RESUMO
Letrozole is a potent aromatase inhibitor and superior to other defined selective estrogen receptor modulators such as tamoxifen in treating hormone-responsive postmenopausal breast cancer patients. Patients who receive this drug may become insensitive to the effects of estrogen deprivation induced by letrozole. Letrozole has known side effects on bone metabolism due to systemic ablation of estrogen production. The purpose of this study was to examine the therapeutic efficacy of hyaluronic acid-bound letrozole nanoparticles (HA-Letr-NPs) in restoring sensitivity to letrozole-resistant (LTLT-Ca) cells. To target letrozole to LTLT-Ca cells, hyaluronic acid-bound letrozole nanoparticles were prepared by nanoprecipitation using biodegradable PLGA-PEG co-polymer. Binding specificity of HA to CD44 on the cell surface was analyzed in vitro using FITC-CD44 Ab and CD44 siRNA by flow cytometry. Effects on in vitro cytotoxicity and aromatase enzymatic activity of HA-Letr-NPs were performed in MCF-7 breast cancer cells, MCF-7 cells over-expressing aromatase (MCF-7/Aro), and LTLT-Ca cells resistant to letrozole. Preclinical efficacy of HA-Letr-NPs was examined in mice using LTLT-Ca xenograft tumors. HA-Letr-NPs were restricted to a maximum size of 100 nm. The in vitro drug release assay showed that the highest released concentration of letrozole occurred after 23 hours at 37 degrees C in phosphate-buffered saline. HA-Letr-NPs on MCF-7/Aro and LTLT-Ca cells showed an IC50 of 2 microM and 5 microM, respectively. HA-Letr-NPs were more efficacious in inhibiting tumor growth, reducing in vitro cellular and in vivo tumor aromatase enzyme activity more than the corresponding Letr-NPs or letrozole. HA-Letr-NPs restored and maintained a prolonged sensitivity and targeted delivery of letrozole in letrozole-resistant tumors in vivo.
Assuntos
Antineoplásicos/farmacologia , Ácido Hialurônico/química , Nanopartículas , Nitrilas/farmacologia , Triazóis/farmacologia , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Western Blotting , Resistencia a Medicamentos Antineoplásicos , Citometria de Fluxo , Letrozol , Camundongos , Microscopia Eletrônica de Varredura , Nitrilas/química , Interferência de RNA , Triazóis/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A majority of breast cancers are hormone-responsive, and require estrogen for growth, and respond to hormonal therapy that blocks estrogen receptor action. Breast tumors with low levels of or completely lacking estrogen receptor fail to respond to antiestrogen therapy yet require estrogen for tumor initiation. To address the importance of local estrogen in oncogene-mediated breast tumorigenesis, we have crossed MMTV-aromatase with MMTV-HER2/neu and examined the incidence of breast cancer in double transgenic mice in comparison with parental strains. Double transgenic mice show normal mammary development and express both transgenes at similar levels to that of parental strains. Tumor incidence in double transgenic mice (<5%) decreased compared to HER2/neu mice (>65%). In addition to a significant decrease in tumorigenesis, these mice expressed ERalpha as well as high levels of ERbeta along with decreased levels of cyclin D1 and phosphorylated pRb among other changes. Furthermore, experiments using THC (ERalpha-agonist and ERbeta-antagonist) clearly demonstrate the critical role of ERbeta in HER2/neu-mediated tumorigenesis. These studies provide the first genetic evidence that estrogen receptor, mainly ERbeta than ERalpha and its dependent changes play an important role in regulating mammary tumorigenesis. These findings provide further evidence for development and testing of novel therapeutic approaches based on selective regulation of estrogen receptors (ERalpha and beta)-dependent actions for the treatment and prevention of breast cancers.
Assuntos
Aromatase/metabolismo , Transformação Celular Neoplásica/metabolismo , Regulação Enzimológica da Expressão Gênica , Neoplasias Mamárias Animais/enzimologia , Neoplasias Mamárias Animais/patologia , Modelos Biológicos , Receptor ErbB-2/metabolismo , Animais , Aromatase/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Ciclina D1/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Feminino , Masculino , Neoplasias Mamárias Animais/genética , Camundongos , Camundongos Transgênicos , Fosforilação , Receptor ErbB-2/genética , Receptores de Estrogênio/agonistas , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Proteína do Retinoblastoma/metabolismoRESUMO
Aromatase transgenic mice exhibit hyperplastic and dysplastic changes, attesting to the importance of local estrogen in breast carcinogenesis. These mice also show increased levels of the estrogen receptor alpha and beta (ERalpha, ERbeta) suggesting that this receptor may play an important role in the initiation of estrogen-mediated mammary hyperplasia observed in these mice. To address the specific role of ERalpha in the mammary development and in the induction of estrogen-mediated hyperplasia in aromatase transgenic mice, we have generated MMTV-aromatase x ERalpha knockout cross (referred as aromatase/ERKO). Even though ERbeta is expressed in aromatase/ERKO mice, lack of ERalpha leads to impaired mammary growth in these mice. The data suggest that ERalpha plays an important role in the mammary gland development as well as in the induction of mammary hyperplasia in aromatase transgenic mice. Lack of ERalpha expression in the aromatase/ERKO mice resulted in a decrease in the expression of Cyclin D1, PCNA and TGFbeta relative to the aromatase parental strain. The studies involving aromatase/ERKO mice show that lack of ERalpha results in impaired mammary development even in the presence of continuous tissue estrogen, suggesting estrogen/ERalpha-mediated actions are critical for mammary development and carcinogenesis.
Assuntos
Aromatase/genética , Receptor alfa de Estrogênio/fisiologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/patologia , Animais , Aromatase/metabolismo , Ciclina D1/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Hiperplasia/metabolismo , Hiperplasia/patologia , Masculino , Glândulas Mamárias Animais/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Breast cancers amplified for the tyrosine kinase receptor Her-2/neu constitute ~30% of advanced breast cancer cases, and are characterized by hormone independence and aggressive growth, implicating this pathway in breast oncogenesis. The induction of Her-2/neu leads to tumor development in 60% of transgenic mice. We have previously examined the effects of estrogen in the MMTV-Her-2/neu background by generating the MMTV-Her-2/neu x aromatase double transgenic mouse strain. MMTV-Her-2/neu x aromatase mice developed fewer mammary tumors than the Her-2/neu parental strain. Our present data show the induction of several estrogen-related genes, including the tumor suppressors BRCA1 and p53, and a decrease in several angiogenic factors. The phosphorylated forms of MAPK p42/44 and AKT were lower in the MMTV-Her-2/neu x aromatase double transgenic mice compared to the MMTV-Her-2/neu parental strain; conversely, phospho-p38 levels were higher in the double transgenic strain. The ERß-selective antagonist THC reversed these changes. The regulation of these factors by ERß was confirmed in clones of MCF7 breast cancer cells overexpressing Her-2/neu in combination with ERß, suggesting that ERß may play a direct role in regulating MAPK and AKT pathways. In summary, the data suggest that ERß may play a major role in decreasing tumorigenesis and that it may affect breast cancer cell proliferation and survival by altering MAPK and AKT activation as well as modulation of tumor suppressor and angiogenesis factors. Treatment with selective ERß agonist may provide therapeutic advantages for the treatment and prevention of breast cancer.
Assuntos
Aromatase/genética , Receptor beta de Estrogênio/metabolismo , Genes erbB-2 , Animais , Aromatase/metabolismo , Ciclo Celular/genética , Proliferação de Células , Intervalo Livre de Doença , Feminino , Regulação da Expressão Gênica , Genes Supressores de Tumor , Neoplasias Mamárias Animais/mortalidade , Neoplasias Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/mortalidade , Camundongos , Camundongos Transgênicos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosforilação , Transdução de Sinais/genéticaRESUMO
Fractalkine (also known as CX3CL1), a CX3C chemokine, activates and attracts monocytes/macrophages to the site of injury/inflammation. It binds to CX3C receptor 1 (CX3CR1), a pertussis toxin-sensitive G-protein-coupled receptor. In smooth muscle cells (SMCs), fractalkine is induced by proinflammatory cytokines [tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)], which may mediate monocyte adhesion to SMCs. However, the mechanisms underlying its induction are unknown. In addition, it is unlear whether SMCs express CX3CR1. TNF-alpha activated nuclear factor kappaB (NF-kappaB) and induced fractalkine and CX3CR1 expression in a time-dependent manner in rat aortic SMCs. Transient transfections with dominant-negative (dn) inhibitory kappaB (IkappaB)-alpha, dnIkappaB-beta, dnIkappaB kinase (IKK)-gamma, kinase-dead (kd) NF-kappaB-inducing kinase (NIK) and kdIKK-beta, or pretreatment with wortmannin, Akt inhibitor, pyrrolidinecarbodithioc acid ammonium salt ('PDTC') or MG-132, significantly attenuated TNF-alpha-induced fractalkine and CX3CR1 expression. Furthermore, expression of dn TNF-alpha-receptor-associated factor 2 (TRAF2), but not dnTRAF6, inhibited TNF-alpha signal transduction. Pretreatment with pertussis toxin or neutralizing anti-CX3CR1 antibodies attenuated TNF-alpha-induced fractalkine expression, indicating that fractalkine autoregulation plays a role in TNF-alpha-induced sustained fractalkine expression. Fractalkine induced its own expression, via pertussis toxin-sensitive G-proteins, phosphoinositide 3-kinase (PI 3-kinase), phosphoinositide-dependent kinase 1 (PDK1), Akt, NIK, IKK and NF-kappaB activation, and induced SMC cell-cell adhesion and cellular proliferation. Taken together, our results demonstrate that TNF-alpha induces the expression of fractalkine and CX3CR1 in rat aortic SMCs and that this induction is mediated by NF-kappaB activation. We also show that fractalkine induces its own expression, which is mediated by the PI 3-kinase/PDK1/Akt/NIK/IKK/NF-kappaB signalling pathway. More importantly, fractalkine increased cell-cell adhesion and aortic SMC proliferation, indicating a role in initiation and progression of atherosclerotic vascular disease.
Assuntos
Quimiocinas CX3C/metabolismo , Proteínas de Membrana/metabolismo , Músculo Liso Vascular/citologia , NF-kappa B/metabolismo , Prolina/análogos & derivados , Proteínas Serina-Treonina Quinases , Fator de Necrose Tumoral alfa/farmacologia , Androstadienos/farmacologia , Animais , Antineoplásicos/farmacologia , Aorta/citologia , Comunicação Autócrina , Receptor 1 de Quimiocina CX3C , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CX3CL1 , Ensaio de Desvio de Mobilidade Eletroforética , Inibidores Enzimáticos/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Proteínas I-kappa B/metabolismo , Luciferases/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Prolina/farmacologia , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/metabolismo , Ratos , Receptores de Citocinas/metabolismo , Receptores de HIV/metabolismo , Fator 2 Associado a Receptor de TNF , Fator 6 Associado a Receptor de TNF , Tiocarbamatos/farmacologia , Transfecção , WortmaninaRESUMO
It is well established that cytokines can induce the production of chemokines, but the role of chemokines in the regulation of cytokine expression has not been fully investigated. Exposure of rat cardiac-derived endothelial cells (CDEC) to lipopolysaccharide-induced CXC chemokine (LIX), and to a lesser extent to KC and MIP-2, activated NF-kappaB and induced kappaB-driven promoter activity. LIX did not activate Oct-1. LIX-induced interleukin-1beta and tumor necrosis factor-alpha promoter activity, and up-regulated mRNA expression. Increased transcription and mRNA stability both contributed to cytokine expression. LIX-mediated cytokine gene transcription was inhibited by interleukin-10. Transient overexpression of kinase-deficient NF-kappaB-inducing kinase (NIK) and IkappaB kinase (IKK), and dominant negative IkappaB significantly inhibited LIX-mediated NF-kappaB activation in rat CDEC. Inhibition of G(i) protein-coupled signal transduction, poly(ADP-ribose) polymerase, phosphatidylinositol 3-kinase, and the 26 S proteasome significantly inhibited LIX-mediated NF-kappaB activation and cytokine gene transcription. Blocking CXCR2 attenuated LIX-mediated kappaB activation and kappaB-driven promoter activity in rat CDEC that express both CXCR1 and -2, and abrogated its activation in mouse CDEC that express only CXCR2. These results indicate that LIX activates NF-kappaB and induces kappaB-responsive proinflammatory cytokines via either CXCR1 or CXCR2, and involved phosphatidylinositol 3-kinase, NIK, IKK, and IkappaB. Thus, in addition to attracting and activating neutrophils, the ELR(+) CXC chemokines amplify the inflammatory cascade, stimulating local production of cytokines that have negative inotropic and proapoptotic effects.