RESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive solid malignancies. A specific mechanism of its metastasis has not been established. In this study, we investigated whether Neural Wiskott-Aldrich syndrome protein (N-WASP) plays a role in distant metastasis of PDAC. We found that N-WASP is markedly expressed in clinical patients with PDAC. Clinical analysis showed a notably more distant metastatic pattern in the N-WASP-high group compared to the N-WASP-low group. N-WASP was noted to be a novel mediator of epithelial-mesenchymal transition (EMT) via gene expression profile studies. Knockdown of N-WASP in pancreatic cancer cells significantly inhibited cell invasion, migration, and EMT. We also observed positive association of lysyl oxidase-like 2 (LOXL2) and focal adhesion kinase (FAK) with the N-WASP-mediated response, wherein EMT and invadopodia function were modulated. Both N-WASP and LOXL2 depletion significantly reduced the incidence of liver and lung metastatic lesions in orthotopic mouse models of pancreatic cancer. These results elucidate a novel role for N-WASP signaling associated with LOXL2 in EMT and invadopodia function, with respect to regulation of intercellular communication in tumor cells for promoting pancreatic cancer metastasis. These findings may aid in the development of therapeutic strategies against pancreatic cancer.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Humanos , Camundongos , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Transição Epitelial-Mesenquimal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Neoplasias Pancreáticas/patologia , Transdução de Sinais , Proteína da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a free radical scavenger approved for the treatment of amyotrophic lateral sclerosis, a fatal neuromuscular disease. Edaravone is administered as an intravenous infusion over 60 min for several treatment cycles. To ease the burden of patients and caregivers, the oral formulation of edaravone has been developed. The purpose of this study was to evaluate pharmacokinetics and tissue distribution of TEJ-1704, an edaravone oral prodrug, in male Sprague Dawley rats and beagle dogs. Animal experiments were conducted using Sprague Dawley rats and beagle dogs to evaluate pharmacokinetics, tissue distribution, and excretion of TEJ-1704. Blood, tissues, cerebrospinal fluid, urine, and feces samples were collected at designated sampling time after intravenous (IV) or oral (PO) administration of edaravone or TEJ-1704. A modified bioanalysis method was developed to quantify edaravone in samples including plasma, tissues, cerebrospinal fluid, urine, and feces. The bioanalysis method was validated and successfully applied to pharmacokinetics, tissue distribution, and excretion studies of the novel edaravone prodrug. Although plasma Cmax of TEJ-1704 was low, groups administered with TEJ-1704 had high AUCinf, suggesting continuous metabolism of TEJ-1704 into edaravone. Groups treated with TEJ-1704 also showed lower CSF distribution than the control groups. After the administration of TEJ-1704, the majority of edaravone was distributed to the heart, lung, and kidney. It was excreted equally via urine and feces. The pharmacokinetics, tissue distribution, and excretion of TEJ-1704, a novel edaravone oral prodrug, were successfully characterized. Additional studies are needed to fully understand the difference between TEJ-1704 and edaravone and determine the potency of TEJ-1704.
RESUMO
Specific protein 1 (SP1) is associated with aggressive behavior, invasive clinical phenotype and poor clinical outcomes in various cancers. We studied whether SP1 exerts its effect on invasiveness and promotion of the epithelial-mesenchymal transition (EMT) by regulating lysyl oxidase-like 2 (LOXL2) in pancreatic ductal adenocarcinoma (PDAC) cell lines. We showed that silencing of SP1 in MIA Paca-2 cell significantly decreased cell invasion and migration. In MIA Paca-2 cells, silencing of SP1 induced a reduction of LOXL2 expression, whereas LOXL2 silencing did not lead to a decrease in the expression of SP1. Chromatin immunoprecipitation assay demonstrated the binding of SP1 to LOXL2 promoter. Wound healing and transmigration assays also showed that transfection of both SP1 and LOXL2 siRNA induced most significant decrease of cell invasion and migration compared to either SP1 or LOXL2-only silenced cells. Finally, we investigated the prognostic value of SP1 in patients with PDAC and SP1/LOX2 expression was examined by immunochemistry. Univariate and multivariate analyses showed that tumor differentiation and co-expression of SP1 and LOXL2 were independent factors for disease-free survival. In summary, our study demonstrates that SP1 modulates EMT and is involved in tumor invasion and migration of PDAC cells through the regulation of LOXL2.
Assuntos
Aminoácido Oxirredutases/metabolismo , Carcinoma Ductal Pancreático/patologia , Transição Epitelial-Mesenquimal , Neoplasias Pancreáticas/secundário , Fator de Transcrição Sp1/metabolismo , Aminoácido Oxirredutases/genética , Biomarcadores Tumorais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Movimento Celular , Proliferação de Células , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Prognóstico , Estudos Retrospectivos , Fator de Transcrição Sp1/genética , Taxa de Sobrevida , Células Tumorais Cultivadas , Neoplasias PancreáticasRESUMO
Aberrant promoter methylation plays a vital role in colorectal carcinogenesis. However, its role in treatment responses is unclear, especially for metastatic disease. Here, we investigated the association between promoter methylation and treatment outcomes of irinotecan-based chemotherapy in 102 patients with metastatic colorectal cancer. Promoter methylation was examined by methylation-specific polymerase chain reaction for three loci (CHFR, WRN, and SULF2) associated with chemotherapy response and five CpG island methylator phenotype (CIMP)-specific markers (CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1). Association between CHFR methylation and in vitro sensitivity to irinotecan was also evaluated. Promoter methylation of CHFR, WRN, and SULF2 was identified in 16 (15.7%), 24 (23.5%), and 33 (32.4%) patients, respectively. CIMP status was positive in 22 (21.6%) patients. CHFR methylation was associated with a significantly longer time to progression (TTP) (median: 8.77 vs. 4.43 months, P = .019), with trends favoring higher overall survival (OS) (median: 22.83 vs. 20.17 months, P = .300) and response rates (31.3% vs. 17.4%, P = .300). For patients with unmethylated CHFR, TTP (median: 5.60 vs. 3.53, P = .020) and OS (median: 20.57 vs. 9.23, P = .006) were significantly different according to CIMP status. Colorectal cancer cell lines with CHFR methylation demonstrated increased sensitivity to irinotecan. Both CHFR overexpression and combination with 5-aza-2'-deoxycytidine reversed irinotecan sensitivity in CHFR-methylated cell lines, whereas CHFR knockdown in unmethylated cells restored sensitivity to irinotecan. These data suggest that CHFR methylation may be associated with favorable treatment outcomes of irinotecan-based chemotherapy in patients with metastatic colorectal cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas de Ciclo Celular/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Metilação de DNA , Proteínas de Neoplasias/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Regiões Promotoras Genéticas , Ubiquitina-Proteína Ligases/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Biomarcadores Tumorais , Proteínas de Transporte , Linhagem Celular Tumoral , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Humanos , Irinotecano/administração & dosagem , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Resultado do TratamentoRESUMO
Triple-negative breast cancer (TNBC) represents approximately 10-17% of all breast cancers, and patients with TNBC show a poorer short-term prognosis than patients with other types of breast cancer. TNBCs also have a higher tendency for early distant metastasis and cancer recurrence due to induction of the epithelial-mesenchymal transition (EMT). Several recent reports have suggested that inhibitor of apoptosis (IAP) proteins function as regulators of the EMT. However, the roles of these proteins in TNBC are not clear. Accordingly, we investigated the roles of cIAP2 in TNBC. Among eight IAP genes, only cIAP2 was upregulated in TNBC cells compared with that in other breast cancer subtypes. Analysis of TMAs revealed that expression of cIAP2 was upregulated in TNBCs. In vitro studies showed that cIAP2 was highly expressed in TNBC cells compared with that in other types of breast cancer cells. Furthermore, silencing of cIAP2 in TNBC cells induced mesenchymal-epithelial transition (MET)-like processes and subsequently suppressed the migratory ability and invasion capacity of the cells by regulation of Snail through the AKT signaling pathway. In contrast, ectopic expression of cIAP2 in luminal-type breast cancer cells induced activation of the AKT signaling pathway. These results collectively indicated that cIAP2 regulated the EMT in TNBC via activation of the AKT signaling pathway, contributing to metastasis in TNBC. Our study proposes a novel mechanism through which cIAP2 regulates the EMT involving AKT signaling in TNBC cells. We suggest that cIAP2 may be an attractive candidate molecule for the development of targeted therapeutics in the future.
RESUMO
Colorectal cancer (CRC) is one of the most dangerous types of malignant tumors, and cancer metastasis is a major factor in the failure of CRC therapy. Recently, LOXL2 (lysyl oxidase-like 2) has been shown to represent a regulator of epithelial-mesenchymal transition (EMT) in different cancer types. However, LOXL2 has not been reported to be involved in CRC metastasis. In this study, we demonstrated that LOXL2 expression is strongly correlated with the rate of CRC metastasis, it participates in the regulation of EMT-related molecule expression in CRC cells in vitro, and it is involved in migratory potential alterations. Additionally, tissue microarray analysis of CRC patients showed an increase in the probability of developing CRC distant metastasis and a decrease in the survival rate of patients with high LOXL2 expression. The results obtained in this study indicate that LOXL2 is involved in the development and progression of CRC metastasis, and therefore, its expression levels may represent a useful prognostic marker.
RESUMO
Hexokinase 2 (HK2) is a rate-determining enzyme in aerobic glycolysis, a process upregulated in tumor cells. HK2 expression is controlled by various transcription factors and epigenetic alterations and is heterogeneous in hepatocellular carcinomas (HCCs), though the cause of this heterogeneity is not known. DNA methylation in the HK2 promoter CpG island (HK2-CGI) and its surrounding regions (shore and shelf) has not previously been evaluated, but may provide clues about the regulation of HK2 expression. Here, we compared HK2 promoter methylation in HCCs and adjacent non-cancerous liver tissues using a HumanMethylation450 BeadChip array. We found that, while the HK2-CGI N-shore was hypomethylated, thereby enhancing HK2 expression, the HK2-CGI was itself hypermethylated in some HCCs. This hypermethylation suppressed HK2 expression by inhibiting interactions between HIF-1α and a hypoxia response element (HRE) located at -234/-230. HCCs that were HK2negative and had distinct promoter CGI methylation were denoted as having a HK2-CGI methylation phenotype (HK2-CIMP), which was associated with poor clinical outcome. These findings indicate that HK2-CGI N-shore hypomethylation and HK2-CGI hypermethylation affect HK2 expression by influencing the interaction between HIF 1α and HRE. HK2-CGI hypermethylation induces HK2-CIMP and could represent a prognostic biomarker for HCC.
Assuntos
Carcinoma Hepatocelular/genética , Ilhas de CpG/genética , Metilação de DNA , Hexoquinase/genética , Neoplasias Hepáticas/genética , Regiões Promotoras Genéticas , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Hexoquinase/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , PrognósticoRESUMO
Lysyl oxidase-like 2 (LOXL2) is associated with invasiveness and metastasis in cancer. We analyzed the prognostic impact of LOXL2 in pancreatic cancer patients and investigated the role of LOXL2 in pancreatic cancer cell lines. Immunohistochemical analysis was performed in samples from 80 patients and showed LOXL2 expression in 81.2% of patients with pancreatic cancer. Regarding recurrence patterns, LOXL2-positive tumors showed a significantly higher rate of distant recurrence. The 1-year and 3-year disease-free survival rates were 84.6% and 0.0%, respectively, for LOXL2-negative patients, and 27.8 % and 0.0 %, respectively, for LOXL2-positive patients. On univariate analysis, combined resection of major vessels, depth of invasion, tumor stage, and LOXL2- positive status were significant factors for poor prognosis. After identification of LOXL2 expression in pancreatic cancer cell lines, LOXL2-silenced and LOXL2-overexpressed cell lines were used to perform transwell invasion and transendothelial migration assays.In vitro studies indicated that LOXL2 silencing in MIA PaCa-2 and PANC-1 cells induced a mesenchymal-epithelial transition (MET)-like process associated with decreased invasive and migratory properties. LOXL2 overexpression in AsPC-1 and BxPC-3 cells enhanced the epithelial-mesenchymal transition (EMT)-like process and increased migratory and invasive activity. These clinical and preclinical data confirm that higher LOXL2 expression is associated with the invasiveness of pancreatic cancer cells and the low survival rate of pancreatic cancer patients. Our results suggest the clinical value of LOXL2 as a therapeutic target in pancreatic cancer.
Assuntos
Aminoácido Oxirredutases/metabolismo , Neoplasias Pancreáticas/metabolismo , Idoso , Linhagem Celular Tumoral , Movimento Celular , Intervalo Livre de Doença , Células Endoteliais/citologia , Transição Epitelial-Mesenquimal , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Vetores Genéticos , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Metástase Neoplásica , Recidiva Local de Neoplasia , Neoplasias Pancreáticas/patologia , Prognóstico , Estudos Retrospectivos , Resultado do TratamentoRESUMO
The constant presence of the viral genome in Epstein-Barr virus (EBV)-associated gastric cancers (EBVaGCs) suggests the applicability of novel EBV-targeted therapies. The antiviral nucleoside drug, ganciclovir (GCV), is effective only in the context of the viral lytic cycle in the presence of EBV-encoded thymidine kinase (TK)/protein kinase (PK) expression. In this study, screening of the Johns Hopkins Drug Library identified gemcitabine as a candidate for combination treatment with GCV. Pharmacological induction of EBV-TK or PK in EBVaGC-originated tumor cells were used to study combination treatment with GCV in vitro and in vivo. Gemcitabine was found to be a lytic inducer via activation of the ataxia telangiectasia-mutated (ATM)/p53 genotoxic stress pathway in EBVaGC. Using an EBVaGC mouse model and a [125I] fialuridine (FIAU)-based lytic activation imaging system, we evaluated gemcitabine-induced lytic activation in an in vivo system and confirmed the efficacy of gemcitabine-GCV combination treatment. This viral enzyme-targeted anti-tumor strategy may provide a new therapeutic approach for EBVaGCs.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Antivirais/farmacologia , Carcinoma/tratamento farmacológico , Desoxicitidina/análogos & derivados , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Ganciclovir/farmacologia , Herpesvirus Humano 4/efeitos dos fármacos , Terapia de Alvo Molecular , Neoplasias Gástricas/tratamento farmacológico , Animais , Carcinoma/diagnóstico , Carcinoma/genética , Carcinoma/virologia , Linhagem Celular Tumoral , Desoxicitidina/farmacologia , Relação Dose-Resposta a Droga , Reposicionamento de Medicamentos , Indução Enzimática , Infecções por Vírus Epstein-Barr/diagnóstico , Infecções por Vírus Epstein-Barr/virologia , Feminino , Herpesvirus Humano 4/enzimologia , Herpesvirus Humano 4/patogenicidade , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Quinases/biossíntese , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/genética , Neoplasias Gástricas/virologia , Timidina Quinase/biossíntese , Fatores de Tempo , Transfecção , Carga Tumoral/efeitos dos fármacos , Proteínas Virais/biossíntese , Ativação Viral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
We undertook this study to estimate an accurate incidence and spread patterns of occult papillary thyroid carcinoma (PTC) in patients with a preoperative diagnosis of solitary PTC by using whole-specimen mapping of all specimens after a total thyroidectomy. Enrolled prospectively in this whole-thyroid mapping study are 82 consecutive patients who underwent a total thyroidectomy under a preoperative diagnosis of solitary PTC. All thyroidectomy specimens were serially sectioned in 2âmm thickness and whole-thyroid mapping was carried out for additional foci of occult PTC. The frequencies of occult lesions detected in the whole and contralateral lobe were determined, and clinicopathologic factors associated with multifocality were assessed. Whole-thyroid mapping revealed 66 occult PTC lesions missed by preoperative ultrasound in 37 (45.1%) of the 82 patients. The great majority (92.5%) of the occult PTC was smaller than 3âmm in size and 25 patients (30.5%) had contralateral lesions. We found that the male sex was an independent predictor of multifocality (odds ratio (OR), 3.00; 95% CI, 1.11-8.14), adjusting for preoperative findings. Analysis with pathologic parameters showed that the male sex (OR, 5.03; 95% CI, 1.68-15.08) and extrathyroidal extensions (OR, 3.03; 95% CI, 1.03-8.95) were associated with multifocal PTC. However, none of the clinicopathologic factors evaluated predicted contralateral PTC. Our study demonstrates the diagnostic limitations of ultrasound for the detection of multifocal PTC and the need to consider the possibility of occult lesions in the management of solitary PTC, especially in male patients.
Assuntos
Carcinoma/diagnóstico por imagem , Carcinoma/patologia , Neoplasias da Glândula Tireoide/diagnóstico por imagem , Neoplasias da Glândula Tireoide/patologia , Adulto , Idoso , Carcinoma Papilar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Câncer Papilífero da Tireoide , Glândula Tireoide/diagnóstico por imagem , Glândula Tireoide/patologia , Tireoidectomia , Ultrassonografia , Adulto JovemRESUMO
Interferon regulatory factor-5 (IRF-5), a member of the mammalian IRF transcription factor family, is regulated by p53, type I interferon and virus infection. IRF-5 participates in virus-induced TLR-mediated innate immune responses and may play a role as a tumor suppressor. It was suppressed in various EBV-infected transformed cells, thus it is valuable to identify the suppression mechanism. We focused on a promoter CpG islands methylation, a kind of epigenetic regulation in EBV-associated Burkitt's lymphomas (BLs) and gastric carcinomas. IRF-5 is not detected in most of EBV-infected BL cell lines due to hypermethylation of IRF-5 distal promoter (promoter-A), which was restored by a demethylating agent, 5-aza-2'-deoxycytidine. Hypomethylation of CpG islands in promoter-A was observed only in EBV type III latent infected BL cell lines (LCL and Mutu III). Similarly, during EBV infection to Akata-4E3 cells, IRF-5 was observed at early time periods (2 days to 8 weeks), concomitant unmethylation of promoter-A, but suppressed in later infection periods as observed in latency I BL cell lines. Moreover, hypermethylation in IRF-5 promoter-A region was also observed in EBV-associated gastric carcinoma (EBVaGC) cell lines or primary gastric carcinoma tissues, which show type I latent infection. In summary, IRF-5 is suppressed by hypermethylation of its promoter-A in most of EBV-infected transformed cells, especially BLs and EBVaGC. EBV-induced carcinogenesis takes an advantage of proliferative effects of TLR signaling, while limiting IRF-5 mediated negative effects in the establishment of EBVaGCs.
Assuntos
Linfoma de Burkitt/genética , Metilação de DNA , Herpesvirus Humano 4/fisiologia , Fatores Reguladores de Interferon/genética , Regiões Promotoras Genéticas , Neoplasias Gástricas/genética , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular Tumoral , Ilhas de CpG , Decitabina , Epigênese Genética , Herpesvirus Humano 4/isolamento & purificação , Humanos , Análise de Sequência de DNA , Latência ViralRESUMO
Thioridazine, a member of the phenothiazine family, is a powerful anti-anxiety and anti-psychotic drug. It can also suppress the growth of several types of tumor in vitro. In the current study, we evaluated the direct anti-tumor and anti-angiogenic effects of thioridazine in vivo. The injection of thioridazine into human ovarian tumor xenografts in nude mice significantly inhibited tumor growth by ~fivefold, and also decreased tumor vascularity. In addition, thioridazine inhibited the phosphorylation of the signaling molecules downstream of phosphatidylinositol-3'-kinase (PI3K), including Akt, phosphoinositide-dependent protein kinase 1 (PDK1), and mammalian target of rapamycin (mTOR), during ovarian tumor progression via vascular endothelial growth factor receptor 2 (VEGFR-2). These results provide convincing evidence that thioridazine regulates endothelial cell function and subsequent angiogenesis by inhibiting VEGFR-2/PI3K/mTOR signal transduction. Collectively, these results strongly suggest that thioridazine might be a novel anti-tumor and anti-angiogenic agent for use in ovarian cancer.
Assuntos
Neoplasias Ovarianas/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Tioridazina/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Antipsicóticos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Immunoblotting , Imuno-Histoquímica , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização Patológica/prevenção & controle , Neoplasias Ovarianas/irrigação sanguínea , Neoplasias Ovarianas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacosRESUMO
Doxazosin is an α1 adrenergic receptor blocker that also exerts antitumor effects. However, the underlying mechanisms by which it modulates PI3K/Akt intracellular signaling are poorly understood. In this study, we reveal that doxazosin functions as a novel antiangiogenic agent by inhibiting vascular endothelial growth factor (VEGF)-induced cell migration and proliferation. It also inhibited VEGF-induced capillary-like structure tube formation in vitro. Doxazosin inhibited the phosphorylation of VEGF receptor-2 (VEGFR-2) and downstream signaling, including PI3K, Akt, 3-phosphoinositide-dependent protein kinase 1 (PDK1), mammalian target of rapamycin (mTOR), and hypoxia-inducible factor 1 (HIF-1α). However, it had no effect on VEGF-induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Furthermore, doxazosin reduced tumor growth and suppressed tumor vascularization in a xenograft human ovarian cancer model. These results provide evidence that doxazosin functions in the endothelial cell system to modulate angiogenesis by inhibiting Akt and mTOR phosphorylation and interacting with VEGFR-2.
Assuntos
Doxazossina/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Antagonistas de Receptores Adrenérgicos alfa 1/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Imuno-Histoquímica , Camundongos Nus , Neovascularização Patológica/metabolismo , Neovascularização Patológica/prevenção & controle , Neovascularização Fisiológica/efeitos dos fármacos , Neoplasias Ovarianas/irrigação sanguínea , Neoplasias Ovarianas/metabolismo , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Lysyl oxidase-like 2 (LOXL2) is a member of the lysyl oxidase gene family that contributes to the invasiveness and metastasis in tumor progression. However, the role of LOXL2 in cellular signaling is incompletely understood. In this study, we investigated a possible mechanism of LOXL2 function in tumor metastases in vitro, using a human breast carcinoma cell line. Myristoylated alanine-rich C kinase substrate-like 1 (MARCKSL1), a modulator in the regulation of cellular homeostasis, was identified as a LOXL2 interacting protein. We examined the binding domains that are required for the interaction between LOXL2 and MARCKSL1. The scavenger-receptor domain of LOXL2 was shown to interact with the N-terminal domain of MARCKSL1. Luciferase activity was noticeably reduced by the transfection of MARCKSL1 in a dose-dependent manner. In addition, over-expression of LOXL2 activates cell growth by inhibiting MARCKSL1-induced apoptosis. The effect of LOXL2 on cell cycle and apoptosis-related components was also confirmed through the silencing of LOXL2 expression. LOXL2 activates the FAK/Akt/mTOR signaling pathways, and MARCKSL1 suppresses LOXL2-induced oncogenesis. These insights supply evidence that LOXL2 promotes cell proliferation and inhibits apoptotic cell death. Taken together, our results indicate an underlying mechanism for an increase of LOXL2-related activity in breast tumor cells.
Assuntos
Aminoácido Oxirredutases/metabolismo , Proteínas de Membrana/metabolismo , Aminoácido Oxirredutases/antagonistas & inibidores , Aminoácido Oxirredutases/genética , Sequência de Aminoácidos , Apoptose , Proteínas de Ligação a Calmodulina , Linhagem Celular Tumoral , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas dos Microfilamentos , Dados de Sequência Molecular , NF-kappa B/metabolismo , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Human γ-aminobutyrate type A (GABAA) receptor-binding protein (GABARBP), a tumor suppressor protein with apoptotic function, can be inhibited in response to angiogenesis through the PI3K/Akt signaling cascades. Here, we investigated whether GABARBP over-expression could regulate vascular endothelial growth factor (VEGF)/hypoxia-inducible factor-1α (HIF-1α) expression and angiogenic activity in a carcinoma model system. GABARBP dramatically inhibited VEGF-induced endothelial cell proliferation, migration, and tube formation, as well as VEGFR-2 phosphorylation in vitro. At the same time, GABARBP exposed potent anti-angiogenic activity and remarkably down-regulated the levels of VEGF and HIF-1α protein expression, key components for angiogenesis. In addressing its biological molecular mechanism, GABARBP was found to effectively inhibit the phosphorylation of down-stream PI3K components, such as PDK1, Akt, mTOR, TSC-2, p70S6K, and 4E-BP1 by directly binding with VEGFR-2. In contrast, p38/JNK phosphorylation was not suppressed by GABARBP. These findings disclose a novel function of GABARBP in suppressing VEGF and HIF-1α protein expression, which is important for tumor angiogenesis and tumor growth. Thus, our data strongly provides novel biological mechanistic insights into the regulatory function of GABARBP in ovarian tumor progression, and the important of pre-clinical certification of GABARBP as a potential angiogenesis agent targeting ovarian tumorigenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Proteínas Associadas aos Microtúbulos/metabolismo , Neovascularização Patológica/metabolismo , Neoplasias Ovarianas/irrigação sanguínea , Neoplasias Ovarianas/patologia , Fosfatidilinositol 3-Quinase/metabolismo , Fosfoproteínas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Apoptose , Proteínas Reguladoras de Apoptose , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica/patologia , Regulação para Baixo , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neoplasias Ovarianas/metabolismo , Fosforilação , Ligação Proteica , Receptores de GABA-A/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
INTRODUCTION: 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) can reveal the metabolic activity of malignant tumors. Recent advances gained from molecular studies suggest that tumor biology can be a good predictor of prognosis in breast cancer. We compared the ability of maximum standardized uptake values (SUVmax) derived by FDG-PET with tumor burden in predicting tumor recurrence for patients with breast cancer. METHODS: 496 patients with breast cancer who underwent preoperative FDG-PET between April 2004 and May 2009 were retrospectively identified. SUVmax was obtained by FDG-PET, and the cutoff point was defined using a time-dependent receiver operating characteristic curve for recurrence-free survival (RFS). The primary endpoint was RFS. RESULTS: In multivariate analysis for RFS, SUVmax carried independent prognostic significance (hazard ratio, 2.39; 95% confidence interval, 1.20 to 4.76; P = 0.012). When the patients were classified into four groups according to the combined factors of tumor size (≤2 cm versus >2 cm) and SUVmax (<4 versus ≥4), RFS differed significantly (P < 0.001). Similarly, SUVmax had prognostic value in combination with nodal status (negative versus positive) or stage (I versus II and III) (P < 0.001 and P = 0.001, respectively). In hormone receptor-positive disease, SUVmax remained a significant prognostic factor for RFS based on multivariate analysis. CONCLUSIONS: Our results highlight the prognostic value of FDG-PET in prediction of tumor relapse for patients with breast cancer. Particularly in patients with hormone receptor-positive disease, the tumor metabolic information provided by FDG-PET is more significantly correlated with prognosis than tumor burden.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Carcinoma Ductal de Mama/diagnóstico por imagem , Carcinoma Lobular/diagnóstico por imagem , Recidiva Local de Neoplasia/diagnóstico por imagem , Carga Tumoral , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Carcinoma/diagnóstico por imagem , Carcinoma/patologia , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/patologia , Estudos de Coortes , Técnicas de Apoio para a Decisão , Feminino , Fluordesoxiglucose F18 , Humanos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Tomografia por Emissão de Pósitrons , Prognóstico , Compostos Radiofarmacêuticos , Estudos RetrospectivosRESUMO
Lysyl oxidase-like 2 (LOXL2) is associated with invasiveness and metastasis in breast cancer. We analyzed the prognostic impact of LOXL2 for breast cancer patients and investigated the role of LOXL2 in breast cancer cell lines. Immunohistochemical study of LOXL2 expression was done in samples from 309 patients. Survival analysis was performed using log-rank test and Cox regression hazard model. After identification of LOXL2 expression in breast cancer cell lines, we performed matrigel invasion and wound-healing assays with LOXL2-silenced cell lines. In the human study, LOXL2 was expressed in 16.2 % of patients. Comparing the LOXL2-positive versus negative groups, there was a significantly higher proportion of estrogen receptor-negative patients (54.0 vs. 37.0 %, respectively; p = 0.029) and triple-negative patients (34.0 vs. 18.0 %; p = 0.022) in the positive group. In multivariate analysis for overall survival and metastasis-free survival, positive LOXL2 was demonstrated as a poor prognostic factor (HR 2.27 and 2.10, respectively). In vitro study indicated that LOXL2 silencing induces a mesenchymal-epithelial transition-like process in basal cell lines (MDA-MB-231 and BT549) associated with decreased invasive and migratory properties. These clinical and preclinical data confirm that higher LOXL2 expression is associated with invasiveness of basal-like breast cancer cells and lower survival of breast cancer patients. Our results suggest the clinical value of LOXL2 as a therapeutic target in breast cancer.
Assuntos
Aminoácido Oxirredutases/análise , Neoplasias da Mama/química , Carcinoma/química , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/análise , Adulto , Aminoácido Oxirredutases/biossíntese , Aminoácido Oxirredutases/genética , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Carcinoma/genética , Carcinoma/mortalidade , Carcinoma/patologia , Carcinoma in Situ/química , Carcinoma in Situ/genética , Carcinoma in Situ/mortalidade , Carcinoma in Situ/patologia , Linhagem Celular Tumoral , Movimento Celular , Colágeno , Intervalo Livre de Doença , Combinação de Medicamentos , Transição Epitelial-Mesenquimal , Feminino , Humanos , Hibridização In Situ , Estimativa de Kaplan-Meier , Laminina , Pessoa de Meia-Idade , Terapia Neoadjuvante , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias Primárias Múltiplas/química , Neoplasias Primárias Múltiplas/genética , Neoplasias Primárias Múltiplas/mortalidade , Neoplasias Primárias Múltiplas/patologia , Tumor Filoide/química , Tumor Filoide/genética , Tumor Filoide/mortalidade , Tumor Filoide/patologia , Prognóstico , Modelos de Riscos Proporcionais , Proteoglicanas , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Análise de Sobrevida , Análise Serial de Tecidos , Neoplasias de Mama Triplo Negativas/química , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/mortalidade , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
In this current work, we investigated whether BLU could enhance pro-apoptotic activity of chemotherapeutic drugs in ovarian carcinoma cells. A combination with a chemotherapeutic drug showed an additive effect, and this additive effect was supplemented by the enhancement of caspase-3 and -9 activities. BLU and paclitaxel induced cell cycle arrest in the G2/M phase through the reduction of cyclin dependent kinase 1, cyclin B1, while promoting both p16 and p27 expression. In addition, both BLU and paclitaxel enhanced the expression of the pro-apoptotic protein Bax together with the suppression of anti-apoptotic protein Bcl-2, a protein which is well-known for its function as a regulator in protecting cells from apoptosis. As expected, the Bax and p21 activities were enhanced by BLU or paclitaxel, while a combination of BLU and paclitaxel were additively promoted, whereas Bcl-xL and NF-κB including Bcl-2 activity were inactivated. This study has yielded promising results, which evidence for the first time that BLU could suppress the growth of carcinoma cells. Furthermore, both BLU and paclitaxel inhibited the phosphorylation of signaling components downstream of phosphoinositide 3-kinase, such as 3-phosphoinositide-dependent protein kinase 1, and Akt. Also, BLU plus paclitaxel decreased phosphorylation of p70 ribosomal S6 kinase, as well as decreasing the phosphorylation of glycogen synthase kinase-3ß, which is one of the representative targets of the mammalian target of rapamycin signaling cascade. These results provide evidence that BLU enhances G2/M cell cycle arrest and apoptotic cell death through the up-regulation of Bax, p21 and p53 expression.
Assuntos
Apoptose/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Paclitaxel/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Transformação Celular Neoplásica/genética , Proteínas do Citoesqueleto , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Immunoblotting , Luciferases/genética , Luciferases/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transfecção , Proteínas Supressoras de Tumor/genética , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Thioridazine is a type of anti-psychotic drug that also includes anti-tumor activity. In this study, we assessed the effects of thioridazine, as a novel anti-angiogenic agent, on the suppression of angiogenesis-mediated cell proliferation. Thioridazine was found to inhibit growth in ovarian cancer cells (OVCAR-3 and 2774), but did not possess any inhibitory effects on normal cell types such as HOSE-E6E7, MCF-10A, MRC-5, and BEAS-2B. Thioridazine also suppressed vascular endothelial growth factor (VEGF)-stimulated HUVEC migration in a dose-time-dependent manner. We also showed that being treated with thioridazine inhibited VEGF-stimulated proliferation, invasion, and capillary-like structure tube formation in vitro. Thioridazine suppressed phosphorylation of the signaling regulators downstream of the focal adhesion kinase (FAK) through αvß3 integrin, which also include Akt, phosphoinositide-dependent protein kinase 1 (PDK-1), mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6K), but had no effect on VEGF-stimulated extracellular signal-regulated kinase (ERK) phosphorylation. We found the molecular mechanism of thioridazine to be a novel anti-angiogenic protein. These results provide evidence for the regulation of endothelial cell functions that are relevant to angiogenesis through the suppression of the αvß3/FAK/mTOR signaling pathway.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica , Serina-Treonina Quinases TOR/metabolismo , Tioridazina/farmacologia , Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Integrina alfaVbeta3/metabolismo , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Transdução de SinaisRESUMO
Recently, thioridazine (10-[2-(1-methyl-2-piperidyl) ethyl]-2-methylthiophenothiazine), a well-known anti-psychotic agent was found to have anti-cancer activity in cancer cells. However, the molecular mechanism of the agent in cellular signal pathways has not been well defined. Thioridazine significantly increased early- and late-stage apoptotic fraction in cervical and endometrial cancer cells, suggesting that suppression of cell growth by thioridazine was due to the induction of apoptosis. Cell cycle analysis indicated thioridazine induced the down-regulation of cyclin D1, cyclin A and CDK4, and the induction of p21 and p27, a cyclin-dependent kinase inhibitor. Additionally, we compared the influence of thioridazine with cisplatin used as a control, and similar patterns between the two drugs were observed in cervical and endometrial cancer cell lines. Furthermore, as expected, thioridazine successfully inhibited phosphorylation of Akt, phosphorylation of 4E-BP1 and phosphorylation of p70S6K, which is one of the best characterized targets of the mTOR complex cascade. These results suggest that thioridazine effectively suppresses tumor growth activity by targeting the PI3K/Akt/mTOR/p70S6K signaling pathway.