Effect of a hyaluronan synthase suppressor, 4-methylumbelliferone, on B16F-10 melanoma cell adhesion and locomotion

Hyaluronan (HA) is a ubiquitous, major component of the extracellular matrix. It is involved in cell adhesion and locomotion, and hence in tumor metastasis. We have previously reported that 4-methylumbelliferone (MU) inhibits HA synthesis and may be a useful tool for examining the functions of HA. We here demonstrate that the formation of cell surface HA by melanoma cells and its release into the culture medium are inhibited by MU. Adhesion and locomotion assays revealed that the adhesion and locomotion of melanoma cells were dose-dependently inhibited by MU. Conversely, treatment with exogenous HA enhanced both adhesion and locomotion. Thus, preventing the formation of cell surface HA reduced both the adhesion and locomotion of melanoma cells, suggesting that MU may act as an inhibitor of tumor metastasis.     

bFGF induces changes in hyaluronan synthase and hyaluronidase isoform expression and modulates the migration capacity of fibrosarcoma cells

Background

Hyaluronan (HA) a glycosaminoglycan, is capable of transmitting extracellular matrix derived signals to regulate cellular functions. In this study, we investigated whether the changes in HT1080 and B6FS fibrosarcoma cell lines HA metabolism induced by basic fibroblast growth factor (bFGF) are correlated to their migration.

Methods

Real-time PCR, in vitro wound healing assay, siRNA transfection, enzyme digestions, western blotting and immunofluorescence were utilized.

Results

bFGF inhibited the degradation of HA by decreasing hyaluronidase-2 expression in HT1080 cells (p = 0.0028), increased HA-synthase-1 and -2 expression as we previously found and enhanced high molecular weight HA deposition in the pericellular matrix. Increased endogenous HA production (p = 0.0022) and treatment with exogenous high molecular weight HA (p = 0.0268) correlated with a significant decrease of HT1080 cell migration capacity. Transfection with siHAS2 and siHAS1 showed that mainly HAS1 synthesized high molecular weight HA regulates HT1080 cell motility. Induced degradation of the HA content by hyaluronidase treatment and addition of low molecular weight HA, resulted in a significant stimulation of HT1080 cells' motility (p < 0.01). In contrast, no effects on B6FS fibrosarcoma cell motility were observed.

Conclusions

bFGF regulates, in a cell-specific manner the migration capability of fibrosarcoma cells by modulating their HA metabolism.HA metabolism is suggested to be a potential therapeutic target in fibrosarcoma.     

Different adhesion inhibiting activities of antisera against plasma membranes of liver and Morris hepatoma 7777

Cell-substratum adhesion of rat hepatocytes was inhibited by antisera raised against plasma membranes of liver (anti-liver antiserum) and Morris hepatoma 7777 (anti-hepatoma antiserum). Similar concentrations of both antisera inhibited adhesion on collagen. Anti-liver antiserum also inhibited the adhesion of hepatocytes on plastic, whereas anti-hepatoma antiserum was only able to inhibit the adhesion on collagen completely. These results suggest the existence of at least two different adhesion-involved molecules. Cells adhere to plastic by means of both molecules, whereas adhesion on collagen is mediated by only one of them. The results further suggest that hepatoma cells lost the molecule involved in adhesion on plastic.     

4-Methylumbelliferone inhibits hyaluronan synthesis by depletion of cellular UDP-glucuronic acid and downregulation of hyaluronan synthase 2 and 3

Hyaluronan accumulation on cancer cells and their surrounding stroma predicts an unfavourable disease outcome, suggesting that hyaluronan enhances tumor growth and spreading. 4-Methylumbelliferone (4-MU) inhibits hyaluronan synthesis and retards cancer spreading in experimental animals through mechanisms not fully understood. These mechanisms were studied in A2058 melanoma cells, MCF-7 and MDA-MB-361 breast, SKOV-3 ovarian and UT-SCC118 squamous carcinoma cells by analysing hyaluronan synthesis, UDP-glucuronic acid (UDP-GlcUA) content, and hyaluronan synthase (HAS) mRNA levels. The maximal inhibition in hyaluronan synthesis ranged 22-80% in the cell lines tested. Active glucuronidation of 4-MU produced large quantities of 4-MU-glucuronide, depleting the cellular UDP-GlcUA pool. The maximal reduction varied between 38 and 95%. 4-MU also downregulated HAS mRNA levels: HAS3 was 84-60% lower in MDA-MB-361, A2058 and SKOV-3 cells. HAS2 was the major isoenzyme in MCF-7 cells and lowered by 81%, similar to 88% in A2058 cells. These data indicate that both HAS substrate and HAS2 and/or HAS3 mRNA are targeted by 4-MU. Despite different target point sensitivities, the reduction of hyaluronan caused by 4-MU was associated with a significant inhibition of cell migration, proliferation and invasion, supporting the importance of hyaluronan synthesis in cancer, and the therapeutic potential of hyaluronan synthesis inhibition.     

Study of hyaluronan synthase inhibitor, 4-methylumbelliferone derivatives on human pancreatic cancer cell (KP1-NL)

The structure of 4-methylumbelliferone (MU) consists of coumarin with 4-methyl group and 7-hydroxy group. MU inhibits HA synthesis and pericellular HA matrix formation. In this study, we used 10 MU derivatives which have hydroxy groups and methyl groups at various positions of coumarin to investigate a more effective HA inhibitor than MU. First, human pancreatic cancer cell (KP1-NL) growth assay was analyzed by Alamar Blue to determine the non-toxic concentration of MU derivatives, and the inhibitory effect on HA synthesis in the cell cultures was analyzed by HA measuring kit. Next, cell surfaces of cancer cells were analyzed by particle-exclusion assay. In conclusion, both hydroxy and methyl groups are necessary for HA inhibition by MU, and two hydroxy groups inhibited HA synthesis more strongly than MU.     

Effect of cell growth rate and dose fractionation on chemically-induced ouabain-resistant mutations in Chinese hamster V79 cells.

Chinese hamster V79 cells were grown in medium containing either 10% or 2% FCS during the expression time following exposure to MNNG. The lower serum concentration was used to reduce the rate of cell replication, thereby allowing more time for DNA repair prior to "fixation" of the mutagenic lesion. In addition, fractionated and continuous exposures to MNNG and MAM, respectively, were carried out to determine their effect on the number of induced ouabain-resistant mutants. The results indicated that lowering the rate of cell growth effectively reduces the mutation frequency at low, but not at high doses of MNNG. Fractionated doses of MNNG result in a potentiation of their mutagenic effects compared to single doses. Also, continuous exposures to MAM result in an exponential increase in the mutation frequency. Collectively, these results suggest the importance of a repair process in Chinese hamster V79 cells which is dependent upon cell growth rate and the dose of the mutagen for its effectiveness.     

Recovery in the survival capacity of ultraviolet-irradiated 3T3 mouse cells at G0 cannot be solely dependent on the excision of pyrimidine dimers

Mouse cells (3T3 line) excised at most 20% of the pyrimidine dimers introduced into their DNA by a dose of short-wavelength ultraviolet radiation that allows a significant fraction of the cells to survive. When irradiation was delivered at the pre-replicative stage, a significant repair of lethal events was observed, as the cells progressed toward S phase. The recovery in survival cannot be accounted for solely by excision of pyrimidine dimers. Therefore, either another lesion produced by ultraviolet radiation is critical in terms of lethality, or the dimer, which may trigger the lethal event, becomes no longer an obstacle for the replication system after a certain period of time.     

The functional molecular mass of the Pasteurella hyaluronan synthase is a monomer

Hyaluronan (HA), a linear polysaccharide composed of β1,3-GlcNAc-β1,4-GlcUA repeats, is found in the extracellular matrix of vertebrate tissues as well as the capsule of several pathogenic bacteria. All known HA synthases (HASs) are dual-action glycosyltransferases that catalyze the addition of two different sugars from UDP-linked precursors to the growing HA chain. The bacterial hyaluronan synthase, PmHAS from Gram-negative Pasteurella multocida, is a 972-residue membrane-associated protein. Previously, the Gram-positive Streptococcus pyogenes enzyme, SpHAS (419 residues), and the vertebrate enzyme, XlHAS1 (588 residues), were found to function as monomers of protein, but the PmHAS is not similar at the protein sequence level and has quite different enzymological properties. We have utilized radiation inactivation to measure the target size of recombinant full-length and truncated PmHAS. The target size of HAS activity was confirmed using internal enzyme standards of known molecular weight. We found that the Pasteurella HA synthase protein functions catalytically as a monomer. Functional truncated soluble PmHAS also behaves as a polypeptide monomer as assessed by gel filtration chromatography and light scattering.     

Effects of culture conditions on glutathione content in A549 cells

The effects of varying culture conditions on glutathione content in A549 (human type II lung tumor derived) cells were examined. Parameters studied were growth time, serum concentration, and the presence or absence of a mixture of insulin, transferrin, and selenous acid. Glutathione content increased with serum concentration. When cells were grown with serum, glutathione increased sharply 24 hours after passage and decreased thereafter. Insulin, transferrin, and selenous acid had little effect on cell growth or glutathione content. Replacement of media with fresh media containing 10% serum did not prevent the growth dependent decrease in glutathione. These results demonstrate that glutathione content in A549 cells is strongly affected by culture conditions.     

Glycosaminoglycans mimetics potentiate the clonogenicity, proliferation, migration and differentiation properties of rat mesenchymal stem cells

Successful use of stem cell-based therapeutic products is conditioned by transplantation of optimized cells in permissive microenvironment. Mesenchymal stem cell (MSC) fates are tightly regulated by humoral factors, cellular interactions and extracellular matrix (ECM) components, such as glycosaminoglycans (GAG), which are complex polysaccharides with structural heterogeneity. During osteogenesis, a temporally controlled expression of particular GAG species is required to interact with specific growth promoting and differentiating factors to regulate their biological activities. As a comparative tool to study natural GAG, we used structurally and functionally related synthetic GAG mimetics. One of these compounds [OTR₄₁₂₀] was previously shown to stimulate bone repair in rat models. Here, we demonstrate that structurally distinct GAG mimetics stimulate differentially clonogenicity, proliferation, migration and osteogenic phenotype of MSC in vitro, according to their specific chemical signature, underlying the role of sulfate and acetyl groups in specific interactions with heparin binding factors (HBF). These effects are dependent on FGF-2 interactions since they are inhibited by a FGF receptor 1 signaling pathway blocker. These data suggest that the in vivo [OTR₄₁₂₀] bone regenerative effect could be due to its ability to induce MSC migration and osteogenic differentiation. To conclude, we provide evidences showing that GAG mimetics may have great interest for bone regeneration therapy and represent an alternative to exogenous growth factor treatments to optimize potential therapeutic properties of MSC.     

Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells

RNF8 is a nuclear protein having an N-terminal forkhead-associated (FHA) domain and a C-terminal RING-finger (RF) domain. Depletion of RNF8 caused cell growth inhibition and cell cycle arrest at not only S but also G2/M phases. In addition, cell death was frequently observed in RNF8-depleted cells. Analyses of time-lapse microscopy revealed that the cells died in mitosis and interphase. To elucidate the RNF8 function in M phase, the Plk1 content in RNF8-depleted cells was examined. The amount of RNF8 decreased time-dependently, whereas Plk1 reciprocally increased by transfection of RNF8 siRNA. Protein contents of RNF8 and Plk1 among various cell lines were also compared. RNF8 in normal cell lines was much higher than that in many cancer cell lines. Conversely, Plk1 in normal cell lines was lower than in cancer cell lines. These results suggest that RNF8 is downregulated in many cancer cells and inversely correlated with Plk1.     

Transferrin uptake in Trypanosoma cruzi is impaired by interference on cytostome-associated cytoskeleton elements and stability of membrane cholesterol, but not by obstruction of clathrin-dependent endocytosis

Transferrin uptake by Trypanosoma cruzi epimastigotes occurs mainly through the cytostome/cytopharynx. Here, we present evidences for the association of sterol-rich membrane domains with the transferrin endocytic site. Assays using pharmacological treatments to disrupt clathrin-coated pits and hinder caveolae formation showed no association between transferrin uptake and clathrin-dependent endocytosis, but indicated that cholesterol stability in membrane domains is essential for the endocytosis of transferrin. Furthermore, it was observed a connection between the integrity of cytoskeleton elements at the cytopharynx and the function of the cytostome. Our data show that T. cruzi epimastigotes depend on a specialized pathway for transferrin uptake, which is cholesterol-dependent, clathrin-independent, and closely associated with the structural stability of the cytostome/cytopharynx cytoskeleton.     

A Monomeric Photoconvertible Fluorescent Protein for Imaging of Dynamic Protein Localization

The use of green-to-red photoconvertible fluorescent proteins (FPs) enables researchers to highlight a subcellular population of a fusion protein of interest and to image its dynamics in live cells. In an effort to enrich the arsenal of photoconvertible FPs and to overcome the limitations imposed by the oligomeric structure of natural photoconvertible FPs, we designed and optimized a new monomeric photoconvertible FP. Using monomeric versions of Clavularia sp. cyan FP as template, we employed sequence-alignment-guided design to create a chromophore environment analogous to that shared by known photoconvertible FPs. The designed gene was synthesized and, when expressed in Escherichia coli, found to produce green fluorescent colonies that gradually switched to red after exposure to white light. We subjected this first-generation FP [named mClavGR1 (monomeric Clavularia-derived green-to-red photoconvertible 1)] to a combination of random and targeted mutageneses and screened libraries for efficient photoconversion using a custom-built system for illuminating a 10-cm Petri plate with 405-nm light. Following more than 15 rounds of library creation and screening, we settled on an optimized version, known as mClavGR2, that has eight mutations relative to mClavGR1. Key improvements of mClavGR2 relative to mClavGR1 include a 1.4-fold brighter red species, 1.8-fold higher photoconversion contrast, and dramatically improved chromophore maturation in E. coli. The monomeric status of mClavGR2 has been demonstrated by gel-filtration chromatography and the functional expression of a variety of mClavGR2 chimeras in mammalian cells. Furthermore, we have exploited mClavGR2 to determine the diffusion kinetics of the membrane protein intercellular adhesion molecule 1 both when the membrane is in contact with a T-lymphocyte expressing leukocyte-function-associated antigen 1 and when it is not. These experiments clearly establish that mClavGR2 is well suited for rapid photoconversion of protein subpopulations and subsequent tracking of dynamic changes in localization in living cells.     

Leishmania major: recruitment of Gr-1+ cells into draining lymph nodes during infection is important for early IL-12 and IFN gamma production

The production of interleukin-12 and interferon-γ is a key event for controlling leishmaniasis. Here, we tested the hypothesis that after murine infection with Leishmania major, cell migration into draining lymph nodes is crucial for early production of those cytokines. We showed that inflammatory cells carrying the marker of recently migrated cells, the Gr-1 antigen, including polymorphonuclear and mononuclear cells, migrate rapidly into the site of promastigote infection and, subsequently, into draining lymph nodes. Treatment with RB6-8C5 monoclonal antibody reduced local inflammation and migration of Gr-1+ cells into the draining lymph nodes. This reduction was associated with a decrease of interleukin-12 production by draining lymph node cells from BALB/c mice but not C57BL/6 mice. Additionally, interferon-γ was also reduced in both mouse strains after depletion of Gr-1+ cells, suggesting that these cells are important for early interleukin-12 and interferon-γ production. Our findings suggest that recently migrated myeloid cells, more than resident cells, are the major source of the early IL-12 production after L. major infection.     

Concanavalin A-mediated agglutinability of Balbc3T3 cells grown in media supplemented with different phosphatidylcholines

$\text{Balb}\text{c3T3}$ cells were grown near confluency in media supplemented with 10% fetal calf serum and than exposed for 24 hours to media containing different phosphatidylcholines bound to delipidated fetal calf serum. Compared to cells grown in regular media, 3T3 cells exposed to media containing dioleoyl-phosphatidylcholine dramatically increased their agglutinability by Concanavalin A. Exposure to several other phosphatidylcholines had no effect.