Dysfunctional autophagy induced by the pro-apoptotic natural compound climacostol in tumour cells

Abstract

Autophagy occurs at a basal level in all eukaryotic cells and may support cell survival or activate death pathways. Due to its pathophysiologic significance, the autophagic machinery is a promising target for the development of multiple approaches for anti-neoplastic agents. We have recently described the cytotoxic and pro-apoptotic mechanisms, targeting the tumour suppressor p53, of climacostol, a natural product of the ciliated protozoan Climacostomum virens. We report here on how climacostol regulates autophagy and the involvement of p53-dependent mechanisms. Using both in vitro and in vivo techniques, we show that climacostol potently and selectively impairs autophagy in multiple tumour cells that are committed to die by apoptosis. In particular, in B16-F10 mouse melanomas climacostol exerts a marked and sustained accumulation of autophagosomes as the result of dysfunctional autophagic degradation. We also provide mechanistic insights showing that climacostol affects autophagosome turnover via p53-AMPK axis, although the mTOR pathway unrelated to p53 levels plays a role. In particular, climacostol activated p53 inducing the upregulation of p53 protein levels in the nuclei through effects on p53 stability at translational level, as for instance the phosphorylation at Ser15 site. Noteworthy, AMPKα activation was the major responsible of climacostol-induced autophagy disruption in the absence of a key role regulating cell death, thus indicating that climacostol effects on autophagy and apoptosis are two separate events, which may act independently on life/death decisions of the cell. Since the activation of p53 system is at the molecular crossroad regulating both the anti-autophagic action of climacostol and its role in the apoptosis induction, it might be important to explore the dual targeting of autophagy and apoptosis with agents acting on p53 for the selective killing of tumours. These findings also suggest the efficacy of ciliate bioactive molecules to identify novel lead compounds in drug discovery and development.

Natural Function and Structural Modification of Climacostol, a Ciliate Secondary Metabolite

“…In particular, experiments performed on human tumor squamous carcinoma A431 cells, and human promyelocytic leukemia HL60 cells demonstrated that climacostol exerts its action by inhibiting cell growth and triggering a mitochondrion-dependent apoptotic program. Subsequent extensive in vitro screenings and in vivo experiments confirmed the previous observations [4,21,[29][30][31].…”

“…Some of us recently reported on how climacostol regulates autophagy and the involvement of p53-dependent mechanisms [30]. Essentially, our data indicated that the protozoan toxin potently and selectively impairs autophagy in multiple tumor cells that are committed to dying by apoptosis.…”

“…The results indicated that cell viability was negatively affected by the two analogues with a comparable strength (Table 1), and both AN1 and AN2 displayed similar or even lower potencies when compared to climacostol [31]. Furthermore, as in the case of various tumor cells exposed to climacostol [4,29,30], immunostaining techniques revealed that B16-F10 melanoma cells expressed high levels of active caspase 3 after AN1 and AN2 treatment ( Figure 6), thus demonstrating the activation of an apoptotic pathway induced by both analogues. The choice to introduce the aforementioned moieties into the aromatic ring of climacostol was supported by the observation that similar modification performed on some polyphenols and phenolic lipids resulted in a significant improvement of their cytotoxic and antimicrobial activity [37,38].…”

“…The results indicated that cell viability was negatively affected by the two analogues with a comparable strength (Table 1), and both AN1 and AN2 displayed similar or even lower potencies when compared to climacostol [31]. Furthermore, as in the case of various tumor cells exposed to climacostol [4,29,30], immunostaining techniques revealed that B16-F10 melanoma cells expressed high levels of active caspase 3 after AN1 and AN2 treatment ( Figure 6), thus demonstrating the activation of an apoptotic pathway induced by both analogues. Table 1.…”

“…Recent investigations on the mechanisms of action of climacostol demonstrated that it reduces the viability/proliferation of melanoma cells, causing rapidly occurring DNA damage, also inducing the intrinsic apoptotic pathway characterized by the dissipation of the mitochondrial membrane potential, the translocation of Bax to the mitochondria, the release of cytochrome c from the mitochondria, and the activation of caspase 9-dependent cleavage of caspase 3 [29,30]. Additionally, persistent inhibition of the growth of melanoma allografts as well as a reduction in the number of viable and proliferating tumor cells was achieved after intratumoral injections of the toxin [4,29].…”