Abstract
BackgroundRecent studies indicated that histone deacetylase inhibitors (HDACi), a class of anticancer agents, are in addition to their ability of apoptosis induction also capable of provoking autophagy. Promoted by the treatment of malignant uterine sarcoma cells with the HDACi suberoylanilide hydroxamic acid (SAHA), we previously demonstrated predominant dose-dependent activation of autophagy in ESS-1 cells, but prevalent induction of apoptosis in MES-SA cells.MethodsIn order to extend our previous studies, SAHA-treated ESS-1 and MES-SA cells were monitored for protein expression to reveal differences in known markers of apoptosis explaining the different cytotoxic responses. Further analysis of the identified candidate protein included cell rescue experiments by gene transfer followed by subsequent screening of cells for induction of apoptosis and autophagy by immunoblotting, caspase activity as well as LC3 and MDC/PI staining. LDH release assays were performed to assess the amount of cell-mediated cytotoxicity.ResultsIn our search for responsible autophagic regulatory genes upstream of mammalian target of rapamycin (mTOR), we now discovered that, in contrast to MES-SA cells, a TP53-637C>T nonsense mutation located in the transactivating domain of the oncogenic suppressor p53 causes loss of its protein and consequently reduced PUMA induction in ESS-1 cells. Upon re-introduction of wild-type TP53, SAHA-treated ESS-1 cells underwent immediate apoptotic cell death as supported by upregulation of PUMA and caspase-9 as well as by activation of caspases-3 and -7 and PARP-1 cleavage. Concurrent downregulation of autophagy was noticed by upregulated mTor and phospho-mTOR expression as well as monitoring autophagosome formation employing LC3 and MDC staining. Previously, cytoplasmic master regulatory activities of the oncogenic suppressor p53 in inhibiting autophagy and triggering apoptosis were unravelled. Accordingly, p53-deficiency could explain both, the previously documented apoptosis resistance and prevailing SAHA-induced autophagy in ESS-1 cells. Using MES-SA cells with RNAi-silenced p53 expression and several p53-deficient tumor cell lines undergoing SAHA-induced autophagy, we could generally validate our finding suggesting an inhibitory role for p53 in the autophagic pathway in response to SAHA treatment.ConclusionsConclusively, these results could identify cytoplasmic p53 protein as a molecular switch that directly mediates the cytotoxic response of SAHA and thus open new therapeutic avenues.
Highlights
Recent studies indicated that histone deacetylase inhibitors (HDACi), a class of anticancer agents, are in addition to their ability of apoptosis induction capable of provoking autophagy
We previously demonstrated that suberoylanilide hydroxamic acid (SAHA) can, in addition to mitochondria-mediated apoptosis, promote caspaseindependent autophagic cell death [9,10,11] which offers an advantage in overcoming apoptotic resistant tumor cells
By screening several important pro-apoptotic key molecules, we found that expression of the tumor suppressor protein p53 was completely undetectable in endometrial stroma sarcoma (ESS)-1 cells when compared to the detectable and abundant expression in MES-SA cells
Summary
Recent studies indicated that histone deacetylase inhibitors (HDACi), a class of anticancer agents, are in addition to their ability of apoptosis induction capable of provoking autophagy. In various tumor cell lines, treatment with HDACi most frequently induces apoptosis by sequential activation of a series of cysteinedependent aspartate-directed proteases, called caspases [4, 5]. We previously demonstrated that SAHA can, in addition to mitochondria-mediated apoptosis, promote caspaseindependent autophagic cell death [9,10,11] which offers an advantage in overcoming apoptotic resistant tumor cells. This coincided with decreased expression of the autophagic key molecular determinant mTOR, a master regulator of cellular metabolism which is a therapeutic target for anticancer treatment. Upon conjugation to ubiquitin-like conjugation systems, cytosolic LC3 (termed LC3-I) gets incorporated to the growing autophagosome structure ( termed LC3-II) and acts as a marker [16, 17]
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