Takamitsu Okada

Antitumor effects of histone deacetylase inhibitor on Ewing's family tumors

A chimeric protein, EWS‐Fli1, identified in most Ewings family tumors (EFTs) has been shown to be associated with the tumorigenicity of EFTs. We have previously reported that p21Waf1/Cip1 expression was inhibited by EWS‐Fli1 in EFTs. Histone deacetylase inhibitors (HDACIs) are known to up‐regulate p21Waf1/Cip1 expression in various cells and show promise as a cancer therapy. Here, we demonstrate the possible involvement of EWS‐Fli1 in the activities of both histone acetylation and deacetylation, as well as the potential use of HDACIs as an antitumor agent for EFTs. A novel HDACI, FK228, strongly induced p21Waf1/Cip1 expression, leading to the hypophosphorylation of retinoblastoma protein (Rb) in EFT cells. Results indicated that EWS‐Fli1 deregulated histone acetylation through both the repression of histone acetyltransferase (HAT) and the enhancement of histone deacetylase (HDAC) activities in EFT cells. FK228 treatment blocked both of the abnormal functions of EWS‐Fli1. Expressions of EWS‐Fli1 protein and mRNA were also inhibited by HDACIs. We suggest that HDACIs might inhibit the expression of EWS‐Fli1 via the suppression of the EWS promoter activity. FK228 demonstrated potent growth inhibitory effects on EFT cells at nanomolar concentrations, as well as an apparent distinction in the apoptotic effects between EFT and normal cells. Moreover, intraperitoneal administration of FK228 significantly inhibited tumor growth and induced apoptosis in EFTs in vivo. These results suggest that HDACI might be a promising reagent for use in molecular‐based chemotherapy against EFTs.

The Possible Role of EWS-Fli1 in Evasion of Senescence in Ewing Family Tumors

The chromosomal translocation t(11;22) yields the EWS-Fli1 fusion gene and is associated with oncogenesis of Ewing family tumors (EFT). In this study, using the RNA interference method, we show that EWS-Fli1-targeting small interfering RNAs (siRNA) depleted EWS-Fli1 protein and caused growth inhibition in EFT cells with the accumulation of p27 protein and the down-regulation of Skp2 protein in dose-dependent, time-dependent, and sequence-specific manners. Depletion of EWS-Fli1 subacutely elicited a senescence-like phenotype, but not apoptosis, in EFT cells. Furthermore, not only the knockdown of p27, but also the forced expression of Skp2, reduced the expression levels of p27 protein and partially rescued senescence-like phenotype caused by EWS-Fli1-targeting siRNAs. The accumulation of p27 protein in EWS-Fli1-depleted cells inhibited cdk2 kinase activity and was related to the stability of p27 protein, which resulted from a decrease in Skp2 protein. Immunohistochemical analysis of p27 and Skp2 proteins in EFT samples revealed that there was an inverse relationship between the expression profiles of p27 and Skp2 proteins. These findings indicate an important role of EWS-Fli1 in the prevention of senescence, leading to the unlimited growth and oncogenesis of EFT cells through a decrease in the stability of p27 protein due to increased action of Skp2-mediated 26S proteasome degradation.

The Effects of Histone Deacetylase Inhibitors on the Induction of Differentiation in Chondrosarcoma Cells

Purpose: Histologically, chondrosarcomas represent the degree of chondrogenic differentiation, which is associated with the prognosis of the disease. Histone acetylation and deacetylation play key roles in the regulation of chondrocytic differentiation. Here, we describe the antitumor effects of histone deacetylase (HDAC) inhibitors as differentiating reagents on chondrosarcomas. Experimental Design: We examined the effects of a HDAC inhibitor, depsipeptide, on the growth of chondrosarcoma cell lines. We also investigated the modulation of the expression levels of extracellular matrix genes and the induction of phenotypic change in chondrosarcoma cells treated with depsipeptide. Finally, we examined the antitumor effect of depsipeptide on chondrosarcoma in vivo. Results: Depsipeptide inhibited the growth of chondrosarcoma cells by inducing cell cycle arrest and/or apoptosis. HDAC inhibitors increased the expression of the α1 chain of type II collagen (COL2A1) gene due to the enhanced histone acetylation in the promoter and enhancer. Depsipeptide also up-regulated the expressions of aggrecan and the α2 chain of type XI collagen (COL11A2) mRNA in a dose-dependent manner. Moreover, long-term treatment with a low dose of depsipeptide resulted in the induction of differentiation into hypertrophic phenotype, as shown by the increment of the α1 chain of type X collagen (COL10A1) expression in chondrosarcoma cells. In vivo studies and histologic analyses confirmed that depsipeptide significantly inhibited tumor growth and induced differentiation into the hypertrophic and mineralized state in chondrosarcoma cells. Conclusions: These results strongly suggest that HDAC inhibitors may be promising reagents for use as a differentiating chemotherapy against chondrosarcomas.

Involvement of P‐glycoprotein and MRP1 in resistance to cyclic tetrapeptide subfamily of histone deacetylase inhibitors in the drug‐resistant osteosarcoma and Ewing's sarcoma cells

Despite recent improvements in multimodal therapies for osteosarcoma (OS) and Ewings family of tumors (EFTs), the prognosis of relapsed cases remains very poor because of the resistance to chemotherapy. Histone deacetylase inhibitors (HDACIs), including members of the cyclic tetrapeptide family such as FK228 and apicidin, are novel antitumor agents that can induce cell cycle arrest and apoptosis in various cancer cells. HDACIs also exhibit potent antitumor effects on OS and EFTs. However, to date there have been no studies to our knowledge reporting the effects of HDACIs on drug‐resistant OS and EFTs. Here, we demonstrated that FK228 and apicidin exhibited strong resistance in doxorubicin‐resistant clones of OS and EFTs expressing P‐glycoprotein (P‐gp) and multidrug resistance‐associated protein 1 (MRP1) and that P‐gp and MRP1 might play a crucial role in the resistance mechanism to FK228 and apicidin. A P‐gp inhibitor (verapamil) and an MRP1 inhibitor (MK571) could independently reverse the resistance to FK228 and apicidin in the drug‐resistant clones. Moreover, the combination of verapamil and MK571 could enhance HDACI‐induced cell number reduction in drug‐resistant clones to a similar extent as that in their parental clones. Although these findings suggest the difficulty in treating drug‐resistant tumors expressing P‐gp and/or MRP1 with these HDACIs, the combination of P‐gp and MRP1 inhibitors might reverse the resistance to the HDACIs in the treatment of those tumors. Because HDACIs are potent and promising antitumor drugs and seem to be close to clinical use, it is necessary to pay attention to the resistance mechanisms against HDACIs.

Cyclin-dependent kinase inhibitor, flavopiridol, induces apoptosis and inhibits tumor growth in drug-resistant osteosarcoma and Ewing's family tumor cells

Multimodal therapies play important roles in the treatment of osteosarcoma (OS) and Ewings family of tumors (EFTs), two most frequent malignant bone tumors. Although the clinical outcome of primary OS and EFTs is greatly improved, the relapsed cases often are associated with multidrug resistance of the tumors and the prognosis of these patients is still poor. Flavopiridol, a pan cyclin‐dependent kinase (CDK) inhibitor is a novel antitumor agent that can induce cell cycle arrest and apoptosis in many cancer cells. However, there have been no studies about the effects of flavopiridol on drug‐resistant OS and EFTs. Here, we demonstrated that flavopiridol induced the cleavage of poly‐ADP‐ribose polymerase (PARP) in a time and dose dependent manner in adriamycin‐resistant OS and EFTs cells expressing P‐glycoprotein (P‐gp) and multidrug resistance‐associated protein 1 (MRP1) as effectively as in their parental cells. Our data also showed that flavopiridol caused the release of mitochondrial cytochrome c and the activation of caspase‐9, caspase‐8 and caspase‐3, with an increase ratio of the proapoptotic protein level (Bax) to the antiapoptotic protein level (Bcl‐2 and Bcl‐XL), while apoptosis was inhibited by pan caspase inhibitor (Z‐VAD‐FMK) and caspase‐3 inhibitor (Z‐DEVD‐FMK), not by caspase‐8 inhibitor (Z‐IETD‐FMK). The treatment with flavopiridol further inhibited the tumor growth in mouse models of the drug‐resistant OS and EFTs. These results suggest that flavopiridol might be promising in clinical therapy for the relapsed OS and EFTs.

Transactivation of cyclin E gene by EWS-Fli1 and antitumor effects of cyclin dependent kinase inhibitor on Ewing's family tumor cells

Chromosomal translocation t(11; 22)(q24; q12) is detected in approximately 90% of Ewings family tumors (EFTs) including Ewings sarcoma and primitive neuroectodermal tumor. This results in the formation of the EWS‐Fli1 fusion gene, which produces EWS‐Fli1 fusion protein. This chimerical gene product acts as an aberrant transcriptional activator, which may be responsible for the tumorigenesis of EFTs. We have previously reported that cyclin E expression was upregulated in EFT cells and in EWS‐Fli1 transformed fibroblastic cells. However, the mechanism of the overexpression of cyclin E by EWS‐Fli1 is still unknown. In our study, we investigated the mechanism of transactivation of the cyclin E gene in EFT cells. We found that EWS‐Fli1 enhanced the activity of the cyclin E gene promoter partially through E2F binding sites in the promoter. In addition, the basic transcriptional factor, Sp1, might also be involved in the transactivation of the cyclin E gene by EWS‐Fli1. To study the biological significance of cyclin E overexpression in EFT cells, we used flavopiridol, a pan‐cyclin‐dependent kinase (CDK) inhibitor and found that flavopiridol efficiently suppressed the growth of EFT cells in vitro and in vivo by the inhibition of cyclinE/CDK2 kinase activity and the induction of apoptosis. These results suggest that targeting of the cyclin/CDK complex may provide new insight into treatment of EFTs.

Focal adhesion kinase is activated in invading fibrosarcoma cells and regulates metastasis.

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is overexpressed in several human cancers, and induces survival, proliferation and motility of cells in culture. Phosphorylation of FAK has been studied extensively in vitro, but little is known about its regulation during tumor invasion in vivo. In the current study, green fluorescent protein (GFP) was expressed stably in an invasive murine fibrosarcoma cell line for the purpose of discrimination between tumor and normal cells. Under fluorescence microscopy, the tumor was highly fluorescent, and the margin between the tumor and normal tissue was clearly demarcated. Using this invasion model, we showed localization of pY397-FAK expression in the infiltrative edge of tumors. We reproduced local invasion in vivo using a tumor tissue culture method in a three dimensional collagen gel. Phosphorylation of FAK is also upregulated in invading fibrosarcoma cells under in vitro conditions. Expression of the FAK C-terminal domain termed FRNK (FAK-related non-kinase) in 2472 cells decreased FAK phosphorylation without changing total FAK levels. FRNK inhibited the motility of 2472 cells, and reduced invasion in vitro. Although FRNK did not affect cell growth, it inhibited experimental metastases in syngenic mice. These results demonstrate that the phosphorylation of FAK might be specifically upregulated in invading fibrosarcoma cells and regulate their invasion and metastasis.

Asymmetric six-strand core sutures enhance tendon fatigue strength and the optimal asymmetry

Under cyclic loading, we recorded the fatigue strength of a six-strand tendon repair with different symmetry in the lengths of suture purchase in two stumps of 120 dental rolls and in 30 porcine tendons. First, the strengths of the repairs with 1, 2, 3, 4 and 5 mm asymmetry were screened using the dental rolls. The asymmetric core suture repairs were then made with a Kessler repair of equal suture purchase (10 mm) in two tendon stumps, and shifting two other Kessler repairs by 1, 3 or 5 mm, respectively, along the longitudinal axis of the tendon in relation to the first (symmetric) Kessler repair. The core repairs with 3 mm or more asymmetry in suture purchases in two tendon ends showed significantly greater fatigue strength and significantly smaller gaps compared with 1 mm asymmetry in core suture repair. Our results support that asymmetric placement of core sutures in two tendon ends favour resisting gapping at the repair site and 3 mm or more asymmetry is needed to produce such beneficial effects.

In vivo dynamic acromiohumeral distance in shoulders with rotator cuff tears

Background: There are no previous studies on the acromiohumeral distance in shoulders with large‐to‐massive full‐thickness rotator cuff tears. In this study, the acromiohumeral distance in rotator cuff tear and healthy shoulders was measured using 3D‐to‐2D model‐to‐image registration techniques. Methods: The dynamic glenohumeral kinematics during scapular plane abduction and axial rotation were analyzed in 11 rotator cuff tear patients and 10 healthy control subjects. Periodic radiographic images of scapular plane abduction and axial rotation were taken using a flat‐panel radiograph image detector. Movements of the shoulder joint were assessed using radiographic images and computed tomography‐derived digitally reconstructed radiographs. The acromiohumeral distance was defined as the shortest 3D distance between the acromion and the proximal humerus. Findings: For scapular plane abduction, the rotator cuff tear group had significantly smaller acromiohumeral distance than the control group at 15°, 30°, 45°, 60°, 75°, 135°, and 150° of humeral abduction (P < 0.05 at each measured angle). For axial rotation in the adducted position, the rotator cuff tear group had significantly smaller acromiohumeral distance than the control group at each point between −20° and 40° of glenohumeral external rotation (P < 0.05 at each measured angle). Interpretation: The minimum measured acromiohumeral distance was 0.9 mm in the rotator cuff tear shoulders and 2.1 mm in the healthy shoulders at 90° of scapular plane abduction. The findings are of clinical relevance because quantitative evaluation of the dynamic acromiohumeral distances in rotator cuff tear and healthy shoulders might provide important insight into subacromial impingement. HighlightsAcromiohumeral distance in rotator cuff tear and healthy shoulders was measured.3D‐to‐2D model‐to‐image registration techniques were performed.Acromiohumeral distance was significantly smaller in rotator cuff tear shoulders.Quantitative evaluation of acromiohumeral distance might provide important insight.

Brodie’s Abscess of the Radius in a Child

We herein report an unusual case of Brodies abscess of the radius in a child. A 13-year-old boy presented with pain on his right distal forearm. A plain radiograph showed an 8 cm translucent lesion in the distal radius. MRI showed a penumbra sign on the T1-weighted image, hyperintensity on T2-weighted images, and ring enhancement on the contrast-enhanced T1 image. 18F-FDG PET/CT images showed an uptake at the margin of the radius. Curettage and iliac cancellous bone grafting were undertaken for Brodies abscess. Bacteriological examinations were found to be negative, however, the pathologic diagnosis showed chronic osteomyelitis. Eight months after surgery, the patient was asymptomatic and there was no sign of recurrence of infection. For Brodies abscess in a child, thorough debridement is mandatory in addition to cancellous bone grafting. Brodies abscess should be considered in the differential diagnosis of a patient who presents with forearm pain and exhibit the radiolucent osteolytic lesion on simple radiography.