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1.
Nat Commun ; 12(1): 6941, 2021 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-34862367

RESUMO

mRNA expression of the DLC1 tumor suppressor gene is downregulated in many lung cancers and their derived cell lines, with DLC1 protein levels being low or absent. Although the role of increased EZH2 methyltransferase in cancer is usually attributed to its histone methylation, we unexpectedly observed that post-translational destabilization of DLC1 protein is common and attributable to its methylation by cytoplasmic EZH2, leading to CUL-4A ubiquitin-dependent proteasomal degradation of DLC1. Furthermore, siRNA knockdown of KRAS in several lines increases DLC1 protein, associated with a drastic reduction in cytoplasmic EZH2. Pharmacologic inhibition of EZH2, CUL-4A, or the proteasome can increase the steady-state level of DLC1 protein, whose tumor suppressor activity is further increased by AKT and/or SRC kinase inhibitors, which reverse the direct phosphorylation of DLC1 by these kinases. These rational drug combinations induce potent tumor growth inhibition, with markers of apoptosis and senescence, that is highly dependent on DLC1 protein.


Assuntos
Antineoplásicos/farmacologia , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteínas Ativadoras de GTPase/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Supressoras de Tumor/metabolismo , Animais , Antineoplásicos/uso terapêutico , Benzodioxóis/farmacologia , Benzodioxóis/uso terapêutico , Compostos de Boro/farmacologia , Compostos de Boro/uso terapêutico , Linhagem Celular Tumoral , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteínas Ativadoras de GTPase/genética , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Glicina/análogos & derivados , Glicina/farmacologia , Glicina/uso terapêutico , Células HEK293 , Compostos Heterocíclicos com 3 Anéis/farmacologia , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Mutagênese Sítio-Dirigida , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Estabilidade Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Exp Eye Res ; 190: 107890, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31811823

RESUMO

PURPOSE: The avascular cornea, trabecular meshwork (TM), and lens obtain iron, an essential biometal, from the aqueous humor (AH). The mechanism by which this exchange is regulated, however, is unclear. Recently we reported that non-pigmented ciliary epithelial cells express ferroportin (Fpn) (Ashok, 2018b), an iron export protein modulated by hepcidin, the master regulator of iron homeostasis secreted mainly by the liver. Here, we explored whether ciliary epithelial and other cells in the anterior segment synthesize hepcidin, suggesting local regulation of iron exchange at this site. METHODS: Human and bovine eyes were dissected to isolate the ciliary body (CB), corneal endothelial (CE), TM, lens epithelial (LE), and outer epithelial cell layer of the iris. Total mRNA and protein lysates were processed to evaluate the synthesis and expression of hepcidin, the iron regulatory peptide hormone, Fpn, the only known iron export protein, ceruloplasmin (Cp), a ferroxidase necessary for iron export, transferrin receptor (TfR), a major iron uptake protein, and ferritin, a major iron storage protein. A combination of techniques including reverse transcription polymerase chain reaction (RT-PCR) of total mRNA, Western blotting of protein lysates, and immunofluorescence of fixed tissue sections were used to accomplish these goals. RESULTS: RT-PCR of isolated tissue samples revealed hepcidin-specific mRNA in the CB, TM, CE, and LE of the bovine eye. Western blotting of protein lysates from these tissues showed reactivity for hepcidin, Fpn, ferritin, and TfR. Western blotting and immunohistochemistry of similar tissues isolated from cadaveric human eyes showed expression of hepcidin, Fpn, and Cp in these samples. Notably, Fpn and Cp were expressed on the basolateral membrane of non-pigmented ciliary epithelial cells, facing the AH. CONCLUSIONS: Synthesis and expression of hepcidin and Fpn in the ciliary epithelium suggests local regulation of iron transport from choroidal plexus in the ciliary body to the AH across the blood-aqueous barrier. Expression of hepcidin and Fpn in CE, TM, and LE cells indicates additional regulation of iron exchange between the AH and cornea, TM, and lens, suggesting autonomous regulation of iron homeostasis in the anterior segment. Physiological and pathological implications of these observations are discussed.


Assuntos
Segmento Anterior do Olho/metabolismo , Anti-Infecciosos/metabolismo , Hepcidinas/biossíntese , Adulto , Idoso , Animais , Western Blotting , Proteínas de Transporte de Cátions/metabolismo , Bovinos , Ceruloplasmina/metabolismo , Corpo Ciliar/metabolismo , Eletroforese em Gel de Poliacrilamida , Endotélio Corneano/metabolismo , Células Epiteliais/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Hepcidinas/genética , Humanos , Iris/metabolismo , Cristalino/metabolismo , Masculino , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Malha Trabecular/metabolismo
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