Indirubin-3′-Monoxime Blocks Vascular Smooth Muscle Cell Proliferation by Inhibition of Signal Transducer and Activator of Transcription 3 Signaling and Reduces Neointima Formation In Vivo

Abstract

Objective-Our goal was to examine the influence of indirubin-3Ј-monoxime (I3MO), a natural product-derived cyclin-dependent kinase inhibitor, on vascular smooth muscle cell (VSMC) proliferation in vitro, experimentally induced neointima formation in vivo, and related cell signaling pathways. Methods and Results-I3MO dose-dependently inhibited platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation by arresting cells in the G 0 /G 1 phase of the cell cycle as assessed by 5-bromo-2Ј-deoxyuridine incorporation and flow cytometry. PDGF-induced activation of the kinases Akt, Erk1/2, and p38 MAPK was not affected. In contrast, I3MO specifically blocked PDGF-, interferon-␥-, and thrombin-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3). Human endothelial cells (EA.hy926) responded to I3MO with increased endothelial nitric oxide synthase activity as assessed via Key Words: indirubin Ⅲ vascular smooth muscle cell Ⅲ proliferation Ⅲ neointima Ⅲ STAT3 R estenosis is the major factor hampering the beneficial effect of angioplasty and stenting. Vascular smooth muscle cell (VSMC) proliferation, next to local vascular inflammation, is a critical factor in neointima formation and vascular lumen loss during restenosis. Thus, one current strategy to maintain proper vascular function after angioplasty is to inhibit VSMC proliferation by targeting cell cycle regulation, eg, by drug-eluting stents. 2,3 Two products, the rapamycin-eluting Cypher stent and the paclitaxel-eluting Taxus stent, were approved by the US Food and Drug Administration in 2003 and 2004, respectively. Although successfully introduced into the market, there are now concerns about an increased risk of late stent thrombosis. 4 Thus, compounds inhibiting neointima formation with mechanisms differing from those of rapamycin and paclitaxel 5 may have the potential to be efficient with fewer side effects.Indirubin, a red isomer of indigo, is the active ingredient of the traditional Chinese medicinal formulation Danggui Longhui Wan, used against chronic myelocytic leukemia. 6 Enzyme-based in vitro studies have indicated that indirubin and its derivatives are potent inhibitors of the cyclindependent kinases (CDKs) 1, 2, 4, and 5. 6 -9 Furthermore, different indirubin derivatives showed antitumor activity in several human cancer cells. 6,10 -14 Moreover, for indirubin-3Ј-monoxime (I3MO), an in vitro antiinflammatory and anticancer activity had been described on the basis of the inhibition of tumor necrosis factor-induced nuclear factor B activation and the subsequent suppression of antiapoptotic and proproliferative gene expression. 14 The aim of this study was to investigate the potential use of I3MO in the prevention of restenosis. We therefore analyzed a possible effect on platelet-derived growth factor (PDGF)- MethodsAn expanded Methods section can be found in the Supplemental Data, available online at http://atvb.ahajournals.org. Cell CultureVSMCs were isolated from male Sprague-Dawley rat thoracic aortas by enzyma...

An Indirubin Derivative, Indirubin-3′-Monoxime Suppresses Oral Cancer Tumorigenesis through the Downregulation of Survivin

“…I3M has been shown to have a broad spectrum of anti-tumorigenic activities in many human cancer cells [12]. In this study, we confirmed that I3M possesses a number of anti-tumorigenic activities in vitro , and we identified a novel anti-tumorigenic role for I3M in vivo .…”

“…Whereas indirubin itself has poor solubility, a low absorption rate, and significant gastrointestinal toxicity, synthetic indirubin-3′-monoxime (I3M) has better pharmacological properties and reduced toxicity. Furthermore, compared with indirubin, I3M inhibits many additional protein kinases as well as STAT3 signaling, and it has been shown to have anti-proliferative effects in vascular smooth muscle cells [10]–[12]. Recently, Indirubin-3′-oxime also have been reported induces mitochondrial dysfunction and triggers growth inhibition and cell cycle arrest in human neuroblastoma cells [13].…”

12/15-Lipoxygenase Contributes to Platelet-derived Growth Factor-induced Activation of Signal Transducer and Activator of Transcription 3

“…We showed previously that the small molecule indirubin-3′-monoxime (I3MO) inhibits VSMC proliferation induced by PDGF in vitro and reduces vessel narrowing in vivo (1). The antiproliferative effect of I3MO, a derivate of the naturally occurring compound indirubin used in traditional Chinese medicine against cancer (2), was linked with the selective inhibition of signal transducer and activator of transcription 3 (STAT3).…”

“…STAT3 phosphorylation at Tyr 705 and activation seem to be pivotal for VSMC proliferation (3, 4). Other PDGF-activated mitogenic kinases, like Akt and ERK1/2, were not influenced by I3MO (1). How I3MO provokes this selective action has, however, not been resolved.…”

Piperine Congeners as Inhibitors of Vascular Smooth Muscle Cell Proliferation

“…Paclitaxel (taxol) was used as a reference compound, and inhibited the proliferation of VSMC and HUVECtert with IC 50 = 108 nM and 4 nM, respectively [46]. 5-bromo-2′-deoxyuridine (BrdU) incorporation assay: Quiescent VSMCs were pre-treated for 30 min with compound, or vehicle (0.1 % DMSO) as indicated and stimulated with PDGF (20 ng/mL) for 2 h. Then BrdU was added to estimate de novo DNA synthesis in VSMCs [47][48][49]. 22 h later, the BrdU incorporation was quantified according to the manufacturerʼs instructions (Roche Diagnostics).…”