Largazole, a novel potent histone deacetylase inhibitor, inhibits liver fibrosis and hepatocellular carcinoma both in vitro and in vivo

Abstract

Background and aims: Histone deacetylase (HDAC) plays a significant role in gene expressions regulation by modulating chromatin structure. Inhibition of HDAC is reported to be one of the potential targeted therapies for cancer. Largazole, a novel HDAC inhibitor (HDACi) synthesized by our group, demonstrated potent and selective biological activities in the inhibition of the growth of many transformed and nontransformed human and murine cell lines in vitro. The purpose of this study was to investigate the effects of Largazole against liver fibrosis and hepatocellular carcinoma (HCC). Methods: In vitro effects of Largazole were examined using hepatic stellate cells (HSC), HCC cell lines and human umbilical vein endothelial cells (HUVECs). In vivo effects of Largazole in liver fibrosis or HCC were studied using a mouse liver fibrotic model induced by CCl4 or an ectopic HCC model established in nude mice, respectively. Results: Largazole exhibited potent activity against HDAC enzymes in the nanomolar range. Largazole augmented acetylation of histone H3 (H3) and histone H4 (H4) in both HSC and HCC cells. Largazole directly inhibited the activation of HSC by inhibition of cell proliferation, and the expression of collagen and α-smooth muscle actin (α-SMA). The direct inhibition of the activation of HSC was due to its HDAC inhibitory activity as the antifibrotic effect of Largazole was significantly decreased in cells with HDAC1, HDAC2 and HDAC3 knockdown. Furthermore, Largazole induced apoptosis of HSC. Largazole inhibited the expression of transforming growth factor β receptor 2 (TGFβR2), and reduced the phosphorylation of Smad2 and Akt induced by TGFβ1. Largazole also inhibited the expression of vascular endothelial growth factor (VEGF) and its receptors. VEGF-induced proliferation of HSC and activation of Akt and p38MAPK were also suppressed by Largazole. In vivo, Largazole reduced the expression of collagen I, α-SMA and tissue inhibitor of metallpproteinase-1 in the developed fibrosis induced by CCl₄, which were associated with increased acetylation of H3 and H4. Largazole also induced HSC undergoing apoptosis in vivo. Furthermore, Largazole inhibited angiogenesis in vivo evidenced by the reduced expression of CD34, VEGF and VEGFR. In addition to its antifibrotic activity, Largazole reduced inflammatory activity in CCl4-induced liver fibrosis. In HCC, Largazole inhibited the cell proliferation of HCC cell lines and HUVECs in a dose-dependent manner. Largazole induced HCC cell cycle arrest and cell apoptosis. Largazole also inhibited the expression of both TGFβR1 and TGFβR2 in HCC cells, and reduced the phosphorylation of Smad2 and Akt induced by TGFβ1. Moreover, Largazole downregulated the expression of VEGF, its receptor and hypoxia-inducible factor-1 α (HIF-1α) in both HCC cells and HUVECs. Largazole also reduced phosphorylation of Akt and p38MAPK induced by VEGF. In vivo, administration of Largazole led to a significant suppression of tumor volume of HCC xenografts in nude mice. Furthermore, Largazole inhibited angiogenesis in vivo as evidenced by the reduced microvessel formation, as well as downregulated expression of VEGF and VEGF receptor. Conclusions: Our finding revealed a novel role of Largazole in the treatment of liver fibrosis and HCC. Through multiple mechanisms, Largazole could be a potentially effective antifibrotic and antitumor agent.