Abstract
4971
Ecteinascidin 743 (ET-743) is a marine product that exhibits chemotherapeutic activity against liposarcoma and leiomyosarcoma. ET-743 has been suggested to inhibit pregnane xenobiotic receptor (PXR) transcriptional activity based on its ability to down-regulate target genes CYP3A4 and MDR1. Cellular localization of PXR is important for its transcriptional regulatory properties. Translocation of PXR into the nucleus is a determining factor for trans-activation of target genes such as CYP3A4 and MDR1. In this study we investigated the mechanism of PXR antagonism by ET-743 in osteosarcoma and HepG2 cells. The expression of CYP3A4, MDR1, PKC and phospho-PKCα/βII proteins in cells exposed to ET-743 was measured by western blot analysis. The catalytic activity of CYP3A4 was measured using 7-benzyl-4(trifluoromethyl) coumarin as a probe substrate. Cytoxicity was determined using the trypan blue exclusion method after exposure of cells to ET-743, doxorubicin, etoposide or ifosfomide alone or in various combinations. Confocal microscopy was used to determine the effect of ET-743 on the localization of PXR. Our results indicate that there is a significant dose-dependent accumulation of PXR in the cytosol after ET-743 exposure; this accumulation correlated with decreases in the expression levels of expressed CYP3A4 and MDR1. Additionally, ET-743 caused down-regulation of total PKC and PKC α/βII phosphorylated protein. PKC is also known to regulate MDR1 expression. The proteosome inhibitor MG132 partially restored the expression levels of the MDR1 protein. Standard chemotherapeutic agents used for the treatment of sarcoma include doxorubicin, ifosfamide and etoposide. We tested the hypothesis that ET-743 mediated down-regulation of CYP3A4 and MDR1 will sensitize osteosarcoma and HepG2 cells to selected chemotherapeutic agents. The sensitivity of these cells to standard drugs were enhanced following the initial exposure of cells to ET-743 for 24 hrs prior to chemotherapeutic agents. We determined that this synergistic effect correlated best with the dose-dependent accumulation of PXR in the cytoplasm leading to down-regulation of CYP3A4 and MDR1. In conclusion, we have shown for the first time that ET-743 induces the accumulation and localization of PXR in the cytosol which correlates with increased sensitvity of cells to selected agents. Our findings have elucidated a mechanism by which ET-743 may antagonize the PXR transcriptional activation of target genes CYP3A4 and MDR1. These results provide a rationale for further clinical investigation on the role of intra-tumoral PXR localization in drug resistance. Studies are in progress to determine the role of PKC and the ubiquitin pathway in ET-743 antagonism of PXR and down regulation of CYP3A4 and MDR1. This work was supported by the University of Michigan Cancer Center program grant # 004712.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]
