Functional Autophagic Flux Regulates AgNP Uptake And The Internalized Nanoparticles Determine Tumor Cell Fate By Temporally Regulating Flux

Leena Fageria,1 Vishakha Bambroo,1 Angel Mathew,1 Sudeshna Mukherjee,1 Rajdeep Chowdhury,1 Surojit Pande2 1Department of Biological Sciences, Pilani Campus, BITS, Pilani, Rajasthan 333031, India; 2Department of Chemistry, Pilani Campus, BITS, Pilani, Rajasthan 333031, IndiaCorrespondence: Surojit Pa...

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Autores principales:	Fageria L, Bambroo V, Mathew A, Mukherjee S, Chowdhury R, Pande S
Formato:	article
Lenguaje:	EN
Publicado:	Dove Medical Press 2019
Materias:	silver nanoparticles endocytosis autophagy ros lysosomes Medicine (General) R5-920
Acceso en línea:	https://doaj.org/article/fc1428f0d02f4a668194d16faaf496d5
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spelling	oai:doaj.org-article:fc1428f0d02f4a668194d16faaf496d52021-12-02T06:56:49ZFunctional Autophagic Flux Regulates AgNP Uptake And The Internalized Nanoparticles Determine Tumor Cell Fate By Temporally Regulating Flux1178-2013https://doaj.org/article/fc1428f0d02f4a668194d16faaf496d52019-11-01T00:00:00Zhttps://www.dovepress.com/functional-autophagic-flux-regulates-agnp-uptake-and-the-internalized--peer-reviewed-article-IJNhttps://doaj.org/toc/1178-2013Leena Fageria,1 Vishakha Bambroo,1 Angel Mathew,1 Sudeshna Mukherjee,1 Rajdeep Chowdhury,1 Surojit Pande2 1Department of Biological Sciences, Pilani Campus, BITS, Pilani, Rajasthan 333031, India; 2Department of Chemistry, Pilani Campus, BITS, Pilani, Rajasthan 333031, IndiaCorrespondence: Surojit PandeDepartment of Chemistry, Pilani Campus, BITS, Pilani, Rajasthan 333031 Email spande@pilani.bits-pilani.ac.inRajdeep ChowdhuryDepartment of Biological Sciences, Pilani Campus, BITS, Pilani, Rajasthan 333031 Email rajdeep.chowdhury@pilani.bits-pilani.ac.inBackground: Silver nanoparticles (AgNPs) are known to induce the conserved, cellular, homeostatic process- autophagy in tumor cells. Previous studies primarily focus on the pro-survival role of autophagy post AgNP exposure in tumor cells, but seldom on its role in AgNP uptake, or on the functional significance of autophagy temporal dynamics. Our study sheds more light on the extensive crosstalk that exists between AgNP and autophagy, which can be critical to the improvement of AgNP-induced therapeutic effects.Methods: β-cyclodextrin (β-CD) coated AgNPs of two different sizes were synthesized by nucleation method and characterized by transmission electron microscopy. Fluorescence microscopy and flow cytometry were used to probe intracellular uptake of AgNPs. Endocytic mechanism of AgNPs was classically analyzed through use of various endocytosis inhibitors. Autophagy was evaluated by immunoblot and fluorescence microscopy. Additionally, immunoblot was performed to monitor Janus Kinase (JNK) signalling, ubiquitination of proteins, expression of endo-lysosomal and apoptotic markers in correlation to AgNP-induced autophagy.Results: The intra-cellular route of entry for the small NPs (&sim;9 nm; ss-AgNPs) was different than the large NPs (&sim;19 nm; ls-AgNPs) studied. However, irrespective of their unique route of entry an inhibition of autophagic flux by chloroquine (CQ) reduced uptake of both the AgNPs. In contrary, rapamycin (Rapa), an autophagy inducer enhanced it. Importantly, JNK activation was required for autophagy induction and AgNP uptake. Furthermore, effect of AgNPs on autophagy showed temporal dependency. An enhanced autophagic flux was noted at early time points; however, prolonged exposure resulted in inhibition of flux marked by increase in Rab7, LC3B-II and p62 proteins. Inhibition of flux was associated with lysosomal dysfunction, decreased LAMP1 expression and an increased accumulation of ubiquitinated (Ub) proteins. This resulted in heightened reactive oxygen species (ROS) and consequent cytotoxicity.Conclusion: In this study, we observed that a functional autophagic flux aids AgNP uptake, but AgNPs in turn, overtime, inhibits flux and endo-lysosomal function. We provide critical, novel insights into crosstalk between AgNP and autophagy which can be vital to future AgNP-based therapy development.Keywords: silver nanoparticles, endocytosis, autophagy, ROS, lysosomesFageria LBambroo VMathew AMukherjee SChowdhury RPande SDove Medical Pressarticlesilver nanoparticlesendocytosisautophagyroslysosomesMedicine (General)R5-920ENInternational Journal of Nanomedicine, Vol Volume 14, Pp 9063-9076 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	silver nanoparticles endocytosis autophagy ros lysosomes Medicine (General) R5-920
spellingShingle	silver nanoparticles endocytosis autophagy ros lysosomes Medicine (General) R5-920 Fageria L Bambroo V Mathew A Mukherjee S Chowdhury R Pande S Functional Autophagic Flux Regulates AgNP Uptake And The Internalized Nanoparticles Determine Tumor Cell Fate By Temporally Regulating Flux
description	Leena Fageria,1 Vishakha Bambroo,1 Angel Mathew,1 Sudeshna Mukherjee,1 Rajdeep Chowdhury,1 Surojit Pande2 1Department of Biological Sciences, Pilani Campus, BITS, Pilani, Rajasthan 333031, India; 2Department of Chemistry, Pilani Campus, BITS, Pilani, Rajasthan 333031, IndiaCorrespondence: Surojit PandeDepartment of Chemistry, Pilani Campus, BITS, Pilani, Rajasthan 333031 Email spande@pilani.bits-pilani.ac.inRajdeep ChowdhuryDepartment of Biological Sciences, Pilani Campus, BITS, Pilani, Rajasthan 333031 Email rajdeep.chowdhury@pilani.bits-pilani.ac.inBackground: Silver nanoparticles (AgNPs) are known to induce the conserved, cellular, homeostatic process- autophagy in tumor cells. Previous studies primarily focus on the pro-survival role of autophagy post AgNP exposure in tumor cells, but seldom on its role in AgNP uptake, or on the functional significance of autophagy temporal dynamics. Our study sheds more light on the extensive crosstalk that exists between AgNP and autophagy, which can be critical to the improvement of AgNP-induced therapeutic effects.Methods: β-cyclodextrin (β-CD) coated AgNPs of two different sizes were synthesized by nucleation method and characterized by transmission electron microscopy. Fluorescence microscopy and flow cytometry were used to probe intracellular uptake of AgNPs. Endocytic mechanism of AgNPs was classically analyzed through use of various endocytosis inhibitors. Autophagy was evaluated by immunoblot and fluorescence microscopy. Additionally, immunoblot was performed to monitor Janus Kinase (JNK) signalling, ubiquitination of proteins, expression of endo-lysosomal and apoptotic markers in correlation to AgNP-induced autophagy.Results: The intra-cellular route of entry for the small NPs (&sim;9 nm; ss-AgNPs) was different than the large NPs (&sim;19 nm; ls-AgNPs) studied. However, irrespective of their unique route of entry an inhibition of autophagic flux by chloroquine (CQ) reduced uptake of both the AgNPs. In contrary, rapamycin (Rapa), an autophagy inducer enhanced it. Importantly, JNK activation was required for autophagy induction and AgNP uptake. Furthermore, effect of AgNPs on autophagy showed temporal dependency. An enhanced autophagic flux was noted at early time points; however, prolonged exposure resulted in inhibition of flux marked by increase in Rab7, LC3B-II and p62 proteins. Inhibition of flux was associated with lysosomal dysfunction, decreased LAMP1 expression and an increased accumulation of ubiquitinated (Ub) proteins. This resulted in heightened reactive oxygen species (ROS) and consequent cytotoxicity.Conclusion: In this study, we observed that a functional autophagic flux aids AgNP uptake, but AgNPs in turn, overtime, inhibits flux and endo-lysosomal function. We provide critical, novel insights into crosstalk between AgNP and autophagy which can be vital to future AgNP-based therapy development.Keywords: silver nanoparticles, endocytosis, autophagy, ROS, lysosomes
format	article
author	Fageria L Bambroo V Mathew A Mukherjee S Chowdhury R Pande S
author_facet	Fageria L Bambroo V Mathew A Mukherjee S Chowdhury R Pande S
author_sort	Fageria L
title	Functional Autophagic Flux Regulates AgNP Uptake And The Internalized Nanoparticles Determine Tumor Cell Fate By Temporally Regulating Flux
title_short	Functional Autophagic Flux Regulates AgNP Uptake And The Internalized Nanoparticles Determine Tumor Cell Fate By Temporally Regulating Flux
title_full	Functional Autophagic Flux Regulates AgNP Uptake And The Internalized Nanoparticles Determine Tumor Cell Fate By Temporally Regulating Flux
title_fullStr	Functional Autophagic Flux Regulates AgNP Uptake And The Internalized Nanoparticles Determine Tumor Cell Fate By Temporally Regulating Flux
title_full_unstemmed	Functional Autophagic Flux Regulates AgNP Uptake And The Internalized Nanoparticles Determine Tumor Cell Fate By Temporally Regulating Flux
title_sort	functional autophagic flux regulates agnp uptake and the internalized nanoparticles determine tumor cell fate by temporally regulating flux
publisher	Dove Medical Press
publishDate	2019
url	https://doaj.org/article/fc1428f0d02f4a668194d16faaf496d5
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_version_	1718399672203083776

Functional Autophagic Flux Regulates AgNP Uptake And The Internalized Nanoparticles Determine Tumor Cell Fate By Temporally Regulating Flux

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