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High-throughput screening for natural compound-based autophagy modulators reveals novel chemotherapeutic mode of action for arzanol.

ABSTRACT: Autophagy is an intracellular recycling pathway with implications for intracellular homeostasis and cell survival. Its pharmacological modulation can aid chemotherapy by sensitizing cancer cells toward approved drugs and overcoming chemoresistance. Recent translational data on autophagy modulators show promising results in reducing tumor growth and metastasis, but also reveal a need for more specific compounds and novel lead structures. Here, we searched for such autophagy-modulating compounds in a flow cytometry-based high-throughput screening of an in-house natural compound library. We successfully identified novel inducers and inhibitors of the autophagic pathway. Among these, we identified arzanol as an autophagy-modulating drug that causes the accumulation of ATG16L1-positive structures, while it also induces the accumulation of lipidated LC3. Surprisingly, we observed a reduction of the size of autophagosomes compared to the bafilomycin control and a pronounced accumulation of p62/SQSTM1 in response to arzanol treatment in HeLa cells. We, therefore, speculate that arzanol acts both as an inducer of early autophagosome biogenesis and as an inhibitor of later autophagy events. We further show that arzanol is able to sensitize RT-112 bladder cancer cells towards cisplatin (CDDP). Its anticancer activity was confirmed in monotherapy against both CDDP-sensitive and -resistant bladder cancer cells. We classified arzanol as a novel mitotoxin that induces the fragmentation of mitochondria, and we identified a series of targets for arzanol that involve proteins of the class of mitochondria-associated quinone-binding oxidoreductases. Collectively, our results suggest arzanol as a valuable tool for autophagy research and as a lead compound for drug development in cancer therapy.

SUBMITTER: Deitersen J

PROVIDER: S-EPMC8167120 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Previous studies from our laboratory and others have demonstrated that in addition to altering chromatin acetylation and conformation, histone deacetylase inhibitors (HDACi) disrupt the acetylation status of numerous transcription factors and other proteins. A whole genome yeast deletion library screen was used to identify components of the transcriptional apparatus that modulate the sensitivity to the hydroxamic acid-based HDACi, CG-1521. RESULTS: Screening 4852 haploid Saccharomyces cerevisiae deletion strains for sensitivity to CG-1521 identifies 407 sensitive and 80 resistant strains. Gene ontology (GO) enrichment analysis shows that strains sensitive to CG-1521 are highly enriched in processes regulating chromatin remodeling and transcription as well as other ontologies, including vacuolar acidification and vesicle-mediated transport. CG-1521-resistant strains include those deficient in the regulation of transcription and tRNA modification. Components of the SAGA histone acetyltransferase (HAT) complex are overrepresented in the sensitive strains, including the catalytic subunit, Gcn5. Cell cycle analysis indicates that both the wild-type and gcn5? strains show a G1 delay after CG-1521 treatment, however the gcn5? strain displays increased sensitivity to CG-1521-induced cell death compared to the wild-type strain. To test whether the enzymatic activity of Gcn5 is necessary in the response to CG-1521, growth assays with a yeast strain expressing a catalytically inactive variant of the Gcn5 protein were performed and the results show that this strain is less sensitive to CG-1521 than the gcn5? strain. CONCLUSION: Genome-wide deletion mutant screening identifies biological processes that affect the sensitivity to the HDAC inhibitor CG-1521, including transcription and chromatin remodeling. This study illuminates the pathways involved in the response to CG-1521 in yeast and provides incentives to understand the mechanisms of HDAC inhibitors in cancer cells. The data presented here demonstrate that components of the SAGA complex are involved in mediating the response to CG-1521. Additional experiments suggest that functions other than the acetyltransferase activity of Gcn5 may be sufficient to attenuate the effects of CG-1521 on cell growth.

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High-throughput screening for natural compound-based autophagy modulators reveals novel chemotherapeutic mode of action for arzanol.

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