Johanna Chluba

SMYD3 Promotes Cancer Invasion by Epigenetic Upregulation of the Metalloproteinase MMP-9

Upregulation of the matrix metalloproteinase (MMP)-9 plays a central role in tumor progression and metastasis by stimulating cell migration, tumor invasion, and angiogenesis. To gain insights into MMP-9 expression, we investigated its epigenetic control in a reversible model of cancer that is initiated by infection with intracellular Theileria parasites. Gene induction by parasite infection was associated with trimethylation of histone H3K4 (H3K4me3) at the MMP-9 promoter. Notably, we found that the H3K4 methyltransferase SMYD3 was the only histone methyltransferase upregulated upon infection. SMYD3 is overexpressed in many types of cancer cells, but its contributions to malignant pathophysiology are unclear. We found that overexpression of SMYD3 was sufficient to induce MMP-9 expression in transformed leukocytes and fibrosarcoma cells and that proinflammatory phorbol esters further enhanced this effect. Furthermore, SMYD3 was sufficient to increase cell migration associated with MMP-9 expression. In contrast, RNA interference-mediated knockdown of SMYD3 decreased H3K4me3 modification of the MMP-9 promoter, reduced MMP-9 expression, and reduced tumor cell proliferation. Furthermore, SMYD3 knockdown also reduced cellular invasion in a zebrafish xenograft model of cancer. Together, our results define SMYD3 as an important new regulator of MMP-9 transcription, and they provide a molecular link between SMYD3 overexpression and metastatic cancer progression.

Leukemic cell xenograft in zebrafish embryo for investigating drug efficacy

Zebrafish were proposed as an alternative to mammalian models to assess the efficacy and toxicity of antileukemic drugs. Due to the limited number of transgenic zebrafish leukemia models, we explored human leukemic cell xenograft in zebrafish embryos. Human leukemic cell lines and blast cells sorted from patients with acute myelogenous leukemia were injected 48 hours post-fertilization and remained in the circulation of zebrafish embryos for several days without affecting their development. Imatinib and oxaphorines did not demonstrate any toxicity on normal zebrafish embryos and decreased the leukemic burden in animals xenografted with sensitive leukemic cell lines. Two other molecules, all-trans retinoic acid and the translation inhibitor 4EGI-1, demonstrated teratogenic effects at concentrations shown to be efficient in vitro, which precluded investigation of their antileukemic activity in such models. Altogether, xenografted leukemic cells in zebrafish embryos are a pharmacologically relevant model for screening non-teratogenic drugs.

Polyelectrolyte multilayer films modulate cytoskeletal organization in chondrosarcoma cells

The aim of this study was to evaluate polyelectrolyte multilayer films as interfaces for implants. Polyelectrolyte multilayers were built up with different terminating layers by alternate deposition of oppositely charged polyelectrolytes on which chondrosarcoma (HCS-2/8) cells were grown in the presence of serum. Films formed by an increasing number of layers were investigated. The terminating layer was made of one of the following polyelectrolytes: poly-sodium-4-styrenesulfonate (PSS), poly-L-glutamic acid (PGA), poly-allylamine hydrochloride (PAH), or poly(L-lysine) (PLL). Cell viability, inflammatory response, adherence, and cytoskeletal organization were studied. Induction of interleukin-8 (IL-8) secretion was detected on PAH and PLL ending polyelectrolyte films. Early cellular adherence was enhanced with PGA, PAH, PLL, and, to a lower extent, PSS terminating layers. Adherence was independent of the number of layers constituting the films. The presence of actin filaments and vinculin focal adhesion spots was observed on PSS or PAH ending films. They were respectively partially and totally absent on PGA and PLL terminating multilayer architectures. For PLL ending films, vinculin and actin organization was clearly dependent on the number of deposited layers. The results of this study suggest that PSS ending multilayered films constitute a good interfacial micro-environment at the material surface for HCS-2/8 cells.

The biofilm mode of life boosts the anti-inflammatory properties of Lactobacillus

The predominant form of life for microorganisms in their natural habitats is the biofilm mode of growth. The adherence and colonization of probiotic bacteria are considered as essential factors for their immunoregulatory function in the host. Here, we show that Lactobacillus casei ATCC334 adheres to and colonizes the gut of zebrafish larvae. The abundance of pro‐inflammatory cytokines and the recruitment of macrophages were low when inflammation was induced in probiotic‐fed animals, suggesting that these bacteria have anti‐inflammatory properties. We treated human macrophage‐differentiated monocytic THP‐1 cells with supernatants of L. casei ATCC334 grown in either biofilm or planktonic cultures. TNF‐α production was suppressed and the NF‐κB pathway was inhibited only in the presence of supernatants from biofilms. We identified GroEL as the biofilm supernatant compound responsible, at least partially, for this anti‐inflammatory effect. Gradual immunodepletion of GroEL demonstrated that the abundance of GroEL and TNF‐α were inversely correlated. We confirmed that biofilm development in other Lactobacillus species affects the immune response. The biofilms supernatants of these species also contained large amounts of GroEL. Thus, our results demonstrate that the biofilm enhances the immunomodulatory effects of Lactobacillus sp. and that secreted GroEL is involved in this beneficial effect.

Survival analysis of rats implanted with porous titanium tracheal prosthesis

BACKGROUND Surgical treatment of a malignancy in the trachea may lead to a long resection that has to be reconstructed with an artificial prosthesis. However, most of the available prostheses encounter inflammatory rejection and mechanical constraint problems. To improve tracheal rehabilitation a porous titanium prosthesis was developed. The aim of this study was to test the biocompatibility of this novel material. METHODS Seventeen rats had a partial tracheal prosthesis made of porous titanium inserted in the cervical trachea. The histologic analysis of the tissue surrounding the prosthesis was performed in 11 surviving animals after a period of 15 to 41 days. RESULTS Fibroblast colonization of titanium pores and a ciliary cylindrical epithelial layer developed on the endoluminal side of the prosthesis and the inflammatory reaction was minimal. CONCLUSIONS The results of this short-term study validate, from surgical and histologic standpoints, the usefulness of a porous titanium tracheal prosthesis.

Biofilms of Lactobacillus plantarum and Lactobacillus fermentum: Effect on stress responses, antagonistic effects on pathogen growth and immunomodulatory properties.

Few studies have extensively investigated probiotic functions associated with biofilms. Here, we show that strains of Lactobacillus plantarum and Lactobacillus fermentum are able to grow as biofilm on abiotic surfaces, but the biomass density differs between strains. We performed microtiter plate biofilm assays under growth conditions mimicking to the gastrointestinal environment. Osmolarity and low concentrations of bile significantly enhanced Lactobacillus spatial organization. Two L. plantarum strains were able to form biofilms under high concentrations of bile and mucus. We used the agar well-diffusion method to show that supernatants from all Lactobacillus except the NA4 isolate produced food pathogen inhibitory molecules in biofilm. Moreover, TNF-α production by LPS-activated human monocytoid cells was suppressed by supernatants from Lactobacillus cultivated as biofilms but not by planktonic culture supernatants. However, only L. fermentum NA4 showed anti-inflammatory effects in zebrafish embryos fed with probiotic bacteria, as assessed by cytokine transcript level (TNF-α, IL-1β and IL-10). We conclude that the biofilm mode of life is associated with beneficial probiotic properties of lactobacilli, in a strain dependent manner. Those results suggest that characterization of isolate phenotype in the biofilm state could be additional valuable information for the selection of probiotic strains.

The Impact of Tumor Nitric Oxide Production on VEGFA Expression and Tumor Growth in a Zebrafish Rat Glioma Xenograft Model

To investigate the effect of nitric oxide on tumor development, we established a rat tumor xenograft model in zebrafish embryos. The injected tumor cells formed masses in which nitric oxide production could be detected by the use of the cell-permeant DAF-FM-DA (diaminofluorophore 4-amino-5-methylamino-2’-7’-difluorofluorescein diacetate) and DAR-4M-AM (diaminorhodamine-4M). This method revealed that nitric oxide production could be co-localized with the tumor xenograft in 46% of the embryos. In 85% of these embryos, tumors were vascularized and blood vessels were observed on day 4 post injection. Furthermore, we demonstrated by qRT-PCR that the transplanted glioma cells highly expressed Nos2, Vegfa and Cyclin D1 mRNA. In the xenografted embryos we also found increased zebrafish vegfa expression. Glioma and zebrafish derived Vegfa and tumor Cyclin D1 expression could be down regulated by the nitric oxide scavenger 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide or CPTIO. We conclude that even if there is a heterogeneous nitric oxide production by the xenografted glioma cells that impacts Vegfa and Cyclin D1 expression levels, our results suggest that reduction of nitric oxide levels by nitric oxide scavenging could be an efficient approach to treat glioma.

zHSF1 modulates zper2 expression in zebrafish embryos

ABSTRACT HSF1 is a transcription factor that plays a key role in circadian resetting by temperature. We have used zebrafish embryos to decipher the roles of zHsf1, heat and light on zper2 transcription in vivo. Our results show that heat shock (HS) stimulated zper2 expression in the dark but has no cumulative effect combined with light. After light exposition, zper2 expression was 2.7 fold increased threefold in the hsf1-morphants in comparison to control embryos. Our results show that zHsf1 plays a positive role in HS-driven expression of zper2 in the dark but seems to act as an attenuator in the presence light.