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Advances in targeted therapies for hepatocellular carcinoma in the genomic era. form Tiotropium Bromide colonies at low cell density. Conclusion: Resminostat shifts mesenchymal cells towards a more epithelial phenotype, lower invasive and stemness properties, which may contribute to the sensitization to sorafenib-induced apoptosis. was first evaluated in multiple myeloma cells. Inhibition of HDACs 1,3 and 6 was demonstrated at nanomolar concentration and the efficacy to abrogate cell growth and to induce apoptosis at micromolar concentration [11]. Safety of resminostat and the recommended dose were evaluated in a phase I study in patients with advanced solid tumors [12] and confirmed in a group of Japanese patients [13]. The recently finished SHELTER study, a phase I/II clinical trial in patients no longer responding to sorafenib, compared the anti-tumor efficacy of resminostat alone or in combination with sorafenib. Safety of mono- and combination therapy was demonstrated and the combination therapy revealed an advantage in terms of overall survival and time to Tiotropium Bromide progression. Moreover, the transcription factor zinc finger protein 64 (ZFP64) was identified as a prognostic and putative predictive factor for overall survival [14]. In a follow-up study, a phase I/II clinical trial of sorafenib alone or in combination with resminostat as first line therapy in Asian patients did not reveal overall survival benefits of the combination treatment. However, stratification of patients, based on HBV, platelet counts or non-prior treatment showed favourable results [15]. The molecular mechanism that could explain the synergism between resminostat and sorafenib has not been explored yet. In previous studies, we described that lack of response to sorafenib in HCC cells correlates with a mesenchymal phenotype and the expression of the stem-related gene CD44 [16]. Therefore, here we performed an analysis of the effects of resminostat on the mesenchymal and stemness phenotype in HCC cells as a possible mechanism of sensitization to sorafenib-induced cell apoptosis. RESULTS HCC cells are sensitive to resminostat induced cell death For this study we have selected three representative HCC cell lines with differences in their epithelial/mesenchymal phenotype. We have selected an epithelial Hep3B cell line, with high expression of an epithelial marker E-cadherin and a marker of tight junctions, Zonula occludens-1 (ZO-1), organised in cell membranes. On the other hand, two mesenchymal HCC cell lines were used: HLE and HLF, with high expression of a mesenchymal marker vimentin and a presence of stress fibres (F-actin) (Figure ?(Figure1A).1A). A mesenchymal phenotype, characterised by low Tiotropium Bromide expression of E-cadherin (and (Figure ?(Figure1B)1B) moreover correlates with the expression of cancer stem cell markers and (Figure ?(Figure1C).1C). Correspondingly, the epithelial phenotype with high expression, low expression of and low expression of EMT-inducing transcription factors (Figure ?(Figure1B)1B) correlates with high expression of cancer stem cell markers and (Figure ?(Figure1C1C). Open in a separate window Figure 1 Characterization of HCC cell lines used in OBSCN the study(A). Immunofluorescence analysis of E-cadherin (green), ZO-1 (green), vimentin (green), F-actin (red) and DAPI (blue). Scale bar =25 m. (B+C). mRNA expression levels detected by qRT-PCR normalized to housekeeping gene and EMT-inducing transcription factors with a concomitant down-regulation of EMT-inducing transcription factors and up-regulation and a significant down-regulation of expression without downregulation of or (Figure ?(Figure5A).5A). Western blot analysis revealed a tendency of increased E-cadherin expression and decreased vimentin expression (Figure ?(Figure5B).5B). Moreover, by immunofluorescence analysis we confirmed vimentin downregulation (Figure ?(Figure5C).5C). During EMT, cells lose the compact, well-arranged, epithelial structure and gain a spindle-like morphology. During this process, cells lose the expression of ZO-1 protein, among others, that is responsible for tight junctions between cells. Therefore, it was interesting to note that resminostat treatment in HLF cells increased the expression of ZO-1 and re-organised ZO-1 to cell membranes, suggesting a formation of tight junctions and cell clusters, and therefore a more epithelial phenotype. Indeed, when we analyzed the % of individual cells in 3D setting (embedded in collagen I), we could observe a decrease of individual cells and an increase of cells in cell clusters after resminostat treatment (Figure ?(Figure5D).5D). Similarly, mesenchymal phenotype is connected with higher migration and invasive capacities. We evaluated Tiotropium Bromide HLF cells treated with resminostat in an invasive growth assay, in which cells need to invade trough collagen I in a 3D setting. In fact, resminostat treatment decreased their invasive ability (Figure ?(Figure5E5E and Supplementary Figure 8). Open in a separate.