Molecular Mechanisms Of Histone Deacetylase Inhibitors Induced Mitotic Catastrophe In Tumor Cells

Background:Histone deacetylase inhibitor (HDACi) is a promising class of anticancer agents with high efficiency on inhibiting tumor growth, inducing cell differentiation and/or apoptosis. The possible mechanisms for HDACi to kill cancer cells are related with epigenetic regulation of gene expression and inhibition of heat shock protein 90 (Hsp90 )chaperone function.The chromosomal passenger complex (CPC), is a group of protein consisting of Incenp, Survivin, Dasra (Borealin), and the kinase Aurora B. CPC plays important roles in key mitotic events, including kinetochore formation, the spindle assembly checkpoint, assembly of stable bipolar spindle and chromosome bi- directly orientation, and cytokinesis.Hsp90 is the important molecular chaperone of the cells, its function involving the modulation of protein conformation, stability and kinases activities. Previous studies show that CPC member Aurora B and Survivin both are the client protein of Hsp90. Our previous data suggested that HDACi can enhance the acetylation of Hsp90 and interrupt its chaperone function, which leads the depletion of several client proteins including Survivn via ubiquitin-proteasome pathway.Our preliminary data also showed that HDACi treatment induced abnormal mitosis in several tumor cell lines. It is rational to assume that HDACi may exert its effect on inducing mitotic catastrophe via affecting the localization and expression of CPC proteins. To confirm our hypothesis and elucidate the possible mechanisms, the effect of HDACi on the localization and expression of CPC proteins was detected, and the influence of FK228 on centromere epigenetic modification, assembly of centromere and the spindle checkpoint function was detected. We got the following Results and conclusion:1. HDACi induce mitotic catastrophe in tumor cellsAfter treatment with HDACi, including FK228, TSA and SAHA respectively, the morphological changes of non small cell lung cancer cells and prostate cancer cells were observed with Wright-Giemsa stain. The results showed that after drugs exposure, the cells exhibited remarkable cytoplasm crimple and studded with increased mutinuclear cells, the mitosis cycle was arrested and show more prometaphase cells, and less anaphase and telophases cells followed FK228 exposure. Immunofluorescence microscopy confirmed that FK228 induced the mitotic catastrophe. Typical features including mitotic spindle disorganization, failed chromosome segregation such as lagging chromosome and multinucleated cells formation.2. FK228 influents the centromere localization and expression of CPC proteins and reduces the Aurora B kinase activity.The results of immunofluorescence stain showed that FK228 treatment for 12 hours, obviously disrupted the the centromere localization of CPC members including Survivin, AuroraB, INCENP and Borealin. The AuroraB kinase activity was also inhibited as the phosphorylation of its two substrates, H3( Ser10) and CENP-A (ser7) at kinetochores was reduced.3. The FK228-induced disruption of CPC centromere localization is not caused by its effect on inhibiting Hsp90 chaperone function.Western blotting results revealed FK228 depleted both Survivin and Aurora B proteins, followed FK228 treatment for 24hour, Survivin began to decline and disappeared at 48hour. Compared with Surivivn, the depletion of Aurora B protein appeared late until 48 hour. Meanwhile, immunofluorescence experiment showed that FK228-induced decline of Survivin at the centromere appeared as early as 12 hour. It indicated that the effect of FK228 on inhibiting the localization of Survivin at the centromeres happened earlier than its effect on depleting the protein. That is to say the lost of CPC proteins from centromeres is not caused by the downregulation of Survivin and Aurora B proteins through interrupting Hsp90 chaperone function.4. FK228 remarkable changes the epigenetic modification markers of pericentromere.The centromere (CEN) is a specific chromosomal locus that embedded in heterochromatin and contains blocks of histone H3 nucleosomes interspersed with the histone H3 variant CENP-A. CEN chromatin has unique epigenetic characteristics, which are distinct from that of both euchromatin and flanking heterochromatin, including hypoacetylation of histones; absence of H3 Lys4 dimethylation (H3 Lys4-diMe); and H3 Lys9-triMe is enriched in pericentric regions. The specific histone modifications in CEN may contribute to the diverse properties and function of the centromere.Followed FK228 treatment, we found that the epigenetics modification markers of centromere have been changed, including:(1) FK228 increased the acetylation of histone H3.(2) FK228 inhibited the expression of H3K9Me3 at the centromenere, but had no influence on the expression of H3K9Me2.(3) FK228 changed the binary 'methyl/phos switch' through inhibiting the H3K9Me3 and H3Ser10pho modification, which can inhibit the releasing of HP1 from metaphase chromosome and increase the accumulation of HP1 on pericentromere.(4) Decreased phosphorylation of CENP-A.5. FK228 affects the assembly and function of centromere. The centromere function including kinetochore formation, spindle-mediated movements, sister cohesion and a mitotic checkpoint. FK228 changed the epigenetic modification of pericentromere and centromere, which led us to further analyze whether these changes would influence the assembly and function of centromere. We got the following results:(1) FK228 inhibited the kinetochore proteins CENP-E and CENP-F located in outer plate of the cetromere.(2) FK228 had no effect on the localization of kinetochore inner plate protein CENP-A, but decreased the phosphorylation at ser7, an important modification for its function.(3) FK228 disrupted the attachment between centromere and microtubes during mitosis via inhibiting the kinetochore function.6.FK228 inhibits the function of spindle checkpoint(1) FK228 inhibited the localization of checkpoint proteins Bubl and Bub R1 at thecentromere.(2) FK228 inhibits the activation of mitotic checkpoint on either losing attachment or tension.In this study, we proved for the first time that the mechanisms for FK228-induced cancer cells mitotic catastrophe are related with its effect on changing epigenetic modification of centromere and pericentromere. These epigenetic changes inhibit the releasing of HP1 from metaphase chromosome, disrupt the kinetochore assembly and the localization of CPC proteins at the centromere, and finally inactivate the function of centromere and mitotic checkpoint.