Preclinical evaluation of zoledronate using an in vitro mimetic cellular model for breast cancer metastatic bone disease.

BACKGROUND: The interactions between metastatic breast cancer cells and host cells of osteoclastic lineage in bone microenvironment are essential for osteolysis. In vitro studies to evaluate pharmacological agents are mainly limited to their direct effects on cell lines. To mimic the communication between breast cancer cells and human osteoclasts, a simple and reproducible cellular model was established to evaluate the effects of zoledronate (zoledronic acid, ZOL), a bisphosphonate which exerts antiresorptive properties. METHODS: Human precursor osteoclasts were cultured on bone-like surfaces in the presence of stimuli (sRANKL, M-CSF) to ensure their activation. Furthermore, immature as well as activated osteoclasts were co-cultured with MDA-MB-231 breast cancer cells. TRAP5b and type I collagen N-terminal telopeptide (NTx) were used as markers. Osteoclasts' adhesion to bone surface and subsequent bone breakdown were evaluated by studying the expression of cell surface receptors and certain functional matrix macromolecules in the presence of ZOL. RESULTS: ZOL significantly suppresses the precursor osteoclast maturation, even when the activation stimuli (sRANKL and M-SCF) are present. Moreover, it significantly decreases bone osteolysis and activity of MMPs as well as precursor osteoclast maturation by breast cancer cells. Additionally, ZOL inhibits the osteolytic activity of mature osteoclasts and the expression of integrin ß3, matrix metalloproteinases and cathepsin K, all implicated in adhesion and bone resorption. CONCLUSIONS: ZOL exhibits a beneficial inhibitory effect by restricting activation of osteoclasts, bone particle decomposition and the MMP-related breast cancer osteolysis. GENERAL SIGNIFICANCE: The proposed cellular model can be reliably used for enhancing preclinical evaluation of pharmacological agents in metastatic bone disease.

Efficient TGFß-induced epithelial-mesenchymal transition depends on hyaluronan synthase HAS2.

Epithelial-mesenchymal transition (EMT) is a developmental program, which can be adopted by cancer cells to increase their migration and ability to form metastases. Transforming growth factor ß (TGFß) is a well-studied inducer of EMT. We demonstrate that TGFß potently stimulates hyaluronan synthesis via upregulation of hyaluronan synthase 2 (HAS2) in NMuMG mammary epithelial cells. This stimulatory effect requires the kinase active type I TGFß receptor and is dependent on Smad signaling and activation of the p38 mitogen-activated protein kinase. Knockdown of HAS2 inhibited the TGFß-induced EMT by about 50%, as determined by the phase contrast microscopy and immunostaining using the EMT marker ZO-1. Furthermore, real-time PCR analysis of the EMT markers fibronectin, Snail1 and Zeb1 revealed decreased expressions upon HAS2 suppression, using specific small interfering RNA (siRNA) for HAS2. Removal of the extracellular hyaluronan by Streptomyces hyaluronidase or inhibiting the binding to its cell surface receptor CD44 by blocking antibodies, did not inhibit TGFß-induced EMT. Interestingly, HAS2 suppression completely abolished the TGFß-induced cell migration, whereas CD44 knockdown did not. These observations suggest that TGFß-dependent HAS2 expression, but not extracellular hyaluronan, has an important regulatory role in TGFß-induced EMT.

Expression of matrix macromolecules and functional properties of EGF-responsive colon cancer cells are inhibited by panitumumab.

The epidermal growth factor receptor (EGFR) is a member of the HER family receptors and its activation induced by its natural ligand EGF results in colon cancer growth and progression. Panitumumab (pmAb) is a fully human IgG2 anti-EGFR antibody that blocks the EGFR actions. In the present study, we evaluated the effects of pmAb on the EGF-mediated cellular responses in a panel of colon cancer cells (HCT-8, HT-29, DLD-1 and HCT-116). HCT-1116 and DLD-1 cells showed no significant EGF-dependent cell proliferation; HT-29 and HCT-8 exhibited an EGF-dependent proliferation, with HCT-8 cells to be the most responsive with significant EGFR phosphorylation upon treatment with EGF. The effects of pmAb were then evaluated in the most EGF-responsive cells, HCT-8. In that respect, pmAb impedes the signaling cascade mediated by EGFR intracellular phosphorylation and activity of focal adhesion kinase (FAK) as well as the EGF-induced invasive and migratory potential of colon cancer cells. At the level of matrix effectors implicated in colon cancer progression we report that pmAb is a potent inhibitor of constitute and EGF-mediated gene expression of certain matrix effectors, such as membrane-type 1 metalloproteinase (MT1-MMP), extracellular metalloproteinases inducer (EMMPRIN), urokinase plasminogen activator (uPA) and syndecan-4. The obtained data demonstrated that pmAb is a specific blocker of EGF-mediated EGFR activation, resulting in a significant inhibition of colon cancer cell proliferation in early stages of growth, migration and invasiveness as well as of matrix effector implicated in cancer progression.

Expression of matrix macromolecules and functional properties of breast cancer cells are modulated by the bisphosphonate zoledronic acid.

BACKGROUND: The extracellular matrix (ECM) components play key roles in the multistep process of cancer growth and progression. Preclinical and clinical data show that bisphosphonates (BPs) may exert direct or indirect antitumoral effects. Despite proven efficiency in cancer treatment, the mechanism by which BPs can interfere with cancer progression remains elusive. METHODS: We investigated the effects of the third generation BP, zoledronate (zoledronic acid, Zometa®), in the expression of ECM macromolecules as well as the functional properties (proliferation, adhesion, migration and invasion) in two breast cancer cell lines (MDA-MB-231 and MCF-7) with different metastatic potentials. RESULTS: The data highlight that zoledronate effectively inhibits growth of breast cancer cells, functional invasion migration and adhesion to various matrices. At the level of ECM interacting molecules, the expression of specific heparan sulfate proteoglycans implicated in cancer progression, such as syndecan-1, -2 and glypican-1 is downregulated, whereas syndecan-4 expression is upregulated upon treatment with zoledronate. The levels of integrins ανß3, ανß5 and α5ß1 were significantly reduced following treatment with zoledronate which is in accordance with the reduced cell adhesion on various ECM matrices. The expression of hyaluronan and its receptor CD44 was also significantly suppressed. Moreover, ZOL suppressed the expression of metalloproteinases MMP-2, -9, the membrane type MT1- and MT2-MMP, whereas it increased the expression of their endogenous tissue inhibitors. CONCLUSIONS AND GENERAL SIGNIFICANCE: The obtained results demonstrate that zoledronate is a critical modulator of ECM gene expression and powerful anticancer agent inhibiting growth, migration and the matrix-associated invasion of breast cancer cells.

Targeting the tumor proteasome as a mechanism to control the synthesis and bioactivity of matrix macromolecules.

Extracellular matrices (ECMs) are dynamic structures that provide cells not only with a structural support but, importantly, exhibit significant functional roles in the control of key cellular events such as adhesion, migration, proliferation, differentiation, and survival. In tumors, matrix effectors such as proteoglycans (PGs) and matrix metalloproteinases (MMPs) constitute major regulators of the interactions between tumor cells and their microenvironment and, therefore, they have been identified as potential molecular targets that are expected to advance the pharmacological treatment of cancer. ECMs composition is highly affected by cells through intrinsic regulatory mechanisms, such as the ubiquitin-proteasome system (UPS). Proteasome is a major cellular protease complex that controls the concentration and turnover of molecules in ECMs, including certain types of PGs, MMPs and collagens, and consequently, in the tumor microenvironment. Furthermore, proteasome activity is regulated by PG-derived intracellular glycosaminoglycan moieties revealing a critical inter-dependence of these compounds. Since ECMs renewal and degradation can be tightly regulated by proteasome activities, its modulation may be considered as a novel strategy to control the properties of tumor microenvironment. Currently, there are several proteasome inhibitors targeting distinct molecular pathways either approved or in clinical trials for the treatment of multiple cancers. In this review, the novel approach of targeting the proteasome to selectively regulate the synthesis and the bioactivity of certain matrix PGs and MMPs is presented and discussed.

Imbalance of MMP-2 and MMP-9 expression versus TIMP-1 and TIMP-2 reflects increased invasiveness of human testicular germ cell tumours.

The histological classification of testicular germ cell tumours (TGCTs) to seminoma or non-seminomatous germ cell tumours is at present the main criterion for the clinical outcome and selection of the treatment strategy. In view of the need to identify novel prognostic biomarkers for TGCTs, we investigated the expression of the matrix metalloproteinases MMP-2 and MMP-9 in testicular tumour tissues and cell lines of both seminoma and non-seminoma origin. Immunohistochemistry and zymography analysis of tumoural tissues showed significantly higher levels of MMP-2 and MMP-9 compared with normal testis with the active forms detected only in the tumour tissues. Three cell lines representative of the different tumour types, JKT-1 seminoma, NCCIT teratocarcinoma and NTERA2/D1 embryonal carcinoma were also evaluated for their expression of these MMPs using qPCR and zymography and for their invasive properties. The more invasive non-seminomatous teratocarcinoma and embryonal cells expressed considerably more MMP-2 and MMP-9 compared with seminoma cells exhibiting lower invasiveness. Furthermore, an inverse relation was observed between invasiveness and the expression of endogenous inhibitors TIMP-1 and TIMP-2. The MMP inhibitor Marimastat inhibited invasion in all cell lines, the highest inhibition was observed in the more invasive NTERA2/D1 and NCCIT cells, which presented the highest ratio of MMP-2 and MMP-9 vs. TIMP-1 and TIMP-2. These results highlight the importance of MMP-2 and MMP-9 in the invasiveness of testicular tumours and suggest that their levels, vs. those of TIMP-1 and TIMP-2, may represent potential biomarkers for testicular malignancy.

Pathophysiology and pharmacological targeting of tumor-induced bone disease: current status and emerging therapeutic interventions.

Bone disease is a common complication of metastatic solid tumors but also of primary hematological malignancies such as multiple myeloma. Our understanding of the molecular mechanisms underlying the development of bone disease by solid tumors and multiple myeloma has been significantly improved. A complex inter-dependence exists between bone disease and malignant cell growth, creating a vicious cycle of extensive bone destruction and tumor progression. Although myeloma and solid tumors share a number of common molecular pathogenetic mechanisms, they involve distinct pathophysiological pathways, resulting in osteoclastic bone resorption and inhibition of bone formation. In this review, we analyze the molecular mechanisms, involved in tumor-induced bone disease and discuss the current therapeutic approaches and the most recent clinical developments of emerging targeted therapies.

Glycosaminoglycans as key molecules in atherosclerosis: the role of versican and hyaluronan.

Cardiovascular disease is the largest cause of death in Western societies and it primarily results from atherosclerosis of large and medium-sized vessels. Atherosclerosis leads to myocardial infarction, when it occurs in the coronary arteries, or stroke, when it occurs in the cerebral arteries. Pathological processes involved in macrovascular disease include the accumulation of lipids which are retained by extracellular matrix (ECM) molecules, especially by the chondroitin sulfate/dermatan sulfate (CS/DS) proteoglycans (CS/DSPGs), such as versican, biglycan and decorin. The sulfation pattern of CS is a key player in protein interactions causing atherosclerosis. Several studies have shown that lipoproteins bind CSPGs via their glycosaminoglycan chains. Galactosaminoglycans, such as CS and DS, bind low density lipoproteins (LDL), affecting the role of these molecules in the arterial wall. In this article, the role of CS and versican in atherosclerosis and hyaluronan in atherogenesis as well as the up to date known mechanisms that provoke this pathological condition are presented and discussed.

The importance of c-Kit and PDGF receptors as potential targets for molecular therapy in breast cancer.

Molecular therapies target key functional molecules in order to halter viable operation of cancer cells. Receptor tyrosine kinases (RTKs) constitute attractive targets, as quite often their abnormal signaling has been associated with tumor development and growth. Overexpression of growth factor receptors, including IGF, EGF, TGF-alpha, SCF and PDGF receptors, has been associated with poor prognosis in breast cancer. Therefore, a number of RTKs are already targets for novel designed drugs, which involve tyrosine kinase inhibitors and monoclonal antibodies. Despite the fact that c-Kit and PDGF-R have been effective targets in a number of cancers, the experimental results in breast have not yet clarified their importance. The expression and function of c-Kit in breast cancer is a quite controversial subject. Several studies propose that the loss of c-Kit expression has been associated with tumor progress, whereas other reports indicate not only its expression but also the implication of c-Kit in breast cancer. On the other hand, the expression of PDGF-R in breast cancer is not in question. A number of inhibitors against tyrosine kinases are currently in trials as to demonstrate their importance in breast cancer treatment. Imatinib (STI571), which is a selective tyrosine kinase inhibitor and particularly of c-Kit and PDGF-R, exhibited encouraging results in respect to its inhibitory effect in cell growth and invasion potential in a panel of human breast cancer cell lines. In this review, the importance of RTKs in human cancer and of c-Kit and PDGF-R as molecular targets in breast cancer treatment, in the view of their expression profiles and the in vitro effects of STI571 is discussed.

Versican but not decorin accumulation is related to metastatic potential and neovascularization in testicular germ cell tumours.

AIMS: To investigate the expression of versican and decorin in patients with testicular germ cell tumours (GCTs) and to correlate this with the clinicopathological findings. Matrix proteoglycans versican and decorin are frequently overexpressed in various malignancies and are involved in the progression of cancer. METHODS AND RESULTS: Overexpression of versican and decorin was detected in GCTs by immunoblotting. Immunohistochemical staining for proteoglycans was performed on 71 cases of paraffin-embedded tissues. In most of the cases increased decorin and versican stromal staining was demonstrated. In both seminomas and non-seminomatous germ cell tumours (NSGCTs) strong staining of decorin was not found to be related to any of the clinicopathological variables. Accumulation of versican was found to be associated with vascular and lymphatic invasion, nodal metastasis and disease stage in seminomas and NSGCTs and, in addition, with tumour size and distant metastasis only in NSGCTs. Additionally, only the deposition of versican was linearly correlated with the number of microvessels in the tumour stroma in GCTs. CONCLUSIONS: Ectopic versican and decorin expression is a frequent feature in GCTs. Versican but not decorin accumulation in GCTs is related to metastatic potential and neovascularization and might be a useful marker for testicular malignancy.

The increased accumulation of structurally modified versican and decorin is related with the progression of laryngeal cancer.

Versican and decorin, two proteoglycans (PGs) with contradictory roles in the pathophysiology of cancer, comprise important stromal components in many tumor types and play a crucial role in the progression of cancer. In this study, we provide direct evidence for a significant and stage-related accumulation of versican and decorin in the tumor-associated stroma of laryngeal squamous cell carcinoma (LSCC) in comparison to normal larynx. Both PGs were found to be co-localized within the peritumorous stroma. In addition, the accumulated versican and decorin were markedly modified on both protein core and glycosaminoglycan (GAG) levels. Decorin, which was present under both glycanated and non-glycanated forms, perceptibly increased with the progression of LSCC, compared to the normal larynx. Tumor-associated glycanated decorin was found to contain significant amounts of dermatan sulfate (DS) sequences. Versican was also found to undergo stage-related structural modifications since a marked heterogeneity of protein cores was observed, being intense in late stage of laryngeal cancer. The increased accumulation of both versican and decorin was associated with a significant stage-related increase of the molar ratio of Delta di-mono4S to Delta di-mono6S up to approximately threefold in LSCC compared to the normal ones. The modified chemical structure of both PGs could be associated with the degree of aggressiveness of laryngeal squamous cell carcinomas.

Transforming growth factor-beta as a key molecule triggering the expression of versican isoforms v0 and v1, hyaluronan synthase-2 and synthesis of hyaluronan in malignant osteosarcoma cells.

Versican, a large sized chondroitin-sulphate proteoglycan (PG), and its binding partner, hyaluronan (HA), are extracellular matrix (ECM) components that play an essential role in transformed cell behavior. Expression of certain versican isoforms has been implicated in cell migration and proliferation of cancer cells and, on the other hand, disruption of HA synthesis by inhibiting hyaluronan synthase-2 (HAS2) expression in osteosarcoma cells by suppressing cell proliferation, invasiveness and motility. Considering that growth factors, such as TGF-beta, bFGF and PDGF-BB, are important regulators for the expression of the ECM macromolecules, in this study we examined the effect of these growth factors on the expression of the various versican isoforms, HA synthases as well as HA synthesis by MG-63 osteosarcoma cells and normal human osteoblastic periodontal ligament cells (hPDL). Real-time PCR and metabolic labelling followed by fine HPLC analysis coupled to radiochemical detection were the methods utilized. It was found that, contrary to normal hPDL cells, osteosarcoma MG-63 cells do not constitutively express the versican isoforms V0 and V1. Exogenous addition of TGF-beta2 stimulated the versican transcript levels mainly by forcing osteosarcoma cells to express V1 and V0 isoforms. PDGF-BB and bFGF had only minor effects in these cells. In hPDL cells a strong stimulation of the V3 transcript by all growth factors was observed. TGF-beta2 was also the major stimulator of HAS2 isoform expression as well as hyaluronan synthesis in osteosarcoma cells, while PDGF-BB exerted dominant influence on HAS2 isoform expression and hyaluronan biosynthesis by osteoblasts. The obtained results show for the first time that TGF-beta2 triggers the malignant phenotype pattern of versican and hyaluronan expression in human osteosarcoma cells and indicate that this growth factor may account for the metastatic potential of these cells.

Immunization with specific polysaccharide antigen reduces alterations in corneal proteoglycans during experimental slime-producing Staphylococcus epidermidis keratitis.

PURPOSE: Staphylococcus epidermidis is a leading cause of bacterial keratitis associated with corneal damage. Corneal integrity is closely associated with matrix macromolecules, such as proteoglycans (PGs) and collagen. The aim of this study was to examine whether active immunization (AI) using a major immunogenic polysaccharide determinant of slime (20-kDa PS) as antigen, and passive immunization (PI) after administration of specific antibodies toward 20-kDa PS affect the distribution of PGs as well as corneal lesions in an experimental model of slime-producing S. epidermidis keratitis. METHODS: For AI, seven rabbits were immunized with 20-kDa PS, whereas for PI, seven rabbits received specific antibodies against 20-kDa PS. Lesions were graded clinically for a 21-day period. Levels of 20-kDa PS antibodies in serum and aqueous humor in both immunization groups were determined by ELISA. The distribution of certain extracellular matrix PGs during corneal healing was analyzed immunohistochemically. RESULTS: Levels of specific anti-20-kDa PS antibodies in serum and aqueous humor obtained after either AI or PI were significantly higher as compared with those in the respective nonimmunized control groups (p<0.001). Clinical grading showed that both AI and PI rabbits had a significantly less corneal damage as compared with infected nontreated rabbits. Immunohistochemical analyses for PGs exhibited significant differences to the wounded regions as compared with noninfected corneal tissue. Accumulation of keratan sulfate PGs and decorin was observed in the corneal stroma of infected rabbits and of heparan sulfate PGs around the new-formed vessels. This phenomenon was significantly reduced in immunized animals in accordance with macroscopically decreased corneal damage observed in these animals. CONCLUSIONS: Results of this study suggest a key role of 20-kDa PS and its antibodies as prophylactic and therapeutic agents in keratitis caused by slime-producing S. epidermidis.

Signal transduction by IL-2 and its receptors as target in treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic and destructive arthropathy with systemic features, the etiopathogenesis of which remains unclear. It is characterized by relapsing and remitting inflammation and hyperplasia of synovial cells. Proinflammatory cytokines, such as interleukin-2 (IL-2), play an important role in maintaining cartilage damage and severe destruction of the joints due to an uncontrolled activation of cellular immunity. An imbalance between proinflammatory and anti-inflammatory mediators is likely to contribute to the chronicity of the disease. Therefore, insight into the activation state of T-cells in different stages of the disease may be important to understand pathogenetic mechanisms underlying RA and could be a lead for the design of future therapeutic strategies. Because of the central role of the IL-2/IL-2 receptor (IL-2R) system in mediation of the immune system, monitoring and manipulation of this system has important diagnostic and therapeutic implications. New approaches in RA therapy with anticytokine agents, which block cytokines and their receptors, are now used as antirheumatic drugs in clinical practice.

Genistein suppresses the invasive potential of human breast cancer cells through transcriptional regulation of metalloproteinases and their tissue inhibitors.

Progression of breast cancer implicates the degradation of extracellular matrix (ECM) by metallo-proteinases (MMPs), a process with important consequences on the growth and invasiveness of cancer cells in adjacent and distant sites. The isoflavone, genistein--a natural inhibitor of protein tyrosine kinase pathway--inhibits the growth of a wide range of cancer cells in vitro. The aim of this study was to investigate: i) the expression of mRNAs encoded for MMPs and their endogenous inhibitors (TIMPs) associated with pathogenesis and metastatic potential of breast cancer cells; and ii) the effect of genistein on the transcription of MMPs and TIMPs and the invasive potential of breast cancer cells. Gene expression at transcriptional level was examined in cell cultures of two epithelial breast cancer cell lines, the high invasive (ER-negative) MDA-MB-231 and the low invasive (ER-positive) MCF-7, as well as the normal mammary cells (MCF-12A) following RNA isolation and reversed transcriptase polymerase chain reaction (RT-PCR). The inhibitory effect of genistein on functional invasiveness was examined by a cell invasion assay. Cell cycle distribution showed that genistein arrested breast cancer MDA-MB-231, MCF-7 and BT-20 cells in the G2/M phase. Both normal and breast cancer cell lines express the genes of MMP-2, -9, MT1-, MT2-, MT3-MMP and TIMP-1, -2 and -3. MCF-7 express notably less MMPs than MDA-MB-231 cell line. The addition of genistein resulted in down-regulation of the transcription of all MMP genes in MDA-MB-231 and most of MMPs in MCF-7 cells. The inhibitory effect of genistein on MMPs was functionally confirmed, since it significantly reduced the invasion properties of cancer cells in vitro. The obtained results indicate that genistein may be of great value in prevention of breast cancer cell metastasis, since it represents both a transcriptional modulator of genes involved in this pathogenetic process and a suppressor of breast cancer cell invasiveness.

STI571 as a potent inhibitor of growth and invasiveness of human epithelial breast cancer cells.

STI571, a specific tyrosine kinase inhibitor, exhibits a substantial therapeutic activity in patients with chronic myeloid leukaemia and gastrointestinal stromal tumors. In this study we examined the activity of STI571 on the growth and invasiveness of three human epithelial breast cancer cell lines of low (MCF-7) and high (ZR-75-1 and MDA-MB-231) invasive potential. Growth of all cell lines in serum-containing medium was significantly inhibited by STI571 in a dose-dependent manner, with an average IC50 of approximately 5-6 microM. Flow cytometric analysis revealed that this effect is characterized by an accumulation of all breast cancer cell types tested in the G2/M-phase of the cell cycle with a concomitant decrease of the percentage of cells in the S-phase. Interestingly, no increase in apoptosis was observed, indicating that the effect of this kinase inhibitor is cytostatic rather than cytotoxic. In addition, STI571 exerts a significant inhibition effect on the invasion of the highly invasive breast cancer cell lines ZR-75-1 and MDA-MB-231. These results encourage further preclinical investigations on the mechanisms underlying the inhibitory effects of STI571, which may be of great value in breast cancer treatment.

Expression of MMPs and TIMPs genes in human breast cancer epithelial cells depends on cell culture conditions and is associated with their invasive potential.

Growth and invasiveness of breast cancer cells in adjacent and distant sites is associated with the expression of metalloproteinases (MMPs), which are capable of degrading almost all extracellular matrix macromolecules of supporting stroma. In order to identify markers useful for monitoring breast cancer pathogenesis and metastatic potential, we examined the expression of mRNAs encoded for MMPs and their endogenous inhibitors (TIMPs) in a panel of four epithelial breast cancer cell lines of high (MDA-MB-231 and ZR-75-1) and low (MCF-7 and BT-20) metastatic potential, and their expression was compared with that of normal mammary cells (MCF-12A). Expression patterns were evaluated using cell cultures in serum-containing and serum-free media. Gene expression studies were performed following cell cultures, RNA isolation, reversed transcription and polymerase chain reaction. Both normal and breast cancer cells express MMPs and TIMPs at various levels, depending on cell type and culture conditions. Comparison of their mRNA levels from serum-containing media showed that MMP-9, MT2-MMP and TIMP-1 are highly expressed in all cancer cells as compared to normal ones, whereas MMP-1 and -7 are overexpressed only in breast cancer cells of high invasion potential. In serum-free cultures, the highly metastatic cells retain the overexpression profile for MMP-1 and -7. Furthermore, MT2-MMP and TIMP-1 were constitutively expressed and they can also be correlated with cancer cells, whereas constitutive expression of MMP-9 was similar in normal and cancer cells. The results of this study indicate that the expression of MMPs is dependent on the culture conditions, i.e. the growth factors present in serum-containing media. Furthermore, data suggest that, independently of cell culture conditions, the expression of MT2-MMP may be associated with malignant transformation of mammary cells and the overexpression of MMP-1 and -7 with the highly metastatic potential of epithelial breast cancer cells.

Versican undergoes specific alterations in the fine molecular structure and organization in human aneurysmal abdominal aortas.

Versican is the major matrix proteoglycan in aortic wall and participates in various biological functions of the tissue. In the present study the molecular characteristics of versican isolated from normal human aorta as well as those of versican expressed in aneurysmal aortic tissue were examined. Versican was isolated by combined anion-exchange and gel permeation chromatography and was further characterized by high-performance liquid chromatography, polyacrylamide gel electrophoresis and immunoblotting. In both tissues versican is exclusively substituted with chondroitin sulfate chains, in contrast to other human tissues where both chondroitin and dermatan sulfate chains are attached onto versican core proteins. Except for the significant decrease in the concentration of versican in the aneurysmal tissue, this PG undergoes specific alterations in the aneurysmal tissue. The molecular size of versican isolated from diseased tissue is decreased with a simultaneous increase in the ratio of glycosaminoglycan to protein in this tissue. The latter reflect the extensive fragmentation of versican in the diseased tissue and most probably the generation of shorter peptides enriched to glycosaminoglycan chains. Although the size of chondroitin sulfate chains is identical in both versican preparations, a significant increase in the percentage of 6-sulfated disaccharides is observed in chondroitin sulfate chains of versican in aneurysmal aortas, which is accompanied by decrease in 4-sulfated and non-sulfated units.

Keratan sulphate in cerebrum, cerebellum and brainstem of sheep brain.

Keratan sulphate was identified in sheep brain. We describe here the isolation and partial characterization of keratan sulphate from cerebrum, cerebellum and brainstem of young sheep brains. The galactosaminoglycan was isolated by using ion-exchange chromatography and gel filtration after exhaustive digestion with papain of the delipidated tissues, followed by alkaline borohydride degradation and chondroitinase ABC and heparinases I, II and III treatment. The material isolated by ion-exchange chromatography from each tissue was eluted as single but polydispersed peak from Sephadex G-75, with average molecular masses 8.4, 7.9 and 8.8 kDa for cerebrum, cerebellum and brainstem, respectively. Keratanase I and II totally degraded keratan sulphate from cerebrum and brainstem, but only partially that from cerebellum. The content of keratan sulphate was found to be about 215, 173 and 144 microg/g dry delipidated tissue for cerebrum, brainstem and cerebellum, respectively.