroitin sulfate A (CSA) supported little or no binding. Chondroitinase treatment of wild-type CHO cells reduced binding by up to 90%. Soluble CSA inhibited binding to CHO cells by 99.2 +/- 0.2% at 10 mg/ml and by 72.5 +/- 3.8% at 1 mg/ml, whereas a range of other glycosaminoglycans such as heparan sulfate had no effect. Parasite lines selected for increased binding to CHO cells and most patient isolates bound specifically to immobilized CSA. We conclude that P. falciparum can express or expose proteins at the surface of the infected erythrocyte that mediate specific binding to CSA. This mechanism of adherence may contribute to the pathogenesis of P. falciparum malaria, but has wider implications as an example of an infectious agent with the capacity to bind specifically to cell-associated or immobilized CS.

Topics: Animals; Cell Adhesion; Cell Adhesion Molecules; Cells, Cultured; Child; CHO Cells; Chondroitin Sulfates; Cricetinae; Cricetulus; Endothelium, Vascular; Erythrocytes; Glycosylation; Heparitin Sulfate; Host-Parasite Interactions; Humans; Malaria, Falciparum; Melanoma; Phosphatidylethanolamines; Plasmodium falciparum; Receptors, Cell Surface; Tumor Cells, Cultured; Umbilical Veins

Glycosaminoglycans were metabolically labeled in subconfluent cultures of highly metastatic 7Gp122 and poorly metastatic IC8 variants and of the low metastatic parental M4Be human melanoma cell line. Proteoglycans were separated by DEAE Trisacryl chromatography from the culture medium, from the heparin extract of the cell layer and from the heparin-extracted cell residue lyzed with detergents. Glycosaminoglycans were released from the proteoglycans by reductive alkaline hydrolysis and heparan sulfate (HS) was detected by deaminative cleavage with nitrous acid. Expressed on cell protein basis, the labeled HS content in the medium and in the cell layer decreased with increasing metastatic ability. The extraction of HS with heparin from the 7Gp122 cells indicated that this variant was enriched in (polypeptide bound) HS non inserted into the plasma membrane, compared with the low metastatic IC8 and M4Be cells. The HS fraction in heparin extract and in the heparin-extracted cell residue exhibited molecular mass heterogeneity on gel permeation chromatography and it contained HS fragments. Scission with nitrous acid followed by molecular sieve chromatography of the degradation products indicated that the tetra- and disaccharide repeats separated by the N-sulfated glucosamine residue were present in about equal amounts and constituted 60% of the HS chains in the IC8 and M4Be cells. HS from 7Gp122, IC8 and M4Be cells did not bind antithrombin III with high affinity but it was capable of binding bFGF in in vitro assay.

Topics: Antithrombin III; Chondroitin Sulfates; Fibroblast Growth Factor 2; Heparitin Sulfate; Humans; Melanoma; Neoplasm Metastasis; Tumor Cells, Cultured

Here we describe a two-step procedure for purification of human tenascin from conditioned medium of the SK-MEL-28 human melanoma cell line. The first step consists in passing the conditioned media through two chromatography columns connected in sequence. The first is a large capacity gelatin--Sepharose affinity chromatography column (to remove fibronectin), the second, over which the unbound material from the first column flows directly, is a hydroxyapatite chromatography column. Under these conditions, all tenascin present in the conditioned medium binds to the hydroxyapatite chromatography column from which it is then eluted by a 5-300 mM sodium phosphate gradient. With this step, we obtain a crude tenascin preparation, concentrated about 20 times with respect to the starting conditioned medium, and in which tenascin represents more than 50% of the total protein. The second step consists of two sequential precipitations with 6% and 12.8% poly(ethylene glycol). After this step, tenascin is more than 95% pure and does not show any contamination of chondroitin-sulfate-containing proteoglycans that are known to bind to it. From 21 medium we obtain about 3-4 mg tenascin which corresponds to a yield of about 40-50%. This procedure gives a higher yield, is simpler with respect to procedures previously described, avoids the exposure of the protein to denaturing agents or harsh conditions and could be used for purification of tenascin from the conditioned media of other cell lines. Thus, this procedure may represent a simple and useful tool for the preparation of tenascin to study its biological functions.

Topics: Cell Adhesion Molecules, Neuronal; Cell Line; Chondroitin Sulfates; Chromatography; Chromatography, Affinity; Culture Media; Durapatite; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix Proteins; Humans; Hydroxyapatites; Macromolecular Substances; Melanoma; Molecular Weight; Neoplasm Proteins; Tenascin

Heparan sulphate (HS) and chondroitin sulphate (CS) proteoglycans (PGs) frequently have opposite biologic functions in cell-matrix adhesion as well as in the regulation of cell proliferation. Data revealed that sulphated glycosaminoglycans (sGAGs) (sugar chains of PGs) are differently expressed in tumor cells characterized by different metastatic potential; the more metastatic cells contain a higher HS/CS ratio. As the proliferative capacity of tumor cells is also frequently altered in parallel with their metastatic potential, it was not clear whether observed PG alterations reflect changes in cell proliferation or metastatic potential. The cell-associated PG expression and sGAG biosynthesis was studied in tumor cells of human melanoma lines characterized by different experimental metastatic potential to the mouse liver but similar in vitro/in vivo proliferation rates. Using antibodies against PGs we found different expression of PG epitopes in melanoma lines, except from the melanoma antigen. Unlike the low CSPG (melCSPG) metastatic melanoma cells, the cell line with high metastatic capacity contained a higher proportion of positive cells for surface-HSPG without the coexpression of certain cartilage-type CSPG epitopes (recognized by MAb HSFPG 529) as well as by an increased pericellular HS/CS ratio due to intracellular accumulation/retention of CS. Immunocytochemistry of adherent cells revealed HSPGs at substrate-attached membrane areas only in cases of highly metastatic melanoma cells. These data further support our view that the absolute or relative dominance of HSPGs over CSPGs at the cell surface of metastatic tumor cells can be considered a marker of a more metastatic phenotype.

Topics: Cell Membrane; Chondroitin Sulfates; Epitopes; Flow Cytometry; Fluorescent Antibody Technique; Glycosaminoglycans; Heparitin Sulfate; Humans; Melanoma; Proteoglycans; Tissue Distribution; Tumor Cells, Cultured

Topics: Chondroitin Sulfates; Chromatography, Gel; Glycosaminoglycans; Heparitin Sulfate; Humans; Melanoma; Tumor Cells, Cultured

We have reported previously that the production of a tumor cell factor that stimulates synthesis of fibroblast collagenase is influenced by a fibroblast-deposited matrix component, possibly heparan sulfate-proteoglycan. In this study, binding sites for heparin and heparan sulfate on mouse B-16 melanoma cells have been demonstrated. Binding of 3H-heparin and 35S-heparan sulfate has been shown to occur to whole cells, isolated membranes, and to a component(s) of detergent extracts of the membranes. Scatchard analysis of binding of 3H-heparin yielded a Kd of 2-5 x 10(-8) M and a Bmax of 0.5 x 10(7) heparin molecules bound per cell. Binding of 35S-heparan sulfate was of at least an order of magnitude lower affinity than heparin, but the Bmax was similar to that for heparin. Competition studies showed that 35S-heparan sulfate binding was inhibited totally by heparin and heparan sulfate and partially by dermatan sulfate, but no inhibition was obtained with hyaluronate or chondroitin sulfate. Binding of 3H-heparin was inhibited totally by heparin but to different extents by preparations of heparan sulfate from different tissue sources. The heparin/heparan sulfate binding activity is a protein(s) because it is destroyed by treatment with trypsin. Binding of 3H-heparin to transblots of the detergent extract of the B-16 cell membranes indicated that at least part of the binding activity is a 14,000-dalton protein.

Topics: Animals; Binding Sites; Cell Line; Chondroitin Sulfates; Dermatan Sulfate; Glycosaminoglycans; Heparin; Heparitin Sulfate; Hyaluronic Acid; Kinetics; Melanoma; Membranes; Receptors, Cell Surface

Changes in glycosaminoglycan composition occurring during the cell cycle were determined in B16-F10 cells sorted flow cytometrically with respect to DNA content. Incorporation of 35S-sulfate into heparan sulfate and chondroitin sulfate of unsorted and G1,S, and G2 +M sorted cells was determined following chondroitinase ABC or nitrous acid treatment; the incorporation into surface material was measured as the difference between the radioactivity of control and trypsin-treated cells. Incorporation of 35S-sulfate and 3H-glucosamine into cetyl pyridinium chloride (CPC)-precipitable material was characterized before and after chondroitinase or nitrous acid treatment by Sephadex G50 chromatography. Long-term (48 h) and short-term (1 h) labeling studies demonstrate that (a) the amount of total cellular chondroitin sulfate is greater than that of heparan sulfate, with larger amounts of unsulfated heparan than chondroitin being present; (b) the rate of turnover of heparan sulfate is greater than that of chondroitin sulfate; (c) greatest short-term incorporation of 3H-glucosamine into CPC-precipitable material occurs during S phase; and (d) the rate of turnover of both heparan sulfate and chondroitin sulfate is decreased in S phase relative to G1 and G2 + M.

Topics: Cell Cycle; Cell Line; Chondroitin; Chondroitin Sulfates; Flow Cytometry; Glucosamine; Glycosaminoglycans; Heparitin Sulfate; Humans; Interphase; Isotope Labeling; Melanoma; Sulfur Radioisotopes; Tritium

The differential adhesion of cultured mammalian clonal cell lines to components of the extracellular matrix was examined by kinetic adhesion and long-term growth assays. Uniform artificial matrices were prepared by air drying collagen Type I solution (C) onto a microtiter well and then air drying a solution containing a single glycosaminoglycan (GAG): hyaluronic acid (HA), chondroitin sulfate-4 (CHS-4), or chondroitin sulfate-6 (CHS-6). The adhesion of [3H]thymidine-prelabeled cells suspended in fibronectin (FN) depleted medium was measured at 2 and 6 hr. Neuroblastoma (N18, Lan 1) and melanoma (B16, G361, S91) cell lines exhibited a significantly greater percentage of cells adhering to one or more C-GAG matrices compared with C matrices. Maximal adhesion at 2 hr was to C-HA. In contrast at 2 hr, two glial, two epithelial, and one fibroblastic cell line showed unchanged or significantly decreased binding to C-GAG compared with C matrices. Further experiments using a neuroblastoma (N18) and a glioma (C6) cell line indicated that the adhesion patterns were not altered either by the method of dissociation from the tissue culture dish, preincubation with exogenous GAG, or the addition of exogenous fibronectin. Assays of N18 and C6 adhesion to matrices made from a non-GAG polyanionic compound, polygalacturonic acid (PGA), did not yield the same adhesion patterns as C-HA matrices. Long-term growth studies of a neuroblastoma (N18) melanoma (S91), and glioma (C6) cell line on nonuniform matrices deliberately prepared with GAG-rich and GAG-poor regions complemented the observations from the kinetic adhesion assays. N18 and S91 cells did not grow on areas which did not contain GAG by toluidine blue staining. However, the C6 cells did not grow on areas which did strongly stain for GAG. A quantitative analysis of the long term growth of N18 and C6 cells substantiated these observations. All these data indicate that the cellular phenotype may be correlated with matrix adhesion. Neuroblastomas and melanomas have a greater affinity for GAG-containing matrices while glial, epithelial, and fibroblastic cells appear to have a greater or equal affinity for collagen matrices.

Topics: Animals; Cell Adhesion; Cell Line; Chondroitin Sulfates; Clone Cells; Collagen; Edetic Acid; Epithelial Cells; Fibroblasts; Fibronectins; Glioma; Glycosaminoglycans; Humans; Hyaluronic Acid; Melanoma; Mice; Neuroblastoma; Phenotype; Rats

The major glycosaminoglycans isolated from B16 mouse melanoma tumors after Pronase digestion were shown to be a family of chondroitin 4-sulfates with different degrees of sulfation and a wide molecular-weight range. Ultracentrifugation data gave molecular weight values as high as 88 000, in contrast to that of costal cartilage chondroitin 4-sulfate which is about 14 000. A mucin-type sialoglycopeptide, isolated from the tumors by cetylpyridinium chloride precipitation of the Pronase digest, was shown to contain O-glycosylically linked tetra- and tri-saccharides consisting of sialic acid, galactose, and N-acetylgalactosamine. The sialoglycoprotein, which on Pronase digestion gave rise to the glycopeptide, was isolated from the tumor by extraction with lithium diiodosalicylate and affinity chromatography on a wheat-germ agglutinin-Sepharose 4B column. It was homogeneous on the basis of gel filtration on Sepharose 4B and Sephadex G-200 columns, lectin affinity, and ion-exchange chromatography. The compounds isolated from the B16 mouse melanoma tumors are similar to those produced by the cultured melanoma cells, which suggests that the latter compounds are not artifacts of the culture system.

Topics: Amino Acids; Animals; Chondroitin Sulfates; Glycosaminoglycans; Melanoma; Mice; Neoplasms, Experimental; Sialoglycoproteins

The treatment of chondroitin sulfate isolated from cultured B16 mouse melanoma cells with 0.04 M HCl at 100 degrees C for 90 min released up to 45% of O-sulfate residues as free inorganic sulfate. In addition to the release of inorganic sulfate, extensive degradation of this polysaccharide as well as of cartilage chondroitin sulfate, pig rib cartilage proteoglycan, heparin and hyaluronic acid was also evident under these conditions. The above hydrolysis conditions are used for characterizing 35S-labeled heparan sulfates synthesized by cultured cells and to calculate ratio of N- and O-sulfates in these molecules. Our results suggest that caution is necessary in interpreting the results of mild acid hydrolysis of glycosaminoglycans.

Topics: Animals; Cell Line; Chondroitin; Chondroitin Sulfates; Heparin; Hyaluronic Acid; Melanoma; Mice; Neoplasms, Experimental; Sulfuric Acids

The mucopolysaccharides produced by B16 mouse melanoma cells have been isolated in milligram quantities from the spent media in which the cells were grown in the presence of 2-amino-2-deoxy-D-glucose-t and [35S]-sulfate. The mucopolysaccharides obtained by precipitation with cetylpyridinium chloride from the Pronase digest of the media were further purified by gel filtration, ion-exchange chromatography, and treatment with nucleases. The major components were identified as chondroitin-4-sulfates by identification of the hexosamine as 2-amino-2-deoxy-D-galactose, and by digestibility with hyaluronidases, chondroitinase AC, and chondro-4-sulfatase. The o.r.d. curve and i.r. spectra of these components also confirmed their similarity to chondroitin-4-sulfate from cartilage. The molecular weight of the polysaccharide chains was estimated to be in the range 90,000-120,000 by sedimentation equilibrium analysis.

Topics: Carbohydrates; Cell Line; Chondroitin; Chondroitin Sulfates; Hexosamines; Melanoma; Molecular Weight; Optical Rotatory Dispersion; Spectrophotometry, Infrared

Melanosomes are present in abundant numbers in HPM-73 melanoma cells maintained in surum-containing cultures. However, withholding of serum for 3 days caused severe retardation of development of these organelles. Addition of chondroitin-4-sulphate (0.4 mg/ml) to serum-containing culture media for 3 days resulted in greater numbers of premelanosomes and melanosomes relative to mitochondria. The same amount of chrondroitin-4-sulphate (0.4 mg/ml) added to serum-free cultures also resulted in an increased ratio of premelanosomes and melanosomes to mitochondria but, within the 3-day period many of these organelles did not attain the pattern of maturity evident in melanoma cells reared in serum-supplemented media. By exploitation of slowed rates of development of melanosomes in cultures (especially, but not exclusively, in serum-free samples) it was possible to examine various early developmental forms of these organelles. It was found that in this cell line mitochondria appear to be involved in the process of melanosome formation.

Topics: Cell Line; Chondroitin Sulfates; Culture Media; Endoplasmic Reticulum; Golgi Apparatus; Melanins; Melanoma; Melanophores; Microscopy, Electron; Mitochondria; Morphogenesis