Involvement of RPL11 in the enhancement of P53 stability by a podophyllum derivative, a topoisomerase II inhibitor†

In previous work we presented experimental and theoretical evidence that D-3F or 4-N-(2-Amino-3-fluoropyridine)-4-deoxidation-4′-demethylepipofophyllotoxin induced G2/M phase arrest and apoptosis, purportedly by increasing the expression of P53. However, the precise mechanism of D-3F action is currently unknown. Here, we investigated the mechanism by which D-3F treatment induces increased expression of P53. This study showed that D-3F definitively inhibited the activity of topoisomerase II in a dose-dependent manner and resulted in DNA damage. The results were in overall agreement with modelling and docking studies performed on D-3F. In addition, D-3F increased the levels of P53 and P21 in HeLa cells in a dose-dependent manner, this in turn prolonged the half-life of P53. Taken together, these data suggested that D-3F-mediated transient enhancement of P53 stabilisation may be critical for the P53/P21 signalling pathway leading to G2/M phase arrest on HeLa cells. Furthermore, D-3F downregulated the phosphorylation of E3 ubiquitin-protein ligase murine double minute 2 (Mdm2) at Ser166, inhibited Mdm2-mediated ubiquitination of P53, and released 60S ribosomal protein L11 (RPL11) from the nucleolus into the nucleoplasm. To conclude, the topoisomerase II inhibitor D-3F, causes P53 to accumulate in HeLa cell lines by enhancing its stability as a result of DNA-damage induced RPL11 relocalisation and subsequent blocking of the P53-Mdm2 feedback loop.