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

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.

Figure 1

Chemical structure of MI-63.

Figure 2

MI-63 treatments of HCT-116 p53 +/+ and HCT-116 p53 −/− cells. (A) MTT analysis indicates decreased cell proliferation with MI-63 in cells with wild-type p53. (B) A measure of 50 μg of total protein from HCT-116 cell lysates was subjected to SDS polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membrane. Membranes were blotted with p53, p21^WAF1, cleaved caspase-3, and GAPDH antibodies.

Figure 3

The effect of MI-63 on the cell viability of ERMS/ARMS cells. (A, B) Rhabdomyosarcoma cells with wild-type p53, RH36 and RH18, showing decreased cell proliferation when treated with MI-63. (C, D) Rhabdomyosarcoma cells with mutated p53, RD2 and RH30, showing decreased cell proliferation when treated with MI-63.

Figure 4

Baseline levels of MDM2 and p53 expression in wild-type p53, RH18 and RH36, and mutant p53, RH30 and RD2, rhabdomyosarcoma cells.

Figure 5

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.

Figure 6

Western blot analysis of ERMS/ARMS cells treated with MI-63. (A, B) Rhabdomyosarcoma cells with wild-type p53, RH36 and RH18, indicate increased p53, downstream targets, p21^WAF1 and Bax, as well as cleaved caspase-3 and cleaved PARP, at 20, 30, and 40 h after treatment with 10 μM of MI-63. (C, D) Rhabdomyosarcoma cells with mutated p53, RD2 and RH30, indicate no increase of p53, downstream target p21^WAF1, cleaved caspase-3, and cleaved PARP at 24 and 36 h after treatment with 10 μM of MI-63.

Figure 7

MI-63 treatments of HSMM cells. (A) MTT analysis shows minimal changes in cell proliferation when normal human skeletal muscle cells (HSMM) are treated with MI-63. (B) Western blot analysis of HSMM cells grown in SkBM-2 medium with growth factors. (C) Western blot analysis of HSMM cells grown in DMEM supplemented with 10% FBS. A measure of 50 μg of total protein from HSMM cell lysates was subjected to SDS PAGE and transferred to PVDF membrane. Membranes were blotted with p53, p21^WAF1, Bax, cleaved caspase-3, cleaved PARP, and GAPDH antibodies. The results show an increase in p53 protein and p21^WAF1 at higher doses and prolonged exposure; however, there is no apparent reactivation of apoptosis.

Figure 8

A comparison of the effects of MI-63 and Nutlin-3 on the viability of RH36 cells. Treatment with MI-63 results in a greater decrease in cell proliferation/viability.

Figure 9

Inhibition of cellular proliferation in RH-36 cells by treatment with (A) doxorubicin, (B) MI-63, or (C) a combination of both, as measured by MTT analysis. (D) A CI-effect plot: CI values calculated from proliferation experiments were plotted against the effects (fa) of five different concentrations of the drugs.