FIGURE 2.
Ability of alanine-substituted RasGAP317–326 peptides to inhibit cell migration and sensitize cancer cells against cisplatin-induced apoptosis. A, U2OS cells were grown to confluence and were pretreated for 3 h with 20 μm of the indicated TAT-coupled peptides. The cells were then subjected to wound healing scratch assays. The graph displays the percentage of migration over untreated cells. Asterisks and number signs denote statistical significant differences after one-way ANOVA (asterisk: different from untreated condition; number sign: different from 317–326 treatment; n = 7 independent experiments). Error bars indicate mean ± 95% confidence intervals. B, U2OS cells were cultured overnight and were subsequently treated for 22 h with 20 μm of the indicated TAT-coupled peptides in the presence or in the absence of 30 μm cisplatin. The number of pyknotic nuclei was then scored and reported as the percentage of apoptosis. The light gray region displays the level of apoptosis induced by cisplatin alone, and the dark gray region shows the apoptosis sensitization zone. Asterisks and number signs denote statistical significant differences after one-way ANOVA with the cisplatin-only treatment and with the cisplatin + 317–326 treatment, respectively (n = 6 independent experiments). Error bars indicate mean ± 95% confidence intervals, C, schematic representation of the importance of each amino acid within the wild-type sequence for the apoptosis sensitization, adhesion promotion, and migration inhibition processes. D, comparison of the dose-dependent effect of 317–326 on apoptosis sensitization, adhesion (prevention of detachment), and inhibition of migration. Each experiment was performed as described in earlier figures and panels except that here all the responses are reported as the percentage of the maximum observed effect. Error bars indicate mean ± 95% confidence intervals.