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MI-63: a novel small-molecule inhibitor targets MDM2 and induces apoptosis in embryonal and alveolar rhabdomyosarcoma cells with wild-type p53.

Canner JA, Sobo M, Ball S, Hutzen B, DeAngelis S, Willis W, Studebaker AW, Ding K, Wang S, Yang D, Lin J - Br. J. Cancer (2009)

Bottom Line: Treatment with MI-63 reduced cell viability by 13.4% and by <1%, respectively, at 72 h in both RH36 and RH18 cell lines expressing wild-type p53.MI-63 was compared with Nutlin-3, a known MDM2 inhibitor, and was found to be more potent in the inhibition of cell proliferation/viability.These results indicate that MI-63 is a potent therapeutic agent for RMS cells expressing wild-type p53 protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Nationwide Children's Hospital, Columbus, OH, USA.

ABSTRACT

Background: Interruption of the role of p53s as a tumour suppressor by MDM2 may be one of the mechanisms by which cancer cells evade current therapy. Blocking the inhibition of wild-type p53 by MDM2 in cancer cells should reactivate p53's tumour suppressor functions and enhance current cancer treatments. MI-63 is a novel non-peptide small molecule that has shown strong binding affinity (K(i)=3 nM) for MDM2; however, its effects on paediatric cancer cells and the specific mechanism of tumour suppressor reactivation have not been evaluated.

Methods: Rhabdomyosarcoma (RMS), the most common childhood soft tissue sarcoma, expresses either wild-type or mutant p53 protein. We examined the inhibitory effects of MI-63 in embryonal RMS (ERMS) and alveolar RMS (ARMS) cell lines expressing wild-type or mutated p53.

Results: Treatment with MI-63 reduced cell viability by 13.4% and by <1%, respectively, at 72 h in both RH36 and RH18 cell lines expressing wild-type p53. In contrast, RH30 and RD2 cells expressing p53 mutants are resistant to MI-63 treatment. An increased expression of p53, p21(WAF1), and Bax protein was observed after treatment with MI-63 in RMS cells with wild-type p53, and apoptosis was confirmed by cleaved PARP and caspase-3 expression. However, RD2 and RH30 RMS cells, as well as human normal skeletal muscle cells, showed a minimal increase in p53 signalling and no induction of cleaved PARP and caspase-3. MI-63 was compared with Nutlin-3, a known MDM2 inhibitor, and was found to be more potent in the inhibition of cell proliferation/viability. Further, synergy was observed when MI-63 was used in combination with doxorubicin.

Conclusion: These results indicate that MI-63 is a potent therapeutic agent for RMS cells expressing wild-type p53 protein.

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The effect of MI-63 on p53 half-life in RH36 cells. Blocking MDM2 activity inhibits p53 degradation resulting in an increased p53 half-life.
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fig5: The effect of MI-63 on p53 half-life in RH36 cells. Blocking MDM2 activity inhibits p53 degradation resulting in an increased p53 half-life.

Mentions: The baseline levels of p53 and MDM2 were assessed in RH18 and RH36 cells with wild-type p53 and in RH30 and RD2 cells expressing mutant p53, which have higher levels of p53 (Figure 4). The half-life of p53 in wild-type cells is short (Cinelli et al, 1998). This is in part due to the role of MDM2 as an E3 ubiquitin ligase, mediating a proteasomal degradation of p53 (Honda et al, 1997; Fuchs et al, 1998). The degradation of the p53 protein by MDM2 is inhibited in RH36 cells after treatment with MI-63, resulting in an increased p53 half-life (Figure 5). Both wild-type p53-expressing cell lines were treated with 10 μM of MI-63. Western blot analysis indicated an induction of p53. RH36 cells were treated for 20, 30, and 40 h. Although baseline levels of p53 were present in the non-treated cells, levels increased after treatment. Antibodies for p21WAF1 were strongly positive when compared with those with no treatment (Figure 6A). The apoptotic pathway was reactivated, displayed by the increased presence of Bax protein by 30 h after treatment, and by the presence of both cleaved caspase-3 and cleaved PARP. RH18 cells expressing wild-type p53 were treated for 24 and 36 h and the findings were comparable (Figure 6B). p53 proteins increased from baseline with exposure to MI-63. Induction of the p21WAF1 protein was evident. Baseline levels of Bax protein were unchanged; however, downstream apoptosis proteins, cleaved caspase-3, and cleaved PARP were increased by 36 h (Figure 6B). In contrast, western blot analyses of both RD2 and RH30 cells expressing mutated p53 treated with MI-63 showed no change from untreated cells when p53, p21WAF1, Bax, cleaved caspase-3, and cleaved PARP proteins were evaluated (Figures 6C and D). These results indicate that MI-63 selectively induces apoptosis in rhabdomysarcoma cells expressing wild-type p53 but not in rhabdomysarcoma cells expressing mutated p53.


MI-63: a novel small-molecule inhibitor targets MDM2 and induces apoptosis in embryonal and alveolar rhabdomyosarcoma cells with wild-type p53.

Canner JA, Sobo M, Ball S, Hutzen B, DeAngelis S, Willis W, Studebaker AW, Ding K, Wang S, Yang D, Lin J - Br. J. Cancer (2009)

The effect of MI-63 on p53 half-life in RH36 cells. Blocking MDM2 activity inhibits p53 degradation resulting in an increased p53 half-life.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2736841&req=5

fig5: The effect of MI-63 on p53 half-life in RH36 cells. Blocking MDM2 activity inhibits p53 degradation resulting in an increased p53 half-life.
Mentions: The baseline levels of p53 and MDM2 were assessed in RH18 and RH36 cells with wild-type p53 and in RH30 and RD2 cells expressing mutant p53, which have higher levels of p53 (Figure 4). The half-life of p53 in wild-type cells is short (Cinelli et al, 1998). This is in part due to the role of MDM2 as an E3 ubiquitin ligase, mediating a proteasomal degradation of p53 (Honda et al, 1997; Fuchs et al, 1998). The degradation of the p53 protein by MDM2 is inhibited in RH36 cells after treatment with MI-63, resulting in an increased p53 half-life (Figure 5). Both wild-type p53-expressing cell lines were treated with 10 μM of MI-63. Western blot analysis indicated an induction of p53. RH36 cells were treated for 20, 30, and 40 h. Although baseline levels of p53 were present in the non-treated cells, levels increased after treatment. Antibodies for p21WAF1 were strongly positive when compared with those with no treatment (Figure 6A). The apoptotic pathway was reactivated, displayed by the increased presence of Bax protein by 30 h after treatment, and by the presence of both cleaved caspase-3 and cleaved PARP. RH18 cells expressing wild-type p53 were treated for 24 and 36 h and the findings were comparable (Figure 6B). p53 proteins increased from baseline with exposure to MI-63. Induction of the p21WAF1 protein was evident. Baseline levels of Bax protein were unchanged; however, downstream apoptosis proteins, cleaved caspase-3, and cleaved PARP were increased by 36 h (Figure 6B). In contrast, western blot analyses of both RD2 and RH30 cells expressing mutated p53 treated with MI-63 showed no change from untreated cells when p53, p21WAF1, Bax, cleaved caspase-3, and cleaved PARP proteins were evaluated (Figures 6C and D). These results indicate that MI-63 selectively induces apoptosis in rhabdomysarcoma cells expressing wild-type p53 but not in rhabdomysarcoma cells expressing mutated p53.

Bottom Line: Treatment with MI-63 reduced cell viability by 13.4% and by <1%, respectively, at 72 h in both RH36 and RH18 cell lines expressing wild-type p53.MI-63 was compared with Nutlin-3, a known MDM2 inhibitor, and was found to be more potent in the inhibition of cell proliferation/viability.These results indicate that MI-63 is a potent therapeutic agent for RMS cells expressing wild-type p53 protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Nationwide Children's Hospital, Columbus, OH, USA.

ABSTRACT

Background: Interruption of the role of p53s as a tumour suppressor by MDM2 may be one of the mechanisms by which cancer cells evade current therapy. Blocking the inhibition of wild-type p53 by MDM2 in cancer cells should reactivate p53's tumour suppressor functions and enhance current cancer treatments. MI-63 is a novel non-peptide small molecule that has shown strong binding affinity (K(i)=3 nM) for MDM2; however, its effects on paediatric cancer cells and the specific mechanism of tumour suppressor reactivation have not been evaluated.

Methods: Rhabdomyosarcoma (RMS), the most common childhood soft tissue sarcoma, expresses either wild-type or mutant p53 protein. We examined the inhibitory effects of MI-63 in embryonal RMS (ERMS) and alveolar RMS (ARMS) cell lines expressing wild-type or mutated p53.

Results: Treatment with MI-63 reduced cell viability by 13.4% and by <1%, respectively, at 72 h in both RH36 and RH18 cell lines expressing wild-type p53. In contrast, RH30 and RD2 cells expressing p53 mutants are resistant to MI-63 treatment. An increased expression of p53, p21(WAF1), and Bax protein was observed after treatment with MI-63 in RMS cells with wild-type p53, and apoptosis was confirmed by cleaved PARP and caspase-3 expression. However, RD2 and RH30 RMS cells, as well as human normal skeletal muscle cells, showed a minimal increase in p53 signalling and no induction of cleaved PARP and caspase-3. MI-63 was compared with Nutlin-3, a known MDM2 inhibitor, and was found to be more potent in the inhibition of cell proliferation/viability. Further, synergy was observed when MI-63 was used in combination with doxorubicin.

Conclusion: These results indicate that MI-63 is a potent therapeutic agent for RMS cells expressing wild-type p53 protein.

Show MeSH
Related in: MedlinePlus