Mechanical forces influence homeostasis in virtually every tissue [1–2]. Tendon, constantly exposed to variable mechanical force, is an excellent model in which to study the conversion of mechanical stimuli into a biochemical response [3–5]. Here we show in a mouse model of acute tendon injury and in vitro that physical forces regulate the release of active transforming growth factor (TGF)-β from the extracellular matrix (ECM). The quantity of active TGF-β detected in tissue exposed to various levels of tensile loading correlates directly with the extent of physical forces. At physiological levels, mechanical forces maintain, through TGF-β/Smad2/3-mediated signaling, the expression of Scleraxis (Scx), a transcription factor specific for tenocytes and their progenitors. The gradual and temporary loss of tensile loading causes reversible loss of Scx expression, whereas sudden interruption, such as in transection tendon injury, destabilizes the structural organization of the ECM and leads to excessive release of active TGF-β and massive tenocyte death, which can be prevented by the TGF-β type I receptor inhibitor SD208. Our findings demonstrate a critical role for mechanical force in adult tendon homeostasis. Furthermore, this mechanism could translate physical force into biochemical signals in much broader variety of tissues or systems in the body.

No abstract

Tendon is a dense connective tissue that transmits high mechanical forces from skeletal muscle to bone. The transcription factor scleraxis (Scx) is a highly specific marker of both precursor and mature tendon cells (tenocytes). Mice lacking scx exhibit a specific and virtually complete loss of tendons during development. However, the functional contribution of Scx to wound healing in adult tendon has not yet been fully characterized. Here, using ScxGFP-tracking and loss-of-function systems, we show in an adult mouse model of Achilles tendon injury that paratenon cells, representing a stem cell antigen-1 (Sca-1)–positive and Scx-negative progenitor subpopulation, display Scx induction, migrate to the wound site, and produce extracellular matrix (ECM) to bridge the defect, whereas resident tenocytes exhibit a delayed response. Scx induction in the progenitors is initiated by transforming growth factor β (TGF-β) signaling. scx-deficient mice had migration of Sca-1–positive progenitor cell to the lesion site but impaired ECM assembly to bridge the defect. Mechanistically, scx-null progenitors displayed higher chondrogenic potential with up-regulation of SRY-box 9 (Sox9) coactivator PPAR-γ coactivator-1α (PGC-1α) in vitro, and knock-in analysis revealed that forced expression of full-length scx significantly inhibited Sox9 expression. Accordingly, scx-null wounds formed cartilage-like tissues that developed ectopic ossification. Our findings indicate a critical role of Scx in a progenitor-cell lineage in wound healing of adult mouse tendon. These progenitor cells could represent targets in strategies to facilitate tendon repair. We propose that this lineage-regulatory mechanism in tissue progenitors could apply to a broader set of tissues or biological systems in the body.

We investigated copy number aberrations in 29 primary tumors and 12 cell lines of esophageal squamous cell carcinoma (ESC) using comparative genomic hybridization. In the primary tumors, the most common sites of copy number gains were 3q26.3-27 (45%), 8q24 (41%), 5p15 (38%), Xq27-28 (38%), 14q32 (31%), 11q13 (28%), and 20q13.3 (28%). High-level gains (HLGs) indicative of gene amplifications were identified at 11q13 in two cases, and in one case each at 2q33-34, 3q25-29, 5p15.1-15.2, 7q21-22, 11p11.2, 12p11.2-12, and 13q34. Recurrent losses were observed only at 9p13(17.2%). In the 12 ESC cell lines, the most common sites of HLGs were 5p15.1-15.3 (four cases), 11q13 (four cases), 8q24.1-24.2 (three cases), 20q13.2-13.3 (three cases), 3q26.3 (two cases), and 7p15-22 (two cases). Less frequent HLGs (one case each) were observed at 2p16-22, 3q25, 7p12-14, 7q21-22, 9q34, 10q21, 11p11.2, 14q13-14, 14q31-32, 15q22-26, and 17p11.2. Chromosomes and chromosome arms that showed frequent losses in the cultured lines were 18q (58%), 4 (50%), 9p (50%), and 3p (42%). These findings provide evidence for a number of previously unknown genomic aberrations in ESC, suggesting target regions for positional cloning of genes relevant to carcinogenesis in the esophagus. In particular, we identified a significant amplification of the DPI gene (TFDPI), a transcription factor that forms heterodimers with E2FI, in the single primary tumor that exhibited HLG at 13q34.

Bisphosphonates (BPs) are widely used to treat bone diseases and also appear to possess direct antitumour activity. We have previously reported that third-generation BPs such as zoledronic acid (ZOL) and minodronic acid (YM529) synergistically augment the effects of anticancer agents in various cancer cells. Recently, we have also reported the antitumour effects of YM529 on murine osteosarcoma cells. As YM529 has not been clinically available, we herein focused on the anti-osteosarcoma effects of ZOL which is clinically available. In addition to ZOL alone, we evaluated the concurrent or sequential combined effects of ZOL with other anticancer agents against murine osteosarcoma cell lines. ZOL showed almost same anti-osteosarcoma activity compared with YM529 and more sensitive growth inhibitory effects against osteosarcoma cells than normal cells. Moreover, ZOL acted synergistically in vitro when administered concurrently with paclitaxel (PAC) or gemcitabine (GEM), not only in wild-type osteosarcoma cells but also in P-glycoprotein (P-gp)-overexpressing osteosarcoma cells, which were much less sensitive against each anticancer agent. Furthermore, 24 h of ZOL pretreatment significantly augmented the sensitivity of doxorubicin (DOX), PAC or GEM against osteosarcoma cells. These findings suggest that combined administration of ZOL with other anticancer agents may improve the osteosarcoma treatment.

Fifty-three patients with benign bone tumours were treated with curettage and filling with a purified betatricalcium phosphate (β-TCP). Recurrences occurred in two cases. There was neither a postoperative infection nor adverse reaction due to the material. Postoperative fractures did not occur in any patients. Radiographically, complete resorption of the material and bone remodelling were achieved in 23 cases (43%). Of these 23 cases, there was a statistical correlation between the filling volume and the time taken for complete resorption (p<0.05). We concluded that purified β-TCP was an ideal bone graft substitute for the treatment of benign bone tumours because of its good biocompatibility and resorption characteristics.Résumé 53 patients présentant une tumeur bénigne des os ont été traités par curetage et greffe par du beta-tricalcium phosphate (β-TCP) pur. La récidive de la tumeur n'a été observée que dans deux cas. Il n'y a eu aucune infection et aucune réaction négative dues au matériel. Nous n'avons pas constaté de fractures post-opératoires. Sur le plan radiographique, nous avons assisté à une résorption complète de la tumeur et remodelage osseux chez 23 patients (43%). Pour ces 23 patients, il existe une corrélation statistique non significative entre le volume du substitut et le temps pour sa complète résorption (p<0.05).Nous pouvons en conclure que le β-TCP est un substitut osseux idéal pour le traitement des tumeurs osseuses bénignes qu'il présente une bonne bio compatibilité et des caractéristiques satisfaisantes en terme de résorption.

Sulforaphane (SFN), a naturally occurring isothiocyanate, is an attractive agent because of its potent anticancer effects. SFN suppresses the proliferation of various cancer cells in vitro and in vivo. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is also one of the most promising candidates for cancer therapeutics owing to its ability to selectively induce apoptosis in tumor cells. In this study, we report that SFN enhances TRAIL-induced apoptosis in human osteosarcoma cells, Saos2 and MG63. The apoptosis induced by co-treatment with SFN and TRAIL was markedly blocked by a dominant negative form of the TRAIL receptor or caspase inhibitors. The combined use of SFN and TRAIL effectively induced Bid cleavage and the activation of caspases 8, 10, 9 and 3 at ineffective concentrations for each agent. SFN upregulated the expression of death receptor 5 (DR5), a receptor for TRAIL, at mRNA and protein levels in a dose-dependent manner. In addition, the SFN-mediated sensitization to TRAIL was reduced by DR5 siRNA, suggesting that the sensitization was at least partially mediated through the induction of DR5 expression. Furthermore, SFN sensitized TRAIL-induced apoptosis in a p53-independent manner. On the other hand, SFN neither induced DR5 protein expression or enhanced TRAIL-induced apoptosis in normal human peripheral blood mononuclear cells. Thus, combined treatment with SFN and TRAIL might be a promising therapy for osteosarcoma.

Abstract. Sulforaphane (SFN), a naturally occurring isothiocyanate, is an attractive agent due to its potent anticancer effects. SFN suppresses the proliferation of various cancer cells in vitro and in vivo. In this study, we report that SFN inhibited the proliferation of cultured murine osteosarcoma LM8 cells. Twenty micromolar SFN completely inhibited the growth of LM8 cells and caused G 2 /M-phase arrest. SFN induced the expression of p21 WAF1/CIP1 protein causing the cell cycle arrest in a dose-dependent manner. SFN induced apoptosis which was characterized by the appearance of cells with sub-G 1 DNA content and the cleavage and activation of caspase-3. We showed that SFN induced the growth arrest and up-regulated the expression of p21 WAF1/CIP1 protein in a p53-independent manner in human osteosarcoma MG63 cells. We found that intraperitoneal administration of SFN (1 or 2 mg, 5 times/week) significantly inhibited the growth of LM8 xenografts to <30% of the controls in a preclinical animal model without causing any toxicity. In osteosarcoma cells, our findings provide in vivo evidence for the efficacy of SFN against the advanced growth of tumor. We showed that SFN induces cell cycle arrest and apoptosis in osteosarcoma cells and inhibits tumor xenograft growth. Furthermore, SFN is a potent inducer of p21 WAF1/CIP1 in osteosarcoma cells. These results raise the possibility that SFN may be a promising candidate for molecular-targeting chemotherapy against osteosarcoma.