Autophagy is an essential process for cell survival that involves lysosomal degradation of cell organelles and cytoplasmic proteins characterized by the formation of autophagosomes. These structures fuse with functional lysosomes followed by degradation of proteins and organelles catalyzed by lysosomal enzymes. Autophagy is imperative for the survival of a cell during starvation states and maintains cellular viability. Cancer cells are known to have greater levels of autophagy compared to normal human cells owing to increased metabolic and nutritional demand in these rapidly proliferating cells. This mechanism forms the basis for cancer therapy using substances or molecules that disrupt autophagy in cancer cells. Molecules such as 3-methyladenine, wortmannin, bafilomycin A1, and hydroxychloroquine serve as autophagy inhibitors, although these molecules have different modes of action.
Sang-Hyun Park, Insu Shin and Professor Injae Shin from Yonsei University together with, Dr. Gun-Hee Kim and Dr. Sung-Kyun Ko from the Anticancer Agent Research Center at Korea Research Institute of Bioscience and Biotechnology identified a novel autophagy inhibitor with anticancer properties for the treatment of different cancerous lesions and assessed the mechanism of action of this small molecule autophagy inhibitor. The authors used HeLa cells to demonstrate the inhibitory effects of autophazole (Atz) against autophagy in cancerous cells. The research team findings showed that Atz induces a rise in p62 and LC3-II indicating disruption of autophagy in the cells. The key findings of the new study, are now published in peer-reviewed journal ChemBioChem.
The authors further explored the mechanism underlying inhibition of autophagy by Atz. Authors observed that Atz impairs lysosomal function by accumulating in the lysosomes and stimulating lysosomal membrane permeabilization (LMP). Induction of LMP disrupts lysosomal pH and promotes leakage of cathepsin B into the cytosol. A novel finding in their study is that Atz does not hinder the fusion of autophagosomes with lysosomes and does not influence the levels of calcium ions in the cytosol. These features of Atz are significantly distinct from other autophagy inhibitors.
Another important finding by the research team is the role of Atz in inducing apoptosis in cancerous cells. LMP promotes the release of lysosomal cathepsin B into the cytosol. This enzyme catalyzes the conversion of a pro-apoptotic protein called Bid into truncated Bid (tBid). The tBid triggers the translocation of Bax protein to the outer mitochondrial membrane with subsequent mitochondrial outer membrane permeabilization (MOMP). This is followed by a chain of processes that result in the formation of an apoptosome and the activation of caspase-9 and caspase-3. Activation of caspases leads to apoptotic cell death of the cancer cells.
The authors tried to identify the target protein(s) of the autophagy inhibitor Atz. They used several advanced techniques including cellular thermal shift assay (CETSA), affinity chromatography, and drug affinity responsive target stability (DARTS) to determine the target molecule/structure of Atz. The two suspected targets were Hsp70 and acid sphingomyelinase (ASM) since these molecules promote lysosomal stability in neoplastic cells. However, results didn’t support the involvement of ASM and Hsp70.
To sum up, Professor Injae Shin and colleagues has successfully identified a novel autophagy inhibitor – autophazole – which is also capable of inducing caspase-mediated apoptosis. The primary mechanism of action of Atz is to stimulate LMP which leads to the development of nonfunctional lysosomes as well as activation of caspases mediated by the release of cathepsin B. Further research in autophazole in testing in animal disease models and possibly in clinical translation will bring a new novel cancer therapy to patients.
Park, Sang‐Hyun & Shin, Insu & Kim, Gun-Hee & Ko, sung-kyun & Shin, Injae. (2021). An Autophagy‐Disrupting Small Molecule Promotes Cancer Cell Death via Caspase Activation. ChemBioChem. 10.1002/cbic.202100398.