We have recently proposed a macromolecular prodrug strategy for improved cancer chemotherapy based on two features (Kratz, F.; et al. J. Med. Chem 2000, 43, 1253-1256.): (a) rapid and selective binding of thiol-reactive prodrugs to the cysteine-34 position of endogenous albumin after intravenous administration and (b) release of the albumin-bound drug in the acidic environment at the tumor site due to the incorporation of an acid-sensitive bond between the drug and the carrier. To investigate this therapeutic strategy in greater depth, four (maleinimidoalkanoyl)hydrazone derivatives of doxorubicin were synthesized differing in the length of the aliphatic spacer (1, -(CH(2))(2)-; 2, -(CH(2))(3)-; 3, -(CH(2))(5)-; 4, -(CH(2))(7)-). The albumin-binding doxorubicin prodrugs, especially the (6-maleimidocaproyl)hydrazone derivative of doxorubicin (3), are rapidly and selectively bound to the cysteine-34 position of endogenous albumin. 3 was distinctly superior to the parent compound doxorubicin in three animal tumor models (RENCA, MDA-MB 435, and MCF-7) with respect to antitumor efficacy and toxicity.
The tumour suppressor HIPK2 is an important regulator of cell death induced by DNA damage, but how its activity is regulated remains largely unclear. Here we demonstrate that HIPK2 is an unstable protein that colocalizes and interacts with the E3 ubiquitin ligase Siah-1 in unstressed cells. Siah-1 knockdown increases HIPK2 stability and steady-state levels, whereas Siah-1 expression facilitates HIPK2 polyubiquitination, degradation and thereby inactivation. During recovery from sublethal DNA damage, HIPK2, which is stabilized on DNA damage, is degraded through a Siah-1-dependent, p53-controlled pathway. Downregulation of Siah-1 inhibits HIPK2 degradation and recovery from damage, driving the cells into apoptosis. We have also demonstrated that DNA damage triggers disruption of the HIPK2-Siah-1 complex, resulting in HIPK2 stabilization and activation. Disruption of the HIPK2-Siah-1 complex is mediated by the ATM/ATR pathway and involves ATM/ATR-dependent phosphorylation of Siah-1 at Ser 19. Our results provide a molecular framework for HIPK2 regulation in unstressed and damaged cells.
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