RESUMEN
The initiation of primary cilium assembly entails the docking of ciliary vesicles presumably derived from the Golgi complex to the distal end of the mother centriole. Distal appendages, which anchor the mother centriole to the plasma membrane, are thought to be involved in the docking process. However, little is known about the molecular players and mechanisms that mediate the vesicle-centriole association. Here we report that coiled-coil domain containing 41 (CCDC41) is required for the docking of ciliary vesicles. CCDC41 specifically localizes to the distal end of the mother centriole and interacts with centrosomal protein 164 (Cep164), a distal appendage component. In addition, a pool of CCDC41 colocalizes with intraflagellar transport protein 20 (IFT20) subunit of the intraflagellar transport particle at the Golgi complex. Remarkably, knockdown of CCDC41 inhibits the recruitment of IFT20 to the centrosome. Moreover, depletion of CCDC41 or IFT20 inhibits ciliogenesis at the ciliary vesicle docking step, whereas intraflagellar transport protein 88 (IFT88) depletion interferes with later cilium elongation steps. Our results suggest that CCDC41 collaborates with IFT20 to support the vesicle-centriole association at the onset of ciliogenesis.
Asunto(s)
Centriolos/metabolismo , Cilios/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Centriolos/ultraestructura , Centrosoma/metabolismo , Aparato de Golgi/metabolismo , Humanos , Hibridación in Situ , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Epitelio Pigmentado de la Retina , Pez CebraRESUMEN
BACKGROUND: Breast cancer is the most common type of malignancy in women worldwide. Euphorbia humifusa Willd (EuH) is a plant that is widely used as a traditional medicine. However, no systemic studies on the anti-cancer effects of EuH have been reported. The aim of this study is to evaluate the anti-metastatic effect of the EuH. METHODS: Ethyl acetate fraction was prepared from EuH methanol extracts (EA/EuH). Inhibitory effect of EA/EuH on cell migration was determined using an in vitro scratch-wound healing assay. The anti-invasive activity was determined by in vitro three-dimensional spheroid culture system and in vivo syngenic experimental lung metastasis experiment. Gene expression profiles were analyzed by using RT-PCR, real-time PCR, and luciferase reporter assay systems. RESULTS: Ethyl acetate fraction from the EuH extract (EA/EuH) inhibited the migration and invasive capabilities of highly metastatic MDA-MB-231 breast cancer cells and attenuated syngeneic lung metastasis of mouse 4 T1 breast cancer cells in vivo. Mechanistically, EA/EuH decreased tumor necrosis factor alpha (TNFα)-induced matrix metalloproteinase (MMP)-9 mRNA expression through the inhibition of NF-κB activity in MDA-MB-231 cells. CONCLUSION: EuH may be beneficial in the prevention of invasion and metastasis of early stage breast cancer and can be served as an anti-metastatic agent or adjuvant therapy against metastatic breast cancer.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Euphorbia/química , Metaloproteinasa 9 de la Matriz/metabolismo , Extractos Vegetales/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Antineoplásicos/química , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Femenino , Humanos , Ratones , Invasividad Neoplásica , Extractos Vegetales/química , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cyclin-dependent kinase inhibitor (CDKN1A), often referred to as p21(Waf1/Cip1) (p21), is induced by a variety of environmental stresses. Transcription factor ELK-1 is a member of the ETS oncogene superfamily. Here, we show that ELK-1 directly trans-activates the p21 gene, independently of p53 and EGR-1, in sodium arsenite (NaASO(2))-exposed HaCaT cells. Promoter deletion analysis and site-directed mutagenesis identified the presence of an ELK-1-binding core motif between -190 and -170 bp of the p21 promoter that confers inducibility by NaASO(2). Chromatin immunoprecipitation and electrophoretic mobility shift analyses confirmed the specific binding of ELK-1 to its putative binding sequence within the p21 promoter. In addition, NaASO(2)-induced p21 promoter activity was enhanced by exogenous expression of ELK-1 and reduced by expression of siRNA targeted to ELK-1 mRNA. The importance of ELK-1 in response to NaASO(2) was further confirmed by the observation that stable expression of ELK-1 siRNA in HaCaT cells resulted in the attenuation of NaASO(2)-induced p21 expression. Although ELK-1 was activated by ERK, JNK, and p38 MAPK in response to NaASO(2), ELK-1-mediated activation of the p21 promoter was largely dependent on ERK. In addition, EGR-1 induced by ELK-1 seemed to be involved in NaASO(2)-induced expression of BAX. This supports the view that the ERK/ELK-1 cascade is involved in p53-independent induction of p21 and BAX gene expression.
Asunto(s)
Arsenitos/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Elementos de Respuesta , Compuestos de Sodio/farmacología , Proteína X Asociada a bcl-2/biosíntesis , Proteína Elk-1 con Dominio ets/metabolismo , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Activación Enzimática/efectos de los fármacos , Humanos , Queratinocitos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteína X Asociada a bcl-2/genética , Proteína Elk-1 con Dominio ets/genéticaRESUMEN
Purpose: We compared the radiation dose and image quality between the 2nd generation and the 3rd generation dual-source single-energy (DSSE) and dual-source dual-energy (DSDE) CT of the abdomen. Materials and Methods: We included patients undergoing follow-up abdominal CT after partial or radical nephrectomy in the first 10 months of 2019 (2nd generation DS CT) and the first 10 months of 2020 (3rd generation DS CT). We divided the 320 patients into 4 groups (A, 2nd generation DSSE CT; B, 2nd generation DSDE CT; C, 3rd generation DSSE CT; and D, 3rd generation DSDE CT) (n = 80 each) matched by sex and body mass index. Radiation dose and image quality (objective and subjective qualities) were compared between the groups. Results: The mean size-specific dose estimation of 3rd generation DSDE CT group was significantly lower than that of the 2nd generation DSSE CT (42.5%, p = 0.013) and 2nd generation DSDE CT (46.9%, p = 0.015) groups. Interobserver agreement was excellent for the overall image quality (intraclass correlation coefficient [ICC]: 0.8867) and image artifacts (ICC: 0.9423). Conclusion: Our results showed a considerable reduction in the radiation dose while maintaining high image quality with 3rd generation DSDE CT as compared to the 2nd generation DSDE CT and 2nd generation DSSE CT.
RESUMEN
NANOG is a homeodomain-containing transcription factor that is essential for the maintenance of pluripotency and self-renewal in embryonic stem cells. However, the molecular mechanisms underlying the regulation of NANOG expression in human cells remain largely unknown. Here, we investigated the role of Tcf/Lef response elements located in the enhancer of the human NANOG gene. We found that forced expression of Lef1 or ß-catenin stimulated human NANOG promoter activity, while shRNA-mediated knockdown of ß-catenin reduced Lef1-induced NANOG promoter activation. Deletion or mutation of the Tcf/Lef element within the enhancer region of the human NANOG gene completely abrogated Lef1-induced NANOG promoter activity. The results of a chromatin immunoprecipitation assay demonstrated that Lef1 and ß-catenin bind to the Tcf/Lef element in the enhancer region of the NANOG gene. Forced expression of GSK-3ß inhibited basal, Lef1-induced, and ß-catenin-induced NANOG promoter activity, while treatment with the GSK-3ß inhibitor SB216763 resulted in the accumulation of ß-catenin and NANOG protein. Furthermore, Dvl-1-induced NANOG promoter activity was abrogated by the expression of ß-catenin shRNA. Stable overexpression of Dvl-1 caused ß-catenin and NANOG to accumulate. These results indicate that the Tcf/Lef response element in the enhancer region of the human NANOG gene is able to stimulate NANOG gene transcription.
Asunto(s)
Elementos de Facilitación Genéticos , Proteínas de Homeodominio/genética , Regiones Promotoras Genéticas , Factores de Transcripción TCF/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Dishevelled , Genes Reporteros , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Células HEK293 , Humanos , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Proteína Homeótica Nanog , Fosfoproteínas/metabolismo , Transcripción Genética , Activación Transcripcional , beta Catenina/metabolismoRESUMEN
The gallbladder (GB) is a rare site of renal cell carcinoma (RCC) metastasis. To the best of our knowledge, only a few reports of CT findings of GB metastasis exist in the literature. Herein, we report a case of histologically proven GB metastasis of RCC in a 55-year-old male who underwent CT for an intraluminal polypoid mass simulating a primary GB lesion.
RESUMEN
Glial cell line-derived neurotrophic factor (Gdnf) promotes neurite outgrowth and survival of neuronal cells, but its transcriptional regulation is poorly understood. Here, we sought to investigate the mechanism underlying fibroblast growth factor-2 (FGF2) induction of Gdnf expression in astrocytes. We found that FGF2 stimulation of rat astrocytes induced expression of Egr-1 at a high level. Sequence analysis of the rat Gdnf gene identified three overlapping Egr-1-binding sites between positions -185 and -163 of the rat Gdnf promoter. Transfection studies using a series of deleted Gdnf promoters revealed that these Egr-1-binding sites are required for maximal activation of the Gdnf promoter by FGF2. Chromatin immunoprecipitation analysis indicated that Egr-1 binds to the Gdnf promoter. Furthermore, the induction of Gdnf expression by FGF2 is strongly attenuated both in C6 glioma cells stably expressing Egr-1-specific small interfering RNA and in primary cultured astrocytes from the Egr-1 knock-out mouse. Additionally, we found that stimulation of the ERK and JNK pathways by FGF2 is functionally linked to Gdnf expression through the induction of Egr-1. These data demonstrate that FGF2-induced Gdnf expression is mediated by the induction of Egr-1 through activation of the ERK and JNK/Elk-1 signaling pathways.
Asunto(s)
Astrocitos/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/fisiología , Factor 2 de Crecimiento de Fibroblastos/fisiología , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Activación Transcripcional , Animales , Sitios de Unión , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , Ratas , Transducción de Señal , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
Curcumin, a constituent of turmeric, has anti-inflammatory, anti-carcinogenic, and chemopreventive effects in several animal tumor models. The expression of P-glycoprotein (P-gp), encoded by the mdr gene, is often associated with multidrug resistance (MDR) to unrelated chemotherapeutic drugs in cancer cells. Here, we demonstrate that curcumin down-regulates P-gp expression in multidrug-resistant L1210/Adr cells. Transfection with a series of 5'-deleted constructs of the mdr1b gene promoter indicated that a proximal region between -205 and +42 of the sequence was responsible for the suppression of promoter activity by curcumin. This response might be associated with the inhibition of the phosphatidyinositol 3-kinase (PI3K)/Akt/nuclear factor-kappa B (NF-kappa B) signaling pathway by curcumin. Moreover, curcumin reversed the MDR of the L1210/Adr cells. Thus, curcumin can contribute to the reversal of the MDR phenotype, probably due to the suppression of P-gp expression via the inhibition of the PI3K/Akt/NF-kappa B signaling pathway.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Antineoplásicos Fitogénicos/farmacología , Curcumina/farmacología , Leucemia L1210/tratamiento farmacológico , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Antibióticos Antineoplásicos/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Línea Celular Tumoral , Curcumina/uso terapéutico , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Doxorrubicina/farmacología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Leucemia L1210/enzimología , Leucemia L1210/metabolismo , Ratones , FN-kappa B/antagonistas & inhibidores , Células 3T3 NIH , Inhibidores de las Quinasa Fosfoinosítidos-3 , Regiones Promotoras Genéticas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Eliminación de Secuencia , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Transfección , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Expression of protein kinase C-delta (PKCdelta) is up-regulated by apoptosis-inducing stimuli. However, very little is known about the signaling pathways that control PKCdelta gene transcription. In the present study, we demonstrate that JNK stimulates PKCdelta gene expression via c-Jun and ATF2 in response to the anticancer agent doxorubicin (DXR) in mouse lymphocytic leukemia L1210 cells. Luciferase reporter assays showed that DXR-induced activation of the PKCdelta promoter was enhanced by ectopic expression of JNK1, c-Jun, or ATF2, whereas it was strongly reduced by expression of dominant negative JNK1 or by treatment with the JNK inhibitor SP600125. Furthermore, point mutations in the core sequence of the c-Jun/ATF2 binding site suppressed DXR-induced activation of the PKCdelta promoter. Our results suggest an additional role for a JNK signaling cascade in DXR-induced PKCdelta gene expression.
Asunto(s)
Factor de Transcripción Activador 2/fisiología , Antibióticos Antineoplásicos/farmacología , Doxorrubicina/farmacología , Proteína Quinasa 8 Activada por Mitógenos/fisiología , Proteína Quinasa C-delta/metabolismo , Proteínas Proto-Oncogénicas c-jun/fisiología , Animales , Antracenos/farmacología , Apoptosis , Línea Celular Tumoral , Ratones , Mutación , Regiones Promotoras Genéticas , Proteína Quinasa C-delta/genética , Proteínas Proto-Oncogénicas c-jun/antagonistas & inhibidores , Transducción de Señal/fisiología , Transcripción GenéticaRESUMEN
Tamoxifen (TAM) is a synthetic non-steroidal anti-estrogen compound that is widely used as an effective chemotherapeutic agent for treatment and prevention of breast cancer. Unfortunately, prolonged treatment with TAM causes TAM-responsive tumors to become TAM resistant through an as-yet-unknown mechanism. To develop novel anti-breast cancer agents that are therapeutically superior to TAM, we must first fully understand the biological effects of TAM. In this study, we found that TAM treatment of MDA-MB-361 breast cancer cells activated p21Waf1/Cip1 gene transcription independently of p53. Furthermore, TAM-induced p21Waf1/Cip1 promoter activity was enhanced by transient expression of the gene encoding Early Growth Response-1 (Egr-1) protein, a transcription factor that plays an important role in cell growth and differentiation. The TAM-induced p21Waf1/Cip1 promoter activity was blocked by the expression of small interfering RNA (siRNA) targeted to Egr-1 mRNA. In addition, induction of Egr-1 expression by TAM occurred at the transcriptional level via Ets-domain transcription factor Elk-1 through the JNK and p38 mitogen-activated protein (MAP) kinase pathways. Inhibition of the JNK and p38 MAP kinase signals inhibited Egr-1-mediated p21Waf1/Cip1 promoter activity. We conclude that TAM stimulation of p21Waf1/Cip1 gene transcription in MDA-MB-361 cells depends largely on Elk-1-mediated Egr-1 expression induced by activation of the JNK and p38 MAP kinase pathways.
Asunto(s)
Neoplasias de la Mama/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Tamoxifeno/farmacología , Transcripción Genética/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Antineoplásicos Hormonales/farmacología , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Modelos Genéticos , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Activación Transcripcional/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
Biodegradable and physically transient optics represent an emerging paradigm in healthcare devices by harnessing optically active system and obviating issues with chronic uses. Light emitting components that can efficiently interact with their environments have advantages of high sensitivity, visibility, and wireless operation. Here, we report a novel combination of silk biopolymer and optically active organic dyes resulting in versatile fluorescent silk nanofibers (FSNs). FSNs generated by the electrospinning method exhibit attractive functions of the doped organic dyes along with programming the system that physically disappear at prescribed time. Red-green-blue (RGB) fluorescent nanofibrous mats, eco-friendly and transient fluorescent chemosensors for acid vapor detection, and disposable membranes for nutrition delivery were successfully demonstrated using FSNs. These functions introduced using four water soluble dyes: rhodamine B, sodium fluorescein, stilbene 420, and riboflavin. The FSN with sodium fluorescein especially, showed a sensing capability for hazardous and volatile hydrochloric acid vapors. Delivering riboflavin (vitamin B2, an important nutrient for skin care) in the FSN to a biological tissue could be observed by tracing the fluorescence of riboflavin.
Asunto(s)
Técnicas Biosensibles/instrumentación , Fibroínas/química , Colorantes Fluorescentes/análisis , Nanofibras/química , Rodaminas/análisis , Piel/metabolismo , Animales , Bombyx/química , Pollos , Técnicas Electroquímicas , Fibroínas/aislamiento & purificación , Fluoresceína/análisis , Fluoresceína/metabolismo , Fluorescencia , Colorantes Fluorescentes/metabolismo , Músculo Esquelético , Nanofibras/ultraestructura , Rodaminas/metabolismo , Riboflavina/análisis , Riboflavina/metabolismo , Estilbenos/análisis , Estilbenos/metabolismoRESUMEN
Aurora kinase A (AURKA) is essential for regulating mitosis and is frequently amplified in various cancer cell types. However, the effect of AURKA inhibition on the induction of apoptosis remains unclear. In the present study, it was reported that treatment with TCS7010, a specific inhibitor of AURKA, resulted in the accumulation of cells in the sub-G0/G1 phase of the cell cycle and increased the percentage of annexin V-binding cells. The cleavage of caspase-2, caspase-7, and poly(ADP-ribose)polymerase (PARP) significantly increased in a time-dependent manner following TCS7010 treatment. In addition, TCS7010 resulted in the production of reactive oxygen species (ROS) and stimulation of the unfolded protein response (UPR), leading to the upregulation of CCAAT/enhancer-binding protein-homologous protein (CHOP), and its downstream target BCL2 like 11 (BIM). Pretreatment with N-acetylcystein, a ROS scavenger, significantly abrogated TCS7010-induced accumulation of CHOP, BIM, cleaved caspase-7 and cleaved PARP. These results suggest that TCS7010 triggers apoptosis through the ROS-mediated UPR signaling pathway.
RESUMEN
(E)-3-(3,5-dimethoxyphenyl)-1-(2-methoxyphenyl)prop-2-en-1-one (DPP23) is a synthetic polyphenol derivative that selectively induces apoptosis in cancer cells through the unfolded protein response pathway. In the present study, we evaluated the effect of DPP23 on tumour invasion and metastasis. Here, we show that DPP23 inhibited tumour necrosis factor alpha (TNFα)-induced motility, F-actin formation, and the invasive capability of MDA-MB-231 cells. DPP23 inhibited NF-κB-dependent MMP-9 expression at the transcriptional level. Akt is involved in the activation of IKK, an upstream regulator of NF-κB. DPP23 inhibited IKK and Akt, and knockdown of Akt2 significantly inhibited TNFα-induced IKK phosphorylation. We found that DPP23 bound to the catalytic domain of Akt2, as revealed by an in silico molecular docking analysis. These results suggest that DPP23 prevents TNFα-induced invasion of highly metastatic MDA-MB-231 breast cancer cells by inhibiting Akt-IKK-NF-κB axis-mediated MMP-9 gene expression. In addition, DPP23 attenuated experimental liver metastasis in a syngenic intrasplenic transplantation model using 4T1 mouse mammary carcinoma cells. Collectively, these results suggest that DPP23 could be used as a potential platform for the prevention of invasion and metastasis of early-stage breast cancer or as an adjuvant for chemo/radiotherapy.
RESUMEN
An artificial three-dimensional (3D) culture system that mimics the tumor microenvironment in vitro is an essential tool for investigating the cross-talk between immune and cancer cells in tumors. In this study, we developed a 3D culture system using an electrospun poly(ε-caprolactone) (PCL) nanofibrous scaffold (NFS). A hybrid NFS containing an uninterrupted network of nano- and submicron-scale fibers (400 nm to 2 µm) was generated by deposition onto a stainless steel mesh instead of an aluminum plate. The hybrid NFS contained multiplanar pores in a 3D structure. Surface-seeded mouse CT26 colon cancer cells and bone marrow-derived dendritic cells (BM-DCs) were able to infiltrate the hybrid NFS within several hours. BM-DCs cultured on PCL nanofibers showed a baseline inactive form, and lipopolysaccharide (LPS)-activated BM-DCs showed increased expression of CD86 and major histocompatibility complex Class II. Actin and phosphorylated FAK were enriched where unstimulated and LPS-stimulated BM-DCs contacted the fibers in the 3D hybrid NFS. When BM-DCs were cocultured with mitoxantrone-treated CT26 cells in a 3D hybrid NFS, BM-DCs sprouted cytoplasm to, migrated to, synapsed with, and engulfed mitoxantrone-treated CT26 cancer cells, which were similar to the naturally occurring cross-talk between these two types of cells. The 3D hybrid NFS developed here provides a 3D structure for coculture of cancer and immune cells.
Asunto(s)
Médula Ósea/crecimiento & desarrollo , Diferenciación Celular , Neoplasias del Colon/patología , Células Dendríticas/citología , Electroquímica/métodos , Nanofibras/química , Poliésteres/química , Animales , Materiales Biocompatibles/química , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Ratones , Ratones Endogámicos BALB C , Ingeniería de Tejidos/métodosRESUMEN
Protein kinase C delta (PKC delta) plays an important role in the regulation of apoptosis in response to diverse anticancer agents. PKC delta is cleaved irreversibly to a catalytically active fragment in response to apoptotic stimuli; however, little information is available about the regulation of PKC delta gene expression. In this study, we found that the amount of steady-state PKC delta mRNA and protein was increased by etoposide in mouse L1210 leukemia cells. The transcriptional rate of the PKC delta gene and the stability of PKC delta mRNA were increased by treatment with etoposide, resulting in the accumulation of PKC delta protein. Rottlerin inhibited etoposide-induced PKC delta gene expression significantly, while Go6976, LY294002 and PD98059 had no effect. Further, both stable and adenovirus-mediated expression of a dominant negative PKC delta(KR) abrogated etoposide-induced PKC delta expression. Etoposide-stimulated PKC delta transcription but not PKC delta mRNA stability was blocked completely by pretreatment with rottlerin. Our data reveal a novel mechanism whereby PKC delta gene is regulated at the transcriptional and post-transcriptional level in the L1210 leukemia cell line.
Asunto(s)
Etopósido/farmacología , Regulación Enzimológica de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Leucemia/genética , Proteína Quinasa C/genética , Transcripción Genética , Acetofenonas/farmacología , Adenoviridae/metabolismo , Animales , Antineoplásicos/farmacología , Benzopiranos/farmacología , Western Blotting , Fraccionamiento Celular , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Ratones , Modelos Biológicos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C-delta , ARN Mensajero/efectos de los fármacos , Regulación hacia ArribaRESUMEN
The Wnt signal transduction pathway plays an important role in organogenesis and carcinogenesis. In an effort to better understand the action of oxidative stress-induced cellular signaling, we investigate the effect of exogenous H2O2 on the Wnt signal pathway. H2O2 decreases the amount of nuclear beta-catenin and Tcf/Lef-dependent transcription. Overexpression of Dvl-1 abrogated H2O2-induced downregulation of beta-catenin. Pretreatment with LiCl or Wnt-3a conditioned medium completely inhibited H2O2-induced release of mitochondrial cytochrome c and DNA fragmentation. These results suggest that H2O2 negatively modulates the Wnt signal pathway through downregulation of beta-catenin.
Asunto(s)
Proteínas del Citoesqueleto/biosíntesis , Regulación hacia Abajo , Peróxido de Hidrógeno/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Transactivadores/biosíntesis , Proteínas Adaptadoras Transductoras de Señales , Línea Celular , Núcleo Celular/metabolismo , Medios de Cultivo Condicionados/farmacología , Citocromos c/metabolismo , Proteínas del Citoesqueleto/metabolismo , Fragmentación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas Dishevelled , Relación Dosis-Respuesta a Droga , Humanos , Peróxido de Hidrógeno/metabolismo , Factor de Unión 1 al Potenciador Linfoide , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Fracciones Subcelulares , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Proteínas Wnt , Proteína Wnt3 , Proteína Wnt3A , beta CateninaRESUMEN
The early growth response gene-1 (Egr-1) is a tumor suppressor which plays an important role in cell growth, differentiation and apoptosis. Egr-1 has been shown to be down-regulated in many types of tumor tissues. Trifluoperazine (TFP), a phenothiazine class of antipsychotics, restored serum-induced Egr-1 expression in several cancer cell lines. We investigated the effect of Egr-1 expression on the TFP-induced inhibition of cell growth. Ectopic expression of Egr-1 enhanced the TFP-induced antiproliferative activity and downregulated cyclin D1 level in U87MG glioma cells. Our results suggest that antipsychotics TFP exhibits antiproliferative activity through up-regulation of Egr-1.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Glioma/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Factores de Transcripción/metabolismo , Trifluoperazina/farmacología , Ciclo Celular , Proliferación Celular/efectos de los fármacos , Ciclina D1/metabolismo , Proteínas de Unión al ADN/genética , Proteína 1 de la Respuesta de Crecimiento Precoz , Expresión Génica , Humanos , Proteínas Inmediatas-Precoces/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Factores de Transcripción/genética , Células Tumorales CultivadasRESUMEN
Many marine triterpene glycosides have in vitro and in vivo activities with very low toxicity, suggesting that they are suitable agents for the prevention and treatment of different diseases, particularly cancer. However, the molecular mechanisms of action of natural marine compounds in cancer, immune, and other various cells are not fully known. This review focuses on the structural characteristics of marine triterpene glycosides and how these affect their biological activities and molecular mechanisms. In particular, the membranotropic and membranolytic activities of frondoside A and cucumariosides from sea cucumbers and their ability to induce cytotoxicity and apoptosis have been discussed, with a focus on structure-activity relationships. In addition, the structural characteristics and antitumor effects of stichoposide C and stichoposide D have been reviewed along with underlying their molecular mechanisms.
RESUMEN
The breast cancer susceptibility gene BRCA1 encodes a nuclear protein, which functions as a tumor suppressor and is involved in gene transcription and DNA repair processes. Many families with inherited breast and ovarian cancers have mutations in the BRCA1 gene. However, only a few studies have reported on the mechanism underlying the regulation of BRCA1 expression in humans. In this study, we investigated the transcriptional regulation of BRCA1 in HeLa cells treated with etoposide. We found that three Egr-1-binding sequences (EBSs) were located at -1031, -1005, and -385 within the enhancer region of the BRCA1 gene. Forced expression of Egr-1 stimulated the BRCA1 promoter activity. EMSA data showed that Egr-1 bound directly to the EBS within the BRCA1 gene. Knockdown of Egr-1 through the expression of a small hairpin RNA (shRNA) attenuated etoposide-induced BRCA1 promoter activity. We conclude that Egr-1 targets the BRCA1 gene in HeLa cells exposed to etoposide.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Proteína BRCA1/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Etopósido/farmacología , Transcripción Genética/efectos de los fármacos , Proteína BRCA1/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/antagonistas & inhibidores , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Células HeLa , Humanos , Regiones Promotoras Genéticas , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , TransfecciónRESUMEN
The natural flavolignan deoxypodophyllotoxin (DPPT) inhibits tubulin polymerization and induces cell cycle arrest at G(2)/M, followed by apoptosis. However, the precise mechanism of DPPT action is currently unknown. Here, we investigated the mechanism by which DPPT treatment of HeLa cervical carcinoma cells induces cell cycle arrest and apoptosis. We show that DPPT treatment inhibits cell viability in a dose-dependent manner and that this reduction in cell viability results from cell cycle arrest at G(2)/M phase, accompanied by an increase in apoptotic cell death. The induction of apoptosis by DPPT was confirmed by visualization of morphologic changes and internucleosomal DNA fragmentation. In addition, DPPT causes p53 and Bax to accumulate, accompanied by activation of DNA damage-sensing kinases, including ataxia-telangiectasia mutated (ATM) kinase and Chk2. Furthermore, DPPT activates caspase-3 and -7, suggesting that caspase-mediated pathways are involved in DPPT-induced apoptosis. Levels of the tumor suppressor PTEN were up-regulated during DPPT treatment, coincident with Akt inhibition. Together, these data suggest that DPPT induces G(2)/M cell-cycle arrest followed by apoptosis through multiple cellular processes, involving the activation of ATM, upregulation of p53 and Bax, activation of caspase-3 and -7, and accumulation of PTEN resulting in the inhibition of the Akt pathway.