Microscopic structural study of collagen aging in isolated fibrils using polarized second harmonic generation

International audiencePolarization resolved second harmonic generation (PSHG) is developed to study, at the microscopic scale, the impact of aging on the structure of type I collagen fibrils in two-dimensional coatings. A ribose-glycated collagen is also used to mimic tissue glycation usually described as an indicator of aging. PSHG images are analyzed using a generic approach of the molecular disorder information in collagen fibrils, revealing significant changes upon aging, with a direct correlation between molecular disorder and fibril diameters

3D collagen type I matrix inhibits the antimigratory effect of doxorubicin

<p>Abstract</p> <p>Background</p> <p>The cell microenvironment, especially extracellular matrix proteins, plays an important role in tumor cell response to chemotherapeutic drugs. The present study was designed to investigate whether this microenvironment can influence the antimigratory effect of an anthracycline drug, doxorubicin, when tumor cells are grown in a matrix of type I collagen, a three-dimensional (3D) context which simulates a natural microenvironment.</p> <p>Methods</p> <p>To this purpose, we studied the migratory parameters, the integrin expression, and the activation state of focal adhesion kinase (FAK) and GTPase RhoA involved in the formation of focal adhesions and cell movement. These parameters were evaluated at non toxic concentrations which did not affect HT1080 cell proliferation.</p> <p>Results</p> <p>We show that while doxorubicin decreased cell migration properties by 70% in conventional two-dimensional (2D) culture, this effect was completely abolished in a 3D one. Regarding the impact of doxorubicin on the focal adhesion complexes, unlike in 2D systems, the data indicated that the drug neither affected β1 integrin expression nor the state of phosphorylation of FAK and RhoA.</p> <p>Conclusion</p> <p>This study suggests the lack of antiinvasive effect of doxorubicin in a 3D environment which is generally considered to better mimic the phenotypic behaviour of cells <it>in vivo</it>. Consistent with the previously shown resistance to the cytotoxic effect in a 3D context, our results highlight the importance of the matrix configuration on the tumor cell response to antiinvasive drugs.</p

The effect of collagen ageing on its structure and cellular behaviour

Collagen is the most important component in extracellular matrix (ECM) and plays a pivotal role in individual tissue function in mammals. During ageing, collagen structure changes, which can detrimentally affect its biophysical and biomechanical properties due to an accumulation of advanced glycation end-products (AGEs). AGEs have been linked to non-enzymatic cross-linking of proteins resulting in the alteration of mechanical properties of the tissue. In this study we investigate the influence of different aged collagens on the mechanical and contractile properties of reconstituted hydrogel constructs seeded with corneal stromal fibroblasts. A non-destructive indentation technique and optical coherence tomography (OCT) are used to determine the elastic modulus and dimensional changes respectively. It is revealed that the youngest collagen constructs have a higher elastic modulus and increased contraction compared to the older collagen. These results provide new insights into the relationship between collagen molecular structures and their biomechanical properties

A microscopic and macroscopic study of aging collagen on its molecular structure, mechanical properties, and cellular response

During aging, collagen structure changes, detrimentally affecting tissues' biophysical and biomechanical properties due to an accumulation of advanced glycation end-products (AGEs). In this investigation, we conducted a parallel study of microscopic and macroscopic properties of different-aged collagens from newborn to 2-yr-old rats, to examine the effect of aging on fibrillogenesis, mechanical and contractile properties of reconstituted hydrogels from these collagens seeded with or without fibroblasts. In addition to fibrillogenesis of collagen under the conventional conditions, some fibrillogenesis was conducted alongside a 12-T magnetic field, and gelation rate and AGE content were measured. A nondestructive indentation technique and optical coherence tomography were used to determine the elastic modulus and dimensional changes, respectively. It was revealed that in comparison to younger specimens, older collagens exhibited higher viscosity, faster gelation rates, and a higher AGE-specific fluorescence. Exceptionally, only young collagens formed highly aligned fibrils under magnetic fields. The youngest collagen demonstrated a higher elastic modulus and contraction in comparison to the older collagen. We conclude that aging changes collagen monomer structure, which considerably affects the fibrillogenesis process, the architecture of the resulting collagen fibers and the global network, and the macroscopic properties of the formed constructs

Resvératrol et cancer. Etude du mécanisme d'action antitumoral du resvératrol au niveau cellulaire. Intérêt en chimiothérapie et production

REIMS-BU Santé (514542104) / SudocSudocFranceF

Microenvironnement cellulaire et réponse de la cellule tumorale au médicament (impact du microenvironnement sur les propriétés anti-migratoires des anthracyclines)

REIMS-BU Santé (514542104) / SudocSudocFranceF

Inhibitory Effect of Nitric Oxide on Chemically Induced Differentiation of Human Leukemic K562 Cells

International audienceThe effect of nitric oxide (NO) was investigated in the human K562 cell line during chemically induced erythroid differentiation. Butyric acid (BA) and the anthracycline antitumour drugs aclarubicin (ACLA) and doxorubicin (DOX) were used as differentiating agents. In all cases, cell hemoglobinization was dose dependently inhibited by NO donors such as sodium nitroprusside (SNP). A 50% inhibition of cell differentiation was obtained with 25 M SNP, which generated less than 2 M nitrite in 3-day culture media. Increasing SNP concentrations led to higher nitrite accumulation (up to 12 M with 1 mM SNP) and total inhibition of cell hemoglobinization, but did not have a significant effect on cell proliferation. As shown by Northern blotting, high concentrations of SNP (1 mM) reduced the expression of-globin and porphobilinogen deaminase, but did not change GATA-1 and NF-E2 mRNA levels in ACLA-and BA-treated cells. In contrast, hemin-induced erythroid differentiation was not affected by the presence of NO donors. Altogether, these results show that NO is able to inhibit cell differentiation induced by some (ACLA, DOX, BA), but not all (hemin), agents. The inhibitory effect of NO seems to take place downstream of the regulation of erythroid gene expression. BIOCHEM PHARMACOL 58;5:773-778, 1999. © 1999 Elsevier Science Inc. KEY WORDS. anthracyclines; butyric acid; erythroid differentiation; hemin; nitric oxide Erythroid differentiation of the human K562 leukemic cell line can be achieved by exposure to several pharmacological agents including hemin [1], BA † [2], and anthra-cyclines such as ACLA and DOX [3]. The molecular mechanisms involved in the differentiation process are quite different depending on the inducer. The transcrip-tional activation of the-globin gene has been demonstrated in BA-and ACLA-induced K562 cells [4, 5]. This has been related to the enhancement of DNA-binding activity and the expression of GATA-1 and NF-E2 transcription factors [6-9]. In addition, the heme synthesis pathway enzymes PBGD [5, 8], erythroid-aminolevulinate dehydratase [10, 11], and eALAS [12] were also shown to be up-regulated at the transcriptional level in response to ACLA and BA. In contrast, DOX-and hemin-induced differentiation did not involve the overexpression of GATA-1 and NF-E2 transcription factors [5, 7]. Moreover, the transcriptional activity of erythroid promoters, which is enhanced by BA and ACLA, is not affected by DOX and hemin [13]. Nitric oxide is synthesized by NO synthases in endothe-lial and inflammatory-activated cells [14]. NO has been demonstrated to modulate the RNA binding of IRP-1 and IRP-2 iron responsive element (IRE) binding proteins [15, 16], which are responsible for the posttranscriptional control of IRE-containing mRNAs related to cellular iron homeostasis (e.g. ferritin, transferrin receptor, and eALAS [16]). Therefore, the study of NO effects on erythroid-specific genes has been mainly restricted to iron homeo-stasis-related genes [17-19]. In addition, NO may disturb cell growth and differentiation by regulating the activity of cellular targets involved in signal transduction, ATP, and oxygen production. Indeed, NO activates the soluble guanylate cyclase and inhibits iron-cluster enzymes including aconitase and mitochondrial complex I and II enzymes [20]. In this work, the effect of NO on chemically induced erythroid differentiation was investigated in K562 cells using NO donors such as SNP, SIN-1, and SNAP. The release of NO from NO donors was shown to inhibit the differentiation of anthracycline-and BA-treated K562 cells with an IC 50 of 25 M for SNP and less than 100 M for SIN-1 and SNAP. Higher doses of SNP were needed to decrease the expression of erythroid mRNAs. In contrast, hemin-induced differentiation was not affected by NO

Activité antiinvasive des anthracyclines antitumorales : effets de l'aclacinomycine sur les propriétés migratoires et invasives des cellules de fibrosarcome humain HT-1080

@Nous avons étudié les effets des anthracyclines antitumorales, en particulier l'aclacinomycine, sur les capacités migratoires et invasives de cellules à fort pouvoir invasif, la lignée de fibrosarcome humain HT-1080. Nous avons démontré que l'aclacinomycine et dans une moindre mesure la doxorubicine inhibent la migration et l'invasion des cellules HT-1080 à des concentrations n'affectant pas la croissance cellulaire. Cette inhibition des propriétés invasives n'implique pas des mécanismes de dégradation protéolytique de la matrice extracellulaire. Par contre, elle met en jeu des modifications au niveau des protéines du cytosquelette et des plaques d'adhésion focale. Nous avons observé un défaut de polarisation cellulaire, une altération des lamellipodes et de son réseau d'actine . En particulier, l'effet antiinvasif est associé à une diminution de l'expression des intégrines b1 et de leur état d'affinité. On note également une diminution de l'expression des tyrosines kinases FAK et Src et l'altération de leur état de phosphorylation. L'ensemble des résultats suggère que l'aclacinomycine pourrait être utilisée en clinique humaine à d'autres fins que son pouvoir cytotoxique.@Aclacinomycin (Aclarubicinâ) is a trisaccharide anthracycline anticancer drug active against a wide variety of solid tumors and haematological malignancies. We have evaluated its antimigrative and antiinvasive properties in a Boyden chamber with or without Matrigel and in wound repair assays. Aclacinomycin was demonstrated to inhibit HT-1080 cell migration and invasion while being more potent than the classical anthracycline doxorubicin. This decrease occurred in a dose-dependent manner and without affecting cell proliferation. Importantly, the antiinvasive effect was not associated to a modification in the production of the matrix-degrading enzymes MMP-2 and MMP-9 but rather to changes in cytoskeletal and focal contact formation. Indeed, the drug reduces cell polarity, impairs the actin-mediated membrane ruffling at the leading edge and decreases b1 integrin expression and activation. Dramatic alterations in the distribution of vinculin and in the expression and phosphorylation state of both FAK and Src kinases were also detected. As a conclusion, these data suggest a novel application for this chemotherapeutic agent due to its ability to reduce tumor cell invasion. Combination of aclacinomycin with MMP inhibitors could have therapeutic potential in preventing tumor metastasis.REIMS-BU Santé (514542104) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF