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Japanese Journal of Cancer Research : Gann logoLink to Japanese Journal of Cancer Research : Gann
. 2002 Jul;93(7):774–782. doi: 10.1111/j.1349-7006.2002.tb01319.x

Troglitazone Induces G1 Arrest by p27Kip1 Induction That Is Mediated by Inhibition of Proteasome in Human Gastric Cancer Cells

Shigeru Takeuchi 1, Toshikatsu Okumura 1, Wataru Motomura 1, Miho Nagamine 1, Nobuhiko Takahashi 1, Yutaka Kohgo ✉
PMCID: PMC5927078  PMID: 12149143

Abstract

We examined in the present study whether human gastric cancer cells express peroxisome proliferator‐activated receptor γ (PPARγ), the effect of PPARy activation by troglitazone, a selective ligand, on cellular growth, and the mechanism of the growth arrest by troglitazone in gastric cancer cells. RT‐PCR, northern blot and western blot analysis demonstrated that all four tested human gastric cancer cell lines, MKN–28, MKN–45, MKN–74 and KATO‐III, expressed PPARγ mRNA and protein. WST–1 assay and flow cytometric analysis revealed that troglitazone inhibited the growth and induced G1 arrest in all four gastric cancer cell lines. To examine the role of p27Kip1, a cyclin‐dependent kinase inhibitor, in the G1 arrest by troglitazone, we determined p27Kip1 protein expression by western blot analysis in gastric cancer cells that had been treated with troglitazone. Troglitazone increased p27Kip1 in all four gastric cancer cell lines. Since it has been reported that the ubiquitin‐proteasome system plays a vital role in the degradation of p27Kip1 protein, we evaluated the hypothesis that inhibition of proteasome mediates the troglitazone‐induced p27Kip1 accumulation. Lactacystin, a proteasome inhibitor, inhibited cell growth and increased p27Kilp1 expression in MKN–74 cells. It was further demonstrated that troglitazone inhibited proteasome activity in a dose‐dependent manner in MKN–74 cells. All these results suggest that troglitazone inhibited proteasome activity, followed by induction of p27Kipl, which arrests cells at the Gl phase of the cell cycle in gastric cancer cells. The troglitazone‐mediated inhibition of the proteasome suggests a novel mechanism for the anti‐proliferative effect of this agent in cancer cells.

Keywords: Troglitazone, PPARy, p27Kipl, Proteasome, Gastric cancer

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REFERENCES

  • 1. ) Mangelsdorf , D. J. , Thummel , C. , Beato , M. , Herrlich , P. , Schutz , G. , Umesono , K. , Blumberg , B. , Kastner , P. , Mark , M. , Chambon , P. and Evans , R. M.The nuclear receptor superfamily: the second decade . Cell , 83 , 835 – 839 ( 1995. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. ) Lemberger , T. , Desvergne , B. and Wahli , W.Peroxisome proliferator‐activated receptors: a nuclear receptor signaling pathway in lipid physiology . Annu. Rev. Cell. Dev. Biol. , 12 , 335 – 363 ( 1996. ). [DOI] [PubMed] [Google Scholar]
  • 3. ) Spiegelman , B. M. and Flier , J. S.Adipogenesis and obesity: rounding out the big picture . Cell , 87 , 377 – 389 ( 1996. ). [DOI] [PubMed] [Google Scholar]
  • 4. ) Sarraf , P. , Mueller , E. , Jones , D. , King , F. , DeAngelo , D. J. , Partridge , J. B. , Holden , S. A. , Chen , L. B. , Singer , S. , Fletcher , C. and Spiegelman , B. M.Differentiation and reversal of malignant changes in colon cancer through PPARy . Nat. Med. , 4 , 1046 – 1052 ( 1998. ). [DOI] [PubMed] [Google Scholar]
  • 5. ) Brockman , J. A. , Gupta , R. A. and DuBois , R. N.Activation of PPARγ leads to inhibition of anchorage‐independent growth of human colorectal cancer cells . Gastroenterology , 115 , 1049 – 1055 ( 1998. ). [DOI] [PubMed] [Google Scholar]
  • 6. ) Kitamura , S. , Miyazaki , Y. , Shinomura , Y. , Kondo , S. , Kanayama , S. and Matsuzawa , Y.Peroxisome proliferator‐activated receptor γ induces growth arrest and differentiation markers of human colon cancer cells . Jpn. J. Cancer Res. , 90 , 75 – 80 ( 1999. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. ) Tontonoz , P. , Singer , S. , Forman , B. M. , Sarraf , P. , Fletcher , J. A. , Fletcher , C. D. , Burn , R. P. , Mueller , E. , Altiok , S. , Oppenheim , H. , Evans , R. M. and Spiegelman , B. M.Terminal differentiation of human liposarcoma cells induced by ligands for peroxisome proliferator‐activated receptor γ and the retinoid X receptor . Proc. Natl. Acad. Sci. USA , 94 , 237 – 241 ( 1997. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. ) Kubota , T. , Koshizuka , K. , Williamson , E. A. , Asou , H. , Said , J. W. , Holden , S. , Miyoshi , I. and Koeffler , H. P.Ligand for peroxisome proliferator‐activated receptor γ (Troglitazone) has potent antitumor effect against human prostate cancer both in vitro and in vivo . Cancer Res. , 58 , 3344 – 3352 ( 1998. ). [PubMed] [Google Scholar]
  • 9. ) Elstner , E. , Muller , C. , Koshizuka , K. , Williamson , E. A. , Park , D. , Asou , H. , Shintaku , P. , Said , J. W. , Heber , D. and Koeffler , H. P.Ligands for peroxisome proliferator‐activated receptor γ and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice . Proc. Natl. Acad. Sci. USA , 95 , 8806 – 8811 ( 1998. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. ) Mueller , E. , Sarraf , P. , Tontonoz , P. , Evans , R. M. , Martin , K. J. , Zhang , M. , Fletcher , C. , Singer , S. and Spiegelman , B. M.Terminal differentiation of human breast cancer through PPARy . Mol. Cell , 1 , 465 – 470 ( 1998. ). [DOI] [PubMed] [Google Scholar]
  • 11. ) Chang , T. H. and Szabo , E.Induction of differentiation and apoptosis by ligands of peroxisome proliferator activated receptor gamma in non‐small cell lung cancer . Cancer Res. , 60 , 1129 – 1138 ( 2000. ). [PubMed] [Google Scholar]
  • 12. ) Tsubouchi , Y. , Sano , H. , Kawahito , Y. , Mukai , S. , Yamada , R. , Kohno , M. , Inoue , K. , Hla , T. and Kondo , M.Inhibition of human lung cancer cell growth by the peroxisome proliferator‐activated receptor‐gamma agonists through induction of apoptosis . Biochem. Biophys. Res. Commun. , 270 , 400 – 405 ( 2000. ). [DOI] [PubMed] [Google Scholar]
  • 13. ) Takahashi , N. , Okumura , T. , Motomura , W. , Fujimoto , Y. , Kawabata , I. and Kohgo , Y.Activation of PPARγ inhibits cell growth and induces apoptosis in human gastric cancer cells . FEBS Lett. , 455 , 135 – 139 ( 1999. ). [DOI] [PubMed] [Google Scholar]
  • 14. ) Motomura , W. , Okumura , T. , Takahashi , N. , Obara , T. and Kohgo , Y.Activation of peroxisome proliferator‐activated receptor γ by troglitazone inhibits cell growth through the increase of p27Kip1 in human pancreatic carcinoma cells . Cancer Res. , 60 , 5558 – 5564 ( 2000. ). [PubMed] [Google Scholar]
  • 15. ) Sugimura , A. , Kiriyama , Y. , Nochi , H. , Tsuchiya , H. , Tamoto , K. , Sakurada , Y. , Ui , M. and Tokumitsu , Y.Troglitazone suppresses cell growth of myeloid leukemia cell lines by induction of p21WAFl/CIPl cyclin‐dependent kinase inhibitor . Biochem. Biophys. Res. Commun. , 261 , 833 – 837 ( 1999. ). [DOI] [PubMed] [Google Scholar]
  • 16. ) Polyak , K. , Lee , M. H. , Erdjument‐Bromage , M. , Koff , A. , Roberts , J. M. , Tempst , P. and Massague , J.Cloning of p27Kip1, a cyclin‐Cdk inhibitor and potential mediator of extracellular antimitogenic signals . Cell , 78 , 59 – 66 ( 1994. ). [DOI] [PubMed] [Google Scholar]
  • 17. ) Toyoshima , H. and Hunter , T.p27, a novel inhibitor of G1 cyclin‐Cdk protein kinase activity, is related to p21 . Cell , 78 , 67 – 74 ( 1994. ). [DOI] [PubMed] [Google Scholar]
  • 18. ) Lehmann , J. M. , Moore , L. B. , Smith‐Oliver , T. A. , Wilkison , W. O. , Willson , T. M. and Kliewer , S. A.An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator‐activated receptor gamma . J. Biol. Chem. , 270 , 12953 – 12956 ( 1995. ). [DOI] [PubMed] [Google Scholar]
  • 19. ) Chomczynski , P. and Sacchi , N.Single‐step method of RNA isolation by acid guanidinium thiocyanate‐phenolchloroform extraction . Anal. Biochem. , 162 , 156 – 159 ( 1987. ). [DOI] [PubMed] [Google Scholar]
  • 20. ) Auboeuf , D. , Rieusset , J. , Fajas , L. , Vallier , P. , Frering , V. , Riou , J. P. , Staels , B. , Auwerx , J. , Laville , M. and Vidal , H.Tissue distribution and quantification of the expression of mRNAs of proliferator‐activated receptors and liver X receptor‐α in humans . Diabetes , 46 , 1319 – 1327 ( 1997. ). [DOI] [PubMed] [Google Scholar]
  • 21. ) Wagner , S. , Bell , W. , Westermann , J. , Logan , R. P. , Bock , C. T. , Trautwein , C. , Bleck , J. S. and Manns , M. P.Regulation of gastric epithelial cell growth by Helicobacter pylori: evidence for a major role of apoptosis . Gastroenterology , 113 , 1836 – 1847 ( 1997. ). [DOI] [PubMed] [Google Scholar]
  • 22. ) Dick , L. R. , Cruikshank , A. , Destree , T. , Grenier , L. , McCormack , T. A. , Melandri , F. D. , Nunes , S. L. , Palombella , V. J. , Parent , L. A. , Plamondon , L. and Stein , R. L.Mechanistic studies on the inactivation of the proteasome by lactacystin in cultured cells . J. Biol. Chem. , 272 , 182 – 188 ( 1997. ). [DOI] [PubMed] [Google Scholar]
  • 23. ) Shirane , M. , Harumiya , Y. , Ishida , N. , Hirai , A. , Miyamoto , C. , Hatakeyama , S. , Nakayama , K. and Kitagawa , M.Down‐regulation of p27 (Kip1) by two mechanisms, ubiquitin‐mediated degradation and proteolytic processing . J. Biol. Chem. , 274 , 13886 – 13893 ( 1999. ). [DOI] [PubMed] [Google Scholar]
  • 24. ) Rao , S. , Porter , D. C. , Chen , X. , Herliczek , T. , Lowe , M. and Keyomarsi , K.Lovastatin‐mediated Gl arrest is through inhibition of the proteasome independent of hydroxymethyl glutaryl‐CoA reductase . Proc. Natl. Acad. Sci. USA , 96 , 7797 – 7802 ( 1999. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. ) Pagano , M. , Tarn , S. W. , Theodoras , A. M. , Beer , R. P. , Del , S. G. , Chau , V. , Yew , P. R. , Draetta , G. F. and Rolfe , M.Role of the ubiquitin‐proteasome pathway in regulating abundance of the cyclin‐dependent kinase inhibitor p27 . Science , 269 , 682 – 685 ( 1995. ). [DOI] [PubMed] [Google Scholar]
  • 26. ) Sato , H. , Ishihara , S. , Kawashima , K. , Moriyama , N. , Suetsugu , H. , Kazumori , H. , Okuyama , T. , Rumi , M. A. , Fukuda , R. , Nagasue , N. and Kinoshita , Y.Expression of peroxisome proliferator‐activated receptor (PPAR) y in gastric cancer and inhibitory effects of PPARγ agonists . Br. J. Cancer , 83 , 1394 – 1400 ( 2000. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. ) Braissant , O. , Foufelle , F. , Scotto , C. , Dauca , M. and Wahli , W.Differential expression of peroxisome proliferator‐activated receptors (PPARs): tissue distribution of PPAR‐alpha, ‐beta, and ‐gamma in the adult rat . Endocrinology , 137 , 354 – 366 ( 1996. ). [DOI] [PubMed] [Google Scholar]
  • 28. ) Akama , Y. , Yasui , W. , Kuniyasu , H. , Yokozaki , H. , Akagi , M. , Tahara , H. , Ishikawa , T. and Tahara , E.Genetic status and expression of the cyclin‐dependent kinase inhibitors in human gastric carcinoma cell lines . Jpn. J. Cancer Res. , 87 , 824 – 830 ( 1996. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. ) Mattar , R. , Yokozaki , H. , Yasui , W. , Ito , H. and Tahara , E.p53 gene mutations in gastric cancer cell lines . Oncology , 11 , 7 – 12 ( 1992. ). [Google Scholar]
  • 30. ) Yokozaki , H. , Kuniyasu , H. , Kitadai , Y. , Nishimura , K. , Todo , H. , Ayhan , A. , Yasui , W. , Ito , H. and Tahara , E.p53 point mutations in primary human gastric carcinomas . J. Cancer Res. Clin. Oncol. , 119 , 67 – 70 ( 1992. ). [DOI] [PubMed] [Google Scholar]
  • 31. ) Harper , J. W. , Adami , G. R. , Wei , N. , Keyomarski , K. and Elledge , S. J.The p21 Cdk‐interacting protein Cip 1 is a potent inhibitor of G1 cyclin‐dependent kinases . Cell , 75 , 805 – 816 ( 1993. ). [DOI] [PubMed] [Google Scholar]
  • 32. ) El‐Deiry , W. S. , Tokino , T. , Velculescu , V. E. , Levy , D. B. , Parsons , R. , Trent , J. M. , Lin , D. , Mercer , W. E. , Kinzler , K. W. and Vogelstein , B.WAF1, a potential mediator of p53 tumor suppression . Cell , 75 , 817 – 825 ( 1993. ). [DOI] [PubMed] [Google Scholar]

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