Docosahexaenoic acid inhibits cancer cell growth via p27Kip1, CDK2, ERK1/ERK2, and retinoblastoma phosphorylation

Abstract

Docosahexaenoic acid (DHA), a PUFA of the n-3 family, inhibited the growth of FM3A mouse mammary cancer cells by arresting their progression from the late-G1 to the S phase of the cell cycle. DHA upregulated p27Kip1 levels by inhibiting phosphorylation of mitogen-activated protein (MAP) kinases, i.e., ERK1/ERK2. Indeed, inhibition of ERK1/ERK2 phosphorylation by DHA, U0126 [chemical MAPK extracellularly signal-regulated kinase kinase (MEK) inhibitor], and MEKSA (cells expressing dominant negative constructs of MEK) resulted in the accumulation of p27Kip1. MAP kinase (MAPK) inhibition by DHA did not increase p27Kip1 mRNA levels. Rather, this fatty acid stabilized p27Kip1 contents and inhibited MAPK-dependent proteasomal degradation of this protein. DHA also diminished cyclin E phosphorylation, cyclin-dependent kinase-2 (CDK2) activity, and phosphorylation of retinoblastoma protein in these cells. Our study shows that DHA arrests cell growth by modulating the phosphorylation of cell cycle-related proteins. Docosahexaenoic acid (DHA), a PUFA of the n-3 family, inhibited the growth of FM3A mouse mammary cancer cells by arresting their progression from the late-G1 to the S phase of the cell cycle. DHA upregulated p27Kip1 levels by inhibiting phosphorylation of mitogen-activated protein (MAP) kinases, i.e., ERK1/ERK2. Indeed, inhibition of ERK1/ERK2 phosphorylation by DHA, U0126 [chemical MAPK extracellularly signal-regulated kinase kinase (MEK) inhibitor], and MEKSA (cells expressing dominant negative constructs of MEK) resulted in the accumulation of p27Kip1. MAP kinase (MAPK) inhibition by DHA did not increase p27Kip1 mRNA levels. Rather, this fatty acid stabilized p27Kip1 contents and inhibited MAPK-dependent proteasomal degradation of this protein. DHA also diminished cyclin E phosphorylation, cyclin-dependent kinase-2 (CDK2) activity, and phosphorylation of retinoblastoma protein in these cells. Our study shows that DHA arrests cell growth by modulating the phosphorylation of cell cycle-related proteins. Abbreviations AAarachidonic acidCDKcyclin-dependent kinaseDHAdocosahexaenoic acidEPAeicosapentaenoic acidES mediumessential seiyaku mediumFCSfetal calf serumMAPmitogen-activated proteinMAPKMAP kinaseMEKMAPK extracellularly signal-regulated kinase kinasePMAphorbol-12-myristate-13-acetatepRbretinoblastoma protein arachidonic acid cyclin-dependent kinase docosahexaenoic acid eicosapentaenoic acid essential seiyaku medium fetal calf serum mitogen-activated protein MAP kinase MAPK extracellularly signal-regulated kinase kinase phorbol-12-myristate-13-acetate retinoblastoma protein During the recent past, there has been an upsurge of information on the role of PUFAs in health and disease (1German J.B. Roberts M.A. Watkins S.M. 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Cell cycle arrest and apoptosis of melanoma cells by docosahexaenoic acid: association with decreased pRb phosphorylation..Cancer Res. 2000; 60: 4139-4145PubMed Google Scholar). The G1 phase of the cell cycle, regulated by stimulatory and inhibitory growth factors, is the most important step with respect to the control of cell proliferation (16Sherr C.J. Cancer cell cycles..Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4961) Google Scholar, 17Sherr C.J. G1 phase progression: cycling on cue..Cell. 1994; 79: 551-555Abstract Full Text PDF PubMed Scopus (2586) Google Scholar). In normal growth-stimulated cells, the levels of cyclin D and cyclin E are increased, respectively, in the mid-G1 and late-G1 phases of the cell cycle (16Sherr C.J. Cancer cell cycles..Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4961) Google Scholar, 17Sherr C.J. G1 phase progression: cycling on cue..Cell. 1994; 79: 551-555Abstract Full Text PDF PubMed Scopus (2586) Google Scholar). Cyclin D and cyclin E associate, respectively, with cyclin-dependent kinase 4/6 (CDK4/6) and CDK2 to form active kinase complexes (16Sherr C.J. Cancer cell cycles..Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4961) Google Scholar). The activity of CDKs is regulated by their specific dephosphorylation and phosphorylation, and by interactions with distinct CDK inhibitors like Cip and Kip family proteins, which inhibit, respectively, cyclin D/CDK4/6 and cyclin E/CDK2 (18Xiong Y. Hannon G.J. Zhang H. Casso D. Kobayashi R. Beach D. p21 is a universal inhibitor of cyclin kinases..Nature. 1993; 366: 701-704Crossref PubMed Scopus (3172) Google Scholar, 19Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases..Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5230) Google Scholar). The activities of cyclin D/CDK4/6 and cyclin E/CDK2 complexes reach their maximum, respectively, in mid-to-late-G1 and late-G1-to-S-phase transition (20Dulic V. Lees E. Reed S.I. Association of human cyclin E with a periodic G1-S phase protein kinase..Science. 1992; 257: 1958-1961Crossref PubMed Scopus (794) Google Scholar). Cyclin D/CDK4/6 and cyclin E/CDK2 complexes phosphorylate retinoblastoma protein (pRb) (21Sheaff R.J. Groudine M. Gordon M. Roberts J.M. Clurman B.E. Cyclin E-CDK2 is a regulator of p27Kip1..Genes Dev. 1997; 11: 1464-1478Crossref PubMed Scopus (791) Google Scholar, 22Zhao J. Dynlacht B. Imai T. Hori T. Harlow E. Expression of NPAT, a novel substrate of cyclin E-CDK2, promotes S-phase entry..Genes Dev. 1998; 12: 456-461Crossref PubMed Scopus (183) Google Scholar). In quiescent cells, pRb is in a hypophosphorylated active form, but after growth stimulation, it starts to become phosphorylated in the mid-G1 phase, and maximal phosphorylation occurs at the late-G1-to-S-phase junction (22Zhao J. Dynlacht B. Imai T. Hori T. Harlow E. Expression of NPAT, a novel substrate of cyclin E-CDK2, promotes S-phase entry..Genes Dev. 1998; 12: 456-461Crossref PubMed Scopus (183) Google Scholar). The increased phosphorylation of pRb leads to its functional inactivation, resulting in the release of S-phase-specific transcription factors that are bound to and sequestered by unphosphorylated pRb during the G1 phase of the cell cycle (23Hengst L. Reed S.I. Translational control of p27Kip1 accumulation during the cell cycle..Science. 1996; 271: 1861-1864Crossref PubMed Scopus (821) Google Scholar). Nevertheless, there is a lack of information on the mechanism of action of these fatty acids in the modulation of cancer cell proliferation. Therefore, the present study was designed to shed light on the role of DHA, the terminal molecule of n-3 PUFA metabolism, in the modulation of mouse mammary FM3A cancer cell growth. Essential seiyaku (ES) medium was procured from Nissui Pharmaceutical Co. (Tokyo, Japan). Unless otherwise stated, all other chemicals, including DHA (22:6n-3) and arachidonic acid (AA, 20:4n-6) were purchased from Sigma (St. Louis, MO). Anti-cyclin D1, anti-cyclin E, anti-p27Kip1, and CDK2 were purchased from Santa Cruz Biotechnology. FUT-175 was kindly supplied by Torii Pharmaceutical Co. (Tokyo, Japan). Anti-phosphorylated anti-total mitogen-activated protein (MAP) kinase (MAPK) (ERK1/ERK2) antibodies were obtained from Cell Signaling. Anti-hypoRb and anti-pRb antibodies were procured from Pharmingen. The dominant negative (S222A) hemagglutinin-1-tagged MAPK extracellularly signal-regulated kinase kinase-1 [MEK-1 (MEKSA)] and constitutively active (S218D/S222D) MEK-1 (MEKDD) constructs were kindly provided by Alain Eychène (Orsay, France) and Stéphane Manenti (Toulouse, France). They were cloned into the pcDNA3 expression vector between the HindIII and XbaI into cells constructs were by and to the of the mammary FM3A cancer cells were in medium with and fetal calf serum at in a containing and as by and D. K. T. of in of mouse FM3A cells with to and Cell PubMed Scopus Google Scholar). Cell were by DHA, in at a of was in to its The cells were for and in the in the medium containing with or DHA at the in the was to cell and to cell mouse mammary FM3A cancer cells were and at in and was France). cell of was with of which was with of The was by to the cells. cells all control and cells were for The control cells were with pcDNA3 The cells were for and for in medium containing with or the cells were in for the cell cycle studies by The of the cell cycle progression was to the of M. D. G. J. of and by to in Google Scholar) by the cell with the of the cells were with the cell the different stages of the cell cycle were from the the provided by the was the to the The of the was by and by the of was with to the was on the and was for p27Kip1 mRNA were as The of the for were as The was in a of containing and containing of and was at an of were by including and cycle The cells were for and in medium containing with or DHA The cells were for as in the the cells were with of containing the in of cell were or at was by the of R.J. with the Full Text PDF PubMed Google Scholar). were by and to were by with at was by anti-p27Kip1, anti-total anti-cyclin D1, anti-cyclin E, and the with antibodies for anti-p27Kip1, for and for all other activity was with France). The cell for were by the with protein by CDK2 kinase were by the phosphorylation of as the of T. T. Y. M. H. Y. acid and docosahexaenoic acid inhibit cell proliferation by inhibiting phosphorylation of Res. PubMed Scopus Google Scholar). The was with and with protein The kinase were at for in the and was by and The hypophosphorylated pRb was by the of to pRb by as by W. arrest in a role for docosahexaenoic J. 2003; PubMed Scopus Google Scholar). In the cells were for and in medium containing with or DHA for different as in the The cells were with for and with in for at were with and in of and for at The cells were and in of in on for cells were with two of which the hypophosphorylated and which pRb protein. of the of to pRb was a are shown as for at of was with France). The of the between was by by a the effects of DHA on cell of this that DHA at and inhibitory effects on the proliferation of FM3A cells is that an n-6 to inhibit cell growth at and DHA at this is the that at a of this fatty acid cell not the effects of DHA were were to this fatty acid its effects on cell cycle cell cycle shows that the of cells in the S phase of the cell cycle was increased in cells. the other DHA of these cells decreased the of cells in the S phase and increased in the phase of the cell cycle. study the for cell cycle the to a role in the phosphorylation of pRb were In p27Kip1 leads to the phosphorylation of pRb and the release of transcription shows that quiescent cells p27Kip1 these cells not a of phosphorylated ERK1/ERK2. of cell proliferation by serum is with p27Kip1 and MAPK of DHA to cells diminished MAPK phosphorylation and upregulated p27Kip1 levels in FM3A cells shows that a MAPK also inhibited of p27Kip1 by inhibiting MAPK is to MAPK of protein kinase V. MAP kinase phosphorylation and inhibitory action of docosahexaenoic 2004; PubMed Scopus Google Scholar). DHA inhibited the of p27Kip1 by inhibiting ERK1/ERK2 phosphorylation in these cells DHA did not an accumulation of p27Kip1 levels in the of In to that MAPK is in the of p27Kip1 FM3A cells with (MEKDD) and dominant negative shows that MAPK phosphorylation in cells diminished p27Kip1 whereas cells expressing MEKSA diminished phosphorylation and increased p27Kip1 levels. of DHA to cells to MAPK phosphorylation and to p27Kip1 levels. In these DHA was at a that is to MAPK DHA was to p27Kip1 levels in FM3A cells, were to this fatty acid its mRNA levels. of cell proliferation increased p27Kip1 mRNA levels for after which levels not cells containing DHA and U0126 did not of p27Kip1 mRNA the cells These observations that inhibition of MAPK phosphorylation not increase p27Kip1 mRNA this an accumulation of p27Kip1 protein as shown in are of p27Kip1 the is the degradation by (18Xiong Y. Hannon G.J. Zhang H. Casso D. Kobayashi R. Beach D. p21 is a universal inhibitor of cyclin kinases..Nature. 1993; 366: 701-704Crossref PubMed Scopus (3172) Google Scholar, 19Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases..Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5230) Google Scholar). a inhibitor of the to inhibit p27Kip1 shows that of FM3A cells with p27Kip1 the cells were with and or DHA, increase in p27Kip1 was the effects of DHA on p27Kip1 the of p27Kip1 in the of the protein synthesis inhibitor the of the p27Kip1 protein. shows that p27Kip1 is in in the cells. in which the control cells, after of were with or with U0126 or of cell were in to the effects of these on p27Kip1 The of p27Kip1 levels that DHA and inhibition by U0126 the degradation of p27Kip1 protein levels in FM3A cells The degradation of p27Kip1 occurs after phosphorylation by CDK2 on Cyclin E is an of and its accumulation is to CDK2 in the late-G1 phase of the cell cycle. phosphorylated CDK2 and its kinase CDK2 protein was increased by serum in FM3A cells, and DHA diminished the phosphorylated form of CDK2 DHA diminished the CDK2 kinase activity in FM3A cells DHA to the expression of cyclin in cells to other of cyclin D in these cells not The phosphorylation of cyclin E was during of FM3A cells, whereas DHA the of the phosphorylation of pRb is for the progression the cell cycle, the of phosphorylation of In cells, pRb in the hypophosphorylated after of stimulation, of which to the form and for not shows that DHA of pRb in FM3A cells. The present study was to the effects of DHA on FM3A mouse mammary cancer cell proliferation. FM3A cells a for cell not to apoptosis T. Y. K. M. Y. Y. of cell by in two of the mouse mammary FM3A cell Res. 1998; PubMed Scopus Google Scholar). In the present DHA, this is the terminal molecule of the n-3 PUFA fatty acids are with fatty after their are and their effects are different from in fatty acid to the effects of DHA, the of its with a of by fetal calf serum at of has also been by other A.P. Juan G. Traganos F. Reinhart L. Connolly J. Rose D.P. Darzynkiewicz Z. Cell cycle arrest and apoptosis of melanoma cells by docosahexaenoic acid: association with decreased pRb phosphorylation..Cancer Res. 2000; 60: 4139-4145PubMed Google Scholar). that DHA inhibited FM3A cell growth at a i.e., the other a PUFA of the n-6 family, did not exert effects at this in FM3A cells. Our observations on the inhibition of cell proliferation are in with the of other (6Singh N.P. Lai H.C. Artemisinin induces apoptosis in human cancer cells..Anticancer Res. 2004; 24: 2277-2280PubMed Google Scholar, 7Heller A.R. Rossel T. Gottschlich B. Tiebel O. Menschikowski M. Litz R.J. Zimmermann T. Koch T. Omega-3 fatty acids improve liver and pancreas function in postoperative cancer patients..Int. J. Cancer. 2004; 111: 611-616Crossref PubMed Scopus (155) Google Scholar, 8Simopoulos A.P. The traditional diet of Greece and cancer..Eur. J. Cancer Prev. 2004; 13: 219-230Crossref PubMed Scopus (86) Google Scholar, H. M. D. Y. J. acid and docosahexaenoic acid mitogen-activated protein kinase activity in PubMed Scopus Google Scholar) have shown that this fatty acid effects on cancer cells. inhibition of cell growth is to its specific action on the cell cycle, cell cycle that there were cells in the S phase of the cell cycle in cells in the cells. the of cells in the phase of the cell cycle was in cells in the cells. Our observations on the specific effects of DHA on the inhibition of cell cycle progression the of A.P. Juan G. Traganos F. Reinhart L. Connolly J. Rose D.P. Darzynkiewicz Z. Cell cycle arrest and apoptosis of melanoma cells by docosahexaenoic acid: association with decreased pRb phosphorylation..Cancer Res. 2000; 60: 4139-4145PubMed Google Scholar), have demonstrated that DHA the growth of human melanoma cells by inhibiting the of these cells into the S phase of the cell cycle. T. T. Y. M. H. Y. acid and docosahexaenoic acid inhibit cell proliferation by inhibiting phosphorylation of Res. PubMed Scopus Google Scholar) have also that DHA and inhibit the proliferation of cells by inhibiting the progression of the cell cycle. has been shown that the of p27Kip1 in the late-G1 phase of the cell cycle S. K. of the cell cycle at the G1-S transition by of cyclin E and Res. PubMed Scopus Google Scholar). that p27Kip1 was in cells, whereas DHA this Manenti S. J.M. The mitogen-activated protein kinase p27Kip1 degradation of E in Full Text Full Text PDF Scopus Google Scholar) have shown that ERK1/ERK2 phosphorylation induces the degradation of p27Kip1 levels protein synthesis in melanoma cells. have demonstrated that DHA phosphorylation of ERK1/ERK2 V. MAP kinase phosphorylation and inhibitory action of docosahexaenoic 2004; PubMed Scopus Google Scholar, acid and docosahexaenoic acid MAP kinase (ERK1/ERK2) in human Res. Full Text Full Text PDF Google Scholar) and proliferation of S. M. containing and fatty acids to and MAP kinase 2004; Full Text Full Text PDF PubMed Scopus (38) Google Scholar) and cells B. Docosahexaenoic acid of signal-regulated and in PubMed Scopus Google Scholar). L.E. C.J. of fatty growth inhibition in human breast cancer Cancer Res. 2005; PubMed Scopus Google Scholar) and H. M. D. Y. J. acid and docosahexaenoic acid mitogen-activated protein kinase activity in PubMed Scopus Google Scholar) have also shown that DHA and other n-3 fatty acids cell growth by with ERK1/ERK2 and inhibition of MAPK phosphorylation is for the of a inhibitor of and a dominant negative MAPK phosphorylation by and a constitutively active that and phosphorylation of ERK1/ERK2 p27Kip1 levels. U0126 and MEKSA inhibited MAPK phosphorylation and upregulated p27Kip1 levels in these cells. DHA inhibited and MAPK phosphorylation and of p27Kip1 levels in FM3A cells. DHA to an accumulation of p27Kip1 in the of the inhibitor DHA and the inhibitor increase p27Kip1 protein by their at the mRNA the mRNA expression in these cells. that DHA and U0126 also not did not the expression of p27Kip1 DHA did not an in the of the inhibitor U0126 on expression of p27Kip1 These observations that the expression of p27Kip1 by DHA and these of the i.e., and have been to in other cells, on cell Manenti S. J.M. The mitogen-activated protein kinase p27Kip1 degradation of E in Full Text Full Text PDF Scopus Google Scholar, S. M. inhibition of cancer cell cycle arrest expression of Res. 2005; PubMed Scopus Google Scholar). of p27Kip1 is a in cell that a S. M. inhibition of cancer cell cycle arrest expression of Res. 2005; PubMed Scopus Google Scholar). DHA the degradation of the p27Kip1 the Indeed, inhibited the degradation of p27Kip1 in FM3A cells. The DHA and inhibitor U0126 did not an accumulation of p27Kip1 levels in the of These observations that of p27Kip1 is the of the of p27Kip1 degradation by this fatty acid and that the in this there are also other of p27Kip1 as E. M. is for degradation of the CDK inhibitor Cell PubMed Scopus Google Scholar), at the of a as did not during the of and N. S. J.M. Manenti S. MAP degradation of p27Kip1 by in melanoma cells. of p27Kip1 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), the of p27Kip1 not by the of p27Kip1 degradation and that after p27Kip1 was the DHA and inhibitor U0126 stabilized p27Kip1 protein and its degradation in the of the ERK1/ERK2 is for p27Kip1 and late-G1 phase N. M. S. K. at a phosphorylation of its protein 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), study that DHA MAPK-dependent degradation of p27Kip1 in with a recent study L.E. C.J. of fatty growth inhibition in human breast cancer Cancer Res. 2005; PubMed Scopus Google Scholar) that MAPK inhibition was to inhibit the proliferation of cell by p27Kip1 levels. is a specific inhibitor of E. its association with cyclin E, is in the of the transition (18Xiong Y. Hannon G.J. Zhang H. Casso D. Kobayashi R. Beach D. p21 is a universal inhibitor of cyclin kinases..Nature. 1993; 366: 701-704Crossref PubMed Scopus (3172) Google Scholar, 19Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases..Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5230) Google Scholar). In DHA did not inhibit cyclin that the cyclin D/CDK4/6 in the but not in G1 phase, is not by DHA (16Sherr C.J. Cancer cell cycles..Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4961) Google Scholar). the other DHA was to inhibit cyclin E phosphorylation and CDK2 levels. DHA inhibited kinase activity of In the E is to phosphorylate this as an (16Sherr C.J. Cancer cell cycles..Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4961) Google Scholar, V. Lees E. Reed S.I. Association of human cyclin E with a periodic G1-S phase protein kinase..Science. 1992; 257: 1958-1961Crossref PubMed Scopus (794) Google Scholar). is that pRb is hypophosphorylated in the phase and in the of pRb in the of pRb and the release of a transcription that induces the S-phase (16Sherr C.J. Cancer cell cycles..Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4961) Google Scholar, V. Lees E. Reed S.I. Association of human cyclin E with a periodic G1-S phase protein kinase..Science. 1992; 257: 1958-1961Crossref PubMed Scopus (794) Google Scholar). In the present that DHA inhibited pRb The inhibition of pRb to activity of CDK2 kinase These observations are in with the of A.P. Juan G. Traganos F. Reinhart L. Connolly J. Rose D.P. Darzynkiewicz Z. Cell cycle arrest and apoptosis of melanoma cells by docosahexaenoic acid: association with decreased pRb phosphorylation..Cancer Res. 2000; 60: 4139-4145PubMed Google Scholar), have that inhibition of cyclin CDK2 kinase activity, and pRb phosphorylation are for the inhibition of the transition of cells from the G1 to the S phase of the cell cycle. In into the mechanism of action of DHA in the of cell proliferation. Our observations that DHA, a PUFA of the n-3 family, cell cycle arrest in the late-G1 to S phase of the cell cycle by inhibiting the MAPK and degradation of p27Kip1 in FM3A cells. DHA also CDK2 kinase activity and of pRb in these cells. was supported by the and by the of and The are to Alain Eychène and Stéphane supplied containing The also to to for and to for