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

Wu J, Wang Y, Liang S, Ma H - Drug Des Devel Ther (2014)

Bottom Line: The objective of this study was to discover small-molecule caspase inhibitors with which to achieve cytoprotective effect.Nineteen compounds were found to have significant cytoprotective effects in cell viability assays.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.

View Article: PubMed Central - PubMed

Affiliation: Reaction Biology Corp, Malvern, PA, USA.

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.

No MeSH data available.


Related in: MedlinePlus

RBC1023 protects STAU-induced cell death in PC-12, U87MG, and SH-SY5Y cells.Notes: PC-12 (A), U87MG (B), and SH-SY5Y (C) cells were treated with DMSO, 20 μM of RBC1023, 1 μM of STAU, or 20 μM of RBC1023 plus 1 μM of STAU, 20 μM of RBC1020 and 20 μM of RBC1020 plus 1 μM of STAU for 24 hours. The cells were measured for viability by Cell Titer-Glo® luminescent assay (Promega Corporation, Fitchburg, WI, USA). *P<0.01 (a highly significant difference between the indicated groups) by Student’s t-test.Abbreviations: DMSO, dimethyl sulfoxide; STAU, staurosporine.
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f2-dddt-8-583: RBC1023 protects STAU-induced cell death in PC-12, U87MG, and SH-SY5Y cells.Notes: PC-12 (A), U87MG (B), and SH-SY5Y (C) cells were treated with DMSO, 20 μM of RBC1023, 1 μM of STAU, or 20 μM of RBC1023 plus 1 μM of STAU, 20 μM of RBC1020 and 20 μM of RBC1020 plus 1 μM of STAU for 24 hours. The cells were measured for viability by Cell Titer-Glo® luminescent assay (Promega Corporation, Fitchburg, WI, USA). *P<0.01 (a highly significant difference between the indicated groups) by Student’s t-test.Abbreviations: DMSO, dimethyl sulfoxide; STAU, staurosporine.

Mentions: Staurosporine is a broad-spectrum inhibitor of protein kinases, and has been widely used in the induction of apoptosis in diverse cellular models.18–22 Staurosporine preferentially activates the mitochondrial apoptotic pathway relying on caspase activation and causes cell death. To confirm the caspase inhibition of the hit compounds in cell-based assay, NIH3T3 cells were treated with 20 μM of each of the 43 hit compounds in the presence or absence of 1 μM staurosporine for 24 hours. Then, cell viability was assessed by Cell Titer-Glo® Luminescent Cell Viability Assay. As shown in Figure 1, while staurosporine treatment alone caused over 90% cell death, 19 compounds showed significant protection against staurosporine-induced cell death in NIH3T3 cells (P<0.01). Several compounds that are toxic to cells alone were removed from the hit list (data not shown). Compound RBC1020 (a caspase-9 selective inhibitor) and RBC1023 (a caspase-3 selective inhibitor) with no cytotoxicity alone demonstrated 7.5-fold and 5.9-fold increases respectively of cell viability compared to cells treated with staurosporine alone, indicating a strong cytoprotective activity of these two compounds. Caspase inhibitors could have potential application in treating neurodegenerative diseases such as Parkinson’s and Alzheimer’s diseases, which are usually associated with neuron cell death. Therefore, we extended the study to further examine the cytoprotective activity of the leads RBC1020 and RBC1023 in several neural origin cell types. PC12, U87MG, and SH-SY5Y cells were treated with DMSO, staurosporine alone, RBC1020 or RBC1023 alone, or RBC1020 or RBC1023 plus staurosporine for 24 hours and then the cell viability was examined. As shown in Figure 2, quantitative measurement of cell viability showed that RBC1023 increased the percentage of staurosporine-treated viable cells from 4% to 66% in PC12 cells, from 8% to 71% in U87MG cells, and from 6% to 47% in SH-SY5Y cells. Interestingly, RBC1020 had no or minimum cytoprotection in these three cell lines. It is still unknown why RBC1020 and RBC1023 showed different effects in different cell lines. Additional studies are needed to investigate the mechanism that causes this difference. These results demonstrated that RBC1023, a selective caspase-3 inhibitor, exhibits strong cytoprotective effect against staurosporine-induced cell death in multiple cell lines.


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

Wu J, Wang Y, Liang S, Ma H - Drug Des Devel Ther (2014)

RBC1023 protects STAU-induced cell death in PC-12, U87MG, and SH-SY5Y cells.Notes: PC-12 (A), U87MG (B), and SH-SY5Y (C) cells were treated with DMSO, 20 μM of RBC1023, 1 μM of STAU, or 20 μM of RBC1023 plus 1 μM of STAU, 20 μM of RBC1020 and 20 μM of RBC1020 plus 1 μM of STAU for 24 hours. The cells were measured for viability by Cell Titer-Glo® luminescent assay (Promega Corporation, Fitchburg, WI, USA). *P<0.01 (a highly significant difference between the indicated groups) by Student’s t-test.Abbreviations: DMSO, dimethyl sulfoxide; STAU, staurosporine.
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f2-dddt-8-583: RBC1023 protects STAU-induced cell death in PC-12, U87MG, and SH-SY5Y cells.Notes: PC-12 (A), U87MG (B), and SH-SY5Y (C) cells were treated with DMSO, 20 μM of RBC1023, 1 μM of STAU, or 20 μM of RBC1023 plus 1 μM of STAU, 20 μM of RBC1020 and 20 μM of RBC1020 plus 1 μM of STAU for 24 hours. The cells were measured for viability by Cell Titer-Glo® luminescent assay (Promega Corporation, Fitchburg, WI, USA). *P<0.01 (a highly significant difference between the indicated groups) by Student’s t-test.Abbreviations: DMSO, dimethyl sulfoxide; STAU, staurosporine.
Mentions: Staurosporine is a broad-spectrum inhibitor of protein kinases, and has been widely used in the induction of apoptosis in diverse cellular models.18–22 Staurosporine preferentially activates the mitochondrial apoptotic pathway relying on caspase activation and causes cell death. To confirm the caspase inhibition of the hit compounds in cell-based assay, NIH3T3 cells were treated with 20 μM of each of the 43 hit compounds in the presence or absence of 1 μM staurosporine for 24 hours. Then, cell viability was assessed by Cell Titer-Glo® Luminescent Cell Viability Assay. As shown in Figure 1, while staurosporine treatment alone caused over 90% cell death, 19 compounds showed significant protection against staurosporine-induced cell death in NIH3T3 cells (P<0.01). Several compounds that are toxic to cells alone were removed from the hit list (data not shown). Compound RBC1020 (a caspase-9 selective inhibitor) and RBC1023 (a caspase-3 selective inhibitor) with no cytotoxicity alone demonstrated 7.5-fold and 5.9-fold increases respectively of cell viability compared to cells treated with staurosporine alone, indicating a strong cytoprotective activity of these two compounds. Caspase inhibitors could have potential application in treating neurodegenerative diseases such as Parkinson’s and Alzheimer’s diseases, which are usually associated with neuron cell death. Therefore, we extended the study to further examine the cytoprotective activity of the leads RBC1020 and RBC1023 in several neural origin cell types. PC12, U87MG, and SH-SY5Y cells were treated with DMSO, staurosporine alone, RBC1020 or RBC1023 alone, or RBC1020 or RBC1023 plus staurosporine for 24 hours and then the cell viability was examined. As shown in Figure 2, quantitative measurement of cell viability showed that RBC1023 increased the percentage of staurosporine-treated viable cells from 4% to 66% in PC12 cells, from 8% to 71% in U87MG cells, and from 6% to 47% in SH-SY5Y cells. Interestingly, RBC1020 had no or minimum cytoprotection in these three cell lines. It is still unknown why RBC1020 and RBC1023 showed different effects in different cell lines. Additional studies are needed to investigate the mechanism that causes this difference. These results demonstrated that RBC1023, a selective caspase-3 inhibitor, exhibits strong cytoprotective effect against staurosporine-induced cell death in multiple cell lines.

Bottom Line: The objective of this study was to discover small-molecule caspase inhibitors with which to achieve cytoprotective effect.Nineteen compounds were found to have significant cytoprotective effects in cell viability assays.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.

View Article: PubMed Central - PubMed

Affiliation: Reaction Biology Corp, Malvern, PA, USA.

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.

No MeSH data available.


Related in: MedlinePlus