Effect of the two drugs was represented by the straight line connecting the two points. If the experimentally determined data points and their confidence interval fall on this line, the drug effects are additive. If the points lie below this line, there is superadditivity, and if the points lie above this line, there is subadditivity. To Nutlin-3 determine whether the interaction between the two drugs was synergistic, additive or antagonistic, the theoretical additive IC50,add was estimated from the dose-response curves of each drug administered individually, considering that the observed effect with the combination was the sum of the individual effects of each component. The theoretical IC50,add value was then compared with the experimental IC50,mix to determine whether there was a statistically significant difference. The interaction index, denoted by c, is an assessment of the degree of synergism or antagonism. The index is defined by the isobolar relationship as follows : c=a/A+b/B where A and B are the doses of drug A and B that give the specified effect, and are the combination doses that produce the same effect. The quantities in the equation are obtained from the dose-response curves of drugs A, B, and their combinations. If c=1, the interaction is additive; if c,1, the interaction is super-additive ; and if c.1, the interaction is sub-additive. The direct and specific activation of the p53 pathway without inducing collateral DNA damage offers a tantalising solution to the shortcomings of current 
therapeutic regimens and appears to be a reasonable approach for GBM therapy in view of the infrequent occurrence of p53 gene mutations. The cumulative evidence of aberrantly increased activity of the primary p53 inhibitor MDM2 in GBM prompted us to examine the effects of targeted inhibition of the MDM2-p53 interaction by the spiro-oxindole analogue ISA27, a recently described smallmolecule inhibitor of MDM2. Little is known about the effects of MDM2 inhibitors on the in vitro growth of GBM cells. Recently, Nutlin-3, the first potent small-molecule inhibitor of MDM2, was reported to be effective at inhibiting GBM cell growth in vitro, suggesting the validity of this experimental approach for the treatment of GBM. In this study, we investigated whether ISA27 affected the growth of GBM cells and explored the intracellular events following ISA27 treatment. The U87MG and U343MG cell lines overexpress MDM2 and maintain wild-type p53 and were chosen as a cell culture model of human GBM. In these cell lines, the primary mechanism of p53 inactivation is the high nuclear MDM2 levels caused by a lack of PTEN, a tumor suppressor protein that normally counteracts MDM2 translocation into the nucleus. The lack of PTEN makes these cell lines a suitable representative model of GBM, as the loss of the PTEN gene locus has been found in up to 80% of GBM cases. This is the first report to demonstrate that ISA27 is a potent inhibitor of GBM cell growth. Previous studies have shown that ISA27 activates p53, resulting in growth inhibition in HEK-293 transformed human embryonic kidney, M14 human melanoma and U937 human monocyte lymphoma cell lines. Our results demonstrate that ISA27 blocks the cell cycle and triggers an apoptotic cell death program in the GBM cells, responses that are similar to those obtained in human M14 melanoma cells. We observed a dose-dependent PLX-4720 918505-84-7 antiproliferative effect of ISA27 in U87MG and U343 cells following short-term treatments with increasing ISA27 concentrations. The specificity of the antiproliferative effects was demonstrated by the accumulation of the p53 protein due to the decreased interaction between p53 and MDM2 and the restoration of p53 function in GBM cells after ISA27 treatment. The reactivation of p53 was suggested by the transcriptional activation of the primary target genes of p53, MDM2 and CDKN1A. The CDKN1A gene encodes the cyclin-dependent kinase inhibitor p21, an essential mediator of p53-induced cell cycle arrest. Exponential growth of U87MG cells was significantly inhibited following long-term ISA27 treatment. The number of viable cells was substantially reduced after 1 day of ISA27 treatment, and this reduction reached almost total growth inhibition after 5 days. Kinetic analyses of the ISA27-induced intracellular effects showed that the decrease in viable cells was mainly due to a G1-phase cell cycle block and cellular senescence during the initial phase of ISA27 treatment. However, apoptotic parameters, such as the dissipation of mitochondrial membrane potential, began to appear.