Background: PRIMA-1met (APR246), a methylated form of PRIMA-1 (p53-reactivation and induction of massive apoptosis-1, APR-017), targets mutant p53 for restoring its wild-type structure and function. We previously demonstrated that PRIMA-1met was efficient in suppressing the growth of colorectal cancer (CRC) cells in a p53-independent manner, and distinctly induced apoptosis mediated by up-regulation of Noxa in p53-mutant cell lines. Here we aimed to the effect of PRIMA-1met on autophagy in different CRC cell lines, to further investigate mechanisms underlying the inhibitory effect in cells with different p53 status.
Methods: 3 CRC cell lines with wild-type p53, 5 lines with mutant p53 and 1 line without p53 were obtained for this study. Using western blotting, acridine orange staining, and transmission electron microscopy detection, we assessed autophagy flux in different cells treated with PRIMA-1met, and detected expression of mTOR/AMPK-ULK1-Vps34 autophagic signaling cascade. We also evaluated cell proliferation of cells with PRIMA-1met treatment by cell counting Kit-8 proliferation assay, compared to combination of PRIAM-1met and 3-Methyladenine. Furthermore, we knocked down Noxa gene by siRNA in different CRC cells, to assess LC3 conversion after administration of PRIMA-1met. Values were expressed as mean + standard error of the mean. Comparison between groups of data was made using one-way analysis of variance.
Results: In this study, we showed that PRIMA-1met induced autophagy in CRC cells independent on p53 status. PRIMA-1met not only promoted autophagic vesicles (AVs) formation and AV-lysosome fusion, but also increased lysosomal degradation. Mechanistically, activation of mTOR/AMPK-ULK1-Vps34 autophagic signaling cascade was important for PRIMA-1met-induced autophagy. Furthermore, autophagy played a crucial role in the inhibitory effect of PRIMA-1met only in cells harboring wild-type p53, which was closely related to the increased Noxa.
Conclusions: Our results indicated that PRIMA-1met induced autophagy in CRC cells regardless of p53 status via activating mTOR/AMPK-ULK1-Vps34 signaling cascade. However, induced autophagy was relevant to the cytotoxicity of PRIMA-1met in cells carrying wild-type p53, along with up-regulation of Noxa. Implying that, PRIMA-1met-based therapy could be an effective strategy for CRC.
Trail registration: Not applicable.