| The coffeic acid benzene ethyl ester (caffeic acid phenethyl ester, CAPE) is a kind of non-cytotoxicity antitumorigenic substance, selectively inhibit the growth of tumour cells, while does not impact the growth of normal cells. CAPE was first refined from the bee glue, which is the main component accounting for its anti-tumor activity, and now, it can be synthesized artificially.Previous studies have revealed that its anti-tumor function could be attributed to its ability to suppress the abnormal Wnt/β-catenin signaling pathway, modulate the expression of apoptosis-associated genes, induce cell apoptosis and inhibit the transcriptional activity of nuclear factor -κB (nuclear factor-κB, NF-κB).As we know, Wnt/β-catenin signaling pathway is a developmental pathway that has been shown to play a role in embryonic development, whose dysfunction has great influence on the survival of cells. To date, it is quite clear that downregulation of Wnt-signaling pathway is the main cause of Alzheimers syndrome; besides, excessive activation of this pathway is closely related with the occurrence of many human tumors. The general Wnt/β-catenin signaling pathway could be schematically described as: dismission of the Wnt→Frz→Dsh→β-catenin degeneration complex→accumulation ofβ-catenin and translocation to the cell nucleus→β-catenin combination with TCF/LEF→gene expression(e.g. c-myc, cyclinD1, VEGF). Translocation ofβ-catenin-Tcf compound to nucleus could initiate this signaling pathway and cause signaling abnormality, which further activate the expression of downstream genes and thus promote cancer developmet.It has been reported that CAPE could interference withβ-catenin in hepatoma and colon cancers, while it's still unclear whether CAPE works in neuroglioma. So we hypothesized that the function of CAPE in neuroglioma could also be related withβ-catenin, since it is a key gene in the upstream of this pathway.Here, the main purpose of research is to reveal the target of CAPE in this pathway in vivo and in vitro; and to detect whether CAPE could influenceβ-catenin, Tcf-4 and target gene c-myc in Wnt/β-catenin pathway of neuroglioma in vitro and in neuroglia mouse models (in vivo).Immunity fluorescence, real-time RT-PCR and western blot methods were employed to analyze the levels ofβ-catenin, Tcf-4 and target gene c-myc, the VEGF in Wnt/β-catenin pathway in neuroglioma. These items were used to evaluate the efficacy of CAPE in neuroglioma, which will help to find the target of CAPE in this pathway. By constructing recombinant adenovirusβ-catenin and siRNA vectors, we investiged the role ofβ-catenin involved in the process of CAPE inhibiting tumor. This study will help to reveal the molecular mechanism of CAPE against neuroglioma, and finally provides the basic evidence for CAPE's clinical use.In the first part of our study, we have sucessfully constructed recombinant Ad-β-catenin and Ad-β-catenin siRNA vectors, which were subsequently used to enhance or suppress the expression ofβ-catenin in neuroglioma U251 cell line and transplanted tumor in athymic mice. The two recombinant vectors could be efficiently transfected to cell line and remain constant in cells and transplanted tumor by detecting mRNA and protein levels ofβ-catenin.In the following part, we observed first that CAPE could induce cell apoptosis using FCM; moreover, overexpressedβ-catenin was suppressed after treatment with CAPE using real-time quantitative PCR; furthermore, we demonstrated that the expression of Tcf-4, c-myc and VEGF were decreased using western-blot assay. Taken together, these results indicated that CAPE could induce U251 cell apoptosis, and this function could attribute to its role againstβ-catenin. From the preliminary results, we may get the conclusion that CAPE could be therapeutically used to treat the neuroglioma. In third part, in an attempt to further confirm that CAPE is also effective against tumor, U251 cell beared athymic mice was used to test the effect of CAPE. First, we observed that all forms ofβ-catenin could be surpressed by treatment with CAPE in U251 cell-beared mice. The expression ofβ-catenin downstream genes, such as, Tcf-4, c-myc and VEGF was also decreased. Moreove, 48.39% tumor inhibition rate can be achieved (>40% is thought to be effective). These in vivo results confirmed that CAPE could suppress the transplanted tumor load and was effective for the treatment of neuroglioma, which was through the inhibition of the abnormal Wnt/β-catenin pathway.In a word, CAPE could induce U251 cell apoptosis, and from the results demonstrated here, this treatment effect could be attributed to its function of suppressing the expression ofβ-catenin.
|
PDF Full Text Request