Elsevier

Experimental Cell Research

Volume 319, Issue 7, 15 April 2013, Pages 982-991
Experimental Cell Research

Research Article
Camptothecin inhibits platelet-derived growth factor-BB-induced proliferation of rat aortic vascular smooth muscle cells through inhibition of PI3K/Akt signaling pathway

https://doi.org/10.1016/j.yexcr.2012.12.024Get rights and content

Abstract

The abnormal proliferation of vascular smooth muscle cells (VSMCs) in arterial wall is a major cause of vascular disorders such as atherosclerosis and restenosis after angioplasty. In this study, we investigated not only the inhibitory effects of camptothecin (CPT) on PDGF-BB-induced VSMC proliferation, but also its molecular mechanism of this inhibition. CPT significantly inhibited proliferation with IC50 value of 0.58 μM and the DNA synthesis of PDGF-BB-stimulated VSMCs in a dose-dependent manner (0.5–2 μM ) without any cytotoxicity. CPT induced the cell cycle arrest at G0/G1 phase. Also, CPT decreased the expressions of G0/G1-specific regulatory proteins including cyclin-dependent kinase (CDK)2, cyclin D1 and PCNA in PDGF-BB-stimulated VSMCs. Pre-incubation of VSMCs with CPT significantly inhibited PDGF-BB-induced Akt activation, whereas CPT did not affect PDGF-receptor beta phosphorylation, extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and phospholipase C (PLC)-γ1 phosphorylation in PDGF-BB signaling pathway. Our data showed that CPT pre-treatment inhibited VSMC proliferation, and that the inhibitory effect of CPT was enhanced by LY294002, a PI3K inhibitor, on PDGF-BB-induced VSMC proliferation. In addition, inhibiting the PI3K/Akt pathway by LY294002 significantly enhanced the suppression of PCNA expression and Akt activation by CPT. These results suggest that the anti-proliferative activity of CPT is mediated in part by downregulating the PI3K/Akt signaling pathway.

Highlights

► CPT inhibits proliferation of PDGF-BB-induced VSMC without cytotoxicity. ► CPT arrests the cell cycle in G0/G1 phase by downregulation of cyclin D1 and CDK2. ► CPT significantly attenuates Akt phosphorylation in PDGF-BB signaling pathway. ► LY294002 enhanced the inhibitory effect of CPT on VSMC proliferation. ► Thus, CPT is mediated by downregulating the PI3K/Akt signaling pathway.

Introduction

The vascular smooth muscle cells (VSMCs) are involved in the development of various pathological states such as neointima formation, restenosis and atherosclerosis. VSMCs are in a non-proliferative quiescent state in normal, showing a well differentiated contractile phonotype, however, this phenotype would be lost after vascular injury, shifting to a synthetic phenotype including the entrance into the cell cycle and proliferation [1], [2]. The dysfunctional VSMCs lead to cell migration, expression of chemo-attractants and adhesion molecules, and extracellular matrix modulation as well as abnormal proliferation followed by the occurrence of pathogenic vascular disorders [3], [4].

Platelet-derived growth factor (PDGF), produced from VSMCs, activated macrophages and endothelial cells, plays an essential role in proliferation of VSMCs. Particularly, PDGF-BB among isoforms of PDGF family including PDGF-AA, -BB, -AB, -CC, and -DD for the activation of VSMCs proliferation [5], [6]. PDGF-BB binds to the PDGF receptor (PDGF-R), and then leads to phosphorylation on multiple tyrosine residues of PDGF-R followed by the activation of three major signal transduction pathways; extracellular signal-regulated kinase (ERK) 1/2, phospholipase C (PLC)-γ1, and phosphatidylinositol 3-kinase (PI3K)/Akt pathways [7], [8]. Consequently, up-regulation of PDGF signal pathway can cause the progression and development of cardiovascular diseases such as hypertension and atherosclerosis. Therefore, the modulation of PDGF signaling pathway in VSMCs can be a good pharmacological strategy for the prevention of atherosclerosis.

Camptothecin (CPT) is derived from Camptotheca acuminata, has a planar pentacyclic ring structure, and has been known an anticancer drug in traditional Chinese medicine (Fig. 1). CPT suppresses DNA synthesis during the cell cycle progression through the inhibition of topoisomerase I [9].

In present study, we investigated the anti-proliferative activity of CPT on PDGF-induced primary cultured rat aortic smooth muscle cells by measuring cell counting and [3H]thymidine incorporation into DNA. In addition, we examined the effects of CPT on PDGF-BB-inducible cell cycle progression in rat VSMCs and on early signal transduction through PDGF-Rβ, ERK1/2, Akt, PLC-γ1, and cell cycle regulatory proteins such as cyclins, CDKs, as part of the early G0/G1 interphase transition.

Section snippets

Materials

CPT was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), dissolved in DMSO, and its final concentration was less than 0.05% in culture medium. The cell culture materials were obtained from Gibco-BRL (Rockville, MD, USA), and other chemical reagents were from Sigma Chemical Co. (St. Louis, MO, USA). [3H]thymidine and liquid cocktail were obtained from American Radiolabeled Chemicals (St. Louis, MO, USA) and Packard Bioscience (Shelton, CT, USA), respectively. LY294002 (Sigma-Aldrich Co.)

Inhibitory effects of CPT on proliferation of PDGF-BB-stimulated VSMCs

To determine whether camptothecin (CPT) inhibits PDGF-BB-stimulated VSMC proliferation, we assessed the anti-proliferative effects of CPT by direct cell counting. VSMCs were pre-incubated in the presence of CPT (0.5–2 μM) in serum-depleted medium for 24 h and then stimulated with 25 ng/mL PDGF-BB for 24 h. Pre-treatment with CPT suppressed the PDGF-BB-stimulated cell numbers in a concentration-dependent manner. The inhibition of PDGF-BB-stimulated cell proliferation by CPT at 0.5, 1 and 2 μM was

Discussion

Proliferation of VSMCs plays an essential role in the progression of atherosclerosis lesion [13]. Inhibition of VSMCs proliferation is a major therapeutic strategy for atherosclerosis-related diseases [14], [15]. Topotecan, one of camptothecin (CPT) derivatives, inhibited human aortic smooth muscle cell proliferation, and irinotecan, the other derivative, suppressed neointimal proliferation in hypercholesterolemic rabbit aortas [16], [17]. In the present study, we investigated the

Conflicts of interest

The authors state no conflict of interest.

Acknowledgment

This study was supported by Konkuk University, and the National Research Foundation of Korea [NRF] grant funded by the Korea government [MEST] (MRC, 2010-0029480).

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    These authors equally contributed to this study.

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