The EWS/Fli-1 fusion gene, a product of the translocation t (11;22, q24;q12), is detected in 85% of Ewing sarcomas and primitive neuroectodermal tumors. It is thought to be a transcriptional activator that plays a significant role in tumorigenesis. In this study, we developed a novel EWS/Fli-1 blockade system using RNA interference and tested its application for inhibiting the proliferation of Ewing sarcoma cells in vitro and the treatment of mouse tumor xenografts in vivo. We designed and synthesized a small interfering RNA (siRNA) possessing an aromatic compound at the 3 0 -end targeting the breakpoint of EWS/Fli-1. As this sequence is present only in tumor cells, it is a potentially relevant target. We found that the siRNA targeting EWS/Fli-1 significantly suppressed the expression of EWS/Fli-1 protein sequence specifically and also reduced the expression of c-Myc protein in Ewing sarcoma cells. We further demonstrated that inhibition of EWS/Fli-1 expression efficiently inhibited the proliferation of the transfected cells but did not induce apoptotic cell death. In addition, the siRNA possessing the aromatic compound at the 3 0 -end was more resistant to nucleolytic degradation than the unmodified siRNA. Administration of the siRNA with atelocollagen significantly inhibited the tumor growth of TC-135, a Ewing sarcoma cell line, which had been subcutaneously xenografted into mice. Moreover, modification of the 3 0 -end with an aromatic compound improved its efficiency in vivo. Our data suggest that specific downregulation of EWS/Fli-1 by RNA interference is a possible approach for the treatment of Ewing sarcoma.The Ewing sarcoma family of tumors (ESFTs) is a group of highly malignant neoplasms that most often affect children and young adults in the first two decades of life. It is the second most common malignant bone tumor and accounts for approximately 10% of all primary bone tumors. Despite aggressive treatment strategies (chemotherapy, radiation therapy and surgery), the long-term disease-free survival rate of patients with ES is still disappointingly low, particularly in poor-risk patients with metastasis. Therefore, identification of new therapeutic targets is urgently needed.The EWS/Fli-1 fusion gene, a product of the translocation t(11;22, q24;q12), is detected in 85% of ESs and primitive neuroectodermal tumors. The EWS/Fli-1 translocation is formed by the N-terminal domain of the RNA-binding protein EWS and the DNA-binding domain of the ETS family transcriptional factor Fli-1. Identification of several breakpoints for both EWS and Fli-1 has demonstrated that the EWS/Fli-1 fusion genes are heterogeneous. Breakpoints of Type 1 (Exon 7 of EWS/Exon 6 of Fli-1) and Type 2 (Exon 7 of EWS/Exon 5 of Fli-1) are most commonly detected in affected patients. [1][2][3] We have reported that the EWS/Fli-1 fusion protein may be a transcriptional activator that plays a significant role in the tumorigenesis of ESFTs. [4][5][6][7] Although substantial studies have reported that antagonism of the EWS fusion gene reduces...
Vascular endothelial growth factor (VEGF)‐A plays an important role in the pathological angiogenesis that occurs in soft‐tissue sarcoma and in about half of Ewing's sarcoma cases, where it is highly overexpressed. EWS/Fli‐1 is considered to be a transcriptional activator and to play a significant role in tumorigenesis of Ewing's sarcoma. However, the relationship between EWS/Fli‐1 and VEGF‐A is still unclear. The aim of this research is to investigate the relationship between EWS/Fli‐1 and VEGF‐A, and to determine whether small interfering RNA (siRNA)‐targeting of VEGF‐A can be developed as a novel treatment for Ewing's sarcoma. Knockdown of EWS/Fli‐1 using siRNA on a Ewing's sarcoma cell line (A673) suppressed VEGF‐A expression, and transfection of EWS/Fli‐1 into a human osteosarcoma cell line increased VEGF‐A expression. To inhibit VEGF‐A secretion from Ewing's sarcoma, we developed a chemically synthesized siRNA that targets VEGF‐A. Transfection of the VEGF siRNA into the Ewing's sarcoma cell line significantly suppressed VEGF‐A secretion by up to 98% in vitro, compared with a control. In vivo, we established Ewing's sarcoma xenograft models and performed intratumoral injection of the siRNA mixed with atelocollagen. We observed that the inhibition of tumor growth occurs in a dose‐dependent manner. Histological examination revealed decreased microvessel density and morphological change around microvessels in the Ewing's sarcoma xenografts treated with the siRNA. It is considered that a combination of chemically synthesized siRNA that targets VEGF‐A and atelocollagen might be a novel and effective option for treating Ewing's sarcoma that secretes VEGF‐A.
The present study demonstrated for the first time that combination of U0126 and LY294002 can augment the cytotoxicity of ActD against Ewing sarcoma cells in vitro and in vivo. Our results indicate that further study on combination of conventional chemotherapies with MEK and PI3K inhibitors may be considered for innovative treatments of Ewing sarcoma patients.
Developmental dysplasia of the hip (DDH) is characterized by anatomical abnormalities of the hip joint, ranging from mild acetabular dysplasia to hip subluxation and eventually dislocation. The mechanism underlying the cartilage degeneration of the hip joints exposed to reduced dynamic loads due to hip dislocation remains unknown. We established a rodent hip dislocation (disarticulation; DA) model of DDH (DA-DDH rats and mice) by swaddling. Expression levels of periostin (Postn) and catabolic factors, such as interleukin-6 (IL-6) and matrix metalloproteinase 3 (Mmp3), increased and those of chondrogenic markers decreased in the acetabular cartilage of the DA-DDH models. Postn induced IL-6 and Mmp3 expression in chondrocytes through integrin αVβ3, focal adhesion kinase, Src, and nuclear factor-κB (NF-κB) signaling. The microgravity environment created by a random positioning machine induced Postn expression in chondrocytes through signal transducer and activator of transcription 3 (STAT3) signaling. IL-6 stimulated Postn expression via STAT3 signaling. Furthermore, cartilage degeneration was suppressed in the acetabulum of Postn−/− DA-DDH mice compared with that in the acetabulum of wild type DA-DDH mice. In summary, reduced dynamic loads due to hip dislocation induced acetabular cartilage degeneration via IL-6 and MMP3 through STAT3/periostin/NF-κB signaling in the rodent DA-DDH models.
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