Inhibition of GSK-3β reverses the pro-apoptotic effect of proadifen (SKF-525A) in HT-29 colon adenocarcinoma cells
Abstract
Proadifen (SKF-525A) is a well-known inhibitor of cytochrome P450 monooxygenases. Besides the prevention of drug metabolism it affects the proliferation of cancer cells, although the mechanisms of possible anti-cancer activity of proadifen have not been fully understood yet. The aim of this study therefore was to evaluate the potential anti-proliferative effect of proadifen on HT-29 colon cancer cells. Our results show that proadifen inhibited the growth of HT-29 cells by the accumulation of cells in the G1 phase of the cell cycle, reduction of metabolic activity and colony formation and by the induction of apoptosis. Analyses of Western blots and flow cytometry revealed time- and dose-dependent phosphatidylserine externalization, caspase-3 activation and PARP cleavage. Intense upregulation of NAG-1 and ATF3 and downregulation of Mcl-1 and Egr-1 were also observed. Further investigation showed that NAG-1 gene silencing by siRNA had no effect on the pro-apoptotic action of proadifen. In contrast, we found that AR-A014418, the specific inhibitor of glycogen synthase kinase-3 β (GSK-3β), significantly decreased proadifen-induced apoptosis. Inactivation of GSK-3β (phosphorylation at serine 9) resulted in changes in phosphatidylserine externalization and caspase-3 activation. These data suggest that GSK-3β is an important factor in the induction of apoptosis in HT-29 colon cancer cells treated with proadifen.
- Publication:
-
Toxicology in Vitro
- Pub Date:
- September 2012
- DOI:
- Bibcode:
- 2012ToxVi..26..775J
- Keywords:
-
- A;
- AR-A014418;
- GSK-3β inhibitor;
- ANOVA;
- analysis of variance;
- ATF3;
- activating transcription factor 3;
- Bax;
- Bcl-2-associated X protein;
- Bcl-2;
- B-cell lymphoma 2;
- Bcl-XL;
- B-cell lymphoma-extra large;
- DMSO;
- dimethyl sulfoxide;
- DOC-Na;
- sodium deoxycholate;
- Egr-1;
- early growth response protein 1;
- FITC;
- fluorescein isothiocyanate;
- FL-1;
- -2;
- -3;
- fluorescence channel-1;
- -2;
- -3;
- GSK-3β;
- glycogen synthase kinase-3 β;
- HEPES;
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
- KLF4;
- Krueppel-like factor 4;
- MCC-555;
- 5-[[6-[(2-fluorophenyl)-methoxy]-2-napthalenyl]-methyl]-2;
- 4-thiazolidinedione;
- Mcl-1;
- induced myeloid leukemia cell differentiation protein;
- MTT;
- (3-[4;
- 5-dimethylthiazol-2-yl]-2;
- 5-diphenyltetrazolium bromide);
- NAG-1;
- non-steroidal anti-inflammatory drug-activated gene-1;
- NP-40;
- nonyl phenoxypolyethoxylethanol;
- NSAIDs;
- non-steroidal anti-inflammatory drugs;
- P;
- proadifen;
- p53;
- protein 53;
- PARP;
- poly (ADP-ribose) polymerase;
- PBS;
- phosphate buffered saline;
- PI;
- propidium iodide;
- PVDF;
- polyvinylidene difluoride;
- RIPA;
- radioimmunoprecipitation assay buffer;
- RT;
- room temperature;
- SD;
- standard deviation;
- SDS;
- sodium dodecyl sulfate;
- siRNA;
- small interfering RNA;
- Sp-1;
- Specificity protein 1;
- TBS;
- tris buffered saline;
- Tris;
- tris(hydroxymethyl)aminomethane;
- TWEEN 20;
- polysorbate surfactant;
- Apoptosis;
- Colon cancer cells;
- GSK-3β;
- HT-29;
- NAG-1;
- Proadifen