Indirubin-3'-monoxime inhibits rat vascular smooth muscle cell proliferation induced by plateled-derived growth factor via the Jak/STAT-pathway

Indirubin, a constituent identified in the traditional Chinese antileukaemic recipe Danggui Longhui Wan, and its derivatives have been shown to be potent cyclin-dependent kinase (CDK)-inhibitors in vitro [1]. CDKs as key regulators of cell cycle progression are promising targets in the treatment of vasculoproliferative disorders, for instance atherosclerosis and restenosis [2]. The aim of the study was therefore to investigate the antiproliferative effect of indirubin-3'-monoxime (I3MO) on plateled-derived growth factor (PDGF-BB)-induced rat vascular smooth muscle cell (RVSMC) growth.

Effects on DNA-synthesis were assessed via BrdU-incorporation after 23h of treatment with PDGF-BB (20 ng/mL) and increasing concentrations of I3MO (0.1–10µM). At a concentration of 3µM, the BrdU-positive labeling index was reduced to control level. Cell cycle analysis in the presence of I3MO showed a significant arrest of RVSMCs in the G0/G1 phase after 16h of stimulation with PDGF-BB. Ongoing treatment over 48h led to DNA strand breaks at high concentrations (10µM), as shown by the detection of propidium-iodide stained nuclei with a sub-diploid DNA-content by flow cytometry. Focusing on the involved signaling pathways for these effects, activation of molecules evidentially participating in proliferation was examined. Western blot analysis revealed that the kinases Akt, Erk1/2 and p38 were activated after 10 minutes of stimulation with PDGF-BB, but the effects were not blocked by I3MO at concentrations of 3 and 5µM. Further investigation of influence on the Jak/STAT pathway, however, indicated a significant inhibition of STAT1 and STAT3(Y705) phosphorylation.

These results demonstrate that I3MO inhibits STAT1 and STAT3(Y705) phosphorylation, suggesting that blocking of the Jak/STAT pathway is at least partially responsible for the antiproliferative activity of the compound.

Acknowlegdements: CNRS, Station Biologique, Amyloïds and Cell Division Cycle, Meijer L.

References: 1. Hoessel, R. et al. (1999), Nat. Cell Biol. 1: 60–67. 2. Dzau, V.J. (2002), Nat. Med. 8: 1249–1256.