Abstract
We have previously demonstrated that the thiazole derivative 3-methylcyclopentylidene-[4-(4′-chlorophenyl)thiazol-2-yl]hydrazone (CPTH6) induces apoptosis and cell cycle arrest in human leukemia cells. The aim of this study was to evaluate whether CPTH6 is able to affect autophagy. By using several human tumor cell lines with different origins we demonstrated that CPTH6 treatment induced, in a dose-dependent manner, a significant increase in autophagic features, as imaged by electron microscopy, immunoblotting analysis of membrane-bound form of microtubule-associated protein 1 light chain 3 (LC3B-II) levels and by appearance of typical LC3B-II-associated autophagosomal puncta. To gain insights into the molecular mechanisms of elevated markers of autophagy induced by CPTH6 treatment, we silenced the expression of several proteins acting at different steps of autophagy. We found that the effect of CPTH6 on autophagy developed through a noncanonical mechanism that did not require beclin-1-dependent nucleation, but involved Atg-7-mediated elongation of autophagosomal membranes. Strikingly, a combined treatment of CPTH6 with late-stage autophagy inhibitors, such as chloroquine and bafilomycin A1, demonstrates that under basal condition CPTH6 reduces autophagosome turnover through an impairment of their degradation pathway, rather than enhancing autophagosome formation, as confirmed by immunofluorescence experiments. According to these results, CPTH6-induced enhancement of autophagy substrate p62 and NBR1 protein levels confirms a blockage of autophagic cargo degradation. In addition, CPTH6 inhibited autophagosome maturation and compounds having high structural similarities with CPTH6 produced similar effects on the autophagic pathway. Finally, the evidence that CPTH6 treatment decreased α-tubulin acetylation and failed to increase autophagic markers in cells in which acetyltransferase ATAT1 expression was silenced indicates a possible role of α-tubulin acetylation in CPTH6-induced alteration in autophagy. Overall, CPTH6 could be a valuable agent for the treatment of cancer and should be further studied as a possible antineoplastic agent.
Highlights
Induction of autophagy may be a double-edged sword, as it is a mechanism leading to cell death when destruction of cell cytoplasm components is activated in a long-lasting way.[3]
Because many lines of evidence suggest a link between apoptosis and autophagy,[15] in this paper we examined the effect of CPTH6 on autophagy in several tumor cell lines with different histotypes
We demonstrated that CPTH6 inhibits basal autophagy in several human tumor cell lines with different origins, indicating a general effect of CPTH6 instead of a cell type-specific phenomenon
Summary
Induction of autophagy may be a double-edged sword, as it is a mechanism leading to cell death when destruction of cell cytoplasm components is activated in a long-lasting way.[3]. A canonical mechanism of nucleation (de novo formation) of autophagosomal membranes depends on beclin-1, which interacts with adaptor protein p150 to stimulate activity of PI3-kinase VPS34.4 The elongation requires Atg-12, Atg-3, Atg-5 and. Atg-7,5 whereas lysosome-associated membrane proteins 1 and 2 (LAMP-1 and LAMP-2) regulate the late step of the autophagic process. Autophagic pathway can be inhibited by pharmacological inhibitors at different steps: PI3-kinase inhibitors block autophagosome formation;[6] microtubule-disrupting agents and endoplasmic reticulum stressors inhibit autophagosome– lysosome fusion;[7,8] and lysosomal proteases inhibitors and acidification modulators strongly reduce final degradation of autophagic cargo inside autolysosome.[9]. Despite the great deal of interest in the regulation of autophagy for therapeutic purposes, there are only few modulators of the autophagic pathway that have shown promising pharmacological value.[10,11,12]
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