Inhibition of p53 by MDM2 is known to have a role in tumour cell proliferation and in increasing oncogenesis. Recent studies have shown that targeted therapy directed at the p53–MDM2 interaction may have a therapeutic benefit (Ding et al, 2006
; Tovar et al, 2006
). In this experiment, we demonstrate that a novel small-molecule inhibitor, MI-63, has the ability to reactivate the wild-type p53 function in paediatric RMS. MI-63 targets the p53–MDM2 interaction, blocking the inhibition of the tumour suppressor and protein ubiquitination (Ding et al, 2006
). After treatment, reactivation of wild-type p53 allows for downstream cell-cycle arrest and initiation of apoptosis in both ARMS and ERMS cell lines. Testing indicates that MI-63 has a minimal apoptotic effect on normal human tissue in vitro
p53 is a tumour suppressor gene that regulates cell cycle, DNA repair, and apoptosis. Although specific mutations of the p53 protein have been described in approximately 50% of human cancers, a disruption of wild-type p53 transcriptional activity can independently contribute to tumourgenesis in other cancer types (Chene, 2004
; Haupt and Haupt, 2004
; Tovar et al, 2006
). MDM2 is a regulator of p53 transcription activity. A direct binding to the p53 transcription activation domain inhibits G1
cell-cycle arrest and apoptosis signalling; however, with cellular stress, p53 activity is enhanced and appropriate signalling occurs (Chen et al, 1996
). Increased activity of MDM2 contributes to defective, wild-type p53 regulation and can interrupt normal tumour suppressor activity, leading to malignant proliferation (Chen et al, 1996
; Keleti et al, 1996
There have been several reports of MDM2 amplification and/or overexpression in human cancers (Keleti et al, 1996
; Momand et al, 1998
; Klein and Vassilev, 2004
; Takahashi et al, 2004
; Levav-Cohen et al, 2005
; Tovar et al, 2006
). Momand et al
examined nearly 4000 tumour samples and reported a 7% frequency of MDM2 amplification, with the highest observed in soft-tissue sarcomas (20%). Evaluation of RMS specifically suggests that an increased MDM2 activity is present in a sub-population of both human tissue samples and in vitro
cell lines contributing to wild-type p53 inactivity (Keleti et al, 1996
; Taylor et al, 2000
; Takahashi et al, 2004
). There seems to be no relationship between MDM2 amplification and p53 mutation, suggesting that MDM2 provides an alternative mechanism for inhibiting wild-type p53 (Keleti et al, 1996
). Further, Taylor et al
demonstrated the presence of wild-type p53 in 19 of 20 ERMS and ARMS tissue samples obtained either at the time of diagnosis or after chemotherapy. These findings draw attention to the p53–MDM2 interaction in RMS, suggesting that blocking MDM2 will reactivate wild-type p53.
The novel small-molecule inhibitor, MI-63, shows potential as an MDM2 antagonist. The potent, non-peptide inhibitor of the p53–MDM2 interaction is designed to mimic previously described hydrophobic residues (Phe19, Trp23, and Leu26), and a newly identified fourth residue (Leu22) in p53 that interacts with the hydrophobic cleft on MDM2 (Ding et al, 2006
). With this novel design, MI-63 shows an increased binding (Ki
) to MDM2, and when compared with previously described non-peptide inhibitors (i.e., Nutlin-3), MI-63 is approximately 12 times more potent (Ding et al, 2006
). Ding et al
described a specific binding to MDM2, an increase in p53 levels, and the increase of downstream target p21WAF1
in adult prostate cancer cells (LNCAP) after treatment. The effect of MI-63 has also been observed in non-Hodgkin's lymphoma cell lines, in which similar results have been reported (Jones et al, 2008
). Our experiment is the first to specifically analyse MI-63 and paediatric RMS (ERMS and ARMS) with wild-type p53.
The results demonstrate the effectiveness of MI-63 against RMS cell lines with wild-type p53. As hypothesised, both ERMS and ARMS cells (RH36 and RH18, respectively) had decreased cell viability after treatment. A previous evaluation of MI-63 demonstrated a reactivation of p53, but only described downstream increases in p21WAF1. We looked at both cell growth arrest and downstream indicators of apoptosis in ERMS and ARMS. Expectedly, p53 and p21WAF1 were elevated on western blot analysis. Pro-apoptotic markers, Bax, cleaved PARP, and cleaved caspase-3, all increased after treatment with MI-63 and may indicate a more comprehensive reactivation of p53. Notably, human cell lines treated similarly had minimal increases in p53 and p21WAF1 at higher doses of MI63; however, there was no evidence of decreased cell viability or apoptosis. Blocking MDM2 in normal cells understandably leads to a reflexive increase in p53, but importantly, this mechanism did not induce death. In vivo studies and phase I trials will better describe the short- and long-term effects of MI-63.
When treating RMS cells with MI-63 in combination with a known chemotherapeutic agent, doxorubicin, synergism was confirmed. Doxorubicin binds and intercalates DNA, inhibiting macromolecular synthesis by blocking the action of DNA topoismerase II. In addition, an inhibition of topoisomerase II prevents replication. As doxorubicin may act in a p53-independent manner, we hypothesised that a combination treatment with MI-63 would potentiate each drug's anti-proliferative effects (Taylor et al, 2000
). The data agree with this supposition, most noted at early time points. Specifically, 20
of doxorubicin, in combination with 2000
MI-63 (day 1), showed a 49% increase in the fraction of cells affected by treatment when compared with the expected additive effect. In vivo
studies, as well as clinical trials, are necessary to fully evaluate combinatorial effects and clinical usage.
In summary, we have shown that the novel small-molecule inhibitor, MI-63, successfully blocks MDM2 and reactivates p53 and downstream cell signalling when tested in vitro. Further, antitumour effects occur with minimal toxicity to normal cells and show synergism when combined with a known chemotherapeutic agent, doxorubicin. In vivo studies on the therapeutic potential of MI-63 are needed. Moving forward, as both RMS and other solid tumours (i.e., neuroblastoma) have wild-type p53 protein, clinical trials may focus on blocking p53–MDM2 interaction in solid tumours as a whole.