ABSTRACT
Triptolide, a diterpene triepoxide derived from Trypterygium wilfordii, is documented to have antitumor activity in a broad range of solid tumors and leukemia. The mechanisms that are involved in triptolide-mediated apoptosis or growth inhibition in cancer cells are not fully understood. We identified a disintegrin and metalloproteinase 10 (ADAM10) as a novel molecular target of triptolide using affinity chromatography and mass spectrometry. The identification was confirmed by western blot analysis using an anti-ADAM10 antibody. The expression of ADAM10 is enhanced in several tumors including leukemia and is involved in malignant cell growth and cancer progression. ADAM10 is a type 1 transmembrane glycoprotein that cleaves several plasma membrane proteins. We show that triptolide, at concentrations in the nM range, resulted in a significant decrease in ADAM10 expression followed by the appearance of ADAM10 cleaved product. Furthermore, triptolide reduced the viability of monocytic leukemic U937 cells. Triptolide treatment of MCF-7 breast cancer cells expressing ectopic ADAM10 or dominant negative ADAM10 (DN ADAM10) resulted in a decreased expression of ADAM10 with a concomitant increase in ADAM10 cleaved products. Moreover, siRNA-mediated knockdown of ADAM10 mRNA significantly affected the growth of MCF-7 cells. Interestingly, the combination of siRNA-mediated knockdown of ADAM10 mRNA expression and triptolide treatment lead to a further reduction in cell growth. Taken together, we provide evidence that ADAM10 is a novel target of triptolide, presenting a novel strategy to inhibit ADAM10 activity in tumorigenesis.
Subject(s)
ADAM Proteins/antagonists & inhibitors , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Antineoplastic Agents, Alkylating/pharmacology , Breast Neoplasms/drug therapy , Diterpenes/pharmacology , Lymphoma, Large B-Cell, Diffuse/drug therapy , Membrane Proteins/antagonists & inhibitors , Phenanthrenes/pharmacology , Protease Inhibitors/pharmacology , ADAM Proteins/metabolism , ADAM10 Protein , Amyloid Precursor Protein Secretases/metabolism , Apoptosis/drug effects , Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Cell Growth Processes/drug effects , Cell Line, Tumor , Chromatography, Affinity , Epoxy Compounds/pharmacology , Humans , Lymphoma, Large B-Cell, Diffuse/enzymology , Lymphoma, Large B-Cell, Diffuse/pathology , Membrane Proteins/metabolism , U937 CellsABSTRACT
Beta-catenin plays a role in intracellular adhesion and regulating gene expression. The latter role is associated with its oncogenic properties. Phosphorylation of beta-catenin controls its intracellular expression but mechanism/s that regulates the nuclear localization of beta-catenin is unknown. We demonstrate that O-GlcNAc glycosylation (O-GlcNAcylation) of beta-catenin negatively regulates its levels in the nucleus. We show that normal prostate cells (PNT1A) have significantly higher amounts of O-GlcNAcylated beta-catenin compared to prostate cancer (CaP) cells. The total nuclear levels of beta-catenin are higher in the CaP cells than PNT1A but only a minimal fraction of the nuclear beta-catenin in the CaP cells are O-GlcNAcylated. Increasing the levels of O-GlcNAcylated beta-catenin in the CaP cells with PUGNAc (O- (2-acetamido-2-deoxy-d-gluco-pyranosylidene) amino-N-phenylcarbamate) treatment is associated with a progressive decrease in the levels of beta-catenin in the nucleus. TOPFlash reporter assay and mRNA expressions of beta-catenin's target genes indicate that O-GlcNAcylation of beta-catenin results in a decrease in its transcriptional activity. We define a novel modification of beta-catenin that regulates its nuclear localization and transcriptional function.
