RESUMO
We have investigated motility in breast cancer cell lines in association with the expression of Transglutaminase type 2 (TG2) as well as upon the administration of Doxorubicin (Dox), an active cytotoxic agent that is employed in chemotherapy. The exposure of MCF-7 cells to the drug increased TG2 levels, triggering epithelial-mesenchymal transition (EMT), thereby supporting cell motility. The effects of Dox on the movement of MCF-7 cells were counteracted by treatment with NC9, a TG2 inhibitor, which induced morphological changes and also reduced the migration of MDA-MB-231 cells exhibiting high levels of TG2. The physical association of TG2 with the cytoskeletal component vimentin appeared pivotal both in drug-treated MCF-7 and in MDA-MB-231 cells and seemed to be independent of the catalytic activity of TG2. NC9 altered the subcellular distribution of TG2 and, consequently, the co-localization of TG2 with vimentin. Furthermore, NC9 induced a nuclear accumulation of TG2 as a prelude to TG2-dependent gene expression modifications. Since enzyme activity can affect both motility and nuclear functions, targeting of this protein could represent a method to improve therapeutic interventions in breast tumors, particularly those to control progression and to limit drug resistance.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Movimento Celular , Espaço Intracelular/metabolismo , Mesoderma/patologia , Proteína 2 Glutamina gama-Glutamiltransferase/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Forma Celular/efeitos dos fármacos , Citoesqueleto/metabolismo , Doxorrubicina/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Fluorescência , Humanos , Invasividade Neoplásica , Proteína 2 Glutamina gama-Glutamiltransferase/genética , Transcrição Gênica , Vimentina/metabolismoRESUMO
Human tissue transglutaminase (hTG2) is a multifunctional enzyme. It is primarily known for its calcium-dependent transamidation activity that leads to formation of an isopeptide bond between glutamine and lysine residues found on the surface of proteins, but it is also a GTP binding protein. Overexpression and unregulated hTG2 activity have been associated with numerous human diseases, including cancer stem cell survival and metastatic phenotype. Herein, we present a series of targeted covalent inhibitors (TCIs) based on our previously reported Cbz-Lys scaffold. From this structure-activity relationship (SAR) study, novel irreversible inhibitors were identified that block the transamidation activity of hTG2 and allosterically abolish its GTP binding ability with a high degree of selectivity and efficiency (kinact/KI > 105 M-1 min-1). One optimized inhibitor (VA4) was also shown to inhibit epidermal cancer stem cell invasion with an EC50 of 3.9 µM, representing a significant improvement over our previously reported "hit" NC9.