Susceptibility of hepatoma-derived cells to histone deacetylase inhibitors is associated with ID2 expression

Abstract

Downregulation of inhibitor of DNA binding 2 (ID2) is associated with poor prognosis in cases of hepatocellular carcinoma (HCC). Therefore, to search for effective antitumor drugs for the treatment of HCC exhibiting poor prognostic indicators, we used two HCC-derived cell lines (HuH-7 and HLE) to alter ID2 levels. Specifically, ID2 expression was knocked down in HuH-7 cells via transfection with ID2-specific small interfering RNAs and separately ID2 was overexpressed in HLE cells via an ID2 expression plasmid vector. To assess the effect of antitumor drugs, MTS assay was performed. Annexin V staining was used to evaluate apoptosis and real-time RT-PCR was used to measure mRNA levels. ID2 knockdown cells were more susceptible to histone deacethylase (HDAC) inhibitors including sodium butyrate (NaB), sodium 4-phenyl-butyrate, tricostatin A, suberoylanilide hydroxamic acid, MS-275, apicidin and HC-toxin. Conversely, cells that overexpressed ID2 were less susceptible than control cells to HDAC inhibitors. NaB-induced apoptosis was inversely correlated with ID2 expression. Expression of the anti-apoptotic mRNA BCL2 was induced by NaB in control cells, but this induction of BCL2 was inhibited by ID2 knockdown and strengthened by ID2 overexpression. Expression of another anti-apoptotic mRNA, BCL2L1, was decreased by NaB administration and then partially recovered. However, in ID2 knockdown cells, BCL2L1 levels did not recover from NaB-induced suppression. ID2 affected the susceptibility of two HCC-derived cell lines to an HDAC inhibitor by regulating the expression of anti-apoptotic genes. Therefore, HDAC inhibitors may be effective for the treatment of HCC for which the prognosis is poor based on ID2 downregulation and ID2 could serve as a marker that is predictive of the clinical response to HDAC inhibitors.

Figure 1

ID2 levels and antitumor activity of NaB. Cells were subjected to an MTS assay 72 h after 20 mM NaB administration; NaB is one of several HDAC inhibitors that had an effect on survival of HCC-derived cells. Cell viability was lower in HCC-derived cells transfected with ID2 knockdown siRNAs than those transfected with control siRNA. Cell viability was higher in HCC-derived cells that overexpressed ID2 than in those transfected with an empty vector. ^*P<0.05 compared with HuH-7/siCont or HLE/pCont.

Figure 2

The antitumor activity of HDAC inhibitors in ID2 knockdown cells. Cells were subjected to an MTS assay to evaluate the effect of ID2 on the antitumor activity of HDAC inhibitors other than NaB. Each HDAC inhibitor had an effect similar to that of NaB (Fig. 1) on the ID2 knockdown cells. ^*P<0.05 compared with HuH-7/siCont.

Figure 3

The antitumor activity of HDAC inhibitors in cells that overexpressed ID2. Cells were subjected to an MTS assay to evaluate the effect of ID2 on the antitumor activity of HDAC inhibitors other than NaB. In cells that overexpressed ID2, each HDAC inhibitor, except SAHA, had an effect similar to that of NaB (Fig. 1). ^*P<0.05 compared with HLE/pCont.

Figure 4

ID2 levels and antitumor activity. Cells were subjected to MTS assay 72 h after administration of the indicated antitumor drugs,. ^†P<0.05 compared with HuH-7/siCont or HLE/pCont.

Figure 5

ID2 levels and apoptosis caused by NaB. Cells were stained with Annexin V/Propidium iodide (PI)/Hoechst 33342 after 20 mM NaB had been administered for 72 h; cells were then assessed by fluorescence microscope. Cells positive for both Annexin V and PI staining were considered to be in the late stage of apoptosis. Cultures containing ID2 knockdown cells had higher percentages of apoptotic cells than did cultures with control siRNA transfected cells. Cultures with ID2 overexpressing cells had lower percentages of apoptotic cells than did cultures containing cells transfected with empty vector. ^*P<0.05 compared with HuH-7/siCont or HLE/pCont.

Figure 6

Changes in expression of anti-apoptotic genes following NaB administration. The mRNA levels of BCL2, BCL2L1 and BAX were measured after 20 mM NaB administration for 0, 6, 24 or 48 h. (A and B) BCL2 mRNA level was induced by NaB administration when compared to the induction in control cells, this NaB-dependent induction was suppressed in ID2 knockdown cells and enhanced in cells that overexpressed ID2. (C and D) The BCL2L1 mRNA level decreased immediately after NaB administration and was then partially restored. In ID2 knockdown cells, the restoration of BCL2L1 expression was largely suppressed relative to that in control cells. ^**Both ID2-targeted cells showed P<0.05 compared with control cells.