Planta Med 2011; 77 - PM135
DOI: 10.1055/s-0031-1282893

Is the inhibition of STAT3 phosphorylation in vascular smooth muscle cells by indirubin-3'-monoxime redox-dependent?

T Blazevic 1, AV Schwaiberger 1, CE Schreiner 1, EH Heiss 1, AG Atanasov 1, VM Dirsch 1
  • 1Department of Pharmacognosy, University of Vienna, Althanstraße 14, A-1090 Vienna, Austria

Indirubin is a natural product found in the traditional Chinese antileukemic recipe, Danggui Longhui Wang.1 Its reported anti-proliferative activity makes it a promising candidate in the treatment of cardiovascular diseases (CVDs). We showed recently that the derivative indirubin-3'-monoxime (I3MO) inhibits the proliferation of vascular smooth muscle cells (VSMC) by inhibition of STAT3 phosphorylation.2 The importance of reactive oxygen species (ROS) in STAT3 activation has been reported3 and oxidative stress has been implicated in many CVDs.4

Here, we examine the role of ROS as a putative target of I3MO acting upstream of STAT3. Employing the fluorescence probes 2',7'-dichlorodihydrofluorescein and Amplex RedTM, I3MO was shown to inhibit PDGF-induced intracellular ROS production and H2O2 release. Interestingly, the compound did not exhibit radical scavenging activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Western blot analyses revealed that STAT3 phosphorylation triggered by exposure of VSMC to exogenous H2O2 is also blunted by I3MO. Furthermore, diphenyliodonium (3µM), an inhibitor of NAD(P)H oxidases (Nox) and other flavoproteins, mimicked the selective effect of I3MO on PDGF-induced STAT3 phosphorylation. Selective downregulation of Nox1 and Nox4 isozymes, using the inhibitory peptide, gp91tat and the siRNA approach, respectively, did not prevent PDGF-induced STAT3 phosphorylation.

This study shows for the first time that I3MO reduces PDGF-induced oxidative stress. Additionally, I3MO-mediated STAT3 inhibition, which was linked to its anti-proliferative effect on VSMCs, was shown to be redox-dependent. Despite being considered the major ROS-source in VSMCs, Nox isozymes, Nox1 and Nox4, are not targets of I3MO responsible for the effect on STAT3 phosphorylation.

Keywords: indirubin, vascular smooth muscle cells, proliferation, atherosclerosis, STAT3, PDGF, reactive oxygen species, NAD(P)H oxidase

Acknowledgement: This work was supported by the Austrian Science Foundation (FWF) (P23317 to E.H.H., NFN S107-BO3 to V.M.D. and P 18982 to V.M.D.)

References: 1. Meijer L et al. (1999) Nature Cell Biol 1: 60–7.

2. Schwaiberger AV et al. (2010) ATVB 30: 2475–81.

3. McCormick J et al. (2006) FASEB J 20: 2115–17.

4. Bronas, UG, Dengel DR (2010) Am J Lifestyle Med 4: 521–534.