Abstract
BackgroundMacroautophagy is a catabolic process that can mediate cell death or survival. Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment (TR) is known to induce autophagy. Here we investigated whether SQSTM1/p62 (p62) overexpression, as a marker of autophagic flux, was related to aggressiveness of human prostate cancer (PCa) and whether autophagy regulated the treatment response in sensitive but not resistant PCa cell lines.MethodsImmunostaining and immunoblotting analyses of the autophagic markers p62 [in PCa tissue microarrays (TMAs) and PCa cell lines] and LC3 (in PCa cell lines), transmission electron microscopy, and GFP-mCherry-LC3 were used to study autophagy induction and flux. The effect of autophagy inhibition using pharmacologic (3-methyladenine and chloroquine) and genetic [(short hairpin (sh)-mediated knock-down of ATG7 and LAMP2) and small interfering (si)RNA-mediated BECN1 knock-down] approaches on TR-induced cell death was assessed by clonogenic survival, sub-G1 DNA content, and annexinV/PI staining by flow cytometry. Caspase-8 activation was determined by immunoblotting.ResultsWe found that increased cytoplasmic expression of p62 was associated with high-grade PCa, indicating that autophagy signaling might be important for survival in high-grade tumors. TR-resistant cells exhibited high autophagic flux, with more efficient clearance of p62-aggregates in four TR-resistant PCa cell lines: C4-2, LNCaP, DU145, and CWRv22.1. In contrast, autophagic flux was low in TR-sensitive PC3 cells, leading to accumulation of p62-aggregates. Pharmacologic (chloroquine or 3-methyladenine) and genetic (shATG7 or shLAMP2) inhibition of autophagy led to cell death in TR-resistant C4-2 cells. shATG7-expressing PC3 cells, were less sensitive to TR-induced cell death whereas those shLAMP2-expressing were as sensitive as shControl-expressing PC3 cells. Inhibition of autophagic flux using chloroquine prevented clearance of p62 aggregates, leading to caspase-8 activation and cell death in C4-2 cells. In PC3 cells, inhibition of autophagy induction prevented p62 accumulation and hence caspase-8 activation.ConclusionsWe show that p62 overexpression correlates with advanced stage human PCa. Pharmacologic and genetic inhibition of autophagy in PCa cell lines indicate that autophagic flux can determine the cellular response to TR by regulating caspase-8 activation. Thus, combining various autophagic inhibitors may have a differential impact on TR-induced cell death.
Highlights
Macroautophagy is a catabolic process that can mediate cell death or survival
Microtubule-associated protein 1 light chain 3 beta (LC3B; called ATG8) is used as a marker of autophagy; it is lipidated upon autophagy induction and it is required for autophagosome formation [4,5]. p62/SQSTM1 (p62) facilitates the degradation of polyubiquitinated substrates by autophagy, causing its own degradation; it is used as an indicator of autophagic degradation [6]
Results p62 is over-expressed in the cytoplasm of advanced human prostate cancer cells To understand the role of autophagy in human PCa progression, we examined the expression of SQSTM1/p62 (p62), a marker of autophagic flux in a tissue microarrays (TMA) of human prostate tissue
Summary
Apo ligand (Apo2L)/ tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment (TR) is known to induce autophagy. Growth factor deprivation, hypoxia, endoplasmic reticulum (ER) stress, and microbial infection can prevent cell death [2]. It can be associated with cell death due to excessive mitophagy, leading to loss of mitochondrial membrane potential (Δψm), caspase activation, and lysosomal membrane permeabilization [3]. Apo2L/TRAIL (TNFSF10; clinical name, dulanermin) belongs to a small subset of pro-apoptotic protein ligands in the tumor necrosis factor (TNF) superfamily. Induction of autophagy in PCa cells has been shown to mediate cell survival, and inhibiting autophagy enhances drug-induced cell death [11,12]
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.