No abstract

Purpose: We previously identified novel thiazole derivatives able to reduce histone acetylation and histone acetyltransferase (HAT) activity in yeast. Among these compounds, 3-methylcyclopentylidene-[4-(4 0 -chlorophenyl)thiazol-2-yl]hydrazone (CPTH6) has been selected and used throughout this study.Experimental Design: The effect of CPTH6 on histone acetylation, cell viability and differentiation, cellcycle distribution, and apoptosis in a panel of acute myeloid leukemia and solid tumor cell lines has been evaluated.Results: Here, we showed that CPTH6 leads to an inhibition of Gcn5 and pCAF HAT activity. Moreover, it inhibits H3/H4 histones and a-tubulin acetylation of a panel of leukemia cell lines. Concentration-and time-dependent inhibition of cell viability, paralleled by accumulation of cells in the G 0 /G 1 phase and depletion from the S/G 2 M phases, was observed. The role of mitochondrial pathway on CPTH6-induced apoptosis was shown, being a decrease of mitochondrial membrane potential and the release of cytochrome c, from mitochondria to cytosol, induced by CPTH6. Also the involvement of Bcl-2 and Bcl-xL on CPTH6-induced apoptosis was found after overexpression of the two proteins in leukemia cells. Solid tumor cell lines from several origins were shown to be differently sensitive to CPTH6 treatment in terms of cell viability, and a correlation between the inhibitory efficacy on H3/H4 histones acetylation and cytotoxicity was found. Differentiating effect on leukemia and neuroblastoma cell lines was also induced by CPTH6.Conclusions: These results make CPTH6 a suitable tool for discovery of molecular targets of HAT and, potentially, for the development of new anticancer therapies, which warrants further investigations.

In addition to act as an antiapoptotic protein, B-cell lymphoma (bcl)-2 can also promote tumor angiogenesis. In this context, we have previously demonstrated that under hypoxia bcl-2 promotes hypoxia-inducible factor-1 (HIF-1)-mediated vascular endothelial growth factor (VEGF) expression in melanoma and breast carcinoma. Here, we report on the role of the BH4 domain in bcl-2 functions, by showing that removal of or mutations at the BH4 domain abrogate the ability of bcl-2 to induce VEGF protein expression and transcriptional activity under hypoxia in human melanoma cells. We have also extended this observation to other human tumor histotypes, such as colon, ovarian and lung carcinomas. The involvement of BH4 on HIF-1a protein expression, stability, ubiquitination and HIF-1 transcriptional activity was also demonstrated in melanoma experimental model. Moreover, we validated the role of the BH4 domain of bcl-2 in the regulation of HIF-1/VEGF axis, demonstrating that BH4 peptide is sufficient to increase HIF-1a protein half-life impairing HIF-1a protein ubiquitination, and to enhance VEGF secretion in melanoma cells exposed to hypoxia. Finally, we found that the mechanism by which bcl-2 regulates HIF-1-mediated VEGF expression does not require BH1 and BH2 domains, and it is independent of antiapoptotic and prosurvival function of bcl-2.

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.