Application Of Proteomics In Antitumor Mechanistic Studies Of Epigenetic Inhibitors

The development of multi-target epigenetic inhibitors is an important research direction in the development of anti-tumor drugs.The combination of DNMT and HDAC inhibitors targeting the major proteases involved in epigenetic regulation showed synergy in tumor therapy.Our previous drug design and synthesis studies have shown that DNMT/HDAC dual target inhibitors show good anti-tumor activity.Based on high-throughput proteomic analysis technology combined with medicinal chemical analysis methods,this paper studied the mechanism of the hydroxamic acid derivative 208,synthesized independently by our group,and proposed possible anti-tumor mechanisms of 208,as a DNMT/HDAC dual target inhibitor.Through medicinal chemical analysis,we found that 208 shows potent DNMT1 inhibitory efficiency and HDAC1/6 inhibitory activity comparable to the HDAC inhibitor SAHA,and can significantly inhibit the proliferation of various tumor cell lines,such as U937 cells.Western blotting results indicated that 208 can cause hyperacetylation of histones H3K9 and H4K8,and activate the transcription of the silenced tumor suppressor gene p16 in tumor cells.In addition,208 can also cause cell cycle arrest in G1 phase and promote apoptosis of U937 cells,ultimately inhibiting tumor cell proliferation.Using label free quantitative proteomic analysis,we compared protein expression in 208-treated and normal(as a blank control)U937 cells,and performed biological functions and pathways analysis on significantly differentially expressed proteins.We set up three groups with different drug treatment time to study the effect of 208 on the proteome of U937 cells over time.The results showed that 208 differentially regulates the expression of proteins over treatment time.Using the DAVID database,we performed the biological functional analysis of significantly differentially expressed proteins and found that the combined inhibition of HDAC and DNMT can affect a variety of biological processes including RNA synthesis and processing,translation,protein transport and DNA repair.We found that 208 significantly down-regulates proteins such as p85?,MEK and CDK4.This result was also confirmed by Western blotting results.These results demonstrate that 208 may induce cell cycle arrest in G1 phase by affecting the expression of CDK4 and its regulators,thereby achieving anti-tumor effects.This study provides novel insights into the underlying mechanisms of action of multi-target epigenetic inhibitors and contributes to structural optimization and further development of these drugs.