Abstract
Pifithrin-α (PFT-α) is a small molecule which has been widely used as a specific inhibitor of p53 transcription activity. However, its molecular mechanism of action remains unclear. PFT-α has also been described to display potent p53-independent activity in cells. In this study, we addressed the mechanism of action of PFT-α. We found that PFT-α failed to prevent the effects of Mdm2 inhibitor Nutlin-3 on cell cycle and apoptosis in several cancer cell lines. However, PFT-α rescued normal primary fibroblasts from growth inhibition by Nutlin-3. PFT-α displayed a very limited effect on p53-dependent transcription upon its activation by Nutlin-3. Moreover, PFT-α inhibitory effect on transcription was highly dependent on the nature of the p53 target gene. PFT-α attenuated post-translational modifications of p53 without affecting total p53 protein level. Finally, we found that PFT-α can decrease the level of intracellular reactive oxygen species through activation of an aryl hydrocarbon receptor (AHR)-Nrf2 axis in a p53-independent manner. In conclusion, PFT-α inhibits only some aspects of p53 function, therefore it should be used with extreme caution to study p53-dependent processes.
Highlights
Pifithrin-α (PFT-α) is a small molecule which has been widely used as a specific inhibitor of p53 transcription activity
As we show that PFT-α can exert an antioxidant effect through the aryl hydrocarbon receptor (AHR)/Nrf[2] pathway independently of p53, our study suggests that its inhibition of p53 transcriptional activity may be due to alterations of p53 post-translational modifications (PTMs) and is highly context- and gene-dependent
The growth of wild type p53 MCF7 breast carcinoma cells was suppressed by Nutlin-3 treatment, whereas it had no effect in p53-depleted (KO) cells, in line with the notion that Nutlin-3 induces cell cycle arrest and/or apoptosis through p53 activation
Summary
Pifithrin-α (PFT-α) is a small molecule which has been widely used as a specific inhibitor of p53 transcription activity. PFT-α has been described to display potent p53-independent activity in cells. We found that PFT-α failed to prevent the effects of Mdm[2] inhibitor Nutlin-3 on cell cycle and apoptosis in several cancer cell lines. PFT-α displayed a very limited effect on p53-dependent transcription upon its activation by Nutlin-3. PFT-α inhibitory effect on transcription was highly dependent on the nature of the p53 target gene. Despite its unclear molecular mechanism and potential off-target effects, PFT-α is still widely used as a specific p53 inhibitor to investigate p53-dependent response, for instance, in autophagy[12], response to drugs[13], DNA damage[14], neurogenesis and angiogenesis[15] or cardiac hypertrophy[16]. The values are reported as relative cell viability normalized to DMSO treatment group and represent the mean ± SD of three replicates
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