An evaluation of the ability of pifithrin-alpha and -beta to inhibit p53 function in two wild-type p53 human tumor cell lines

The small-molecule compound pifithrin-α (PFT-α) has been reported to inhibit p53 function and protect against a variety of genotoxic agents. We show here that PFT-α is unstable in tissue culture medium and is rapidly converted to its condensation product PFT-β. Both compounds showed limited solubility with PFT-α precipitating out of tissue culture medium at concentrations >30 μmol/L. PFT-α and -β exhibited cytotoxic effects in vitro towards two human wild-type p53–expressing tumor cell lines, A2780 ovarian and HCT116 colon (IC50 values for both cell lines were 21.3 ± 8.1 μmol/L for PFT-α and 90.3 ± 15.5 μmol/L for PFT-β, mean ± SD, n = 4). There was no evidence of protection by clonogenic assay with either compound in combination with ionizing radiation. Indeed, there was some evidence that PFT-α enhanced cytotoxicity, particularly at higher concentrations of PFT-α. Neither compound had any effect on p53, p21, or MDM-2 protein expression following ionizing radiation exposure and there was no evidence of any abrogation of p53-dependent, ionizing radiation–induced cell cycle arrest. Similarly, there was no evidence of cellular protection, or of effects on p53-dependent gene transcription, or on translation of MDM-2 or p21 following UV treatment of these human tumor cell lines. In addition, there was no effect on p53 or p21 gene transactivation or p38 phosphorylation after UV irradiation of NIH-3T3 mouse fibroblasts. In conclusion, neither PFT-α nor -β can be regarded as a ubiquitous inhibitor of p53 function, and caution should be exercised in the use of these agents as specific p53 inhibitors.