Predictive factors of mortality of patients with fragility hip fractures at 1 year after discharge: A multicenter, retrospective study in the northern Kyushu district of Japan.

PURPOSE: Fragility hip fractures (FHFs) are associated with a high risk of mortality, but the relative contribution of various factors remains controversial. This study aimed to evaluate predictive factors of mortality at 1 year after discharge in Japan. METHODS: A total of 497 patients aged 60 years or older who sustained FHFs during follow-up were included in this study. Expected variables were finally assessed using multivariable Cox proportional hazards models. RESULTS: The 1-year mortality rate was 9.1% (95% confidence interval: 6.8-12.0%, n = 45). Log-rank test revealed that previous fractures (p = 0.003), Barthel index (BI) at discharge (p = 0.011), and place-to-discharge (p = 0.004) were significantly associated with mortality for male patients. Meanwhile, body mass index (BMI; p = 0.023), total Charlson comorbidity index (TCCI; p = 0.005), smoking (p = 0.007), length of hospital stay (LOS; p = 0.009), and BI (p = 0.004) were the counterparts for females. By multivariate analyses, previous vertebral fractures (hazard ratio (HR) 3.33; p = 0.044), and BI <30 (HR 5.42, p = 0.013) were the predictive variables of mortality for male patients. BMI <18.5 kg/m2 (HR 2.70, p = 0.023), TCCI ≥5 (HR 2.61, p = 0.032), smoking history (HR 3.59, p = 0.018), LOS <14 days (HR 13.9; p = 0.007), and BI <30 (HR 2.76; p = 0.049) were the counterparts for females. CONCLUSIONS: Previous vertebral fractures and BI <30 were the predictive variables of mortality for male patients, and BMI <18.5 kg/m2, TCCI ≥5, smoking history, LOS <14 days, and BI <30 were those for females. Decreased BI is one of the independent and preventable risk factors. A comprehensive therapeutic approach should be considered to prevent deterioration of activities of daily living and a higher risk of mortality.

Characteristics of patients with fragility hip fractures in the northern Kyushu district in Japan: a multicenter prospective registry based on an electronic data capture system.

Osteoporosis has become a worldwide public health problem, in part due to the fact that it increases the risk of fragility hip fractures (FHFs). The epidemiological assessment of FHFs is critical for their prevention; however, datasets for FHFs in Japan remain scarce. This was a multicenter, prospective, observational study in the northern district of Kyushu Island. Inclusion criteria were age > 60 years with a diagnosis of FHF and acquisition of clinical data by an electronic data capture system. Of 1294 registered patients, 1146 enrolled in the study. Nearly one third of patients (31.8%) had a history of previous fragility fractures. The percentage of patients receiving osteoporosis treatment on admission was 21.5%. Almost all patients underwent surgical treatment (99.1%), though fewer than 30% had surgery within 48 h after hospitalization. Bone mineral density (BMD) was evaluated during hospitalization in only 50.4% of patients. The rate of osteoporosis treatment increased from 21.5% on admission to 39.3% during hospitalization. The main reasons that prescribers did not administer osteoporosis treatment during hospitalization were forgetfulness (28.4%) and clinical judgment (13.6%). Age and female ratio were significantly higher in patients with previous FHFs than in those without. There was a significant difference in the rate of osteoporosis treatment or L-spine BMD values in patients with or without previous FHFs on admission. In conclusion, this study confirmed that the evaluation and treatment of osteoporosis and FHFs is still suboptimal in Japan, even in urban districts.

Suberoylanilide hydroxamic acid (SAHA) induces apoptosis or autophagy-associated cell death in chondrosarcoma cell lines.

BACKGROUND: Since chondrosarcoma has a high resistance to conventional chemotherapy and radiotherapy, surgical resection is currently the only effective treatment. Histone deacetylase (HDAC) inhibitor exert anticancer effects, but have not been tested in chondrosarcoma. MATERIALS AND METHODS: We investigated the phenotypic change in chondrosarcoma cells treated with SAHA by cell viability assay, Western blot, flow cytometric analysis and electron microscopy. RESULTS: SAHA inhibited the growth of chondrosarcoma cell lines and induced apoptosis in SW1353 with a cleaved-PARP expression and sub-G1 fragmentation according to flow cytometric analysis. On the other hand, in RCS and OUMS-27, SAHA induced autophagy-associated cell death as shown by the detection of autophagosome-specific protein and specific ultrastructural morphology in the cytoplasm. In addition, SAHA significantly inhibited tumor growth in an in vivo xenograft model. CONCLUSION: These results suggest that SAHA might be a promising agent for performing clinically useful chemotherapy against chondrosarcomas.

The mechanism of cross-resistance to proteasome inhibitor bortezomib and overcoming resistance in Ewing's family tumor cells.

EWS-Fli1 plays important roles in oncogenesis of Ewing's family tumors (EFTs). We have reported that EWS-Fli1 inhibits p21(waf1/cip1) and p27(kip1) expressions, which are degraded by the ubiquitin-proteasome pathway. Bortezomib efficiently up-regulated p21(waf1/cip1) and p27(kip1) expression, and induced apoptosis accompanied by the expression of cleaved-PARP, DR4 and activated caspase-8 in EFT cells. Since most EFTs deaths result from the tumor being resistant to chemotherapeutic drugs, the effects of novel anti-tumor reagents on drug-resistant tumors were next investigated. The results demonstrated that the drug-resistant EFT clones were cross-resistant to bortezomib probably due to the over-expression of the efflux pumps, P-glycoprotein and MRP1. We further investigated whether the efflux pump inhibitors would modulate the effects of bortezomib. The combination of P-gp-specific or MRP1-specific inhibitors could enhance the anti-tumor effects of bortezomib on the drug-resistant clones. These data suggest that bortezomib might be a substrate of P-gp and MRP1. Although bortezomib would be effective on the primary EFTs, it is necessary to pay attention to the resistance to bortezomib in clinical trials for the advanced cases. The combination of bortezomib and the efflux pump inhibitors might be a promising method as a novel molecular target therapy for advanced EFTs.

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 alpha1 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 alpha2 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 alpha1 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.

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.

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 Ewing's 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.

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 2,472 cells decreased FAK phosphorylation without changing total FAK levels. FRNK inhibited the motility of 2,472 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.

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

A chimeric protein, EWS-Fli1, identified in most Ewing's family tumors (EFTs) has been shown to be associated with the tumorigenicity of EFTs. We have previously reported that p21(Waf1/Cip1) expression was inhibited by EWS-Fli1 in EFTs. Histone deacetylase inhibitors (HDACIs) are known to up-regulate p21(Waf1/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 p21(Waf1/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.

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 Ewing's family tumors (EFTs) including Ewing's 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.

The prognostic and therapeutic relevance of p27kip1 in Ewing's family tumors.

PURPOSE: Ewing's family tumors (EFTs) display the characteristic fusion gene EWS-Fli1. We have reported EWS-Fli1 may promote the cell cycle progression accompanied by the suppression of the expression of cyclin-dependent kinase inhibitor p27(kip1) in EFT cells. Here, we describe the prognostic and therapeutic relevance of p27 in EFTs. EXPERIMENTAL DESIGN: We examined tumor samples taken from 21 patients with primary EFTs for the expression of p27 protein immunohistochemically and evaluated its correlation with clinical outcome. We also investigated the usefulness of p27 as a therapeutic strategy in vitro and in vivo using p27 expression adenovirus. Finally, we examined the process of EWS-Fli1-mediated reduction of p27 expression. RESULTS: Immunohistochemical analysis showed that a low expression level of p27 protein was related to poor event-free survival in an univariate analysis and that the expression level of p27 correlated more significantly with patient survival than several clinical factors in a multivariate survival analysis. Overexpression of p27 with the adenoviral vector remarkably inhibited the cell growth in all EFT cells tested and further induced apoptosis in the wild-type p53 EFT cells. In vivo studies demonstrated a reduction in tumor growth of EFT xenograft in nude mice treated with the intratumoral injection of p27-expressing adenovirus. EWS-Fli1 did not significantly affect the p27 promoter activity and p27 mRNA levels. However, the challenge of the proteasome inhibitor caused accumulation of p27 protein in EFT cells. These data strongly suggest EWS-Fli1 might attenuate p27 protein level via activation of the proteasome-mediated degradation pathway. CONCLUSIONS: Our findings provide the first evidence of the prognostic relevance of p27 expression in EFTs. We propose p27 as a novel and powerful therapeutic factor for the molecular target therapy of EFTs.

Identification of p21WAF1/CIP1 as a direct target of EWS-Fli1 oncogenic fusion protein.

Translocation t(11;22) is a karyotypic abnormality detected in over 90% of Ewing's family tumors. This translocation results in the EWS-Fli1 fusion gene, which has been shown to be a potent, single-step transforming gene. We reported previously that suppression of the EWS-Fli1 fusion protein altered the expression of G(1) regulatory cyclins and cyclin-dependent kinase inhibitors both at mRNA and protein levels, resulting in G(1) growth arrest in Ewing's family tumor cell lines. These data suggest that the G(1) regulatory molecules may be targets of the EWS-Fli1 fusion protein, which functions as an aberrant transcription factor. By using electrophoretic mobility shift assays, we show here the direct association of EWS-Fli1 fusion protein with ETS consensus sequences, which are in the promoter of the p21(WAF1/CIP1) gene. Reporter gene assays revealed that the activity of the p21(WAF1/CIP1) promoter is negatively regulated by EWS-Fli1 fusion protein through at least two ETS-binding sites in the promoter. EWS-Fli1 interacted with p300 cotransactivator and suppressed its histone acetyltransferase activity, which may explain the down-regulation of p21(WAF1/CIP1) by EWS-Fli1. In the presence of a histone deacetylase inhibitor, the histone acetyltransferase activity of the Ewing's family tumor cell was recovered resulting in the induction of p21, and the cell growth was dramatically inhibited. These results demonstrated that p21(WAF1/CIP1) might be one of the direct targets of EWS-Fli1, and that p21(WAF1/CIP1) could serve as a target for a molecularly based therapy for Ewing's family tumors.