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Japanese Journal of Cancer Research : Gann logoLink to Japanese Journal of Cancer Research : Gann
. 1995 Sep;86(9):819–827. doi: 10.1111/j.1349-7006.1995.tb03091.x

Inhibition of p53 Protein Phosphorylation by 9‐Hydroxyellipticine: A Possible Anticancer Mechanism

Motoaki Ohashi 1,✉, Emiko Sugikawa 1, Noriyuki Nakanishi 1
PMCID: PMC5920929  PMID: 7591958

Abstract

Abnormality of p53, a tumor suppressor gene, is considered to be a potential cause of malignancy. We found that ellipticine and 9‐hydroxyellipticine (9HE), antitumor alkaloids, caused selective inhibition of p53 protein phospborylation in Lewis lung carcinoma and SW480 (human colon cancer cell line) in a concentration‐dependent manner from 0.1 to 100 μM. 9HE suppressed cdk2 kinase activity concentration‐dependently from 1 to 100 μM. By contrast, the inhibition of p53 protein phosphorylation by elliptinium and elliprabin (N2 substituted derivatives of 9HE) was very weak. A good correlation was observed between p53 phosphorylation inhibition and cytotoxic activity of these agents in terms of concentration‐response relationships, suggesting that inhibition of p53 protein phosphorylation via kinase inhibition may be involved in the anticancer mechanism of these agents. In addition, this study demonstrated that brief exposure to 9HE caused apoptosis of cancer cells. It is suggested that accumulation of dephosphorylated mutant p53 may induce apoptosis.

Keywords: Ellipticine, p53, Phosphorylation inhibition, Apoptosis induction, Anticancer mechanism

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REFERENCES

  • 1. ) Gribble , G. W.Synthesis and antitumor activity of ellipticine alkaloids and related compounds . Alkaloids , 39 , 239 – 352 ( 1990. ). [Google Scholar]
  • 2. ) Kohn , K. W. , Waring , M. J. , Glaubiger , D. and Friedman , C. A.Intercalative binding of ellipticine to DNA . Cancer Res. , 35 , 71 – 76 ( 1975. ). [PubMed] [Google Scholar]
  • 3. ) Zwelling , L. A. , Michaels , S. , Kerrigan , D. , Pommier , Y. and Kohn , K. W.Protein‐associated deoxyribonucleic acid strand breaks produced in mouse leukemia L1210 cells by ellipticine and 2‐methyl‐9‐hydroxy‐ellipticinium . Biochem. Pharmacol. , 31 , 3261 – 3267 ( 1982. ). [DOI] [PubMed] [Google Scholar]
  • 4. ) Multon , E. , Riou , J. , LeFevre , D. , Ahomadegbe , J. and Riou , G.Topoisomerase II mediated DNA cleavage activity induced by ellipticines on the human tumor cell line N417 . Biochem. Pharmacol. , 38 , 2077 – 2086 ( 1989. ). [DOI] [PubMed] [Google Scholar]
  • 5. ) Monnot , M. , Mauffret , O. , Simon , V. , Lescot , E. , Psaume , B. , Saucier , J. , Charra , M. , Belehradek , J. , Jr. and Fermandjian , S.DNA‐drug recognition and effects of topoisomerase II‐mediated cytotoxicity. A three‐mode binding model for ellipticine derivatives . J. Biol. Chem. , 26 , 1820 – 1829 ( 1991. ). [PubMed] [Google Scholar]
  • 6. ) Nigro , J. M. , Baker , S. J. , Presinger , A. C. , Jessup , J. M. , Hostetter , R. , Cleary , K. , Bigner , S. H. , Davidson , N. , Baylin , S. , Devilee , P. , Glover , T. , Collins , F. S. , Weston , A. , Modali , R. , Harris , C. C. and Vogelstein , B.Mutations in the p53 gene occur in diverse human tumour types . Nature , 342 , 705 – 708 ( 1989. ). [DOI] [PubMed] [Google Scholar]
  • 7. ) Hollstein , M. , Sidransky , D. , Vogelstein , B. and Harris , C. C.p53 mutations in human cancers . Science , 253 , 49 – 52 ( 1991. ). [DOI] [PubMed] [Google Scholar]
  • 8. ) Baker , S. J. , Fearon , E. R. , Nigro , J. M. , Hamilton , S. R. , Presinger , A. D. , Jessup , J. M. , van Tuinen , P. , Ledbetter , D. H. , Baker , D. F. , Nakamura , Y. , White , R. and Vogelstein , B.Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas . Science , 244 , 217 – 221 ( 1989. ). [DOI] [PubMed] [Google Scholar]
  • 9. ) Takahashi , T. , Nau , M. M. , Chiba , I. , Birrer , M. J. , Rosenberg , R. K. , Vinocour , M. , Levitt , M. , Pass , H. , Gazdar , A. F. and Minna , J. D.p53: a frequent target for genetic abnormalities in lung cancer . Science , 246 , 491 – 494 ( 1989. ). [DOI] [PubMed] [Google Scholar]
  • 10. ) Levine , A. J. , Momand , J. and Finlay , C. A.The p53 tumour suppressor gene . Nature , 351 , 453 – 456 ( 1991. ). [DOI] [PubMed] [Google Scholar]
  • 11. ) Finlay , C. A. , Hinds , P. W. and Levine , A. J.The p53 proto‐oncogene can act as a suppressor of transformation . Cell , 57 , 1083 – 1093 ( 1989. ). [DOI] [PubMed] [Google Scholar]
  • 12. ) Eliyahu , D. , Michalovitz , D. , Eliyahu , S. , Pinhasi‐Kimhi , O. and Oren , M.Wild‐type p53 can inhibit oncogenemediated focus formation . Proc. Natl. Acad. Sci. USA , 86 , 8763 – 8767 ( 1989. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. ) Montenarh , M.Functional implications of the growthsuppressor/oncoprotein p53 (Review) . Int. J. Oncol. , 1 , 37 – 45 ( 1992. ). [PubMed] [Google Scholar]
  • 14. ) Fields , S. and Jang , S. K.Presence of a potent transcription activating sequence in the p53 protein . Science , 249 , 1046 – 1049 ( 1990. ). [DOI] [PubMed] [Google Scholar]
  • 15. ) Milner , J. , Cook , A. and Mason , J.p53 is associated with P34cdc2 in transformed . EMBO J. , 9 , 2885 – 2889 ( 1990. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. ) Bischoff , J. R. , Friedman , P. N. , Marshak , D. R. , Prives , C. and Beach , D.Human p53 is phosphorylated by p60‐cdc2 and cyclinB‐cdc2 . Proc. Natl. Acad. Sci. USA , 87 , 4766 – 4770 ( 1990. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. ) Addison , C. , Jenkins , J. R. and Sturzbecher , H.The p53 nuclear localization signal is structurally linked to a p34cdc2 kinase motif . Oncogene , 5 , 423 – 427 ( 1990. ). [PubMed] [Google Scholar]
  • 18. ) Sturzbecher , H. , Maimets , T. , Chumakov , P. , Brain , R. , Addison , C. , Simanis , V. , Rudge , K. , Philp , R. , Grimaldi , M. , Court , W. and Jenkins , J. R.p53 interacts with p34cdc2 in mammalian cells: implications for cell cycle control and oncogenesis . Oncogene , 5 , 795 – 801 ( 1990. ). [PubMed] [Google Scholar]
  • 19. ) Lorenz , A. , Herrmann , C. P. E. , Issinger , O. and Montenarh , M.Phosphorylation of wild‐type and mutant phenotypes of p53 by an associated protein kinase . Int. J. Oncol. , 1 , 571 – 579 ( 1992. ). [DOI] [PubMed] [Google Scholar]
  • 20. ) Filhol , O. , Baudier , J. , Delphin , C. , Loue‐Mackenbach , P. , Chambaz , E. M. and Cochet , C.Casein kinase II and the tumor suppressor protein p53 associate in a molecular complex that is negatively regulated upon p53 phosphorylation . J. Biol. Chem. , 267 , 20577 – 20583 ( 1992. ). [PubMed] [Google Scholar]
  • 21. ) Milne , D. M. , Palmer , R. H. and Meek , D. W.Mutation of the casein kinase II phosphorylation site abolishes the anti‐proliferative activity of p53 . Nucleic Acids Res. , 20 , 5565 – 5570 ( 1992. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. ) Lees‐Miller , S. P. , Chen , Y. R. and Anderson , C. W.Human cells contain a DNA‐activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen . Mol. Cell. Biol. , 10 , 6472 – 6481 ( 1990. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. ) Carter , T. , Vancurova , I. , Sun , L. , Lou , W. and DeLeon , S.A DNA‐activated protein kinase from HeLa cell nuclei . Mol. Cell. Biol. , 10 , 6460 – 6471 ( 1990. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. ) Lees‐Miller , S. P. , Sakaguchi , K. , Ullrich , S. J. , Appella , E. and Anderson , C. R.Human DNA‐activated protein kinase phosphorylates serines 15 and 37 in the aminoterminal transactivation domain of human p53 . Mol. Cell. Biol. , 12 , 5041 – 5049 ( 1992. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. ) Yonish‐Rouach , E. , Resnitzky , D. , Lotem , J. , Sachs , L. , Kimchi , A. and Oren , M.Wild‐type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin‐6 . Nature , 352 , 345 – 347 ( 1991. ). [DOI] [PubMed] [Google Scholar]
  • 26. ) Ryan , J. J. , Danish , R. , Gottlieb , C. A. and Clarke , M. F.Cell cycle analysis of p53‐induced cell death in murine erythroleukemia cells . Mol. Cell. Biol. , 13 , 711 – 719 ( 1993. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. ) Ramqvist , T. , Magnusson , K. P. , Wang , Y. , Szekely , L. , Klein , G. and Wiman , K. G.Wild‐type p53 induces apoptosis in a Burkitt lymphoma (BL) line that carries mutant p53 . Oncogene , 8 , 1495 – 1500 ( 1993. ). [PubMed] [Google Scholar]
  • 28. ) Lowe , S. W. , Schmitt , E. M. , Smith , S. W. , Osborne , B. A. and Jacks , T.p53 is required for radiation‐induced apoptosis in mouse thymocytes . Nature , 362 , 847 – 849 ( 1993. ). [DOI] [PubMed] [Google Scholar]
  • 29. ) Lane , D. P.A death in the life of p53 . Nature , 362 , 786 – 787 ( 1993. ). [DOI] [PubMed] [Google Scholar]
  • 30. ) El‐Deiry , W. S. , Harper , W. , O'Connor P. M. , Velculescu V. E. , Canman , C. E. , Jackman , J. , Pietenpol , J. A. , Burrell , M. , Hill , D. E. , Wang , Y. , Wiman , K. G. , Mercer , W. E. , Kastan , M. B. , Kohn , K. W. , Elledge , S. J. , Kinzler , K. W. and Vogelstein , B.Waf 1/cipl is induced in p53‐mediated G1 arrest and apoptosis . Cancer Res. , 54 , 1169 – 1174 ( 1994. ). [PubMed] [Google Scholar]
  • 31. ) Kastan , M. B. , Onyinye , O. , Sidransky , D. , Vogelstein , B. and Craig , R. W.Participation of p53 in the cellular response to DNA damage . Cancer Res. , 51 , 6304 – 6311 ( 1991. ). [PubMed] [Google Scholar]
  • 32. ) Fritsche , M. , Haessler , C. and Brandner , G.Induction of nuclear accumulation of the tumor‐suppressor protein p53 by DNA‐damaging agents . Oncogene , 8 , 307 – 318 ( 1993. ). [PubMed] [Google Scholar]
  • 33. ) Nelson , W. G. and Kastan , M. B.DNA strand breaks: the DNA template alterations that trigger p53‐dependent DNA damage response pathways . Mol. Cell. BioL. , 14 , 1815 – 1823 ( 1994. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. ) Iggo , R. , Gatter , K. , Bartek , J. , Lane , D. and Harris , A.Increased expression of mutant forms of p53 oncogene in primary lung cancer . Lancet , 335 , 675 – 679 ( 1990. ). [DOI] [PubMed] [Google Scholar]
  • 35. ) Marks , J. R. , Davidoff , A. M. , Kerns , B. J. , Humphrey , P. A. , Pence , J. C. , Dodge , R. K. , Clarke‐Pearson , D. L. , Iglehart , J. D. , Bast , R. C. , Jr. and Berchuk , A.Overexpression and mutation of p53 in epithelial ovarian cancer . Cancer Res. , 51 , 2979 – 2984 ( 1991. ). [PubMed] [Google Scholar]
  • 36. ) Dolcetti , R. , Doglioni , C. , Maestro , R. , Gasparotto , D. , Barzan , L. , Pastore , A. , Romanelli , M. and Boiocchi , M.p53 overexpression is an early event in the development of human squamous‐cell carcinoma of the larynx; genetic and prognostic implications . Int. J. Cancer , 52 , 178 – 182 ( 1992. ). [DOI] [PubMed] [Google Scholar]
  • 37. ) Pignatelli , M. , Stamp , G. W. H. , Kafiri , G. , Lane , D. and Bodmer , W. F.Overexpression of p53 nuclear oncoprotein in colorectal adenomas . Int. J. Cancer , 50 , 683 – 688 ( 1992. ). [DOI] [PubMed] [Google Scholar]
  • 38. ) Westa , W. H. , Offerhaus , G. J. A. , Goodman , S. T. , Slebos , R. J. C. , Baas , I. O. , Rodenhuis , S. and Hruban , R. H.Overexpression of the p53 tumor suppressor gene product in primary lung adenocarcinomas is associated with cigarette smoking . Am. J. Surg. Pathol. , 17 , 213 – 220 ( 1993. ). [DOI] [PubMed] [Google Scholar]
  • 39. ) Kakeji , Y. , Korenaga , D. , Tsujitani , S. , Baba , H. , Anai , H. , Maehara , Y. and Sugimachi , K.Gastric cancer with p53 overexpression has high potential for metastasizing to lymphnodes . Br. J. Cancer , 67 , 589 – 593 ( 1993. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40. ) Lassam , N. J. , From , L. and Kahn , H. J.Overexpression of p53 is a late event in the development of malignant melanoma . Cancer Res. , 53 , 2235 – 2238 ( 1993. ). [PubMed] [Google Scholar]
  • 41. ) Jenkins , J. R. , Rudge , K. and Currie , G. A.Cellular immortalization by a cDNA clone encoding the transformation‐associated phosphoprotein p53 . Nature , 312 , 651 – 654 ( 1984. ). [DOI] [PubMed] [Google Scholar]
  • 42. ) Scheidtmann , K. H. and Harber , A.Simian virus large T antigen induces or activates a protein kinase which phosphorylates the transformation‐associated protein p53 . J. Virol. , 64 , 672 – 679 ( 1990. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. ) McClure , J. E. , Noonan , C. A. and Shearer , W. T.Okadaic acid inhibits dephosphorylation of cytoplasmic p53 during lymphocyte activation . Biochem. Biophys. Res. Commun. , 197 , 1578 – 1584 ( 1993. ). [DOI] [PubMed] [Google Scholar]
  • 44. ) Lowe , S. W. , Ruley , H. E. , Jacks , T. and Housman , D. E.p53‐Dependent apoptosis modulates the cytotoxicity of anticancer agents . Cell , 74 , 957 – 967 ( 1993. ). [DOI] [PubMed] [Google Scholar]
  • 45. ) Moreno , S. , Hayles , J. and Nurse , P.Regulation of p34cdc2 protein kinase during mitosis . Cell , 58 , 361 – 372 ( 1989. ). [DOI] [PubMed] [Google Scholar]
  • 46. ) Bradford , M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principal of protein‐dye binding . Anal. Biochem. , 72 , 245 – 248 ( 1976. ). [DOI] [PubMed] [Google Scholar]
  • 47. ) Brizuela , L. , Draetta , G. and Beach , D.p13suc1 acts in the fission yeast cell division cycle as a component of the p34cdc2 protein kinase . EMBO J. , 6 , 3507 – 3514 ( 1987. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48. ) Tsao , Y.‐P. , D'Arpa , P. and Liu , L. F.The involvement of active DNA synthesis in camptothecin‐induced G2 arrest; altered regulation of p34cdc2/cyclin B . Cancer Res. , 52 , 1823 – 1829 ( 1992. ). [PubMed] [Google Scholar]
  • 49. ) Mosmann , T.Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assay . J. Immunol. Methods , 65 , 55 – 63 ( 1983. ). [DOI] [PubMed] [Google Scholar]
  • 50. ) Gannon , J. V. and Lane , D. P.Protein synthesis required to anchor a mutant p53 protein which is temperaturesensitive for nuclear transport . Nature , 349 , 802 – 806 ( 1991. ). [DOI] [PubMed] [Google Scholar]
  • 51. ) Ohashi , M. , Sugikawa , E. , Oda , K. and Nakanishi , N.Inhibition of p53 protein phosphorylation by 9‐hydroxyellipticine: a possible anticancer mechanism . Proc. 16th Int. Cancer Congr. , 165 – 169 ( 1994. ). [DOI] [PMC free article] [PubMed]
  • 52. ) Mogi , Y. , Kato , J. , Horimoto , M. , Takimoto , R. , Murakami , T. , Hirayama , A. , Kohgo , Y. , Watanabe , N. and Niitu , Y.Close correlation between the dephosphorylation of p53 and growth suppression by transforming growth factor‐β1 in nasopharyngeal carcinoma cells transduced with adenovirus early region genes . Jpn. J. Cancer Res. , 85 , 459 – 463 ( 1994. ). [DOI] [PMC free article] [PubMed] [Google Scholar]

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