Based on our hypothesis, we successfully identified genes that enhanced 5-FU- and IFN-α/5-FU-induced cytotoxicity of HCC cells when overexpressed in vitro. The identified genes, especially PRKAG2 and TGFBR2, can serve as prognostic markers for IFN-α/5-FU therapy.
encodes the gamma 2 non-catalytic but regulatory subunit of AMP-activated protein kinase (AMPK), an important cellular homeostasis sensor. Activated AMPK reserves cellular energy content and serves as the key determinant of cell survival in response to pathological energetic, oxidative, and ER stress 
. Indeed, reports documenting that AMPK significantly enhances 5-FU antitumor effects via COX-2 suppression and inhibits the mTOR signaling pathway that regulates tumor growth may support our observations 
. We found that siRNA-mediated knockdown of PRKAG2
strongly inhibited chemosensitivity to 5-FU; however, the effects of adenovirus-mediated overexpression of PRKAG2
on 5-FU were small compared with those of adenovirus-mediated overexpression of TGFBR2
. These results can be explained as follows. PRKAG2
overexpression without the other subunits may be stoichiometrically insufficient to activate the AMPK complex, while its knockdown may be sufficient to suppress its kinase activity. In the present study, PRKAG2
expression was positively correlated with survival period in HCC patients treated with IFN-α/5-FU. Indeed, phosphorylated AMPK expression has been associated with survival period and disease-free survival in HCC and lung cancer patients 
. Taken together, AMPK activation may inhibit tumorigenesis.
TGFBR2 is a type II TGF-β receptor that, upon binding to its ligand, triggers various responses, including proliferation, differentiation, and apoptosis 
. In the present study, TGFB1
mRNA was not increased by the infection with Ad-TGFBR2. This result could be explained as follows: Because TGF-β is a pleiotropic cytokine, the drug-induced TGFB1
expression may provide an advantage for cancer cell survival, possibly in an endocrine fashion in vivo
, such as immunosuppression and angiogenesis 
. Therefore, our observation suggests that TGFBR2
expression level may be a critical factor to determine the fate of the role of TGF-β in cancer cells. However, the discrepancy between the increased chemosensitization to IFN-α/5-FU in vitro
and the negative correlation of TGFBR2
with survival period in patients treated with IFN-α/5-FU remains unclear. The TGF-β signaling pathway reportedly exhibits paradoxical roles of tumor suppression and oncogenesis. It is well known that TGF-β signaling is a potent suppressor of HCC cells 
. A previous report 
showed that TGF-β enhanced the lethal effects of 5-FU in human lung cancer cells; this supports our in vitro
data that IFN-α/5-FU-induced apoptosis was enhanced by TGFBR2
expression. On the contrary, altered TGF-β signaling reportedly plays an important role in HCC progression 
. Indeed, the TGF-β signaling pathway promotes hepatocarcinogenesis in experimental p53-depleted mice 
. Furthermore, epithelial–mesenchymal transition (EMT), the underlying molecular mechanisms of which include the TGF-β pathway, is increasingly being recognized to occur during HCC progression 
. These reports and our findings suggest that TGF-β signaling may be associated with tumor progression or development in patients with advanced HCC, rather than enhancing the antitumor effect of IFN-α/5-FU. Additionally, downregulation of TGFBR2
mRNA has been reported in HepG2 cells transfected with an HCV clone 
, suggesting that a potential benefit of HCV infection may exist. In consistent with this notion, a significantly prolonged survival period during IFN-α/5-FU therapy was observed in patients infected with HCV. Further evaluations are required to determine these associations between HCV infection, TGFBR2
expression, and EMT.
EXT1 expression was reported to be epigenetically silenced in tumors, while the restoration of EXT1 expression in cancer cells induced tumor-suppressive effects 
. However, to our knowledge, little is known about the relationship between EXT1
expression and HCC. In our present study, EXT1
overexpression induced ER stress in HepG2 cells in the presence of 5-FU and IFN-α/5-FU. Induction of ER stress by EXT1
overexpression through the reduction of heparin sulfate N-sulfation has been reported 
. As per these observations, EXT1
may sensitize HCC cells to 5-FU through ER stress, which is induced by alternating heparin sulfate posttranslational modification.
In conclusion, we identified PRKAG2, TGFBR2, and EXT1 as chemosensitizing genes of HCC cells to 5-FU. Furthermore, TGFBR2 and EXT1 overexpression enhanced the anti-tumor effects of IFN-α/5-FU on HCC cells. These genes are promising candidates to enhance the therapeutic effects of IFN-α/5-FU.