Figure 5.

Cisplatin augmented nutlin-3-induced p53 activation and DR5-mediated apoptosis. A2780 cells were pretreated for 4 h with low doses of cisplatin and after removal of cisplatin treated with nutlin-3 overnight. (A) P53, p21 and MDM2 levels were examined using western blotting. This showed that cisplatin (10 μM) combined with nutlin-3 (10 μM) induces a massive upregulation of these proteins. (B) DR5 and DcR2 membrane expression levels were determined by FACS analysis and showed that low doses of cisplatin combined with nutlin-3 (10 μM) enhanced DR5 levels when compared with nutlin-3 alone. DcR2 levels, however, remained unchanged. *P<0.05, **P<0.01 compared with DR5 MFI at 0 μM cisplatin. ^#P<0.05 compared with DR5 MFI in the absence of nutlin-3. (C) rhTRAIL or D269H/E195R was added for 4 h following treatment with cisplatin (1 μM) and nutlin-3 (10 μM), and apoptosis levels were examined with acridine orange. At this low dose, cisplatin additively sensitised A2780 cells to combined nutlin-3 and D269H/E195R exposure. *P<0.05, **P<0.01 compared with untreated control. ^#P<0.05 apoptosis of combination compared with the effect of each single drug from the combination. ^$P<0.05 triple combination compared with all other treatments. (D) Cell survival was measured using an MTT assay following 92 h continuous treatment. Treatment in the absence of rhTRAIL or D269H/E195R is set at 100%. Nutlin-3 in combination with D269H/E195R more potently inhibited cell survival compared with the combination with rhTRAIL (upper panel). Addition of cisplatin (0.25 μM) to the combination of nutlin-3 (1 μM) and D269H/E195R had a stronger effect on cell survival (lower panel). ^#P<0.05, ^##P<0.01 combination compared with single rhTRAIL or D269H/E195R treatment. ^$P<0.05 triple combination compared with D269H/E195R plus cisplatin and with D269H/E195R plus nutlin-3 (1 μM) treatment. Presented data are representative for at least three independent experiments.