Elsevier

Biochemical Pharmacology

Volume 61, Issue 10, 15 May 2001, Pages 1205-1215
Biochemical Pharmacology

Effects of structurally related flavonoids on cell cycle progression of human melanoma cells: regulation of cyclin-dependent kinases CDK2 and CDK11

https://doi.org/10.1016/S0006-2952(01)00583-4Get rights and content

Abstract

We have investigated the effects of a series of flavonoids on cell proliferation and cell cycle distribution in human melanoma cells OCM-1. Among the compounds that potently inhibited OCM-1 cell proliferation, we show that the presence of a hydroxyl group at the 3′-position of the ring B in quercetin and luteolin, correlated to a G1 cell cycle arrest while its absence in kaempferol and apigenin correlated to a G2 block. Genistein with a hydroxyl at 5-position of the ring A arrested cells in G2 while daidzein which lacks it, induced an accumulation of cells in G1. We demonstrate that flavonoids, which induced a cell cycle block in G1, inhibited the activity of CDK2 by 40–60%. By contrast, those which caused an accumulation of cells in G2/M were without effect. On the other hand, while quercetin, daidzein and luteolin did not alter the activity of CDK1, kaempferol, apigenin and genistein inhibited this kinase by 50–70%. We demonstrate that the up-regulation of the CDK inhibitors p27KIP1 and p21CIP1 is likely responsible for the inhibition of CDK2 while inhibition of CDK1 was rather due to the phosphorylation of the kinase on Tyr15 residue.

Introduction

Flavonoids are polyphenolic compounds naturally present in vegetables, fruits and beverages such as tea and wine [1]. People from European countries consume several mg of flavonoids per day in the common diet. A large number of epidemiological studies [2], [3], [4] as well as experiments performed on animal models [5], [6], [7], [8] suggest that these compounds can prevent or inhibit cancer development. They are described as anti-oxydant [9], [10] and anti-angiogenic [7], [11] agents and they have been found to inhibit cell growth in vitro[12], [13].

More than 4,000 different flavonoids have been categorized into flavones, flavonols, flavanones, flavanonols, flavan-3-ols and isoflavones (Fig. 1). They all consist of a benzene ring (A) fused with a pyrone ring (C) that in position 2 or 3 carries a phenyl ring (B) as a substituent. Flavonoids have been shown to inhibit several kinases involved in signal transduction, such as protein kinases C [14], [15], tyrosine kinases [16], [17], PI 3-kinases [15], [18], or S6 kinase [19]. They can also interact with estrogen type II binding sites [20]. However, the precise mechanism responsible for the antitumoral effects of these compounds is not yet clearly understood.

Flavonoids have been found to arrest cell cycle progression either at G1/S or at G2/M boundaries. While flavone, luteolin, or daidzein have been shown to arrest human gastric cancer cells in G1, the isoflavone genistein has been found to block these cells at the G2/M transition [21]. This might be dependent on the flavonoid structure. However, conflicting results have been reported with regard to the stage-specific cell cycle arrest caused by the same compound. For example, quercetin has been shown to block the cell cycle at the G1/S transition in colon and gastric cancer cells as well as in leukemic cells [22], [23], [24]. By contrast, it has been found to cause a G2/M block in breast and laryngeal cancer cell lines or in non-oncogenic fibroblasts [25], [26], [27]. Genistein has been reported to arrest cells in G2 in several cell models [21], [28], [29], [30], [31] while it has been shown to induce both G1 and G2 blocks in BALB/c 3T3 fibroblasts or mouse melanoma cells [32]. Apigenin has been shown to induce a G2/M arrest in neuronal or keratinocyte cell lines [33], [34] while it has been reported to block human diploid fibroblasts in both G1 and G2 [35].

Cell cycle progression is orchestrated by cyclin-dependent kinases (CDKs) whose activity is firstly dependent on association with cyclin subunits [36]. G1 progression and G1/S transition are regulated by CDK4 (and CDK6) which assembles with cyclins D in mid-G1 and CDK2 which combines later with cyclin E. While CDK2 controls S-phase when associated with cyclin A, G2/M transition is regulated by CDK1 in combination with cyclins A and B. Activation of CDKs also needs a complex set of phosphorylations and dephosphorylations on specific residues [36]. In particular, dephosphorylation of CDK1 on Thr14 and Tyr15 residues by the dual-specificity phosphatase CDC25C has been demonstrated to be an absolute requirement for the onset of mitosis [37]. Finally, CDK activity is counterbalanced by a variety of low molecular weight CDK inhibitors (CKIs). Two gene families of mammalian CKIs have been identified, one including p21CIP1 and p27KIP1 and the other one p16INK4A and p15INK4B[38]. The INK4 family specifically binds CDK4 (and CDK6) and inhibits complex formation with cyclins D. The CIP/KIP family seems to function on a broader spectrum of CDKs and inactivates CDK-cyclin complexes by stoechiometric binding.

In this paper, we attempt to establish the possible relationships between the structure of flavonoids and their effect on cell cycle progression in human melanoma cells OCM-1. To our knowledge, such an extensive structure/activity study on a given cell model has never been reported with regard to the cell cycle. In order to define more precisely the mechanism of action of flavonoids, we further investigated their effect on CDK2 and CDK1 activities, shown to control respectively the G1/S and G2/M transitions. We also looked at their ability to up-regulate the CKIs p21CIP1 and p27KIP1 and to alter the phosphorylation state of CDK1.

Section snippets

Cell culture

Human choroidal melanoma cells (spindle-shape OCM-1 cell line) were cultured in RPMI-1640 medium, pH 7.3, supplemented with 100 IU/mL penicillin, 100 μg/mL streptomycin, 2.5 μg/mL amphotericin B, 2 mM l-glutamine, and 5% fetal calf serum (FCS). Cells were grown at 37° under 5% CO2 atmosphere. All culture reagents and media were from Gibco. Culture media were changed every 2–3 days. When they reached confluence, cells were dissociated by 0.05% trypsin-0.02% EDTA and replated at 1:30 dilution.

Cell proliferation measurement

For

Inhibition of OCM-1 cell proliferation by flavonoids

We have investigated the effect of a series of flavonoids on OCM-1 melanoma cell proliferation. The list of the flavonoids was selected among the different chemical classes of this family (Fig. 1), to allow the discovery of potential structure/activity relationships. As shown on Fig. 2, three groups of compounds may be clearly distinguished: a first group of very active compounds (ic50 between 9 and 19 μM) which comprises flavones (luteolin and apigenin), flavonols (quercetin and kaempferol)

Discussion

In the present study, we have established that several structurally related flavonoids were able to strongly inhibit the proliferation of human OCM-1 melanoma cells. We have demonstrated that a ring C with oxo function at position 4 and a C2-C3 double bond was required for maximal anti-proliferative activity. Taxifolin which lacks the C2-C3 double bond and catechin which lacks both the double bond and the C4 oxo group are unable to inhibit cell proliferation. Naringenin and hesperetin which

Acknowledgements

The authors are very indebted to Marie Penary and Brigitte Serres for their excellent technical assistance and to Georges Cassar for the flow cytometry analyses. We acknowledge Dr Maria A Saornil, Valadolid Institute, Spain, for the gift of human choroidal melanoma cells OCM-1. This work was supported by Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Université Paul Sabatier, Région Midi-Pyrénées, Fondation de France and Ligue Nationale

References (52)

  • T Kuzumaki et al.

    Genistein induces p21Cip1/WAF1 expression and blocks the G1 to S phase transition in mouse fibroblast and melanoma cells

    Biochem Biophys Res Commun

    (1998)
  • F Sato et al.

    Apigenin induces morphological differentiation and G2-M arrest in rat neuronal cells

    Biochem Biophys Res Commun

    (1994)
  • M.D Losiewicz et al.

    Potent inhibition of CDC2 kinase activity by the flavonoid L86–8275

    Biochem Biophys Res Commun

    (1994)
  • F Casagrande et al.

    G1 phase arrest by the phosphatidylinositol 3-kinase inhibitor LY294002 is correlated to up-regulation of p27KIP1 and inhibition of G1 CDKs in choroidal melanoma cells

    FEBS Lett

    (1998)
  • F Bai et al.

    Promoter activation and following induction of the p21/WAF1 gene by flavone is involved in G1 phase arrest in A549 lung adenocarcinoma cells

    FEBS Lett

    (1998)
  • K Iwao et al.

    Quercetin inhibited DNA synthesis and induced apoptosis associated with increase in c-fos mRNA level and the upregulation of p21WAF1/CIP1 mRNA and protein expression during liver regeneration after partial hepatectomy

    Biochim Biophys Acta

    (1999)
  • A.I Constantinou et al.

    Genistein induces maturation of cultured human breast cancer cells and prevents tumor growth in nude mice

    Am J Clin Nutr

    (1998)
  • J.M Darbon et al.

    Distinct Chk2 activation pathways are triggered by genistein and DNA-damaging agents in human melanoma cells

    J Biol Chem

    (2000)
  • Middleton EJ, Kandaswami C. The impact of plant flavonoids on mammalian biology: implication for immunity, inflammation...
  • M.J Messina et al.

    Soy intake and cancer riska review of the in vitro and in vivo data

    Nutr Cancer

    (1994)
  • P Knekt et al.

    Dietary flavonoids and the risk of lung cancer and other malignant neoplasms

    Am J Epidemiol

    (1997)
  • A Gescher et al.

    Suppression of tumour development by substances derived from the diet mechanisms and clinical implications

    Br J Clin Pharmacol

    (1998)
  • A.K Verma et al.

    Inhibition of 7,12-dimethylbenz(a)anthracene- and N-nitrosomethylurea-induced rat mammary cancer by dietary flavonol quercetin

    Cancer Res

    (1988)
  • H Wei et al.

    Inhibitory effect of apigenin, a plant flavonoid, on epidermal ornithine decarboxylase and skin tumor promotion in mice

    Cancer Res

    (1990)
  • J.R Zhou et al.

    Inhibition of murine bladder tumorigenesis by soy isoflavones via alterations in the cell cycle, apoptosis, and angiogenesis

    Cancer Res

    (1998)
  • Z.M Shao et al.

    Genistein exerts multiple suppressive effects on human breast carcinoma cells

    Cancer Res

    (1998)
  • Cited by (289)

    View all citing articles on Scopus
    1

    Abbreviations: CDK, cyclin-dependent kinase; CKI, CDK inhibitor; PI 3-kinase, phosphatidylinositol 3-kinase; PKC, protein kinase C; DTT, dithiothreitol; RIPA, radioimmunoprecipitation assay buffer.

    View full text