The thiazole derivative CPTH6 impairs autophagy

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.

Histone deacetylase inhibition synergistically enhances pemetrexed cytotoxicity through induction of apoptosis and autophagy in non-small cell lung cancer

“…To obtain clearer results about the effect of drug combination on autophagic flux, we analysed the distribution and alteration of mRFP-GFP-LC3B fluorescent signals using H1299 cells stably expressing mRFP-EGFP-LC3 reporter (H1299/ptf-LC3) [ 37 , 41 ] (Figure 4 C,D). As GFP but not mRFP fluorescence is lost in acidic compartments, mRFP-GFP-LC3B labels nonacidic autophagosomes as yellow fluorescence (positive for both green and red) but acidic autophagolysosomes as red fluorescence only [ 41 ].…”

“…Human NSCLC established cell lines (H1299, H460, A549, H1650, Calu-1) were cultured in 10% inactivated foetal bovine serum (HyClone, Termoscientific, South Logan, UT) in RPMI medium (Invitrogen, Carlsbad, CA). H1299 short hairpin (sh) Beclin1, shControl, EGFP-LC3B and ptf-LC3 stable clones were generated as previously described [ 37 ] and cultured in the presence of geneticin (800 μg/ml, Sigma-Aldrich, St. Louis, MO).…”

Resveratrol analogue 3,4,4′-trihydroxy-trans-stilbene induces apoptosis and autophagy in human non-small-cell lung cancer cells in vitro

“…The increases in the LC3-II levels and autophagosome accumulation induced by 3,4,4'-THS may be related to either enhanced autophagosome formation due to enhanced autophagic activity or reduced autophagosome degradation due to impaired autophagic flux [18] . To discriminate between these two possibilities, we first evaluated the level of p62, which is involved in the formation of autophagosomes and constitutively degraded by the autophagic pathway via specific binding to LC3 [19] .…”

Histone acetyltransferase inhibitor CPTH6 preferentially targets lung cancer stem-like cells

“…Several molecules with HAT inhibitory activity have been identified and some of them showed to induce cell death preferentially in cancer cells when compared to normal ones [6][7][8][9][10]. Among them, the thiazole derivative 3-methyl-cyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl)]hydrazone (CPTH6), has been characterized by our group as a novel Gcn5 and pCAF HAT inhibitor, able to activate the apoptotic program and to modulate the autophagic flux in a panel of tumor cell lines [11,12]. Cancer stem cells (CSCs) have been proposed as potential culprits not only of the tumor initiation and progression, but also of therapeutic failure and tumor relapse, mainly because of their intrinsic resistance to conventional drugs [13].…”