We have previously demonstrated that the thiazole derivative 3-methylcyclopentylidene-[4-(4 0 -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 a-tubulin acetylation and failed to increase autophagic markers in cells in which acetyltransferase ATAT1 expression was silenced indicates a possible role of a-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. Cell Death and Disease (2013) 4, e524; doi:10.1038/cddis.2013.53; published online 7 March 2013 Subject Category: Cancer Autophagy has been predominantly characterized as a nonselective degradative pathway activated by starvation. In this context, it is a process known to provide metabolic sources to the cell under nutrient depletion or other stresses. However, it has become apparent that autophagy is not solely dedicated to nutrient management, and a nutrient-independent 'basal' autop-hagy has been defined. 1 It occurs at basal levels in most tissues and contributes to the routine turnover of cytoplasmic compo-nents, thus enforcing intracellular quality control. 2 Induction of autophagy may be a double-edged sword, as it is also a mechanism leading to cell death when destruction of cell cytoplasm components is activated in a long-lasting way. 3 Autophagy is triggered by various stimuli, is mediated by a number of proteins encoded by autophagy-related genes (Atgs) and is usually described as a cascade of events such as nucleation, elongation, maturation and fusion. A canonical mechanism of nucleation (de novo formation) of autophago-somal 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 auto-phagosome 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 Recently, the cross-talk between autophagy and apoptosis has been considered as a key factor in the development and treatment of cancer. 3 The two pathways share molecular regulators and, in some cases, are activated by the same stimulus. 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. 10–12 Recently, CPTH6 (3-methylcyclopentylidene-[4-(4 0 -chloro-phenyl)thiazol-2-yl]hydrazone), a newly synthesized molecule