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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 restores NIH3T3 cells from STAU-induced loss of cell viability, ATP contents, and disruption of mitochondrial membrane potential.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 24 hours (A and B), or 6 hours (C). The cell viability was measured by MTT assay (A) and ATP content assay (B), and the mitochondrial membrane potential was measured by JC-10 mitochondrial membrane potential assay (C). One-way analysis of variance indicates: (A) P>0.05 (DMSO versus [vs] RBC1023), P<0.01 (DMSO vs RBC1023 + STAU), and P<0.01 (STAU vs RBC1023 + STAU); (B) P<0.01 (DMSO vs RBC1023), P<0.001 (DMSO vs RBC1023 + STAU), and P<0.001 (STAU vs RBC1023 + STAU); (C) P>0.05 (DMSO vs RBC1023), P>0.05 (DMSO vs RBC1023 + STAU), and P<0.001 (STAU vs RBC1023 + STAU).Abbreviations: ATP, adenosine triphosphate; DMSO, dimethyl sulfoxide; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); STAU, staurosporine; Δψm, mitochondrial membrane potential.
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f5-dddt-8-583: RBC1023 restores NIH3T3 cells from STAU-induced loss of cell viability, ATP contents, and disruption of mitochondrial membrane potential.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 24 hours (A and B), or 6 hours (C). The cell viability was measured by MTT assay (A) and ATP content assay (B), and the mitochondrial membrane potential was measured by JC-10 mitochondrial membrane potential assay (C). One-way analysis of variance indicates: (A) P>0.05 (DMSO versus [vs] RBC1023), P<0.01 (DMSO vs RBC1023 + STAU), and P<0.01 (STAU vs RBC1023 + STAU); (B) P<0.01 (DMSO vs RBC1023), P<0.001 (DMSO vs RBC1023 + STAU), and P<0.001 (STAU vs RBC1023 + STAU); (C) P>0.05 (DMSO vs RBC1023), P>0.05 (DMSO vs RBC1023 + STAU), and P<0.001 (STAU vs RBC1023 + STAU).Abbreviations: ATP, adenosine triphosphate; DMSO, dimethyl sulfoxide; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); STAU, staurosporine; Δψm, mitochondrial membrane potential.

Mentions: To investigate if RBC1023 can rescue staurosporine-induced decrease of cell viability, we performed the MTT assay, in which the tetrazolium dye, MTT, is reduced to formazan by intracellular nicotinamide adenine dinucleotide phosphate (NADPH) oxidoreductases, which correlates with cellular metabolic activity and the number of viable cells present. As shown in Figure 5A, staurosporine treatment for 24 hours reduced the cell viability dramatically. RBC1023 co-treatment significantly rescued the loss of cell viability induced by staurosporine in NIH3T3 cells. We also performed ATP measurement by generating an ATP standard curve using Cell Titer-Glo® luminescent assay. As shown in Figure 5B, RBC1023 clearly restored the loss of ATP contents in cells treated with staurosporine. One-way ANOVA of the MTT and ATP assay data suggested that RBC1023 induces normalization or partial recovery of cell viability and ATP contents. Our results here further confirmed the cytoprotective effect of RBC1023 demonstrated by Cell Titer-Glo® assay.


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 restores NIH3T3 cells from STAU-induced loss of cell viability, ATP contents, and disruption of mitochondrial membrane potential.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 24 hours (A and B), or 6 hours (C). The cell viability was measured by MTT assay (A) and ATP content assay (B), and the mitochondrial membrane potential was measured by JC-10 mitochondrial membrane potential assay (C). One-way analysis of variance indicates: (A) P>0.05 (DMSO versus [vs] RBC1023), P<0.01 (DMSO vs RBC1023 + STAU), and P<0.01 (STAU vs RBC1023 + STAU); (B) P<0.01 (DMSO vs RBC1023), P<0.001 (DMSO vs RBC1023 + STAU), and P<0.001 (STAU vs RBC1023 + STAU); (C) P>0.05 (DMSO vs RBC1023), P>0.05 (DMSO vs RBC1023 + STAU), and P<0.001 (STAU vs RBC1023 + STAU).Abbreviations: ATP, adenosine triphosphate; DMSO, dimethyl sulfoxide; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); STAU, staurosporine; Δψm, mitochondrial membrane potential.
© Copyright Policy
Related In: Results  -  Collection

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

f5-dddt-8-583: RBC1023 restores NIH3T3 cells from STAU-induced loss of cell viability, ATP contents, and disruption of mitochondrial membrane potential.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 24 hours (A and B), or 6 hours (C). The cell viability was measured by MTT assay (A) and ATP content assay (B), and the mitochondrial membrane potential was measured by JC-10 mitochondrial membrane potential assay (C). One-way analysis of variance indicates: (A) P>0.05 (DMSO versus [vs] RBC1023), P<0.01 (DMSO vs RBC1023 + STAU), and P<0.01 (STAU vs RBC1023 + STAU); (B) P<0.01 (DMSO vs RBC1023), P<0.001 (DMSO vs RBC1023 + STAU), and P<0.001 (STAU vs RBC1023 + STAU); (C) P>0.05 (DMSO vs RBC1023), P>0.05 (DMSO vs RBC1023 + STAU), and P<0.001 (STAU vs RBC1023 + STAU).Abbreviations: ATP, adenosine triphosphate; DMSO, dimethyl sulfoxide; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); STAU, staurosporine; Δψm, mitochondrial membrane potential.
Mentions: To investigate if RBC1023 can rescue staurosporine-induced decrease of cell viability, we performed the MTT assay, in which the tetrazolium dye, MTT, is reduced to formazan by intracellular nicotinamide adenine dinucleotide phosphate (NADPH) oxidoreductases, which correlates with cellular metabolic activity and the number of viable cells present. As shown in Figure 5A, staurosporine treatment for 24 hours reduced the cell viability dramatically. RBC1023 co-treatment significantly rescued the loss of cell viability induced by staurosporine in NIH3T3 cells. We also performed ATP measurement by generating an ATP standard curve using Cell Titer-Glo® luminescent assay. As shown in Figure 5B, RBC1023 clearly restored the loss of ATP contents in cells treated with staurosporine. One-way ANOVA of the MTT and ATP assay data suggested that RBC1023 induces normalization or partial recovery of cell viability and ATP contents. Our results here further confirmed the cytoprotective effect of RBC1023 demonstrated by Cell Titer-Glo® assay.

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