RESUMO
A disintegrin-like and metalloprotease with therombospondin type1 motif 8 (ADAMTS8) plays an important role in many malignancies. However, the clinical and biological significance of ADAMTS8 in breast cancer remain unknown. In this study, the clinical data from 1066 breast cancer patients were analyzed by The Cancer Genome Atlas (TCGA) database, and were analyzed using the correlation between ADAMTS8 expression and the clinicopathological features and prognoses. The CCK-8 assay, clone formation assay, flow cytometry and Transwell assay were used to characterize the effects of ADAMTS8 on proliferation, migration and invasion of breast cancer cells. Gene set enrichment analysis (GSEA) and western blotting were used to identify the potential molecular mechanism on how ADAMTS8 exert its biological function. ADAMTS8 overexpression correlated longer overall survival (OS) and progression-free survival (PFS). ADAMTS8 was considered as an independent prognostic factor for OS. ADAMTS8 overexpression inhibited breast cancer cell proliferation, migration and invasion in vitro, and induced G2/M cell cycle arrest. ADAMTS8 was also involved in cell cycle regulation and was associated with the EGFR/Akt signaling pathway. ADAMTS8 knockdown showed the reverse effect. Together, the results showed that ADAMTS8 functioned as a tumor suppressor gene (TGS) and could be a prognostic biomarker for breast cancer.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/patologia , Prognóstico , Linhagem Celular Tumoral , Genes Supressores de Tumor , Transdução de Sinais/genética , Proteínas ADAMTS/genética , Proteínas ADAMTS/metabolismoRESUMO
Breast cancer is the leading cause of cancer death in female. Until now, advanced breast cancer is still lack effective treatment strategies and reliable prognostic markers. In the present article, we introduced the physiologic and pathologic functions and regulation mechanisms of ZBTB28, a tumor suppressor gene, in breast cancer. ZBTB28 is frequently silenced in breast cancer due to promoter CpG methylation, and its expression is positively correlated with breast cancer patient survival. The antineoplastic effect of ZBTB28 in breast cancer was elucidated through a series of in vitro and in vivo measurements, including cell proliferation, apoptosis, cell cycle, epithelial mesenchymal transition (EMT), and growth of xenografts. Furthermore, ZBTB28 can directly regulate IFNAR to activate interferon-stimulated genes and potentiate macrophage activation. Ectopic ZBTB28 expression in breast cancer cells was sufficient to downregulate CD24 and CD47 to promote phagocytosis of macrophages, demonstrating that ZBTB28 was beneficial for the combination treatment of anti-CD24 and anti-CD47. Collectively, our results reveal a mode of action of ZBTB28 as a tumor suppressor gene and suggest that ZBTB28 is an important regulator of macrophage phagocytosis in breast cancer, holding promise for the development of novel therapy strategies for breast cancer patients.
Assuntos
Neoplasias da Mama/genética , Antígeno CD24/genética , Antígeno CD47/genética , Fagocitose , Receptor de Interferon alfa e beta/genética , Proteínas Repressoras/genética , Animais , Neoplasias da Mama/imunologia , Antígeno CD24/imunologia , Antígeno CD47/imunologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Ativação de Macrófagos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Receptor de Interferon alfa e beta/imunologia , Proteínas Repressoras/imunologia , Células THP-1RESUMO
Lung cancer is the most frequently diagnosed cancer worldwide. Epigenetic regulation contributes to lung cancer pathogenesis. The ADAMTS18 tumor suppressor gene is inactivated in some cancers, but its involvement in lung cancer has not been shown. Immunohistochemistry, quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and methylation-specific PCR were used to assay ADAMTS18 expression and promoter methylation in lung tumor tissues and adjacent tissues. Cell viability, transwell, and wound-healing assays, as well as flow cytometry were used to characterize the biological activity of ADAMTS18. The influence of ADAMTS18 on protein expression was assayed using western blots analysis, and its effect on chemosensitivity was assayed by the response to cisplatin. We found that ADAMTS18 was silenced in lung cancer cells by promoter methylation. Demethylation by the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine, with or without the histone deacetylase inhibitor trichostatin A, restored ADAMTS18 expression. Compared with normal lung tissue, ADAMTS18 in lung tumors was frequently methylated. Overexpression of ADAMTS18 in lung cancer cells inhibited cell proliferation, migration, and invasiveness and induced G0/G1 cell cycle arrest. Furthermore, ADAMTS18 suppressed epidermal growth factor receptor/protein kinase B (EGFR/AKT) signaling, which sensitized lung cancer cells to cisplatin. Thus, our results demonstrated that the tumor suppressor gene ADAMTS18 was downregulated in lung cancer by promoter CpG methylation, and it promoted sensitivity to cisplatin via EGFR/AKT signaling. Our study suggests that ADAMTS18 promoter methylation is a potential epigenetic biomarker for early detection of lung cancer and warrants investigation as a therapeutic target for early-stage lung cancer.
Assuntos
Proteínas ADAMTS/genética , Metilação de DNA/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Regiões Promotoras Genéticas/genética , Células A549 , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cisplatino/farmacologia , Ilhas de CpG/efeitos dos fármacos , Ilhas de CpG/genética , Metilação de DNA/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Epigênese Genética/efeitos dos fármacos , Epigênese Genética/genética , Receptores ErbB/genética , Fase G1/efeitos dos fármacos , Fase G1/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Inativação Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Invasividade Neoplásica/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
In this study, we found that the phospholipase C delta1 (PLCD1) protein expression is reduced in colorectal tumor tissues compared with paired surgical margin tissues. PLCD1-promoted CpG methylation was detected in 29/64 (45%) primary colorectal tumors, but not in nontumor tissues. The PLCD1 RNA expression was also reduced in three out of six cell lines, due to PLCD1 methylation. The ectopic expression of PLCD1 resulted in inhibited proliferation and attenuated migration of colorectal tumor cells, yet promoted colorectal tumor cell apoptosis in vitro. We also observed that PLCD1 suppressed proliferation and promoted apoptosis in vivo. In addition, PLCD1 induced G1/S phase cell cycle arrest. Furthermore, we found that PLCD1 led to the downregulation of several factors downstream of ß-catenin, including c-Myc and cyclin D1, which are generally known to be promoters of tumorigenesis. This downregulation was caused by an upregulation of E-cadherin in colorectal tumor cells. Our findings provide insights into the role of PLCD1 as a tumor suppressor gene in colorectal cancer (CRC), and demonstrate that it plays significant roles in proliferation, migration, invasion, cell cycle progression, and epithelial-mesenchymal transition. On the basis of these results, tumor-specific methylation of PLCD1 could be used as a novel biomarker for early detection and prognostic prediction in CRC.
Assuntos
Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal , Inativação Gênica , Fosfolipase C delta/metabolismo , Idoso , Animais , Apoptose/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Células Clonais , Neoplasias Colorretais/genética , Metilação de DNA/genética , Desmetilação , Regulação para Baixo/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Fase G1/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosfolipase C delta/genética , Regiões Promotoras Genéticas , Fase S/genética , Transdução de Sinais , beta Catenina/metabolismoRESUMO
BACKGROUND: Tumor chemo-resistance is a hallmark of malignant tumors as well as the major cause of poor survival rates in lung cancer. Transmembrane-4 L-six family member-1 (TM4SF1), an antigen that serves as an oncogene, mainly affects tumor invasion and metastasis. We investigated the roles of TM4SF1 in non-small-cell lung cancer progression, particularly in the regulation of chemo-sensitivity. METHODS: TM4SF1 was silenced by small interfering RNA transfection.TM4SF1 expression in cell lines and tissues were determined by Quantitative Real-time PCR. MTS, clonogenic, Transwell assay, Flow cytometry verified cell function. By RT-PCR, Western blot, the mechanisms were studied. RESULTS: TM4SF1 was upregulated in both lung cancer cell lines and tissues, compared with 293 T epithelial cells. Analysis of online databases revealed that high expression of TM4SF1 is associated with the older patient age, smoking habits, and poor patient survival and outcome. Knockdown of TM4SF1 substantially inhibited tumor cell growth, migration, and invasion, and enhanced the chemo-sensitivity of the lung cancer cell lines A549 and H1299 to cisplatin and paclitaxel. Furthermore, the silencing of TM4SF1 induced lung cancer cell apoptosis and arrested cells at the G2/M phase. These results suggest that TM4SF1 is associated with lung cancer progression and appears to be required for tumor cell growth, maintenance of chemo-resistance and metastasis. We further found that TM4SF1 exerts these effects in part by regulating the expression of the discoidin domain receptor DDR1 and its downstream target, the Akt/ERK/mTOR pathway, and consequently alters cell sensitivity to chemo-reagents and contributes to invasion and metastasis. CONCLUSIONS: These findings demonstrate that TM4SF1 may serve as a prognostic factor for lung cancer chemo-response and patient outcome.
Assuntos
Antígenos de Superfície/biossíntese , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Receptor com Domínio Discoidina 1/metabolismo , Neoplasias Pulmonares/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Neoplasias/biossíntese , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Células A549 , Adulto , Idoso , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Células HEK293 , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/patologiaRESUMO
Chromosome region 3p12-14 is an important tumour suppressor gene (TSG) locus for multiple cancers. ADAMTS9, a member of the metalloprotease large family, has been identified as a candidate 3p14.2 TSG inactivated by aberrant promoter CpG methylation in several carcinomas, but little known about its expression and function in breast cancer. In this report, ADAMTS9 expression and methylation was analysed in breast cancer cell lines and tissue samples. ADAMTS9 RNA was significantly down-regulated in breast cancer cell lines (6/8). After treating the cells with demethylation agent Aza and TSA, ADAMTS9 expression was dramatically increased. Bisulphite genomic sequencing and methylation-specific PCR detected promoter methylation, which was associated with decreased ADAMTS9 expression. Hypermethylation was also detected in 130/219 (59.4%) of primary tumours but only in 4.5% (2/44) of paired surgical margin tissues. Ectopic expression of ADAMTS9 in tumor cells induced significant growth suppression, cell cycle arrest at the G0/G1 phase, enhanced apoptosis and reduced cell migration and invasion. Conditioned culture medium from ADAMTS9-transfected BT549 cells markedly disrupted tube formation ability of human umbilical vein endothelial cell (HUVEC) in Matrigel. Furthermore, ADAMTS9 inhibited AKT signaling and its downstream targets (MDM2, p53, p21, p27, E-cadherin, VIM, SNAIL, VEGFA, NFκB-p65 and MMP2). In addition, we demonstrated, for the first time, that ADAMTS9 inhibits AKT signaling, through suppressing its upstream activators EGFR and TGFß1/TßR(I/II) in breast cancer cells. Our results suggest that ADAMTS9 is a TSG epigenetically inactivated in breast cancer, which functions through blocking EGFR- and TGFß1/TßR(I/II)-activated AKT signaling.
Assuntos
Proteína ADAMTS9/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Cromossomos Humanos Par 3/metabolismo , Ilhas de CpG/genética , Metilação de DNA/genética , Regiões Promotoras Genéticas , Proteína ADAMTS9/genética , Adulto , Apoptose/genética , Neoplasias da Mama/irrigação sanguínea , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ensaio Tumoral de Célula-TroncoRESUMO
BACKGROUND/AIMS: Interferon consensus sequence-binding protein 8 (IRF8) belongs to a family of interferon (IFN) regulatory factors that modulates various important physiological processes including carcinogenesis. As reported by others and our group, IRF8 expression is silenced by DNA methylation in both human solid tumors and hematological malignancies. However, the role of IRF8 in lung carcinoma remains elusive. In this study, we determined IRF8 epigenetic regulation, biological functions, and the signaling pathway involved in non-small cell lung cancer (NSCLC). METHODS: IRF8 expression were determined by Q- PCR. MSP and A+T determined promotor methylation. MTS, clonogenic, Transwell assay, Flow cytometry, three-dimensional culture and AO/EB stain verified cell function. In vivo tumorigenesis examed the in vivo effects. By Chip-QPCR, RT-PCR, Western blot and Immunofluorescence staining, the mechanisms were studied. RESULTS: IRF8 was significantly downregulated in lung tumor tissues compared with adjacent non-cancerous tissues. Furthermore, methylation-specific PCR analyses revealed that IRF8 methylation in NSCLC was a common event, and demethylation reagent treatment proved that downregulation of IRF8 was due to its promoter CpG hypermethylation. Clinical data showed that the IRF8 methylation was associated with tumor stage, lymph node metastasis status, patient outcome, and tumor histology. Exogenous expression of IRF8 in the silenced or downregulated lung cancer cell lines A549 and H1299 at least partially restored the sensitivity of lung cancer cells to apoptosis, and arrested cells at the G0/G1 phase. Cell viability, clonogenicity, and cell migration and invasive abilities were strongly inhibited by restored expression of IRF8. A three-dimensional culture system demonstrated that IRF8 changed the cells to a more spherical phenotype. Moreover, ectopic expression of IRF8 enhanced NSCLC chemosensitivity to cisplatin. Furthermore, as verified by Chip-qPCR, immunofluorescence staining, and western blotting, IRF8 bound to the T-cell factor/lymphoid enhancer factor (TCF /LEF) promoter, thus repressing ß-catenin nuclear translocation and its activation. IRF8 significantly disrupted the effects of Wnt agonist, bml284, further suggesting its involvement in the Wnt/ß-catenin pathway. CONCLUSION: IRF8 acted as a tumor suppressor gene through the transcriptional repression of ß-catenin-TCF/LEF in NSCLC. IRF8 methylation may serve as a potential biomarker in NSCLC prognosis.
Assuntos
Fatores Reguladores de Interferon/metabolismo , Via de Sinalização Wnt , Idoso , Animais , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular/efeitos dos fármacos , Cisplatino/farmacologia , Metilação de DNA , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Fatores Reguladores de Interferon/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Fator 1 de Ligação ao Facilitador Linfoide/química , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Regiões Promotoras Genéticas , Proteínas Wnt/agonistas , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismoRESUMO
BACKGROUND/AIMS: ADAMTS (disintegrin-like and metalloproteinase with thrombospondin motifs) proteins are extracellular zinc metalloproteinases that play an important role in extracellular matrix assembly and degradation, connective tissue structuring, angiogenesis, and cell migration. Multiple studies suggest that ADAMTS proteins (e.g. ADAMTS9) can act as tumor suppressors. In gastric, esophageal, and nasopharyngeal carcinomas ADAMTS9 is frequently down-regulated by promoter methylation. Whether ADAMTS9 can function as a tumor suppressor gene (TSG) in colorectal cancer is still unclear. METHODS: We performed immunohistochemistry, RT-PCR, and qRT-PCR, to examine the expression of ADAMTS9 in colorectal cancer cell lines and primary colorectal cancer tissues. Methylation-specific PCR was also carried out to investigate the promoter methylation status of ADAMTS9. We also explored the functions of ADAMTS9 in colorectal cancer cell lines through in vitro experiments. RESULTS: ADAMTS9 expression was down-requlated or silenced in 83.3% (5/6) of colorectal cancer cell lines, and frequently repressed in 65.6% (21/32) of colorectal cancer tissues. Down-regulation of ADAMTS9 was partially due to promoter methylation. Exogenous expression of ADAMTS9 in colorectal cancer cell lines inhibited cell proliferation and migration through the regulation of cell cycle and apoptosis. In addition, ADAMTS9 prevented the activation of Akt, and its downstream targets in colorectal cancer cell lines. CONCLUSION: Our findings suggest ADAMTS9 is a TSG in colorectal cancer.
Assuntos
Proteína ADAMTS9/metabolismo , Neoplasias Colorretais/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína ADAMTS9/antagonistas & inibidores , Proteína ADAMTS9/genética , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Ilhas de CpG , Metilação de DNA , Regulação para Baixo , Transição Epitelial-Mesenquimal , Pontos de Checagem da Fase G1 do Ciclo Celular , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Células HCT116 , Humanos , Imuno-Histoquímica , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Recent studies suggest that paired box 5 (PAX5) is down-regulated in multiple tumours through its promoter methylation. However, the role of PAX5 in non-small cell lung cancer (NSCLC) pathogenesis remains unclear. The aim of this study is to examine PAX5 expression, its methylation status, biological functions and related molecular mechanism in NSCLC. We found that PAX5 was widely expressed in normal adult tissues but silenced or down-regulated in 88% (7/8) of NSCLC cell lines. PAX5 expression level was significantly lower in NSCLC than that in adjacent non-cancerous tissues (P = 0.0201). PAX5 down-regulation was closely associated with its promoter hypermethylation status and PAX5 expression could be restored by demethylation treatment. Frequent PAX5 promoter methylation in primary tumours (70%) was correlated with lung tumour histological types (P = 0.006). Ectopic expression of PAX5 in silenced lung cancer cell lines (A549 and H1975) inhibited their colony formation and cell viability, arrested cell cycle at G2 phase and suppressed cell migration/invasion as well as tumorigenicity in nude mice. Restoration of PAX5 expression resulted in the down-regulation of ß-catenin and up-regulation of tissue inhibitors of metalloproteinase 2, GADD45G in lung tumour cells. In summary, PAX5 was found to be an epigenetically inactivated tumour suppressor that inhibits NSCLC cell proliferation and metastasis, through down-regulating the ß-catenin pathway and up-regulating GADD45G expression.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Fator de Transcrição PAX5/metabolismo , beta Catenina/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Metilação de DNA , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Fator de Transcrição PAX5/genética , Regiões Promotoras Genéticas , Ligação Proteica , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genéticaRESUMO
Dickkopf-related protein 3 (DKK3) is an antagonist of Wnt ligand activity. Reduced DKK3 expression has been reported in various types of cancers, but its functions and related molecular mechanisms in breast tumorigenesis remain unclear. We examined the expression and promoter methylation of DKK3 in 10 breast cancer cell lines, 96 primary breast tumours, 43 paired surgical margin tissues and 16 normal breast tissues. DKK3 was frequently silenced in breast cell lines (5/10) by promoter methylation, compared with human normal mammary epithelial cells and tissues. DKK3 methylation was detected in 78% of breast tumour samples, whereas only rarely methylated in normal breast and surgical margin tissues, suggesting tumour-specific methylation of DKK3 in breast cancer. Ectopic expression of DKK3 suppressed cell colony formation through inducing G0/G1 cell cycle arrest and apoptosis of breast tumour cells. DKK3 also induced changes of cell morphology, and inhibited breast tumour cell migration through reversing epithelial-mesenchymal transition (EMT) and down-regulating stem cell markers. DKK3 inhibited canonical Wnt/ß-catenin signalling through mediating ß-catenin translocation from nucleus to cytoplasm and membrane, along with reduced active-ß-catenin, further activating non-canonical JNK signalling. Thus, our findings demonstrate that DKK3 could function as a tumour suppressor through inducing apoptosis and regulating Wnt signalling during breast tumorigenesis.
Assuntos
Apoptose/fisiologia , Neoplasias da Mama/patologia , Epigênese Genética , Inativação Gênica , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Transdução de Sinais , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Bases , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Quimiocinas , Metilação de DNA , Primers do DNA , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Reação em Cadeia da Polimerase , Regiões Promotoras GenéticasRESUMO
INTRODUCTION: Aberrant activation of Wnt/ß-catenin signaling plays an important role in the pathogenesis of breast cancer. DACT1 (Dapper/Frodo) has been identified as involved in antagonizing Wnt/ß-catenin signaling through interacting with Dishevelled (Dvl), a central mediator of Wnt signaling, whereas its role in breast tumorigenesis remains unclear. METHODS: We examined DACT1 expression in breast cancer cell lines and primary tumors with semiquantitative or quantitative RT-PCR and immunochemistry, and further evaluated the promoter methylation of DACT1 with methylation-specific PCR (MSP). We also explored the tumor-suppressive functions of DACT1 in vivo and in vitro, and its related mechanism in breast cancer. RESULTS: We identified DACT1 as a methylated target in our breast cancer epigenome study. Here, we further investigated DACT1 expression in multiple breast cell lines and primary tumors, and further studied its function and molecular mechanisms. We found that DACT1 expression was silenced in eight (88.9%) of nine breast cancer cell lines, and its protein levels were obviously reduced in breast tumors compared with paired surgical-margin tissues. Promoter CpG methylation of DACT1 was detected in five (55.6%) of nine breast cancer cell lines and 40 (29.9%) of 134 primary tumors, but not in surgical-margin tissues and normal breast tissues. Demethylation treatment of breast cancer cell lines restored DACT1 expression along with promoter demethylation, suggesting that an epigenetic mechanism mediates DACT1 silencing in breast cancer. Functional assays showed that ectopic expression of DACT1 could inhibit breast tumor cell proliferation in vivo and in vitro through inducing apoptosis, and further suppress tumor cell migration through antagonizing the Wnt/ß-catenin signaling pathway. CONCLUSIONS: Our study demonstrates that DACT1 could function as a tumor suppressor but was frequently downregulated in breast cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células , Inativação Gênica , Proteínas Nucleares/metabolismo , Via de Sinalização Wnt , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose , Western Blotting , Metilação de DNA , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Nus , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-Tronco , Cicatrização , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although mangiferin which is a naturally occurring glucosylxanthone has exhibited promising anticancer activities, the detailed molecular mechanism of mangiferin on cancers still remains enigmatic. In this study, the anticancer activity of mangiferin was evaluated in breast cancer cell line-based in vitro and in vivo models. We showed that mangiferin treatment resulted in decreased cell viability and suppression of metastatic potential in breast cancer cells. Further mechanistic investigation revealed that mangiferin induced decreased matrix metalloproteinase (MMP)-7 and -9, and reversal of epithelial-mesenchymal transition (EMT). Moreover, it was demonstrated that mangiferin significantly inhibited the activation of ß-catenin pathway. Subsequent experiments showed that inhibiting ß-catenin pathway might play a central role in mangiferin-induced anticancer activity through modulation of MMP-7 and -9, and EMT. Consistent with these findings in vitro, the antitumor potential was also verified in mangiferin-treated MDA-MB-231 xenograft mice where significantly decreased tumor volume, weight and proliferation, and increased apoptosis were obtained, with lower expression of MMP-7 and -9, vimentin and active ß-catenin, and higher expression of E-cadherin. Taken together, our study suggests that mangiferin might be used as an effective chemopreventive agent against breast cancer.
Assuntos
Antineoplásicos Fitogênicos , Neoplasias da Mama/tratamento farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Metaloproteinases da Matriz/biossíntese , Transdução de Sinais/efeitos dos fármacos , Xantonas/farmacologia , beta Catenina/fisiologia , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Dependovirus/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Imuno-Histoquímica , Indicadores e Reagentes , Metaloproteinase 7 da Matriz/biossíntese , Metaloproteinase 7 da Matriz/fisiologia , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/fisiologia , Camundongos , Camundongos PeladosRESUMO
The use of anticancer therapeutic agents is limited largely by their severe toxicity to normal tissues. The development of novel agents with tumor-specific cell-killing and effective gene delivery properties is thus very desirable. We used human adenovirus serotype 5 (AdHu5) as a vehicle to deliver the apoptin gene to specifically target gastric cancer in a recombinant gene delivery approach. AdHu5-apoptin is a safe and efficacious agent for the treatment of gastric cancer (GC). Our results show that apoptin protein encoded by the apoptin gene delivered via AdHu5 significantly inhibited the proliferation of SGC-7901 GC cells. Apoptin reduced the clone number by more than 75% and resulted in cell cycle arrest in the G2/M phase for 48% of the GC cells. It also induced cleavage of caspase-3, caspase-7, and caspase-9 in the GC cells. Intratumoral and peritumoral in vivo injection of AdHu5-apoptin significantly suppressed tumor growth and induced apoptosis in xenogeneic tumors in mice. The apoptosis induced by AdHu5-apoptin was independent of anti-apoptotic Bcl-2 and Bcl-xL proteins and the p53 pathway. Taken together, our results show that AdHu5-apoptin has great potential as a therapeutic agent for effective treatment of gastric tumors.
Assuntos
Adenovírus Humanos/genética , Apoptose/genética , Proteínas do Capsídeo/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Neoplasias Gástricas/genética , Proteína Supressora de Tumor p53/genética , Adenovírus Humanos/classificação , Animais , Apoptose/fisiologia , Western Blotting , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/fisiologia , Caspases/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Vetores Genéticos/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sorotipagem , Neoplasias Gástricas/patologia , Neoplasias Gástricas/terapia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
BACKGROUND: The incidence of colorectal cancer (CRC) has increased in recent years. Identification of accurate tumor markers has become the focus of CRC research. Early and frequent DNA methylation tends to occur in cancer. Thus, identifying accurate methylation biomarkers would improve the efficacy of CRC treatment. Neuroglobin (NGB) is involved in neurological and oncological diseases. However, there are currently no reports on epigenetic regulation involvement of NGB in CRC. RESULTS: NGB was downregulated or silenced in majority CRC tissues and cell lines. The hypermethylation of NGB was detected in tumor tissue, but no or a very low methylation frequency in normal tissues. Overexpression of NGB induced G2/M phase arrest and apoptosis, suppressed proliferation, migration, invasion in vitro, and inhibited CRC tumor growth and angiogenesis in vivo. Isobaric tag for relative and absolute quantitation (Itraq)-based proteomics identified approximately 40% proteins related to cell-cell adhesion, invasion, and tumor vessel formation in the tumor microenvironment, among which GPR35 was proved critical for NGB-regulated tumor angiogenesis suppression in CRC. CONCLUSIONS: NGB, an epigenetically silenced factor, inhibits metastasis through the GPR35 in CRC. It is expected to grow into a potential cancer risk assessment factor and a valuable biomarker for early diagnosis and prognosis assessment of CRC.
Assuntos
Neoplasias Colorretais , Metilação de DNA , Humanos , Neuroglobina/genética , Neuroglobina/metabolismo , Epigênese Genética , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Biomarcadores/metabolismo , Regulação Neoplásica da Expressão Gênica , Proliferação de Células , Microambiente Tumoral , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Our previous studies found that Zinc-finger protein 382 (ZNF382) played as a tumor suppressor gene in esophageal and gastric cancers, and a positive correlation between the high expression of ZNF382 and better outcome in breast cancer patients. However, the biological roles and mechanisms of ZNF382 in breast cancer remains unclear. We detected ZNF382 expression by reverse-transcription PCR (RT-PCR) and real-time quantitative PCR (qRT-PCR) in breast cancer cells and tissues, and explored the impacts and mechanisms of ectopic ZNF382 expression in breast cancer cells in vitro and in vivo, respectively. Our results revealed that ZNF382 was significantly down-regulated in breast cancer tissues compared with adjacent non-cancer tissues. Restoration of ZNF382 expression in silenced breast cancer cells not only inhibited tumor cell colony formation, viability, migration and invasion, and epithelial-mesenchymal-transition (EMT), but also induced apoptosis and G0/G1 arrest. In conclusion, ZNF382 could induce G0/G1 cell cycle arrest through inhibiting CDC25A signaling, and, inhibit cell migration, invasion and EMT by antagonizing ZEB1 signaling in breast cancer cells.
RESUMO
AIM: Zinc finger and BTB domain-containing protein 28 (ZBTB28) is a potential tumor suppressor for some cancers. However, its epigenetic regulation and functions in renal cell carcinoma (RCC) remain to be elucidated. METHODS: The expression of ZBTB28 mRNA was analyzed by semi-quantitative reverse transcription polymerase chain reaction (PCR) in nine RCC cell lines and normal kidney tissues. Methylation status of ZBTB28 promoter was assessed by methylation-specific PCR in RCC cell lines, primary RCC, tumors and adjacent tissues. The involvement of ZBTB28 in cell proliferation and migration was investigated. RESULTS: ZBTB28 promoter was hypermethylated in 88.9% (8/9) of RCC cell lines with reduced ZBTB28 mRNA expression, and could be reversed by DNA methyltransferase inhibitors. The methylation of ZBTB28 promoter was detected in 73.5% (36/49) of primary RCC tissues, compared with 7.1% (1/14) in normal tissues. Overexpression of ZBTB28 significantly inhibited RCC cell proliferation and migration, and induced apoptosis. Further analyses revealed that ZBTB28 upregulation could inhibit multiple oncogenic signaling transduction pathways. CONCLUSION: ZBTB28 is frequently silenced by promoter methylation in RCC pathogenesis and functions as a novel tumor suppressive gene. ZBTB28 may be a potential target for the development of RCC therapeutic strategies.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Metilação de DNA/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , Rim/metabolismo , Rim/patologia , Neoplasias Renais/genética , Neoplasias Renais/patologia , RNA Mensageiro/metabolismoRESUMO
Aberrant protocadherins (PCDHs) expression trigger tumor invasion and metastasis. PCDH20 anti-tumor functions in various tumor have been identified. Tumor suppression is due to Wnt/ß-catenin pathway antagonism and may be suppressed caused by PCDH20 downregulation through promotor methylation, whereas PCDH20 effects and regulation mechanism in esophageal squamous cell carcinoma (ESCC) remains elusive. We analyzed PCDH20 effects on ESCC and underlying action mechanisms for PCDH20. We test PCDH20 expression in ESCC tissues and cells by semi-quantitative PCR (RT-PCR) and q-PCR (real-time quantitative polymerase chain reaction). MSP (methylation-specific PCR) was carried out to assess the methylation of PCDH20 in ESCC cells and tissues. Anti-tumor effects of PCDH20 in vitro were assessed by clone formation assay, CCK8 assay, Transwell assay, and flow cytometry. Nude mice tumorigenicity was used to assess PCDH20 anti-tumor effect in vivo. Online database, qPCR, and Western blotting were used to identify the downregulation of MAP3K9 by PCDH20, associated with AKT/ß-catenin signaling inactivation. We found that PCDH20 expression was dramatically attenuated in esophageal cancer tissues and cells, maybe due to promotor methylation, and ectopic PCDH20 expression suppressed ESCC malignant biological phenotypes. PCDH20 exerted anti-tumor effects by MAP3K9 downregulation, which suppressed AKT/ß-catenin signaling in ESCC cells. Conclusion: PCDH20 was a tumor suppressor gene, which antagonized AKT/ß-catenin signaling pathway in ESCC by decreasing MAP3K9.
RESUMO
The RGD motif on the extracellular matrix or cell surface, together with its integrin receptors, constitutes a major recognition system for cell adhesion. There are several erythrocyte major membrane skeletal proteins, e.g., α spectrin, ankyrin, and protein 4.2, that bear an RGD motif. However, it is not known whether the RGD/integrin recognition system is utilized in the erythrocyte-macrophage adhesion during erythrophagocytosis. Here we report that the RGD motif of ankyrin, but not others, is recognized by the α(v)ß(3) integrin receptor. In addition, the RGD motif of ankyrin, a peripheral membrane protein, can be externalized onto the cell surface when erythrocytes are incubated with calcium and sheared both at physiological levels. Furthermore, the erythrocyte-macrophage adhesion can be specifically inhibited by ankyrin and/or α(v)ß(3). Thus, externalization of ankyrin followed by RGD/integrin recognition may be a novel mechanism by which erythrocytes adhere to macrophages preceding phagocytosis.
Assuntos
Anquirinas/metabolismo , Citofagocitose , Membrana Eritrocítica/fisiologia , Eritrócitos/fisiologia , Integrina alfaVbeta3/metabolismo , Oligopeptídeos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Adesão Celular , Células Cultivadas , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Humanos , Macrófagos/fisiologia , Camundongos , Microscopia de Fluorescência , Dados de Sequência MolecularRESUMO
Oxygen transport behavior in erythrocyte suspension or in hemoglobin solution was studied as a potential therapeutic model for the clinical treatment of blood loss, and this can also provide physiological data with which to evaluate blood substitutes. In the present project, we examined the in vitro kinetics of hemoglobin binding to and releasing oxygen, to provide detailed oxygen-flux measurements for unmodified hemoglobin solutions and erythrocyte suspensions in human, as well as other vertebrates. An in vitro method was used, based on a widely used artificial system, with the oxygen saturation level being detected in real time. Results from this study indicated that the kinetic curves of human erythrocyte suspensions and hemoglobin solutions were either S-shaped or hyperbolic, respectively. Based on these curves, the significance of T(50) emerged in our investigation, where T(50) is defined as the time needed for 50% hemoglobin to be saturated with oxygen, and reflects the efficiency with which hemoglobin carries oxygen. This parameter may be used to diagnose blood diseases, and could be a standard for evaluating blood substitutes. In this study, we also compared the T(50) of 4 species of vertebrates, and found that it shows a distinct efficiency of oxygen binding related to species, and potentially reveals the evolutionary function of hemoglobin and its possible adaptation to the environment.
Assuntos
Eritrócitos/metabolismo , Hemoglobinas/metabolismo , Oxigênio/sangue , Oxigênio/metabolismo , Animais , Anuros , Substitutos Sanguíneos/metabolismo , Carpas , Galinhas , Humanos , Cinética , Ligação Proteica , Coelhos , Transporte Respiratório , Soluções , SuspensõesRESUMO
BACKGROUND: Mitogen-activated protein kinase 10 (Mapk10) is a member of the c-jun N-terminal kinases (jnk) subgroup in the MAPK superfamily, and was proposed as a tumor suppressor inactivated epigenetically. Its role in hepatocellular carcinoma (HCC) has not yet been illustrated. We aimed to investigate the expression and epigenetic regulation of mapk10 as well as its clinical significance in HCC. RESULTS: Mapk10 was expressed in almost all the normal tissues including liver, while we found that the protein expression of MAPK10 was significantly downregulated in clinical samples of HCC patients compared with these levels in adjacent normal tissues (29/46, P < 0.0001). Clinical significance of MAPK10 expression was then assessed in a cohort of 59 HCC cases, which indicated its negative expression was significantly correlated with advanced tumor stage (P = 0.001), more microsatellite nodules (P = 0.025), higher serum AFP (P = 0.001) and shorter overall survival time of HCC patients. Methylation was further detected in 58% of the HCC cell lines we tested and in 66% of primary HCC tissues by methylation-specific PCR (MSP), which was proved to be correlated with the silenced or downregulated expression of mapk10. To get the mechanisms more clear, the transcriptional silencing of mapk10 was reversed by pharmacological demethylation, and ectopic expression of mapk10 in silenced HCC cell lines significantly inhibited the colony formation ability, induced apoptosis, or enhanced the chemosensitivity of HCC cells to 5-fluorouracil. CONCLUSION: Mapk10 appears to be a functional tumor suppressor gene frequently methylated in HCC, which could be a valuable biomarker or a new diagnosis and therapy target in a clinical setting.