Cytoprotective effect of selective small-molecule caspase inhibitors against staurosporine-induced apoptosis

Abstract

Caspases are currently known as the central executioners of the apoptotic pathways. Inhibition of apoptosis and promotion of normal cell survival by caspase inhibitors would be a tremendous benefit for reducing the side effects of cancer therapy and for control of neurodegenerative disorders such as Parkinson’s, Alzheimer’s, and Huntington’s diseases. The objective of this study was to discover small-molecule caspase inhibitors with which to achieve cytoprotective effect. We completed the high-throughput screening of Bionet’s 37,500-compound library (Key Organics Limited, Camelford, Cornwall, UK) against caspase-1, -3, and -9 and successfully identified 43 initial hit compounds. The 43 hit compounds were further tested for cytoprotective activity against staurosporine-induced cell death in NIH3T3 cells. Nineteen compounds were found to have significant cytoprotective effects in cell viability assays. One of the compounds, RBC1023, was demonstrated to protect NIH3T3 cells from staurosporine-induced caspase-3 cleavage and activation. RBC1023 was also shown to protect against staurosporine-induced impairment of mitochondrial membrane potential. DNA microarray analysis demonstrated that staurosporine treatment induced broad global gene expression alterations, and RBC1023 co-treatment significantly restored these changes, especially of the genes that are related to cell growth and survival signaling such as Egr1, Cdc25c, cdkn3, Rhob, Nek2, and Taok1. Collectively, RBC1023 protects NIH3T3 cells against staurosporine-induced apoptosis via inhibiting caspase activity, restoring mitochondrial membrane potential, and possibly upregulating some cell survival-related gene expressions and pathways.

Keywords:

• cell death
• caspase inhibition
• mitochondria
• RBC1023

Supplementary material

Table S1 Molecular functions of the top 200 upregulated and downregulated clustering genes in the heat map

Acknowledgments

We gratefully acknowledge Drs Kurumi Horiuchi and Yuan Wang for their early works in this project; Mr John Tobias from the Molecular Profiling Facility at University of Pennsylvania School of Medicine for performing the DNA microarray analysis; and Dr Honglin Zhou at University of Pennsylvania School of Medicine for providing the SHSY-5Y cell line and conducting the cell viability assay of SHSY-5Y cells in his lab. This work was partially supported by NIH Small Business Innovation Research grants 5R44DE017485 and 5R44CA140166 to HM.

Disclosure

The authors report no conflicts of interest in this work.