RESUMO
Leucine-rich repeat-containing G-protein-coupled receptor (LGR5) and LGR6 mark epithelial stem cells in normal tissues and tumors. They are expressed by stem cells in the ovarian surface and fallopian tube epithelia from which ovarian cancer arises. High-grade serous ovarian cancer is unique in expressing unusually high levels of LGR5 and LGR6 mRNA. R-spondins are the natural ligands for LGR5 and LGR6 to which they bind with nanomolar affinity. To target stem cells in ovarian cancer, we used the sortase reaction to site-specifically conjugate the potent cytotoxin monomethyl auristatin E (MMAE) via a protease sensitive linker to the two furin-like domains of RSPO1 (Fu1-Fu2) that mediate its binding to LGR5 and LGR6 and their co-receptors Zinc And Ring Finger 3 and Ring Finger Protein 43 via a protease-cleavable linker. An immunoglobulin Fc domain added to the N-terminal end served to dimerize the receptor-binding domains so that each molecule carries two MMAE. The resulting molecule, FcF2-MMAE, demonstrated: 1) selective LGR5-dependent low nanomolar cytotoxicity against ovarian cancer cells in vitro; 2) selectivity that was dependent on binding to both the LGR receptors and ubiquitin ligase co-receptors; 3) favorable stability and plasma pharmacokinetic properties when administered intravenously with an elimination half-life of 29.7 hours; 4) selective inhibition of LGR5-rich as opposed to isogenic LGR5-poor tumors in vivo; and, 5) therapeutic efficacy in three aggressive wild-type human ovarian cancer xenograft models. These results demonstrate the successful use of the Fu1-Fu2 domain of RSPO1 as a drug carrier and the ability of FcF2-MMAE to target cells in tumors that express stem cell markers. SIGNIFICANCE STATEMENT: FcF2-MMAE is a novel cancer therapeutic that exploits the high-affinity binding domains of RSPO1 to target monomethyl auristatin E to tumor stem cells that express LGR5. FcF2-MMAE has low nanomolar LGR5-dependent cytotoxicity in vitro, favorable pharmacokinetics, and differential efficacy in an isogenic LGR5-poor versus LGR5-rich ovarian cancer xenograft model when given on a weekly schedule.
Assuntos
Neoplasias Ovarianas , Receptores Acoplados a Proteínas G , Feminino , Humanos , Leucina , Neoplasias Ovarianas/tratamento farmacológico , Peptídeo Hidrolases , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/metabolismo , Trombospondinas/metabolismoRESUMO
LGR5 and LGR6 mark epithelial stem cells in many niches including the ovarian surface and fallopian tube epithelia from which ovarian cancer arises. Human ovarian cancers express these receptors at high levels and express one of their ligands, RSPO1, at levels uniquely higher than all other tumor types except mesothelioma. Reasoning that these receptors are also important to tumor stem cells, arming the LGR binding domain of RSPO1 with a cytotoxin may permit depletion of the tumor stem cells. The Fu1-Fu2 receptor binding domain of RSPO1 (R1FF), containing a sortase recognition sequence at the C-terminal end, was produced in bacteria and a single molecule of MMAE was attached to each R1FF through a val-cit-PAB linker using the sortase reaction, thus producing a homogeneous population of armed molecules. R1FF-MMAE demonstrated (1) selective LGR-dependent binding, uptake, and cytotoxicity; (2) low nM cytotoxicity to multiple types of human tumor cell lines in vitro; (3) favorable plasma pharmacokinetic properties when administered iv with an elimination half-life of 27.8 h; (4) favorable absorption from the peritoneal cavity; and (5) therapeutic activity in aggressive xenograft models of ovarian cancer in the absence of any weight loss or other adverse events. These results demonstrate that the Fu1-Fu2 domain of RSPO1 can be exploited to deliver a potent cytotoxin to tumor cells that express the LGR4-6 family of stem cell receptors.
Assuntos
Receptores de Superfície Celular/metabolismo , Células-Tronco/metabolismo , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Meia-Vida , Humanos , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Cobre/metabolismo , Integrina alfaV/metabolismo , Antineoplásicos/uso terapêutico , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Cisplatino/uso terapêutico , Transportador de Cobre 1 , Resistencia a Medicamentos Antineoplásicos , Humanos , Neoplasias/tratamento farmacológico , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: The EphA2 receptor, which is expressed in many types of cancer, is activated by two different mechanisms. Activation by engagement with one of its ephrin ligands is anti-oncogenic whereas phosphorylation of S897 by AKT increases migration, invasion and metastasis. Down-regulation of claudin-4 (CLDN4) produces a loss of E-cadherin and increased ß-catenin signaling and a phenotype similar to that produced by oncogenic activation of EphA2, suggesting that CLDN4 may serve to restrain the pro-oncogenic signaling of EphA2. RESULTS: We found that constitutive knockdown of CLDN4 was associated with a 4.5-fold increase in EphA2 mRNA and a 2.5-fold increase in EphA2 protein which was reversible by re-expression of CLDN4. Knockdown of EphA2 blocked the migratory phenotype induced by loss of CLDN4. Knockdown of CLDN4 resulted in a 5.8-fold increase in pEphA(S897), the oncogenic form of the receptor, as well as partial mislocalization of the excess EphA2 to the interior of the cell. Forced expression of E-cadherin did not reduce total EphA2 or pEphA(S897) whereas re-expression of CLDN4 restored localization and reduced EphA2 and pEphA(S897) even in cells not expressing E-cadherin. Transient siRNA-mediated knockdown of EphA2 and ß-catenin, and inhibition of PI3K by LY294002, demonstrated that increased pEphA(S897) in the CLDN4 knockdown cells was attributable to an increase in the level of active dephospho-ß-catenin upstream of PI3K and AKT. CONCLUSIONS: We conclude that CLDN4 serves to restrain pro-oncogenic signaling from EphA2 by limiting the activity of ß-catenin and PI3K and preventing phosphorylation of EphA2 on S897 by AKT. This suggests that interventions directed at enhancing the level or functional activity of CLDN4 may be of therapeutic interest.
Assuntos
Claudina-4/metabolismo , Receptor EphA2/metabolismo , beta Catenina/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Claudina-4/genética , Técnicas de Silenciamento de Genes , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Receptor EphA2/genética , CicatrizaçãoRESUMO
BACKGROUND: Injury due to myocardial infarction (MI) is largely irreversible. Once an infarct has occurred, the clinical goal becomes limiting remodeling, preserving left ventricular function, and preventing heart failure. Although traditional approaches (e.g., ß-blockers) partially preserve left ventricular function, novel strategies are needed to limit ventricular remodeling post-MI. OBJECTIVES: The aim of this study was to determine the role of glycogen synthase kinase-3α (GSK-3α) in post-MI remodeling. METHODS: Mice with cardiomyocyte-specific conditional deletion of Gsk3α and littermate controls underwent sham or MI surgery. Heart function was assessed using serial M-mode echocardiography. RESULTS: Gsk3α deletion in the heart markedly limits remodeling and preserves left ventricular function post-MI. This is due at least in part to dramatic thinning and expansion of the scar in the control hearts, which was less in the heart of knockout (KO) mice. In contrast, the border zone in the KO mice demonstrated a much thicker scar, and there were more viable cardiomyocytes within the scar/border zone. This was associated with less apoptosis and more proliferation of cardiomyocytes in the KO mice. Mechanistically, reduced apoptosis was due, at least in part, to a marked decrease in the Bax/Bcl-2 ratio, and increased cardiomyocyte proliferation was mediated through cyclin E1 and E2F-1 in the hearts of the KO mice. CONCLUSIONS: Taken together, these findings show that reducing GSK-3α expression in cardiomyocytes limits ventricular remodeling and preserves cardiac function post-MI. Specifically targeting GSK-3α could be a novel strategy to limit adverse remodeling and heart failure.
Assuntos
Deleção de Genes , Quinase 3 da Glicogênio Sintase/genética , Insuficiência Cardíaca/genética , Infarto do Miocárdio/complicações , Miócitos Cardíacos/metabolismo , Disfunção Ventricular Esquerda/genética , Remodelação Ventricular/genética , Animais , Apoptose , Proliferação de Células , DNA/genética , Modelos Animais de Doenças , Quinase 3 da Glicogênio Sintase/metabolismo , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/fisiopatologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Miocárdica , Infarto do Miocárdio/genética , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/patologia , Disfunção Ventricular Esquerda/etiologia , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
The αV integrin is expressed in most cancer cells where it regulates a diverse array of cellular functions essential to the initiation, progression and metastasis of solid tumors. However, little is known about how αV integrin modulates cellular sensitivity to chemotherapeutic agents, particularly the platinum drugs. In this study, we found that down-regulation of αV sensitized human M21 cells to cisplatin (cDDP) through up-regulation of the copper influx transporter CTR1. Cells selected for low αV integrin expression (M21L) were more sensitive to cDDP, accompanied by increase in CTR1 mRNA and CTR1 protein levels, more intracellular cDDP accumulation and cDDP DNA adduct formation. Basal copper (Cu) content, Cu uptake, and Cu cytotoxicity were also increased. Transfection of a luciferase reporter construct containing the hCTR1 promoter sequence revealed an increase of the hCTR1 transcription activity in M21L cells. The basis for the increased hCTR1 transcription was related to an increase in the steady-state level of Sp1, a transcription factor known to drive hCTR1 expression. These results indicate that the αV integrin modulates sensitivity of human cells to the cytotoxic effect of cDDP by regulating expression of the Cu transporter CTR1, and introduce the concept that αV expression is linked to Cu homeostasis.
RESUMO
Studies from our lab have shown that decreasing myocardial G protein-coupled receptor kinase 2 (GRK2) activity and expression can prevent heart failure progression after myocardial infarction. Since GRK2 appears to also act as a pro-death kinase in myocytes, we investigated the effect of cardiomyocyte-specific GRK2 ablation on the acute response to cardiac ischemia/reperfusion (I/R) injury. To do this we utilized two independent lines of GRK2 knockout (KO) mice where the GRK2 gene was deleted in only cardiomyocytes either constitutively at birth or in an inducible manner that occurred in adult mice prior to I/R. These GRK2 KO mice and appropriate control mice were subjected to a sham procedure or 30 min of myocardial ischemia via coronary artery ligation followed by 24 hrs reperfusion. Echocardiography and hemodynamic measurements showed significantly improved post-I/R cardiac function in both GRK2 KO lines, which correlated with smaller infarct sizes in GRK2 KO mice compared to controls. Moreover, there was significantly less TUNEL positive myocytes, less caspase-3, and -9 but not caspase-8 activities in GRK2 KO mice compared to control mice after I/R injury. Of note, we found that lowering cardiac GRK2 expression was associated with significantly lower cytosolic cytochrome C levels in both lines of GRK2 KO mice after I/R compared to corresponding control animals. Mechanistically, the anti-apoptotic effects of lowering GRK2 expression were accompanied by increased levels of Bcl-2, Bcl-xl, and increased activation of Akt after I/R injury. These findings were reproduced in vitro in cultured cardiomyocytes and GRK2 mRNA silencing. Therefore, lowering GRK2 expression in cardiomyocytes limits I/R-induced injury and improves post-ischemia recovery by decreasing myocyte apoptosis at least partially via Akt/Bcl-2 mediated mitochondrial protection and implicates mitochondrial-dependent actions, solidifying GRK2 as a pro-death kinase in the heart.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Miocárdio/enzimologia , Animais , Apoptose , Citocromos c/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Quinase 2 de Receptor Acoplado a Proteína G/genética , Hemodinâmica/efeitos dos fármacos , Isoproterenol/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , Regulação para Cima , Proteína bcl-X/metabolismoRESUMO
The mechanisms that control intracellular adhesion are central to the process of invasion and metastasis. Claudin-3 (CLDN3) and claudin-4 (CLDN4) are major structural molecules of the tight junctions that link epithelial cells. Our prior work has demonstrated that knockdown of the expression of either CLDN3 or CLDN4 produces marked changes in the phenotype of ovarian carcinoma cells including increases in growth rate in vivo, migration, invasion, metastasis, and drug resistance, similar to those produced by the epithelial-to-mesenchymal transition (EMT). We postulated that these changes may result from the ability of CLDN3 or CLDN4 to suppress EMT. In this study we found that knockdown of either CLDN3 or CLDN4 increased cell size and resulted in flattened morphology. While knockdown of CLDN3 or CLDN4 did not alter the expression of vimentin, it significantly down-regulated the level of E-cadherin and up-regulated N-cadherin expression. Conversely, over-expression of CLDN3 or CLDN4 in a cell line that does not express endogenous CLDN3 or CLDN4 decreased N-cadherin expression. Re-expression of E-cadherin in the CLDN3 or CLDN4 knockdown cells reduced migration, invasion and tumor growth in vivo. Loss of either CLDN3 or CLDN4 resulted in activation of the PI3K pathway as evidenced by increased Akt phosphorylation, elevated cellular PIP3 content and PI3K activity as well as up-regulation of the mRNA and protein levels of the transcription factor Twist. Taken together, these findings suggest that CLDN3 and CLDN4 function to sustain an epithelial phenotype and that their loss promotes EMT.
Assuntos
Claudina-3/metabolismo , Claudina-4/metabolismo , Animais , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Claudina-3/antagonistas & inibidores , Claudina-3/genética , Claudina-4/antagonistas & inibidores , Claudina-4/genética , Transição Epitelial-Mesenquimal , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , Transdução de Sinais , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Regulação para CimaRESUMO
BACKGROUND: Vitamin D plays a role in cancer development and acts through the vitamin D receptor (VDR). Although African-Americans have the lowest levels of serum vitamin D, there is a dearth of information on VDR gene polymorphisms and breast cancer among African-Americans and Hispanics. This study examines whether VDR gene polymorphisms are associated with breast cancer in these cohorts. METHODS: Blood was collected from 232 breast cancer patients (Cases) and 349 non-cancer subjects (Controls). Genotyping for four polymorphic variants of VDR (FokI, BsmI, TaqI and ApaI) was performed using the PCR-RFLP method. RESULTS: An increased association of the VDR-Fok1 f allele with breast cancer was observed in African-Americans (OR = 1.9, p = 0.07). Furthermore, the FbTA, FbtA and fbtA haplotypes were associated with breast cancer among African-Americans (p<0.05). Latinas were more likely to have the VDR-ApaI alleles (Aa or aa) (p = 0.008). The VDR-ApaI aa genotype was significantly associated with poorly-differentiated breast tumors (p = 0.04) in combined Cases. Kaplan-Meier survival analysis showed decreased 5-year disease-free-survival (DFS) in breast cancer patients who had the VDR-Fok1 FF genotype (p<0.05). The Cox regression with multivariate analysis revealed the independent predictor value of the VDR-FokI polymorphism for DFS. The other three variants of VDR (BsmI, TaqI and ApaI) were not associated with disease outcome. CONCLUSIONS: VDR haplotypes are associated with breast cancer in African-Americans, but not in Hispanic/Latinas. The VDR-FokI FF genotype is linked with poor prognosis in African-American women with breast cancer.
Assuntos
Alelos , Negro ou Afro-Americano , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Haplótipos , Hispânico ou Latino , Polimorfismo de Fragmento de Restrição , Receptores de Calcitriol/genética , Adulto , Neoplasias da Mama/sangue , Neoplasias da Mama/etnologia , Neoplasias da Mama/terapia , Estudos de Casos e Controles , Intervalo Livre de Doença , Feminino , Humanos , Pessoa de Meia-Idade , Receptores de Calcitriol/metabolismo , Taxa de Sobrevida , Vitamina D/sangueRESUMO
Claudin-3 (CLDN3) and claudin-4 (CLDN4) are the major structural molecules that form tight junctions (TJs) between epithelial cells. We found that knockdown of the expression of either CLDN3 or CLDN4 produced marked changes in the phenotype of ovarian cancer cells, including an increase in resistance to cisplatin (cDDP). The effect of CLND3 and CLDN4 on cDDP cytotoxicity, cDDP cellular accumulation, and DNA adduct formation was compared in the CLDN3- and CLDN4-expressing parental human ovarian carcinoma 2008 cells and CLDN3 and CLDN4 knockdown sublines (CLDN3KD and CLDN4KD, respectively). Knockdown of CLDN3 or CLDN4 rendered human ovarian carcinoma 2008 cells resistant to cDDP in both in vitro culture and in vivo xenograft model. The net accumulation of platinum (Pt) and the Pt-DNA adduct levels were reduced in CLDN3KD and CLDN4KD cells. The endogenous mRNA levels of copper influx transporter CTR1 were found to be significantly reduced in the knockdown cells, and exogenous expression of CTR1 restored their sensitivity to cDDP. Reexpression of an shRNAi-resistant CLDN3 or CLDN4 up-regulated CTR1 levels, reversed the cDDP resistance, and enhanced TJ formation in the knockdown cells. Baseline copper (Cu) level, Cu uptake, and Cu cytotoxicity were also reduced in CLDN3KD and CLDN4KD cells. Cu-dependent tyrosinase activity was also markedly reduced in both types of CLDN knockdown cells when incubated with the substrate l-DOPA. These results indicate that CLDN3 and CLDN4 affect sensitivity of the ovarian cancer cells to the cytotoxic effect of cDDP by regulating expression of the Cu transporter CTR1.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Transporte de Cátions/metabolismo , Cisplatino/farmacologia , Claudina-3/metabolismo , Claudina-4/metabolismo , Animais , Proteínas de Transporte de Cátions/genética , Linhagem Celular Tumoral , Claudina-3/genética , Claudina-4/genética , Cobre/metabolismo , Transportador de Cobre 1 , Resistencia a Medicamentos Antineoplásicos , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Monofenol Mono-Oxigenase/metabolismo , Transplante de Neoplasias , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , RNA Mensageiro/metabolismo , Junções Íntimas/fisiologia , Transplante HeterólogoRESUMO
The extent of tight junction (TJ) formation is one of many factors that regulate motility, invasion, and metastasis. Claudins are required for the formation and maintenance of TJs. Claudin-3 (CLDN3) and claudin-4 (CLDN4) are highly expressed in the majority of ovarian cancers. We report here that CLDN3 and CLDN4 each serve to constrain the growth of human 2008 cancer xenografts and limit metastatic potential. Knockdown of CLDN3 increased in vivo growth rate by 2.3-fold and knockdown of CLDN4 by 3.7-fold in the absence of significant change in in vitro growth rate. Both types of tumors exhibited increase in birth rate as measured by Ki67 staining and decrease in death rate as reflected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Knockdown of either claudin did not alter expression of other TJ protein but did reduce TJ formation as measured by transepithelial resistance and paracellular flux of dextran, enhance migration and invasion in in vitro assays, and increase lung colonization following intravenous injection. Knockdown of CLDN3 and CLDN4 increased total lung metastatic burden by 1.7-fold and 2.4-fold, respectively. Loss of either CLDN3 or CLDN4 resulted in down-regulation of E-cadherin mRNA and protein, increased inhibitory phosphorylation of glycogen synthase kinase-3ß (GSK-3ß), and activation of ß-catenin pathway signaling as evidenced by increases in nuclear ß-catenin, the dephosphorylated form of the protein, and transcriptional activity of ß-catenin/T-cell factor (TCF). We conclude that both CLDN3 and CLDN4 mediate interactions with other cells in vivo that restrain growth and metastatic potential by sustaining expression of E-cadherin and limiting ß-catenin signaling.
Assuntos
Claudina-3/metabolismo , Claudina-4/metabolismo , Neoplasias das Tubas Uterinas/patologia , Neoplasias Pulmonares/secundário , Neoplasias Ovarianas/patologia , Neoplasias do Colo do Útero/patologia , Apoptose , Western Blotting , Caderinas/genética , Caderinas/metabolismo , Adesão Celular , Movimento Celular , Proliferação de Células , Claudina-3/antagonistas & inibidores , Claudina-3/genética , Claudina-4/antagonistas & inibidores , Claudina-4/genética , Impedância Elétrica , Neoplasias das Tubas Uterinas/genética , Neoplasias das Tubas Uterinas/metabolismo , Feminino , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Junções Íntimas , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Cicatrização , beta Catenina/genética , beta Catenina/metabolismoRESUMO
BACKGROUND: Obesity and diabetes mellitus adversely affect postischemic heart remodeling via incompletely understood mechanisms. C1q/tumor necrosis factor-related protein-3 (CTRP3) is a newly identified adipokine exerting beneficial metabolic regulation, similar to adiponectin. The aim of the present study was to determine whether CTRP3 may regulate postischemic cardiac remodeling and cardiac dysfunction, and, if so, to elucidate the underlying mechanisms. METHODS AND RESULTS: Male adult mice were subjected to myocardial infarction (MI) via left anterior descending coronary artery occlusion. Both the effect of MI on endogenous CTRP3 expression/production and the effect of exogenous CTRP3 (adenovirus or recombinant CTRP3) replenishment on MI injury were investigated. MI significantly inhibited adipocyte CTRP3 expression and reduced the plasma CTRP3 level, reaching a nadir 3 days after MI. CTRP3 replenishment improved survival rate (P<0.05), restored cardiac function, attenuated cardiomyocyte apoptosis, increased revascularization, and dramatically reduced interstitial fibrosis (all P<0.01). CTRP3 replenishment had no significant effect on cardiac AMP-activated protein kinase phosphorylation but significantly increased Akt phosphorylation and expression of hypoxia inducing factor-1α and vascular endothelial growth factor. Surprisingly, treatment of human umbilical vascular endothelial cells with CTRP3 did not directly affect nitric oxide production or tube formation. However, preconditioned medium from CTRP3-treated cardiomyocytes significantly enhanced human umbilical vascular endothelial cell tube formation, an effect blocked by either pretreatment of cardiomyocytes with a PI3K inhibitor or pretreatment of human umbilical vascular endothelial cells with a vascular endothelial growth factor inhibitor. Finally, the protective effect of adipocyte-conditioned medium against hypoxia-induced cardiomyocyte injury is significantly blunted when CTRP3 is knocked down. CONCLUSION: CTRP3 is a novel antiapoptotic, proangiogenic, and cardioprotective adipokine, the expression of which is significantly inhibited after MI.
Assuntos
Adipocinas/fisiologia , Indutores da Angiogênese/uso terapêutico , Proteínas Reguladoras de Apoptose/fisiologia , Cardiotônicos/uso terapêutico , Isquemia Miocárdica/prevenção & controle , Adipocinas/antagonistas & inibidores , Adipocinas/uso terapêutico , Indutores da Angiogênese/metabolismo , Animais , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/uso terapêutico , Cardiotônicos/metabolismo , Técnicas de Silenciamento de Genes/métodos , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Distribuição AleatóriaRESUMO
It is generally accepted that the intercalated disc (ICD) required for mechano-electrical coupling in the heart consists of three distinct junctional complexes: adherens junctions, desmosomes and gap junctions. However, recent morphological and molecular data indicate a mixing of adherens junctional and desmosomal components, resulting in a 'hybrid adhering junction' or 'area composita'. The α-catenin family member αT-catenin, part of the N-cadherin-catenin adhesion complex in the heart, is the only α-catenin that interacts with the desmosomal protein plakophilin-2 (PKP2). Thus, it has been postulated that αT-catenin might serve as a molecular integrator of the two adhesion complexes in the area composita. To investigate the role of αT-catenin in the heart, gene targeting technology was used to delete the Ctnna3 gene, encoding αT-catenin, in the mouse. The αT-catenin-null mice are viable and fertile; however, the animals exhibit progressive cardiomyopathy. Adherens junctional and desmosomal proteins were unaffected by loss of αT-catenin, with the exception of the desmosomal protein PKP2. Immunogold labeling at the ICD demonstrated in the αT-catenin-null heart a preferential reduction of PKP2 at the area composita compared with the desmosome. Furthermore, gap junction protein Cx43 was reduced at the ICD, including its colocalization with N-cadherin. Gap junction remodeling in αT-catenin-knockout hearts was associated with an increased incidence of ventricular arrhythmias after acute ischemia. This novel animal model demonstrates for the first time how perturbation in αT-catenin can affect both PKP2 and Cx43 and thereby highlights the importance of understanding the crosstalk between the junctional proteins of the ICD and its implications for arrhythmogenic cardiomyopathy.
Assuntos
Arritmias Cardíacas/fisiopatologia , Cardiomiopatia Dilatada/patologia , Junções Comunicantes/metabolismo , Ventrículos do Coração/fisiopatologia , Isquemia Miocárdica/complicações , Miócitos Cardíacos/metabolismo , alfa Catenina/deficiência , Junções Aderentes/metabolismo , Animais , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/patologia , Caderinas/metabolismo , Cardiomiopatia Dilatada/etiologia , Cardiomiopatia Dilatada/fisiopatologia , Conexina 43/deficiência , Conexina 43/metabolismo , Desmossomos/metabolismo , Modelos Animais de Doenças , Eletrocardiografia , Junções Comunicantes/patologia , Ventrículos do Coração/patologia , Camundongos , Camundongos Knockout , Mutação , Traumatismo por Reperfusão Miocárdica , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/patologia , Placofilinas/deficiência , Placofilinas/metabolismo , alfa Catenina/genética , alfa Catenina/metabolismoRESUMO
BACKGROUND: The T-box transcription factor TBX3 is necessary for early embryonic development and for the normal development of the mammary gland. Homozygous mutations, in mice, are embryonic lethal while heterozygous mutations result in perturbed mammary gland development. In humans, mutations that result in the haploinsufficiency of TBX3 causes Ulnar Mammary Syndrome (UMS) characterized by mammary gland hypoplasia as well as other congenital defects. In addition to its role in mammary gland development, various studies have also supported a role for Tbx3 in breast cancer development. TBX3 is over-expressed in various breast cancer cell lines as well as cancer tissue and has been found to contribute to breast cancer cell migration. Previous studies have suggested that TBX3 contributes to cancer development by its ability to bypass senescence by repressing the expression of p14(ARF)-tumor suppressor. Although many studies have shown that a dysregulation of TBX3 expression may contribute to cancer progression, no direct evidence shows TBX3 causes breast cancer. RESULTS: In this study, we created doxycycline inducible double transgenic mice (MMTV-rtTA;tet-myc-TBX3-IRES-Luciferase) to test whether TBX3 over-expression can induce tumor formation within the mammary gland. Although over-expression of TBX3, alone, did not induce tumor formation it did promote accelerated mammary gland development by increasing mammary epithelial cell proliferation. We also show that TBX3 directly binds to and represses NFκBIB, an inhibitor of the NF-κB pathway known to play a role in regulating cell proliferation. Lastly, we also show that the over-expression of TBX3 is associated with an increase in mammary stem-like cells. CONCLUSIONS: Overall, our data suggests that over-expression of TBX3 may contribute to breast cancer development by promoting accelerated mammary gland development through the inhibition of the NF-κB pathway and stimulation of both mammary epithelial cell and stem-like cell proliferation.
Assuntos
Regulação da Expressão Gênica , Glândulas Mamárias Animais/patologia , Células-Tronco/metabolismo , Proteínas com Domínio T/genética , Animais , Feminino , Hiperplasia , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Animais , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas com Domínio T/metabolismoRESUMO
Previous studies from our group and others have shown that increased circulatory levels of the ligand insulin-like growth factor 1 (IGF-1) and decreased levels of the predominant IGF-1 binding protein 3 (IGFBP-3) are associated with an increased incidence of breast cancer and poor outcome. Some studies suggest that, in addition to the influence of environmental factors on the levels of IGF-1 and IGFBP-3, alterations in their gene polymorphisms may play a significant role in the risk of cancer. In this study, we investigated the association between gene polymorphisms along the IGF axis and breast cancer, including the IGF-1 (CA) dinucleotide repeat, IGFBP-3 A-202C single nucleotide polymorphism, and the 2-bp deletion and (AGG)n repeat polymorphisms in the IGF type 1 receptor (IGF-IR). A total of 654 subjects, including both African-American and Hispanic/Latino subjects, were screened for various gene polymorphisms. IGF gene polymorphism genotyping was performed by PCR-GeneScan and PCR-RFLP methods. Our results demonstrated a significant association between the non-19/non-19 IGF-1 (CA)n polymorphism and breast cancer (OR = 1.75; 95% CI = 1.07-2.88; P = 0.027). Furthermore, absence of the wild-type-19 allele and alleles <(CA)19 were strongly associated with breast cancer (OR = 1.82; 95% CI = 1.20-2.77; P = 0.005 and OR = 1.70; 95% CI = 1.19-2.43; P = 0.003, respectively). The association of the non-19/non-19 polymorphism with breast cancer was also more significant in premenopausal women (P = 0.04). We did not find any significant association of the IGFBP-3 polymorphism with breast cancer. In the case of IGF-1R polymorphisms, the only significant trend was in the (AGG)5 allele; however, the frequency of this allele was very rare. In summary, our study demonstrated a significant association of IGF-1 polymorphisms and breast cancer. Future studies are necessary to understand the mechanistic value of these polymorphisms in breast cancer risk.
Assuntos
Negro ou Afro-Americano/genética , Neoplasias da Mama/genética , Hispânico ou Latino/genética , Fator de Crescimento Insulin-Like I/genética , Polimorfismo Genético , Adulto , Alelos , Feminino , Genótipo , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Pessoa de Meia-Idade , Receptor IGF Tipo 1/genética , Estados Unidos/etnologiaRESUMO
PURPOSE: Luteinizing hormone receptor (LHR) is found in abundance on human ovarian, breast, endometrial and prostate carcinomas but at only low levels on non-gonadal tissues. To selectively kill LHR-expressing tumors, granzyme B (GrB) was linked to a protein in which both chains of human chorionic gonadotropin were yoked together (YCG). METHODS: GrB-YCG was expressed and secreted from insect Sf9 cells. Its GrB enzymatic activity and binding affinity for hLHR were then characterized. The differential cytotoxicity of GrB-YCG versus GrB alone was tested in a panel of LHR-expressing tumor cells by SRB assay, and the mechanisms involved in the cell death were investigated by confocal fluorescence microscopy, flow cytometry, and western blot analysis. RESULTS: GrB-YCG was successfully expressed and secreted from Sf9 insect cells and purified from cell culture supernatants. The serine protease activity of GrB-YCG was equivalent to that of human recombinant GrB. An in vitro hormone binding assay revealed that the GrB-YCG molecule also retained the ability to bind to the LHR receptor with an affinity similar to that of native hCG. Upon cell binding, GrB-YCG was rapidly internalized into LHR-expressing human ovarian cancer cells and produced selective and potent tumor cell killing by inducing apoptosis through activation of caspase-3. CONCLUSIONS: These results validate LHR as a therapeutic target and indicate that delivery of the human pro-apoptotic enzyme GrB to tumor cells by yoked hCG has substantial selectivity and therapeutic potential for human tumors that express high levels of LHR such as ovarian carcinomas.
Assuntos
Gonadotropina Coriônica/química , Sistemas de Liberação de Medicamentos , Granzimas/administração & dosagem , Neoplasias Ovarianas/tratamento farmacológico , Receptores do LH/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Caspase 3/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Gonadotropina Coriônica/genética , Gonadotropina Coriônica/metabolismo , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Granzimas/metabolismo , Granzimas/farmacologia , Humanos , Camundongos , Microscopia Confocal , Neoplasias Ovarianas/patologia , Ligação Proteica , Receptores do LH/genética , SpodopteraRESUMO
RATIONALE: coronary artery ligation to induce myocardial infarction (MI) in mice is typically performed by an invasive and time-consuming approach that requires ventilation and chest opening (classic method), often resulting in extensive tissue damage and high mortality. We developed a novel and rapid surgical method to induce MI that does not require ventilation. OBJECTIVE: the purpose of this study was to develop and comprehensively describe this method and directly compare it to the classic method. METHODS AND RESULTS: male C57/B6 mice were grouped into 4 groups: new method MI (MI-N) or sham (S-N) and classic method MI (MI-C) or sham (S-C). In the new method, heart was manually exposed without intubation through a small incision and MI was induced. In the classic method, MI was induced through a ventilated thoracotomy. Similar groups were used in an ischemia/reperfusion injury model. This novel MI procedure is rapid, with an average procedure time of 1.22 ± 0.05 minutes, whereas the classic method requires 23.2 ± 0.6 minutes per procedure. Surgical mortality was 3% in MI-N and 15.9% in MI-C. The rate of arrhythmia was significantly lower in MI-N. The postsurgical levels of tumor necrosis factor-α and myeloperoxidase were lower in new method, indicating less inflammation. Overall, 28-day post-MI survival rate was 68% with MI-N and 48% with MI-C. Importantly, there was no difference in infarct size or post-MI cardiac function between the methods. CONCLUSIONS: this new rapid method of MI in mice represents a more efficient and less damaging model of myocardial ischemic injury compared with the classic method.
Assuntos
Vasos Coronários/cirurgia , Modelos Animais de Doenças , Infarto do Miocárdio/etiologia , Projetos de Pesquisa/normas , Animais , Ligadura/efeitos adversos , Ligadura/métodos , Masculino , Métodos , Camundongos , Camundongos Endogâmicos C57BL , Fatores de TempoRESUMO
Trastuzumab treatment has improved the overall survival of HER2-overexpressing breast cancer patients. However, many of these patients will eventually become resistant to treatment. The mechanisms that contribute to resistance to trastuzumab are unknown. In this study, we tested the hypothesis that targeting of the FKHR transcription factor FOXO1A in HER2-overexpressing breast tumor cells can overcome the trastuzumab resistance in vitro. We have shown that overexpression of HER2 leads to activation of phosphatidylinositol 3-kinase (PI3K)/Akt pathway and subsequent inactivation of FOXO1A in HER2-overexpressing breast cancer cells SKBR3, BT474, and MCF7-HER2. In wild-type SKBR3 and BT474 cells, trastuzumab downregulates active Akt and increases FOXO1A expression that leads to increase in p27(kip1) and decrease in cyclin D1 and finally inhibits cell proliferation. In contrast, the effect of trastuzumab was eliminated by the reduction of FOXO1A in HER2-overexpressing cells with constitutively active Akt1 (SKBR3/AA28 and BT474/AA9). The downregulation of FOXO1A resulted in nuclear export of p27(kip1). Blocking the constitutively active Akt by a specific Akt/protein kinase B signaling inhibitor-2 (API-2) significantly increased FOXO1A expression and rendered the cells more responsive to trastuzumab-induced growth inhibition. Reactivation of FOXO1A by stable or transient transfection also restored the growth-inhibitory effects of trastuzumab in SKBR3/AA28, BT474/AA9, and MCF7-HER2 cells. Knocking down FOXO1A by small interfering RNA resulted in reducing trastuzumab-induced growth inhibition. In summary, trastuzumab can inhibit proliferation of HER2-overexpressing breast cancer cells by reactivating FOXO1A through inhibition of the PI3K/Akt pathway. FOXO1A may therefore serve as a target for HER2-overexpressing breast tumors.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Anticorpos Monoclonais Humanizados , Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Cromonas/farmacologia , Regulação para Baixo , Sinergismo Farmacológico , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Feminino , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptor ErbB-2/metabolismo , Transfecção , TrastuzumabRESUMO
In order to explore the transfection and expression of hRI gene on human umbilical blood stem cells, and observe it's effect on the tumor growth. After enriching human umbilical cord blood CD34+ cells with a high-gradient magnetic cell sorting system (MACS), transfected them with supernatant of retrovirus containing human Ribonuclease inhibitor (hRI) cDNA. Hematopoietic progenitor clonogenic assay and PCR were used to evaluate transfection efficiency, and Western-blot and immune fluorescence were used to evaluate the expression quantity of hRI gene after transfection. Observe the effect of RI on the growth of melonoma in B16C57BL mice. The results showed that human umbilical blood CD34+ cells were highly purified by MACS, which made the purity of human umbilical blood CD34+ cells average 96.15%. hRI can be transfected on umbilical blood CD34+ cells, and the transfection efficiency was 35%. The positive expression of hRI gene on transfected CD34+ cells is identified by Western-blot and immune fluorescence assay. Mice injected with transfected CD34+ cells show a significant restraint of the tumor growth, a lower efficiency of tumor formation, a lower weight of the tumor and a longer incubation period of tumor formation with respect to the control groups. The results demonstrated the capacity of RI to inhibited the tumor growth by blocking the vasculature in tumor.