RESUMEN
Microphthalmia-associated transcription factor (MITF) is expressed in melanomas and has a critical role in melanocyte development and transformation. Because inhibition of MITF inhibits cell growth in melanoma, MITF is a potential therapeutic target molecule. Here, we report the identification of CH6868398, which has a novel chemical structure and suppresses MITF expression at the protein level in melanoma cells. CH6868398 showed cell growth inhibition activity against MITF-dependent melanoma cells both with and without BRAF mutation and also exhibited anti-tumor efficacy in a melanoma xenograft model. Because selective BRAF inhibitors are standard therapeutics for BRAF-mutated melanoma, we investigated the effect of CH6868398 with a BRAF inhibitor, PLX4720, on cell growth inhibition. The addition of CH6868398 enhanced the cell growth inhibition activity of PLX4720 in melanoma cell lines. Furthermore, combination of CH6868398 and PLX4720 efficiently suppressed MITF protein and enhanced cleavage of Caspase3 and poly (ADP-ribose) polymerase (PARP) in melanoma cell lines. These data support the therapeutic potential of CH6868398 as an anti-melanoma agent that reduces MITF protein levels in combination with BRAF inhibitors.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Melanoma/tratamiento farmacológico , Factor de Transcripción Asociado a Microftalmía/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Humanos , Indoles/farmacología , Melanoma/enzimología , Melanoma/genética , Melanoma/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Inhibidores de Proteínas Quinasas/administración & dosificación , Distribución Aleatoria , Sulfonamidas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Although treatment of melanoma with BRAF inhibitors and immune checkpoint inhibitors achieves a high response rate, a subset of melanoma patients with intrinsic and acquired resistance are insensitive to these therapeutics, so to improve melanoma therapy other target molecules need to be found. Here, we screened our chemical library to identify an anti-melanoma agent and examined its action mechanisms to show cell growth inhibition activity. METHODS: We screened a chemical library against multiple skin cancer cell lines and conducted ingenuity pathway analysis (IPA) to investigate the mechanisms of CH5552074 activity. Suppression of microphthalmia-associated transcription factor (MITF) expression levels was determined in melanoma cells treated with CH5552074. Cell growth inhibition activity of CH5552074 was evaluated in MITF-dependent melanoma cell lines. RESULTS: We identified an anti-melanoma compound, CH5552074, which showed remarkable cell growth inhibition activity in melanoma cell lines. The IPA results suggested that CH5552074-sensitive cell lines had activated MITF. In further in vitro studies in the melanoma cell lines, a knockdown of MITF with siRNA resulted in cell growth inhibition, which showed that CH5552074 inhibited cell growth by reducing the expression level of MITF protein. CONCLUSIONS: These results suggest that CH5552074 can inhibit cell growth in melanoma cells by reducing the protein level of MITF. MITF inhibition by CH5552074 would be an attractive option for melanoma treatment.
Asunto(s)
Antineoplásicos/farmacología , Imidazoles/farmacología , Melanoma/tratamiento farmacológico , Factor de Transcripción Asociado a Microftalmía/antagonistas & inhibidores , Neoplasias Cutáneas/tratamiento farmacológico , Tiazoles/farmacología , División Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Análisis por Micromatrices , Factor de Transcripción Asociado a Microftalmía/genética , ARN Interferente Pequeño/genéticaRESUMEN
Studies on the mechanisms of decidualization and endometriosis are often hampered by lack of primary endometrial cells. To facilitate in vitro studies, we established a human endometrial stromal cell line, KC02-44D, immortalized with human telomerase reverse transcriptase. Upon exposure to ovarian stimuli, KC02-44D cells showed similar cytoskeletal marker or gene expression and biochemical phenotype to primary endometrial stromal cells. KC02-44D would be useful for studies of human endometrial function and its associated pathologies.
Asunto(s)
Línea Celular , Endometrio/citología , Células del Estroma/fisiología , Bucladesina/farmacología , Antígenos CD13/metabolismo , Decidua/efectos de los fármacos , Decidua/fisiología , Regulación hacia Abajo , Estradiol/farmacología , Femenino , Humanos , Interleucina-1beta/farmacología , Acetato de Medroxiprogesterona/farmacología , Persona de Mediana Edad , Neprilisina/metabolismo , Receptores de Estrógenos/biosíntesis , Receptores de Progesterona/biosíntesis , Células del Estroma/efectos de los fármacos , Vimentina/metabolismoRESUMEN
The M5 muscarinic acetylcholine receptor (M5R) has been shown to play a crucial role in mediating acetylcholine-dependent dilation of cerebral blood vessels. We show that male M5R-/- mice displayed constitutive constriction of cerebral arteries using magnetic resonance angiography in vivo. Male M5R-/- mice exhibited a significantly reduced cerebral blood flow (CBF) in the cerebral cortex, hippocampus, basal ganglia, and thalamus. Cortical and hippocampal pyramidal neurons from M5R-/- mice showed neuronal atrophy. Hippocampus-dependent spatial and nonspatial memory was also impaired in M5R-/- mice. In M5R-/- mice, CA3 pyramidal cells displayed a significantly attenuated frequency of the spontaneous postsynaptic current and long-term potentiation was significantly impaired at the mossy fiber-CA3 synapse. Our findings suggest that impaired M5R signaling may play a role in the pathophysiology of cerebrovascular deficits. The M5 receptor may represent an attractive novel therapeutic target to ameliorate memory deficits caused by impaired cerebrovascular function.
Asunto(s)
Encéfalo/metabolismo , Arterias Cerebrales/metabolismo , Trastornos Cerebrovasculares/metabolismo , Trastornos del Conocimiento/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Receptor Muscarínico M5/genética , Acetilcolina/metabolismo , Animales , Encéfalo/irrigación sanguínea , Encéfalo/fisiopatología , Arterias Cerebrales/patología , Arterias Cerebrales/fisiopatología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Corteza Cerebral/fisiopatología , Circulación Cerebrovascular/genética , Trastornos Cerebrovasculares/genética , Trastornos Cerebrovasculares/fisiopatología , Trastornos del Conocimiento/genética , Trastornos del Conocimiento/fisiopatología , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Hipocampo/patología , Hipocampo/fisiopatología , Potenciación a Largo Plazo/genética , Angiografía por Resonancia Magnética , Masculino , Trastornos de la Memoria/genética , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Degeneración Nerviosa/genética , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/fisiopatología , Células Piramidales/metabolismo , Células Piramidales/patología , Transmisión Sináptica/genética , Vasoconstricción/genéticaRESUMEN
Interactions between ectodermal and mesenchymal extracellular signaling pathways regulate hair follicle (HF) morphogenesis and hair cycling. Bone morphogenetic proteins (BMPs) are known to be important in hair follicle development by affecting the local cell fate modulation. To study the role of BMP signaling in the HF, we disrupted Bmpr1a, which encodes the BMP receptor type IA (BMPR1A) in an HF cell-specific manner, using the Cre/loxP system. We found that the differentiation of inner root sheath, but not outer root sheath, was severely impaired in mutant mice. The number of HFs was reduced in the dermis and subcutaneous tissue, and cycling epithelial cells were reduced in mutant mice HFs. Our results strongly suggest that BMPR1A signaling is essential for inner root sheath differentiation and is indispensable for HF renewal in adult skin.