Vels may not be as significant for G1/S phase transition in Rb-null cells as in Hep3B cells. Thus, the extent of its effect may be differently manifest, depending on the genetic background of the HCC cells. Levels of cyclin D1, p-Rb795 and p-Rb807/811 were all reduced in VRK1-depleted cells, which is consistent with an earlier report MS 275 manufacturer showing that cyclin D1 and p-Rb levels declined upon VRK1 downregulation in fibroblasts. The reduction in the levels of cyclin D1 was attributed to a decrease in its gene’s transcription, which was in turn related to decreased CREB phosphorylation in VRK1-depleted HCC cells. We also observed that both p21 and p27 were upregulated upon VRK1 knockdown, but that p53 levels were unchanged. VRK1 thus appears to regulate p21 expression independently of p53, though the mechanism by which VRK1 affects expression of p21 and p27 remains to be clarified. Taken together, these findings indicate that VRK1 AIC316 price depletion causes arrest or delay of G1/S transition, leading to a significant increase in the HCC cell fraction in G1 phase. This suggests the retardation of HCC cell growth induced by VRK1 depletion reflects interference with the cell cycle. We suggest that VRK1 is a potential therapeutic target for treatment of HCC, based on its levels in our PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19861045 clinical samples and its role in cell proliferation. It has also been proposed that in lung cancer and rhabdomyosarcoma, the VRK1 gene is a “druggable target gene” whose function in tumors is distinguishable from that in healthy tissue. In the present study, the VRK1 inhibitor luteolin effectively inhibited HCC cell proliferation. Although several kinase inhibitors have been shown to suppress VRK1 kinase activity, the inhibitory mechanism has not been determined. Our group recently reported that luteolin specifically binds to the catalytic domain of VRK1 to inhibit the kinase activity. By inhibiting VRK1, luteolin reduced HCC cell growth in vitro and in vivo, and also induced apoptosis in HCC cells. Consistent with our results, the anti-cancer effects of luteolin have also been demonstrated in several HCC cell lines. For example, luteolin inhibited HGF-induced HepG2 cell invasion and mediated increases in intracellular ROS levels in Huh-7 cells. In the present study, however, we did not observe significant induction of apoptosis upon VRK1 depletion by VRK1 siRNA. The extent of its different effect on apoptotic induction between VRK1 inhibition with luteolin 
treatment and VRK1 depletion by VRK1 siRNA could be explained by multiple molecular target of luteolin or/and the residual VRK1 expression in cells transfected with VRK1 siRNAs. Addressing this issue will require development of more specific and potent inhibitors of VRK1. Aberrant VRK1 expression has been reported in colon cancer and lung cancer tissues. Similarly, Oncotarget we observed higher levels of VRK1 in HCC tumor tissue and cell lines than in corresponding non-tumorous liver tissue and normal liver cells. VRK1 expression is known to be dependent on the p53 status. Induction of p53 expression using UV light or transfection with the plasmid encoding p53 downregulated VRK1 levels, which is consistent with the inverse correlation between VRK1 and p53 levels reported previously. Moreover, VRK1 accumulated in lung tumors expressing mutant p53 with an altered regulatory loop. In contrast to those earlier reports, we did not find an inverse correlation between VRK1 and p53 levels in HCC cell lines; nonetheless, the regulatio.Vels may not be as significant for G1/S phase transition in Rb-null cells as in Hep3B cells. Thus, the extent of its effect may be differently manifest, depending on the genetic background of the HCC cells. Levels of cyclin D1, p-Rb795 and p-Rb807/811 were all reduced in VRK1-depleted cells, which is consistent with an earlier report showing that cyclin D1 and p-Rb levels declined upon VRK1 downregulation in fibroblasts. The reduction in the levels of cyclin D1 was attributed to a decrease in its gene’s transcription, which was in turn related to decreased CREB phosphorylation in VRK1-depleted HCC cells. We also observed that both p21 and p27 were upregulated upon VRK1 knockdown, but that p53 levels were unchanged. VRK1 thus appears to regulate p21 expression independently of p53, though the mechanism by which VRK1 affects expression of p21 and p27 remains to be clarified. Taken together, these findings indicate that VRK1 depletion causes arrest or delay of G1/S transition, leading to a significant increase in the HCC cell fraction in G1 phase. This suggests the retardation of HCC cell growth induced by VRK1 depletion reflects interference with the cell cycle. We suggest that VRK1 is a potential therapeutic target for treatment of HCC, based on its levels in our PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19861045 clinical samples and its role in cell proliferation. It has also been proposed that in lung cancer and rhabdomyosarcoma, the VRK1 gene is a “druggable target gene” whose function in tumors is distinguishable from that in healthy tissue. In the present study, the VRK1 inhibitor luteolin effectively inhibited HCC cell proliferation. Although several kinase inhibitors have been shown to suppress VRK1 kinase activity, the inhibitory mechanism has not been determined. Our group recently reported that luteolin specifically binds to the catalytic domain of VRK1 to inhibit the kinase activity. By inhibiting VRK1, luteolin reduced HCC cell growth in vitro and in vivo, and also induced apoptosis in HCC cells. Consistent with our results, the anti-cancer effects of luteolin have also been demonstrated in several HCC cell lines. For example, luteolin inhibited HGF-induced HepG2 cell invasion and mediated increases in intracellular ROS levels in Huh-7 cells. In the present study, however, we did not observe significant induction of apoptosis upon 
VRK1 depletion by VRK1 siRNA. The extent of its different effect on apoptotic induction between VRK1 inhibition with luteolin treatment and VRK1 depletion by VRK1 siRNA could be explained by multiple molecular target of luteolin or/and the residual VRK1 expression in cells transfected with VRK1 siRNAs. Addressing this issue will require development of more specific and potent inhibitors of VRK1. Aberrant VRK1 expression has been reported in colon cancer and lung cancer tissues. Similarly, Oncotarget we observed higher levels of VRK1 in HCC tumor tissue and cell lines than in corresponding non-tumorous liver tissue and normal liver cells. VRK1 expression is known to be dependent on the p53 status. Induction of p53 expression using UV light or transfection with the plasmid encoding p53 downregulated VRK1 levels, which is consistent with the inverse correlation between VRK1 and p53 levels reported previously. Moreover, VRK1 accumulated in lung tumors expressing mutant p53 with an altered regulatory loop. In contrast to those earlier reports, we did not find an inverse correlation between VRK1 and p53 levels in HCC cell lines; nonetheless, the regulatio.