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1.
STAR Protoc ; 3(2): 101453, 2022 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-35707683

RESUMO

Intracellular vesicles such as lysosomes contain micromolar to millimolar concentrations of Zn2+, and disturbing lysosomal Zn2+ homeostasis via lysosomal Zn2+ release leads to mitochondria damage and consequent lytic cell death. Methods have been developed to image cellular Zn2+ dynamics. Here, we present a protocol using GZnP3, a genetically encoded fluorescent Zn2+ indicator, to assess lysosomal Zn2+ release in cultured cells by fluorescence microscopy imaging. For complete details on the use and execution of this protocol, please refer to Du et al. (2021) or Minckley et al. (2019).


Assuntos
Lisossomos , Zinco , Morte Celular , Células Cultivadas , Lisossomos/genética , Mitocôndrias/genética , Zinco/metabolismo
2.
Cell Rep ; 37(3): 109848, 2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34686351

RESUMO

During tumor progression, lysosome function is often maladaptively upregulated to match the high energy demand required for cancer cell hyper-proliferation and invasion. Here, we report that mucolipin TRP channel 1 (TRPML1), a lysosomal Ca2+ and Zn2+ release channel that regulates multiple aspects of lysosome function, is dramatically upregulated in metastatic melanoma cells compared with normal cells. TRPML-specific synthetic agonists (ML-SAs) are sufficient to induce rapid (within hours) lysosomal Zn2+-dependent necrotic cell death in metastatic melanoma cells while completely sparing normal cells. ML-SA-caused mitochondria swelling and dysfunction lead to cellular ATP depletion. While pharmacological inhibition or genetic silencing of TRPML1 in metastatic melanoma cells prevents such cell death, overexpression of TRPML1 in normal cells confers ML-SA vulnerability. In the melanoma mouse models, ML-SAs exhibit potent in vivo efficacy of suppressing tumor progression. Hence, targeting maladaptively upregulated lysosome machinery can selectively eradicate metastatic tumor cells in vitro and in vivo.


Assuntos
Antineoplásicos/farmacologia , Lisossomos/efeitos dos fármacos , Melanócitos/efeitos dos fármacos , Melanoma/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Neoplasias Cutâneas/tratamento farmacológico , Canais de Potencial de Receptor Transitório/agonistas , Zinco/metabolismo , Animais , Morte Celular , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Lisossomos/metabolismo , Lisossomos/patologia , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/genética , Melanoma/metabolismo , Melanoma/secundário , Camundongos Nus , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Fatores de Tempo , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismo , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
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