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.

Figure 1

CPTH6 treatment induces autophagic markers under basal conditions. (A) Western blot analysis of LC3B-I to LC3B-II conversion in the indicated cell lines after 72 h of treatment with CPTH6. HSP72/73 is shown as a loading and transferring control. Western blots representative of three independent experiments with similar results are shown. LC3B-II levels were quantified by densitometric analyses and fold increase relative to untreated cells are presented. (B) Representative images of fluorescence microscopy and (C) quantification of cells positive for autophagosomal structures in H1299 cells stably expressing EGFP-LC3B protein untreated (black) or treated with CPTH6 (100 μM, white) for the indicated times. The results represent the average±S.E.M. of three independent experiments. *P values were calculated between untreated and treated cells, P<0.05. (D) TEM analysis in H1299 cells (a) untreated and (b–d) treated with CPTH6 (100 μM, 24 h). Untreated cells showed their typical ultrastructural features with well-preserved cytoplasm, indented nucleus with prominent nucleolus and dispersed chromatin. After CPTH6 treatment, numerous vesicles containing material, including organelles, in various degrees of degradation are observed in the cytoplasm (b, see arrows). At higher magnification (c), myelin-like structures and not well-delimited autophagosomes induced by CPTH6 treatment were visible (box in the picture). Numerous lipid highly osmiophilic granules (see arrows) were also very often observed in the treated cells (d). N, nucleus

Figure 2

CPTH6 induces a block of basal autophagy. (a) Quantification of cells positive for autophagosomal structures in M14 cells stably expressing EGFP-LC3B protein after 48 h of treatment with CPTH6 (50 μM), bafilomycin A1 (Baf A1, 2.5 nM), serum starvation or 3-methyladenine (3-MA, 1 mM), alone or in combination. The results represent the average±S.E.M. of three independent experiments. (b) Western blot analysis of LC3B-I to LC3B-II conversion in H1299 cells after treatment with CPTH6 (100 μM) alone or in combination with chloroquine (CQ, 25 μM) or bafilomycin A1 for the indicated times. (c) Representative images of immunofluorescence of p62 protein and (d) quantification of cells positive for p62/EGFP-LC3B colocalization in M14 EGFP-LC3B-expressing cells after CPTH6 treatment (50 μM, 72 h) or serum starvation (48 h). (e) Western blot analysis of p62 protein in U-937 cell lines after serum starvation (48 h). (f) Western blot analysis of NBR1 and p62 proteins in the indicated cell lines after 72 h of treatment with CPTH6. (g) Western blot analysis of HIF-1α, p62, phospho-IkBα (ser34/ser36) and ubiquitin in U-937 cell line after treatment with MG132 (5 μM, 6 h) or CPTH6 (100 μM, 48 h). (b, and e–g) Western blots representative of three independent experiments with similar results are shown. HSP72/73 is shown as loading and transferring control. (b and f) LC3B-II, p62 and NBR1 levels were quantified by densitometric analyses and fold increase relative to untreated cells is presented. (a and d) The results represent the average±S.E.M. of three independent experiments. *P-values were calculated between untreated and treated cells, P<0.05

Figure 3

Atg-7, but not Beclin-1, is required for CPTH6-induced autophagic features. (a–c) Western blot analysis of LC3B-I to LC3B-II conversion, Beclin-1, p62, Atg-7 and LAMP-2 proteins in H1299 cells stably expressing (a) control short hairpin RNA (shCont), short hairpin RNA directed against Beclin-1 (shBECL #1 and #2), or transiently transfected with control RNA interference (siCont), or RNA interference directed against (b) Atg-7 (siAtg-7) or (c) LAMP-2 (siLAMP-2) after CPTH6 treatment (100 μM, 24 h). Western blots representative of three independent experiments with similar results are shown. HSP72/73 is shown as a loading and transferring control. LC3B-II and p62 levels were quantified by densitometric analyses and fold increase relative to untreated cells are presented. (d) Representative images of fluorescence microscopy and (e) quantification of cells positive for autophagosomal structures in H1299 cells stably expressing EGFP-LC3B protein and transiently transfected with shCont, shBECL, siCont, siAtg-7 or siLAMP-2 after treatment with CPTH6 (100 μM, 24 h). The results represent the average±S.E.M. of three independent experiments. P-values were calculated between *untreated and treated cells, or ^#control (siCont/shCont) and silenced cells, P<0.05

Figure 4

CPTH6 blocks autophagy at the late stage. (a) Representative images of immunofluorescence of LAMP-2 protein in M14 EGFP-LC3B-expressing cells after treatment with CPTH6 (50 μM, 72 h) or serum starvation (48 h). (b) Western blot analysis of cathepsin B and cathepsin D proteins in U-937 cells after CPTH6 treatment (48 h). Western blots representative of three independent experiments with similar results are shown. HSP72/73 is shown as a loading and transferring control. (c) Quantification of acid phosphatase activity of U-937 whole-cell extracts or lysosomal fractions obtained after CPTH6 treatment (100 μM, 24 h). The results represent the average±S.D. of two independent experiments and are expressed as optical density (OD) at 540 nm

Figure 5

ATAT1 protein expression is required for CPTH6 effect on autophagy. (a) Western blot analysis of acetylated α-tubulin and HDAC6 proteins in the indicated cell lines after treatment with CPTH6 (100 μM, 48 h). (b and c) Western blot analysis of LC3B-I to LC3B-II conversion, HDAC6 and acetylated α-tubulin proteins in H1299 cells transiently transfected with control RNA interference (siCont), or RNA interference directed against HDAC6 (siHDAC6) or ATAT1 (siATAT1), after treatment with CPTH6 (100 μM, 24 h). LC3B-II levels were quantified by densitometric analyses and fold increase relative to untreated cells is presented. (a–c) Western blots representative of three independent experiments with similar results are shown. HSP72/73 is shown as a loading and transferring control. (d) Representative images of fluorescence microscopy and (e) quantification of cells positive for autophagosomal structures in H1299 cells stably expressing EGFP-LC3B protein and transiently transfected with siHDAC6 or siATAT1 after treatment with CPTH6 (100 μM, 24 h). The results represent the average±S.E.M. of three independent experiments. P-values were calculated between *untreated and treated cells or between ^#control (siCont) and silenced cells, P<0.05

Figure 6

CPTH derivatives elicit the same effect of CPTH6 on autophagic flux. (a) Western blot analysis of LC3B-I to LC3B-II conversion and p62 protein in U-937 cell line after treatment with CPTH2 (50 μM, 48 h), CPTH9 or CPTH11 (5 μM, 24 h). Western blots representative of three independent experiments with similar results are shown. HSP72/73 is shown as a loading and transferring control. LC3B-II and p62 levels were quantified by densitometric analyses and fold increase relative to untreated cells is presented. (b) Representative images of fluorescence microscopy and (c) quantification of cells positive for autophagosomal structures in H1299 EGFP-LC3B-expressing cells after 48 h of treatment with CPTH2 (50 μM). (d) Representative images of immunofluorescence of p62 protein and (e) quantification of cells positive for p62/EGFP-LC3B colocalization in M14 EGFP-LC3B-expressing cells after CPTH2 treatment (50 μM, 48 h). (c and e) The results represent the average±S.E.M. of three independent experiments. *P-values were calculated between untreated and treated cells, P<0.05

Figure 7

The effect of CPTH6 on autophagic flux was dependent on the stimulus used to induce autophagy. (a) Western blot analysis of LC3B-I to LC3B-II conversion and p62 protein in H1299 cells after 6 h of treatment with CPTH6 (100 μM) alone or in combination with serum starvation. (b) Western blot analysis of LC3B-I to LC3B-II conversion and p62 protein in H1299 cells after treatment with chloroquine (CQ, 25 μM) alone or in combination with serum starvation (48 h). (c) Representative images of fluorescence microscopy and (d) quantification of cells positive for mRFP (red) and mRFP/GFP (yellow) autophagosomal structures in H1299 cells stably transfected with ptf-LC3B vector after 6 h of treatment with CPTH6 (100 μM) or chloroquine (CQ, 25 μM) alone or in combination with serum starvation. The results represent the average±S.E.M. of three independent experiments. *P-values were calculated between untreated and treated cells, P<0.05. (e) Western blot analysis of LC3B-I to LC3B-II conversion and p62 protein in H1299 cell line after 2 h of treatment with CPTH6 (100 μM) alone or in combination with RAD001 (1 μM). (a, b and e) Western blots representative of three independent experiments with similar results are shown. HSP72/73 is shown as a loading and transferring control. LC3B-II and p62 levels were quantified by densitometric analyses and fold increase relative to untreated cells is presented