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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 inhibits STAU-induced caspase-3 cleavage and activation in NIH3T3 cells.Notes: NIH3T3 cells were treated with DMSO, 20 μM of RBC1023, 1 μM of STAU, or 20 μM of RBC1023 plus 1 μM of STAU for 6 hours. Caspase activity was measured by Apo-ONE® Homogeneous Caspase-3/7 Assay (Promega Corporation, Fitchburg, WI, USA) (A), and processing of caspase-3 was detected by Western blotting with anti-caspase-3 antibody (B). Positions of the intact and cleaved forms of capase-3 are indicated by arrows. α-tubulin was used as a control for protein loading. One-way analysis of variance indicates: P>0.05 (DMSO versus [vs] RBC1023), P>0.05 (DMSO vs RBC1023 + STAU), and P<0.01 (STAU vs RBC1023 + STAU).Abbreviations: DMSO, dimethyl sulfoxide; STAU, staurosporine.
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f4-dddt-8-583: RBC1023 inhibits STAU-induced caspase-3 cleavage and activation in NIH3T3 cells.Notes: NIH3T3 cells were treated with DMSO, 20 μM of RBC1023, 1 μM of STAU, or 20 μM of RBC1023 plus 1 μM of STAU for 6 hours. Caspase activity was measured by Apo-ONE® Homogeneous Caspase-3/7 Assay (Promega Corporation, Fitchburg, WI, USA) (A), and processing of caspase-3 was detected by Western blotting with anti-caspase-3 antibody (B). Positions of the intact and cleaved forms of capase-3 are indicated by arrows. α-tubulin was used as a control for protein loading. One-way analysis of variance indicates: P>0.05 (DMSO versus [vs] RBC1023), P>0.05 (DMSO vs RBC1023 + STAU), and P<0.01 (STAU vs RBC1023 + STAU).Abbreviations: DMSO, dimethyl sulfoxide; STAU, staurosporine.

Mentions: To confirm RBC1023 as a caspase inhibitor in cell-based assays, we examined the caspase activity/activation-inducing effect in NIH3T3 cells that were treated with staurosporine in the presence or absence of RBC1023. As shown in Figure 4A, 6-hour staurosporine treatment induced significant caspase activation in NIH3T3 cells, as evaluated by Apo-ONE® Homogeneous Caspase-3/7 Assay using Z-DEVD-rhodamine 110 as a caspase substrate. Treatment of NIH3T3 cells with staurosporine for 6 hours also resulted in cleavage/activation of caspase-3, as was evident by the appearance of a 17 kD proteolytic product of caspase-3 as determined by Western blot analysis (Figure 4B). RBC1023 dramatically inhibited staurosporine-induced caspase-3 cleavage and activation (Figure 4B). These results confirmed that RBC1023 is a caspase inhibitor in cells.


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 inhibits STAU-induced caspase-3 cleavage and activation in NIH3T3 cells.Notes: NIH3T3 cells were treated with DMSO, 20 μM of RBC1023, 1 μM of STAU, or 20 μM of RBC1023 plus 1 μM of STAU for 6 hours. Caspase activity was measured by Apo-ONE® Homogeneous Caspase-3/7 Assay (Promega Corporation, Fitchburg, WI, USA) (A), and processing of caspase-3 was detected by Western blotting with anti-caspase-3 antibody (B). Positions of the intact and cleaved forms of capase-3 are indicated by arrows. α-tubulin was used as a control for protein loading. One-way analysis of variance indicates: P>0.05 (DMSO versus [vs] RBC1023), P>0.05 (DMSO vs RBC1023 + STAU), and P<0.01 (STAU vs RBC1023 + STAU).Abbreviations: DMSO, dimethyl sulfoxide; STAU, staurosporine.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4043716&req=5

f4-dddt-8-583: RBC1023 inhibits STAU-induced caspase-3 cleavage and activation in NIH3T3 cells.Notes: NIH3T3 cells were treated with DMSO, 20 μM of RBC1023, 1 μM of STAU, or 20 μM of RBC1023 plus 1 μM of STAU for 6 hours. Caspase activity was measured by Apo-ONE® Homogeneous Caspase-3/7 Assay (Promega Corporation, Fitchburg, WI, USA) (A), and processing of caspase-3 was detected by Western blotting with anti-caspase-3 antibody (B). Positions of the intact and cleaved forms of capase-3 are indicated by arrows. α-tubulin was used as a control for protein loading. One-way analysis of variance indicates: P>0.05 (DMSO versus [vs] RBC1023), P>0.05 (DMSO vs RBC1023 + STAU), and P<0.01 (STAU vs RBC1023 + STAU).Abbreviations: DMSO, dimethyl sulfoxide; STAU, staurosporine.
Mentions: To confirm RBC1023 as a caspase inhibitor in cell-based assays, we examined the caspase activity/activation-inducing effect in NIH3T3 cells that were treated with staurosporine in the presence or absence of RBC1023. As shown in Figure 4A, 6-hour staurosporine treatment induced significant caspase activation in NIH3T3 cells, as evaluated by Apo-ONE® Homogeneous Caspase-3/7 Assay using Z-DEVD-rhodamine 110 as a caspase substrate. Treatment of NIH3T3 cells with staurosporine for 6 hours also resulted in cleavage/activation of caspase-3, as was evident by the appearance of a 17 kD proteolytic product of caspase-3 as determined by Western blot analysis (Figure 4B). RBC1023 dramatically inhibited staurosporine-induced caspase-3 cleavage and activation (Figure 4B). These results confirmed that RBC1023 is a caspase inhibitor in cells.

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