Michiro Susa

MicroRNA-199a-3p Is Downregulated in Human Osteosarcoma and Regulates Cell Proliferation and Migration

microRNAs (miRNA, miR) play an important role in cancer cell growth and migration; however, the potential roles of miRNAs in osteosarcoma remain largely uncharacterized. By applying a miRNA microarray platform and unsupervised hierarchical clustering analysis, we found that several miRNAs have altered expression levels in osteosarcoma cell lines and tumor tissues when compared with normal human osteoblasts. Three miRNAs, miR-199a-3p, miR-127-3p, and miR-376c, were significantly decreased in osteosarcoma cell lines, whereas miR-151-3p and miR-191 were increased in osteosarcoma cell lines in comparison with osteoblasts. Transfection of precursor miR-199a-3p into osteosarcoma cell lines significantly decreased cell growth and migration, thus indicating that the inhibition effect is associated with an increase in the G1-phase and a decrease of the S-phase cell population. In addition, we observed decreased mTOR and Stat3 expression in miR-199a-3p transfected cells. This study provides new insights for miRNAs in osteosarcoma and suggests that miR-199a-3p may play a functional role in osteosarcoma cell growth and proliferation. Restoring miR-199a-3ps function may provide therapeutic benefits in osteosarcoma. Mol Cancer Ther; 10(8); 1337–45. ©2011 AACR.

Doxorubicin loaded Polymeric Nanoparticulate Delivery System to overcome drug resistance in osteosarcoma

BackgroundDrug resistance is a primary hindrance for the efficiency of chemotherapy against osteosarcoma. Although chemotherapy has improved the prognosis of osteosarcoma patients dramatically after introduction of neo-adjuvant therapy in the early 1980s, the outcome has since reached plateau at approximately 70% for 5 year survival. The remaining 30% of the patients eventually develop resistance to multiple types of chemotherapy. In order to overcome both the dose-limiting side effects of conventional chemotherapeutic agents and the therapeutic failure incurred from multidrug resistant (MDR) tumor cells, we explored the possibility of loading doxorubicin onto biocompatible, lipid-modified dextran-based polymeric nanoparticles and evaluated the efficacy.MethodsDoxorubicin was loaded onto a lipid-modified dextran based polymeric nano-system. The effect of various concentrations of doxorubicin alone or nanoparticle loaded doxorubicin on KHOS, KHOSR2, U-2OS, and U-2OSR2 cells was analyzed. Effects on drug retention, immunofluorescence, Pgp expression, and induction of apoptosis were also analyzed.ResultsDextran nanoparticles loaded with doxorubicin had a curative effect on multidrug resistant osteosarcoma cell lines by increasing the amount of drug accumulation in the nucleus via Pgp independent pathway. Nanoparticles loaded with doxorubicin also showed increased apoptosis in osteosarcoma cells as compared with doxorubicin alone.ConclusionLipid-modified dextran nanoparticles loaded with doxorubicin showed pronounced anti-proliferative effects against osteosarcoma cell lines. These findings may lead to new treatment options for MDR osteosarcoma.

Effects of Siltuximab on the IL-6 Induced Signaling Pathway in Ovarian Cancer

Purpose: To explore potential therapeutic strategies for interrupting the interleukin-6 (IL-6) signaling pathway, we measured IL-6 expression in ovarian cancer tissues, and evaluated the effects of a monoclonal anti-IL-6 antibody; siltuximab (CNTO 328), on levels of IL-6–induced Stat3 phosphorylation, Stat3 nuclear translocation, and Stat3 downstream antiapoptotic genes. We then looked for enhancing paclitaxel sensitivity in multidrug-resistant ovarian cancer cell lines. Experimental Design: Expressions of IL-6 in ovarian cancer patient specimens were assessed by immunohistochemistry. Effects of siltuximab on IL-6–induced activation of Stat3 in an ovarian cancer cell line were determined by Western blot and real-time analysis of Stat3 nucleocytoplasmic translocation. Influence of combination of siltuximab and paclitaxel on tumor growth was evaluated in a xenograft mouse mode in vivo. Results: Metastatic and drug-resistant recurrent tumors have significantly higher IL-6 expression when compared with the matched primary tumors. Siltuximab specifically suppressed IL-6–induced Stat3 phosphorylation and Stat3 nuclear translocation. Treatment with siltuximab significantly decreased the levels of Stat3 downstream proteins such as MCL-1, Bcl-XL, and survivin. Treatment with siltuximab reduced expression of multiple IL-6–induced genes in these cell lines. Furthermore, siltuximab increased the cytotoxic effects of paclitaxel in a paclitaxel resistant ovarian cancer cell line in vitro, but combination therapy with siltuximab did not have a significant effect on paclitaxel resistant tumor growth in vivo. Conclusions: These results show that siltuximab effectively block the IL-6 signaling pathways and IL-6–induced gene expression. Blockage of IL-6 signaling may provide benefits for the treatment of ovarian cancer. Clin Cancer Res; 16(23); 5759–69. ©2010 AACR.

Activation of signal transducer and activator of transcription 3 (Stat3) pathway in osteosarcoma cells and overexpression of phosphorylated-Stat3 correlates with poor prognosis

Stat3 expression in cancer may have important prognostic and therapeutic value, but there has been no reports correlating Stat3 expression with prognosis in patients with osteosarcoma. The goal of this study is to correlate patient prognosis with the expression of Stat3 in osteosarcoma tissue and determine the effectiveness of blocking this pathway in osteosarcoma cell lines by Stat3 inhibitor, CDDO‐Me. We examine the expression levels of Stat3 and pStat3 in osteosarcoma cell lines and primary tissues by Western blot analysis. We also evaluate the levels of pStat3 expression in osteosarcoma tissue microarray (TMA) by immunohistochemistry. We use clinical data to determine the impact of levels of Stat3 expression on patient prognosis. Finally, we evaluated the effect of CDDO‐Me on the inhibition of activated Stat3 pathway in osteosarcoma cell lines using MTT assay and Western blot analysis. Stat3 is observed to be activated in osteosarcoma tissues as well as in cultured cell lines. Overexpression of pStat3 is associated with poor prognosis. CDDO‐Me inhibits the growth of osteosarcoma cell lines and induces apoptosis as well. Our results suggest that Stat3 may be a prognostic indicator and potential therapeutic target for osteosarcoma. Blocking the pathway of Stat3 may lead to develop new therapeutic strategies against osteosarcoma.

Lentiviral shRNA screen of human kinases identifies PLK1 as a potential therapeutic target for osteosarcoma

We describe an optimized systematic screen of known kinases using osteosarcoma cell lines (KHOS and U-2OS) and a lentiviral-based short hairpin RNA (shRNA) human kinase library. CellTiter 96(R)AQueous One Solution Cell Proliferation Assay was used to measure cell growth and survival. We identified several kinases, including human polo-like kinase (PLK1), which inhibit cell growth and induce apoptosis in osteosarcoma cells when knocked down. cDNA rescue and synthetic siRNA assays confirm that the observed phenotypic changes result from the loss of PLK1 gene expression. Furthermore, a small molecule inhibitor to PLK1 inhibited osteosarcoma cell growth and induced apoptosis. Western blot analysis confirmed that PLK1 is highly expressed and activated in several osteosarcoma cell lines as well as in resected tumor samples. Immunohistochemistry analysis showed that patients with high PLK1 tumor expression levels correlated with significantly shorter survival than patients with lower levels of tumor PLK1 expression. These results demonstrate the capability and feasibility of a high-throughput screen with a large collection of lentiviral kinases and its effectiveness in identifying potential drug targets. The development of more potent inhibitors that target PLK1 may open doors to a new range of anti-cancer strategies in osteosarcoma.

Oleanane triterpenoid CDDO-Me induces apoptosis in multidrug resistant osteosarcoma cells through inhibition of Stat3 pathway

BackgroundThe activation of signal transducer and activator of transcription 3 (Stat3) pathway correlates with tumor growth, survival, drug resistance and poor prognosis in osteosarcoma. To explore the potential therapeutic values of this pathway, we assessed both the expression and the activation of Stat3 pathway in several pairs of multidrug resistant (MDR) osteosarcoma cell lines, and tissues. To explore the potential therapeutic values of this pathway, we analyzed the ability of the synthetic oleanane triterpenoid, C-28 methyl ester of 2-cyano-3,12-dioxoolen-1,9-dien-28-oic acid (CDDO-Me), to inhibit Stat3 expression and activation as well as its effects on doxorubicin sensitivity in osteosarcoma cells.MethodsExpression of Stat3, phosphorylated Stat3 (pStat3) and Stat3 targeted proteins, including Bcl-XL, Survivin and MCL-1 were determined in drug sensitive and MDR osteosarcoma cell lines and tissues by Western blot analysis. The effect of CDDO-Me on osteosarcoma cell growth was evaluated by MTT and apoptosis by PARP cleavage assay and caspase-3/7 activity.ResultsStat3 pathway was activated in osteosarcoma tissues and in MDR cell lines. CDDO-Me inhibited growth and induced apoptosis in osteosarcoma cell lines. Treatment with CDDO-Me significantly decreased the level of nuclear translocation and phosphorylation of Stat3. The inhibition of Stat3 pathway correlated with the suppression of the anti-apoptotic Stat3 targeted genes Bcl-XL, survivin, and MCL-1. Furthermore, CDDO-Me increased the cytotoxic effects of doxorubicin in the MDR osteosarcoma cell lines.ConclusionsStat3 pathway is overexpressed in MDR osteosarcoma cells. CDDO-Me significantly inhibited Stat3 phosphorylation, Stat3 nuclear translocation and induced apoptosis in osteosarcoma. This study provides the framework for the clinical evaluation of CDDO-Me, either as monotherapy or perhaps even more effectively in combination with doxorubicin to treat osteosarcoma and overcome drug resistance.

Metastatic Patterns of Myxoid/Round Cell Liposarcoma: A Review of a 25-Year Experience

Myxoid/round cell liposarcoma (MRCL), unlike other soft tissue sarcomas, has been associated with unusual pattern of metastasis to extrapulmonary sites. In an attempt to elucidate the clinical features of MRCL with metastatic lesions, 58 cases, from the medical database of Keio University Hospital were used for the evaluation. 47 patients (81%) had no metastases, whereas 11 patients (11%) had metastases during their clinical course. Among the 11 patients with metastatic lesions, 8 patients (73%) had extrapulmonary metastases and 3 patients (27%) had pulmonary metastases. Patients were further divided into three groups; without metastasis, with extrapulmonary metastasis, and with pulmonary metastasis. When the metastatic patterns were stratified according to tumor size, there was statistical significance between the three groups (P = 0.028). The 8 cases with extrapulmonary metastases were all larger than 10 cm. Similarly, histological grading had a significant impact on metastatic patterns (P = 0.027). 3 cases with pulmonary metastatic lesions were all diagnosed as high grade. In conclusion, large size and low histological grade were significantly associated with extrapulmonary metastasis.

Detection of HEY1‐NCOA2 fusion by fluorescence in‐situ hybridization in formalin‐fixed paraffin‐embedded tissues as a possible diagnostic tool for mesenchymal chondrosarcoma

Mesenchymal chondrosarcoma (MC) is an extremely rare subtype of chondrosarcoma. A tumor specific fusion gene, HEY1‐NCOA2 fusion, was recently identified in this tumor. The finding raises the possibility that the diagnosis of MC can be improved by examining the fusion gene. In the present study, we aimed to evaluate the efficacy of fluorescence in situ hybridization (FISH) in detecting HEY1‐NCOA2 fusion for the diagnosis of MC. Specimens from 10 patients diagnosed with MC were used for the study. Dual‐color FISH was performed using two different probes that specifically hybridize to HEY1 and NCOA2, respectively. Fusion signals were identified in all but two specimens, in which no signal was detected, presumably because of inadequate sample preparation. In accordance with results of a previous study, FISH analysis was highly sensitive in detecting HEY1‐NCOA2 fusion in adequately prepared MC samples. The current study adds further support for the use of HEY1‐NCOA2 fusion as a valid diagnostic marker for MC.

A novel multi-kinase inhibitor pazopanib suppresses growth of synovial sarcoma cells through inhibition of the PI3K-AKT pathway†

Synovial sarcoma is an aggressive soft tissue sarcoma with only a modest response to conventional cytotoxic agents. In the present study, we evaluated the potential antitumor effects of a novel anti‐angiogenesis agent, pazopanib, against synovial sarcoma cells. We found that pazopanib directly inhibited the growth of synovial sarcoma cells by inducing G1 arrest. Multiplex analyses revealed that the PI3K‐AKT pathway was highly suppressed in pazopanib‐sensitive synovial sarcoma cells. Furthermore, administration of pazopanib highly suppressed the tumor growth in a xenograft model. Taken together, these results suggest pazopanib as a possible agent against synovial sarcoma and may warrant further clinical studies.

Multidrug Resistance Reversal Agent, NSC77037, Identified with a Cell-Based Screening Assay

The development of multidrug resistance (MDR) remains a significant obstacle in treating cancer patients with chemotherapy. To identify small-molecule compounds that can reverse MDR, the authors used a cell-based screening assay with an MDR ovarian cancer cell line. Incubating MDR cells with a sublethal concentration of paclitaxel in combination with each of 2000 small-molecule compounds from the National Cancer Institute Diversity Set Library, they identified NSC77037. The cytotoxic activity of NSC77037 and the duration of its effect were evaluated in vitro using a panel of cancer cell lines expressing permeability glycoprotein (Pgp), multiple drug resistance protein 1 (MRP 1), and breast cancer resistance protein (BCRP). The mechanism of its effects was further analyzed by assessing the retention of calcein and Pgp-ATPase activity. The relative potency of MDR reversal by NSC77037 was significantly higher than that of frequently used MDR reversal agents such as verapamil and cyclosporine A. NSC77037 reversed Pgp without reversing MRP or BCRP-mediated MDR. NSC77037, at a concentration of >10 µM, moderately inhibited the proliferation of both sensitive and resistant cell lines, but the inhibitory effect of NSC77037 was not altered by coincubation with the Pgp inhibitor verapamil, suggesting that NSC77037 itself is not a substrate of Pgp. NSC77037 directly inhibited the function of Pgp in a dose-dependent manner, but it did not alter the protein expression level of Pgp. The use of NSC77037 to restore sensitivity to chemotherapy or to prevent resistance could be a potential treatment strategy for cancer patients.