5 '-Nitro-indirubinoxime induces G1 cell cycle arrest and apoptosis in salivary gland adenocarcinoma cells through the inhibition of Notch-1 signaling

Abstract

Background: 5'-Nitro-indirubinoxime (5'-NIO) is a new derivative of indirubin that exhibits anti-cancer activity in a variety of human cancer cells. However, its mechanism has not been fully clarified. Methods: Human salivary gland adenocarcinoma (SGT) cells were used in this study. Western blot and RTPCR analyses were performed to determine cellular Notch levels. The cell cycle stage and level of apoptosis were analyzed using flow cytometry analysis. Results: 5'-NIO significantly inhibited the mRNA levels of Notch-1 and Notch-3 and their ligands (Delta1, 2, 3, and Jagged-2) in SGT cells. Immunocytochemistry analysis showed that 5'-NIO specifically decreased the level of Notch-1 in the nucleus. In addition, 5'-NIO induced G1 cell cycle arrest by reducing levels of CDK4 and CDK6 in SGT cells. Using flow cytometry and immunoblotting analysis, we found that 5'-NIO induces apoptosis following the secretion of cytochrome c and the activation of caspase-3 and caspase-7. Intracellular Notch-1 overexpression led to a decrease in Cl phase arrest and an inhibition of 5'-NIO-induced apoptosis. Conclusion: These observations suggest that 5'-NIO induces cell cycle arrest and apoptosis by down-regulating Notch-1 signaling. General significance: This study identifies a new mechanism of 5'-NIO-mediated anti-tumor properties. Thus, 5'-NIO could be used as a candidate for salivary gland adenocarcinoma therapeutics. (C) 2009 Elsevier B.V. All rights reserved.