FIGURE 5.

Z_HPV16E61235 inhibits the proliferation of HPV16-positive cell lines by binding to and blocking the intracellular activity of the HPV16 E6 oncoprotein. (A) p53 was up-regulated in a dose-dependent manner by treatment with Z_HPV16E61235 in CaSki cells. (B) The effect of Z_HPV16E61235 on the intracellular binding between HPV16 E6 and p53 was assessed by immunoprecipitating the E6/E6AP/p53 trimeric complex using an anti-p53 antibody bound to Protein A/G agarose beads from CaSki cells treated for 24 h. A parallel negative control assay was run for each group by incubating cell lysates with control IgG. The bar graph represents the amount of E6 bound to the relative amount of immunoprecipitated p53 after the quantification of p53 and E6 protein bands with ImageJ software. Data are presented as the mean ± SD of three independent experiments. **P < 0.01. (C) CaSki cells were treated with 10 μM Z_HPV16E61235 for the indicated periods and the expression of p53 target genes, including PUMA, BAX and p21, was evaluated by Western blotting. Cells without any treatment (Mock) or treated with 10 μM Z_WT for 48 h were used as negative controls. GAPDH served as an internal reference standard. (D) The effects of Z_HPV16E61235 and Z_HPV16E7384 alone or in combination on the viability of HPV16-positive cancer cells (TC-1, CaSki), HPV18-positive cervical cancer cells (HeLa229) and HPV-negative cancer cells (C666-1) were assessed by CCK-8 assay after 48 h of treatment with the indicated concentrations; these cells were compared to Z_WT-treated cells. Data are shown as the mean ± SD of three independent experiments. (E–F) Colony formation assays of HPV16-positive TC-1 and CaSki cells or HPV18-positive HeLa229 cells following treatment with 2.5 μM test affibody molecules for 14 days. The Z_WT affibody and medium groups were set as controls. **P < 0.01, ***P < 0.001 vs. the control group. ^#P < 0.05 vs. the Z_HPV16E61235 or Z_HPV16E7384 alone treatment group.