RESUMO
New targeted therapy approaches for certain subtypes of breast cancer, such as triple-negative breast cancers and other aggressive phenotypes, are desired. High levels of the mitotic checkpoint kinase Mps1/TTK have correlated with high histologic grade in breast cancer, suggesting a potential new therapeutic target for aggressive breast cancers (BC). Novel small molecules targeting Mps1 were designed by computer assisted docking analyses, and several candidate compounds were synthesized. These compounds were evaluated in anti-proliferative assays of a panel of 15 breast cancer cell lines and further examined for their ability to inhibit a variety of Mps1-dependent biological functions. The results indicate that the lead compounds have strong anti-proliferative potential through Mps1/TTK inhibition in both basal and luminal BC cell lines, exhibiting IC50 values ranging from 0.05 to 1.0µM. In addition, the lead compounds 1 and 13 inhibit Mps1 kinase enzymatic activity with IC50 values from 0.356µM to 0.809µM, and inhibited Mps1-associated cellular functions such as centrosome duplication and the spindle checkpoint in triple negative breast cancer cells. The most promising analog, compound 13, significantly decreased tumor growth in nude mice containing Cal-51 triple negative breast cancer cell xenografts. Using drug discovery technologies, computational modeling, medicinal chemistry, cell culture and in vivo assays, novel small molecule Mps1/TTK inhibitors have been identified as potential targeted therapies for breast cancers.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Proteínas de Ciclo Celular/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/uso terapêutico , Animais , Feminino , Humanos , Camundongos , Camundongos Nus , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologiaRESUMO
Human serum albumin (HSA)-coated lipid nanoparticles (HSA-LNPs) loaded with phrGFP-targeted siRNA (HSA-LNPs-siRNA) were prepared and evaluated for gene downregulation effect in phrGFP-transfected breast cancer cells and the corresponding xenograft tumor model. HSA-LNPs-siRNA were successfully prepared with a particle size of 79.5±5.5 nm. In phrGFP-transfected MCF-7 cells, HSA-LNPs-siRNA significantly decreased cell fluorescence even in the presence of fetal bovine serum (FBS). Moreover, cell fluorescence and phrGFP mRNA expression were significantly downregulated by HSA-LNPs-siRNA in phrGFP-transfected MCF-7, MDA-MB-231, and SK-BR-3 cells in comparison with control or HSA-LNPs-siRNA (scrambled). In phrGFP-transfected MCF-7 xenograft tumor model, tumor fluorescence was significantly decreased after three IV administrations of HSA-LNPs-siRNA at a dose of 3 mg/kg in comparison with siRNA alone. HSA-LNPs-siRNA demonstrated a superior pharmacokinetic profile in comparison with siRNA at a dose of 1mg/kg. These results show that the novel nonviral carrier, HSA-LNPs, may be used for the delivery of siRNA to breast cancer cells. FROM THE CLINICAL EDITOR: Targeted delivery of siRNA to cancer cells may be a viable anti-cancer strategy with low toxicity. In this study the novel nonviral carrier, human serum albumin-coated lipid nanoparticles (HSA-LNP) were demonstrated as an efficient delivery agent of siRNA to breast cancer cells.
Assuntos
Neoplasias da Mama/genética , Metabolismo dos Lipídeos , Nanopartículas , RNA Interferente Pequeno/genética , Albumina Sérica/metabolismo , Sequência de Bases , Neoplasias da Mama/metabolismo , Primers do DNA , Feminino , Humanos , Células MCF-7RESUMO
Nonionic surfactant vesicles, or SPANosomes (SPs), comprised of cationic lipid and sorbitan monooleate (Span 80) were synthesized and evaluated as small interfering RNA (siRNA) vectors. The SPs had a mean diameter of less than 100 nm and exhibited excellent colloidal stability. The SP/siRNA complexes possessed a slightly positive zeta potential of 12 mV and demonstrated a high siRNA incorporation efficiency of greater than 80%. Cryogenic transmission electron microscopy (cryo-TEM) imaging of the SP/siRNA indicated a predominantly core-shell structure. The SP/siRNA complexes were shown to efficiently and specifically silence expression of both green fluorescent protein (GFP) (66% knockdown) and aromatase (77% knockdown) genes in breast cancer cell lines. In addition, the cellular trafficking pathway of the SP/siRNA was investigated by confocal microscopy using molecular beacons as probes for cytosolic delivery. The results showed efficient endosomal escape and cytosolic delivery of the siRNA cargo following internalization of the SP/siRNA complexes. In conclusion, Span 80 is a potent helper lipid, and the SPs are promising vehicles for siRNA delivery.
Assuntos
Técnicas de Silenciamento de Genes , Hexoses/química , Lipossomos/química , RNA Interferente Pequeno/administração & dosagem , Tensoativos/química , Transfecção/métodos , Aromatase/genética , Linhagem Celular Tumoral , Microscopia Crioeletrônica , Ácidos Graxos Monoinsaturados/química , Citometria de Fluxo , Proteínas de Fluorescência Verde/antagonistas & inibidores , Proteínas de Fluorescência Verde/genética , Humanos , Lipossomos/síntese química , Microscopia Eletrônica de Transmissão , Nanopartículas , Sondas de Oligonucleotídeos/análise , Tamanho da Partícula , Polietilenoglicóis/química , Compostos de Amônio Quaternário/química , Vitamina E/análogos & derivados , Vitamina E/químicaRESUMO
PURPOSE: Estrogen receptor alpha (ERalpha) expression has previously been evaluated in lens epithelial cells (LEC). However, its function in the lens has not been determined. One potential function may be its interaction with the catalytic subunit of telomerase (TERT), which is present in normal LEC and higher in LEC that have undergone epithelial to mesenchymal transition (EMT). ERalpha is known to play a role in EMT, a process that may also involve TERT. METHODS: A commercially available transcription factor array was used to evaluate potential interactions between TERT and other proteins in normal and cataractous LEC. Based on these findings, ERalpha protein and mRNA expressions were measured using western blot analysis, immunohistochemical staining, and quantitative reverse transcription polymerase chain reaction (RT-PCR). Co-immunoprecipitation assays were used to evaluate the interaction of TERT with ERalpha as well as their phosphorylation in normal and cataractous LEC. RESULTS: The transcription factor array suggested that TERT interacted with ERalpha via the estrogen response element (ERE) in cataractous LEC but not in normal LEC. Expression of ERalpha protein and mRNA increased in cataractous LEC compared with normal LEC. Co-immunoprecipitation assays confirmed the interaction of TERT with ERalpha in cataractous LEC while no interaction was found in normal LEC. LEC that have undergone EMT, e.g., cataracts, are rapidly proliferating and migrating along the posterior lens capsule. CONCLUSIONS: ERalpha is known to play a role in EMT, and our data suggests that TERT and phosphorylated protein kinase B (pAkt) may be involved in the regulation of this process in cataractous LEC.
Assuntos
Catarata/enzimologia , Catarata/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Receptor alfa de Estrogênio/metabolismo , Cristalino/patologia , Telomerase/metabolismo , Animais , Western Blotting , Catarata/genética , Extratos Celulares , Cães , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Regulação da Expressão Gênica , Imuno-Histoquímica , Imunoprecipitação , Cristalino/metabolismo , Fosforilação , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Aromatase expression and enzyme activity in breast cancer patients is greater in or near the tumor tissue compared with the normal breast tissue. Complex regulation of aromatase expression in human tissues involves alternative promoter sites that provide tissue-specific control. Previous studies in our laboratories suggested a strong association between aromatase (CYP19) gene expression and the expression of cyclooxygenase (COX) genes. Additionally, nonsteroidal anti-inflammatory drugs (NSAIDs) and COX selective inhibitors can suppress CYP19 gene expression and decrease aromatase activity. Our current hypothesis is that pharmacological regulation of aromatase and/or cyclooxygenases can act locally to decrease the biosynthesis of estrogen and may provide additional therapy options for patients with hormone-dependent breast cancer. Two pharmacological approaches are being developed, one involving mRNA silencing by selective short interfering RNAs (siRNA) molecules and the second utilizing small molecule drug design. In the first approach, short interfering RNAs were designed against either human aromatase mRNA or human COX-2 mRNA. Treatment of breast cancer cells with siAROMs completely masked the aromatase enzyme activity. Treatment with COX-2 siRNAs decreased the expression of COX-2 mRNA; furthermore, the siCOX-2-mediated decrease also resulted in suppression of CYP19 mRNA. The small molecule drug design approach focuses on the synthesis and biological evaluation of a novel series of sulfonanilide analogs derived from the COX-2 selective inhibitors. The compounds suppress aromatase enzyme activity in SK-BR-3 breast cancer cells in a dose and time-dependent manner, and structure activity analysis does not find a correlation between aromatase suppression and COX inhibition. Real-time PCR analysis demonstrates that the sulfonanilide analogs decrease aromatase gene transcription in breast cells. Thus, these results suggest that the siRNAs and novel sulfonanilides targeting aromatase expression may be valuable tools for selective regulation of aromatase in breast cancer.
Assuntos
Inibidores da Aromatase/toxicidade , Aromatase/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/toxicidade , Anilidas/química , Anilidas/farmacologia , Aromatase/genética , Inibidores da Aromatase/química , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/genética , Inibidores de Ciclo-Oxigenase/química , Dinoprostona/metabolismo , Ativação Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Estrutura Molecular , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Multidrug resistance (MDR) is a major impediment to successful cancer chemotherapy. P-glycoprotein (P-gp), the product of the multidrug resistance 1 (MDR1) gene, acts as an efflux pump and prevents sufficient intracellular accumulation of several anticancer agents, thus, playing a major role in MDR. Tamoxifen (Tam), ICI 182 780 (ICI) and Adriamycin (Adr) alone or with (-)-gossypol-enriched cottonseed oil [(-)-GPCSO] possible effects on cell growth inhibition and regulation of MDR1, mRNA and P-gp expression were examined in both an MDR human breast cancer cell line, MCF-7/Adr cells, and primary cultured human breast cancer epithelial cells (PCHBCEC). MATERIALS AND METHODS: Cells were treated with 0.05% of (-)-GPCSO either in the absence or presence of either 0.1 microM Tam, ICI or Adr for 24 h. RESULTS: Using the non-radioactive cell proliferation MTS assay, none of these chemotherapeutic agents alone inhibited MCF-7/Adr cell and PCHBCEC proliferation; meanwhile, the combination of 0.1 microM Tam, ICI or Adr with 0.05% (-)-GPCSO significantly reduced MCF-7/Adr cell growth by approximately 34%, 32% and 23%, respectively, of that of the vehicle-treated cells. For PCHBCEC, the combination of 0.05% (-)-GPCSO with 0.1 microM of Tam, ICI and Adr reduced cell growth to about 94%, 90%, and 71% respectively, of the vehicle treated PCHBCEC. Furthermore, (-)-GPCSO inhibited MDR1/P-gp expression in both MCF- 7/Adr and PCHBCEC in a dose-dependent manner. Our results provide insight into the MDR-reversing potential of (-)-GPCSO in human breast cancer cells resistant to current chemotherapeutic agents.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Óleo de Sementes de Algodão/farmacologia , Genes MDR/efeitos dos fármacos , Gossipol/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Óleo de Sementes de Algodão/administração & dosagem , Óleo de Sementes de Algodão/química , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Estradiol/administração & dosagem , Estradiol/análogos & derivados , Estradiol/farmacologia , Fulvestranto , Expressão Gênica/efeitos dos fármacos , Gossipol/administração & dosagem , Gossipol/química , Humanos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Tamoxifeno/administração & dosagem , Tamoxifeno/farmacologia , Células Tumorais CultivadasRESUMO
In normal prostate, keratinocyte growth factor (KGF), also known as fibroblast growth factor-7 (FGF-7) serves as a paracrine growth factor synthesized in stromal cells that acts on epithelial cells through its receptor, KGFR. KGF and KGFR were found in human cancer epithelial cells as well as stromal cells. Since KGF expressed in epithelial cells of benign prostatic hyperplasia (BPH) and in prostate cancer, it has been suggested that KGF might act as an autocrine factor in BPH and prostate cancer. To investigate the roles of KGF in cancerous stroma, primary cultured human prostate cancer stromal cells (PCSCs) were isolated and evaluated. These PCSCs possessed estrogen receptors and KGFR, but not androgen receptor as determined by RT-PCR and Western blot, respectively. KGF exhibited mitogenic and anti-apoptotic effects that correlated with induction of cyclin-D1, Bcl-2, Bcl-xL and phospho-Akt expression in PCSCs, where treatment with KGF antiserum abolished cell proliferation and anti-apoptotic protein expression. PCSCs exposed to KGF for various time periods resulted in phosphorylation of Akt and subsequent up-regulation of Bcl-2. KGF modulated dynamic protein expression indicated that KGF triggered cell cycle machinery and then activated anti-apoptotic actions in PCSCs. Cell proliferation analysis indicated that tamoxifen or ICI 182,780 reduced cell viability in a dose-dependent manner; however, KGF prevented this inhibition, which further demonstrated KGF triggered anti-apoptotic machinery through activating Bcl-2 and phospho-Akt expression. In summary, KGF has an autocrine effect and serves as a survival factor in primary cultured human prostate cancer stromal cells.
Assuntos
Fator 7 de Crescimento de Fibroblastos/farmacologia , Neoplasias da Próstata/patologia , Células Estromais/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Imuno-Histoquímica , Masculino , Receptores de Estrogênio/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Células Estromais/citologiaRESUMO
BACKGROUND: Conjugated linoleic acid (CLA), a naturally occurring compound found in ruminants products, has been shown to possess anticancer properties in vivo and in vitro. There are several CLA isomers in ruminant-produced foods, among which t10, c12-CLA and c9, t11-CLA are the most potent. Protein tyrosine phosphatase gamma (PTPgamma) has been implicated as a tumor suppressor gene in kidney and lung cancers. Our previous results indicated that estradiol-17beta (E2)-induced suppression of PTPgamma may play a role in mammary tumorigenesis. MATERIALS AND METHODS: The effects of t10, c12-CLA and c9, t11-CLA on PTPgamma mRNA expression in human breast epithelial cells and stromal cells, isolated from surgical specimens of mammoplasty and breast cancer patients, were detected and quantified by RT-PCR RESULTS: The PTPgamma mRNA expression was lower in cancer than in normal breast cells. Both t10, c12-CLA and c9, t11-CLA significantly (p < 0.05) increased the PTPgamma mRNA levels in primary cultured normal breast epithelial cells, normal breast stromal cells and breast cancer epithelial cells, but not in breast cancer stromal cells. t10, c12-CLA appeared to be the most active isomer in estrogen receptor a (ERalpha)-positive human breast cancer epithelial cells. CONCLUSION: The results indicate that dietary CLA might serve as a chemo-preventive and chemo-therapeutic agent in human breast cancers by up-regulating the estrogen-regulated tumor suppressor gene, PTPgamma expression.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Ácidos Linoleicos Conjugados/farmacologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas Tirosina Fosfatases/biossíntese , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Receptor alfa de Estrogênio/biossíntese , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Supressores de Tumor , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas Tirosina Fosfatases/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores , Células Estromais/efeitos dos fármacos , Células Estromais/enzimologia , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Both estrogen receptor-alpha (ER-alpha) and progesterone receptor (PR) are good prognostic factors and indicators of benefit from endocrine therapy in breast cancer patients. The relationship of the ER-alpha and PR status and the difference in clinical benefit from endocrine therapy in breast cancer is currently unclear. It has been suggested that keratinocyte growth factors (KGFs) are important regulatory factors in breast cancer. Our laboratory has demonstrated that KGF can act as an estromedin for the stimulation of breast cancer cell growth. Also, KGF stimulates aromatase activity in primary cultured human breast cells. This enzyme is a key to the conversion of androgens to estrogens. In the present study, ER-alpha, two estrogen-regulated genes, PR and PTPgamma, KGF and their relationship with endocrine resistance in human breast cancer cells were investigated. MATERIALS AND METHODS: MCF-7 cells were treated with KGF (1, 5, 10, 20 ng/ml), KGF-13 (0.1, 1, 10 microM) or vehicles as control for 24 hours. KGF-13 is a potential receptor-binding pentapeptide constructed using the KGF peptide residues 101-105 (RTVAV) as a template, located within the beta 4--beta 5 loop. Total RNA were isolated and real-time PCR was employed to identify ER-alpha, PR and PTPgamma gene expressions in response to KGF and KGF-13. Western blot analysis was used to verify the levels of ER-alpha and PR protein, whereas immunohistochemical staining was used to detect PTPgamma expression in MCF-7 cells. To determine the response of MCF-7 cells to endocrine therapy, MCF-7 was treated with either 20 ng/ml KGF or 10 microM KGF-13 combined with 1, 3 and 5 microM of 4-hydroxytamoxifen (4OH-Tam). A non-radioactive cell proliferation assay was applied to determine the growth rate of MCF-7 cells. The results of real-time PCR and the cell proliferation assay were analyzed by Student's t-test and p-values of less than 0.05 were considered statistically significant. RESULTS: Our data showed that KGF significantly suppressed ER-alpha, PR and PTPgamma expression in MCF-7 cells. KGF suppressed ER-alpha, PR and PTPgamma mRNA to a maximal inhibition at 20 ng/ml by 88%, 57% and 61%, respectively. Western blot analysis and immunohistochemical staining confirmed the down-regulation of ER-alpha, PR and PTPgamma by KGF in protein levels. Ten microM KGF-13 also decreased ER-alpha expression. Ten microM KGF-13 significantly decreased ER-alpha, PR and PTPgamma mRNA expressions by 51%, 57% and 67%, respectively. These KGF-13-mediated mRNA down-regulations were also observed in protein levels. In a cell proliferation assay, 4OH-Tam (3, 5 microM) induced MCF-7 cell death. KGF and KGF-13 alone did not stimulate MCF-7 cell growth. KGF significantly disrupted 4OH-Tam cell killing effects by 1.2- and 1.3-fold at 4OH-Tam concentrations of 3 microM and 5 microM, respectively. KGF-13 significantly disrupted 4OH-Tam cell killing effects by 1.2- and 1.7-fold at 4OH-Tam concentrations of 3 microM and 5 microM, respectively. CONCLUSION: Our results suggested that not only ER-alpha and PR but also PTPgamma could be potential bio-makers for growth factor-induced endocrine resistant in breast cancer. KGF might increase the endocrine resistance via decreasing ER-alpha, PR and PTPgamma as well. Moreover, the functional analysis of KGF-13 implied possible applications of using short receptor-binding peptides derived from intact KGF as breast cancer therapeutic agents. Thus, our experimental data provided evidence of KGF-induced anti-hormone resistance in human breast cancer and suggested novel strategies for breast cancer via interference with KGF signaling.
Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Fator 7 de Crescimento de Fibroblastos/metabolismo , Tamoxifeno/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/biossíntese , Humanos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Proteínas Tirosina Fosfatases/biossíntese , Proteínas Tirosina Fosfatases/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores , Receptores de Progesterona/biossíntese , Transdução de Sinais , Tamoxifeno/análogos & derivadosRESUMO
BACKGROUND: Gossypol, a natural polyphenolic compound present in cottonseeds, possesses antiproliferative and pro-apoptotic effects in in vivo and in vitro models. There are two enantiomers, (+)-gossypol and (-)-gossypol, the latter being a more potent inhibitor of cancer cell growth. Here, the effect of bovine serum albumin (BSA) and dextran-coated charcoal-treated fetal bovine serum (DCC-FBS)-containing medium on the ability of (-)-gossypol to inhibit the growth of human prostate cancer cells was studied. MATERIALS AND METHODS: BSA- and DCC-FBS-supplemented medium were used to examine the influence of serum proteins on the antiproliferative effects of (-)-gossypol in DU-145 cells, a human prostate cancer cell line. The viability of the DU-145 cells was determined by CellTiter 96 Aqueous assay. The expressions of mRNA and protein for the cell cycle regulators, cyclin-D1, Rb, CDK, p21 and TGF-beta, were determined by RT-PCR and Western blot analyses, respectively. RESULTS: (-)-Gossypol caused growth suppression of the DU-145 cells. In comparison with BSA-supplemented medium, DCC-FBS blocked the antiproliferative effects of (-)-gossypol at 1 and 2.5 microM, but not at 5 microM. Furthermore, (-)-gossypol treatment down-regulated cyclin-D1, Rb, CDK4 and CDK6, and up-regulated p21 and TGF-beta1 at the mRNA and/or protein levels. CONCLUSION: The data suggested that (-)-gossypol-suppressed prostate cancer cell growth may be influenced through cell cycle regulators, which may lead to better prognosis. We further speculate that (-)-gossypol might serve as a chemotherapeutic agent for human prostate cancer patients.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Gossipol/farmacologia , Neoplasias da Próstata/patologia , Soroalbumina Bovina/farmacologia , Soro/metabolismo , Animais , Western Blotting , Bovinos , Carvão Vegetal/química , Ciclina D1/genética , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dextranos/química , Humanos , Masculino , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Neoplasias da Próstata/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Soro/química , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Células Tumorais CultivadasRESUMO
BACKGROUND: Conjugated linoleic acid (CLA), a naturally occurring linoleic acid isomer found in ruminant-produced foods, has the potential to serve as an effective chemopreventive nutriceutical factor for breast cancer prevention based upon previous published studies. There are several CLA isomers in ruminant-produced food products, among which t10,c12-CLA and c9,t11-CLA are more potent. Expression of cyclooxygenase 2 (COX-2) in mammary tumors has been correlated with poor prognosis. Prostaglandin E2 (PGE2) is a major COX-2 product in various cancers and, as in humans, PGE2 concentrations in canine tumor tissues were frequently elevated. Moreover, a PGE2 receptor subtype, EP2, is highly expressed in mammary tumors. Thus, various studies have implicated the important role of PGE2 and EP2 in COX-2-regulated tumor development. MATERIALS AND METHODS: Mammary tumor and normal mammary tissues were both collected from a female dog with mammary tumor. Both malignant and normal mammary tissues were subjected to isolation of epithelial and stromal cells. The effects of t10,c12-CLA and c9,t11-CLA on proliferation, as well as COX-2 and EP2 protein expression in canine mammary normal and cancerous cells, were detected by CellTiter 96 AQueous assay and Western blot assay, respectively. RESULTS: Both t10,c12-CLA and c9,t11-CLA not only suppressed malignant mammary cell growth, but also exerted inhibitory effects on tumor-associated non-malignant mammary cells. Similarly, both t10,c12-CLA and c9,t11-CLA suppressed EP2 protein expression in both normal and malignant mammary cells. t10,c12-CLA was more effective in decreasing COX-2 protein expression in malignant mammary cells, while, in contrast, c9,t11-CLA down-regulated COX-2 protein expression in both normal and malignant mammary cells. CONCLUSION: The results indicate that the dietary component CLA regulates COX-2 and EP2 protein expression in both malignant mammary cells and cells from the tumor-associated stromal compartment. In turn, this may suppress PGE2 signaling, leading to better prognosis. We further speculate that the knowledge obtained from canine studies may also be beneficial to study human breast cancer.
Assuntos
Dinoprostona/metabolismo , Doenças do Cão/tratamento farmacológico , Ácidos Linoleicos Conjugados/farmacologia , Neoplasias Mamárias Animais/tratamento farmacológico , Neoplasias Mamárias Animais/metabolismo , Animais , Ciclo-Oxigenase 2/biossíntese , Inibidores de Ciclo-Oxigenase 2/farmacologia , Cães , Feminino , Receptores de Prostaglandina E/antagonistas & inibidores , Receptores de Prostaglandina E/biossíntese , Receptores de Prostaglandina E Subtipo EP2 , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Protein tyrosine phosphatase gamma (PTPgamma) is a member of the receptor-like family of tyrosine phosphatases and has been implicated as a tumor suppressor gene in kidney and lung cancers. Based on our previous findings, we hypothesize that PTPgamma is a potential estrogen-regulated tumor suppressor gene in human breast cancer. To examine the effects of PTPgamma on growth of MCF-7 human breast cancer cells and compare the estrogenic responses of human breast cells with different PTPgamma expression levels, we established several stably transfected MCF-7 cell lines expressing different levels of PTPgamma, which were confirmed by RT-PCR and immunostaining. In our work, we used the antisense construct to breakdown endogenous PTPgamma level in MCF-7 cells. The results from doubling time assay suggested that PTPgamma is capable of inhibiting MCF-7 breast cancer cell growth. We further demonstrated that PTPgamma is able to inhibit anchorage-independent growth of breast cancer cells in soft agar and reduce the estrogenic responses of MCF-7 cell proliferation to estradiol-17beta (E(2)) and zeranol (Z, a nonsteroidal growth promoter with estrogenic activity). Our data suggest that PTPgamma may function as an important modulator in regulating the process of tumorigenesis in human breast.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas do Tecido Nervoso/genética , Proteínas Tirosina Fosfatases/genética , Sequência de Bases , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Primers do DNA , Estradiol/farmacologia , Estrogênios não Esteroides/farmacologia , Feminino , Regulação Enzimológica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores , Recombinação Genética , Transfecção , Células Tumorais Cultivadas , Zeranol/farmacologiaRESUMO
Aromatase expression and enzyme activity in breast cancer patients is greater in or near the tumor tissue compared with the normal breast tissue. Regulation of aromatase expression in human tissues is quite complex, involving alternative promoter sites that provide tissue-specific control. Previous studies in our laboratories suggested a strong association between aromatase (CYP19) gene expression and the expression of cyclooxygenase (COX) genes. Our hypothesis is that higher levels of COX expression result in higher levels of prostaglandin E2 (PGE2), which in turn increases CYP19 expression through increases in intracellular cyclic AMP levels. This biochemical mechanism may explain the beneficial effects of non-steroidal anti-inflammatory drugs (NSAIDs) on reducing the risks of breast cancer. The effects of NSAIDs (ibuprofen, piroxicam, and indomethacin), a COX-1 selective inhibitor (SC-560), and COX-2 selective inhibitors (celecoxib, niflumic acid, nimesulide, NS-398, and SC-58125) on aromatase activity and CYP19 expression were investigated in breast cancer cell culture systems. Dose-dependent decreases in aromatase activity were observed following treatment with an NSAID or COX inhibitor, with the most effective agents being COX selective inhibitors. Real time PCR analysis of aromatase gene expression showed a significant decrease in mRNA levels in treated cells when compared to vehicle control. These results suggest that the effect of COX inhibitors on aromatase occurs at the transcriptional level. To further probe these interactions, short interfering RNAs (siRNA) were designed against either human CYP19 mRNA or human COX-2 mRNA. Treatment of breast cancer cells with aromatase siRNAs suppressed CYP19 mRNA and aromatase enzyme activity. Finally, treatment with COX-2 siRNAs downregulated the expression of COX-2 mRNA; furthermore, the siCOX-2-mediated suppression of COX-2 also resulted in suppression of aromatase mRNA. In summary, pharmacological regulation of aromatase and cyclooxygenases can act locally in an autocrine fashion to decrease the biosynthesis of estrogen and may provide additional therapy options for patients with hormone-dependent breast cancer.
Assuntos
Aromatase/genética , Neoplasias da Mama/enzimologia , Inibidores de Ciclo-Oxigenase/farmacologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Aromatase/biossíntese , Comunicação Autócrina , Neoplasias da Mama/genética , Células Cultivadas , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Feminino , Humanos , Proteínas de Membrana , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Prostaglandina-Endoperóxido Sintases/genética , Biossíntese de Proteínas , Interferência de RNA , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND: Benign prostatic hyperplasia (BPH) is an age-dependent prostatic disease in human males and dogs. The prostatic stromal estrogen level of health control and BPH patients increases significantly with age, while the dihydrotestosterone (DHT) level is not connected with age. Moreover, experimentally estrogens have induced BPH in the presence of androgens. Our aim was to investigate the effects of age on the proliferation and estrogen receptor beta (ER/3) mRNA of canine prostatic epithelial and stromal cells. MATERIALS AND METHODS: Epithelial and stromal cells were isolated from canine prostatic tissues. The proliferation of these cell types from dogs of different ages was assessed by thymidine incorporation assay, while the expression and identification of ERbeta mRNA were performed by RT-PCR and DNA sequence. RESULTS: Prostatic epithelial cells isolated from 1-year-old dogs exhibited a greater proliferative activity than those of 4-year-old dogs. In contrast, the prostatic stromal cells from 4-year-old dogs proliferated more rapidly than the cells from 1-year-old dogs. ERbeta mRNA expression was detected in the canine prostatic epithelial and stromal cells, decreasing with age. The partial DNA sequence showed that the canine ERbeta sequence shares 90.0%, 87.0% and 83.0% of its nucleotide homology with human, rat and mouse ERbeta, respectively. CONCLUSION: The decrease in the expression of ERbeta in prostatic cells with age reduces its negative control over the androgen receptor, associated with the overgrowth of canine prostatic stromal cells, which further induces the development of canine BPH.
Assuntos
Receptor beta de Estrogênio/biossíntese , Próstata/citologia , Próstata/metabolismo , RNA Mensageiro/biossíntese , Fatores Etários , Animais , Sequência de Bases , Processos de Crescimento Celular/fisiologia , Células Cultivadas , Cães , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Receptor beta de Estrogênio/genética , Masculino , Dados de Sequência Molecular , Próstata/fisiologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/citologia , Células Estromais/metabolismo , Células Estromais/fisiologiaRESUMO
BACKGROUND: Conjugated linoleic acid (CLA), a naturally occurring compound found in ruminant dairy and beef products, has been shown to possess anti-cancer ability in vivo and in vitro. There are several CLA isomers in ruminant-produced food products, among which t10,c12-CLA and c9,t11-CLA are most potent. Vascular endothelial growth factor-A (VEGF-A) has been implicated as an angiogenesis-activating cytokine. Our previous results indicated that CLA induced suppression of VEGF-A in MCF-7 cells, which may be one of CLA's anticancer mechanisms. MATERIALS AND METHODS: The effects of 10,c12-CLA and c9,t11-CLA on VEGF-A mRNA and protein expression in MCF-7 cells, which were co-cultured with human breast stromal cells isolated from breast tissues of surgical specimens of mammoplasty and breast cancer patients, were detected by RT-PCR and Western blot analysis. RESULTS: VEGF-A mRNA and protein expressions were significantly (p < 0.05) elevated in co-cultured MCF-7 cells in comparison with cultured MCF-7 cells alone. Normal human breast stromal cells contribute greater effects in increasing VEGF-A protein expression in MCF-7 cells. Both t10,c12-CLA and c9,t11-CLA significantly (p < 0.05) decreased VEGF-A mRNA and protein levels in co-cultured MCF-7 cells. t10,c12-CLA appeared to be the more active isomer than c9, t11-CLA. CONCLUSION: The results indicate that dietary CLA might serve as a chemo-therapeutic agent in human breast cancers by down-regulating VEGF-A expression.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Comunicação Celular/fisiologia , Ácidos Linoleicos Conjugados/farmacologia , Células Estromais/citologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Técnicas de Cocultura , Regulação para Baixo , Humanos , Isomerismo , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
PURPOSE: To confirm that lens epithelial cells (LEC) synthesize 17ß-estradiol, active estrogen, and to identify the pathway(s) by which normal and cataractous LEC synthesize 17ß-estradiol. METHODS: ELISA was used to measure estradiol in aqueous humor; immunohistochemical staining was used to localize estradiol, testosterone and sulfatase; tritiated water release assay was used to measure aromatase activity; and qRT-PCR was used to quantify expression of aromatase and sulfatase in normal and cataractous canine and human LEC. RESULTS: Canine eyes with and without cataracts had no differences in aqueous humor estradiol levels; however, cataractous LEC had more intense immunoreactivity for estradiol than normal LEC. There were little to no differences in canine sulfatase protein and mRNA expression when normal and cataractous LEC were compared. qRT-PCR demonstrated that canine cataractous LEC had significantly higher expression of aromatase; this was confirmed with the tritiated water release assay. Similar to dogs, human cataracts had both sulfatase and aromatase mRNA expression. CONCLUSIONS: Normal and cataractous LEC can synthesize estradiol by the sulfatase pathway; however, cataractous LEC appear to use the aromatase pathway as well. Because no differences in aqueous humor estradiol levels were detected, we suspect that estradiol synthesized by the sulfatase pathway is secreted into the aqueous humor; whereas, estradiol synthesized by the aromatase pathway is used for, as yet unknown, intracrine purposes.
Assuntos
Catarata/metabolismo , Estradiol/biossíntese , Cristalino/metabolismo , Animais , Western Blotting , Catarata/patologia , Células Cultivadas , Modelos Animais de Doenças , Cães , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Cristalino/patologiaRESUMO
We have reported PTP gamma expression was downregulated by 17 beta-estradiol (E2) and Zeranol (Z) and that PTP gamma may function as an estrogen-regulated cancer suppressor in human breast. We utilized RT-PCR to examine expression of estrogen receptor alpha (ER alpha) and beta (ER beta) mRNA in MCF-7 and MDA-MB-231 cells and to investigate the regulation of PTP gamma expression by E2 and Z in the absence or presence of ICI 182,780 (ICI) in both cells, and immunohistochemistry to examine ER alpha and ER beta protein in normal and cancerous human breast. Results show that MCF-7 express both ER alpha and ER beta, and MDA-MB-231 express only ER beta. Both E2 and Z (30 nM; 24 h) suppressed PTP gamma by approximately 56% in MCF-7 cells and these effects were completely blocked by 1 mM of ICI. In contrast, E2, Z and ICI had no effects on PTP gamma expression in MDA-MB-231 cells. Interestingly, both E2 and Z suppressed PTP gamma by approximately 45% in MDA-MB-231 cells transfected with ER alpha, and these effects were completely blocked by 100 nM of ICI. Both RT-PCR and immunohistochemical staining showed that ER alpha expression was significantly higher in cancerous human breast than in normal breast, while ER beta was higher in normal human breast than in cancerous breast. In combination with our previous findings of greater PTP gamma expression levels in normal human breast than cancerous breast, current results show that lower PTP gamma was associated with higher ER alpha in cancerous human breast tissues. In conclusion, results indicate that Z induces estrogenic effects in human breast relative of PTP gamma expression and the estrogenic down-regulation of PTP gamma expression in human breast is associated with ER alpha.
Assuntos
Mama/metabolismo , Estradiol/análogos & derivados , Estrogênios/farmacologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas Tirosina Fosfatases/biossíntese , Receptores de Estrogênio/fisiologia , Mama/efeitos dos fármacos , Mama/enzimologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Estradiol/farmacologia , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Fulvestranto , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores , Receptores de Estrogênio/biossíntese , Receptores de Estrogênio/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Zeranol/farmacologiaRESUMO
BACKGROUND: Gossypol (GP) is a potent antifertility agent contained in cottonseed and other parts of cotton plants. We have shown that GP inhibits in vitro growth of Dunning rodent prostate cancer cells (MAT-LyLu), PC3, MCF-7 and primary cultured human prostate cells, as well as the in vivo tumor growth of the MAT-LyLu cell line after implantation into Copenhagen rats. MATERIALS AND METHODS: GP's effects on O2 consumption/CO2 production of PC3 cells were studied using the Micro-Oxymax respirometer. The effects of GP on oxidative phosphorylation in PC3 cells were determined by the succinic dehydrogenase assay. RESULTS: GP at the concentration of 1.0 microM reduced DNA synthesis by 25.6%, 54.6% and 81.25% after treatment times of 24, 28, and 32 hours, respectively, while GP at 2 microM reduced DNA synthesis by 78.57%, 81.44% and 83.72% after treatment durations of 24, 28 and 32 hours, respectively. GP at the concentration of 2.0 microM decreased significantly CO2 production by 37.5%, 42.4% and 44.7% and O2 consumption by 6.4%, 6.9% and 7.9% in PC3 cells after the different periods of treatment (24, 28 and 32 hours, respectively). GP at the concentration of 1.0 microM did not significantly inhibit O2 consumption/CO2 production of PC3 cells. GP also reduced the activity of mitochondrial succinic dehydrogenase in PC3 cells to 31% of control at the concentration of 1.0 microM and 15% of control at the concentration of 2.0 microM. CONCLUSION: The inhibitory action of GP on O2 consumption/CO2 production of PC3 cells may be due to disruption of oxidative phosphorylation by inhibition of mitochondrial succinic dehydrogenase.
Assuntos
Dióxido de Carbono/metabolismo , Gossipol/farmacologia , Inibidores do Crescimento/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , DNA de Neoplasias/biossíntese , Humanos , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Oxigênio/antagonistas & inibidores , Oxigênio/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Succinato Desidrogenase/metabolismo , Células Tumorais CultivadasRESUMO
Natural gossypol (GP), a polyphenolic pigment in cottonseed, is a racemic mixture of two enantiomers, (+)GP and (-)GP. Our aim was to compare the abilities of (+/-)GP, (+)GP and (-)GP to reduce proliferation of breast cancerous epithelial cells (cEC) and cancerous stromal cells (cSC). Proliferation was measured by 3H-thymidine uptake. Results showed that (+)GP had no effect on both cEC and cSC. In contrast, in both cell types, (+/-)GP and (-)GP significantly inhibited proliferation. (+/-)GP caused reductions of 15, 46 and 82% at 25, 5.0 and 7.5 microM, respectively, in cEC, and reductions of 17, 28, 39 and 56% at 2.0, 3.0, 4.0 and 5.0 microM, respectively, in cSC. (-)GP induced reductions of 33, 89 and 98% at 2.5, 5.0 and 7.5 microM, respectively, in cEC, and reductions of 29, 51, 64 and 72% at 2.0, 3.0, 4.0 and 5.0 microM, respectively, in cSC. By RT-PCR, we found that 3 microM of (+/-)GP and (-)GP decreased cyclin D1 mRNA expression in both cell types (52% and 71%, respectively, in cEC; and 47% and 71%, respectively, in cSC), and increased transforming growth factor beta (TGFbeta) mRNA expression in both cell types (93% and 130%, respectively, in cEC; and 45% and 89%, respectively, in cSC). Interestingly, (-)GP was significantly more potent than (+/-)GP. These results show that (-)GP is the major inhibitory component of (+/-)GP, (-)GP is the more potent inhibitor of cancerous breast cell growth, and the inhibitory activity of (-)GP and (+/-)GP is related to the reduction of the cell cycle regulator, cyclin D1, and the induction of the cell proliferation inhibitor, TGFbeta.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Gossipol/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Ciclina D1/biossíntese , Ciclina D1/genética , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores do Crescimento/farmacologia , Humanos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Estereoisomerismo , Células Estromais/efeitos dos fármacos , Células Estromais/patologia , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genética , Células Tumorais CultivadasRESUMO
BACKGROUND: Racemic gossypol [(+/-)-GP], a naturally occurring polyphenolic yellow pigment present in cottonseed products, inhibits in vitro proliferation of Dunning prostate cancer cells (MAT-LyLu), human prostate cancer cells derived from a bone marrow metastasis (PC3), MCF-7 and primary cultured human prostate cells. (+/-)-GP also has the ability to inhibit the metastasis of lung and lymph nodes of the androgen-independent rodent prostate cancer cell line, MAT-LyLu, after implantation into Copenhagen rats. MATERIALS AND METHODS: The effects of (+/-)-GP on the proliferation of human prostate cancer PC3 cells were determined by thymidine incorporation assay and doubling-time (DT) determination. The mechanisms of action of (+/-)-GP on the proliferation of PC3 cells were determined by RT-PCR analysis, ELISA assay and Western blot analysis. RESULTS: The results show that (+/-)-GP caused reductions in DNA synthesis and prolonged the DTs in PC3 cells. RT-PCR and ELISA results show that (+/-)-GP elevate the mRNA expression and protein secretion of transforming growth factor beta1 (TGFbeta1) in PC3 cells. Consistent with these findings, (+/-)-GP has been shown to decrease the cyclin D1 mRNA expression and protein expression in PC3 cells. Furthermore, the growth inhibition of PC3 cells by conditioned media collected from the (+/-)-GP-treated-PC3 cells was completely reversed by addition of 25 microg/ml of mouse monoclonal anti-TGFbeta1, -beta2, -beta3 antibody, suggesting the involvement of TGFbeta1 in (+/-)-GP-induced growth inhibition of PC3 cells. CONCLUSION: These results indicate that the inhibitory effects of (+/-)-GP on the proliferation of human prostate cancer PC3 cells are associated with induction of TGFbeta1, which in turn influences the expression of the cell cycle-regulatory protein, cyclin D1, in prostate cancer cells.