The Reversal Effects On Epithelial-mesenchymal Transition Of Trichostatin A, A Histone Deacetylase Inhibitor, In Tumor Cells And The Investigation Of The Initial Molecular Mechanisms

Background and Objective:Histone deacetylase inhibitors(HDACIs) are now considered to be promising anti-cancer agents and some of them are near clinical stage or already on the market. As reported, their effects are correlated with the transcriptional regulation of specific cancer-related genes. Trichostatin A(TSA) is the representative of four kinds of classical HDACIs, which inhibits HDACs in a noncompetitive and reversible way. TSA could inhibit proliferation and induce differentiation in a variety of cancer cells such as colorectal, prostate, neuroblastoma and skin cancer cells. And its molecular mechanisms for inhibiting proliferation and inducing differentiation have been studied a lot and relatively well known. Also, some studies have observed TSA could suppress invasion and migration in prostate cancer cells. But the underlying molecular mechanisms are rarely studied and still not clear. It is well-known that Epithelial–mesenchymal transition(EMT) importantly contributes to invasion and migration of tumor cells. The indeed clinical evidence reveal that regulators of EMT in tumor cells are closely associated with poor outcomes and tumor aggressiveness. On the basis of the introduction above, we want to know whether EMT reversal were the crucial factors by which TSA exerted anti-invasion and anti-metastasis effects. And we also want to investigate the involved molecular mechanism. Therefore, we here firstly investigated more molecular evidence of TSA-induced EMT reversal effects and then validated TSA-mediated anti-invasion and anti-metastasis effects. Given that transcription factors have been regarded as a crucial factor in EMT regulation, further study investigated the expression changes of transcription factors and the possible mechanisms induced by TSA. These findings will help to better understand the molecular mechanisms for anti-cancer potentials of HDACIs. Methods:This studies included the TSA mediated EMT reversal effects in tumor cells and the involved mechanism, which were carried out by following four aspects:EMT related biomarkers, invasion and metastasis capability, EMT related transcription factors, Chromatin level. 1. In SW480, PC3 cells, we firstly detected the expression of E-cadherin and Vimentin inm RNA and protein levels. Then we detected the expression of E-cadherin on the cellmembrane by confocal microscopy. 2. We firstly detected the anti-proliferation effects of TSA in SW480, PC3 cells to determine alow concentration which make no difference to cell proliferation. Then we detected the TSAinduced changes of invasion and migration abilities in SW480, PC3 cells by transwellinvasion and migration assay. 3. In m RNA and protein levels, we firstly examined the expression of Slug in SW480 and PC3cells after treatment with TSA, then detected the change of Slug and E-cadherin protein indifferent time points. Furthermore, SW480 and PC3 cells were transfected with pc DNA-Slugand then cells with pc DNA-Slug were treated with or without TSA, finally the expression ofE-cadherin, Vimentin and Slug were detected in m RNA and protein levels. 4. We performed CHIP assay to detect the changes of HDAC1, HDAC2, HDAC3, HDAC4 onthe promoter of Slug gene. Results: 1. TSA led to increase of E-cadherin and decrease of Vimentin in m RNA and protein levels,which implied an increase of epithelial biomarker and a decrease of mesenchymal biomarker.Through confocal microscopy scanning, the up-regulation of E-cadherin was observedvisually on the cellular membrane visually by TSA treatment. 2. After treatment with TSA, the invasive cells that had spread through the filter and adheredthe underside decreased significantly, as well as the migrated cells in the lower chamber.Meanwhile, treatment with TSA suppressed the growth of the SW480 and PC3 cells withsimilar efficacies and 100 n M TSA showed no inhibitory activity on cell proliferation within48 h, suggesting that the decreased invasive and migrated cells by TSA were not due to itsanti-proliferation effects. 3. TSA significantly suppressed Slug in m RNA and protein levels. Meanwhile, after treatmentwith TSA, the expression changes of Slug protein was earlier than the changes of E-cadherinprotein. Furthermore, in m RNA and protein levels, Slug, Vimentin were up-regulated andE-cadherin was down-regulated in SW480 and PC3 cells after being transfected withpc DNA-Slug. 4. TSA specifically induced the increase of HDAC1 and HDAC2 on promoters of Slug whileTSA did not show any changes in the total amount of HDACs in SW480 and PC3 cells. Conclusions and Significance: 1. Our data suggested that TSA could induce the EMT reversal and the suppression effects ofinvasion and migration in tumor cells, which were the most direct effects against progressionof malignant tumors. 2. Further study revealed that TSA could reverse epithelial–mesenchymal transition in SW480and PC3 cells through suppressing Slug. TSA-induced expression changes of EMTbiomarkers and transcription factors both validated its EMT reversal effects. It would providea theoretical basis for a reasonable guide of HDAC inhibitors in anti-tumor clinical use 3. We found that the amount of HDAC1, HDAC2 associated with promoter of Slug was higherin TSA treated cells compared to control cells, which would be responsible fordown-regulation of Slug. However, it have been reported that HDACIs usually have directeffects on histone deacetylases and elevate the acetylation level of HDACs to regulate theexpression and activity of numerous protein. It is rare that HDACIs induce increase ofHDACs on the promoter of target gene, which might be an indirect and complex process, andmore attentions should be paid on these similar effects.