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  • Dual role of autophagy on docetaxel-sensitivity in prostate cancer cells.

Dual role of autophagy on docetaxel-sensitivity in prostate cancer cells.

Cell death & disease (2018-09-01)
Riccardo Cristofani, Marina Montagnani Marelli, Maria Elena Cicardi, Fabrizio Fontana, Monica Marzagalli, Patrizia Limonta, Angelo Poletti, Roberta Manuela Moretti
ABSTRACT

Prostate cancer (PC) is one of the leading causes of death in males. Available treatments often lead to the appearance of chemoresistant foci and metastases, with mechanisms still partially unknown. Within tumour mass, autophagy may promote cell survival by enhancing cancer cells tolerability to different cell stresses, like hypoxia, starvation or those triggered by chemotherapic agents. Because of its connection with the apoptotic pathways, autophagy has been differentially implicated, either as prodeath or prosurvival factor, in the appearance of more aggressive tumours. Here, in three PC cells (LNCaP, PC3, and DU145), we tested how different autophagy inducers modulate docetaxel-induced apoptosis. We selected the mTOR-independent disaccharide trehalose and the mTOR-dependent macrolide lactone rapamycin autophagy inducers. In castration-resistant PC (CRPC) PC3 cells, trehalose specifically prevented intrinsic apoptosis in docetaxel-treated cells. Trehalose reduced the release of cytochrome c triggered by docetaxel and the formation of aberrant mitochondria, possibly by enhancing the turnover of damaged mitochondria via autophagy (mitophagy). In fact, trehalose increased LC3 and p62 expression, LC3-II and p62 (p62 bodies) accumulation and the induction of LC3 puncta. In docetaxel-treated cells, trehalose, but not rapamycin, determined a perinuclear mitochondrial aggregation (mito-aggresomes), and mitochondria specifically colocalized with LC3 and p62-positive autophagosomes. In PC3 cells, rapamycin retained its ability to activate autophagy without evidences of mitophagy even in presence of docetaxel. Interestingly, these results were replicated in LNCaP cells, whereas trehalose and rapamycin did not modify the response to docetaxel in the ATG5-deficient (autophagy resistant) DU145 cells. Therefore, autophagy is involved to alter the response to chemotherapy in combination therapies and the response may be influenced by the different autophagic pathways utilized and by the type of cancer cells.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-LC3 antibody produced in rabbit, ~1 mg/mL, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Protease Inhibitor Cocktail, for use with mammalian cell and tissue extracts, DMSO solution
Sigma-Aldrich
3-Methyladenine, autophagy inhibitor
Sigma-Aldrich
Chloroquine diphosphate salt, powder or crystals, 98.5-101.0% (EP), meets EP testing specifications
Sigma-Aldrich
MISSION® esiRNA, targeting human ATG5
Sigma-Aldrich
Rapamycin from Streptomyces hygroscopicus, ≥95% (HPLC), powder
Sigma-Aldrich
TRI Reagent®, For processing tissues, cells cultured in monolayer or cell pellets
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Phosphate buffered saline, tablet
Sigma-Aldrich
ANTI-FLAG® M2 antibody, Mouse monoclonal, 1 mg/mL, clone M2, affinity isolated antibody, buffered aqueous solution (50% glycerol, 10 mM sodium phosphate, and 150 mM NaCl, pH 7.4)
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Anti-α-Tubulin antibody, Mouse monoclonal, clone DM1A, purified from hybridoma cell culture