Characterization of SAHA-Induced Cell Death: The Role of Autophagy and Apoptosis.
Authors: Lam Lam H. , Jiang Xuejun 
Published: October 2019

Abstract 1566

Poster Board I-590

Autophagy is a cellular pathway in which proteins and organelles are degraded and recycled. It is a steady state process, but can be up-regulated by a number of different intra and extracellular stimuli including endoplasmic reticulum stress, starvation, and hypoxia. Its molecular pathway has been well described in the yeast system through complementation experiments. These earlier studies have defined a number of ATG (autophagy) genes involved in autophagy. Recently, mammalian homologs of many of the yeast ATG genes have been identified and studies are underway to better characterize their roles in autophagy. Histone deacetylase (HDAC) inhibitors, such as suberoylanilide hydroxamic acid (SAHA), are a new class of targeted therapeutic agents that have anticancer effects. Our laboratory has shown that SAHA can trigger both mitochondria-mediated apoptosis and caspase-independent cell death. The significance of the latter finding is that SAHA can potentially treat malignant cells with apoptotic defects. However, the exact mechanism by which cell death occurs in an apoptotic defective cell is unclear. Moreover, whether cell death involves the autophagy pathway remains to be determined. Using mouse embryonic fibroblasts (MEF), which are defective in either apoptosis (Apaf-1 -/-) or autophagy (ATG5 -/-), we have begun to analyze what mechanisms of cell death are being triggered by SAHA treatment. Specifically, we hope to address the possibility, and the extent to which, SAHA utilizes the autophagy pathway to carry out this effect. Through the use of RNA-interference to knock-down the expression levels of ATG5 proteins, we were able to create a cell line that is defective in both apoptosis and autophagy, in efforts to test how SAHA treatment affects cell lines impaired in both pathways. Apoptosis and autophagy activities can be routinely assessed using specific markers by western blot, immuno-fluorescence, and also through assays developed and used routinely in the laboratory (e.g. caspase activity assays to measure induction of apoptosis). As previously reported in cells with defective apoptosis, treatment with SAHA appears to induce autophagic cell death. Using wildtype MEFs, we found that treatment with SAHA could directly induce autophagic activity as demonstrated by the presence of autophagosome structures on EM imaging. Interestingly, in cells with defective autophagy, treatment with SAHA induced apoptotic cell death, providing evidence that SAHA was capable of directly inducing cell death by either apotosis or autophagy, if one pathway was defective. Surprisingly we found that in cells defective in both pathways, where we were expecting SAHA would not have any effect, treatment with SAHA still induced cell death by some unknown mechanism. Our current findings lead us to the hypothesis that SAHA can directly induce cell death by not only induction of either apoptosis or autophagy in cells defective in one or the other pathways, but also by some as of yet unknown and possibly novel cell death mechanism if both pathways are impaired. Future studies will address deciphering the molecular components of this possibly novel cell death pathway. In addition, attempts will be made to distinguish if other HDAC inhibitors can induce cell death by mechanisms similar to SAHA.

Disclosures

No relevant conflicts of interest to declare.