Rottlerin inhibits the nuclear factor κB/Cyclin-D1 cascade in MCF-7 breast cancer cells

Abstract

In the course of a project aimed to clarify the molecular mechanisms by which phorbol 12-myristate 13-acetate (PMA)-activated forms of protein kinase C (PKC) promote growth arrest in an MCF-7 cell line, we found that the PKCδ inhibitor Rottlerin was able by itself to block cell proliferation. In the current study, we investigated further the antiproliferative response to Rottlerin. Western blotting analysis of cytoplasmic/nuclear extracts showed that the drug did not prevent either extracellular signal-regulated kinase (ERK) activation by PMA or Akt phosphorylation, but did interfere with the NFκB activation process (both basal and PMA-stimulated), by lowering the levels of phospho-IκBα and preventing p65 nuclear migration. The growth arrest evoked by Rottlerin was not mediated by cell-cycle inhibitors p21 and p27 but was accompanied by a dramatic fall in the cyclin-D1 protein, the levels of which were not altered by the pan-PKC inhibitor GF 109203X, thus excluding a PKC-mediated mechanism in the Rottlerin effect. The parallel drop in cyclin-D1 mRNA suggested a down-regulation of the gene caused by the inhibition of nuclear factor-kappa B (NFκB), which occurs via a PKC-, Akt-, ERK- and mitochondrial uncoupling-independent mechanism. We provide preliminary evidence that the interference on the NFκB activation process likely occurs at the level of calcium/calmodulin-dependent protein kinase II (CaMKII), a known Rottlerin target. Indeed the drug prevented calcium-induced CaMKII autophosphorylation which, in turn, led to decreased NFκB activation.

Introduction

In the course of a project aimed to clarify the molecular mechanisms by which phorbol ester-activated PKCs promote growth arrest in an MCF-7 breast cancer cell line, we unexpectedly found that the PKCδ inhibitor Rottlerin was able to block the basal proliferation rate, in the absence of evident PKCδ activation.

Rottlerin is a pharmacological inhibitor of PKCs with selectivity for PKCδ (Gschwendt et al., 1994a). In addition to PKCs, Rottlerin inhibits the activity of a number of unrelated kinases, such as Akt/PKB and CaMKs (Davies et al., 2000). CaMKIII (Gschwendt et al., 1994b) and I/II (Cho et al., 2000) activity is suppressed by Rottlerin with potency similar to that of PKCδ. Moreover, Rottlerin has mitochondrial uncoupling properties that cause ATP depletion and inhibition of cellular processes controlled by phosphorylated molecules (Soltoff, 2001). Rottlerin has also been reported to potentiate the effects of antineoplastic drugs through inhibition of ERK and Akt phosphorylation and down-regulation of crucial cell-cycle proteins, such as cyclins and cdks (Jane et al., 2006).

Cyclin-D1 is a crucial player in the progression of mammalian cells through the G1 phase. In a large majority of breast cancer cases, cyclin-D1 is overexpressed and its levels correlate to negative prognosis (Gillett et al., 1994, Kenny et al., 1999). Transcription factors such as Ap-1, Sp-1 and NFκB are known to regulate the cyclin-D1 gene transcription (Guttridge et al., 1999, Nagata et al., 2001), thus the Ras/ERK and the Phosphoinositide kinase-3 (PI3K)/Akt pathways are generally relevant in cyclin-D1 induction. The Ras/ERK pathway promotes cyclin-D1 gene transcription via Ap-1 and Sp-1. In fact, Ap-1 is one major target of the ERK cascade (Balmanno and Cook, 1999), and Sp-1 can be phosphorylated by ERK in two major sites, both critical for Sp-1 transcriptional activity (Milanini-Mongiat et al., 2002). Moreover, the cytoplasmic target of ERK, the p90^rsk1, can activate NFκB by phosphorylation of IκBα on Ser-32 (Schouten et al., 1997). Also Akt activates NFκB, through phosphorylation of IKK on Thr-23 (Ozes et al., 1999, Ouyang et al., 2006).

In addition, NFκB is also a target of CaMKII. In fact PMA-induced activation of NFκB is dependent on CaMKII, and inhibitors of CaMKII, other than Rottlerin (such as KN93), inhibit IκB phosphorylation (Hughes et al., 2001). Moreover, CaMKII is a modulator of NFκB activation in lymphocytes (Ishiguro et al., 2007) and in cardiomyocytes (Kashiwase et al., 2005).

With this rationale in mind, we investigated proliferation and viability of MCF-7 cells after Rottlerin treatment and searched for changes in key molecules potentially involved in the observed cell-cycle arrest, such as cyclin-D1, and the cyclin-dependent kinase inhibitors p21 and p27. The major upstream signalling pathways, such as ERK, Akt and NFκB cascades were also investigated. We provide evidence that Rottlerin blocks MCF-7 cell proliferation through a mechanism PKC-, ERK-, p21/p27- and Akt-independent involving the sequential inhibition of NFκB/cyclin-D1.