Induced-Expression Of Wnt Signaling Pathway Inhibitor FrpHE And DKK-1

Objective Wnt signaling pathway is closely related to tumor, its inappropriate activation involving in episode process of human multicancer, therefore, it may be a new target to restrain or block its inappropriate activation for anticancer therapies. Endogenous antagonist has been found at present to be able to antagonize Wnt signaling pathway, but endogenous regulated mechanism remains yet unclear. This study aims at the expression of Wnt signaling pathway inhibitor FrpHE and DKK-1 induced by ectogenous factor. In the study, we used p53, adriamycin, hydrocamptothecin, cisplatin as the ectogenous factors to detect their capability of regulating wnt signaling pathway as well as its inhabitors FrpHE, and DKK-1.Methods Human hepatocarcinom cells lines (HepSB) were transfected with an ectogenesis p53 (Adp53) to observe the action of p53 on wnt signaling pathway by fluorescence-activated cell sorting (FACS) and RT-PCR.HepG2 and Lovo cell lines (wild p53) were transfected with p53 antisense ODN to detect the expression of wnt signaling pathway and wnt inhibitor FrpHE and DKK-1 after p53 were blocked. In various p53 status cell lines Hep3B,Lovo,U251 cells were treated with chemoagents, FrpHE, DKK-1 gene mRNA expression levels were detected by RT-PCR.Results l.The study revealed that p53 protein expression emerged in more than 96% of Hep3B cells 24h and 48h after Adp53transfection, with its average fluorescent intensity increased by 50 times by FACS.2. The RT-PCR results: (1) FrpHE and DKK-1 were induced by p53. Our results demonstrated obvious increase of mRNA 20h after the Hep3B cells were transfected with ectogenetic Adp53, FrpHE and DKK-1 mRNA, the peak 32h after the transfection and then decline expression (which, however was higher than that in the controls) 36, 40h after the transfection. On the other hand, FrpHE mRNA had the highest expression when FrpHE and DKK-1 was transfected with Adp53 at the dose of 5pfu/cell and the expression began to decline at the dosage of 50pfu/cell even though it was higher than that in the control group, while DKK-1 mRNA had the apparently growing express at the dose of 0.05pfu/cell and then the highest expression at the dose of 50pfu/cell.(2) FrpHE and DKK-1 were down-regulated by blocked normal p53 in tumor cells. The results demonstrated that FrpHE mRNA expression in HepG2 cells was declined in 24h and remained markedly unchanged in 48h and 72h, and it was not expressed in Lovo cells, though. DKK-1 mRNA expressions in HepG2, Lovo cells were declined in 24h and remained markedly unchanged in 48h and 72h in 48h and 72h.(3) FrpHE and DKK-1 were up-regulated by chemoagents. Our study showed that FrpHE mRNA expressions in HepG2 and Hep3B cells were increased compared with the control groups, but nil in Lovo and U251 cells. DKK-1 mRNA expressions in HepG2, Hep3B, Lovo and U251 cells were increased compare with control groups.3. 3 -catenin, the key factor of Wnt signaling pathway was sharply inhibited by p53. The FACS results demonstrated that the expression of P -catenin was declined in Hep3B cells after transfection with Adp53. (3 -catenin expression hoisted but p53 gene expression was declined in HepG2 and Lovo cells after transfection with antisense p53 ODN. p53 expression hoisted in HepG2, Lovo and U251 cells by chemotherapy.Conclusion Wnt signaling pathway inhibitor FrpHE and DKK-1, induced by anticancer p53 and chemoagents like adriamycin, hydroxycamptothecin, cisplatin, antagonized Wnt signaling pathway. No inhibitor FrpHE was expressed in Lovo and U251, indicating it varied with some tumor cells inhibitor FrpHE.