RESUMO
Osteosarcoma (OS) is one of the most aggressive bone tumors worldwide. Emerging documents have shown that long noncoding RNAs (lncRNAs) elicit crucial regulatory functions in the process of tumorigenesis. LncRNA DLGAP1-AS2 is recognized as a regulator in several types of cancers, but its biological functions and molecular mechanisms in OS remain to be elucidated. RT-qPCR and In situ hybridization (ISH) were used to evaluate DLGAP1-AS2 expression in OS samples. Western blotting was used for the measurement of the protein levels of hexokinase 2 (HK2) and epithelial-mesenchymal transition (EMT)-related markers. The proliferation of OS cells was determined using a CCK-8 assay and EdU assay. TUNEL assay and flow cytometry were performed to assess OS cell apoptosis. Glucose metabolism in vitro assays were used. The binding relations among miR-451a, HK2, and DLGAP1-AS2 were validated by luciferase reporter assay. The cellular distribution of DLGAP1-AS2 in OS cells was determined by FISH and subcellular fractionation assays. Mouse xenograft models were established to perform the experiments in vivo. We found that DLGAP1-AS2 expression was upregulated in OS tissues and cells. Downregulation of DLGAP1-AS2 expression suppressed the malignancy of OS cells by restraining cell proliferation, the EMT process, invasiveness, migration, and aerobic glycolysis and accelerating apoptotic behaviors. Of note, silenced DLGAP1-AS2 restrained tumor growth and metastasis in vivo. However, DLGAP1-AS2 overexpression accelerated the progression of OS. We further found that DLGAP1-AS2 upregulation was induced by hypoxia and low glucose. Additionally, DLGAP1-AS2 bound to miR-451a to upregulate HK2 expression. Rescue assays revealed that the DLGAP1-AS2/miR-451a/HK2 axis contributed to OS cell malignancy by promoting aerobic glucose metabolism. Overall, these findings revealed a new regulatory pathway where DLGAP1-AS2 upregulated HK2 expression by sponging miR-451a to accelerate OS development.
Assuntos
Osteossarcoma , RNA Longo não Codificante , Efeito Warburg em Oncologia , Humanos , Linhagem Celular Tumoral , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Hexoquinase/metabolismo , Camundongos Endogâmicos BALB C , MicroRNAs/metabolismo , Osteossarcoma/genética , Osteossarcoma/fisiopatologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
Signal transducer and activator of transcription 3 (STAT3) dysregulation has been characterized in canine OS, with previous data suggesting that constitutive STAT3 activation contributes to survival and proliferation in OS cell lines in vitro. Recently, the contribution of STAT3 to tumour metabolism has been described across several tumour histologies, and understanding the metabolic implications of STAT3 loss may elucidate novel therapeutic approaches with synergistic activity. The objective of this work was to characterize metabolic benchmarks associated with STAT3 loss in canine OS. STAT3 expression and activation was evaluated using western blotting in canine OS cell lines OSCA8 and Abrams. STAT3 was deleted from these OS cell lines using CRISPR-Cas9, and the effects on proliferation, invasion and metabolism (respirometry, intracellular lactate) were determined. Loss of STAT3 was associated with decreased basal and compensatory glycolysis in canine OS cell lines, without modulation of cellular proliferation. Loss of STAT3 also resulted in diminished invasive capacity in vitro. Interestingly, the absence of STAT3 did not impact sensitivity to doxorubicin in vitro. Our data demonstrate that loss of STAT3 modulates features of aerobic glycolysis in canine OS impacting capacities for cellular invasions, suggesting a role for this transcription factor in metastasis.
Assuntos
Neoplasias Ósseas , Doenças do Cão , Osteossarcoma , Animais , Cães , Apoptose , Neoplasias Ósseas/fisiopatologia , Neoplasias Ósseas/veterinária , Linhagem Celular Tumoral , Proliferação de Células , Doenças do Cão/fisiopatologia , Osteossarcoma/fisiopatologia , Osteossarcoma/veterinária , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Deleção de GenesRESUMO
Circular RNAlipoprotein receptor 6 (circLRP6) serves a role in promoting the tumorigenesis of retinoblastoma, esophageal squamous cell cancer and oral squamous cell carcinoma; however, whether circLRP6 demonstrates the same effect in osteosarcoma (OS) is yet to be fully elucidated. The present study aimed to analyze the expression, role and potential molecular mechanism of circLRP6 in OS. The expression levels of circLRP6, microRNA (miR)1413p, histone deacetylase 4 (HDAC4) and high mobility group protein 1 (HMGB1) were evaluated by reverse transcription-quantitative PCR in OS tissues and cell lines. Cell Counting Kit8, Transwell and Matrigel assays were conducted to evaluate cell proliferation, migration and invasion, respectively. Western blotting was also performed to determine HDAC4 and HMGB1 protein expression levels. Bioinformatics and dualluciferase reporter assays were used to predict and analyze the interactions between circLRP6 and miR1413p, miR1413p and HDAC4, as well as between miR1413p and HMGB1. Additionally, RNA immunoprecipitation was performed to verify the association between circLRP6 and miR1413p. The results confirmed that circLRP6 was highly expressed in OS tissues and cell lines. In addition, circLRP6 negatively regulated the expression of miR1413p and, in turn, miR1413p negatively regulated HDAC4 and HMGB1 expression. Functional assays revealed that circLRP6 knockdown inhibited the proliferation, migration and invasion of OS cells, whereas the inhibition of miR1413p or the overexpression of either HDAC4 or HMGB1 partly reversed the inhibitory effect of circLRP6 knockdown. In summary, the present study determined that circLRP6 knockdown inhibited the proliferation, migration and invasion of OS cells by regulating the miR1413p/HDAC4/HMGB1 axis.
Assuntos
Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Osteossarcoma/metabolismo , Adolescente , Adulto , Criança , Feminino , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Osteossarcoma/fisiopatologia , RNA Circular/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
Osteosarcoma represents one of the most common bone tumours in dogs. It commonly occurs in the proximal humerus, the most affected anatomic site. Until recently, amputation or limb-sparing surgery leading to an arthrodesis coupled with chemotherapy were the only available treatments, but they often lead to complications, reduced mobility and highly impact dog's quality of life. Prototypes of both articulated and monobloc (no mobility) patient-specific endoprostheses have been designed to spare the limb afflicted with osteosarcoma of the proximal humerus. This study focuses on the biomechanical effects of endoprostheses and shoulder muscle kinematics. For each of the endoprosthesis designs, a minimal number of muscles needed to ensure stability and a certain degree of joint movement during walking is sought. A quasi-static study based on an optimization method, the minimization of the sum of maximal muscle stresses, was carried out to assess the contribution of each muscle to the shoulder function. The identification of the most important muscles and their impact on the kinematics of the prosthetic joint lead to an improvement of the endoprosthesis design relevance and implantation feasibility.
Assuntos
Neoplasias Ósseas , Doenças do Cão , Úmero , Locomoção , Músculo Esquelético , Osteossarcoma , Próteses e Implantes , Escápula , Articulação do Ombro , Animais , Fenômenos Biomecânicos , Neoplasias Ósseas/fisiopatologia , Neoplasias Ósseas/cirurgia , Doenças do Cão/fisiopatologia , Doenças do Cão/cirurgia , Cães , Úmero/fisiopatologia , Úmero/cirurgia , Masculino , Músculo Esquelético/fisiopatologia , Músculo Esquelético/cirurgia , Osteossarcoma/fisiopatologia , Osteossarcoma/cirurgia , Desenho de Prótese , Escápula/fisiopatologia , Escápula/cirurgia , Articulação do Ombro/fisiopatologia , Articulação do Ombro/cirurgiaRESUMO
Hypoxia/oxygen-sensing signally is closely associated with many tumor progressions, including osteosarcoma (OS). Previous research principally focused on the function of hypoxia-inducible factor (HIF)-1α and HIF-2α as the major hypoxia-associated transcription factors in OS, however, the role of HIF-3α has not been investigated. Our study found that HIF-3α was upregulated in OS tissues and cell lines. HIF-3α overexpression facilitated cell proliferation and invasion, and inhibited apoptosis, whereas HIF-3α knockdown showed the opposite results. Chromatin immunoprecipitation analysis revealed that lysine demethylase 3A (KDM3A) expression was transcriptionally activated by HIF-3α under hypoxia, and KDM3A occupied the SRY-box transcription factor 9 (SOX9) gene promoter region through H3 lysine 9 dimethylation (H3K9me2). Additionally, rescue results revealed that KDM3A or SOX9 overexpression reversed the effects of HIF-3α silence on cell functions. The Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway inhibitor cucurbitacin I suppressed the promotive effects of HIF-3α overexpression on cell proliferation, invasion and TAK2/STAT3 pathway. Finally, OS cell line MG-63 transfected with HIF-3α short hairpin RNA (HIF-3α shRNA) were subcutaneously injected into nude mice, and the results found that HIF-3α knockdown significantly inhibited the xenograft tumor growth of OS in vivo. In conclusion, this study reveals that HIF-3α promotes OS progression in vitro and in vivo by activating KDM3A-mediated SOX9 promoter demethylation, which may provide a potential therapeutic mechanism for OS.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias Ósseas/fisiopatologia , Histona Desmetilases com o Domínio Jumonji/metabolismo , Osteossarcoma/fisiopatologia , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOX9/metabolismo , Animais , Apoptose/fisiologia , Proliferação de Células/fisiologia , Feminino , Humanos , Masculino , Metilação/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Transdução de Sinais/fisiologiaRESUMO
This study aimed to elucidate the interactions between osteosarcoma (OS) and M1 macrophages infiltrated into the tumor microenvironment and to explore the underlying mechanisms whereby M1 macrophages influence the growth of OS, so that novel treatments of OS can be developed. A transwell co-culture system, an indirect conditioned medium culture system and two orthotopic bearing OS models were established to assess for the interplay between M1 macrophages and OS. We found that the co-culture of M1 macrophages with OS cells significantly inhibited the growth of the tumor cells by inducing apoptosis. Furthermore, HSPA1L secreted by M1 macrophages exerted this anti-tumor effect through the IRAK1 and IRAK4 pathways. LGALS3BP secreted by OS cells bound to the ligand LGALS3 on M1 macrophages and thereby induced the secretion of Hspa11 via Akt phosphorylation. In vivo experiments demonstrated that the culture supernatant of OS-stimulated M1 macrophages significantly inhibited the growth of OS, whereas silencing Lgals3bp promoted the progression of OS. In conclusion, OS modifies the phenotype of tumor-associated macrophages (TAMs) and thereby influences the apoptosis of OS cells through soluble factors. The modulation of TAMs may be a promising and effective therapeutic approach in OS.
Assuntos
Exocitose/fisiologia , Macrófagos/fisiologia , Osteossarcoma/fisiopatologia , Animais , Apoptose , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , FenótipoRESUMO
PURPOSE: Osteosarcoma (OS) universally exhibits heterogeneity and cisplatin (CDDP) resistance. Although the Wee1/CDC2 and nuclear factor кB (NF-κB) pathways were reported to show abnormal activation in some tumor cells with CDDP resistance, whether there is any concrete connection is currently unclear. We explored it in human OS cells. MATERIALS AND METHODS: Multiple OS cell lines were exposed to a Wee1 inhibitor (AZD1775) and CDDP to assess the half-maximal inhibitory concentration values. Western blot, coimmunoprecipitation, confocal immunofluorescence, cell cycle, and Cell Counting Kit-8assays were performed to explore the connection between the Wee1/CDC2 and NF-κB pathways and their subsequent physiological contribution to CDDP resistance. Finally, CDDP-resistant PDX-OS xenograft models were established to confirm that AZD1775 restores the antitumor effects of CDDP. RESULTS: A sensitivity hierarchy of OS cells to CDDP and AZD1775 exists. In the highly CDDP-tolerant cell lines, Wee1 and RelA were physically crosslinked, which resulted in increased abundance of phosphorylated CDC2 (Y15) and RelA (S536) and consequent modulation of cell cycle progression, survival, and proliferation. Wee1 inhibition restored the effects of CDDP on these processes in CDDP-resistant OS cells. In addition, animal experiments with CDDP-resistant PDX-OS cells showed that AZD1775 combined with CDDP not only restored CDDP efficacy but also amplified AZD1775 in inhibiting tumor growth and prolonged the median survival of the mice. CONCLUSION: Simultaneous enrichment of molecules in the Wee1/CDC2 and NF-κB pathways and their consequent coactivation is a new molecular mechanism of CDDP resistance in OS cells. OS with this molecular signature may respond well to Wee1 inhibition as an alternative treatment strategy.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Osteossarcoma/fisiopatologia , Transdução de Sinais , Animais , Proteína Quinase CDC2/antagonistas & inibidores , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Osteossarcoma/genética , Proteínas Tirosina Quinases/antagonistas & inibidoresRESUMO
Osteosarcoma (OS) is the most prevalent malignant bone tumor in children and young adults. There is an urgent need for a novel biomarker related to the prognosis of OS. We performed a meta-analysis incorporating six independent datasets and performed a survival analysis with one independent dataset GSE21257 in the GEO database for gene screening. The results revealed that one potential biomarker related to OS survival, POGZ was the most significantly upregulated gene. We also verified that the POGZ was overexpressed in clinical samples. The survival analysis revealed that POGZ is associated with a poor prognosis in OS. Moreover, flow cytometry analysis of isolated OS cells demonstrated that OS cells were arrested in the G1 phase after POGZ knockdown. The RNA-seq results indicated that POGZ was co-expressed with CCNE1 and CCNB1. Pathway analysis showed that genes associated with high expression levels of POGZ were related to the cell cycle pathway. A cell model was constructed to detect the effects of POGZ. After POGZ knockdown, OS cell proliferation, invasion and migration were all decreased. Therefore, POGZ is an important gene for evaluating the prognosis of OS patients and is a potential therapeutic target.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/diagnóstico , Osteossarcoma/diagnóstico , Transposases/metabolismo , Adulto , Biomarcadores Tumorais/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/fisiopatologia , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Masculino , Invasividade Neoplásica/fisiopatologia , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Osteossarcoma/fisiopatologia , Prognóstico , Transposases/genética , Adulto JovemRESUMO
BACKGROUND: Trichinella spiralis (T. spiralis) is a parasite occurring worldwide that has been proven to have antitumour ability. However, studies on the antitumour effects of cross antigens between the tumour and T. spiralis or antibodies against cross antigens between tumours and T. spiralis are rare. METHODS: To study the role of cross antigens between osteosarcoma and T. spiralis, we first screened the cDNA expression library of T. spiralis muscle larvae to obtain the cross antigen gene tumour protein D52 (TPD52), and prepared fusion protein TPD52 and its antiserum. The anti-osteosarcoma effect of the anti-TPD52 antiserum was studied using cell proliferation and cytotoxicity assays as well as in vivo animal models; preliminary data on the mechanism were obtained using western blot and immunohistochemistry analyses. RESULTS: Our results indicated that TPD52 was mainly localized in the cytoplasm of MG-63 cells. Anti-TPD52 antiserum inhibited the proliferation of MG-63 cells and the growth of osteosarcoma in a dose-dependent manner. The tumour inhibition rate in the 100 µg treatment group was 61.95%. Enzyme-linked immunosorbent assay showed that injection of anti-TPD52 antiserum increased the serum levels of IFN-γ, TNF-α, and IL-12 in nude mice. Haematoxylin and eosin staining showed that anti-TPD52 antiserum did not cause significant pathological damage. Apoptosis of osteosarcoma cells was induced by anti-TPD52 antiserum in vivo and in vitro. CONCLUSIONS: Anti-TPD52 antiserum exerts an anti-osteosarcoma effect by inducing apoptosis without causing histopathological damage.
Assuntos
Anticorpos Anti-Helmínticos/administração & dosagem , Antígenos de Helmintos/imunologia , Osteossarcoma/tratamento farmacológico , Osteossarcoma/imunologia , Trichinella spiralis/imunologia , Triquinelose/imunologia , Animais , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/genética , Apoptose/efeitos dos fármacos , Reações Cruzadas , Citocinas/genética , Citocinas/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Osteossarcoma/genética , Osteossarcoma/fisiopatologia , Trichinella spiralis/genética , Triquinelose/genética , Triquinelose/parasitologiaRESUMO
BACKGROUND: Neoadjuvant chemotherapy in patients with primary osteosarcoma improves survival rates, but it also causes side effects in various organs including bone. Low bone mineral density (BMD) can occur owing partly to chemotherapy or limited mobility. This can cause a higher risk of fractures compared with those who do not receive such treatment. Changes in BMD alone cannot explain the propensity of fractures. Studying microarchitectural changes of bone might help to understand the effect. QUESTIONS/PURPOSES: (1) Do patients who were treated for osteosarcoma (more than 20 years previously) have low BMD? (2) Do these patients experience more fractures than controls who do not have osteosarcoma? (3) What differences in bone microarchitecture are present between patients treated for high-grade osteosarcoma and individuals who have never had osteosarcoma? METHODS: We contacted 48 patients who were treated for osteosarcoma and who participated in an earlier study. These patients underwent multimodal treatment including chemotherapy more than 20 years ago. Of the original patient group, 60% (29 of 48) were missing, leaving 40% (19 of 48) available for inclusion in this study; all 19 agreed to participate. There were nine men and 10 women with a mean age of 46 ± 4 years and a mean time from surgery to examination of 28 ± 3 years. BMD was measured by dual-energy x-ray absorptiometry, and any fracture history was assessed using a questionnaire. Additionally, high-resolution peripheral quantitative CT was performed to compare the groups in terms of microarchitectural changes, such as cortical and trabecular area, cortical and trabecular thickness, cortical porosity, and endocortical perimeter. Participants in the control group were selected from a cohort consisting of a population-based random sample of 499 healthy adult women and men. Osteoporosis or low BMD was not an exclusion criterion for entering this study; however, the patients in the control group were selected based on a normal BMD (that is, T score > -1.0 at both the spine and hip). Also, the participants were matched based on age and sex. Differences between patients and controls were assessed using the Wilcoxon rank sum test for continuous variables and a chi-square test for categorical variables. A multiple regression analysis was performed. Model assumptions were checked using histograms and quantile-quantile plots of residuals. RESULTS: Twelve of 19 patients who were treated for osteosarcoma had either osteopenia (eight patients) or osteoporosis (four patients). More patients with osteosarcoma reported sustaining fractures (11 of 19 patients) than did control patients (2 of 19 controls; p < 0.001). Among all microarchitectural parameters, only the endocortical perimeter was increased in patients compared with the control group (75 ± 15 mm versus 62 ± 18 mm; p = 0.04); we found no differences between the groups in terms of cortical and trabecular area, cortical and trabecular thickness, or cortical porosity. CONCLUSION: Although patients who were treated for osteosarcoma had osteopenic or osteoporotic BMD and a higher proportion of patients experienced fractures than did patients in the control group, we could not confirm differences in microarchitectural parameters using high-resolution peripheral quantitative CT. Therefore, it seems that bone geometry and microstructural parameters are not likely the cause of the increased proportion of fractures observed in our patients who were treated for osteosarcoma. Until we learn more about the bone changes associated with chemotherapy in patients with osteosarcoma, we recommend that patients undergo regular BMD testing, and we recommend that physicians consider osteoporosis treatment in patients with low BMD. These data might provide the impetus for future multicenter prospective studies examining the association between chemotherapy and bone microarchitecture. LEVEL OF EVIDENCE: Level III, therapeutic study.
Assuntos
Doenças Ósseas Metabólicas/induzido quimicamente , Fraturas Ósseas/induzido quimicamente , Terapia Neoadjuvante/efeitos adversos , Osteoporose/induzido quimicamente , Osteossarcoma/terapia , Absorciometria de Fóton , Adulto , Densidade Óssea , Osso Esponjoso/diagnóstico por imagem , Osso Esponjoso/fisiopatologia , Osso Esponjoso/ultraestrutura , Terapia Combinada , Osso Cortical/diagnóstico por imagem , Osso Cortical/fisiopatologia , Osso Cortical/ultraestrutura , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Osteossarcoma/fisiopatologia , Tomografia Computadorizada por Raios XRESUMO
MG132 is a potent, reversible, and cell-permeable 20S proteasome inhibitor and it is derived from a Chinese medicinal plant. The purpose of this study is to investigate the anticancer effects of MG132 against human osteosarcoma U2OS cells. We first performed MTT and colony formation assays to investigate the anti-proliferative effects of MG132. The results demonstrated that MG132 suppressed the proliferation of U2OS cells. Furthermore, we found that treatment with MG132 increased apoptosis and induced DNA damage in U2OS cells. Additionally, zymography, wound healing, and invasion assays showed that MG132 suppressed the enzymatic activity of matrix metalloproteinases, cell migration, and invasion, respectively of U2OS cells. Furthermore, western blotting assay was performed to investigate the apoptotic signaling pathways in MG132-treated U2OS cells. Our results showed that MG132 downregulated the expression of antiapoptotic proteins, including CDK2, CDK4, Bcl-xL, and Bcl-2, whereas it upregulated the expression of proapoptotic proteins, including p21, p27, p53, p-p53 (ser15, ser20, and ser46), cleaved forms of caspase-3, caspase-7, caspase-9, and PARP, and FOXO3 in U2OS cells. These results demonstrated that MG132 activated apoptotic signaling pathways in U2OS cells. Interestingly, MG132 downregulated the phosphorylation of Akt and Erk. Taken together, our results suggest that MG132 has anticancer effects in U2OS cells. Therefore, MG132 may be a potential therapeutic agent for the treatment of osteosarcoma.
Assuntos
Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/tratamento farmacológico , Linhagem Celular Tumoral/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Osteossarcoma/tratamento farmacológico , Inibidores de Proteassoma/uso terapêutico , Neoplasias Ósseas/fisiopatologia , Humanos , Medicina Tradicional Chinesa , Osteossarcoma/fisiopatologiaRESUMO
Dysregulation of long noncoding RNA (lncRNA) is frequently involved in the progression and development of osteosarcoma. LncRNA RUSC1-AS1 is reported to be upregulated and acts as an oncogene in hepatocellular carcinoma, cervical cancer and breast cancer. However, its role in osteosarcoma has not been studied yet. In the present study, we investigated the role of RUSC1-AS1 in osteosarcoma both in vitro and in vivo. The results showed that the expression of RUSC1-AS1 was significantly upregulated in osteosarcoma cell line U2OS and HOS compared to that in human osteoblast cell line hFOB1.19. Similar results were found in human samples. Silencing RUSC1-AS1 by siRNA significantly inhibited U2OS and HOS cell proliferation and invasion, measured by CCK-8 and transwell assay. Besides, knockdown of RUSC1-AS1 increased cell apoptosis in osteosarcoma cell lines. In addition, RUSC1-AS1 promoted the epithelial-mesenchymal transition (EMT) process of osteosarcoma cells. In vivo experiments confirmed that RUSC1-AS1 knockdown had an inhibitory effect on osteosarcoma tumor growth. Mechanically, we showed that RUSC1-AS1 directly binds to and inhibits miR-340-5p and activates the PI3K/AKT signaling pathway. In conclusion, our study demonstrated that RUSC1-AS1 promoted osteosarcoma development both in vitro and in vivo through sponging to miR-340-5p and activating the PI3K/AKT signaling pathway. Therefore, RUSC1-AS1 becomes a potential therapeutic target for osteosarcoma.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , MicroRNAs/metabolismo , Osteossarcoma/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Antissenso/genética , RNA Longo não Codificante/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Osteossarcoma/metabolismo , Osteossarcoma/fisiopatologia , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Antissenso/metabolismo , Transdução de SinaisRESUMO
Cellular adaptation to a hypoxic microenvironment is essential for tumour progression and is largely mediated by HIF-1α and hypoxia-regulated factors, including CXCR4, VEGF-A and GLUT-1. In human osteosarcoma, hypoxia is associated with resistance to chemotherapy as well as with metastasis and poor survival, whereas little is known about its role in canine osteosarcoma (cOSA). This study aimed primarily to evaluate the prognostic value of several known hypoxic markers in cOSA. Immunohistochemical analysis for HIF-1α, CXCR4, VEGF-A and GLUT-1 was performed on 56 appendicular OSA samples; correlations with clinicopathological features and outcome was investigated. The second aim was to investigate the in vitro regulation of markers under chemically induced hypoxia (CoCl2). Two primary canine osteosarcoma cell lines were selected, and Western blotting, immunofluorescence and qRT-PCR were used to study protein and gene expression. Dogs with high-grade OSA (35.7%) were more susceptible to the development of metastases (P = 0.047) and showed high HIF-1α protein expression (P = 0.007). Moreover, HIF-1α overexpression (56%) was correlated with a shorter disease-free interval (DFI; P = 0.01), indicating that it is a reliable negative prognostic marker. The in vitro experiments identified an accumulation of HIF-1α in cOSA cells after chemically induced hypoxia, leading to a significant increase in GLUT-1 transcript (P = 0.02). HIF-1α might be a promising prognostic marker, highlighting opportunities for the use of therapeutic strategies targeting the hypoxic microenvironment in cOSA. These results reinforce the role of the dog as a comparative animal model since similar hypoxic mechanisms are reported in human osteosarcoma.
Assuntos
Neoplasias Ósseas/veterinária , Hipóxia Celular/fisiologia , Doenças do Cão/fisiopatologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Osteossarcoma/veterinária , Animais , Biomarcadores Tumorais/análise , Neoplasias Ósseas/química , Neoplasias Ósseas/fisiopatologia , Linhagem Celular Tumoral , Doenças do Cão/patologia , Cães , Feminino , Regulação Neoplásica da Expressão Gênica , Transportador de Glucose Tipo 1/análise , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Imuno-Histoquímica/veterinária , Masculino , Metástase Neoplásica/fisiopatologia , Osteossarcoma/química , Osteossarcoma/fisiopatologia , Prognóstico , Receptores CXCR4/análise , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
BACKGROUND Avicularin is a plant-derived flavonoid used in traditional Chinese medicine to treat conditions that include ankle fracture. Bradykinin stimulated MG-63 human osteoblastic osteosarcoma cells has previously been studied in an in vitro model. This study aimed to investigate the effects of avicularin on bradykinin-treated MG-63 human osteoblastic osteosarcoma cells in vitro. MATERIAL AND METHODS MG-63 cells were treated with increasing concentrations of avicularin for 48 hours, followed by 1 µM of bradykinin for 24 h. The MTT assay was used to measure cell viability. Quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to measure the expression of inflammatory mediators, interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-alpha (TNF-alpha) mRNA and protein, respectively. The expression of oxidative stress factors, malondialdehyde (MDA), superoxide dismutase (SOD), and catalase were measured. Western blot and qRT-PCR were performed to determine p38, p65, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) protein levels and mRNA expression, respectively. RESULTS Avicularin had no cytotoxic effect on MG-63 cells. Avicularin significantly upregulated the expression levels IL-1ß, IL-6, and TNF-alpha in the bradykinin treated group in a dose-dependent manner. Avicularin reduced the level of MDA and the activity of SOD and catalase in the bradykinin treated MG-63 cells, reduced p-p38, p-p65, iNOS, and COX-2 expression, and decreased the p-p38/p38 ratio and the p-p65/p65 ratio in bradykinin treated MG-63 cells in a dose-dependent manner. CONCLUSIONS Avicularin reduced the expression of inflammatory cytokines and the levels of markers of oxidative stress in MG-63 human osteoblastic osteosarcoma cells in vitro.
Assuntos
Flavonoides/farmacologia , Osteossarcoma/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Bradicinina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , China , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Humanos , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Osteoblastos/metabolismo , Osteossarcoma/fisiopatologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Osteosarcomas are the most frequent primary bone sarcomas, affecting mainly children, adolescents, and young adults, and with a second peak of incidence in elderly individuals. The current therapeutic management, a combined regimen of poly-chemotherapy and surgery, still remains largely insufficient, as patient survival has not improved in recent decades. Osteosarcomas are very heterogeneous tumors, both at the intra- and inter-tumor level, with no identified driver mutation. Consequently, efforts to improve treatments using targeted therapies have faced this lack of specific osteosarcoma targets. Nevertheless, these tumors are inextricably linked to their local microenvironment, composed of bone, stromal, vascular and immune cells and the osteosarcoma microenvironment is now considered to be essential and supportive for growth and dissemination. This review describes the different actors of the osteosarcoma microenvironment and gives an overview of the past, current, and future strategies of therapy targeting this complex ecosystem, with a focus on the role of extracellular vesicles and on the emergence of multi-kinase inhibitors.
Assuntos
Terapia de Alvo Molecular , Osteossarcoma/patologia , Osteossarcoma/terapia , Microambiente Tumoral , Animais , Remodelação Óssea , Humanos , Sistema Imunitário/patologia , Células-Tronco Mesenquimais/patologia , Osteossarcoma/imunologia , Osteossarcoma/fisiopatologiaRESUMO
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/metabolismoRESUMO
Osteosarcoma is an often highly malignant mesenchymal tumor. By definition, osteosarcoma cells are able to form osteoid, which can mature into tumor bone. Osteosarcoma metastasizes preferentially to the lung. In Europe, the incidence is between 2 and 5 new diagnoses per 1,000,000 people per year. The underlying mechanisms for osteosarcoma formation are not well understood. However, previous radiotherapy or exposition to nuclear radiation increase the risk of osteosarcoma. Patients are usually treated with a neoadjuvant chemotherapy, followed by complete surgical resection of the tumor and post-surgical chemotherapy, which leads to a five-year survival rate of approximately 70% for all stages. Scientific publications in recent years have shown that expression of the cell surface protein interleukin-11 receptor (IL-11R) correlates with a worse prognosis for patients. The IL-11R is activated by its ligand, the cytokine IL-11. IL-11 activates several intracellular signaling cascades within its target cells and is known to be an important regulator of bone homeostasis. Patients with dysfunctional IL-11 signaling display different forms of craniosynostosis. IL-11 induces proliferation of osteosarcoma cell lines in vitro, and the IL-11 signaling cascade was further used to reduce tumor growth and lung metastasis in preclinical mouse models of primary intratibial osteosarcoma. This article gives a comprehensive overview of the frequency, classification, and etiology of osteosarcoma and describes the basic biology of the cytokine IL-11. Furthermore, it summarizes current knowledge about the functional role of IL-11 in osteosarcoma and lists possible therapeutic opportunities.
Assuntos
Neoplasias Ósseas/fisiopatologia , Interleucina-11/fisiologia , Osteossarcoma/fisiopatologia , HumanosRESUMO
Extraskeletal osteosarcoma are rare malignant mesenchymal neoplasms of soft tissues representing around 1% of all soft tissue. The exact mechanism of tumorigenesis of primary breast osteosarcoma is still unclear. However, most of the cases develop without a recognized etiologic factor. Primary osteosarcoma of the breast is often initially misdiagnosed as breast fibroadenoma. Different imaging modalities and pathology play important role in differentiating breast osteosarcoma from other benign and malignant lesions of the breast resulting in dramatic change in the management.
Assuntos
Diagnóstico por Imagem/métodos , Osteossarcoma/diagnóstico por imagem , Osteossarcoma/fisiopatologia , Mama/diagnóstico por imagem , Mama/fisiopatologia , Diagnóstico Diferencial , Feminino , HumanosRESUMO
After surgical resection for a bone tumor, the uncleared bone tumor cells can multiply and cause recurrence of the bone tumor. It is worthwhile to design a scaffold that kills the remaining bone tumor cells and repairs bone defects that were given rise to by surgical resection. Additionally, it is extremely important to consider the function of angiogenesis in the process of bone regeneration because the newly formed blood vessels can offer the nutrients for bone regeneration. In this work, a novel metal-organic framework Cu-TCPP nanosheets interface-structured ß-tricalcium phosphate (TCP) (Cu-TCPP-TCP) scaffold was successfully prepared through integrating a 3D-printing technique with an in-situ growth method in a solvothermal system. Owing to the excellent photothermal effect of Cu-TCPP nanosheets, Cu-TCPP-TCP scaffolds that were illuminated by near-infrared (NIR) light demonstrated photothermal performance, which was well regulated through varying the contents of Cu-TCPP nanosheets, and the ambient humidity and power density of NIR light. When cultured with osteosarcoma cells, Cu-TCPP-TCP scaffolds killed a significant quantity of osteosarcoma cells through released heat energy after exposure to NIR light with power density 1.0 W cm-2 and duration 10 min. Similarly, Cu-TCPP-TCP scaffolds ablated subcutaneous bone tumor tissues on the backs of naked mice and suppressed their growth because of the heat energy transformed from NIR light. I n-vitro studies found that Cu-TCPP-TCP scaffolds ably supported the attachments of both human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HUVECs), and significantly stimulated expressions of osteogenesis differentiation-related genes in HBMSCs and angiogenesis differentiation-related genes in HUVECs. After implanting Cu-TCPP-TCP scaffolds into the bone defects of rabbits, they effectively promoted bone regeneration. Thus, the integration of the bone-forming bioactivity of TCP scaffolds with the photothermal properties of Cu-TCPP nanosheets and angiogenesis activity of Cu ions makes Cu-TCPP-TCP scaffolds multifunctional, representing a new horizon to develop biomaterials for simultaneously curing bone tumors and repairing bone defects.
Assuntos
Neoplasias Ósseas/terapia , Células-Tronco Mesenquimais/citologia , Estruturas Metalorgânicas/química , Nanoestruturas/química , Osteossarcoma/terapia , Alicerces Teciduais/química , Animais , Bioimpressão , Neoplasias Ósseas/fisiopatologia , Regeneração Óssea , Transplante Ósseo , Fosfatos de Cálcio/química , Proliferação de Células , Cobre/química , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Fisiológica , Osteossarcoma/fisiopatologia , Impressão Tridimensional , Coelhos , Engenharia TecidualRESUMO
OBJECTIVE: To report demographic characteristics of a contemporary population of dogs with appendicular osteosarcoma and assess the relationship between demographic characteristics, site distribution, and phylogenetic breed clusters. DESIGN: Retrospective case series. METHODS: A search of the Veterinary Medical Database was performed for dogs with appendicular osteosarcoma as a new diagnosis. Entries were reviewed for the sex, neuter status, age at diagnosis, breed, affected limb, and tumor location. The reported breed for purebred dogs was used to categorize each dog into one of five phylogenetic groups based on microsatellite analysis. RESULTS: 744 client-owned dogs were included in the study. Study dogs were represented by a male-to-female ratio of 0.95:1.0, the majority of which (80.9%) were neutered. Most dogs were diagnosed between 7-10 years of age. The majority (77.8%) of dogs were large or giant-breed dogs. Purebred dogs comprised 80.4% of the population. The most common purebred breed affected by OS was the Rottweiler (17.1%). The most common phylogenetic group represented was Mastiff-Terrier (M-T, 26.3%). OS was more commonly located in the forelimb (64.2%) versus the hindlimb (35.8%), and the humerus was the most common site (20.9%). The distribution of age groups and tumor locations were significantly different between phylogenetic clusters. The distribution of age groups and neuter status were significantly different between size groups. CONCLUSIONS AND SIGNIFICANCE: The demographic data of canine appendicular OS are similar to previous reports. The data on phylogenetic associations can guide future studies aimed at evaluating the genomic mutations that contribute to OS development and its biological behavior.