Masataka Hirotsu

Inhibition of Notch pathway prevents osteosarcoma growth by cell cycle regulation

The study shows constitutive activation of the Notch pathway in various types of malignancies. However, it remains unclear how the Notch pathway is involved in the pathogenesis of osteosarcoma. We investigated the expression of the Notch pathway molecules in osteosarcoma biopsy specimens and examined the effect of Notch pathway inhibition. Real-time PCR revealed overexpression of Notch2, Jagged1, HEY1, and HEY2. On the other hand, Notch1 and DLL1 were downregulated in biopsy specimens. Notch pathway inhibition using γ-secretase inhibitor and CBF1 siRNA slowed the growth of osteosarcomas in vitro. In addition, γ-secretase inhibitor-treated xenograft models exhibited significantly slower osteosarcoma growth. Cell cycle analysis revealed that γ-secretase inhibitor promoted G1 arrest. Real-time PCR and western blot revealed that γ-secretase inhibitor reduced the expression of accelerators of the cell cycle, including cyclin D1, cyclin E1, E2, and SKP2. On the other hand, p21cip1 protein, a cell cycle suppressor, was upregulated by γ-secretase inhibitor treatment. These findings suggest that inhibition of Notch pathway suppresses osteosarcoma growth by regulation of cell cycle regulator expression and that the inactivation of the Notch pathway may be a useful approach to the treatment of patients with osteosarcoma.

Smoothened as a new therapeutic target for human osteosarcoma

BackgroundThe Hedgehog signaling pathway functions as an organizer in embryonic development. Recent studies have demonstrated constitutive activation of Hedgehog pathway in various types of malignancies. However, it remains unclear how Hedgehog pathway is involved in the pathogenesis of osteosarcoma. To explore the involvement of aberrant Hedgehog pathway in the pathogenesis of osteosarcoma, we investigated the expression and activation of Hedgehog pathway in osteosarcoma and examined the effect of SMOOTHENED (SMO) inhibition.ResultsTo evaluate the expression of genes of Hedgehog pathway, we performed real-time PCR and immunohistochemistry using osteosarcoma cell lines and osteosarcoma biopsy specimens. To evaluate the effect of SMO inhibition, we did cell viability, colony formation, cell cycle in vitro and xenograft model in vivo. Real-time PCR revealed that osteosarcoma cell lines over-expressed Sonic hedgehog, Indian hedgehog, PTCH1, SMO, and GLI. Real-time PCR revealed over-expression of SMO, PTCH1, and GLI2 in osteosarcoma biopsy specimens. These findings showed that Hedgehog pathway is activated in osteosarcomas. Inhibition of SMO by cyclopamine, a specific inhibitor of SMO, slowed the growth of osteosarcoma in vitro. Cell cycle analysis revealed that cyclopamine promoted G1 arrest. Cyclopamine reduced the expression of accelerators of the cell cycle including cyclin D1, cyclin E1, SKP2, and pRb. On the other hand, p21cip1 wprotein was up-regulated by cyclopamine treatment. In addition, knockdown of SMO by SMO shRNA prevents osteosarcoma growth in vitro and in vivo.ConclusionsThese findings suggest that inactivation of SMO may be a useful approach to the treatment of patients with osteosarcoma.

Role of GLI2 in the growth of human osteosarcoma

The Hedgehog pathway functions as an organizer in embryonic development. Aberrant activation of the Hedgehog pathway has been reported in various types of malignant tumours. The GLI2 transcription factor is a key mediator of Hedgehog pathway but its contribution to neoplasia is poorly understood. To establish the role of GLI2 in osteosarcoma, we examined its expression by real‐time PCR using biopsy tissues. To examine the function of GLI2, we evaluated the growth of osteosarcoma cells and their cell cycle after GLI2 knockdown. To study the effect of GLI2 activation, we examined mesenchymal stem cell growth and the cell cycle after forced expression of GLI2. We found that GLI2 was aberrantly over‐expressed in human osteosarcoma biopsy specimens. GLI2 knockdown by RNA interferences prevented osteosarcoma growth and anchorage‐independent growth. Knockdown of GLI2 promoted the arrest of osteosarcoma cells in G1 phase and was accompanied by reduced protein expression of the cell cycle accelerators cyclin D1, SKP2 and phosphorylated Rb. On the other hand, knockdown of GLI2 increased the expression of p21cip1. In addition, over‐expression of GLI2 promoted mesenchymal stem cell proliferation and accelerated their cell cycle progression. Finally, evaluation of mouse xenograft models showed that GLI2 knockdown inhibited the growth of osteosarcoma in nude mice. Our findings suggest that inhibition of GLI2 may represent an effective therapeutic approach for patients with osteosarcoma. Copyright

Tumour formation by single fibroblast growth factor receptor 3-positive rhabdomyosarcoma-initiating cells

Background:The hypothesis that malignant tumours are generated by rare populations of cancer stem cells that are more tumourigenic than other cancer cells has gained increasing credence. The objective of this study was to identify and characterise a subpopulation of human sarcoma-initiating cells.Methods:We examined established rhabdomyosarcoma cell lines by flow cytometry. Tumourigenesis was examined by xenograft models. Real-time PCR and immunohistochemistry were performed to examine the gene expression using cell lines and biopsy specimens.Results:Rhabdomyosarcoma cell lines included small populations of fibroblast growth factor receptor 3 (FGFR3)-positive cells. FGFR3-positive KYM-1 and RD cells were more strongly tumourigenic than FGFR3-negative cells. In addition, xenoengraftment of 33% of single FGFR3-positive KYM-1 cells yielded tumour formation. Stem cell properties of FGFR3-positive cells were further established by real-time PCR, which demonstrated upregulation of undifferentiated cell markers and downregulation of differentiation markers. We showed that in the absence of serum, addition of basic fibroblast growth factor maintained and enriched FGFR3-positive cells. On the other hand, ciliary neurotrophic factor reduced the proportion of FGFR3-positive cells. Real-time PCR and immunohistochemical examination revealed that embryonal rhabdomyosarcoma patient biopsy specimens were found to over-express FGFR3.Conclusions:Our findings suggest that rhabdomyosarcoma cell lines include a minor subpopulation of FGFR3-positive sarcoma-initiating cells, which can be maintained indefinitely in culture and which is crucial for their malignancy.

Pathological femoral fractures due to osteomalacia associated with adefovir dipivoxil treatment for hepatitis B: a case report

We present a case of a 62-year-old man who underwent total hip arthroplasty for treatment of pathologic femoral neck fracture associated with adefovir dipivoxil-induced osteomalacia. He had a 13-month history of bone pain involving his shoulders, hips, and knee. He received adefovir dipivoxil for treatment of lamivudine-resistant hepatitis B virus infection for 5 years before the occurrence of femoral neck fracture. Orthopedic surgeons should be aware of osteomalacia and pathological hip fracture caused by drug-induced renal dysfunction, which results in Fanconi’s syndrome.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1600344696739249

Predictive factors of mortality and deterioration in performance of activities of daily living after hip fracture surgery in Kagoshima, Japan

Given that different hospitals achieve different outcomes, optimal evaluation of treatment outcomes in the local community requires evaluation of many institutions in that area. We carried out a prospective multicenter cohort study in Kagoshima Prefecture to identify factors that contribute to deterioration in activities of daily living performance and patient mortality 1 year after surgical treatment of hip fractures.

A Kirschner wire as a transverse-axis guide to improve acetabular cup positioning

Purpose. To compare cup-positioning accuracy in total hip arthroplasty (THA) with or without use of a Kirschner wire as a transverse-axis guide for pelvic alignment. Methods. Records of 18 men and 73 women (mean age, 60 years) who underwent primary THA with (n=49) or without (n=42) use of a Kirschner wire as a transverse-axis guide for pelvic alignment were reviewed. A 2.4-mm Kirschner wire as a transverse-axis guide was inserted to the anterior superior iliac spine and was parallel to a line linking the left and right anterior superior iliac spine. The safe zone for cup positioning was defined as 30° to 50° abduction and 10° to 30° anteversion. Of the 5 operative surgeons, 2 were classified as experienced (total surgical volume >300) and 3 as inexperienced (total surgical volume of <50). The proportion of patients with the cup in the safe zone was compared in patients with or without use of the transverse-axis guide and in experienced and inexperienced surgeons. Results. For inexperienced surgeons, the use of the transverse-axis guide significantly improved the proportion of patients with the cup in the safe zone from 90% to 100% for abduction, from 50% to 82.4% for anteversion, and from 40% to 82.4% for both. Patients with the cup inside or outside the safe zone were comparable in terms of body height, weight, BMI, subcutaneous fat thickness, incision length, and acetabular cup size. Conclusion. The use of the transverse-axis guide improved the accuracy of cup positioning by inexperienced surgeons.

Clinical Outcome of Arthroscopic Partial Resection of Hip Acetabular Labrum Tear: A Case Report

51歳男性．1年程前より，誘因なく右股関節痛が出現し，平成18年10月当科紹介受診．股関節内外旋での疼痛認めるも，単純レントゲンにて明らかな異常を認めなかった．腰椎を含め，他部位に異常を認めず，臨床所見より股関節唇損傷を疑い，MRI，CT arthrographyを行った．その結果，いずれにおいても関節唇の一部に関節唇損傷を疑う所見を認めた．また，関節内局麻注入にて一時的な症状改善を認め，症状，画像を総合的に判断し，最終的に関節唇損傷と診断し，関節鏡を行った．鏡視にて前外側の関節唇損傷を認め，鏡視下関節唇切除術を行った．術後早期より，歩行時の疼痛などの症状の改善を認め，経過良好である．股関節唇損傷は症状やCT arthrography，MRI，関節内局麻注入などで診断されることが多いが，画像上明らかな異常所見がない例も少なくない．原因が明らかでない股関節痛が持続する症例では，関節唇損傷も考慮に入れて，関節鏡を用いた．診断治療を行うことは有効な方法であると考える．