Suppression of autophagy enhances preferential toxicity of paclitaxel to folliculin-deficient renal cancer cells

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Abstract

Background

Paclitaxel, a widely used chemotherapeutic drug, can induce apoptosis in variety of cancer cells. A previous study has shown preferential toxicity of paclitaxel to FLCN-deficient kidney cancer cell line, UOK257. In this report, we investigate the cellular and molecular mechanism of paclitaxel-induced autophagy and apoptosis in renal cancer cells with and without FLCN expression.

Methods

Two pairs of cell lines were used: FLCN siRNA-silenced ACHN cell line (ACHN-5968) and scrambled ACHN cell line (ACHN-sc); FLCN-null UOK257 cell line and UOK257-2 cell line restored with ectopic expression of FLCN. Autophagy was examined by western blot, GFP-LC3, transmission electron microscopy, and MDC assay. Cell viability and apoptosis were detected using MTT assay, DAPI stain and TUNEL assay. After inhibition of autophagy with 3-Methyladenine (3-MA) or Beclin 1 siRNA, cell viability and apoptosis were measured by MTT assay and TUNEL assay.

Results

After paclitaxel treatment, a dose-dependent decrease in cell viability and increase in apoptosis were observed in FLCN-deficient UOK257 and ACHN-5968 cells compared to their FLCN-expressing counterparts, suggesting that renal cancer cells without FLCN were more sensitive to paclitaxel. Enhanced autophagy was found to be associated with paclitaxel treatment in FLCN-deficient RCC cells. The MAPK pathway was also identified as a key pathway for the activation of autophagy in these kidney cancer cells. Inhibition of phosphorylated ERK with ERK inhibitor U0126 showed a significant decrease in autophagy. Furthermore, after inhibition of autophagy with 3-Methyladenine (3-MA) or Beclin 1 siRNA, apoptosis induced by paclitaxel was significantly increased in FLCN-deficient UOK257 and ACHN-5968 cells.

Conclusions

Preferential toxicity of paclitaxel to FLCN-deficient kidney cancer cells is associated with enhanced autophagy. Suppression of autophagy further enhances paclitaxel-induced apoptosis in FLCN-deficient renal cancer cells. Our results suggest that paclitaxel combined with an autophagy inhibitor might be a potentially more effective chemotherapeutic approach for FLCN-deficient renal cancer.

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Most cited references 26

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In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

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Andrew Thorburn, Jacob Gump (2011)

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Author and article information

Contributors

Qi Zhang

Shuhui Si

Sue Schoen

Jindong Chen

Xun-Bo Jin

Guan Wu

Journal

Journal ID (nlm-ta): J Exp Clin Cancer Res

Journal ID (iso-abbrev): J. Exp. Clin. Cancer Res

Title: Journal of Experimental & Clinical Cancer Research : CR

Publisher: BioMed Central

ISSN (Print): 0392-9078

ISSN (Electronic): 1756-9966

Publication date Collection: 2013

Publication date (Electronic): 4 December 2013

Volume: 32

Issue: 1

Page: 99

Affiliations

[1 ]Minimally Invasive Urology Center, Provincial Hospital Affiliated to Shandong University, Jinan 250021, China

[2 ]Department of Urology, University of Rochester Medical Center, 601 Elmwood Avenue, Box 656, 14642 Rochester, NY, USA

[3 ]Pathology, University of Rochester Medical Center, Rochester, NY, USA

[4 ]Wilmot Cancer Center, University of Rochester Medical Center, Rochester, NY, USA

Article

Publisher ID: 1756-9966-32-99

DOI: 10.1186/1756-9966-32-99

PMC ID: 3879005

PubMed ID: 24305604

SO-VID: d605e8a7-0346-4f84-be9d-5151ed677f11

License:

This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Suppression of autophagy enhances preferential toxicity of paclitaxel to folliculin-deficient renal cancer cells

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Abstract

Background

Methods

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Karger: Oncology

Most cited references 26

Guidelines for the use and interpretation of assays for monitoring autophagy.

How to interpret LC3 immunoblotting.

Autophagy and apoptosis: what is the connection?

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