RESUMO
One of the greatest unmet needs hindering the successful treatment of nasopharyngeal carcinomas (NPCs) is for representative physiological and cost-effective models. Although Epstein-Barr virus (EBV) infection is consistently present in NPCs, most studies have focused on EBV-negative NPCs. For the first time, we established and analyzed three-dimensional (3D) spheroid models of EBV-positive and EBV-negative NPC cells and compared these to classical two-dimensional (2D) cultures in various aspects of tumor phenotype and drug responses. Compared to 2D monolayers, the 3D spheroids showed significant increases in migration capacity, stemness characteristics, hypoxia and drug resistance. Co-culture with endothelial cells, which mimics essential interactions in the tumor microenvironment, effectively enhanced spheroid dissemination. Furthermore, RNA sequencing revealed significant changes at the transcriptional level in 3D spheroids compared to expression in 2D monolayers. In particular, we identified known (VEGF, AKT and mTOR) and novel (Wnt-ß-catenin and Eph-ephrin) cell signaling pathways that are activated in NPC spheroids. Targeting these pathways in 3D spheroids using FDA-approved drugs was effective in monoculture and co-culture. These findings provide the first demonstration of the establishment of EBV-positive and EBV-negative NPC 3D spheroids with features that resemble advanced and metastatic NPCs. Furthermore, we show that NPC spheroids have potential use in identifying new drug targets.
Assuntos
Infecções por Vírus Epstein-Barr , Neoplasias Nasofaríngeas , Linhagem Celular Tumoral , Células Endoteliais/metabolismo , Efrinas , Herpesvirus Humano 4/metabolismo , Humanos , Carcinoma Nasofaríngeo/tratamento farmacológico , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/genética , Transdução de Sinais , Microambiente Tumoral , beta Catenina/genética , beta Catenina/metabolismoRESUMO
BACKGROUND: Gamabufotalin (CS-6), a major bufadienolide of Chansu, has been used for cancer therapy due to its desirable metabolic stability and less adverse effect. However, the underlying mechanism of CS-6 involved in anti-tumor activity remains poorly understood. METHODS: The biological functions of gamabufotalin (CS-6) were investigated by migration, colony formation and apoptosis assays in NSCLC cells. The nuclear localization and interaction between transcriptional co-activator p300 and NF-κB p50/p65 and their binding to COX-2 promoter were analyzed after treatment with CS-6. Molecular docking study was used to simulate the interaction of CS-6 with IKKß. The in vivo anti-tumor efficacy of CS-6 was also analyzed in xenografts nude mice. Western blot was used to detect the protein expression level. RESULTS: Gamabufotalin (CS-6) strongly suppressed COX-2 expression by inhibiting the phosphorylation of IKKß via targeting the ATP-binding site, thereby abrogating NF-κB binding and p300 recruitment to COX-2 promoter. In addition, CS-6 induced apoptosis by activating the cytochrome c and caspase-dependent apoptotic pathway. Moreover, CS-6 markedly down-regulated the protein levels of COX-2 and phosphorylated p65 NF-κB in tumor tissues of the xenograft mice, and inhibited tumor weight and size. CONCLUSIONS: Our study provides pharmacological evidence that CS-6 exhibits potential use in the treatment of COX-2-mediated diseases such as lung cancer.
Assuntos
Bufanolídeos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Ciclo-Oxigenase 2/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Venenos de Anfíbios/química , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/genética , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Transplante de Neoplasias , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Conformação Proteica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Overexpression of cyclooxygenase 2 (COX-2) is frequently found in early and advanced lung cancers. However, the precise regulatory mechanism of COX-2 in lung cancers remains unclear. Here we identified cleavage and polyadenylation specific factor 4 (CPSF4) as a new regulatory factor for COX-2 and demonstrated the role of the CPSF4/COX-2 signaling pathway in the regulation of lung cancer growth and progression. Overexpression or knockdown of CPSF4 up-regulated or suppressed the expression of COX-2 at mRNA and protein levels, and promoted or inhibited cell proliferation, migration and invasion in lung cancer cells. Inhibition or induction of COX-2 reversed the CPSF4-mediated regulation of lung cancer cell growth. Cancer cells with CPSF4 overexpression or knockdown exhibited increased or decreased expression of p-IKKα/ß and p-IκBα, the translocation of p50/p65 from the cytoplasm to the nucleus, and the binding of p65 on COX-2 promoter region. In addition, CPSF4 was found to bind to COX-2 promoter sequences directly and activate the transcription of COX-2. Silencing of NF-κB expression or blockade of NF-κB activity abrogated the binding of CPSF4 on COX-2 promoter, and thereby attenuated the CPSF4-mediated up-regulation of COX-2. Moreover, CPSF4 was found to promote lung tumor growth and progression by up-regulating COX-2 expression in a xenograft lung cancer mouse model. CPSF4 overexpression or knockdown promoted or inhibited tumor growth in mice, while such regulation of tumor growth mediated by CPSF4 could be rescued through the inhibition or activation of COX-2 signaling. Correspondingly, CPSF4 overexpression or knockdown also elevated or attenuated COX-2 expression in tumor tissues of mice, while treatment with a COX-2 inducer LPS or a NF-κB inhibitor reversed this elevation or attenuation. Furthermore, we showed that CPSF4 was positively correlated with COX-2 levels in tumor tissues of lung cancer patients. Simultaneous high expression of CPSF4 and COX-2 proteins predicted poor prognosis of patients with lung cancers. Our results therefore demonstrated a novel mechanism for the transcriptional regulation of COX-2 by CPSF4 in lung cancer, and also offer a potential therapeutic target for lung cancers bearing aberrant activation of CPSF4/COX-2 signaling.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/enzimologia , Proliferação de Células , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Ciclo-Oxigenase 2/metabolismo , Neoplasias Pulmonares/enzimologia , NF-kappa B/metabolismo , Células A549 , Animais , Sítios de Ligação , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular , Fator de Especificidade de Clivagem e Poliadenilação/genética , Ciclo-Oxigenase 2/genética , Progressão da Doença , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas I-kappa B/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos Nus , Inibidor de NF-kappaB alfa , NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Invasividade Neoplásica , Fosforilação , Regiões Promotoras Genéticas , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Fator de Transcrição RelA/metabolismo , Transcrição Gênica , Transfecção , Carga TumoralRESUMO
Cyclooxygenase-2 (COX-2) is highly expressed in tumor cells and has been regarded as a hallmarker for cancers, but the excise regulatory mechanism of COX-2 in tumorigenesis remains largely unknown. Here, we pulled down and identified a novel COX-2 regulator, heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1), which could specifically bind to COX-2 core promoter and regulate tumor growth in non-small-cell lung cancers (NSCLCs). Knockdown of hnRNPA2/B1 by shRNA or siRNA downregulated COX-2 expression and prostaglandin E2 (PGE2) production, and suppressed tumor cell growth in NSCLC cells in vitro and in vivo. Conversely, overexpression of hnRNPA2/B1 up-regulated the levels of COX-2 and PGE2 and promoted tumor cell growth. We also showed that hnRNPA2/B1 expression was positively correlated with COX-2 expression in NSCLC cell lines and tumor tissues, and the up-regulated expression of hnRNPA2/B1 and COX-2 predicted worse prognosis in NSCLC patients. Furthermore, we demonstrated that the activation of COX-2 expression by hnRNPA2/B1 was mediated through the cooperation with p300, a transcriptional co-activator, in NSCLC cells. The hnRNPA2/B1 could interact with p300 directly and be acetylated by p300. Exogenous overexpression of p300, but not its histone acetyltransferase (HAT) domain deletion mutation, augmented the acetylation of hnRNPA2/B1 and enhanced its binding on COX-2 promoter, thereby promoted COX-2 expression and lung cancer cell growth. Collectively, our results demonstrate that hnRNPA2/B1 promotes tumor cell growth by activating COX-2 signaling in NSCLC cells and imply that the hnRNPA2/B1/COX-2 pathway may be a potential therapeutic target for human lung cancers.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Ciclo-Oxigenase 2/biossíntese , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Regulação para Cima , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/genética , Indução Enzimática , Feminino , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas , Transdução de Sinais/genéticaRESUMO
Cyclooxygenase-2 (COX-2) plays an important role in lung cancer development and progression. Using streptavidin-agarose pulldown and proteomics assay, we identified and validated Ku80, a dimer of Ku participating in the repair of broken DNA double strands, as a new binding protein of the COX-2 gene promoter. Overexpression of Ku80 up-regulated COX-2 promoter activation and COX-2 expression in lung cancer cells. Silencing of Ku80 by siRNA down-regulated COX-2 expression and inhibited tumor cell growth in vitro and in a xenograft mouse model. Ku80 knockdown suppressed phosphorylation of ERK, resulting in an inactivation of the MAPK pathway. Moreover, CBP, a transcription co-activator, interacted with and acetylated Ku80 to co-regulate the activation of COX-2 promoter. Overexpression of CBP increased Ku80 acetylation, thereby promoting COX-2 expression and cell growth. Suppression of CBP by a CBP-specific inhibitor or siRNA inhibited COX-2 expression as well as tumor cell growth. Tissue microarray immunohistochemical analysis of lung adenocarcinomas revealed a strong positive correlation between levels of Ku80 and COX-2 and clinicopathologic variables. Overexpression of Ku80 was associated with poor prognosis in patients with lung cancers. We conclude that Ku80 promotes COX-2 expression and tumor growth and is a potential therapeutic target in lung cancer.
Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Antígenos Nucleares/metabolismo , Ciclo-Oxigenase 2/biossíntese , Proteínas de Ligação a DNA/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Adenocarcinoma/enzimologia , Adenocarcinoma/genética , Adenocarcinoma de Pulmão , Animais , Antígenos Nucleares/genética , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Ciclo-Oxigenase 2/genética , Proteínas de Ligação a DNA/genética , Progressão da Doença , Xenoenxertos , Humanos , Autoantígeno Ku , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Camundongos , Regiões Promotoras Genéticas , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Distribuição Aleatória , TransfecçãoRESUMO
Melatonin is a hormone identified in plants and pineal glands of mammals and possesses diverse physiological functions. Fisetin is a bio-flavonoid widely found in plants and exerts antitumor activity in several types of human cancers. However, the combinational effect of melatonin and fisetin on antitumor activity, especially in melanoma treatment, remains unclear. Here, we tested the hypothesis that melatonin could enhance the antitumor activity of fisetin in melanoma cells and identified the underlying molecular mechanisms. The combinational treatment of melanoma cells with fisetin and melatonin significantly enhanced the inhibitions of cell viability, cell migration and clone formation, and the induction of apoptosis when compared with the treatment of fisetin alone. Moreover, such enhancement of antitumor effect by melatonin was found to be mediated through the modulation of the multiply signaling pathways in melanoma cells. The combinational treatment of fisetin with melatonin increased the cleavage of PARP proteins, triggered more release of cytochrome-c from the mitochondrial inter-membrane, enhanced the inhibition of COX-2 and iNOS expression, repressed the nuclear localization of p300 and NF-κB proteins, and abrogated the binding of NF-κB on COX-2 promoter. Thus, these results demonstrated that melatonin potentiated the anti-tumor effect of fisetin in melanoma cells by activating cytochrome-c-dependent apoptotic pathway and inhibiting COX-2/iNOS and NF-κB/p300 signaling pathways, and our study suggests the potential of such a combinational treatment of natural products in melanoma therapy.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Proteína p300 Associada a E1A/metabolismo , Flavonoides/farmacologia , Melatonina/farmacologia , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase 2/farmacologia , Sinergismo Farmacológico , Flavonóis , Humanos , Melanoma/patologiaRESUMO
Upregulated expression and activation of human telomerase reverse transcriptase (hTERT) is a hallmarker of lung tumorigenesis. However, the mechanism underlying the aberrant hTERT activity in lung cancer cells remains poorly understood. In this study, we found the transcriptional co-activator CBP as a new hTERT promoter-binding protein that regulated hTERT expression and tumor growth in lung adenocarcinoma cells using a biotin-streptavidin-bead pulldown technique. Chromatin immunoprecipitation assay verified the immortalized cell and tumor cell-specific binding of CBP on hTERT promoter. Overexpression of exogenous CBP upregulated the expression of the hTERT promoter-driven luciferase and endogenous hTERT protein in lung cancer cells. Conversely, inhibition of CBP by CBP-specific siRNA or its chemical inhibitor repressed the expression of hTERT promoter-driven luciferase and endogenous hTERT protein as well as telomerase activity. Moreover, inhibition of CBP expression or activity also significantly reduced the proliferation of lung cancer cells in vitro and tumor growth in an xenograft mouse model in vivo. Immunohistochemical analysis of tissue microarrays of lung cancers revealed a positive correlation between CBP and hTERT. Importantly, the patients with high CBP and hTERT expression had a significantly shorter overall survival. Furthermore, CBP was found to interact with and acetylate transactivator Sp1 in lung cancer cells. Inhibition of CBP by CBP-specific siRNA or its chemical inhibitor significantly inhibited Sp1 acetylation and its binding to the hTERT promoter. Collectively, our results indicate that CBP contributes to the upregulation of hTERT expression and tumor growth, and overexpression of CBP predicts poor prognosis in human lung cancers.