RESUMEN
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is activated by environmental contaminants such as dioxins and polycyclic aromatic hydrocarbons. Following ligand binding, AhR binds to xenobiotic responsive elements and modulates the transcription of AhR target genes. Multiple studies have shown that AhR plays important roles in a range of cancer cells and is attracting attention as a therapeutic target for cancer treatment. We have previously reported that AhR agonists inhibit tumorsphere formation in an AhR-dependent manner in the MCF-7 breast cancer cell line. In the present study, we found that FDI-6, an inhibitor of the transcription factor Forkhead Box M1 (FOXM1) induced the mRNA expression of AhR target genes, nuclear translocation of AhR, and transcriptional activity of AhR. In addition, FDI-6 dose-dependently reduced the mRNA expression of FOXM1-regulated genes in AhR-expressing MCF-7 cells, although not in AhR-deficient MCF-7 cells. Furthermore, FDI-6 was found to suppress tumorsphere formation via the AhR in MCF-7 cells and HepG2 human liver cancer cell line. On the basis of the findings of this study, we show that FDI-6, a FOXM1 inhibitor, functions as an AhR agonist, and suppresses tumorsphere formation via the AhR.
Asunto(s)
Citocromo P-450 CYP1A1 , Receptores de Hidrocarburo de Aril , Humanos , Línea Celular Tumoral , Citocromo P-450 CYP1A1/genética , Proteína Forkhead Box M1/genética , Ligandos , Células MCF-7 , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity of dioxins and polycyclic aromatic hydrocarbons. Recent studies have suggested that AhR is involved in cancer immunity. In the present study, we examined whether AhR regulates the expression of immune checkpoint genes in breast cancer cells. We discovered that the mRNA expression of V-set domain containing T cell activation inhibitor 1 (VTCN1) that negatively regulates T cell immunity was upregulated by AhR agonists in breast cancer cell lines, MCF-7 and T47D. Furthermore, AhR knockout or knockdown experiments clearly demonstrated that upregulation of VTCN1 gene expression by 3-methylcholanthrene was AhR dependent. Luciferase reporter and chromatin immunoprecipitation assays revealed that this upregulation of VTCN1 gene expression was induced by the recruitment of AhR to the AhR responsive element in the VTCN1 gene promoter in MCF-7 cells. Taken together, AhR directly regulates VTCN1 gene expression in MCF-7 cells.
Asunto(s)
Neoplasias de la Mama , Receptores de Hidrocarburo de Aril , Inhibidor 1 de la Activación de Células T con Dominio V-Set , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Neoplasias de la Mama/genética , Femenino , Expresión Génica , Humanos , Células MCF-7 , Metilcolantreno/toxicidad , Receptores de Hidrocarburo de Aril/genética , Inhibidor 1 de la Activación de Células T con Dominio V-Set/genéticaRESUMEN
Breast cancer is the most commonly diagnosed cancer among women worldwide. Despite a variety of drugs available for the treatment of patients with breast cancer, drug resistance remains a significant clinical problem. Therefore, there is an urgent need to develop drugs with new mechanisms of action. Camalexin is the main indole phytoalexin in Arabidopsis thaliana and other crucifers. Camalexin inhibits the proliferation of various cancer cells. However, the mechanism by which camalexin inhibits cell proliferation remains unclear. In this study, we found that camalexin inhibited cell proliferation and migration of breast cancer cell lines. Furthermore, camalexin also suppressed breast cancer stem cell-derived mammosphere formation. We previously reported that the ligand-activated transcription factor aryl hydrocarbon receptor (AhR) agonist suppresses mammosphere formation. Several compounds with indole structures are known to act as AhR agonists. Therefore, we hypothesized that the inhibition of mammosphere formation by camalexin may involve AhR activation. We found that camalexin increased the nuclear translocation of AhR, AhR-mediated transcriptional activation, and expression of AhR target genes. In addition, camalexin suppressed mammosphere formation in AhR-expressing breast cancer cells more than in the breast cancer cells that lacked AhR expression. Taken together, the data demonstrate that camalexin is a novel AhR agonist and that the inhibition of cell proliferation, migration, and mammosphere formation by camalexin involves the activation of AhR. Our findings suggest that camalexin, an AhR agonist, may be a novel therapeutic agent for breast cancer.
Asunto(s)
Neoplasias de la Mama , Receptores de Hidrocarburo de Aril , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Indoles/farmacología , Receptores de Hidrocarburo de Aril/genética , Sesquiterpenos , Tiazoles , FitoalexinasRESUMEN
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates various toxicological and biological functions. We reported previously that 3-methylcholanthrene (3MC), an exogenous AhR agonist, inhibited tumorsphere (mammosphere) formation from breast cancer cell lines, while the endogenous AhR agonist, indirubin, very weakly inhibited this process. However, the difference in inhibition mechanism of mammosphere formation by 3MC or indirubin is still unknown. In this study, we established AhR-re-expressing (KOTR-AhR) cells from AhR knockout MCF-7 cells using the tetracycline (Tet)-inducible gene expression systems. To identify any difference in inhibition of mammosphere formation by 3MC or indirubin, RNA-sequencing (RNA-seq) experiments were performed using KOTR-AhR cells. RNA-seq experiments revealed that cell division cycle 20 (CDC20), which regulates the cell cycle and mitosis, was decreased by 3MC, but not by indirubin, in the presence of AhR expression. Furthermore, the mRNA and protein levels of CDC20 were decreased by 3MC in MCF-7 cells via the AhR. In addition, mammosphere formation was suppressed by small interfering RNA-mediated CDC20 knockdown compared to the negative control in MCF-7 cells. These results suggest that AhR activation by 3MC suppresses mammosphere formation via downregulation of CDC20 expression in breast cancer cells. This study provides useful information for the development of AhR-targeted anti-cancer drugs.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas Cdc20/metabolismo , Regulación hacia Abajo , Metilcolantreno/farmacología , Receptores de Hidrocarburo de Aril/metabolismo , Esferoides Celulares/patología , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Indoles/farmacología , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismo , Transcriptoma/genéticaRESUMEN
The aryl hydrocarbon receptor (AhR) regulates expression of genes encoding drug/xenobiotic metabolizing enzymes. Cytochrome P450 (CYP) 3A5 is involved in drug metabolism. However, regulation of CYP3A5 gene expression is not yet well understood. In this study, we aimed to investigate the effect of the ligands of AhR on CYP3A5 gene expression. CYP3A5 mRNA expression was induced by the polycyclic aromatic hydrocarbons (PAHs) such as 3-methylcholanthrene (3MC) and benzo[a]pyrene in HepG2 cells. To determine whether the PAHs induced CYP3A5 gene expression via AhR, we generated AhR knockout (AhR KO) HepG2 cells. CYP3A5 mRNA expression was not induced by 3MC treatment in AhR KO cells. In addition, we generated AhR rescue cells from AhR KO cells and evaluated CYP3A5 mRNA expression. We found that CYP3A5 mRNA expression was induced by 3MC treatment in AhR rescue cells. Taken together, these results demonstrated that CYP3A5 mRNA expression was induced by PAHs via AhR in HepG2 cells. Our findings suggest that ligand-activated AhR affects CYP3A5-mediated drug metabolism.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Hidrocarburos Policíclicos Aromáticos/toxicidad , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Células Hep G2 , Humanos , Ligandos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear protein that is activated by binding to DNA lesions and catalyzes poly(ADP-ribosyl)ation of nuclear acceptor proteins, including PARP1 itself, to recruit DNA repair machinery to DNA lesions. When excessive DNA damage occurs, poly(ADP-ribose) (PAR) produced by PARP1 is translocated to the cytoplasm, changing the activity and localization of cytoplasmic proteins, e.g., apoptosis-inducing factor (AIF), hexokinase, and resulting in cell death. This cascade, termed parthanatos, is a caspase-independent programmed cell death distinct from necrosis and apoptosis. In contrast, PARP1 is a substrate of activated caspases 3 and 7 in caspase-dependent apoptosis. Once cleaved, PARP1 loses its activity, thereby suppressing DNA repair. Caspase cleavage of PARP1 occurs within a nuclear localization signal near the DNA-binding domain, resulting in the formation of 24-kDa and 89-kDa fragments. In the present study, we found that caspase activation by staurosporine- and actinomycin D-induced PARP1 autopoly(ADP-ribosyl)ation and fragmentation, generating poly(ADP-ribosyl)ated 89-kDa and 24-kDa PARP1 fragments. The 89-kDa PARP1 fragments with covalently attached PAR polymers were translocated to the cytoplasm, whereas 24-kDa fragments remained associated with DNA lesions. In the cytoplasm, AIF binding to PAR attached to the 89-kDa PARP1 fragment facilitated its translocation to the nucleus. Thus, the 89-kDa PARP1 fragment is a PAR carrier to the cytoplasm, inducing AIF release from mitochondria. Elucidation of the caspase-mediated interaction between apoptosis and parthanatos pathways extend the current knowledge on mechanisms underlying programmed cell death and may lead to new therapeutic targets.
Asunto(s)
Factor Inductor de la Apoptosis/metabolismo , Apoptosis , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Proteolisis , Factor Inductor de la Apoptosis/genética , Transporte Biológico Activo , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 7/genética , Caspasa 7/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Células HeLa , Humanos , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli Adenosina Difosfato Ribosa/genéticaRESUMEN
Aryl hydrocarbon receptor (AhR) and androgen receptor (AR) are ligand-activated transcription factors with profound cross-talk between their signal transduction pathways. Previous studies have shown that AhR agonists activate the transcription of AR-regulated genes in an androgen-independent manner; however, the underlying mechanism remains unclear. To decipher this mechanism, we evaluated the effects of 3-methylcholanthrene (3MC), a potent AhR agonist, on the transcription of AR-regulated genes in three AR-expressing cell lines. 3MC induced the expression of not only three representative AR-regulated chromosomal genes but also the exogenous AR-responsive luciferase reporter gene. No significant difference in the 3MC-induced luciferase activity was detected in the presence of SKF-525A, a non-specific inhibitor of CYP enzymes. The androgenic effects of 3MC were diminished by AhR and AR knockdown. Following 3MC treatment, the amount of nuclear AhR and AR increased synchronously. Co-immunoprecipitation revealed that AhR and AR formed a complex in the nucleus of cells treated with 3MC. AR was recruited to the proximal promoter and distal enhancer regions of the PSA gene upon the addition of 3MC. We propose that AhR activated by 3MC forms a complex with unliganded AR which translocates from the cytoplasm to the nucleus. Nuclear AR now binds the transcriptional regulatory region of AR-regulated genes and activates the transcription.
RESUMEN
Triple-negative breast cancer (TNBC) is associated with poor prognosis, because of no effective targeted therapy. In the present study, we demonstrated the crucial role of the aryl hydrocarbon receptor (AhR) in mediating the effects of the chemotherapeutic agent doxorubicin (DOX) in the chemotherapeutic sensitivity of TNBC. Firstly, we established AhR knockout (KO) MDA-MB 231 TNBC cells. The cytotoxic effects of DOX were more pronounced in AhR KO cells than in parental cells. In addition, our results indicated that AhR KO cells showed downregulated expression of DOX-metabolism enzyme, aldo-keto reductase (AKR) 1C3, relative to those of parental cells. Furthermore, AhR was found to enhance AKR1C3 promoter reporter activity, suggesting that AKR1C3 mRNA transcription is activated by AhR. Additionally, our findings confirmed that the downregulation of AKR1C3 expression enhanced DOX sensitivity in MDA-MB 231â¯cells. Finally, AhR and AKR1C3 expression were positively correlated in human breast cancer. Taken together, our results suggested that AhR is involved in DOX sensitivity by regulating AKR1C3 expression in TNBC cells.
Asunto(s)
Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas/genética , Doxorrubicina/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Receptores de Hidrocarburo de Aril/genética , Neoplasias de la Mama Triple Negativas/genética , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Femenino , Técnicas de Inactivación de Genes , Humanos , Receptores de Hidrocarburo de Aril/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Polycyclic aromatic hydrocarbons (PAHs) and dioxins are ubiquitous environmental pollutants and activate the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. It has been reported that testosterone represses 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced transcription of the cytochrome P450 (CYP) 1A1 gene in LNCaP cells. In this study, we investigated the mechanism for the repression of 3-methylcholanthrene (3MC)-induced transcription of AhR-regulated genes, CYP1A1, CYP1A2, CYP1B1, and AhR repressor (AhRR), by 5alpha-dihydroteststerone (DHT) in LNCaP and T47D cells, which are androgen receptor (AR)- and AhR-positive. Real-time PCR analysis showed that DHT repressed 3MC-induced mRNA expression of the CYP1 family and AhRR genes. DHT repressed 3MC-induced luciferase activity in an AhR response element-driven luciferase reporter assay in LNCaP and T47D cells. The inhibitory effect of DHT was abolished by knockdown of AR protein with siRNA. The protein levels of AhR and AhR nuclear translocator (Arnt), the AhR-dimerizing partner, were not affected by DHT. Co-immunoprecipitation assay showed that DHT significantly facilitated the complex formation between AR and AhR in 3MC-treated cells. These results suggest that complex formation between activated AR and AhR plays an important role in the suppression of 3MC-induced transcription of CYP1 family genes by DHT.
Asunto(s)
Andrógenos/farmacología , Dihidrotestosterona/farmacología , Metilcolantreno/toxicidad , Transcripción Genética/efectos de los fármacos , Hidrocarburo de Aril Hidroxilasas/efectos de los fármacos , Hidrocarburo de Aril Hidroxilasas/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Citocromo P-450 CYP1A1/efectos de los fármacos , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/efectos de los fármacos , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1B1 , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunoprecipitación , Luciferasas/efectos de los fármacos , Luciferasas/metabolismo , Masculino , Reacción en Cadena de la Polimerasa , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas Represoras/efectos de los fármacos , Proteínas Represoras/genéticaRESUMEN
We have previously identified Ntan1 as a magnetism response gene by differential display screening in cultured rat hippocampal neurons. Ntan1 mRNA was ubiquitously expressed in all the mouse tissues examined but relatively abundant in brain, retina and testis. Ntan1 mRNA expression was detectable in the embryonic 12-day mouse brain and gradually increased with ageing. In situ hybridization analysis showed high localization of Ntan1 mRNA in pyramidal cell layer of CA region and granular cell layer of dentate gyrus in the hippocampus, and Purkinje and granular cell layers in the cerebellum, respectively. Ntan1 mRNA expression was significantly increased about two-fold 12 h after brief exposure for 15 min to magnetism at 100 mT with a gradual decrease thereafter in cultured mouse hippocampal neurons. When embryonic 12-day-old or newborn mice were successively exposed to magnetic fields at 100 mT for 2 h, four times per day until the postnatal seventh day, Ntan1 mRNA was significantly increased about 1.5-2-fold in the hippocampus in vivo. The mice exposed to magnetic fields under the same condition showed significantly decreased locomotor activity. These results suggest that magnetic exposure affects higher order neural functions through modulation of genes expression.
Asunto(s)
Amidohidrolasas/genética , Química Encefálica/genética , Campos Electromagnéticos , Animales , Animales Recién Nacidos , Conducta Animal/efectos de la radiación , Northern Blotting , Células Cultivadas , Femenino , Expresión Génica/efectos de la radiación , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos ICR , Actividad Motora/fisiología , Neuronas/efectos de la radiación , Embarazo , ARN Complementario/biosíntesis , ARN Complementario/genética , ARN Complementario/metabolismo , Distribución TisularRESUMEN
The vitamin D receptor (VDR) is believed to mediate different biologic actions of vitamin D3, an active metabolite of vitamin D, through regulation of gene expression after binding to specific DNA-response element (VDRE) on target genes. To further understand roles of both vitamin D3 and VDR in the central nervous system, we examined VDRE binding in nuclear extracts prepared from discrete rat brain regions and cultured rat cortical neurons by electrophoretic mobility shift assay. The highest activity of VDRE binding was found in the cerebellum among other brain regions examined, but sequence specific by taking into consideration the efficient competition with excess unlabeled VDRE but not with mutated VDRE. On in situ hybridization analysis, cells stained for VDR mRNA were abundant in neuron-enriched areas of cerebral cortex, hippocampus and cerebellar cortex in the mouse brain. Chronic treatment of vitamin D3 increased the expression of microtubule-associated protein-2, growth-associated protein-43 and synapsin-1 in cultured rat cortical neurons, suggesting a trophic role of vitamin D3 in differentiation and maturation of neurons. Neuronal cell death by brief glutamate exposure was significantly protected in cultured cortical neurons chronically treated with vitamin D3. Parallel studies showed that VDR mRNA was significantly upregulated 12-24 hr after brief glutamate exposure in cultured neurons chronically treated with vitamin D3, but not in those with vehicle alone. Our results suggest that vitamin D3 may play a role in mechanisms relevant to protective properties against the neurotoxicity of glutamate through upregulation of VDR expression in cultured rat cortical neurons.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Colecalciferol/farmacología , Ácido Glutámico/toxicidad , Neurotoxinas/antagonistas & inhibidores , Receptores de Calcitriol/genética , Animales , Animales Recién Nacidos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Células Cultivadas , Colecalciferol/metabolismo , Citoprotección/efectos de los fármacos , Citoprotección/fisiología , Esquema de Medicación , Proteína GAP-43/efectos de los fármacos , Proteína GAP-43/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Proteínas Asociadas a Microtúbulos/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/metabolismo , Fármacos Neuroprotectores/farmacología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Elementos de Respuesta/efectos de los fármacos , Elementos de Respuesta/genética , Sinapsinas/efectos de los fármacos , Sinapsinas/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Metabotropic glutamate receptors (mGluRs) are a class of G-protein-coupled receptors that possess a seven transmembrane region involved in the modulation of excitatory synaptic transmission in the nervous system. mGluR orthologs have been identified in Drosophila, Caenorhabditis elegans, and higher organisms. Drosophila possesses two mGluR genes, DmGluRA and DmXR. We screened the Dictyostelium genome data base using the ligand binding domain of rat mGluR1 as bait, and identified a new receptor, DdmGluPR, belonging to the mGluR family. Similar to Drosophila DmXR, the residues of mGluRs involved in the binding of the alpha-carboxylic and alpha-amino groups of glutamate were well conserved in DdmGluPR, but the residues interacting with the gamma-carboxylic group of glutamate were not. The phylogenetic analysis suggests that DdmGluPR diverged after the mGluR family-GABA(B) receptors split but before mGluR family divergence. DdmGluPR mRNA was expressed in vegetative cells and throughout starvation-induced development, but the level of the expression was relatively high until 4 h after starvation. DdmGluPR was localized to the plasma membrane of axenically grown Ax-2 cells expressed as a green fluorescent protein fusion protein. DdmGluPR-null cells grew faster at high cell density and reached higher densities than wild-type cells. DdmGluPR-null cells exhibited delayed aggregates formation upon starvation and impaired chemotaxis toward cAMP. Although expressions of cAR1 and aca, cAMP-signaling components, were rapidly induced and peaked at 2-4 h in wild-type cells, DdmGluPR-null cells displayed sustained and peaked at 8 h of the expressions of these genes. Our findings suggest the involvement of DdmGluPR in the early development of Dictyostelium discoideum.