Cancer Letters

Cancer Letters

Volume 298, Issue 2, 8 December 2010, Pages 167-175
Cancer Letters

Luteolin induces G1 arrest in human nasopharyngeal carcinoma cells via the Akt–GSK-3β–Cyclin D1 pathway

https://doi.org/10.1016/j.canlet.2010.07.001Get rights and content

Abstract

Luteolin, a plant flavonoid is known to possess multiple biological activities such as anti-inflammation, anti-allergy, anti-oxidant as well as anti-cancer. At present, the anti-proliferative potential of luteolin has not been fully understood. In this study, we focused on the effect of luteolin on cell cycle regulation in human nasopharyngeal carcinoma (NPC) cells. First, we found that luteolin inhibited cell cycle progression at G1 phase and prevented entry into S phase in a dose- and time-dependent manner. Next, it was found that luteolin treatment led to down-regulation of cyclin D1 via enhanced protein phosphorylation and proteasomal degradation, leading to reduced CDK4/6 activity and suppression of retinoblastoma protein (Rb) phosphorylation, and subsequently inhibition of the transcription factor E2F-1. In search of the molecular mechanisms underlying luteolin-mediated cyclin D1 down-regulation, it was found that luteolin was capable of suppressing Akt phosphorylation and activation, resulting in de-phosphorylation and activation of glycogen synthase kinase-3β (GSK-3β). Activated GSK-3β then targeted cyclin D1, causing phosphorylation of cyclin D1 at Thr286 and subsequent proteasomal degradation. The above findings were reinforced by the fact that luteolin was able to abrogate the effect of insulin on the Akt/GSK-3β/Cyclin D1 pathway, resulting in suppression of insulin-induced cell proliferation. Since Akt is often over-activated in many human cancers including NPC, it is thus believed that data from this study support the potential application of luteolin as a chemotherapeutic or chemopreventive agent in human cancer.

Introduction

Flavonoids are polyphenolic compounds ubiquitously present in plants including fruits and vegetables. There is growing evidence and interest in the health benefits of flavonoids due to their biological activities such as anti-oxidant, anti-inflammatory and anti-cancer [1], [2]. Among these activities, the anti-cancer effect of flavonoids has been extensively studied [3], [4]. Many types of dietary flavonoids are able to inhibit cancer cell proliferation, induce cancer cell death by apoptosis and cell cycle arrest by targeting key intracellular molecules and pathways [5], [6]. For instance, the anti-proliferative activity of flavonoids on tumor cell growth has been linked to their effects on numerous intracellular biochemical pathways including the cyclins – cyclin-dependent kinases (CDKs) network [7].

Cyclins are essential components of the cell cycle machinery; each binds and activates specific types of cyclin-dependent kinases (CDKs). Progression through the G1 phase of the cell cycle requires both cyclin D and cyclin E to activate CDK4/6 and CDK2, respectively [8]. The cyclin D1–CDK4/6 complexes formed during G1 phase phosphorylate retinoblastoma (Rb) protein and activate the transcriptional factor E2F-1 which initiates the transcription of key cell cycle regulators such as cyclins E and A and in the process, driving cells into the S phase [9], [10]. Therefore, it has been well established that cyclin D plays a crucial role in the progression of cell cycle from G1 to S phase and the down-regulation of cyclin D will lead to cell cycle arrest at G1 [11], [12].

The phosphoinositide 3-kinase (PI3K)/Akt pathway is known to play a major role in cell cycle progression during the G1/S transition [13]. Amongst various substrates of Akt, several of them are involved in cell cycle regulation, including GSK (glycogen synthase kinase)-3β, the forkhead transcription factors, CDK inhibitors p21WAF1 and p27KIP1[14]. Akt is capable of phosphorylating GSK-3β at Ser9 and subsequently inhibiting its kinase activity. Active GSK-3β phosphorylates cyclin D1 at Thr286 that triggers its subsequent ubiquitination and degradation by proteasomes [15], [16]. Therefore, the Akt–GSK-3β–Cyclin D1 signaling pathway appears to be crucial in regulating the cell cycle at G1/S transition.

Luteolin (3′,4′,5′,7′-tetrahydroxyflavone), a member of the flavonoid family which usually exists in the glycosylated forms, is commonly found in celery, green peppers, perilla leaf, camomile and chrysanthemum tea [17]. It exhibits a wide spectrum of pharmacologic properties ranging from anti-cancer, anti-oxidant, anti-inflammatory and anti-allergic properties [18], [19], [20]. At present, the anti-cancer property of luteolin has been evaluated mainly on its ability to induce apoptosis [19]. For instance, luteolin is capable of directly inducing apoptotic cell death in numerous human cancer cells [21], [22], [23], [24], [25], [26] and sensitizing cancer cells to chemotherapeutics or biotherapeutic agents [27], [28], [29], [30], [31]. However, relatively little is known about the anti-proliferative activity of luteolin. Thus, in this study, we focused on the effect of luteolin on cell cycle regulation. Data from this study demonstrate that luteolin induces G1 arrest in human nasopharyngeal carcinoma cells by down-regulating cyclin D1, which subsequently leads to suppression of the E2F-1 transcriptional activity. We further identified the molecular mechanism in which luteolin down-regulates cyclin D1 through the inhibition of the Akt–GSK-3β signaling pathway. Data from this study thus expand the spectrum of the anti-cancer potential of luteolin and support its potential application in cancer prevention and therapy.

Section snippets

Chemicals and reagents

Luteolin, insulin, lithium chloride (LiCl), DMSO, camptothecin, MG132 as well as other common chemicals were purchased from Sigma (St. Louis, MO, USA). Cycloheximide (CHX), anti-cyclin D1 and anti-α-tubulin were purchased from Santa Cruz (Santa Cruz, CA, USA). Anti-cyclin A, anti-cyclin E, anti-Rb, anti-pRb Ser780, anti-Akt, anti-pAkt Ser473, anti-pCyclin D1 Thr286, anti-GSK-3β, anti-pGSK-3β Ser9, anti-ubiquitin, horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse secondary

Luteolin induces cell cycle arrest at G1 phase in a dose- and time-dependent manner

The effects of luteolin on the cell cycle progression in two nasopharyngeal carcinoma cell lines, HK1 and CNE2, were determined by flow cytometry with anti-BrdU–FITC and 7-AAD staining. In HK1 cells, treatment with various concentrations of luteolin for 24 h resulted in a dose-dependent increase in the percentage of cells in G0/G1 phase and a concomitant reduction of cell numbers in S phase (Fig. 1A, upper panel). Higher concentrations of luteolin (50 and 100 μM) almost completely abolished the S

Discussion

At present, the anti-cancer potential of luteolin is mainly based on its ability to induce apoptosis in cancer cells [19]. However, relatively little is known about the effect of luteolin on cell cycle regulation. Several earlier reports have found that luteolin induces cell cycle arrest either at G1 by down-regulating cellular protein levels of CDK4 and CDK2 [33], [34], or G2/M arrest by the inhibition of cdc2 and up-regulation of p21CIP1[30]. In the present study, we identified the molecular

Conflicts of interest

The authors would like to state that there are no conflicts of interest related to this work.

Acknowledgements

This study was supported by research grants from the National Medical Research Council (NMRC) and Biomedical Research Council (BMRC) to H.M. Shen and the Singapore Polytechnic R&D Funding to C.S. Ong. J. Zhou is supported by a research scholarship by the National University of Singapore.

References (46)

  • Y. Li et al.

    Recent advance in the research of flavonoids as anticancer agents

    Mini Rev. Med. Chem.

    (2007)
  • A. Kale et al.

    Cancer phytotherapeutics: role for flavonoids at the cellular level

    Phytother. Res.

    (2008)
  • R.P. Singh et al.

    Natural flavonoids targeting deregulated cell cycle progression in cancer cells

    Curr. Drug Targets

    (2006)
  • A.J. Obaya et al.

    Regulation of cyclin-Cdk activity in mammalian cells

    Cell Mol. Life Sci.

    (2002)
  • C. Giacinti et al.

    RB and cell cycle progression

    Oncogene

    (2006)
  • C. Genovese et al.

    Cell cycle control and beyond: emerging roles for the retinoblastoma gene family

    Oncogene

    (2006)
  • M. Malumbres et al.

    Cell cycle, CDKs and cancer: a changing paradigm

    Nat. Rev. Cancer

    (2009)
  • S.W. Blain

    Switching cyclin D-Cdk4 kinase activity on and off

    Cell Cycle

    (2008)
  • J. Liang et al.

    Multiple roles of the PI3K/PKB (Akt) pathway in cell cycle progression

    Cell Cycle

    (2003)
  • P. Blume-Jensen et al.

    Oncogenic kinase signalling

    Nature

    (2001)
  • J.A. Diehl et al.

    Glycogen synthase kinase-3β regulates cyclin D1 proteolysis and subcellular localization

    Genes Dev.

    (1998)
  • J.A. Diehl et al.

    Inhibition of cyclin D1 phosphorylation on threonine-286 prevents its rapid degradation via the ubiquitin–proteasome pathway

    Genes Dev.

    (1997)
  • M. Lopez-Lazaro

    Distribution and biological activities of the flavonoid luteolin

    Mini Rev. Med. Chem.

    (2009)
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