Histone Acetyltransferase MOF-mediated Anti-cancer Mechanism Of Chemotherapy Drugs In Human Bladder Cancer Cells

The exposed N-terminal tails of nucleosomes can undergo a variety of covalent modifications,including acetylation,methylation,phosphorylation,ubiquitination and glycosylation.These histone post-translational modifications affect the affinity between histone and DNA through altering the nucleosome structure,participating in most of the cellular life processes such as gene transcription regulation,DNA damage repair and genome stability maintenance.Histone acetylation is one of the most important epigenetic modifications,and its dynamic equilibrium is regulated by both histone acetyltransferases(HATs)and histone deacetyltransferases(HDACs).Males absent on the first(MOF)is a member of the MYST family,as a catalytic subunit forming MOF/ male-specific lethal(MSL)and MOF/ non-specific lethal(NSL)complexes in human cells.The former is responsible for the acetylation of histone H4K16 sites,and the latter can acetylate histone H4K16,H4K5 and H4K8 sites.A large number of research data suggest that MOF and its enzyme activity may be involved in tumorigenesis and the development of various tumors.So far,the role of MOF and its enzyme activity in bladder cancer has been poorly reported.In China,bladder cancer is the most common malignant tumor of genitourinary system.It is easy to recur and metastasize after surgical excision,which brings great difficulty to the treatment of bladder cancer.In this paper,a series of cellular and biochemical experiments were carried out to observe the protein levels of MOF/H4K16 ac in bladder cancer tissues,to explore the influence of MOF on the anti-cancer effect of camptothecin(CPT),mitomycin C(MMC)and gemcitabine(GEM),and to elucidate MOF-mediated anti-cancer mechanism of GEM in human bladder cancer cells.First,to know about the expression of MOF in bladder cancer,the MOF protein level and histone H4K16 acetylation were detected by immunohistochemistry in 56 cases of primary bladder cancer.Unfortunately,in our limited case studies,we did not find statistically significant differences between bladder cancer tissues and normal tissues.Interestingly,when we treated bladder cancer cells with anti-bladder cancer chemotherapy drugs CPT,MMC and GEM,it was found that the endogenous MOF protein expression and histone H4K16/K5/K8 acetylation were decreased in a dose-dependent manner.Similarly,exogenous MOF protein and histone H4 acetylation levels were down-regulated in CPT or GEM treated T24 cells.Subsequent luciferase reporter assay confirmed that the above chemotherapy drugs directly inhibit the transcriptional activity of MOF,indicating these drugs may target MOF gene expression and alter the histone H4K16/K5/K8 acetylation.In order to find out the interaction between chemotherapy drugs and MOF,we over-expressed or knocked down MOF in bladder cancer cells and carried out CCK-8test,colony formation assay,scratch test,transwell assay and western blot assay to observe the influence of MOF on anti-bladder cancer effect of chemotherapy drugs.Data show that exogenous MOF reduced the cell viability,whereas inhibition of MOF gene expression promoted cell proliferation,suggesting that MOF enhances the chemosensitivity of T24 bladder cancer cells.In addition,exogenous MOF inhibits cell migration in cooperation with CPT/GEM.Compared to GEM only group,the expression of the epithelial cell maker E-cadherin was up-regulated,while the mesenchymal cell makers N-cadherin and Vimentin were down-regulated in MOF+GEM group,indicating that MOF may be involved in the anti-cancer mechanism of these chemotherapy drugs in human bladder cancer cells.To further confirm the MOF-mediated anti-cancer mechanism of chemotherapy drugs in human blader cancer cells,we used flow cytometry and western blot assay to investigate the possibility of MOF participating in the anti-cancer mechanism of chemotherapy drugs.The results show that exogenous MOF resulted in G1 phase arrest in combination with GEM and promoted GEM-induced cell death.Furthermore,western blot analysis exhibited that exogenous MOF not only reduced Cyclin A in expression level,but also lead to the increase of GEM-induced ?H2AX and cleaved Parp1 as well as the decrease of Bcl2 expression.These results suggest that the anti-bladder cancer effect of the chemotherapy drug GEM is at least partially mediated by MOF.In summary,CPT,MMC and GEM inhibit the HAT activity of the MOF/NSL complex by regulating the expression of MOF,resulting in decreased global acetylation levels of histone H4K16,H4K5 and H4K8 in bladder cancer cells.Based on a series of experiments,we have demonstrated that directly interfering with the expression level of MOF can affect the proliferation and cell cycle of bladder cancer cells,and that MOF in combination with CPT/GEM has a synergistic inhibitory effect on bladder cancer cells.These results provide experimental basis for further elucidating the anti-bladder cancer mechanism and developing targeted therapy drugs for bladder cancer.