Ten hepatocellular carcinoma tissues and corresponding normal tissues were obtained from Yonsei University, School of Medicine, Seoul. Informed consent was provided in compliance with the Declaration of Helsinki, and the study was approved by the Institutional Review of Board of Songeui Campus, College of Medicine, The Catholic University of Korea (IRB approval no. CUMC11U010).

The human HCC cell lines, HepG2 (wt p53) and SNU-182 (mt p53), and the non-small lung carcinoma cell line A549 (wt p53) were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA). Cell lines were maintained in RPMI-1640 medium (Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO, USA) and 100 units/ml of penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA).

Small interfering RNA duplexes (siRNAs) specific to SIRT1 (sense, 5′-GGAUAGAGCCUCACAUGCA-3′; anti-sense, 5′-UGCTUGUGAGGCUCUAUCC-3′) and DBC1 (sense, 5′-CAGCUUGCAUGACUACUUU-3′; antisense, 5′-AAAGUAGUCAUGCAAGCUG-3′) and negative control siRNA were purchased from Ambion (Austin, TX, USA). SIRT1 expression plasmid, pcDNA 3.1-SIRT1-myc-His was kindly donated by Dr. Kouzarides (University of Cambridge, UK). For gene silencing, siRNAs against SIRT1 and DBC1 were transfected at final concentrations of 50 and 200 nM, respectively. Two micrograms of mock (empty vector) and SIRT1 expression plasmids were used to induce SIRT1 overexpression. All transfections were performed using Lipofectamine™ 2000 reagents (Invitrogen) according to the manufacturer’s instructions. To induce DNA damage, cells were treated with 20 μM etoposide (Sigma) for 12 h.

Tissue proteins and cell lysates were prepared using Radio Immunoprecipitation (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate) supplemented with 1 mM phenylmethane-sulfonylfluoride (PMSF, Sigma) and 1X complete protease inhibitor cocktail tablets (Roche, Mannheim, Germany). Proteins (10 μg) were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (PVDF, Bio-Rad Laboratories, Hercules, CA, USA). Proteins were analyzed using anti-SIRT1, -p53 and -GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-DBC1 (Bethyl Laboratories, Montgomery, TX, USA) and anti-acetylated p53 (Cell Signaling, Danvers, MA, USA). An ECL Plus western blotting detection system (Millipore, Billerica, MA, USA) was used to detect signals. Membranes were evaluated using a LAS 3000 image analyzer (Fuji Photo Film, Japan). For immunoprecipitations, cells were lysed with sodium deoxycholate-free RIPA buffer. Cell lysates (1∼2 mg) were incubated with 1∼2 μg anti-His (Applied Biological Materials, BC, Canada) or anti-SIRT1 at 4°C overnight, and then rotated with 50 μl protein-A-agarose (Millipore) at 4°C for 4 h. Samples were analyzed by western blotting with appropriate primary antibodies.

SNU-182 cells were seeded into 6-well plates and transfected with each siRNA. Twenty-four hours later, cells were treated with 20 μM etoposide for 12 h. After etoposide treatment, cells were incubated with 0.5 mg/ml MTT for 1 h at 37°C for the indicated times. The formazan crystals produced were dissolved with 500 μl DMSO and absorbances were read at 570 nm using a VICTOR3™ Multilabel Plate Reader (PerkinElmer, Foster City, CA, USA). All measurements were performed in triplicate and each experiment was repeated at least three times.

It was recently suggested that SIRT1 is expressed at very low levels in normal liver tissue, but that it is overexpressed in HCC cell lines and in a subset of HCCs, and that it is essential for tumorigenesis in HCC (17). Conversely, an analysis of SIRT1 mRNA levels in HCC patients by real-time qPCR revealed that average SIRT1 mRNA levels in tumor and non-tumoral liver tissues do not differ significantly, which suggests that the tumor-specific overexpression of SIRT1 is regulated in a transcription-independent manner (17). In addition, it has been demonstrated that SIRT1 activity is negatively regulated by DBC1 by direct binding (6,7). However, how SIRT1 is regulated by DBC1 in liver cancer remains unclear. Therefore, to explore this association in liver cancer, we examined SIRT1 and DBC1 gene expressions available from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (accession numbers GSE14520 and GSE25097) in a large cohort of HCC patients. As shown previously, SIRT1 and DBC1 gene expression was not significantly different in the two large HCC cohorts (Fig. 1A and B). We then examined SIRT1 protein overexpression by using western blotting in 10 randomly selected human HCC tissues and paired non-cancerous surrounding liver tissues. As expected, SIRT1 was markedly upregulated in HCCs compared to non-cancerous tissues (Fig. 1C). Notably, we also observed that DBC1 protein was highly overexpressed in the same HCCs. Since DBC1 has been reported to function as a negative regulator of SIRT1 in other cancers, we had expected an inverse correlation between DBC1 and SIRT1. Thus, we investigated the association between DBC1 and SIRT1 expression in a liver cancer cell line by immunoprecipitation and western blot analysis.

As shown in Fig. 2A, to elucidate whether DBC1 is associated with SIRT1, we introduced His-tagged SIRT1 expression plasmid into SNU-182 cells. In the following immunoprecipitation with anti-His antibody, endogenous DBC1 was detected by western blotting. SIRT1 was simultaneously detected in the same anti-His immunoprecipitates. In addition, endogenous DBC1 and SIRT1 were also co-immunoprecipitated in SNU-182 cells (Fig. 2B). These results demonstrated that DBC1 interacts with SIRT1 in liver cancer cells in vitro.

Next, to explain the biological consequences of the aberrant expressions of SIRT1 and DBC1 in hepatocarcinogenesis, SIRT1 and DBC1 expression was abrogated using the RNA interference-mediated protein knockdown method in HepG2 and SNU-182 cells (two different HCC cell lines). As previously observed (17), SIRT1 depletion resulted in a significant reduction in tumor cell growth in both cell lines (Fig. 3A and B). Notably, when DBC1 protein was inactivated by its siRNA, both cell lines exhibited significantly less cell viability. However, double knockdown of SIRT1 and DBC1 did not exhibit a synergistic effect on cell viability (Fig. 3A and B). These results suggest that DBC1 does not negatively regulate SIRT1, but that it does contribute to the neoplastic property of liver cancer cells.

In previous studies, inactivation of DBC1 by siRNA attenuated etoposide-induced p53 acetylation by inhibiting SIRT1 deacetylase activity in A549 and U2OS cell lines (6,7). To determine whether DBC1 inhibits p53, we analyzed acetylated p53 in the absence or presence of DBC1 protein in liver cancer cells. Initially, to confirm the suppressive effect of DBC1 on p53 acetylation by SIRT1, we performed etoposide-induced hyperacetylation of p53 in A549 cells. As shown in Fig. 4A, DBC1 silencing reduced the acetylation status of p53 in response to etoposide treatment in A549 cells (Fig. 3B). However, inactivation of DBC1 by its siRNA did not affect the etoposide-mediated p53 hyperacetylation status in SNU-182 cells (Fig. 4B). These results suggest that DBC1 does not functionally influence SIRT1 activity in liver cancer cells.