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Are all food ingredients, dietary supplement ingredients and even foods, required to meet the same safety standards? Are they all equally safe? If so, then why do the various categories have different expressions describing their safety, such as "reasonable certainty of no harm" for food ingredients and "reasonable expectation of no harm" for dietary supplement ingredients? The basis for these different expressions is that they are not standards of safety, but standards of proof of safety. Just as in criminal vs. civil courts, the threshold for proving guilt or fault is different, so too are there differences between various categories of consumer products regulated by the US Food and Drug Administration. This manuscript describes the threshold requirements for each standard, as well as to the identity of the decision makers on what is safe, their credentials as decision makers and the databases mandated for their use.
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Introduction: The domestic cat (Felis catus) is one of the most common pets. Worldwide, approximately one in five adults are sensitive to cat allergens. The major cat allergen is the secretoglobulin Fel d 1, which is primarily produced in the salivary and sebaceous glands. Chickens produce IgY antibodies, which are similar in structure to mammalian IgG. When chickens are exposed to Fel d 1, anti-Fel d 1-specific IgY (AFD1) is produced and is naturally concentrated in egg yolk. The aim of this study was to evaluate the tolerability, effects on growth and food consumption, and potential adverse effects of a chicken egg product ingredient containing AFD1 in kittens. Methods: This was a blinded, controlled study. Twenty-seven (27) eight-week old kittens were randomly assigned to three feeding groups containing 0 ppm AFD1 (Group 0), 8 ppm AFD1 (Group 1), and 16 ppm AFD1 (Group 2) for 84 days. Veterinary exams and bloodwork were performed on Day 42 and Day 84, and body weight and body condition score (BCS) were monitored weekly. Results: Throughout the study, there were no signs of nutritional deficiency or adverse clinical events in any of the subjects. Administration of a chicken egg product ingredient containing AFD1 in the diet (whether in coating or combination of coating and top dress) had no significant effect on body weight nor food consumption, and all subjects maintained a healthy Body Condition Score (BCS) throughout the study. Moreover, there were no biologically significant differences in the mean clinical chemistry and hematology parameters. Discussion: This study demonstrated that a diet formulated to contain up to 16 ppm AFD1, included in the coating and the top-dress of dry kitten food, was well tolerated, promoted adequate growth, and exhibited no adverse effects.
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Specificity protein (Sp) transcription factors (TFs) Sp1, Sp3 and Sp4, and the orphan nuclear receptor 4A1 (NR4A1) are highly expressed in pancreatic tumors and Sp1 is a negative prognostic factor for pancreatic cancer patient survival. Results of knockdown and overexpression of Sp1, Sp3 and Sp4 in pancreatic and other cancer lines show that these TFs are individually pro-oncogenic factors and loss of one Sp TF is not compensated by other members. NR4A1 is also a pro-oncogenic factor and both NR4A1 and Sp TFs exhibit similar functions in pancreatic cancer cells and regulate cell growth, survival, migration and invasion. There is also evidence that Sp TFs and NR4A1 regulate some of the same genes including survivin, epidermal growth factor receptor, PAX3-FOXO1, α5- and α6-integrins, ß1-, ß3- and ß4-integrins; this is due to NR4A1 acting as a cofactor and mediating NR4A1/Sp1/4-regulated gene expression through GC-rich gene promoter sites. Several studies show that drugs targeting Sp downregulation or NR4A1 antagonists are highly effective inhibitors of Sp/NR4A1-regulated pathways and genes in pancreatic and other cancer cells, and the triterpenoid celastrol is a novel dual-acting agent that targets both Sp TFs and NR4A1.
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Neoplasias Pancreáticas , Fatores de Transcrição , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Oncogenes , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição Sp/genética , Fatores de Transcrição Sp/metabolismo , Fatores de Transcrição/genéticaRESUMO
Nuclear receptor 4A1 (NR4A1, Nur77) is overexpressed in rhabdomyosarcoma (RMS), and inactivation of NR4A1 (siNR4A1) or treatment with the NR4A1 antagonist 1,1-bis(3'-indoly)-1-(p-hydroxy-phenyl)methane (DIM-C-pPhOH) has antiproliferative and proapoptotic effects on RMS cells. However, the mechanism by which NR4A1 inhibition exerts these effects is poorly defined. Here, we report that NR4A1 silencing or inhibition resulted in accumulation of reactive oxygen species (ROS) and ROS-dependent induction of the tumor suppressor-like cytokine IL24 in RMS cells. Mechanistically, NR4A1 was found to regulate the expression of the proreductant genes thioredoxin domain-containing 5 (TXNDC5) and isocitrate dehydrogenase 1 (IDH1), which are downregulated in RMS cells following NR4A1 knockdown or inhibition. Silencing TXNDC5 and IDH1 also induced ROS accumulation and IL24 expression in RMS cells, suggesting that NR4A1 antagonists mediate their antiproliferative and apoptotic effects through modulation of proreductant gene expression. Finally, cotreatment with the antioxidant glutathione or IL24-blocking antibody reversed the effects of NR4A1 inhibition, demonstrating the importance of both ROS and IL24 in mediating the cellular responses. IMPLICATIONS: Overall, these data elucidate the mechanism by which NR4A1 inhibition functions to inhibit the proliferation, survival, and migration of RMS cells.
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Regulação Neoplásica da Expressão Gênica , Interleucinas/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rabdomiossarcoma/terapia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Regulação para Baixo , Glutationa/uso terapêutico , Humanos , Indóis/uso terapêutico , Interleucinas/antagonistas & inibidores , Interleucinas/genética , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/antagonistas & inibidores , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Fenóis/uso terapêutico , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologiaRESUMO
BACKGROUND: Nuclear receptor 4A1 (NR4A1) is overexpressed in mammary tumors, and the methylene-substituted bis-indole derivative 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) acts as an NR4A1 antagonist (inverse agonist) and inhibits NR4A1-regulated pro-oncogenic pathways/genes in breast and other cancer cells. METHODS: Buttressed analogs of DIM-C-pPhOH were synthesized by condensation of the substituted p-hydroxybenzaldehydes with indole. Breast cancer cell growth, survival, and migration assays were carried out by cell counting, Annexin V staining, and Boyden chamber assays, respectively. Changes in RNA and protein expression were determined by RT-PCR and western blots, respectively. Analysis of RNAseq results was carried out using Ingenuity Pathway Analysis, and in vivo potencies of NR4A1 antagonists were determined in athymic nude mice bearing MDA-MB-231 cells in an orthotopic model. RESULTS: Ingenuity Pathway analysis of common genes modulated by NR4A1 knockdown or treatment with DIM-C-pPhOH showed that changes in gene expression were consistent with the observed decreased functional responses, namely inhibition of growth and migration and increased apoptosis. DIM-C-pPhOH is rapidly metabolized and the effects and potencies of buttressed analogs of DIM-C-pPhOH which contain one or two substituents ortho to the hydroxyl groups were investigated using NR4A1-regulated gene/gene products as endpoints. The buttressed analogs were more potent than DIM-C-pPhOH in both in vitro assays and as inhibitors of mammary tumor growth. Moreover, using 1,1-bis(3'-indolyl)-1-(3-chloro-4-hydroxy-5-methoxyphenyl)methane (DIM-C-pPhOh-3-Cl-5-OCH3) significant tumor growth inhibition was observed at doses as low as 2 mg/kg/d which was at least an order of magnitude more potent than DIM-C-pPhOH. CONCLUSIONS: These buttressed analogs represent a more potent set of second generation NR4A1 antagonists as inhibitors of breast cancer.
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Antineoplásicos/farmacologia , Indóis/farmacologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/antagonistas & inibidores , Fenóis/farmacologia , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Humanos , Indóis/química , Camundongos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Fenóis/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Alveolar rhabdomyosarcoma (ARMS) patients have a poor prognosis, and this is primarily due to overexpression of the oncogenic fusion protein PAX3-FOXO1. Results of RNA-sequencing studies show that PAX3-FOXO1 represses expression of interleukin-24 (IL24), and these two genes are inversely expressed in patient tumors. PAX3-FOXO1 also regulates histone deacetylase 5 (HDAC5) in ARMS cells, and results of RNA interference studies confirmed that PAX3-FOXO1-mediated repression of IL24 is HDAC5-dependent. Knockdown of PAX3-FOXO1 decreases ARMS cell proliferation, survival, and migration, and we also observed similar responses in cells after overexpression of IL24, consistent with results reported for this tumor suppressor-like cytokine in other solid tumors. We also observed in double knockdown studies that the inhibition of ARMS cell proliferation, survival, and migration after knockdown of PAX3-FOXO1 was significantly (>75%) reversed by knockdown of IL24. Adenoviral-expressed IL24 was directly injected into ARMS tumors in athymic nude mice, and this resulted in decreased tumor growth and weight. Because adenoviral IL24 has already successfully undergone phase I in clinical trials, this represents an alternative approach (alone and/or combination) for treating ARMS patients who currently undergo cytotoxic drug therapies.
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Proteína Forkhead Box O1/metabolismo , Interleucinas/metabolismo , Fator de Transcrição PAX3/metabolismo , Rabdomiossarcoma Alveolar/tratamento farmacológico , Rabdomiossarcoma Alveolar/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Camundongos Nus , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Rabdomiossarcoma Alveolar/patologia , Regulação para CimaRESUMO
TGFß induces migration of lung cancer cells (A549, H460, and H1299), dependent on activation of c-Jun N-terminal kinase (JNK1), and is inhibited by the JNK1 inhibitor SP600125. Moreover, TGFß-induced migration of the cells is also blocked by the nuclear export inhibitor leptomycin B (LMB) and the orphan nuclear receptor 4A1 (NR4A1) ligand 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (CDIM8), which retains NR4A1 in the nucleus. Subsequent analysis showed that the TGFß/TGFß receptor/PKA/MKK4 and -7/JNK pathway cascade phosphorylates and induces nuclear export of NR4A1, which in turn forms an active complex with Axin2, Arkadia (RNF111), and RNF12 (RLIM) to induce proteasome-dependent degradation of SMAD7 and enhance lung cancer cell migration. Thus, NR4A1 also plays an integral role in mediating TGFß-induced lung cancer invasion, and the NR4A1 ligand CDIM8, which binds nuclear NR4A1, represents a novel therapeutic approach for TGFß-induced blocking of lung cancer migration/invasion. IMPLICATIONS: Effective treatment of TGFß-induced lung cancer progression could involve a number of agents including the CDIM/NR4A1 antagonists that block not only TGFß-induced migration, but several other NR4A1-regulated prooncogenic genes/pathways in lung cancer cell lines.
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Neoplasias Pulmonares/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Células A549 , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Antracenos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Ácidos Graxos Insaturados/farmacologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genéticaRESUMO
Specificity protein (Sp) transcription factors (TFs) such as Sp1 are critical for early development but their expression decreases with age and there is evidence that transformation of normal cells to cancer cells is associated with upregulation of Sp1, Sp3, and Sp4, which are highly expressed in cancer cells and tumors. Sp1 is a negative prognostic factor for pancreatic, colon, glioma, gastric, breast, prostate, and lung cancer patients. Functional studies also demonstrate that Sp TFs regulate genes responsible for cancer cell growth, survival, migration/invasion, inflammation and drug resistance, and Sp1, Sp3 and Sp4 are also nononcogene addiction (NOA) genes and important drug targets. The mechanisms of drug-induced downregulation of Sp TFs and pro-oncogenic Sp-regulated genes are complex and include ROS-dependent epigenetic pathways that initially decrease expression of the oncogene cMyc. Many compounds such as curcumin, aspirin, and metformin that are active in cancer prevention also exhibit chemotherapeutic activity and these compounds downregulate Sp TFs in cancer cell lines and tumors. The effects of these compounds on downregulation of Sp TFs in normal cells and the contribution of this response to their chemopreventive activity have not yet been determined. Cancer Prev Res; 11(7); 371-82. ©2018 AACR.
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Antineoplásicos/farmacologia , Desenvolvimento de Medicamentos/métodos , Neoplasias/prevenção & controle , Fatores de Transcrição Sp/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Terapia de Alvo Molecular/métodos , Neoplasias/genética , Neoplasias/mortalidade , Neoplasias/patologia , Prognóstico , Fatores de Transcrição Sp/metabolismoRESUMO
Metastasis-associated lung adenocarcinoma transcript-1 (MALAT-1) is a long non-coding RNA (lncRNA) that is a negative prognostic factor for patients with pancreatic cancer and several other tumors. In this study, we show that knockdown of MALAT-1 in Panc1 and other pancreatic cancer cell lines decreases cell proliferation, survival and migration. We previously observed similar results for the lncRNAs HOTTIP and HOTAIR in Panc1 cells; however, RNAseq comparison of genes regulated by MALAT-1 shows minimal overlap with HOTTIP/HOTAIR-regulated genes. Analysis of changes in gene expression after MALAT-1 knockdown shows that this lncRNA represses several tumor suppressor-like genes including N-myc downregulated gene-1 (NDRG-1), a tumor suppressor in pancreatic cancer that is also corepressed by EZH2 (a PRC2 complex member). We also observed that Specificity proteins Sp1, Sp3 and Sp4 are overexpressed in Panc1 cells and Sp knockdown or treatment with small molecules that decrease Sp proteins expression also decrease MALAT-1 expression. We also generated Kras-overexpressing p53L/L;LSL-KrasG12DL/+;p48Cre+/- (p53L/L/KrasG12D) and p53L/+;LSLKrasG12DL/+;p48Cre+/- (p53L/+/KrasG12D) mice which are p53 homo- and heterozygous, respectively. These mice rapidly develop pancreatic ductal adenocarcinoma-like tumors and were crossed with MALAT-1-/- mice. We observed that the loss of one or two MALAT-1 alleles in these Ras overexpressing mice does not significantly affect the time to death; however, the loss of MALAT-1 in the p53-/+ (heterozygote) mice slightly increases their lifespan.
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Biomarcadores Tumorais/genética , Neoplasias Pancreáticas/patologia , RNA Longo não Codificante/genética , Animais , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias Pancreáticas/genéticaRESUMO
Piperlongumine is a natural product found in the plant species Piper longum, and this compound exhibits potent anticancer activity in multiple tumor types and has been characterized as an inducer of reactive oxygen species (ROS). Treatment of Panc1 and L3.6pL pancreatic, A549 lung, 786-O kidney, and SKBR3 breast cancer cell lines with 5 to 15 µmol/L piperlongumine inhibited cell proliferation and induced apoptosis and ROS, and these responses were attenuated after cotreatment with the antioxidant glutathione. Piperlongumine also downregulated expression of Sp1, Sp3, Sp4, and several pro-oncogenic Sp-regulated genes, including cyclin D1, survivin, cMyc, EGFR and hepatocyte growth factor receptor (cMet), and these responses were also attenuated after cotreatment with glutathione. Mechanistic studies in Panc1 cells showed that piperlongumine-induced ROS decreased expression of cMyc via an epigenetic pathway, and this resulted in downregulation of cMyc-regulated miRNAs miR-27a, miR-20a, and miR-17 and induction of the transcriptional repressors ZBTB10 and ZBTB4. These repressors target GC-rich Sp-binding sites to decrease transactivation. This pathway observed for piperlongumine in Panc1 cells has previously been reported for other ROS-inducing anticancer agents and shows that an important underlying mechanism of action of piperlongumine is due to downregulation of Sp1, Sp3, Sp4, and pro-oncogenic Sp-regulated genes. Cancer Prev Res; 10(8); 467-77. ©2017 AACR.
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Antineoplásicos/farmacologia , Dioxolanos/farmacologia , Espécies Reativas de Oxigênio , Fatores de Transcrição Sp/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Fatores de Transcrição Sp/biossínteseRESUMO
Transforming growth factor ß (TGF-ß)-induced migration of triple-negative breast cancer (TNBC) cells is dependent on nuclear export of the orphan receptor NR4A1, which plays a role in proteasome-dependent degradation of SMAD7. In this study, we show that TGF-ß induces p38α (mitogen-activated protein kinase 14 [MAPK14]), which in turn phosphorylates NR4A1, resulting in nuclear export of the receptor. TGF-ß/p38α and NR4A1 also play essential roles in the induction of epithelial-to-mesenchymal transition (EMT) and induction of ß-catenin in TNBC cells, and these TGF-ß-induced responses and nuclear export of NR4A1 are blocked by NR4A1 antagonists, the p38 inhibitor SB202190, and kinase-dead [p38(KD)] and dominant-negative [p38(DN)] forms of p38α. Inhibition of NR4A1 nuclear export results in nuclear export of TGF-ß-induced ß-catenin, which then undergoes proteasome-dependent degradation. TGF-ß-induced ß-catenin also regulates NR4A1 expression through formation of the ß-catenin-TCF-3/TCF-4/LEF-1 complex on the NR4A1 promoter. Thus, TGF-ß-induced nuclear export of NR4A1 in TNBC cells plays an essential role in cell migration, SMAD7 degradation, EMT, and induction of ß-catenin, and all of these pathways are inhibited by bis-indole-derived NR4A1 antagonists that inhibit nuclear export of the receptor and thereby block TGF-ß-induced migration and EMT.
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Transporte Ativo do Núcleo Celular/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Fator de Crescimento Transformador beta/genética , Neoplasias de Mama Triplo Negativas/patologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Feminino , Humanos , Imidazóis/farmacologia , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/biossíntese , Complexos Multiproteicos/biossíntese , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/antagonistas & inibidores , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Fosforilação , Piridinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , Proteína Smad7/metabolismo , Fator de Transcrição 4 , Proteína 1 Semelhante ao Fator 7 de Transcrição/metabolismo , Fatores de Transcrição/metabolismo , beta Catenina/metabolismoRESUMO
ß1-Integrin is highly expressed and is a negative prognostic factor for colon and pancreatic cancer patients and the gene plays a functional role in cell migration and invasion. In this study, we demonstrate that ß1-integrin expression is regulated in pancreatic and colon cancer cells by the pro-oncogenic orphan nuclear receptor 4A1 (NR4A1, Nur77, TR3) and knockdown of this receptor by RNA interference decreases ß1-integrin protein and mRNA expression, α5-integrin, and also expression of ß1-integrin-dependent phosphorylation of FAK (pFak). Knockdown of NR4A1 also decreased migration and fibronectin-induced adhesion in pancreatic (Panc1, L3.6 pL, and MiaPaCa2) and colon (RKO and SW480) cancer cells. 1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methane (C-DIM) compounds containing p-hydroxy (DIM-C-pPhOH) and p-carbomethoxy (DIM-C-pPhCO2 Me) groups are NR4A1 ligands that act as antagonists for this receptor. Treatment of pancreatic and colon cancer cells with DIM-C-pPhOH or DIM-C-pPhCO2 Me mimics the effects of NR4A1 knockdown and decreases ß1-integrin expression, ß1-integrin regulated genes and responses including migration and adhesion. The results demonstrate a novel method for targeting ß1-integrin in colon and pancreatic cancer cells and indicate possible clinical applications for C-DIM/NR4A1 antagonists for pancreatic and colon cancer therapy.
Assuntos
Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica , Integrina beta1/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/antagonistas & inibidores , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Neoplasias Pancreáticas/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Indóis/farmacologia , Fenóis/farmacologia , Fenilacetatos/farmacologiaRESUMO
It was recently demonstrated the penfluridol inhibited breast tumor growth and metastasis and this was associated with downregulation of α6- and ß4-integrins. In this study, we observed the penfluridol induced reactive oxygen species (ROS) and this was the primary mechanism of action. Penfluridol-mediated growth inhibition, induction of apoptosis, and inhibition of breast cancer cell migration was attenuated after cotreatment with glutathione. Penfluridol also downregulated Sp transcription factors Sp1, Sp3, and Sp4 through epigenetic downregulation of cMyc and cMyc-regulated miRNAs (miR27a and miR20a/miR17) and induction of the miR-regulated Sp transcriptional repressors ZBTB10 and ZBTB4. α6- and ß4-integrins as well as α5- and ß1-integrins are Sp-regulated genes that are also coregulated by the orphan nuclear receptor NR4A1 and these integrins can be targeted by agents such as penfluridol that suppress Sp1, Sp3, and Sp4 and also by NR4A1 antagonists. Mol Cancer Ther; 16(1); 205-16. ©2016 AACR.
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Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Integrinas/genética , Penfluridol/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição Sp/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Genes myc , Humanos , MicroRNAs/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Dedos de ZincoRESUMO
The antineoplastic agent benzyl isothiocyanate (BITC) acts by targeting multiple pro-oncogenic pathways/genes, including signal transducer and activator of transcription 3 (STAT3); however, the mechanism of action is not well known. As reported previously, BITC induced reactive oxygen species (ROS) in Panc1, MiaPaCa2, and L3.6pL pancreatic cancer cells. This was accompanied by induction of apoptosis and inhibition of cell growth and migration, and these responses were attenuated in cells cotreated with BITC plus glutathione (GSH). BITC also decreased expression of specificity proteins (Sp) Sp1, Sp3, and Sp4 transcription factors (TFs) and several pro-oncogenic Sp-regulated genes, including STAT3 and phospho-STAT3 (pSTAT3), and GSH attenuated these responses. Knockdown of Sp TFs by RNA interference also decreased STAT3/pSTAT3 expression. BITC-induced ROS activated a cascade of events that included down-regulation of c-Myc, and it was also demonstrated that c-Myc knockdown decreased expression of Sp TFs and STAT3 These results demonstrate that in pancreatic cancer cells, STAT3 is an Sp-regulated gene that can be targeted by BITC and other ROS inducers, thereby identifying a novel therapeutic approach for targeting STAT3.
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Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Isotiocianatos/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fatores de Transcrição Sp/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/uso terapêutico , Fatores de Transcrição Sp/genética , Fatores de Transcrição Sp/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The orphan nuclear receptor NR4A1 is expressed in tumors from rhabdomyosarcoma (RMS) patients and Rh30 and RD RMS cell lines, and we used RNA interference (RNAi) to investigate the role of this receptor in RMS cells. Knockdown of NR4A1 in Rh30 cells decreased cell proliferation, induced Annexin V staining and induced polyADPribose polymerase (PARP) cleavage and these results were similar to those observed in other solid tumors. Previous studies show that NR4A1 regulates expression of growth promoting/pro-survival genes with GC-rich promoters, activates mTOR through suppression of p53, and maintains low oxidative stress by regulating expression of isocitrate dehydrogenase 1 (IDH1) and thioredoxin domain containing 5 (TXNDC5). Results of RNAi studies demonstrated that NR4A1 also regulates these pathways and associated genes in RMS cells and thereby exhibits pro-oncogenic activity. 1,1-Bis(3-indolyl)-1-(p-substituted phenyl)methane (C-DIM) analogs containing p-hydroxyl (DIM-C-pPhOH) and p-carboxymethyl (DIM-C-pPhCO2Me) substituents are NR4A1 ligands that decreased NR4A1-dependent transactivation in RMS cells and inhibited RMS cell and tumor growth and induced apoptosis. Moreover, the effects of NR4A1 knockdown and the C-DIM/NR4A1 antagonists were comparable as inhibitors of NR4A1-dependent genes/pathways. Both NR4A1 knockdown and treatment with DIM-C-pPhOH and DIM-C-pPhCO2Me also induced ROS which activated stress genes and induced sestrin 2 which activated AMPK and inhibited mTOR in the mutant p53 RMS cells. Since NR4A1 regulates several growth-promoting/pro-survival pathways in RMS, the C-DIM/NR4A1 antagonists represent a novel mechanism-based approach for treating this disease alone or in combination and thereby reducing the adverse effects of current cytotoxic therapies.
Assuntos
Indóis/farmacologia , Terapia de Alvo Molecular/métodos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/antagonistas & inibidores , Fenóis/farmacologia , Fenilacetatos/farmacologia , Rabdomiossarcoma/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Nus , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Interferência de RNA , Rabdomiossarcoma/genética , Rabdomiossarcoma/metabolismo , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Specificity protein (Sp) transcription factor (TF) Sp1 is overexpressed in multiple tumors and is a negative prognostic factor for patient survival. Sp1 and also Sp3 and Sp4 are highly expressed in cancer cells and in this study, we have used results of RNA interference (RNAi) to show that the three TFs individually play a role in the growth, survival and migration/invasion of breast, kidney, pancreatic, lung and colon cancer cell lines. Moreover, tumor growth in athymic nude mice bearing L3.6pL pancreatic cancer cells as xenografts were significantly decreased in cells depleted for Sp1, Sp3 and Sp4 (combined) or Sp1 alone. Ingenuity Pathway Analysis (IPA) of changes in gene expression in Panc1 pancreatic cancer cells after individual knockdown of Sp1, Sp3 and Sp4 demonstrates that these TFs regulate genes and pathways that correlated with the functional responses observed after knockdown but also some genes and pathways that inversely correlated with the functional responses. However, causal IPA analysis which integrates all pathway-dependent changes in all genes strongly predicted that Sp1-, Sp3- and Sp4-regulated genes were associated with the pro-oncogenic activity. These functional and genomic results coupled with overexpression of Sp transcription factors in tumor vs. non-tumor tissues and decreased Sp1 expression with age indicate that Sp1, Sp3 and Sp4 are non-oncogene addiction (NOA) genes and are attractive drug targets for individual and combined cancer chemotherapies.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Neoplasias/genética , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3/genética , Fator de Transcrição Sp4/genética , Animais , Linhagem Celular Tumoral , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Neoplasias/patologiaRESUMO
Overexpression of the nuclear receptor 4A1 (NR4A1) in breast cancer patients is a prognostic factor for decreased survival and increased metastasis, and this has been linked to NR4A1-dependent regulation of transforming growth factor ß (TGF-ß) signaling. Results of RNA interference studies demonstrate that basal migration of aggressive SKBR3 and MDA-MB-231 breast cancer cells is TGF-ß independent and dependent on regulation of ß1-integrin gene expression by NR4A1 which can be inhibited by the NR4A1 antagonists 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) and a related p-carboxymethylphenyl [1,1-bis(3'-indolyl)-1-(p-carboxymethylphenyl)methane (DIM-C-pPhCO2Me)] analog. The NR4A1 antagonists also inhibited TGF-ß-induced migration of MDA-MB-231 cells by blocking nuclear export of NR4A1, which is an essential step in TGF-ß-induced cell migration. We also observed that NR4A1 regulates expression of both ß1- and ß3-integrins, and unlike other ß1-integrin inhibitors which induce prometastatic ß3-integrin, NR4A1 antagonists inhibit expression of both ß1- and ß3-integrin, demonstrating a novel mechanism-based approach for targeting integrins and integrin-dependent breast cancer metastasis.
Assuntos
Neoplasias da Mama/patologia , Indóis/farmacologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/antagonistas & inibidores , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Fenóis/farmacologia , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Feminino , Humanos , Integrina beta1/metabolismo , Integrina beta3/metabolismo , Camundongos Endogâmicos BALB C , Fenilacetatos/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The orphan nuclear receptor 4A1 (NR4A1) is overexpressed in mammary tumors and breast cancer cell lines. The functional activity of this receptor was investigated by RNA interference with oligonucleotides targeted to NR4A1 (siNR4A1) and by treatment with NR4A1 antagonists. Breast cancer cells were treated with NR4A1 antagonists or transfected with siNR4A. Effects on cell proliferation and apoptosis as well as specific genes associated with these responses were investigated in MCF-7, SKBR3, and MDA-MB-231 cells, and in athymic nude mice bearing MDA-MB-231 cells as xenografts. Transfection of MCF-7, MDA-MB-231, and SKBR3 breast cancer cells with siNR4A1 decreased cell proliferation and induced apoptosis in these cell lines. Transfection of breast cancer cells with siNR4A1 also decreased expression of Sp-regulated genes including survivin, bcl-2, and epidermal growth factor receptor, inhibited mTOR signaling in MCF-7 cells that express WT p53, and activated oxidative and endoplasmic reticulum stress through downregulation of thioredoxin domain-containing 5 and isocitrate dehydrogenase 1. 1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes (C-DIMs) are NR4A1 ligands that act as NR4A1 antagonists. Treatment with selected analogs also inhibited breast cancer cell and tumor growth and induced apoptosis. The effects of C-DIM/NR4A1 antagonists were comparable to those observed after NR4A1 knockdown. Results with siNR4A1 or C-DIMs/NR4A1 antagonists in breast cancer cells and tumors were similar to those previously reported in pancreatic, lung, and colon cancer cells. They demonstrate the potential clinical applications of NR4A1 antagonists in patients with tumors that overexpress this receptor.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Indóis/farmacologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/antagonistas & inibidores , Fenóis/farmacologia , RNA Interferente Pequeno/genética , Animais , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The two major types of rhabdomyosarcoma (RMS) are predominantly diagnosed in children, namely embryonal (ERMS) and alveolar (ARMS) RMS, and patients are treated with cytotoxic drugs, which results in multiple toxic side effects later in life. Therefore, development of innovative chemotherapeutic strategies is imperative, and a recent genomic analysis suggested the potential efficacy of reactive oxygen species (ROS)-inducing agents. Here, we demonstrate the efficacy of the potent histone deacetylase (HDAC) inhibitors, panobinostat and vorinostat, as agents that inhibit RMS tumor growth in vivo, induce apoptosis, and inhibit invasion of RD and Rh30 RMS cell lines. These effects are due to epigenetic repression of cMyc, which leads to decreased expression of cMyc-regulated miRs-17, -20a, and -27a; upregulation of ZBTB4, ZBTB10, and ZBTB34; and subsequent downregulation of Sp transcription factors. We also show that inhibition of RMS cell growth, survival and invasion, and repression of Sp transcription factors by the HDAC inhibitors are independent of histone acetylation but reversible after cotreatment with the antioxidant glutathione. These results show a novel ROS-dependent mechanism of antineoplastic activity for panobinostat and vorinostat that lies outside of their canonical HDAC-inhibitory activity and demonstrates the potential clinical utility for treating RMS patients with ROS-inducing agents.