The Mechanism For Inducing Cell Differentiation And Its Effect On Hsp90 Chaperone Function Of HDAC Inhibitor Depsipeptide

Background:Heat shock proteins (Hsps) are kinds of important molecular chaperone of the cells, except for involving the modulation of protein conformation, stability and kinases activities, these proteins could offer protection when cells in various stress condition. Among the Hsps, Hsp90 is a chaperon protein with special client proteins including oncogenic proteins (mutant p53, Bcr-abl), kinase proteins(v-Src, ErbB2, Raf-l,Akt,Cdk4, Cdk6, et al); cell membrane growth factor receptors(EGFR, PDGFR,IGFR, et al) and nucleus receptors( androgen receptor, estrogen receptor). Since all these client proteins play important roles in tumor proliferation, chemotherapy resistance and anti-apoptotic potential, to deplete all these client protein from the cells by disrupting the chaperone function of Hsp90 has obvious advantage for anticancer activity.Histone deacetylase inhibitors(HDACi) are a kind of novel anti-cancer agents with high efficiency and low cytotoxicity. HDACi can inhibit cell proliferation, induce differentiation and/or apoptotic cell death, but their accurate mechanism remains undefined. Although HDACi can enhance the histone acetylation which leads to change chromatin conformation and active some genes transcription, the key genes which mediate their biological activities have not been conformed yet even with cDNA microarray detection. On the other hand, it was shown that there is no correlation between enhanced histone acetylation and cell growth inhibition. People try to investigate other possible mechanism for HDACi. On 2002, it was reported forthe first time that HDACi can interrupt Hsp90 chaperone function by enhanced Hsp90 acetylation which leads to depletion of several client proteins and inducement of apoptosis in lung cancer cells.v-Srv is one of the client proteins of Hsp90 that was recognized previously, and its cognate c-Src expressed in eukaryocyte is an important intracellular non-receptor tyrosine kinase, participating in the modulation of multiple signal transduction pathways involving cell proliferation and anti-apoptosis, and also has effects on cell cytoskeleton formation and cell adhesion to extracellular matrix(ECM). Up to date, people have found that HDACi can induce Src-transformed cells differentiation, but the mechanism is still unclear. In this study, we try to set up Src-transformed cell model, and to investigate the effect of HDACi depsipeptide(DP) on cells differentiaion and Hsp90 chaperone function, especially on Src protein expression and its binding ability with Hsp90. These data may help to de novo elucidate the mechanism of HDACi on cell differentiation.Experiment design and results:1. Set up transformed cell model by Src gene transfectionBy using stable gene transfection technique, the plasmid containing wild type Src gene was transfected into NIH-3T3 cells, cell clones with Src overexpression were obtained after G418 selection. Compared with mock transfectant, the 3T3-Src cells showed enhanced potential on cell proliferation, clone formation on soft-agar and adhesive ability with extracellular matrix.2. Investigate the effect of HDACi depsipeptide(DP) on 3T3-Src cellsAfter exposing with DP for various time, 3T3-mock and 3T3-Src cells both showed growth inhibition, cell cycle blocked in G0/G1 phase and time-dependent cell apoptosis. DP could change the cells shape of 3T3-Src from spindle to flat, with actin stress fiber bundles reorganized. The expression of actin-binding proteingelsolin and cell adhesive potential with extracellular matrix were also changed following DP treatment. DP can also upregulate CDK inhibitor p21 expression and enhance histone H3 acetylation, but unlike other HDACi, DP did not show any effect on increasing tubulin acetylation, which suggested DP can not inhibit HDAC6 activity.3. Investigate the effect of DP on Hsp90 chaperon function and the mechanism of DP on inducing cell differentiation and apoptosisAfter treatment with DP for various times, the enhanced acetylation of Hsp90 can be detected in 3T3-Src cells. Like the Hsp90 inhibitor Geldanamycin(GA), DP can deplete several Hsp90 client proteins from the cell, including c-Src, Raf-l,Akt and Cdk4 kinases, which indicated Hsp90 chaperone function has been interrupted. The activation of two important cell survival pathways of ERK and PDK/AKT were blocked which directly mediated cell apoptosis. The changes for endogenous and the exogenous Src protein were different, like GA, DP could effectively decrease endogenous Src protein level, but visibly increase exogenous Src expression which can be explained by its effect on active CMV promotor of the transfected vector. The interesting phenomenon was that, although DP inducing the exogenous Src protein overexpression, the increased Src could not bind with Hsp90 so accelerated its degradation. The possible mechanism may involve the enhanced acetylation of Hsp90 and Src protein as well. To our knowledge, this is the first time to report that DP can acetylate both members of the Hsp90-client protein complex. This find may help to elucidate clearly why HDACi can interrupt Hsp90 chaperone function. In conclusion, the mechanism for DP inducing Src transformed cell differentiation related with interdicting Hsp90 function and speeding Src degradation.Conclusion:1. HDAC inhibitor DP can induce Src transformed cell differentiation, inhibit the cell proliferation, and change their morphological and adhesive characteristics.