József Dudás

Proteoglycans and tumor progression: Janus-faced molecules with contradictory functions in cancer.

Understanding the details of the molecular mechanism of tumor dissemination revealed that several proteoglycan species are involved in the process but their role can be described as Janus-faced. One level of proteoglycan alterations is at the expression of their genes coding for the core protein. Characteristically, in progressing tumors two patterns emerged: loss or neoexpression of surface proteoglycans (PG) depending on the initial expression pattern of the cell type of origin. The situation is similarly complex concerning the changes of glycosaminoglycan (GAG) of the PG during tumor progression. This is due to the fact that the majority of PGs involved is hybrid molecule meaning that their core protein can be glycanated both with chondroitin and heparan sulfate. However, such an alteration in glycanation of PG may fundamentally change the function of the molecule, especially the one operating at the cell surface. Among the extracellular PGs, decorin emerged as inhibitor of progression while perlecan as a promoter of the process. Analysis of the available data indicate that during metastatization tumor cells must express at least one cell surface HSPG species from the syndecan-glypican-CD44v3 group. Furthermore, the HS-chain of these proteoglycan(s) carry important molecular signatures (suphution or epimerization patterns). Experimental data suggest that tumor cell surface heparan sulfate (PG) may provide a target for specific anti-metastatic interventions.

Effect of heparin and liver heparan sulphate on interaction of HepG2-derived transcription factors and their cis-acting elements: altered potential of hepatocellular carcinoma heparan sulphate.

Proteoglycan assembly in malignant tumours is subject to profound changes. The significance of these alterations is not well understood; especially, their role in nuclear regulation is a topic for debate. The capacity of heparin and liver carcinoma heparan sulphate (HS) to alter DNA-transcription factor interactions has been studied to provide further evidence concerning the regulatory potential of glycosaminoglycan (GAG) in the nucleus. Experiments both in vitro and in vivo indicated that heparin and HS are capable of inhibiting the interaction of transcription factors with their consensus oligonucleotide elements. Among five transcription factors studied, AP-1, SP-1, ETS-1 and nuclear factor kappaB proved to be sensitive to heparin and heparan sulphate, whereas TFIID was hardly inhibited in either in vitro or in vivo systems. Interestingly, HS from peritumoral liver was five times more effective than heparin. Liver carcinoma HS was less effective than liver HS, but its activity was comparable with that of heparin. These results indicate that the structural differences of GAG chains strongly influence their biological behaviour. The loss of their recognized functional activity in malignant tumours might promote the development of uncontrolled growth and gene expression favouring the neoplastic process.

Expression of decorin, transforming growth factor-beta1, tissue inhibitor metalloproteinase 1 and 2, and type IV collagenases in chronic hepatitis

Decorin is a small extracellular matrix proteoglycan. It binds and modulates transforming growth factor (TGF)-beta 1 action, the major stimulator of fibrogenesis. Its role in the pathogenesis of human liver cirrhosis is unknown. Therefore, we studied the relationship of the 2 proteins in normal human liver and in 43 chronic hepatitis and liver cirrhosis specimens. To understand the mechanism that maintains matrix deposition in stage IV hepatitis, we studied expression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2, as well as the activities of type IV collagenases. Gene expression was analyzed on messenger RNA and protein level by morphologic and biochemical approaches. Decorin proved to be an early marker of fibrogenesis, and its deposition increased parallel to that of TGF-beta 1 and to inflammatory activity. Liver fibrosis progressed despite high temporospatial expression of decorin with TGF-beta 1. Neither decorin nor TGF-beta 1 protein deposition increased further in cirrhosis with low inflammatory activity, suggesting that impaired extracellular matrix catabolism rather than active production plays a role in this stage. This possibility was supported by high message levels of metalloproteinase inhibitors, no 72-kd collagenase activities, and low 92-kd collagenase activities.

Upregulation of heme oxygenase-1 gene by turpentine oil-induced localized inflammation: involvement of interleukin-6.

Heme oxygenase-1 (HO-1) is the inducible isoform of an enzyme family responsible for heme degradation and was suggested to be involved in the acute phase response in the liver. However, the mechanisms of the HO-1 regulation under inflammatory conditions are poorly understood. Therefore, the purpose of the current work was to study the expression of HO-1 in the liver and other organs of rats with a localized inflammation after intramuscular injection of turpentine oil (TO). Since interleukin-6 (IL-6) is known to be a principal mediator of inflammation, the levels of this cytokine were also estimated in the animal model used. HO-1 and IL-6 expression was evaluated by Northern blot, in situ hybridization, Western blot, immunohistochemistry and enzyme-linked immunosorbent assay. In the liver and injured muscle, the HO-1 mRNA levels were dramatically increased 4–6u2009h after TO administration. HO-1 protein levels in the liver were elevated starting from 6–12u2009h after the treatment. In other internal organs such as the heart, kidney and large intestine, only a slight induction of HO-1 mRNA was observed. IL-6-specific transcripts appeared only in the injured muscle and were in accordance with serum levels of IL-6. In turn, temporal expression of IL-6 in the muscle and circulatory IL-6 levels correlated well with HO-1 expression in the liver and injured muscle. In the liver of control rats HO-1 protein was detected in Kupffer cells, while in TO-injected rats also hepatocytes became strongly HO-1 positive. Conversely, in the injured muscle, HO-1 immunoreactivity was attributed only to macrophages. Our data demonstrate that during localized inflammation HO-1 expression was rapidly and strongly induced in macrophages of injured muscle and in hepatocytes, and IL-6 derived from injured muscle seems to be responsible for the HO-1 induction in the liver.

Inhibition of DNA topoisomerase I activity by heparan sulfate and modulation by basic fibroblast growth factor.

Eukaryotic DNA topoisomerase I catalyzes changes in the superhelical state of duplex DNA by transiently breaking single strands thereby allowing relaxation of both positively and negatively supercoiled DNA. Topoisomerase I is a nuclear enzyme localized at active sites of transcription, and abnormal levels of the enzyme have been observed in a variety of neoplasms. Because the enzyme binds heparin and, given the presence of heparan sulfate within the nuclei of mammalian cells, we sought to investigate the interaction between topoisomerase I and sulfated glycosaminoglycans isolated from normal and neoplastic human liver. The results demonstrated that low concentrations (∼100 nM) of heparan sulfate from normal liver but not from its malignant counterpart effectively blocked relaxation of supercoiled DNA driven by either purified holoenzyme or topoisomerase I activity present in nuclear extracts of three malignant cell lines. Heparin acted at even lower (∼10 nM) concentrations. Moreover, we show that basic fibroblast growth factor could interfere with this heparan sulfate/heparin-driven inhibition and that both basic fibroblast growth factor and heparin-binding sites co-localized in the nuclei of U937 leukemic cells. Our results suggest that DNA topoisomerase I activity may be modulated in vivo by specific heparan sulfate moieties present in normal cells but markedly reduced or absent in their transformed counterparts.

Tumor cell and carcinoma-associated fibroblast interaction regulates matrix metalloproteinases and their inhibitors in oral squamous cell carcinoma

Co-culture of periodontal ligament (PDL) fibroblasts and SCC-25 oral squamous carcinoma cells (OSCC), results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs). Paracrin circuits between CAFs and OSCC cells were hypothesized to regulate the gene expression of matrix remodeling enzymes in their co-culture, which was performed for 7 days, followed by analysis of the mRNA/protein expression and activity of metalloproteinases (MMPs), their tissue inhibitors (TIMPs) and other relevant genes. Interleukin1-β, transforming growth factor-β1, fibronectin and αvβ6 integrin have shown to be involved in the regulation of the MMP and TIMP gene expression in co-culture of CAFs and tumor cells. In addition, these cells also cooperated in activation of MMP pro-enzymes. It is particularly interesting that the fibroblast-produced inactive MMP-2 has been activated by the tumor-cell-produced membrane-type 1 matrix metalloproteinase (MT1-MMP). The crosstalk between cancer- and the surrounding fibroblast stromal-cells is essential for the fine tuning of cancer cells invasivity.

Differential responses of fibroblasts, non-neoplastic epithelial cells, and oral carcinoma cells to low-level laser therapy

Low-level laser therapy (LLLT) is used in the treatment of chemoradiotherapy- or radiotherapy-induced oropharyngeal mucositis (ORM). In head and neck cancer, tumor cells may lie in the LLLT irradiation field, and LLLT might promote tumor progression. We therefore investigated the effect of LLLT on proliferation, cell cycle distribution, and apoptosis in a human oral carcinoma cell line (SCC-25), non-malignant epithelial cells (BEAS-2B), and fibroblasts in vitro. The cell lines were subjected to LLLT on three consecutive days for 15xa0min. Cell proliferation was assessed using the MTT assay, cell cycle distribution by flow cytometry and propidium-iodide DNA staining, and apoptosis using an Annexin V-FITC assay. Controls were sham-treated, but not exposed to the laser treatment. LLLT treatment resulted in increased fibroblast proliferation (pu2009<u20090.001), whereas decreased cell proliferation was observed after LLLT treatment of BEAS-2B (pu2009=u20090.003) and SCC-25 cells (pu2009<u20090.001). In SCC-25 cells, an increased percentage of S-phase cells and decreased percentage of G1-phase cells were observed (pu2009<u20090.001). Moreover, a proapoptotic effect of LLLT was observed in SCC-25 cells (pu2009=u20090.02). LLLT did not exhibit a tumor-promoting effect in this in vitro study.

Cytokine-induced neutrophil chemoattractant-1 is released by the noninjured liver in a rat acute-phase model

The source of serum cytokine-induced neutrophil chemoattractant (CINC-1) and consequences of its presence in the tissue of synthesis have not been clearly elucidated under acute-phase situation. To pursue this question, turpentine oil (TO) was intramuscularly injected into rats, and RNA and local protein levels of acute-phase cytokines and of CINC-1 were studied in the TO injected gluteal muscle, as well as in noninjured muscle, in the liver, kidney, lung and spleen. The serum levels of acute-phase mediators and of CINC-1 were measured together with total leukocyte subpopulations. Recruitment of inflammatory cells in muscle and in the other organs was investigated by quantitative immunohistochemical methods. The effect of acute-phase mediators, including interferon gamma (IFN-γ) on the synthesis of CINC-1 in cultured hepatocytes was also investigated at the RNA and protein level. We found that the sera of the TO-treated rats contained elevated levels of IL-6, IL-1β and CINC-1. Increased serum levels of IFN-γ were also observed not only in the injured muscle but also and to a higher extent in the liver. However, while neutrophils and mononuclear phagocytes were found in the injured muscle, no inflammatory cells were detected at the non-‘inflamed’ site, namely, the liver or in the other organs. In vitro, treatment of cultured hepatocytes with IL-1β led to elevated CINC-1 gene expression. This was true to a lesser extent upon IL-6 and tumor necrosis factor (TNF-α) exposure. Interestingly, IFN-γ did not effect CINC-1 gene expression. These results indicate that CINC-1 behaves as an acute-phase protein and its expression is inducible in hepatocytes. However, CINC-1-production in the liver does not lead to recruitment of inflammatory cells into the organ.

Tumor-produced, active Interleukin-1 β regulates gene expression in carcinoma-associated fibroblasts

Recently we described a co-culture model of periodontal ligament (PDL) fibroblasts and SCC-25 lingual squamous carcinoma cells, which resulted in conversion of normal fibroblasts into carcinoma-associated fibroblasts (CAFs), and in epithelial–mesenchymal transition (EMT) of SCC-25 cells. We have found a constitutive high interleukin-1β (IL1-β) expression in SCC-25 cells in normal and in co-cultured conditions. In our hypothesis a constitutive IL1-β expression in SCC-25 regulates gene expression in fibroblasts during co-culture. Co-cultures were performed between PDL fibroblasts and SCC-25 cells with and without dexamethasone (DEX) treatment; IL1-β processing was investigated in SCC-25 cells, tumor cells and PDL fibroblasts were treated with IL1-β. IL1-β signaling was investigated by western blot and immunocytochemistry. IL1-β-regulated genes were analyzed by real-time qPCR. SCC-25 cells produced 16 kD active IL1-β, its receptor was upregulated in PDL fibroblasts during co-culture, which induced phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1), and nuclear translocalization of NFκBα. Several genes, including interferon regulatory factor 1 (IRF1) interleukin-6 (IL-6) and prostaglandin-endoperoxide synthase 2 (COX-2) were induced in CAFs during co-culture. The most enhanced induction was found for IL-6 and COX-2. Treatment of PDL fibroblasts with IL1-β reproduced a time- and dose-dependent upregulation of IL1-receptor, IL-6 and COX-2. A further proof was achieved by DEX inhibition for IL1-β-stimulated IL-6 and COX-2 gene expression. Constitutive expression of IL1-β in the tumor cells leads to IL1-β-stimulated gene expression changes in tumor-associated fibroblasts, which are involved in tumor progression.

Remodeling of extracellular matrix by normal and tumor-associated fibroblasts promotes cervical cancer progression.

BackgroundComparison of tissue microarray results of 29 cervical cancer and 27 normal cervix tissue samples using immunohistochemistry revealed considerable reorganization of the fibrillar stroma of these tumors.Preliminary densitometry analysis of laminin-1, α-smooth muscle actin (SMA) and fibronectin immunostaining demonstrated 3.8-fold upregulation of laminin-1 and 5.2-fold increase of SMA in the interstitial stroma, indicating that these proteins and the activated fibroblasts play important role in the pathogenesis of cervical cancer. In the present work we investigated the role of normal and tumor-associated fibroblasts.MethodsIn vitro models were used to throw light on the multifactorial process of tumor-stroma interaction, by means of studying the cooperation between tumor cells and fibroblasts. Fibroblasts from normal cervix and cervical cancers were grown either separately or in co-culture with CSCC7 cervical cancer cell line. Changes manifest in secreted glycoproteins, integrins and matrix metallo-proteases (MMPs) were explored.ResultsWhile normal fibroblasts produced components of interstitial matrix and TGF-β1 that promoted cell proliferation, cancer-associated fibroblasts (CAFs) synthesized ample amounts of laminin-1. The following results support the significance of laminin-1 in the invasion of CSCC7 cells: 1.) Tumor-associated fibroblasts produced more laminin-1 and less components of fibrillar ECM than normal cells; 2.) The production of laminin chains was further increased when CSCC7 cells were grown in co-culture with fibroblasts; 3.) CSCC7 cells were capable of increasing their laminin production; 4.) Tumor cells predominantly expressed integrin α6β4 laminin receptors and migrated towards laminin. The integrin profile of both normal and tumor-associated fibroblasts was similar, expressing receptors for fibronectin, vitronectin and osteopontin. MMP-7 secreted by CSCC7 cells was upregulated by the presence of normal fibroblasts, whereas MMP-2 produced mainly by fibroblasts was activated in the presence of CSCC7 cells.ConclusionsOur results indicate that in addition to degradation of the basement membrane, invasion of cervical cancer is accomplished by the remodeling of the interstitial stroma, which process includes decrease and partial replacement of fibronectin and collagens by a laminin-rich matrix.