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