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Objective To explore the mechanism of hepatitis B virus X protein down-regulating DKK4 and its effect on the proliferation, migration of HCC cell lines. Methods HCC cell lines HepG2 and SMMC7721 cells were infected with adenovirus encoding hepatitis B virus X protein (Ad-HBx), and GFP adenovirus (Ad-GFP) was designed as a control group. We used deacetylase inhibitor (TSA) to treat HCC cell lines and transfected HCC cell lines with small interfering RNA-histone deacetylase 1 (si-HDAC1) and lentivirus overexpressing DKK4. Western blot was used to detect the expression of DKK4, HDAC1 and SIRT1. The proliferation and migration ability of HCC lines were assessed using MTT, crystal violet experiment and Transwell experiment. Results DKK4 expression level was significantly downregulated after Ad-HBx infection (P < 0.05), and its expression level was recovered after TSA treatment (P < 0.05). After silencing HDAC1 with small interfering RNA, the expression of DKK4 could be restored (P < 0.05), the proliferation and migration of HDAC1-silencing or/and DKK4-overexpressing cells decreased (P < 0.05). Conclusion Hepatitis B virus X protein inhibits the expression of DKK4 protein by up-regulating HDAC1 and SIRT1. Silencing HDAC1 and over expressing DKK4 protein could inhibit the proliferation and migration of HCC cell lines infected with Ad-HBx.
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Altered glucose uptake and metabolism is the key characteristic of cancer cells including hepatocellularcarcinoma (HCC). However, role of glucose availability in chemotherapeutic outcome of HCC is unclear. Thepresent study investigates the effect of glucose facilitated sensitization of HCC cells towards doxorubicin(DOX) and sorafenib (SORA). In HCC cells, we observed that hyperglycemic culture condition (HG) isassociated with increased sensitivity towards DOX and SORA. P-glycoprotein (P-gp), a transporter involved indrug efflux, was elevated in HCC cells in NG, rendering them less susceptible to DOX and SORA. Further, thisstudy demonstrated that knockdown of dickkopf protein 4 (DKK4), a Wnt antagonist protein, causes enhancedglucose uptake and reduction in P-gp level rendering HCC cells in NG sensitive to DOX and SORA.Moreover, HG elevates the level of intracellular reactive oxygen species (ROS), which regulates P-gp.Alteration in intracellular ROS did not directly affect regulation of DKK4 in HCC cells. Functional assayssuggest that alterations in DKK4 and P-gp level in HCC cells are dependent on glucose availability andchanges in ROS level because of enhanced glucose utilization, respectively. Collectively, the present studyhighlights direct involvement of glucose-induced ROS, DKK4 and P-gp in altering the sensitivity of HCC cellstowards DOX and SORA.
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OBJECTIVES: The inhibitory effect of Dickkopf (Dkk)-1 on osteoblastic differentiation through blocking Wnt signaling has been well studied. However, the role of other members of the subfamily of Dkks remains unclear. We have examined the role of different Dkks on osteoblastic differentiation of mesenchymal progenitor cells and apoptosis of osteoblasts. METHODS: Osteoblastic differentiation was induced by treatment of Wnt-3a with Dkks or vehicle in C3H10T1/2 cells and alkaline phosphatase (ALP) activity was measured. Serum deprivation induced apoptosis was performed with pre-treatment of Dkks or vehicle in MC3T3-E1 cells and methyl thiazolyl tetrazolium (MTT) assay was done. RESULTS: Dkk-2 at low concentrations (5 and 20 nM) and Dkk-3, -4 at any concentrations (5 to 100 nM) significantly increased Wnt-3a-induced ALP activity, whereas Dkk-2 at high concentration (100 nM) significantly reduced. Treatment of Dkk-2, -3 and -4 at high concentration (100 nM) showed significant decreases of Wnt/beta-catenin transcriptional activity, whereas no effects were seen at low concentration (20 nM). In parallel experiments, treatment of Dkk-1 showed robust dose dependent inhibition not only in ALP activity but also in Wnt/beta-catenin transcriptional activity. Dkk-2, -3 and -4 increased serum deprivation-induced apoptosis in MC3T3-E1 mouse osteoblasts, while Dkk-1 had no effect. CONCLUSIONS: We found that unlike Dkk-1, Dkk-3 and -4 stimulated early osteoblastic differentiation at various concentrations regardless of their inhibitory effects on Wnt/beta-catenin transcriptional activity at high concentration. Dkk-2 had a biphasic effect where the lower doses significantly increased ALP activity while the high dose was inhibitory. Dkk-2, -3 and -4 stimulated osteoblast apoptosis whereas Dkk-1 had no effect.