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International audience; Background Cytotoxic efficacy of anticancer drugs has been widely studied with monolayer-cultured cancer cells. However, the efficacy of drugs under two-dimensional (2D) culture condition usually differs from that of three-dimensional (3D) one. In the present study, an in vitro tumor tissue model was constructed using alginate hydrogel, and in vitro cytotoxic efficacy of two anticancer drugs (cisplatin and DZNep) was investigated in chondrosarcomas, and compared to in vivo response. Methods Three cell lines derived from human chondrosarcomas, CH2879, JJ012 and SW1353, were embedded in alginate hydrogel. Proliferation and survival were assayed by ATP measurement using Cell Titer-Glo luminescent cell viability assay kit, and by counting viable cells in beads. Collagen and COMP expression was determined by RT-PCR. Invasion/migration was estimated by counting cells leaving alginate beads and adhering to culture dish. Then, chondrosarcoma response to cisplatin and DZNep was compared between cells cultured in monolayer or embedded in alginate, and using chondrosarcoma xenografts in nude mice. Results Chondrosarcomas survived at least for 8 weeks, after embedment in alginate. However, only CH2879 cells could proliferate. Also, this cell line is more invasive than SW1353 and JJ012, which was coherent with the grade of their respective primary tumors. Furthermore, the expression of type II collagen was higher in chondrosarcomas cultured in 3D than in 2D. Interestingly, this 3D culture system allows to validate the absence of response of chondrosarcomas to cisplatin, and to predict the efficiency of DZNep to reduce chondrosarcoma growth in vivo. Conclusions This study validates alginate beads as a relevant 3D model to study cancer biology and tumor responses to biological treatments.

International audience; Background/Aims: 3-Deazaneplanocin, DZNep, has been reported to inhibit the EZH2 histone methylase and to induce cell apoptosis in chondrosarcomas (CS). The present study aims to confirm the therapeutic potential of EZH2 inhibitors and investigate the molecular mechanisms of DZNep in chondrosarcomas.Methods: CS cell lines and primary cultures were used. Apoptosis was investigated using PARP cleavage, caspase 3/7 activity, or Apo2.7 expression. S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM) were quantified by UHPLC-MS/MS. Differentially expressed genes in treated-chondrosarcomas and chondrocytes were researched by microarray analysis.Results: DZNep induced apoptosis in chondrosarcomas both in vivo and in vitro. However, this effect was not correlated to EZH2 expression nor activity, and EZH2 knock-down by siRNA did not reduce CS viability. Additionally, the reduction of H3K27me3 induced by GSK126 or tazemetostat (EPZ-6438) did not provoke chondrosarcoma death. However, as expected, DZNep induced SAH accumulation and reduced SAM:SAH ratio. Further, microarray analysis suggests a key role of EGFR in antitumoral effect of DZNep, and pharmacological inhibition of EGFR reduced chondrosarcoma survival.Conclusion: EZH2 is not an adequate target for chondrosarcoma treatment. However, DZNep induces apoptosis in chondrosarcomas in vitro and in vivo, by a mechanism likely mediated though EGFR expression. Consequently, it would be worth initiating clinical trials to evaluating efficiency to S-adenosylhomocysteine hydrolase or EGFR inhibitors in patients with chondrosarcomas.

International audience; Cancer patients display cognitive impairment due, at least partly, to the treatments. Additionally, chemotherapeutic treatments can lead to organ injury, limiting their use, and are likely to have negative impacts on patients' quality of life. The aim of this study was to investigate the toxicity of 3-Deazaneplanocin A (DZNep) on several tissues and organs, as well as on cognitive functions. DZNep is an inhibitor of S-adenosylmethionine-dependent methyltransferase (in particular of the histone methyltransferase EZH2) which showed antitumoral functions in preclinical trials but whose effects on behavior and on organs (side effects) are not known. Chronic injections of DZNep were performed intraperitoneally in male NMRI mice (2 mg/kg; i.p.; three times per week) during 8 weeks. A follow-up of body weight was assessed during all experiments. Histological analysis were performed on several organs. EZH2 expression and H3K27me3 were assayed by western-blot. Several behavioral tests were performed during treatment and 2 weeks after. A particular focus was made on spontaneous locomotor activity, cognitive functions (spontaneous alternation and recognition memory), and anxiety- and depression-related behavior. Hematological modifications were also assessed. Chronic DZNep treatment transiently reduced animal growth. It had no effect on most organs but provoked a reversible splenomegaly, and persistent testis reduction and erythropoiesis. DZNep administration did not alter animal behavior. In conclusion, this study is encouraging for the use of DZNep for cancer treatment. Indeed, it has no effect on animal behavior, conferring an advantageous safety, and induces irreversible side effects limited on testis which are unfortunately found in most chemotherapy treatments.

METHODS. Inflammation was mimicked in vitro by treatment of human articular chondrocytes with interleukin-1β, and in synoviocytes with LPS. Effects of a treatment with a copolymer of fatty acids (ara 3000 beta®) were determined by evaluating MMP production by RT-PCR and ELISA, NO release by Griess assay, and PGE2 expression by ELISA. In addition, in vivo analysis (evolution of weight and edema) were also performed after injection of Freund adjuvant in rats.RESULTS. The copolymer of fatty acids clearly reduced inflammation in joint. In vitro, it impaired IL1 stimulated-MMP production and release, as well as release of NO and PGE2 and activation of NFκB in chondrocytes. Also, in synoviocytes, it reduces the induction of MMP and IL-6. Furthermore, in vivo experiments corroborate the anti-inflammatory effects of ara 3000 beta®, with a reduction of edema, erythema and ankylosis in arthritic rats.CONCLUSION. The results support the hypothesis that a copolymer of fatty acids, such as ara 3000 beta® has anti-inflammatory properties, suggesting a beneficial potential for preventing cartilage degradation associated with chronic inflammatory joint diseases., MÉTHODOLOGIE. Une étude in vitro et in vivo a évalué les effets d’un traitement avec un copolymère d’acides gras (ara 3000 beta®) sur une inflammation des chondrocytes et des synoviocytes.RÉSULTATS. Le copolymère d’acides gras réduit nettement l’inflammation dans l’articulation. In vitro, il limite la production des métalloprotéases matricielles et de l’interleukine 6, la sécrétion d’oxyde nitrique et de prostaglandine E2 et l’activation de NFκB. In vivo, l’ara 3000 beta® diminue l’oedème, l’érythème et l’ankylose.CONCLUSION. Les résultats de cette étude suggèrent qu’un copolymère d’acides gras possède des propriétés antiinflammatoires et freine la dégradation du cartilage associée aux maladies articulaires inflammatoires chroniques

Cartilage engineering is one challenging issue in regenerative medicine. Low oxygen tension or hypoxia inducible factor-1 (HIF-1α) gene therapy are promising strategies in the field of cartilage repair. Previously, we showed that hypoxia and its mediator HIF-1 regulate matrix genes expression (collagens and aggrecan). Here, we investigated the molecular mechanism involved in the regulation of type I collagen (COL1A1) by HIF-1 in human articular chondrocytes. We show that HIF-1α reduces COL1A1 transcription, through a distal promoter (-2300 to -1816 bp upstream transcription initiation site), containing two GC boxes that bind Sp transcription factors (Sp1/Sp3). Sp1 acts as a positive regulator but is not induced by HIF-1. COL1A1 inhibition caused by HIF-1 implies only Sp3, which accumulates and competes Sp1 binding on COL1A1 promoter. Additionally, Sp3 ectopic expression inhibits COL1A1, while Sp3 knockdown counteracts the downregulation of COL1A1 induced by HIF-1. In conclusion, we established a new regulatory model of COL1A1 regulation by HIF-1, and bring out its relationship with Sp3 transcription factor. In a fundamental level, these findings give insights in the mechanisms controlling COL1A1 gene expression. This may be helpful to improve strategies to impair type I collagen expression during chondrocyte differentiation for cartilage engineering. © 2016 IUBMB Life, 68(9):756-763, 2016.

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Life expectancy increases in developed countries and results in a high prevalence of agerelated diseases namely in women. Elderly subjects generate a huge demand on the social and health services linked to their disability and dependence. Besides the own effects of aging, the estrogenic deficiency, especially during menopause, is the major cause of degenerative diseases such as osteoporosis, skin aging, Alzheimer’s disease and osteoarthritis (OA) (Antonicelli et al., 2008; Candore et al., 2006; Elders, 2000; Felson & Nevitt, 1998; Pietschmann et al., 2008; Stovall & Pinkerton, 2008). OA is a worldwide public health problem which may affect different sites of the skeleton. In the Western world, at least 10% of the population has OA symptoms and 80% of the population will be potentially affected after the age of 70 years. Knee OA represents one of the major causes of morbidity and disability in relation to the worsening of quality of life (Elders, 2000). Pathogenesis of knee OA involves multiple factors including gender, weight, and genetics. Although agedependent degenerative pathologies, especially OA, involve complex mechanisms in which the imbalance of the cytokines/growth factors plays a crucial role, it is more and more obvious that estrogens participate, with their receptors and modulators, to all of these diseases affecting connective tissues (Calleja-Agius & Brincat, 2009). Estrogens are steroidal hormones irreversibly synthesized from androgens via a crucial enzyme of steroidogenesis called aromatase, a member of cytochrome P450 superfamily, encoded by the CYP19 gene and being expressed in both sexes of many species (Simpson, 2003). Although non genomic action of estrogens is now recognized but these sexual hormones act often as signaling molecules that exert their effects by binding to estrogen receptors (ER) within the cells. The estrogen-receptor complex interacts then with DNA to change the expression of estrogen-responsive genes. The two known estrogen receptors, ER and ER, are present in numerous tissues other than those associated with reproduction, including bone, liver, heart, brain and cartilage and being selectively expressed in some targets; for instance ER is selectively expressed in lung and intestine. The presence of ER and ER was detected in articular chondrocytes of different species, especially in human, rat, pig, cow and rabbit