Histone acetylation plays an important role in regulating the chromatin structure and is tightly regulated by histone acetyltransferases (HAT) and histone deacetylases (HDAC). Recent studies have demonstrated that several chromatin-regulating proteins can modulate cellular responses to other cytotoxic modalities including ionizing radiation and chemotherapeutic drugs. Even though histone acetylation is one of the key mechanisms of epigenetic regulation, relatively little is known about the cancer therapeutic potential of HAT inhibitors. This is in stark contrast to the well-studied effects of HDAC inhibitors. In this study, we investigated the proton beam-sensitizing effect of C646, a selective small molecule inhibitor of p300 histone acetyltransferase in human pancreatic cancer cells.
Cell viability assay (CCK-8; Dojindo), Clonogenic survival assay, Cell cycle analysis (PI staining). Annexin V-FITC and PI staining, γ-H2AX foci, Western blot, BxPC-3 Xenograft tumor model, TUNEL assay.
AsPC-1, BxPC-3 and Mia-paca2 cells exhibited increase in radiosensitivity when exposed to C646 at all doses of proton beam tested. C646 pre-treatment induce led to increase of sub-G1 population and abolishment of G2/M arrest. Flow cytometry analysis with annexin V staining and Western blot analysis showed that The pre-treatment with C646 significantly enhanced proton-induced apoptotic cell death through the down-regulation of anti-apoptotic molecules. Our in vivo results demonstrate synergistic effects of combination therapy with C646 and proton beam. In BxPC-3 xenograft tumor model, C646 pre-treatment increased proton-induced tumor growth inhibition and apoptotic cell death in tumor tissues. In addition, the combination treatment of C646 with proton beam increased DNA damage and decreased activation of DNA repair pathway compared with proton alone.
Our results suggest that the C646 may increase proton- induced apoptosis through the modulation of various pro- and anti- apoptotic molecules in human pancreatic cancer cells. These results provide proof of principle that the inhibition of histone acetylation by HATis can be exploited to develop new proton-sensitizer.
The authors.
National Research Foundation of Korea (NRF).
All authors have declared no conflicts of interest.