Investigational New Drugs

, Volume 30, Issue 3, pp 889–897 | Cite as

In vitro antitumor mechanism of a novel cyclin-dependent kinase inhibitor CDKI-83

  • Xiangrui Liu
  • Frankie Lam
  • Shenhua Shi
  • Peter M. Fischer
  • Shudong WangEmail author


Cancer is regarded as a proliferative disorder. Inhibition of cyclin-dependent kinases (CDKs), which are the key regulators of the cell-cycle and RNA transcription, represents an attractive strategy for cancer therapy. In this study, we report the cellular mechanistic investigation of CDKI-83, a K i-nanomolar CDK9 inhibitor. The compound shows effective anti-proliferative activity in human tumour cell lines with GI50 <1 μM, and is capable of inducing apoptosis in A2780 human ovarian cancer cells as determined by the activated caspase-3, Annexin V/PI double staining and accumulated cells at the sub-G1 phase of cell-cycle. While A2780 cells were arrested in G2/M phase with CDKI-83 treatment, phosphorylation at Thr320 of PP1α was significantly reduced, indicating CDK1 inhibition. Importantly, this compound reduced phosphorylation at Ser-2 of RNA polymerase II (RNAPII) by inhibiting cellular CDK9 activity, and down-regulated Mcl-1 and Bcl-2. These results suggest that combined inhibition of CDK9 and CDK1 may result in the effective induction of apoptosis and CDKI-83 has the potential to be developed as an anti-cancer agent.


Cyclin-dependent kinase Anti-cancer agent RNAPII transcription PTEF-b CDK9 inhibitor Mcl-1 Bcl-2 



This study was supported in part by the Cancer Research UK grant C21568/A8988. The authors would like to thank Dr Scott Roberts for his technical assistant with RT-PCR.


  1. 1.
    Sausville EA (2002) Complexities in the development of cyclin-dependent kinase inhibitor drugs. Trends Mol Med 8(4 suppl):S32–S37PubMedCrossRefGoogle Scholar
  2. 2.
    Shapiro GI (2006) Cyclin-dependent kinase pathways as targets for cancer treatment. J Clin Oncol 24(11):1770–1783PubMedCrossRefGoogle Scholar
  3. 3.
    Lapenna S, Giordano A (2009) Cell cycle kinases as therapeutic targets for cancer. Nat Rev Drug Discov 8(7):547–566PubMedCrossRefGoogle Scholar
  4. 4.
    Santamaria D, Barriere C, Cerqueira A, Hunt S, Tardy C, Newton K, Caceres JF, Dubus P, Malumbres M, Barbacid M (2007) Cdk1 is sufficient to drive the mammalian cell cycle. Nature 448(7155):811–815PubMedCrossRefGoogle Scholar
  5. 5.
    Ortega S, Prieto I, Odajima J, Martin A, Dubus P, Sotillo R, Barbero JL, Malumbres M, Barbacid M (2003) Cyclin-dependent kinase 2 is essential for meiosis but not for mitotic cell division in mice. Nat Genet 35(1):25–31PubMedCrossRefGoogle Scholar
  6. 6.
    Sims RJ 3rd, Mandal SS, Reinberg D (2004) Recent highlights of RNA-polymerase-II-mediated transcription. Curr Opin Cell Biol 16(3):263–271PubMedCrossRefGoogle Scholar
  7. 7.
    Fujinaga K, Irwin D, Huang Y, Taube R, Kurosu T, Peterlin BM (2004) Dynamics of human immunodeficiency virus transcription: P-TEFb phosphorylates RD and dissociates negative effectors from the transactivation response element. Mol Cell Biol 24(2):787–795PubMedCrossRefGoogle Scholar
  8. 8.
    Yamada T, Yamaguchi Y, Inukai N, Okamoto S, Mura T, Handa H (2006) P-TEFb-mediated phosphorylation of hSpt5 C-terminal repeats is critical for processive transcription elongation. Mol Cell 21(2):227–237PubMedCrossRefGoogle Scholar
  9. 9.
    Kobor MS, Greenblatt J (2002) Regulation of transcription elongation by phosphorylation. Biochim Biophys Acta 1577(2):261–275PubMedGoogle Scholar
  10. 10.
    Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100(1):57–70PubMedCrossRefGoogle Scholar
  11. 11.
    Adams RR, Tavares AAM, Salzberg A, Bellen HJ, Glover DM (1998) Pavarotti encodes a kinesin-like protein required to organize the central spindle and contractile ring for cytokinesis. Genes Dev 12(10):1483–1494PubMedCrossRefGoogle Scholar
  12. 12.
    Storey S (2008) Targeting apoptosis: selected anticancer strategies. Nat Rev Drug Discov 7(12):971–972PubMedCrossRefGoogle Scholar
  13. 13.
    Gelinas C, White E (2005) BH3-only proteins in control: specificity regulates MCL-1 and BAK-mediated apoptosis. Genes Dev 19(11):1263–1268PubMedCrossRefGoogle Scholar
  14. 14.
    Derenne S, Monia B, Dean NM, Taylor JK, Rapp M-J, Harousseau J-L, Bataille R, Amiot M (2002) Antisense strategy shows that Mcl-1 rather than Bcl-2 or Bcl-xL is an essential survival protein of human myeloma cells. Blood 100(1):194–199PubMedCrossRefGoogle Scholar
  15. 15.
    Hussain SR, Cheney CM, Johnson AJ, Lin TS, Grever MR, Caligiuri MA, Lucas DM, Byrd JC (2007) Mcl-1 is a relevant therapeutic target in acute and chronic lymphoid malignancies: down-regulation enhances rituximab-mediated apoptosis and complement-dependent cytotoxicity. Clin Cancer Res 13(7):2144–2150. doi: 10.1158/1078-0432.CCR-06-2294 PubMedCrossRefGoogle Scholar
  16. 16.
    Fischer PM, Gianella-Borradori A (2005) Recent progress in the discovery and development of cyclin-dependent kinase inhibitors. Expert Opin Investig Drugs 14(4):457–477PubMedCrossRefGoogle Scholar
  17. 17.
    Wang S, Fischer PM (2008) Cyclin-dependent kinase 9: a key transcriptional regulator and potential drug target in oncology, virology and cardiology. Trends Pharmacol Sci 29(6):302–313. doi: 10.1016/ PubMedCrossRefGoogle Scholar
  18. 18.
    Byrd JC, Lin TS, Dalton JT, Wu D, Phelps MA, Fischer B, Moran M, Blum KA, Rovin B, Brooker-McEldowney M, Broering S, Schaaf LJ, Johnson AJ, Lucas DM, Heerema NA, Lozanski G, Young DC, Suarez JR, Colevas AD, Grever MR (2007) Flavopiridol administered using a pharmacologically derived schedule is associated with marked clinical efficacy in refractory, genetically high-risk chronic lymphocytic leukemia. Blood 109(2):399–404. doi: 10.1182/blood-2006-05-020735 PubMedCrossRefGoogle Scholar
  19. 19.
    Chen R, Keating MJ, Gandhi V, Plunkett W (2005) Transcription inhibition by flavopiridol: mechanism of chronic lymphocytic leukemia cell death. Blood 106(7):2513–2519PubMedCrossRefGoogle Scholar
  20. 20.
    Chen R, Wierda WG, Chubb S, Hawtin RE, Fox JA, Keating MJ, Gandhi V, Plunkett W (2009) Mechanism of action of SNS-032, a novel cyclin-dependent kinase inhibitor, in chronic lymphocytic leukemia. Blood 113(19):4637–4645. doi: 10.1182/blood-2008-12-190256 PubMedCrossRefGoogle Scholar
  21. 21.
    Wang S, McClue SJ, Ferguson JR, Hull JD, Stokes S, Parsons S, Westwood R, Fischer PM (2001) Synthesis and configuration of the cyclin-dependent kinase inhibitor roscovitine and its enantiomer. Tetrahedron Asymmetr 12(20):2891–2894CrossRefGoogle Scholar
  22. 22.
    McClue SJ, Blake D, Clarke R, Cowan A, Cummings L, Fischer PM, MacKenzie M, Melville J, Stewart K, Wang S, Zhelev N, Zheleva D, Lane DP (2002) In vitro and in vivo antitumor properties of the cyclin dependent kinase inhibitor CYC202 (R-roscovitine). Int J Cancer 102:463–468PubMedCrossRefGoogle Scholar
  23. 23.
    Wang S, Meades C, Wood G, Osnowski A, Anderson S, Yuill R, Thomas M, Mezna M, Jackson W, Midgley C, Griffiths G, Fleming I, Green S, McNae I, Wu SY, McInnes C, Zheleva D, Walkinshaw MD, Fischer PM (2004) 2-Anilino-4-(thiazol-5-yl)pyrimidine CDK inhibitors: synthesis, SAR analysis, X-ray crystallography, and biological activity. J Med Chem 47(7):1662–1675. doi: 10.1021/jm0309957 PubMedCrossRefGoogle Scholar
  24. 24.
    Wang S, Wood G, Meades C, Griffiths G, Midgley C, McNae I, McInnes C, Anderson S, Jackson W, Mezna M, Yuill R, Walkinshaw M, Fischer PM (2004) Synthesis and biological activity of 2-anilino-4-(1H-pyrrol-3-yl) pyrimidine CDK inhibitors. Bioorg Med Chem Lett 14(16):4237–4240. doi: 10.1016/j.bmcl.2004.06.012S0960894X04007656 PubMedCrossRefGoogle Scholar
  25. 25.
    Wang S, Midgley CA, Scaerou F, Grabarek JB, Griffiths G, Jackson W, Kontopidis G, McClue SJ, McInnes C, Meades C, Mezna M, Plater A, Stuart I, Thomas MP, Wood G, Clarke RG, Blake DG, Zheleva DI, Lane DP, Jackson RC, Glover DM, Fischer PM (2010) Discovery of N-Phenyl-4-(thiazol-5-yl)pyrimidin-2-amine aurora kinase inhibitors. J Med Chem. doi: 10.1021/jm901913s Google Scholar
  26. 26.
    Wang S, Griffiths G, Midgley CA, Barnett AL, Cooper M, Grabarek J, Ingram L, Jackson W, Kontopidis G, McClue SJ, McInnes C, McLachlan J, Meades C, Mezna M, Stuart I, Thomas MP, Zheleva DI, Lane DP, Jackson RC, Glover DM, Blake DG, Fischer PM (2010) Discovery and characterization of 2-anilino-4- (thiazol-5-yl)pyrimidine transcriptional CDK inhibitors as anticancer agents. Chem Biol 17(10):1111–1121PubMedCrossRefGoogle Scholar
  27. 27.
    Wang S, Shi S, Zaytsev A, Fischer PM (2009) Pyrimidines, triazines and their use as pharmaceutical agents WO/2009/118567Google Scholar
  28. 28.
    Cheng Y, Prusoff WH (1973) Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22(23):3099–3108PubMedCrossRefGoogle Scholar
  29. 29.
    Nigg EA (2001) Mitotic kinases as regulators of cell division and its checkpoints. Nat Rev Mol Cell Biol 2(1):21–32PubMedCrossRefGoogle Scholar
  30. 30.
    Merino R, Ding L, Veis DJ, Korsmeyer SJ, Nunez G (1994) Developmental regulation of the Bcl-2 protein and susceptibility to cell death in B lymphocytes. EMBO J 13(3):683–691PubMedGoogle Scholar
  31. 31.
    Blagosklonny MV, Alvarez M, Fojo A, Neckers LM (1996) bcl-2 protein downregulation is not required for differentiation of multidrug resistant HL60 leukemia cells. Leuk Res 20(2):101–107PubMedCrossRefGoogle Scholar
  32. 32.
    Berthet C, Aleem E, Coppola V, Tessarollo L, Kaldis P (2003) Cdk2 knockout mice are viable. Curr Biol 13(20):1775–1785PubMedCrossRefGoogle Scholar
  33. 33.
    Barriere C, Santamaria D, Cerqueira A, Galan J, Martin A, Ortega S, Malumbres M, Dubus P, Barbacid M (2007) Mice thrive without Cdk4 and Cdk2. Mol Oncol 1(1):72–83PubMedCrossRefGoogle Scholar
  34. 34.
    Malumbres M, Sotillo R, Santamaria D, Galan J, Cerezo A, Ortega S, Dubus P, Barbacid M (2004) Mammalian cells cycle without the D-type cyclin-dependent kinases Cdk4 and Cdk6. Cell 118(4):493–504PubMedCrossRefGoogle Scholar
  35. 35.
    Cai D, Latham VM Jr, Zhang X, Shapiro GI (2006) Combined depletion of cell cycle and transcriptional cyclin-dependent kinase activities induces apoptosis in cancer cells. Cancer Res 66(18):9270–9280PubMedCrossRefGoogle Scholar
  36. 36.
    Lam LT, Pickeral OK, Peng AC, Rosenwald A, Hurt EM, Giltnane JM, Averett LM, Zhao H, Davis RE, Sathyamoorthy M, Wahl LM, Harris ED, Mikovits JA, Monks AP, Hollingshead MG, Sausville EA, Staudt LM (2001) Genomic-scale measurement of mRNA turnover and the mechanisms of action of the anti-cancer drug flavopiridol. Genome Biol 2(10):RESEARCH0041PubMedCrossRefGoogle Scholar
  37. 37.
    Vassilev LT, Tovar C, Chen S, Knezevic D, Zhao X, Sun H, Heimbrook DC, Chen L (2006) Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1. Proc Natl Acad Sci USA 103(28):10660–10665. doi: 10.1073/pnas.0600447103 PubMedCrossRefGoogle Scholar
  38. 38.
    Chen CY, Shyu AB (1995) AU-rich elements: characterization and importance in mRNA degradation. Trends Biochem Sci 20(11):465–470PubMedCrossRefGoogle Scholar
  39. 39.
    Schiavone N, Rosini P, Quattrone A, Donnini M, Lapucci A, Citti L, Bevilacqua A, Nicolin A, Capaccioli S (2000) A conserved AU-rich element in the 3′ untranslated region of bcl-2 mRNA is endowed with a destabilizing function that is involved in bcl-2 down-regulation during apoptosis. FASEB J 14(1):174–184PubMedGoogle Scholar
  40. 40.
    Rechsteiner M, Rogers SW (1996) PEST sequences and regulation by proteolysis. Trends Biochem Sci 21(7):267–271PubMedGoogle Scholar
  41. 41.
    Reed JC, Tsujimoto Y, Alpers JD, Croce CM, Nowell PC (1987) Regulation of bcl-2 proto-oncogene expression during normal human lymphocyte proliferation. Science 236(4806):1295–1299PubMedCrossRefGoogle Scholar
  42. 42.
    Snowden RT, Sun XM, Dyer MJ, Cohen GM (2003) Bisindolylmaleimide IX is a potent inducer of apoptosis in chronic lymphocytic leukaemic cells and activates cleavage of Mcl-1. Leukemia 17(10):1981–1989PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Xiangrui Liu
    • 1
  • Frankie Lam
    • 1
  • Shenhua Shi
    • 1
  • Peter M. Fischer
    • 1
  • Shudong Wang
    • 1
    Email author
  1. 1.School of Pharmacy and Centre for Biomolecular SciencesUniversity of NottinghamNottinghamUK

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