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1.
Nat Commun ; 11(1): 4480, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32900992

RESUMO

Macroautophagy initiates by formation of isolation membranes, but the source of phospholipids for the membrane biogenesis remains elusive. Here, we show that autophagic membranes incorporate newly synthesized phosphatidylcholine, and that CTP:phosphocholine cytidylyltransferase ß3 (CCTß3), an isoform of the rate-limiting enzyme in the Kennedy pathway, plays an essential role. In starved mouse embryo fibroblasts, CCTß3 is initially recruited to autophagic membranes, but upon prolonged starvation, it concentrates on lipid droplets that are generated from autophagic degradation products. Omegasomes and isolation membranes emanate from around those lipid droplets. Autophagy in prolonged starvation is suppressed by knockdown of CCTß3 and is enhanced by its overexpression. This CCTß3-dependent mechanism is also present in U2OS, an osteosarcoma cell line, and autophagy and cell survival in starvation are decreased by CCTß3 depletion. The results demonstrate that phosphatidylcholine synthesis through CCTß3 activation on lipid droplets is crucial for sustaining autophagy and long-term cell survival.


Assuntos
Autofagia/fisiologia , Colina-Fosfato Citidililtransferase/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Animais , Autofagossomos/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Colina-Fosfato Citidililtransferase/antagonistas & inibidores , Colina-Fosfato Citidililtransferase/genética , Meios de Cultura , Ativação Enzimática , Técnicas de Silenciamento de Genes , Humanos , Gotículas Lipídicas/metabolismo , Camundongos , Modelos Biológicos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Fosfatidilcolinas/metabolismo
2.
Int J Nanomedicine ; 15: 5131-5146, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764941

RESUMO

Background: Gene therapy is considered a novel way to treat osteosarcoma, and microRNAs are potential therapeutic targets for osteosarcoma. miR-214 has been found to promote osteosarcoma aggression and metastasis. Graphene oxide (GO) is widely used for gene delivery for the distinct physiochemical properties and minimal cytotoxicity. Methods: Polyethyleneimine (PEI)-functionalized GO complex was well-prepared and loaded with miR-214 inhibitor at different concentrations. The load efficacy was tested by gel retardation assay and the cy3-labeled fluorescence of cellular uptake. The experiments of wound healing, immunofluorescence staining, Western blot, qRT-PCR and immunohistochemical staining were performed to measure the inhibitory effect of the miR-214 inhibitor systematically released from the complexes against MG63, U2OS cells and xenograft tumors. Results: The systematic mechanistic elucidation of the efficient delivery of the miR-214 inhibitor by GO-PEI indicated that the inhibition of cellular miR-214 caused a decrease in osteosarcoma cell invasion and migration and an increase in apoptosis by targeting phosphatase and tensin homolog (PTEN). The synergistic combination of the GO-PEI-miR-214 inhibitor and CDDP chemotherapy showed significant cell death. In a xenograft mouse model, the GO-PEI-miR-214 inhibitor significantly inhibited tumor volume growth. Conclusion: This study indicates the potential of functionalized GO-PEI as a vehicle for miRNA inhibitor delivery to treat osteosarcoma with low toxicity and miR-214 can be a good target for osteosarcoma therapy.


Assuntos
Grafite/química , MicroRNAs/antagonistas & inibidores , Terapia de Alvo Molecular , Osteossarcoma/tratamento farmacológico , PTEN Fosfo-Hidrolase/metabolismo , Polietilenoimina/química , Polietilenoimina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias Ósseas/patologia , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/genética , Terapia Combinada , Humanos , Camundongos , MicroRNAs/genética , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia
3.
Gene ; 763: 145068, 2020 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-32827680

RESUMO

CircRNAs are reported to exert a significant role in modulating genes in cancers, including osteosarcoma progression. Up to now, the function of circ_0010220 in osteosarcoma is still poorly known. The aim of our work was to figure out the potential mechanism of circ_0010220/miR-503-5p/CDCA4 axis in osteosarcoma progression. Firstly, quantitative RT-qPCR was utilized to measure the expression of circ_0010220 in osteosarcoma cells. Then, osteosarcoma cell proliferation, apoptosis, cell cycle, migration and invasion after loss of circ_0010220 were evaluated using CCK-8, flow cytometry, transwell migration, invasion and tumorigenesis experiments respectively. Circ_0010220 expression was markedly increased in osteosarcoma cells. Additionally, knockdown of circ_0010220 significantly depressed tumor growth. CCK-8 analysis indicated that down-regulation of circ_0010220 inhibited osteosarcoma cells proliferation. Flow cytometry assay showed that knockdown of circ_0010220 induced cell apoptosis and blocked cell cycle in the G1 phase. Meanwhile, cell migration an invasion was reduced by circ_0010220. Furthermore, miR-503-5p was predicted as the target for circ_0010220 and miR-503-5p inhibitors reversed cell growth suppressed through silencing circ_0010220. Then, our study demonstrated that Cell Division Cycle-Associated protein 4 (CDCA4) could be a downstream target of miR-503-5p. Additionally, circ_0010220 down-regulation reduced CDCA4 expression level and the inhibitors of miR-503-5p reversed that. In conclusion, we indicated circ_0010220 can be an important biomarker for osteosarcoma via regulating miR-503-5p and CDCA4.


Assuntos
Neoplasias Ósseas/genética , Proteínas de Ciclo Celular/genética , MicroRNAs/genética , Osteossarcoma/genética , RNA Circular/genética , Animais , Apoptose , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Carcinogênese/genética , Carcinogênese/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Pontos de Checagem da Fase G1 do Ciclo Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/metabolismo , Osteossarcoma/metabolismo , Osteossarcoma/patologia , RNA Circular/metabolismo
4.
Nat Commun ; 11(1): 3531, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669601

RESUMO

Homologous recombination (HR) factors were recently implicated in DNA replication fork remodeling and protection. While maintaining genome stability, HR-mediated fork remodeling promotes cancer chemoresistance, by as-yet elusive mechanisms. Five HR cofactors - the RAD51 paralogs RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3 - recently emerged as crucial tumor suppressors. Albeit extensively characterized in DNA repair, their role in replication has not been addressed systematically. Here, we identify all RAD51 paralogs while screening for modulators of RAD51 recombinase upon replication stress. Single-molecule analysis of fork progression and architecture in isogenic cellular systems shows that the BCDX2 subcomplex restrains fork progression upon stress, promoting fork reversal. Accordingly, BCDX2 primes unscheduled degradation of reversed forks in BRCA2-defective cells, boosting genomic instability. Conversely, the CX3 subcomplex is dispensable for fork reversal, but mediates efficient restart of reversed forks. We propose that RAD51 paralogs sequentially orchestrate clinically relevant transactions at replication forks, cooperatively promoting fork remodeling and restart.


Assuntos
Replicação do DNA , Rad51 Recombinase/metabolismo , Proteína BRCA2/metabolismo , Linhagem Celular Tumoral , Estruturas Cromossômicas/metabolismo , Cromossomos/ultraestrutura , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Recombinação Homóloga , Humanos , Microscopia , Mutagênicos , Mutação , Osteossarcoma/metabolismo , RNA Interferente Pequeno/metabolismo
5.
J Cancer Res Ther ; 16(2): 215-221, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32474504

RESUMO

Objective: Osteosarcoma is a malignant bone tumor and is generally treated with radiotherapy combined with radiosensitizers. The aim of the present study was to investigate the radiosensitization effects of berberine on osteosarcoma cells and the role of Rad51 in radiosensitivity by berberine. Materials and Methods: Cells from the human osteosarcoma cell line MG-63 were exposed to γ-ray irradiation (0, 2, 4, 6, and 8 Gy) and berberine (20 µM). Radiosensitivity was evaluated by determining cell viability using an MTT assay. Flow cytometry was used to determine cell cycle and apoptosis. Real-time PCR and western blot were performed to analyze the mRNA and protein expressions of Rad51. The protein levels of E-cadherin and vimentin were also measured to evaluate the epithelial-mesenchymal transition (EMT) process. Tumor invasion was determined by the Boyden chamber assay. Results: Berberine exacerbated the decline in viability of MG-63 cells exposed to γ-rays irradiation at various concentrations (25, 50, 75, and 100 µmol/L) and induced cell cycle arrest in the G2/M phase as well as apoptosis. The mRNA and protein expressions of Rad51 were significantly decreased by berberine in MG-63 cells. Inhibition of Rad51 by B02 enhanced the radiosensitivity of MG-63 cells. Berberine inhibited their invasive capability as well as increased E-cadherin and decreased vimentin protein levels; this indicated that berberine suppressed the EMT process in MG-63 cells exposed to γ-rays irradiation. Conclusion: Berberine enhances the radiosensitivity of MG-63 osteosarcoma cells. Rad51 is a potential target of berberine in the radiosensitization of osteosarcoma.


Assuntos
Berberina/farmacologia , Pontos de Checagem do Ciclo Celular , Sobrevivência Celular , Transição Epitelial-Mesenquimal , Osteossarcoma/radioterapia , Rad51 Recombinase/antagonistas & inibidores , Radiossensibilizantes/farmacologia , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/radioterapia , Linhagem Celular Tumoral , Humanos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Rad51 Recombinase/metabolismo
6.
Nat Cell Biol ; 22(7): 868-881, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32483387

RESUMO

Osteosarcoma is a type of aggressive malignant bone tumour that frequently metastasizes to lungs, resulting in poor prognosis. However, the molecular mechanisms of lung metastasis of osteosarcoma remain poorly understood. Here we identify exon-intron fusion genes in osteosarcoma cell lines and tissues. These fusion genes are derived from chromosomal translocations that juxtapose the coding region for amino acids 1-38 of Rab22a (Rab22a1-38) with multiple inverted introns and untranslated regions of chromosome 20. The resulting translation products, designated Rab22a-NeoFs, acquire the ability to drive lung metastasis of osteosarcoma. The Rab22a1-38 moiety governs the function of Rab22a-NeoFs by binding to SmgGDS-607, a GTP-GDP exchange factor of RhoA. This association facilitates the release of GTP-bound RhoA from SmgGDS-607, which induces increased activity of RhoA and promotes metastasis. Disrupting the interaction between Rab22a-NeoF1 and SmgGDS-607 with a synthetic peptide prevents lung metastasis in an orthotopic model of osteosarcoma. Our findings may provide a promising strategy for a subset of osteosarcoma patients with lung metastases.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Pulmonares/secundário , Osteossarcoma/patologia , Translocação Genética , Proteínas rab de Ligação ao GTP/metabolismo , Adulto , Animais , Apoptose , Biomarcadores Tumorais/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Movimento Celular , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Osteossarcoma/genética , Osteossarcoma/metabolismo , Prognóstico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem , Proteínas rab de Ligação ao GTP/genética
7.
J Cancer Res Clin Oncol ; 146(7): 1693-1700, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32333142

RESUMO

PURPOSE: Osteosarcoma is the most common bone tumor, mainly affecting adolescents and young adults, and metastatic disease has poor outcomes with a dismal overall survival. Currently, chemotherapy is the standard of care with limited results, finding that new therapies could improve these outcomes. Preclinical and clinical studies have suggested a possible important role of ErbB pathway aberrations in osteosarcoma etiology. The present study shows the effect of afatinib, an irreversible ErbB family blocker in osteosarcoma cell lines. METHODS: Within a panel of human osteosarcoma cell lines, we addressed cell viability assay using afatinib at increasing concentrations. Motility was measured in wound-healing assays and invasion capacity was assessed in Transwell chamber assays. Finally, to monitor ErbB pathway modulation by afatinib and related compounds, we used Western blot analyses. RESULTS: Cell viability inhibition, as well as a reduction of motility and migration of osteosarcoma cell line were observed after treatment with afatinib. Likewise, in the HOS cell line, afatinib decreased phosphorylation of key components in the ErbB signaling pathway. CONCLUSIONS: Afatinib shows relevant antitumor effect in several osteosarcoma cell lines, as it causes a significant impact on cell viability, motility, and migration with a significant decrease in the activation of ErbB pathway activity.


Assuntos
Afatinib/farmacologia , Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Hum Cell ; 33(3): 810-818, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32274658

RESUMO

Circular RNAs (circRNAs) exert pivotal effects on regulating the progression of osteosarcoma (OS). It was found through microarray analysis that circ-0002052 is abnormally expressed in OS, but the role of circ-0002052 in OS remains obscure. The results of this research manifested that relative to that in non-tumor controls, circ-0002052 level was raised in OS tissues. Up-regulated circ-0002052 was associated with advanced stage, tumor size, and metastasis. Additionally, circ-0002052 elevation indicated a low survival rate in OS patients and silencing of circ-0002052 suppressed proliferation, migration, and invasion of OS cells. It was proved that circ-0002052 sponged miR-382 and stimulated STX6 expression, thus activating Wnt/ß-catenin. The function of circ-0002052 reduction in OS cells was effectively reversed by miR-382 suppression. To sum up, it can be concluded that circ-0002052, functioning as a sponge for miR-382, enhances the activation of Wnt/ß-catenin mediated by STX6 to stimulate the progression of OS, and circ-0002052 may be an underlying treatment target and a biomarker for prognosis of osteosarcoma.


Assuntos
Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Osteossarcoma/genética , Osteossarcoma/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , RNA Circular/fisiologia , Linhagem Celular Tumoral , Humanos , Terapia de Alvo Molecular , Osteossarcoma/diagnóstico , Osteossarcoma/terapia , RNA Circular/genética , RNA Circular/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
9.
Phytomedicine ; 69: 153183, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32113150

RESUMO

BACKGROUND: Osteosarcoma (OS) is a significant threat to the lives of children and young adults. Although neoadjuvant chemotherapy is the first choice of treatment for OS, it is limited by serious side-effects and cancer metastasis. ß-Elemonic acid (ß-EA), an active component extracted from Boswellia carterii Birdw., has been reported to exhibit potential anti-inflammatory and anticancer activities. However, the anti-tumor effects and underlying mechanisms on OS as well as pharmacokinetic characteristics of ß-EA remain unknown. PURPOSE: This study was aimed to investigating the anti-tumor effects of ß-EA on human OS, the underlying mechanisms, and the pharmacokinetic and tissue distribution characteristics. STUDY DESIGN AND METHODS: Cell viability and colony formation assays were performed to determine the effect of ß-EA cell on cell proliferation. Apoptosis rates, mitochondrial membrane potential and cell cycle features were analyzed by flow cytometry. qRT-PCR, Western blot, immunofluorescence and immunohistochemical assays were conducted to evaluate the expression levels of genes or proteins related to the pathways affected by ß-EA in vitro and in vivo. Cell migration and invasion were evaluated in wound healing and Transwell chamber assays. The effects and pharmacokinetic characteristics of ß-EA in vivo were evaluated by analyzing tumor suppression, pharmacokinetics and tissue distribution. RESULTS: Explorations indicated that endoplasmic reticulum (ER) stress conditions provoked by ß-EA activated the PERK/eIF2α/ATF4 branch of the unfolded protein reaction (UPR), stimulating C/EBP homologous protein (CHOP)-regulated apoptosis and inducing Ca2+ leakage leading to caspase-dependent apoptosis. Furthermore, ß-EA induced G0/G1 cell cycle arrest and inhibited metastasis of HOS and 143B cells by attenuating Wnt/ß-catenin signaling effects, which included decreased levels of p-Akt(Ser473), p-Gsk3ß (Ser9), Wnt/ß-catenin target genes (c-Myc and CyclinD1) along with a decline in nuclear ß-catenin accumulation. The fast absorption, short elimination half-life, and linear pharmacokinetic characteristics of ß-EA were also revealed. The distribution of ß-EA was detected in the tumor and bone tissues. CONCLUSIONS: Overall, both in vitro and in vivo investigations showed the potential of ß-EA for the treatment of human OS. The pharmacokinetic profile and considerable distribution in the tumor and bone tissues warrant further preclinical or even clinical studies.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Osteossarcoma/tratamento farmacológico , Fenantrenos/farmacologia , Fator 4 Ativador da Transcrição/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacocinética , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Estresse do Retículo Endoplasmático/fisiologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos BALB C , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Distribuição Tecidual , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismo , eIF-2 Quinase/metabolismo
10.
Nat Commun ; 11(1): 1141, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111827

RESUMO

Osteosarcoma, an aggressive malignant cancer, has a high lung metastasis rate and lacks therapeutic target. Here, we reported that chromobox homolog 4 (CBX4) was overexpressed in osteosarcoma cell lines and tissues. CBX4 promoted metastasis by transcriptionally up-regulating Runx2 via the recruitment of GCN5 to the Runx2 promoter. The phosphorylation of CBX4 at T437 by casein kinase 1α (CK1α) facilitated its ubiquitination at both K178 and K280 and subsequent degradation by CHIP, and this phosphorylation of CBX4 could be reduced by TNFα. Consistently, CK1α suppressed cell migration and invasion through inhibition of CBX4. There was a reverse correlation between CK1α and CBX4 in osteosarcoma tissues, and CK1α was a valuable marker to predict clinical outcomes in osteosarcoma patients with metastasis. Pyrvinium pamoate (PP) as a selective activator of CK1α could inhibit osteosarcoma metastasis via the CK1α/CBX4 axis. Our findings indicate that targeting the CK1α/CBX4 axis may benefit osteosarcoma patients with metastasis.


Assuntos
Caseína Quinase Ialfa/metabolismo , Ligases/antagonistas & inibidores , Ligases/metabolismo , Osteossarcoma/patologia , Proteínas do Grupo Polycomb/antagonistas & inibidores , Proteínas do Grupo Polycomb/metabolismo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Caseína Quinase Ialfa/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Ligases/genética , Camundongos , Mutação , Metástase Neoplásica , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Osteossarcoma/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas do Grupo Polycomb/genética , Regiões Promotoras Genéticas , Compostos de Pirvínio/farmacologia , Compostos de Pirvínio/uso terapêutico , Análise de Sobrevida , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos , Fatores de Transcrição de p300-CBP/metabolismo
11.
Technol Cancer Res Treat ; 19: 1533033820909125, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32129151

RESUMO

Osteosarcoma is the most common primary malignant bone tumor in the clinic. It is more common in children and adolescents. It has high malignancy, early metastasis rate, rapid disease progression, and high mortality. Although past years have witnessed the great improvement in the treatments of osteosarcoma, there remains a long way to go. MicroRNAs affect the malignant biological behaviors such as tumor proliferation and metastasis by regulating their target genes. In this study, we investigated the role and mechanism of miR-384 in osteosarcoma. Quantitative real-time polymerase chain reaction assay was performed to detect the expression of miR-384 and insulin-like growth factor binding protein 3 in osteosarcoma tissues and cell lines and established its correlation with osteosarcoma tumor progression and metastasis. To probe whether miR-384 played a tumor suppression role in osteosarcoma, we carried out gain-of-function and loss-of-function assays. Cell Counting Kit-8, cell colony formation, and transwell assays were carried out to determine the cells proliferation and invasion, respectively. Western blot was used to detect the changes of epithelial-mesenchymal transition marker proteins and insulin-like growth factor binding protein 3. MiR-384 was downregulated in osteosarcoma tissues and cell lines. MiR-384 was overexpressed in G292 cells transfected with miR-384 mimics and knocked down in Saos-2 cells with small hairpin RNA targeting miR-384. Ectopic expression of miR-384 inhibited osteosarcoma cell proliferation, colony formation, and invasion. E-cadherin was brought to a decrease whereas N-cadherin and Snail to an increase under the silent expression of miR-384, while overexpression of miR-384 led to an opposite result. MiR-384 could regulate insulin-like growth factor binding protein 3 expression in osteosarcoma. Quantitative polymerase chain reaction and Western blotting results validated that miR-384 knockdown downgrades both messenger RNA and protein levels of insulin-like growth factor binding protein 3 in G292 cells, while miR-384 upregulation exerted an opposite effect in Saos-2 cells. Insulin-like growth factor binding protein 3 was upregulated in osteosarcoma tissues and osteosarcoma cell lines compared with normal ones. Through the bioinformatics database found that the upstream transcriptional regulator of insulin-like growth factor binding protein 3 is MECP2. So miR-384 can directly inhibit MECP2 and then promote the expression of insulin-like growth factor binding protein 3. These results suggested that miR-384 might be a potential therapeutic targets and biomarker in osteosarcoma.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/patologia , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , MicroRNAs/genética , Osteossarcoma/patologia , Adulto , Biomarcadores Tumorais/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Biologia Computacional/métodos , Transição Epitelial-Mesenquimal , Feminino , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Masculino , MicroRNAs/metabolismo , Invasividade Neoplásica , Estadiamento de Neoplasias , Osteossarcoma/genética , Osteossarcoma/metabolismo , Prognóstico , Adulto Jovem
12.
Gene ; 740: 144520, 2020 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-32130980

RESUMO

Osteosarcoma (OS) is the most common primary bone tumor, and mainly occurs in children and early adults. The overall pathogenesis of osteosarcoma remains unclear. Circular RNAs (circRNAs) have been demonstrated to be one of the regulators in tumors as non-coding RNAs. In present study, we identified an elevated expressed circ-0060428 in osteosarcoma cells. The results of CCK-8 and flow cytometry assay showed that circ-0060428 promoted cell proliferation and reduced cell apoptosis of osteosarcoma cells. Bioinformatics analyses predicted the binding site between miR-375 to RPBJ and circ-0060428, and dual luciferase reporter assay verified this prediction. Furthermore, miRNA inhibitor of miR-375 could recover the influence of cell proliferation and apoptosis by knocking down the expression of circ-0060428. Meanwhile, knockdown of both circ-0060428 and RBPJ resulted in a lower apoptosis rate than circ-0060428 alone. Western blot analyses indicated that circ-0060428 regulated the expression of apoptosis related proteins (Bax, Bcl-2, and cleaved-Caspase-3) by upregulating RPBJ expression in osteosarcoma cells. Altogether, we confirmed the up-regulated circ-0060428 could improve the proliferation and survival of osteosarcoma cells by sponging miR-375 to upregulate RBPJ expression. This finding supported a novel clinically biomarker and treatment target for osteosarcoma therapy.


Assuntos
Proliferação de Células/fisiologia , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , MicroRNAs/metabolismo , Osteossarcoma/fisiopatologia , RNA Circular , Linhagem Celular Tumoral , Proliferação de Células/genética , Perfilação da Expressão Gênica , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , MicroRNAs/genética , Osteossarcoma/metabolismo , RNA Circular/genética , RNA Circular/metabolismo
13.
J Vis Exp ; (156)2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-32065171

RESUMO

Methylation-specific probe amplification (MSPA) is a simple and robust technique that can be used to detect relative differences in methylation levels of DNA samples. It is resourceful, requires small amounts of DNA, and takes around 4-5 h of hands-on work. In the presented technique, DNA samples are first denatured then hybridized to probes that target DNA at either methylated or reference sites as a control. Hybridized DNA is separated into parallel reactions, one undergoing only ligation and the other undergoing ligation followed by HhaI-mediated digestion at unmethylated GCGC sequences. The resultant DNA fragments are amplified by PCR and separated by capillary electrophoresis. Methylated GCGC sites are not digested by HhaI and produce peak signals, while unmethylated GCGC sites are digested and no peak signals are generated. Comparing the control-normalized peaks of digested and undigested versions of each sample provides the methylation dosage ratio of a DNA sample. Here, MSPA is used to detect the effects of osteosarcoma-derived extracellular vesicles (EVs) on the methylation status of long interspersed nuclear element-1 (LINE-1) in mesenchymal stem cells. LINE-1s are repetitive DNA elements that typically undergo hypomethylation in cancer and, in this capacity, may serve as a biomarker. Ultracentrifugation is also used as a cost-effective method to separate extracellular vesicles from biological fluids (i.e., when preparing EV-depleted fetal bovine serum [FBS] and isolating EVs from osteosarcoma conditioned media [differential centrifugation]). For methylation analysis, custom LINE-1 probes are designed to target three methylation sites in the LINE-1 promoter sequence and seven control sites. This protocol demonstrates the use of MSPA for LINE-1 methylation analysis and describes the preparation of EV-depleted FBS by ultracentrifugation.


Assuntos
Neoplasias Ósseas/genética , Metilação de DNA , Vesículas Extracelulares/genética , Elementos Nucleotídeos Longos e Dispersos , Células-Tronco Mesenquimais/metabolismo , Osteossarcoma/genética , Reação em Cadeia da Polimerase/métodos , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Meios de Cultivo Condicionados/metabolismo , DNA/genética , Epigênese Genética , Vesículas Extracelulares/metabolismo , Humanos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Regiões Promotoras Genéticas , Células Tumorais Cultivadas
14.
Biochem J ; 477(4): 801-814, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32011652

RESUMO

Autophagy is a critical cellular homeostatic mechanism, the dysfunction of which has been linked to a wide variety of disease states. It is regulated through the activity of specific kinases, in particular Unc-51 like autophagy activating kinase 1 (ULK1) and Phosphatidylinositol 3-kinase vacuolar protein sorting 34 (VPS34), which have both been suggested as potential targets for drug development. To identify new chemical compounds that might provide useful chemical tools or act as starting points for drug development, we screened each protein against the Published Kinase Inhibitor Set (PKIS), a library of known kinase inhibitors. In vitro screening and analysis of the published selectivity profiles of the hits informed the selection of three relatively potent ATP-competitive inhibitors against each target that presented the least number of off-target kinases in common. Cellular assays confirmed potent inhibition of autophagy in response to two of the ULK1 inhibitors and all three of the VPS34 inhibitors. These compounds represent not only a new resource for the study of autophagy but also potential chemical starting points for the validation or invalidation of these two centrally important autophagy kinases in disease models.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/antagonistas & inibidores , Autofagia , Classe III de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Descoberta de Drogas , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Osteossarcoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Osteossarcoma/tratamento farmacológico , Osteossarcoma/metabolismo , Fosforilação , Células Tumorais Cultivadas
15.
Phytomedicine ; 68: 153186, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32088353

RESUMO

BACKGROUND: Osteosarcoma is the most common type of primary malignant bone tumor. This disease has exhibited a progressively lower survival rate over the past several decades, which has resulted in it becoming a main cause of death in humans. Rosmarinic acid (RA), a water-soluble polyphenolic phytochemical, exerts powerful anticancer effects against multiple types of cancer; however, its potential effects on osteosarcoma remain unknown. Hence, the present study investigated the efficacy of RA against osteosarcoma and aimed to clarify the mechanisms underlying this process. METHODS: The effects of RA on cell viability, apoptosis, cell cycle distribution, migration, invasion, and signaling molecules were analyzed by CCK-8 assay, flowcytometric analysis, wound healing assay, Transwell assay, proteomic analysis, and use of shRNAs. RESULTS: RA exerted anti-proliferation and pro-apoptotic effects on U2OS and MG63 osteosarcoma cells. Apoptosis was induced via extrinsic and intrinsic pathways by increasing the Bax/Bcl-2 ratio, triggering the intracellular production of reactive oxygen species (ROS), reducing the mitochondrial membrane potential (MMP), and upregulating the cleavage rates of caspase-8, caspase-9, and caspase-3. Additionally, RA suppressed the migration and invasion of osteosarcoma cells by inhibiting the expression levels of matrix metalloproteinase-2 and -9 (MMP-2 and -9), which are associated with a weakening of the epithelial-mesenchymal transition (EMT). Moreover, proteomic analyses identified DJ-1 as a potential target for RA. Several studies have indicated an oncogenic role for DJ-1 using knockdowns via the lentiviral-mediated transfection of shRNA, which caused the conspicuous suppression of cell proliferation, migration, and invasion as well as the arrest of cell cycle progression. At the molecular level, the expression levels of DJ-1, p-PI3K, and p-Akt were reduced, whereas the protein levels of phosphatase and tensin homologue (PTEN) were increased. CONCLUSION: In conjunction with the high levels of DJ-1 expression in osteosarcoma tissues and cell lines, the present results suggested that RA exhibited anticancer effects in osteosarcoma cells by inhibiting DJ-1 via regulation of the PTEN-PI3K-Akt signaling pathway. Therefore, DJ-1 might be a biological target for RA in osteosarcoma cells.


Assuntos
Neoplasias Ósseas/tratamento farmacológico , Cinamatos/farmacologia , Depsídeos/farmacologia , Osteossarcoma/tratamento farmacológico , Proteína Desglicase DJ-1/antagonistas & inibidores , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Desglicase DJ-1/genética , Proteína Desglicase DJ-1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
16.
Life Sci ; 254: 117392, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32014424

RESUMO

Increasing evidence has uncovered that long noncoding RNAs (lncRNAs) play extremely important roles in numerous steps of gene regulation concerning the progression of tumors. Defined as a kind of lncRNA, DDX11-AS1 has been considered to be closely related to the tumorigenesis of malignancies. Nevertheless, the underlying regulatory role of it in osteosarcoma remains to be analyzed and elucidated. In this research, a dramatically upregulated expression of DDX11-AS1 was detected in osteosarcoma cells. Loss-of-function assays revealed that decreased expression of DDX11-AS1 impaired osteosarcoma cell proliferation, metastasis as well as epithelial-mesenchymal transition (EMT) process. Afterwards, molecular mechanism tests validated that DDX11-AS1 could sponge miR-873-5p to upregulate DDX11 expression in osteosarcoma. Additionally, functional tests delineated that upregulation of miR-873-5p inhibited cell proliferation, metastasis as well as EMT process in osteosarcoma progression. Further, DDX11-AS1 was verified to regulate the mRNA stability of DDX11 through binding with IGF2BP2 in osteosarcoma. Final rescue tests in vitro and in vivo further elucidated that DDX11 overexpression could reversed the DDX11-AS1 downregulation-mediated effect on osteosarcoma progression. To sum up, DDX11-AS1 contributes to osteosarcoma progression via stabilizing DDX11.


Assuntos
Elementos Antissenso (Genética) , Neoplasias Ósseas/patologia , RNA Helicases DEAD-box/genética , DNA Helicases/genética , Osteossarcoma/patologia , Neoplasias Ósseas/metabolismo , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Progressão da Doença , Transição Epitelial-Mesenquimal/genética , Humanos , Osteossarcoma/metabolismo , Ligação Proteica , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/metabolismo , Regulação para Cima
17.
Oncol Rep ; 43(2): 601-608, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31894282

RESUMO

Receptor tyrosine kinase like orphan receptor 2 (ROR2) regulates Wnt5a-induced cell migration by phosphorylating PI3K/Akt and activating RhoA in osteosarcoma. However, the role of Wnt5a signaling and its corresponding receptors in the regulation of osteosarcoma metastasis remains poorly understood. ROR1 monoclonal antibody (mAb) and short hairpin (sh)RNA targeting ROR2 markedly inhibited the activity of dishevelled associated activator of morphogenesis 1 (DAAM1) and RhoA and retarded cell migration in osteosarcoma. ROR1 mAb and ROR2 shRNA destroyed the microfilament formation of osteosarcoma cells. Silencing of DAAM1 (with DAAM1 shRNA) downregulated RhoA activity and inhibited cell migration. The decrease of cell migration caused by DAAM1 shRNA was rescued by wild-type DAAM1 overexpression. DAAM1 and PI3Kα/Akt were parallel signaling pathways mediating osteosarcoma cell migration in response to Wnt5a. It was concluded that Wnt5a promotes osteosarcoma cell migration via ROR1/2 receptors, and then activates DAAM1 and RhoA.


Assuntos
Neoplasias Ósseas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Osteossarcoma/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Proteína Wnt-5a/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Transdução de Sinais
18.
Oncol Rep ; 43(2): 491-502, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31894343

RESUMO

MicroRNA­708­5p (miR­708­5p) and epithelial­â€‹to­mesenchymal transition (EMT) have been widely identified to contribute to the pathogenesis and progression of multiple cancers. However, the connection between miR­708­5p and EMT has not been sufficiently clarified. Therefore, our research aimed to investigate the impact of miR­708­5p on EMT and the metastasis of osteosarcoma (OS). We first analyzed the differentially expressed microRNAs (DEmiRNAs) from the GSE70367 dataset. We found that the expression of miR­708­5p was lower in OS cells. Overexpression of miR­708­5p was able to impair the migration and invasion of OS cells. Moreover, miR­708­5p inhibited EMT of OS cells MG63 and SaOS­2, wherein E­cadherin was increased, and N­cadherin, vimentin, and Snail were decreased. Semaphorin 4C (SEMA4C), mitogen­activated protein kinase kinase kinase 3 (MAP3K3), and zinc finger E­box­binding homeobox 1 (ZEB1) were predicted as target genes of miR­708­5p by bioinformatics method. Only ZEB1, one of the EMT­inducing transcription factors, was validated as the direct target gene of miR­708­5p in OS cells through dual­luciferase reporter assay and Western blot analysis. Knockdown of ZEB1 was found to inhibit the metastasis of MG63 and SaOS­2 cells, whereas ZEB1 over-expression promoted their metastasis. In summary, miR­708­5p impaired the metastasis and EMT of OS, which was found to be mediated by inhibition of ZEB1.


Assuntos
Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , MicroRNAs/genética , Osteossarcoma/genética , Osteossarcoma/patologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Neoplásica , Osteossarcoma/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética
19.
Biochim Biophys Acta Gen Subj ; 1864(4): 129522, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31945406

RESUMO

BACKGROUND: Osteosarcoma (OS) is the most frequent malignant bone tumor, affecting predominantly children and young adults. Metastases are a major clinical challenge in OS. In this context, 20% of OS patients are diagnosed with metastatic OS, but near 80% of all OS patients could present non-detectable micrometastases at the moment of diagnosis. METHODS: Osteogenic differentiation; doxorubicin exclusion assay; fluorescence microscopy; RT-qPCR; proteomic analysis. RESULTS: Our results suggest that metastatic OS cells possess a diminished osteoblastic differentiation potential with a gain of metastatic traits like the capacity to modify intracellular localization of chemodrugs and higher levels of expression of stemness-related genes. On the opposite hand, non-metastatic OS cells possess bone-associated traits like higher osteoblastic differentiation and also an osteoblastic-inducer secretome. OS cells also differ in the nature of their interaction with mesenchymal stem cells (MSCs), with opposites impacts on MSCs phenotype and behavior. CONCLUSIONS: All this suggests that a major trait acquired by metastatic cells is a switch into a stem-like state that could favor its survival in the pulmonary niche, opening new possibilities for personalized chemotherapeutic schemes. GENERAL SIGNIFICANCE: Our work provides new insights regarding differences among metastatic and non-metastatic OS cells, with particular emphasis on differentiation potential, multidrug resistance and interaction with MSCs.


Assuntos
Neoplasias Ósseas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteossarcoma/metabolismo , Antibióticos Antineoplásicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/secundário , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/patologia , Osteossarcoma/tratamento farmacológico , Osteossarcoma/secundário , Fenótipo , Relação Estrutura-Atividade , Células Tumorais Cultivadas
20.
Mater Sci Eng C Mater Biol Appl ; 108: 110460, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31923975

RESUMO

Myelosuppression, gastrointestinal toxicity and hypersensitivities always accompany chemotherapy of osteosarcoma (OS). In addition, the intricate karyotype of OS, the lack of targeted antitumor drugs and the bone microenvironment that provides a protective alcove for tumor cells reduce the therapeutic efficacy of chemotherapy. Here, we developed a multifunctional bone cement loaded with Fe3O4 nanoparticles and the antitumor drug doxorubicin (DOX/Fe3O4@PMMA) for synergistic MH ablation and chemotherapy of OS. The localized intratumorally administered DOX/Fe3O4@PMMA can change from liquid into solid at the tumor site via a polyreaction. The designed multifunctional bone cement was constructed with Fe3O4 nanoparticles, PMMA, and an antitumor drug approved by the U.S. Food and Drug administration (FDA). The injectability, magnetic hyperthermia (MH) performance, controlled drug release profile, and synergistic therapeutic effect of DOX/Fe3O4@PMMA in vitro were investigated in detail. Furthermore, the designed DOX/Fe3O4@PMMA controlled the release of DOX, enhanced the apoptosis of OS tissue, and inhibited the proliferation of tumor cells, demonstrating synergistic MH ablation and chemotherapy of OS in vivo. The biosafety of DOX/Fe3O4@PMMA was also evaluated in detail. This strategy significantly reduced surgical time, avoided operative wounds and prevented patient pain, showing a great clinical translational potential for OS treatment.


Assuntos
Cimentos para Ossos , Neoplasias Ósseas/terapia , Hipertermia Induzida , Nanopartículas de Magnetita , Osteossarcoma/terapia , Animais , Cimentos para Ossos/química , Cimentos para Ossos/farmacologia , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Doxorrubicina/química , Doxorrubicina/farmacologia , Humanos , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Polimetil Metacrilato/química , Polimetil Metacrilato/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
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