Introduction: Inhibition of MDM2 results in activation of p53 signalling. We have investigated the activity of cisplatin (CDDP) or SN-38 in combination with MDM2 inhibitors using antisense MDM2 oligonucleotide (AS MDM2), roscovitine or nutlin-3. Roscovitine (CYC202 or Seliciclib) is a cyclin-dependent kinase inhibitor reported to down-regulate genes involved in chemosensitivity such as MCL-1 (Raje et al, 2005, Blood 106(3),1042) and MDM2 (Lu et al, 2001,Oncogene, 20, 3206). Nutlin-3 is a small molecule antagonist of MDM2 that inhibits p53-MDM2 binding.
Methods: LS174T(p53wt), MCF-7 (p53wt), HCT-116 (p53wt), HCT-116 (p53null), and SW620 (p53mt) cells were treated with MDM2 inhibitors, both alone and in simultaneous combination with CDDP or SN-38 for 48hrs. Cell viability was determined by trypan blue exclusion or by Vialight ATP assay (Cambrex), cell cycle distribution by PI staining of DNA and flow cytometry, and p53, p21 and MDM2 protein levels by western blotting.
Results: An increase in p53 was demonstrated with all anti-MDM2 agents used, confirming inhibition of MDM2. AS MDM2 alone showed no effect on cell number or viability over 48hrs.Roscovtine had limited effect on cell viabilityat the concs used (<40µM). Cells with wild type p53 showed increased sensitivity to nutlin-3 as a single agent (EC50 6.9-8.7 µM cf 16.8-33.9 in p53Mut or p53null cells).
Combining AS MDM2 with EC5 or EC25 concs of CDDP or SN38 resulted in additive effects on cell viability, and additive / antagonistic effects on cell number. Roscovitine with EC5 or EC25 concs of CDDP showed at least an additive effect in all combinations tested. A greater than additive effect was seen in MCF-7 cells treated with 10µM roscovitine and 5µM CDDP (P=0.003), and in LS174T cells with 20µM roscovitine and 25µM CDDP (P=0.0046). Nutlin-3 resulted in additive cytotoxicity when combined with low effective concs of CDDP or SN-38, but may act antagonistically at higher concentrations.
Cell cycle analysis showed a general decrease in G0/G1 cells and an increase in G2/M cells in all treatments with CDDP or SN-38. AS MDM2 alone showed no effect on cell cycle distribution over 24 / 48hrs.An increase in apoptosis was seen in HCT-116 (p53wt) and LS174T cells treated with 40µM roscovitine at 24hrs. Nutlin-3 protected LS174T (p53wt) cells from the cell cycle effects of both CDDP and SN-38 treatments alone by inducing a partial G1 block, possibly explaining the antagonism seen in some combinations. This effect was not seen in SW620 (p53mt) cells after 48hrs.Changes in p21 and MDM2 proteins are currently under investigation.
Conclusions: Combining AS MDM2 with CDDP or SN38 resulted in additive effects on cell viability while roscovitine and CDDP combinations resulted in additive or supra-additive activity. No synergistic interactions were observed when nutlin-3 was used with cytotoxic agents in these cell lines.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA