Pharmacological or transcriptional inhibition of both HDAC1 and 2 leads to cell cycle blockage and apoptosis via p21Waf1/Cip1 and p19INK4d upregulation in hepatocellular carcinoma

Abstract

Objectives: Histone deacetylases (HDACs) are commonly dysregulated in cancer and represent promising therapeutic targets. However, global HDAC inhibitors have shown limited efficacy in the treatment of solid tumours, including hepatocellular carcinoma (HCC). In this study, we investigated the therapeutic effect of selectively inhibiting HDAC1 and 2 in HCC.

Methods: HDAC1 inhibitor Tacedinaline (CI994), HDAC2 inhibitor Santacruzamate A (CAY10683), HDAC1/2 common inhibitor Romidepsin (FK228) and global HDAC inhibitor Vorinostat (SAHA) were used to treat HCC cells. Cell cycle, apoptosis and the protein levels of CDKs and CDKNs were performed to evaluate HCC cell growth. Inhibition of HDAC1/2 by RNAi was further investigated.

Results: Combined inhibition of HDAC1/2 led to HCC cell morphology changes, growth inhibition, cell cycle blockage and apoptosis in vitro and suppressed the growth of subcutaneous HCC xenograft tumours in vivo. p21^Waf1/Cip1 and p19^INK4d , which play roles in cell cycle blockage and apoptosis induction, were upregulated. Inhibition of HDAC1/2 by siRNA further demonstrated that HDAC1 and 2 cooperate in blocking the cell cycle and inducing apoptosis via p19^INK4d and p21^Waf1/Cip1 upregulation. Finally, H3K18, H3K56 and H4K12 in the p19^INK4d and p21^Waf1/Cip1 promoter regions were found to be targets of HDAC1/2.

Conclusions: Pharmacological or transcriptional inhibition of HDAC1/2 increases p19^INK4d and p21^Waf1/Cip1 expression, decreases CDK expression and arrests HCC growth. These results indicated a potential pharmacological mechanism of selective HDAC1/2 inhibitors in HCC therapy.

Figure 1

HDAC1 and 2 expression predicts HCC patient survival, based on data from the TCGA database. (A‐C) Expression levels of HDAC1, 2 and 3 in patients with HCC. (D) The overall survival of 109 patients who were divided into a high HDAC1 expression group (n = 57) and a low HDAC1 expression group (n = 52). (E) The overall survival of 101 patients who were divided into a high HDAC2 expression group (n = 40) and a low HDAC2 expression group (n = 61). (F) The overall survival of 102 patients who were divided into a high HDAC3 expression group (n = 46) and a low HDAC3 expression group (n = 56)

Figure 2

Effect of HDAC inhibitors on the morphology, growth and cell cycle distribution of HCC cells. The experimental groups were the Control, CI994, CAY10683, CI994+ CAY10683, FK228 and SAHA groups. (A) HepG2 and Huh7 cell morphology viewed under a microscope. (B) CCK‐8 staining of HepG2 and Huh7 cells treated with HDAC inhibitors. (C) Effect of HDAC inhibitors on HepG2 and Huh7 cell cycle distribution. (D) Effect of HDAC inhibitors on the expression of key cell cycle‐related proteins in HepG2 and Huh7 cells

Figure 3

Effect of HDAC inhibitors on HCC cell apoptosis. The experimental groups were the Control, CI994, CAY10683, CI994+ CAY10683, FK228 and SAHA groups. (A) and (B) Treating HepG2 and Huh7 cells with FK228 and CI994+CAY10683 had a significant effect on cell apoptosis compared with the other groups. (C) and (D) FK228 and CI994+CAY10683 significantly increased the expression of Bax and decreased the expression of Bcl‐2. The Bax/Bcl‐2 ratio was significantly higher in FK228‐ and CI994+CAY10683‐treated groups than in the control, CI994, CAY10683 and SAHA groups. GAPDH expression served as an internal control

Figure 4

The anti‐tumour effects of HDAC inhibitors on the growth of subcutaneous HCC xenograft tumours in nude mice. (A) Subcutaneous tumours in nude mice were allowed to grow for 4 weeks and were then treated with intratumoural injections of CI994, CAY10683, FK228 or SAHA. (B) Ex vivo tumour images and (C) tumour weight at the end of the experiment. Scale bar, 1 cm. (D) H&E staining and IHC analysis of Ki67 were performed to examine the histopathological alterations and proliferation of HCC tumours injected with HDAC inhibitors

Figure 5

The expression of tumour‐suppressor genes in HepG2 and Huh7 cells. The experimental groups were the Control (no HDACi treatment), CI994, CAY10683, CI994+ CAY10683, FK228 and SAHA groups. (A) and (B) mRNA and protein levels of the tumour‐suppressor genes p21^Waf1/Cip1 and p19^{INK 4d} in HepG2 and Huh7 cells. The mRNA levels are presented as fold changes compared with the control group. The protein expression of p21^Waf1/Cip1 and p19^{INK 4d} was measured by Western blot analysis. GAPDH expression served as an internal control. (C) ChIP assays with monoclonal antibodies against H3K9, H3K18, H3K56, H4K5, H4K12 and H4K16 revealed a significantly higher occupancy of H3K18, H3K56 and H4K12 in the promoter regions of p^21Waf1/Cip1 and p^{19 INK 4d} in FK228‐ and CI994+CAY10683‐treated groups than in the control, CI994, CAY10683 and SAHA groups

Figure 6

Transcriptional HDAC1/2 inhibition leads to cell cycle blockage and apoptosis in liver cancer cells. (A) qRT‐PCR and Western blot analyses of HDAC1 and HDAC2 were performed after siRNA transfection. The levels of HDAC1 and HDAC2 are expressed as ratios to that of GAPDH. (B) Flow cytometric analyses of cell cycle distribution was performed after siRNA transfection. (C) Immunoblotting of CDKs was performed after siRNA transfection. (D) Flow cytometric analyses of apoptosis was performed after siRNA transfection. (E) and (F) The expression of p19^{INK 4d} and p21^Waf1/Cip1 was measured by qPCR and Western blot analyses. GAPDH expression served as an internal control

Figure 7

Proposed model for selective inhibition of HDAC1 and HDAC2 in the regulation of hepatocellular carcinoma proliferation