Long-term ISA27 treatment induces cell cycle arrest and a persistent increase in p21 mRNA levels in U87MG cells.

<p><i>7A) Flow cytometric cell cycle profiling</i>: the figure shows the percentage of untreated and MDM2 inhibitor-treated U87MG cells in G1-, S- and G2/M-phase. <i>7B) p21 mRNA evaluation</i>: ISA27 treatment resulted in a statistically significant increase in p21 mRNA levels at 24, 48 and 72 h.</p

Effect of ISA27, Temozolomide (TMZ) and their fixed-ratio combinations with regard to growth inhibition of U87MG cells.

<p>Data are presented as IC₅₀±S.E.M. Statistical analysis was performed with Student’s <i>t</i>-test.</p>*<p>P<0.05 <i>vs</i> the respective additive group. γ <1 indicates supra-additivity (synergy).</p

ISA27 induces an increase in PUMA mRNA levels, mitochondrial cytochrome c release, and DNA fragmentation.

<p><i>11A) Relative quantification of PUMA mRNA:</i> ISA27 induced a statistically significant increase in PUMA mRNA levels at 24 and 48 h. Nutlin-3 treatment resulted in a statistically significant increase at 72 h. <i>11B) Evaluation of cytosolic cytochrome c content</i>: ISA27-treated cells showed a 25% increase in cytochrome c levels in the cytosolic fraction. Nutlin-3-treated cells did not give statistically significant results. <i>11C) Evaluation of DNA content</i>: Frequency histograms from a representative experiment are shown. ISA27-treated cells showed a significant increase in the percentage of nuclei with hypodiploid DNA content at 72 h compared with control cells. In contrast, Nutlin-3 did not induce significant nuclear DNA fragmentation.</p

ISA27 induces U87MG cell senescence.

<p><i>8A</i>) <i>Representative images of SA-β-Gal-expressing cells:</i> The panel shows the SA-β-Gal-expressing MDM2 inhibitor-treated and untreated cells at 72 h. <i>8B) Percentage of SA-β-Gal-expressing cells:</i> The number of SA-β-Gal-expressing cells was determined with respect to the total cells in each sample (untreated cells, ISA27- and Nutlin-3-treated cells). The percentage of SA-β-Gal-expressing MDM2 inhibitor-treated cells was then calculated with respect to the untreated cells, for which an arbitrary value of 100% was assigned.</p

Synergistic effect of ISA27 and temozolomide on the survival/growth of GBM cells.

<p>Isobologram 2-D showing the interactions between TMZ and ISA27 in MTS viability tests performed in U87MG cells treated for 72 h with TMZ and/or ISA27. The IC₅₀ values for TMZ and ISA27 are shown on the X- and Y-axes, respectively. The isobole of additivity is shown as a solid line drawn between the aforementioned IC₅₀ values of TMZ and ISA27 and connects the X- and Y-axes. The open points (○) on the additivity line depict the theoretical IC_50,add values for total dose expressed as the proportion of TMZ and ISA27 that produced a 50% effect. The solid points (•) depict the experimental IC_50,mix values for total dose expressed as the proportion of TMZ and ISA27 that produced a 50% effect. The experimental IC_50,mix values of the mixture of ISA27 and TMZ for the fixed-ratio combinations of 1∶80, 1∶150 and 1∶1,000 were found to be significantly below the theoretical isoboles of additivity, indicating super-additive (synergy) interactions.</p

U87MG cells did not recover normal growth upon removal of ISA27.

<p>U87MG cells were cultured in the presence of 2.5 µM ISA27 or 10 µM Nutlin-3. After 4 days of MDM2 inhibitor treatment, cell culture medium was replaced with fresh culture medium without the MDM2 inhibitor. The figure shows the number of viable cells that were counted by Trypan blue exclusion after 4 days of MDM2 inhibitor treatment and 1, 2 and 3 days after MDM2 inhibitor removal. Data represent the means of three independent experiments performed in duplicate; bars, SEM.</p

ISA27 induces the dissipation of the mitochondrial membrane potential.

<p><i>10A) Representative dot plots of untreated and MDM2 inhibitor-treated cells:</i> After ISA27 treatment, mitochondrial depolarisation is visible by a decrease and an increase in fluorescence in the FL-2 and FL-1 channels, respectively. After Nutlin-3 treatment, mitochondrial depolarisation is not visible; CCCP is the positive control. <i>10B and C) Time-course analysis of ΔΨm dissipation:</i> Histograms show the mean values of cell percentages either in the UR (polarised mitochondria) or LR (depolarised mitochondria) quadrant of the Δ<i>Ψ</i>m analysis plots derived from three independent experiments.</p

ISA27 inhibits the growth/survival of GBM cell lines expressing wild-type p53.

<p>GBM cells with wild-type p53 (U87MG, U343MG) were exposed to a range of ISA27 or Nutlin-3 concentrations for 24 h. After incubation, GBM cell viability was determined by MTS assay. <i>A and B) Effect of ISA27 on the growth/survival of GBM cell lines expressing wild-type p53:</i> curves for U87MG and U343MG cell samples were generated using a sigmoidal dose-response curve model (GraphPad Prism 4 software) from which the IC₅₀ values were derived. ISA27-treated cells (▾), Nutlin-3-treated cells (▪); <i>C) Effect of ISA27 on the growth/survival of the GBM T98G cell line expressing mutant p53:</i> T98G cells were incubated with increasing concentrations of ISA27 or Nutlin-3 (ranging from 1 µM to 25 µM), and cell viability was measured after 24 h by MTS assay. ISA27-treated cells (<b>▾</b>), Nutlin-3-treated cells (<b>▪</b>). The results are expressed as the percentages of cell viability with respect to the vehicle-treated sample for each cell line; the viability of the vehicle-treated sample was arbitrarily set at 100%. Points, means of three independent experiments performed in duplicate; bars, SEM.</p

siRNA against p21 made ISA27 unable to induce ΔΨm dissipation and PE.

<p>Random siRNA and p21siRNA samples were exposed to a fixed dose of ISA27 (5 µM) or DMSO for 24 h. A) <i>Evaluation of ISA27 effect on the ΔΨm in p21 siRNA samples</i>. The ISA27-treated random siRNA sample showed dissipation of ΔΨm compared to random siRNA sample. The comparison of ΔΨm between p21 siRNA and ISA27-treated p21 siRNA sample did not show statistically significant differences. Upper panel shows representative dot plots of untreated and ISA27-treated random or p21siRNA samples. <i>B) Evaluation of ISA27 effect on PE in p21 siRNA samples.</i> The ISA27-treated random siRNA sample showed a statistical significant increase of early and late apoptosis compared to random siRNA sample. The comparison of early and late apoptosis between p21 siRNA and ISA27-treated p21 siRNA sample did not show statistically significant differences.</p

ISA27 increases p53 protein levels in GBM cell lines.

<p>GBM cells (U87MG, U343MG) were treated with ISA27 or Nutlin-3 for the indicated incubation times, and protein levels of p53 were analysed in whole-cell lysates by Western blot. One representative Western blot is presented (upper panel) for each cell line. The blots show that the antibody to p53 (FL-393; Santa Cruz Biotechnology) recognised a single specific band at approximately 55 kDa, corresponding to the molecular weight of the p53 protein; β-actin is used as the loading control. Densitometric analyses of Western blots (lower panel) demonstrated that ISA27 induced a significant enhancement of p53 protein levels in U87MG or U343MG cells with a maximal effect at the 8 h incubation time.</p