Withaferin A inhibits JAK/STAT3 signaling and induces apoptosis of human renal carcinoma Caki cells

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Abstract

Withaferin A, the active component of Withania somnifera, causes cytotoxicity in a variety of tumor cell lines. In this study, we show that withaferin A inhibits constitutive and IL-6-induced phosphorylation of STAT3 (on Tyr705), but not IFN-γ-induced STAT1 phosphorylation. Withaferin A-induced down-regulation of STAT3 activation is associated with a reduction in Janus-activated kinase 2 (JAK2) activity. Withaferin A also down-regulates the expression of STAT3 regulated genes such as Bcl-xL, Bcl-2, cyclin D1 and survivin. The apoptotic effect of withaferin A in Caki human renal cancer cells was investigated. Withaferin A induced dose-dependent apoptotic cell death in Caki cells, as measured by FACS analysis and PARP cleavage. Furthermore, overexpression of STAT3 attenuated withaferin A-induced apoptosis. Taken together, the present study provides strong evidence that down-regulation of the STAT3 signaling pathway mediates withaferin A-induced apoptosis.

Highlights

Withaferin A inhibits both the constitutive and inducible STAT3 activation. ► Withaferin A effectively inhibits JAK2 phosphorylation. ► Withaferin A suppresses the expression of proteins (survivin, Bcl-2 and Bcl-xL). ► Overexpression of STAT3 attenuated withaferin A-induced apoptosis.

Introduction

Signal transducer and activator of transcription 3 (STAT3), a member of the STAT family, is a key signal transduction protein that mediates the signaling of many cytokines, hormones, growth factors, and oncoproteins [1]. STAT3 is constitutively activated in most human solid tumors. Persistent activation of STAT3 signaling has been demonstrated to influence important processes in tumors such as survival, proliferation, apoptosis, angiogenesis, and invasion [2], [3], [4]. Activated STAT3 binds to consensus sequences (TTCN2–4GAA), known as gamma activated sites (GAS), and modulates the expression of target genes that are involved in various physiological functions including apoptosis (survivin, Bcl-xL, and HSP27), cell cycle regulation (cyclin D1, c-fos, and c-myc), and angiogenesis (VEGF) [5].

Withaferin A, a steroidal lactone purified from the Indian medicinal plant Withania somnifera, exhibits inhibitory effects against several different cancer types [6]. Withaferin A induces apoptosis through reactive oxygen species (ROS) generation [7], Par-4 induction [8] and p38 MAP kinase activation [9]. Withaferin A was also shown to cause FOXO3a- and Bim-dependent apoptosis [10]. In our previous study, we found that withaferin A induced Akt inactivation and endoplasmic reticulum (ER) stress [11], [12]. We also found that the withaferin A sensitized TRAIL-mediated apoptosis through up-regulation of death receptor 5 (DR5) and down-regulation of c-FLIP [13]. However, the cellular and molecular mechanisms underlying withaferin A-induced apoptosis are not fully understood.

In this study, we investigated whether withaferin A could modulate the constitutive and IL-6-inducible STAT3 pathways in Caki cells, leading to the induction of apoptosis. In addition, STAT3 overexpression attenuated withaferin A-induced apoptosis. The present study clearly demonstrates that withaferin A-induced apoptosis is associated, at least in part, with down-regulation of the STAT3 signaling pathway.

Section snippets

Cells and materials

Caki, and MDA-MB 231 were purchased from the American Type Culture Collection (ATCC, Manassas, VA). The cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 20 mM Hepes buffer, and 100 μg/ml gentamicin. Anti-phospho-STAT3 (Tyr705), anti-STAT3, anti-cyclin D1, anti-Bcl-2, anti-Bcl-xL, anti-survivin, anti-PARP, anti-Ref-1 and anti-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-STAT1 (Tyr701), anti-STAT1,

Withaferin A inhibits constitutive STAT3 phosphorylation in Caki cells

We previously reported that withaferin A inhibited expression of Bcl-xL, a target gene of STAT3 [11]. To determine whether withaferin A inhibits STAT3 activation, we incubated Caki cells with various concentrations of withaferin A for 3 h. We examined the activation of STAT3 by Western Blot analysis using an antibody that recognized phosphorylation of STAT at tyrosine 705. As shown in Fig. 1A, withaferin A inhibited constitutive STAT3 phosphorylation of Caki cells in a dose-dependent manner.

Discussion

Withaferin A is reported to have anti-inflammation and anti-tumor properties [17], [18], [19]. However, the molecular mechanism involved in withaferin A-induced apoptosis is not well established. In this study, we investigated the effect of withaferin A on the apoptotic pathway in Caki cells. We provide important evidence, which suggests that withaferin A inhibits STAT3 activation and induces apoptosis in Caki cells. This view is supported by the following findings: (a) withaferin A inhibits

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These authors contributed equally to this work.

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