Dynamic interplay between breast cancer cells and normal endothelium mediates the expression of matrix macromolecules, proteasome activity and functional properties of endothelial cells.

BACKGROUND: Breast cancer-endothelium interactions provide regulatory signals facilitating tumor progression. The endothelial cells have so far been mainly viewed in the context of tumor perfusion and relatively little is known regarding the effects of such paracrine interactions on the expression of extracellular matrix (ECM), proteasome activity and properties of endothelial cells. METHODS: To address the effects of breast cancer cell (BCC) lines MDA-MB-231 and MCF-7 on the endothelial cells, two cell culture models were utilized; one involves endothelial cell culture in the presence of BCCs-derived conditioned media (CM) and the other co-culture of both cell populations in a Transwell system. Real-time PCR was utilized to evaluate gene expression, an immunofluorescence assay for proteasome activity, and functional assays (migration, adhesion and invasion) and immunofluorescence microscopy for cell integrity and properties. RESULTS: BCC-CM decreases the cell migration of HUVEC. Adhesion and invasion of BCCs are favored by HUVEC and HUVEC-CM. HA levels and the expression of CD44 and HA synthase-2 by HUVEC are substantially upregulated in both cell culture approaches. Adhesion molecules, ICAM-1 and VCAM-1, are also highly upregulated, whereas MT1-MMP and MMP-2 expressions are significantly downregulated in both culture systems. Notably, the expression and activity of the proteasome ß5 subunit are increased, especially by the action of MDA-MB-231-CM on HUVEC. CONCLUSIONS AND GENERAL SIGNIFICANCE: BCCs significantly alter the expression of matrix macromolecules, proteasome activity and functional properties of endothelial cells. Deep understanding of such paracrine interactions will help to design novel drugs targeting breast cancer at the ECM level. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

Heparan sulfate proteoglycans and heparin regulate melanoma cell functions.

BACKGROUND: The solid melanoma tumor consists of transformed melanoma cells, and the associated stromal cells including fibroblasts, endothelial cells, immune cells, as well as, soluble macro- and micro-molecules of the extracellular matrix (ECM) forming the complex network of the tumor microenvironment. Heparan sulfate proteoglycans (HSPGs) are an important component of the melanoma tumor ECM. Importantly, there appears to be both a quantitative and a qualitative shift in the content of HSPGs, in parallel to the nevi-radial growth phase-vertical growth phase melanoma progression. Moreover, these changes in HSPG expression are correlated to modulations of key melanoma cell functions. SCOPE OF REVIEW: This review will critically discuss the roles of HSPGs/heparin in melanoma development and progression. MAJOR CONCLUSIONS: We have correlated HSPGs' expression and distribution with melanoma cell signaling and functions as well as angiogenesis. GENERAL SIGNIFICANCE: The current knowledge of HSPGs/heparin biology in melanoma provides a foundation we can utilize in the ongoing search for new approaches in designing anti-tumor therapy. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

Could growth factor-mediated extracellular matrix deposition and degradation offer the ground for directed pharmacological targeting in fibrosarcoma?

The specific organization of the tumor extracellular matrix (ECM) is an intrinsic and basic step in the convoluted pathways of tumorigenesis. Fibrosarcoma is a rare, lethal, malignant tumor originating from fibroblasts, characterized by the formation of an abundant ECM. Fibroblastoid cells undergoing malignant transformation specifically alter composition and organization of their ECM to facilitate growth, survival and invasion. Fibrosarcoma cells were shown to have a high content and turnover of ECM components including hyaluronan, proteoglycans, collagens, fibronectin and laminin. Cell signaling by endogenous growth factors, such as TGFβ, EGF, FGF2, VEGF and IFG-I, is directly correlated to ECM remodeling, stroma formation and fibrosarcoma progression. In this regard, growth factors affect the expression of matrix macromolecules, such as secreted and cell-associated proteoglycans, hyaluronan and its receptors CD44 and RHAMM, as well as the expression and activity of matrix- degrading metalloproteinases, which are of critical importance in tissue remodeling and fibrosarcoma progression. Therefore, therapeutic approaches considering growth factors and their receptors as well as downstream signaling in human cancers may well be pharmacological targets being currently explored. In this article, we focus on growth factor signaling regulating fibrosarcoma cell ECM organization at the level of deposition and degradation of ECM macromolecules, the relation of ECM remodeling with fibrosarcoma cell malignant behaviour as well as the putative strategies for its therapeutic intervention.

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.

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.

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.

Insights into targeting colon cancer cell fate at the level of proteoglycans / glycosaminoglycans.

Proteoglycans (PGs), glycosaminoglycans (GAGs) and hyaluronan as a free GAG, have unique structural characteristics which enable them via specific interactions with matrix proteins and cell surface receptors to regulate key tumor cell functions and thus to affect cancer growth and progression. This article explores the many layers of interdependent signaling among transformed colon epithelial cells, neighboring stromal cells and their respective PGs / GAGs components along the insidious and often deadly route of colon cancer progression. Specifically addressed is the altered deposition of PGs / GAGs by colon cancer cells; the effects of these malignant cells on gene expression and biosynthesis of PGs / GAGs of the surrounding stromal cells and the signaling pathways involved, with the utmost goal to highlight potential therapeutic targets in the rapidly developing field of glycan-based therapy.

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.

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.

Monitoring serum chondroitin sulfate levels in patients submitted to coronary artery bypass surgery.

Glycosaminoglycans (GAGs) are functionally important molecules of the arterial wall and play a crucial role in atherogenesis. Chondroitin sulfate/dermatan sulfate proteoglycans (CS/DSPGs) participate in several biological events through their GAG chains, and are also involved in the development of atherosclerosis. The aim of this study was to compare the pre- and post-operative levels of CS in serum of patients after coronary artery bypass graft surgery using a highly sensitive reversed-polarity capillary electrophoresis method and to investigate the correlation of CS with common biochemical lipid markers. It was found that CS values were significantly higher for all patients post-operatively and, furthermore, CS levels were statistically correlated to apolipoprotein A and B levels. Notably, the pre-operational lipid profile of the patient may be indicative of the values of 4-sulfated CS post-operationally. Furthermore, the obtained results highlight the clinical significance of CS levels in serum, since they may provide complementary information for the latent inflammatory state of the patient.

The role of oligodendrocytes in the molecular pathobiology and potential molecular treatment of cervical spondylotic myelopathy.

Cervical spondylotic myelopathy (CSM) is a very common and debilitating disease; however, its underlying pathocellular process remains uncertain. Attempts have been made to reproduce CSM in experimental animal models in order to deepen the knowledge on the molecular pathobiology of this disease. The up-to-date observations have established the apoptosis of oligodendrocytes (OLGs) as the principal pathocellular process of CSM. Since favorable neurological recovery cannot be obtained in afflicted patients, even after the decompression surgery, elucidation of the apoptotic cascade in OLGs may unveil possible molecular treatments which could inhibit demyelination and ameliorate the neurological deficits. Moreover, additional therapeutic benefits may include improvement of myelin self-repair capability by stimulating OLG progenitor cells to become mature and finally, myelinating OLGs. This review focuses on the factors and mechanisms of crucial importance for developing antiapoptotic treatments. Critical evaluations of the role of OLGs in molecular pathobiology of CSM as well as strategies for potential remyelination of CSM are also provided. The analyses and evaluations of the experimental findings can possibly lead to treatment of CSM as well as to development of novel biopharmacenticals.

Heparin regulates colon cancer cell growth through p38 mitogen-activated protein kinase signalling.

OBJECTIVES: Heparin acts as an extracellular stimulus capable of activating major cell signalling pathways. Thus, we examined the putative mechanisms utilized by heparin to stimulate HT29, SW1116 and HCT116 colon cancer cell growth. MATERIALS AND METHODS: Possible participation of the mitogen-activated protein kinase (MAPK) cascade on heparin-induced HT29, SW1116 and HCT116 colon cancer cell growth was evaluated using specific MAPK cascade inhibitors, Western blot analysis, real-time quantitative PCR and FACS apoptosis analysis. RESULTS: Treatment with a highly specific p38 kinase inhibitor, SB203580, significantly (50-70%) inhibited heparin-induced colon cancer cell growth, demonstrating that p38 MAPK signalling is involved in their heparin-induced proliferative response. This was shown to be correlated with increased (up to 3-fold) phosphorylation of 181/182 threonine/tyrosine residues on p38 MAP kinase. Furthermore, heparin inhibited cyclin-dependent kinase inhibitor p21(WAF1/CIP1) and p53 tumour suppressor gene and protein expression up to 2-fold or 1.8-fold, respectively, and stimulated cyclin D1 expression up to 1.8-fold, in these cell lines through a p38-mediated mechanism. On the other hand, treatment with heparin did not appear to affect HT29, SW1116 and HCT116 cell levels of apoptosis. CONCLUSIONS: This study demonstrates that an extracellular glycosaminoglycan, heparin, finely modulates expression of genes crucial to cell cycle regulation through specific activation of p38 MAP kinase to stimulate colon cancer cell growth.

Targeting epidermal growth factor receptor in solid tumors: critical evaluation of the biological importance of therapeutic monoclonal antibodies.

Numerous cellular pathways have a significant impact in the growth and metastatic potential of tumors. Essential element of such pathways is the epidermal growth factor receptor (EGFR), a member of the HER family of receptor tyrosine kinases. One of the most important issues in cancer, which attracted the attention of clinical oncologists, is the potential use of targeted therapies. EGFR signaling pathway is implicated in the control of cell survival, proliferation, metastasis and angiogenesis. EGFR is, therefore, an appealing target for molecular-targeted cancer therapy as it is expressed in a variety of solid tumors (colorectal, breast, head and neck, etc.). Receptor antagonists that target EGFR have already been of high interest for a number of years. Multiple therapeutic strategies have been developed to target EGFR, including monoclonal antibodies (mAbs), tyrosine kinase inhibitors (TKIs), ligand-toxin conjugates, and antisense oligonucleotides. In particular, mAbs block ligand from binding to the extracellular domain of the receptor. Two mAbs that block EGFR (erbB1), cetuximab and panitumumab, have been approved by FDA. Cetuximab is a chimeric IgG1 anti-EGFR monoclonal antibody, whereas panitumumab is a fully human IgG2 anti-EGFR monoclonal antibody. This review highlights the cellular effects of EGFR blockade by mAbs and their relationship to therapeutic efficacy and biological significance.

Regulation of hyaluronan and versican deposition by growth factors in fibrosarcoma cell lines.

Versican, a large chondroitin sulphate proteoglycan and hyaluronan (HA), a non-sulphated glycosaminoglycan are major constituents of the pericellular matrix. In many neoplastic tissues, changes in the expression of versican and HA affect tumour progression. Here, we analyse the synthesis of versican and hyaluronan by fibrosarcoma cells, and document how the latter is affected by PDGF-BB, bFGF and TGFB2, growth factors endogenously produced by these cells. Fibrosarcoma cell lines B6FS and HT1080 were utilised and compared with normal lung fibroblasts (DLF). The major versican isoforms expressed by DLF and B6FS cells were V0 and V1. Treatment of B6FS cells with TGFB2 showed a significant increase of V0 and V1 mRNAs. Versican expression in HT1080 cells was not significantly affected by any of the growth factors. In addition, TGFB2 treatment increased versican protein in DLF cells. HA, showed approximately a 2-fold and a 9-fold higher production in DLF cells compared to B6FS and HT1080 cells, respectively. In HT1080 cells, HA biosynthesis was significantly increased by bFGF, whereas, in B6FS cells it was increased by TGFB2 and PDGF-BB. Furthermore, analysis of HA synthases (HAS) expression indicated that HT1080 expressed similar levels of all three HAS isoforms in the following order: HAS2> HAS3> HAS1. bFGF shifted that balance by increasing the abundance of HAS1. The major HAS isoform expressed by B6FS cells was HAS2. PDGF-BB and TGFB2 showed the most prominent effects by increasing both HAS2 and HAS1 isoforms. In conclusion, these growth factors modulated, through upregulation of specific HAS isoforms, HA synthesis, secretion and net deposition to the pericellular matrix.

Chondroitin sulfate prevents platelet derived growth factor-mediated phosphorylation of PDGF-Rbeta in normal human fibroblasts severely impairing mitogenic responses.

Platelet-derived growth factor (PDGF) is a major polypeptide mitogen for cells of mesenchymal origin such as fibroblasts. Chondroitin sulfate chains (CS), which are abundant in the extracellular matrix have been shown to physically interact with PDGF-BB modulating its biological function. The aim of the present study was to examine the involvement of CS on PDGF-BB induced proliferative responses and receptor activation in human lung fibroblasts. The addition of exogenous free CS chains caused a significant downregulation of the PDGF-BB mediated mitogenic and chemotactic responses. Similar results were obtained by the increase of endogenous CS biosynthesis after beta-D-xyloside treatment. Furthermore, removal of the membrane-bound CS chains by selective enzymatic treatment significantly increased the proliferative capacity of human fibroblasts. Analysis of PDGF-R phosphorylation in the presence of CS or beta-D-xyloside, revealed a reduction of PDGF-Rbeta phosphorylation in the tyrosine residue 1021. These results demonstrate, for the first time, that CS either soluble or surface bound downregulates the mitogenic responses of PDGF-BB in normal human lung fibroblasts through the reduction of PDGF-Rbeta phosphorylation.

Chondroitin sulfate and heparan sulfate-containing proteoglycans are both partners and targets of basic fibroblast growth factor-mediated proliferation in human metastatic melanoma cell lines.

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.

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.

Expression and distribution of N-acetyl and N-glycolylneuraminic acids in secreted and cell-associated glycoconjugates by two human osteosarcoma cell lines.

N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the dominant sialic acids (Sia) in mammals usually found in the non-reducing terminal of oligosaccharide side chains in glycoproteins and glycolipids. Their expression and distribution pattern have been correlated both with the malignant phenotype and tumor grade of human cancers. The aim of the present study was to determine by reversed-phase HPLC method the amounts of Neu5Ac and Neu5Gc as well as their distribution among the culture media and cell surface of MG-63 and Saos-2 human osteosarcoma cell lines of high and low metastatic potential. It was determined that MG-63 cells produce up to 5-fold more total sialic acid as compared with the Saos 2 cells. Neu5Ac accounts for ca 60% of the total sialic acids secreted by MG-63 cells, whereas Neu5Gc is the predominant sialic acid present on the MG-63 cell membrane. Saos 2 cells secrete considerable amounts of Neu5Ac to culture media. The obtained data indicate that the human osteosarcoma cells express both forms of Sia-containing glycoconjugates; the differences in the amounts of each of the two major Sia types and their distribution may be related to their differences in morphology and/or metastatic potentials.

Protein tyrosine kinase and estrogen receptor-dependent pathways regulate the synthesis and distribution of glycosaminoglycans/proteoglycans produced by two human colon cancer cell lines.

The soy isoflavone genistein can affect cell metabolism by specifically inhibiting protein tyrosine kinase (PTK) and/or interacting with the estrogen receptors (ERs). Glycosaminoglycans (GAG)/proteoglycans (PG) may participate in tumor development and progression. The synthesis of GAG by two human colon cancer cell lines, HT-29 and SW-1116, and the effects of genistein on their production and distribution between culture medium and cell membrane were studied. The mitogenic activity of genistein on both cell lines growth was also examined. Metabolic labeling, sensitive high pressure liquid chromatography (HPLC) techniques and fluorometric cell proliferation assays were utilized. The results demonstrate that both estrogen receptor beta-positive (ERbeta+) cancer cell lines produced hyaluronan (HA), both extracellular and membrane-associated galactosaminoglycans (GalAG) and heparan sulfate (HS), with the HT-29 cells producing all GAG fractions at significantly higher rates. The observed dose-dependent inhibitory effect of genistein on the synthesis of both secreted and cell-associated GAG/PG by the SW-1116 cells, as well as on their growth, was suggestive of a PTK mechanism. On the other hand, the synthesis of GAGs/PGs by HT-29 cells in the presence of genistein was dependent on their type and localization which implies the active participation of the ERs, which was further supported by the observed growth stimulation at low concentrations of genistein.