| External factors: | SN-38 |
| Aging type: | Accelerate |
| Aging characteristic: |
| Category: | Chemical compounds |
| Phenotype: | Colorectal cancer |
| Experiment: | Colony formation assay//Western blot//SA-β-gal activity assay |
| Description: | Clonogenic assays performed on two different colorectal cell lines, LS174T and HCT116, confirmed that the number of colonies was reduced after treatment with sn38.Using western blot analysis, we observed an increase in p21waf1 expression after 48-72 hours of treatment.Using β-galactosidase staining, a known marker of senescence, results indicated that approximately 70% of HCT116 and LS174T cells had entered senescence after 3 days. |
| Regulatory pathway: | Akt//p21-p53 |
| R-EF-Pathway: | Activation//Activation |
| Official symbol(s): | AKT1//CDKN1A-TP53 |
| Pathway experiment: | Flow cytometry//SA-β-gal activity assay//Colony formation assay//Western blot |
| Pathway description: | Using intracellular flow cytometry staining, we observed that around 25% of dividing PLD express the Ser473 phosphorylated form of Akt. By contrast the kinase was almost not activated in the non-dividing PLS subpopulation .Using β-galactosidase staining, we also confirmed using RNA interference that Akt inhibition reduced sn38-mediated senescence as compared to cells transfected with a control siRNA Interestingly, p21waf1 inactivation significantly reduced the number of persistent clones in response to sn38. As expected, p53 was upregulated and phosphorylated on its Ser15 residue following sn38 treatment. Results also confirmed that its inactivation by RNA interference prevented p21waf1 activation.Using western blot analysis, we observed an increase in p21waf1 expression after 48-72 hours of treatment. |
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