Suppression of Osteosarcoma Cell Invasion by Chemotherapy Is Mediated by Urokinase Plasminogen Activator Activity via Up-Regulation of EGR1

Matsunoshita, Yukihiro; Ijiri, Kosei; Ishidou, Yasuhiro; Nagano, Satoshi; Yamamoto, Takuya; Nagao, Hiroshi; Komiya, Setsuro; Setoguchi, Takao

doi:10.1371/journal.pone.0016234

Cited by 28 publications

(21 citation statements)

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“…An invasion assay was performed as previously described . The invasion of cells was measured using BD Matrigel (BD Bioscience, Franklin Lakes, NJ) according to the manufacturer's protocol.…”

Section: Methodsmentioning

confidence: 99%

GLI2 is a novel therapeutic target for metastasis of osteosarcoma

Nagao‐Kitamoto

Nagata

Nagano

et al. 2014

Intl Journal of Cancer

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Aberrant activation of the Hedgehog (Hh) pathway has been reported in several malignancies. We previously demonstrated that knockdown of GLI2 inhibited proliferation of osteosarcoma cells through regulation of the cell cycle. In this study, we analyzed the function of GLI2 in the pathogenesis of osteosarcoma metastasis. Immunohistochemical studies showed that GLI2 was overexpressed in patient osteosarcoma specimens. Knockdown of GLI2 inhibited migration and invasion of osteosarcoma cells. In contrast, the forced expression of constitutively active GLI2 in mesenchymal stem cells promoted invasion. In addition, xenograft models showed that knockdown of GLI2 decreased lung metastasis of osteosarcomas. To examine clinical applications, we evaluated the efficacy of arsenic trioxide (ATO), which is a Food and Drug Administration-approved antitumor drug, on osteosarcoma cells. ATO treatment suppressed the invasiveness of osteosarcoma cells by inhibiting the transcriptional activity of GLI2. In addition, the combination of Hh inhibitors including ATO, vismodegib and GANT61 prevented migration and metastasis of osteosarcoma cells. Consequently, our findings suggested that GLI2 regulated metastasis as well as the progression of osteosarcomas. Inhibition of the GLI2 transcription may be an effective therapeutic method for preventing osteosarcoma metastasis.

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Section: Methodsmentioning

confidence: 99%

GLI2 is a novel therapeutic target for metastasis of osteosarcoma

Nagao‐Kitamoto

Nagata

Nagano

et al. 2014

Intl Journal of Cancer

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“…The p62 expression vector was kindly provided by Dr. Sudo T. 59 Transfection of the expression vector was performed as recommended in the supplier's protocol using FuGENE 6 as previously reported. 60 (Roche Applied Science, 1815091) LC3 turnover assay. MEB5 cells were incubated in normoxic or hypoxic conditions for 48 h in the presence and absence of 10 μM chloroquine (lysosomal inhibitor, Sigma Aldrich, C6628-25) followed by western blot analysis using LC3 antibodies.…”

Section: Methodsmentioning

confidence: 99%

Accumulation of p62 in degenerated spinal cord under chronic mechanical compression

et al. 2011

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Intracellular accumulation of altered proteins, including p62 and ubiquitinated proteins, is the basis of most neurodegenerative disorders. The relationship among the accumulation of altered proteins, autophagy, and spinal cord dysfunction by cervical spondylotic myelopathy has not been clarified. We examined the expression of p62 and autophagy markers in the chronically compressed spinal cord of tiptoe-walking Yoshimura mice. In addition, we examined the expression and roles of p62 and autophagy in hypoxic neuronal cells. Western blot analysis showed the accumulation of p62, ubiquitinated proteins, and microtubule-associated protein 1 light chain 3 (LC3), an autophagic marker, in the compressed spinal cord. Immunohistochemical examinations showed that p62 accumulated in neurons, axons, astrocytes, and oligodendrocytes. Electron microscopy showed the expression of autophagy markers, including autolysosomes and autophagic vesicles, in the compressed spinal cord. These findings suggest the presence of p62 and autophagy in the degenerated compressed spinal cord. Hypoxic stress increased the expression of p62, ubiquitinated proteins, and LC3-II in neuronal cells. In addition, LC3 turnover assay and GFP-LC3 cleavage assay showed that hypoxic stress increased autophagy flux in neuronal cells. These findings suggest that hypoxic stress induces accumulation of p62 and autophagy in neuronal cells. The forced expression of p62 decreased the number of neuronal cells under hypoxic stress. These findings suggest that p62 accumulation under hypoxic stress promotes neuronal cell death. Treatment with 3-methyladenine, an autophagy inhibitor decreased the number of neuronal cells, whereas lithium chloride, an autophagy inducer increased the number of cells under hypoxic stress. These findings suggest that autophagy promotes neuronal cell survival under hypoxic stress. Our findings suggest that pharmacological inducers of autophagy may be useful for treating cervical spondylotic myelopathy patients.

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“…The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate cell proliferation, as previously described (9). Briefly, cells cultured on microplates were incubated with the MTT substrate for 4 h, rinsed with PBS, and subsequently lysed.…”

Section: Methodsmentioning

confidence: 99%

Role of GOLPH3 and GOLPH3L in the proliferation of human rhabdomyosarcoma

Kunigou

Nagao²,

Kawabata³

et al. 2011

Oncol Rep

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Abstract. GOLPH3 was originally identified by proteomic analyses of Golgi proteins localized in the trans-Golgi network. Recently, it was reported that GOLPH3 is up-regulated in various types of malignancies, including melanoma, colon cancer and lung cancer. However, the mechanism through which GOLPH3 is involved in the pathogenesis of rhabdomyosarcoma remains unidentified. In order to explore the function of GOLPH3 and its isoform, GOLPH3L, in the pathogenesis of rhabdomyosarcoma, we investigated the expression and knockdown effects of GOLPH3 and GOLPH3L in human rhabdomyosarcoma. Western blot analysis and real-time PCR revealed that human rhabdomyosarcoma cell lines and biopsy specimens exhibited an increased expression of GOLPH3 and GOLPH3L. GOLPH3 and GOLPH3L knockdown by siRNA prevented the proliferation of human rhabdomyosarcoma cell lines. In addition, double-knockdown of GOLPH3 and GOLPH3L also prevented the proliferation of rhabdomyosarcoma cell lines. Our findings improve the understanding of rhabdomyosarcoma pathogenesis and suggest that the knockdown of GOLPH3 or GOLPH3L may be an effective treatment for rhabdomyosarcoma. IntroductionRhabdomyosarcoma, which is the most common soft-tissue sarcoma in children, includes two major subtypes, alveolar rhabdomyosarcoma and embryonal rhabdomyosarcoma. Approximately 70% of rhabdomyosarcoma patients without metastatic tumors are cured, but the prognosis of patients with metastasis or relapse remains dismal. Although numerous clinical trials have been performed, little improvement has been made for high-risk rhabdomyosarcoma patients, who have a 3-year overall survival of approximately 30% (1,2). Thus, improvements in outcome and in the understanding of the molecular pathways of rhabdomyosarcoma pathogenesis are a priority.GOLPH3 (also known as GOPP1, GPP34, MIDAS or FLJ90675) was originally identified by proteomic analyses of Golgi proteins localized to the trans-Golgi network (3,4). It has been reported that GOLPH3 is dynamically associated with the trans-Golgi matrix, as it rapidly moves from the transGolgi network to the cytosol and localizes in endosomes and at the plasma membrane (5). Recently, it has been reported that GOLPH3 modulates solid tumors through mTOR signaling (6).GOLPH3L (also known as GPP34R, GMx33β or FLJ10687) is an isoform of GOLPH3; these proteins are very homologous to each other and are similarly localized (4). Although the two isoforms are highly homologous in their amino acid sequences, the function of GOLPH3L has not yet been examined.In the present study, we investigated the involvement of GOLPH3 and GOLPH3L in the pathogenesis of human rhabdomyosarcoma. We assessed the expression of GOLPH3 and GOLPH3L in rhabdomyosarcoma and the effect of GOLPH3 or GOLPH3L inhibition on the growth of rhabdomyosarcoma cells. We found that the knockdown of GOLPH3 or GOLPH3L inhibited human rhabdomyosarcoma cell proliferation. Materials and methodsCell culture. The human rhabdomyosarcoma cell lines, KYM-1 and RD, were provided by the Health Science R...

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Suppression of Osteosarcoma Cell Invasion by Chemotherapy Is Mediated by Urokinase Plasminogen Activator Activity via Up-Regulation of EGR1

Cited by 28 publications

References 50 publications

GLI2 is a novel therapeutic target for metastasis of osteosarcoma

GLI2 is a novel therapeutic target for metastasis of osteosarcoma

Accumulation of p62 in degenerated spinal cord under chronic mechanical compression

Role of GOLPH3 and GOLPH3L in the proliferation of human rhabdomyosarcoma

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