Skip to main content
NCBI home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 1996 Jun;118(4):915–922. doi: 10.1111/j.1476-5381.1996.tb15486.x

Antiproliferative and c-myc mRNA suppressive effect of tranilast on newborn human vascular smooth muscle cells in culture.

K Miyazawa 1, S Hamano 1, A Ujiie 1
PMCID: PMC1909533  PMID: 8799562

Abstract

1. Newborn human vascular smooth muscle cells (VSMCs) proliferated faster and were more sensitive to platelet-derived growth factor-BB (PDGF-BB) than those from adults. In this study, we investigated mechanism of the inhibitory effect of tranilast on PDGF-BB-induced proliferation of VSMCs from newborns. 2. Tranilast (30-300 microM) concentration-dependently inhibited the VSMC proliferation in randomly growing cultures stimulated with PDGF-BB. 3. Tranilast (30-300 microM) concentration-dependently inhibited the [3H]-thymidine incorporation into DNA in VSMCs that had been synchronized by 48 h serum depletion and then stimulated by addition of PDGF-BB. However, tranilast had little influence on unscheduled DNA synthesis in quiescent cells or on RNA and protein synthesis, unlike aphidicolin, actimomycin D, and cycloheximide. 4. In synchronized VSMC cultures, tranilast still inhibited the PDGF-BB-induced DNA synthesis even when added 18 h after stimulation of the quiescent cells. The mode of the antiproliferative action of tranilast was different from that of NiCl2, genistein, or staurosporin. In addition, flow cytometry of synchronized VSMCs treated with tranilast revealed a blockade of PDGF-inducible cell-cycle progression at the G1/S checkpoint. 5. Northern blotting showed that tranilast (30-300 microM) concentration-dependently suppressed constitutive c-myc mRNA expression even when added 18 h after PDGF-BB-stimulation of quiescent VSMCs. Tranilast still had an inhibitory effect on the induction of c-myc mRNA when de novo protein synthesis was inhibited by cycloheximide and did not shorten the degradation of c-myc mRNA at the post-transcriptional level, demonstrating that tranilast directly inhibited c-myc mRNA expression at the transcriptional level. 6. These results suggest that the inhibitory effect of tranilast on PDGF-BB-induced proliferation is due to S-phase blockade and may be, at least in part, involved in the direct suppression of c-myc gene expression. Tranilast did not cause cell toxicity and may therefore hold promising potential for the prevention of vascular proliferative diseases.

Full text

PDF
915

Images in this article

918Figure 3
on p.918
919Figure 4
on p.919

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aikawa M., Sivam P. N., Kuro-o M., Kimura K., Nakahara K., Takewaki S., Ueda M., Yamaguchi H., Yazaki Y., Periasamy M. Human smooth muscle myosin heavy chain isoforms as molecular markers for vascular development and atherosclerosis. Circ Res. 1993 Dec;73(6):1000–1012. doi: 10.1161/01.res.73.6.1000. [DOI] [PubMed] [Google Scholar]
  2. Akiyama T., Ishida J., Nakagawa S., Ogawara H., Watanabe S., Itoh N., Shibuya M., Fukami Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem. 1987 Apr 25;262(12):5592–5595. [PubMed] [Google Scholar]
  3. Azuma H., Banno K., Yoshimura T. Pharmacological properties of N-(3',4'-dimethoxycinnamoyl) anthranilic acid (N-5'), a new anti-atopic agent. Br J Pharmacol. 1976 Dec;58(4):483–488. doi: 10.1111/j.1476-5381.1976.tb08614.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bennett M. R., Anglin S., McEwan J. R., Jagoe R., Newby A. C., Evan G. I. Inhibition of vascular smooth muscle cell proliferation in vitro and in vivo by c-myc antisense oligodeoxynucleotides. J Clin Invest. 1994 Feb;93(2):820–828. doi: 10.1172/JCI117036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Biro S., Fu Y. M., Yu Z. X., Epstein S. E. Inhibitory effects of antisense oligodeoxynucleotides targeting c-myc mRNA on smooth muscle cell proliferation and migration. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):654–658. doi: 10.1073/pnas.90.2.654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  7. Clowes A. W., Schwartz S. M. Significance of quiescent smooth muscle migration in the injured rat carotid artery. Circ Res. 1985 Jan;56(1):139–145. doi: 10.1161/01.res.56.1.139. [DOI] [PubMed] [Google Scholar]
  8. Dartsch P. C., Weiss H. D., Betz E. Human vascular smooth muscle cells in culture: growth characteristics and protein pattern by use of serum-free media supplements. Eur J Cell Biol. 1990 Apr;51(2):285–294. [PubMed] [Google Scholar]
  9. Dean M., Levine R. A., Ran W., Kindy M. S., Sonenshein G. E., Campisi J. Regulation of c-myc transcription and mRNA abundance by serum growth factors and cell contact. J Biol Chem. 1986 Jul 15;261(20):9161–9166. [PubMed] [Google Scholar]
  10. Edelman E. R., Simons M., Sirois M. G., Rosenberg R. D. c-myc in vasculoproliferative disease. Circ Res. 1995 Feb;76(2):176–182. doi: 10.1161/01.res.76.2.176. [DOI] [PubMed] [Google Scholar]
  11. Elledge S. J., Richman R., Hall F. L., Williams R. T., Lodgson N., Harper J. W. CDK2 encodes a 33-kDa cyclin A-associated protein kinase and is expressed before CDC2 in the cell cycle. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2907–2911. doi: 10.1073/pnas.89.7.2907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Epstein S. E., Speir E., Unger E. F., Guzman R. J., Finkel T. The basis of molecular strategies for treating coronary restenosis after angioplasty. J Am Coll Cardiol. 1994 May;23(6):1278–1288. doi: 10.1016/0735-1097(94)90368-9. [DOI] [PubMed] [Google Scholar]
  13. Ferns G. A., Raines E. W., Sprugel K. H., Motani A. S., Reidy M. A., Ross R. Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF. Science. 1991 Sep 6;253(5024):1129–1132. doi: 10.1126/science.1653454. [DOI] [PubMed] [Google Scholar]
  14. Fujita H., Shimokado K., Yutani C., Takaichi S., Masuda J., Ogata J. Human neonatal and adult vascular smooth muscle cells in culture. Exp Mol Pathol. 1993 Feb;58(1):25–39. doi: 10.1006/exmp.1993.1003. [DOI] [PubMed] [Google Scholar]
  15. Glagov S. Intimal hyperplasia, vascular modeling, and the restenosis problem. Circulation. 1994 Jun;89(6):2888–2891. doi: 10.1161/01.cir.89.6.2888. [DOI] [PubMed] [Google Scholar]
  16. Grotendorst G. R., Chang T., Seppä H. E., Kleinman H. K., Martin G. R. Platelet-derived growth factor is a chemoattractant for vascular smooth muscle cells. J Cell Physiol. 1982 Nov;113(2):261–266. doi: 10.1002/jcp.1041130213. [DOI] [PubMed] [Google Scholar]
  17. Hann S. R., Thompson C. B., Eisenman R. N. c-myc oncogene protein synthesis is independent of the cell cycle in human and avian cells. 1985 Mar 28-Apr 3Nature. 314(6009):366–369. doi: 10.1038/314366a0. [DOI] [PubMed] [Google Scholar]
  18. Hanson K. D., Shichiri M., Follansbee M. R., Sedivy J. M. Effects of c-myc expression on cell cycle progression. Mol Cell Biol. 1994 Sep;14(9):5748–5755. doi: 10.1128/mcb.14.9.5748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ip J. H., Fuster V., Badimon L., Badimon J., Taubman M. B., Chesebro J. H. Syndromes of accelerated atherosclerosis: role of vascular injury and smooth muscle cell proliferation. J Am Coll Cardiol. 1990 Jun;15(7):1667–1687. doi: 10.1016/0735-1097(90)92845-s. [DOI] [PubMed] [Google Scholar]
  20. Jawien A., Bowen-Pope D. F., Lindner V., Schwartz S. M., Clowes A. W. Platelet-derived growth factor promotes smooth muscle migration and intimal thickening in a rat model of balloon angioplasty. J Clin Invest. 1992 Feb;89(2):507–511. doi: 10.1172/JCI115613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kelly K., Cochran B. H., Stiles C. D., Leder P. Cell-specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth factor. Cell. 1983 Dec;35(3 Pt 2):603–610. doi: 10.1016/0092-8674(83)90092-2. [DOI] [PubMed] [Google Scholar]
  22. Kimchi A. Cytokine triggered molecular pathways that control cell cycle arrest. J Cell Biochem. 1992 Sep;50(1):1–9. doi: 10.1002/jcb.240500102. [DOI] [PubMed] [Google Scholar]
  23. Kobayashi S., Nishimura J., Kanaide H. Cytosolic Ca2+ transients are not required for platelet-derived growth factor to induce cell cycle progression of vascular smooth muscle cells in primary culture. Actions of tyrosine kinase. J Biol Chem. 1994 Mar 25;269(12):9011–9018. [PubMed] [Google Scholar]
  24. Komatsu H., Kojima M., Tsutsumi N., Hamano S., Kusama H., Ujiie A., Ikeda S., Nakazawa M. Study of the mechanism of inhibitory action of tranilast on chemical mediator release. Jpn J Pharmacol. 1988 Jan;46(1):43–51. doi: 10.1254/jjp.46.43. [DOI] [PubMed] [Google Scholar]
  25. Koyama N., Hart C. E., Clowes A. W. Different functions of the platelet-derived growth factor-alpha and -beta receptors for the migration and proliferation of cultured baboon smooth muscle cells. Circ Res. 1994 Oct;75(4):682–691. doi: 10.1161/01.res.75.4.682. [DOI] [PubMed] [Google Scholar]
  26. Lange R. A., Willard J. E., Hillis L. D. Southwestern internal medicine conference: restenosis: the Achilles heel of coronary angioplasty. Am J Med Sci. 1993 Oct;306(4):265–275. doi: 10.1097/00000441-199310000-00010. [DOI] [PubMed] [Google Scholar]
  27. Leclerc G., Isner J. M., Kearney M., Simons M., Safian R. D., Baim D. S., Weir L. Evidence implicating nonmuscle myosin in restenosis. Use of in situ hybridization to analyze human vascular lesions obtained by directional atherectomy. Circulation. 1992 Feb;85(2):543–553. doi: 10.1161/01.cir.85.2.543. [DOI] [PubMed] [Google Scholar]
  28. Li Z., Alavi M. Z., Moore S. The proliferation of neointimal smooth muscle cells cultured from rabbit aortic explants 15 weeks after de-endothelialization by a balloon catheter. Int J Exp Pathol. 1994 Jun;75(3):169–177. [PMC free article] [PubMed] [Google Scholar]
  29. Libby P., O'Brien K. V. Culture of quiescent arterial smooth muscle cells in a defined serum-free medium. J Cell Physiol. 1983 May;115(2):217–223. doi: 10.1002/jcp.1041150217. [DOI] [PubMed] [Google Scholar]
  30. MacLeod D. C., Strauss B. H., de Jong M., Escaned J., Umans V. A., van Suylen R. J., Verkerk A., de Feyter P. J., Serruys P. W. Proliferation and extracellular matrix synthesis of smooth muscle cells cultured from human coronary atherosclerotic and restenotic lesions. J Am Coll Cardiol. 1994 Jan;23(1):59–65. doi: 10.1016/0735-1097(94)90502-9. [DOI] [PubMed] [Google Scholar]
  31. Majesky M. W., Benditt E. P., Schwartz S. M. Expression and developmental control of platelet-derived growth factor A-chain and B-chain/Sis genes in rat aortic smooth muscle cells. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1524–1528. doi: 10.1073/pnas.85.5.1524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Majesky M. W., Reidy M. A., Bowen-Pope D. F., Hart C. E., Wilcox J. N., Schwartz S. M. PDGF ligand and receptor gene expression during repair of arterial injury. J Cell Biol. 1990 Nov;111(5 Pt 1):2149–2158. doi: 10.1083/jcb.111.5.2149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. March K. L., Wilensky R. L., Roeske R. W., Hathaway D. R. Effects of thiol protease inhibitors on cell cycle and proliferation of vascular smooth muscle cells in culture. Circ Res. 1993 Feb;72(2):413–423. doi: 10.1161/01.res.72.2.413. [DOI] [PubMed] [Google Scholar]
  34. Miano J. M., Vlasic N., Tota R. R., Stemerman M. B. Smooth muscle cell immediate-early gene and growth factor activation follows vascular injury. A putative in vivo mechanism for autocrine growth. Arterioscler Thromb. 1993 Feb;13(2):211–219. doi: 10.1161/01.atv.13.2.211. [DOI] [PubMed] [Google Scholar]
  35. Miyazawa K., Kikuchi S., Fukuyama J., Hamano S., Ujiie A. Inhibition of PDGF- and TGF-beta 1-induced collagen synthesis, migration and proliferation by tranilast in vascular smooth muscle cells from spontaneously hypertensive rats. Atherosclerosis. 1995 Dec;118(2):213–221. doi: 10.1016/0021-9150(95)05607-6. [DOI] [PubMed] [Google Scholar]
  36. Pazin M. J., Williams L. T. Triggering signaling cascades by receptor tyrosine kinases. Trends Biochem Sci. 1992 Oct;17(10):374–378. doi: 10.1016/0968-0004(92)90003-r. [DOI] [PubMed] [Google Scholar]
  37. Pukac L. A., Castellot J. J., Jr, Wright T. C., Jr, Caleb B. L., Karnovsky M. J. Heparin inhibits c-fos and c-myc mRNA expression in vascular smooth muscle cells. Cell Regul. 1990 Apr;1(5):435–443. doi: 10.1091/mbc.1.5.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rabbitts P. H., Watson J. V., Lamond A., Forster A., Stinson M. A., Evan G., Fischer W., Atherton E., Sheppard R., Rabbitts T. H. Metabolism of c-myc gene products: c-myc mRNA and protein expression in the cell cycle. EMBO J. 1985 Aug;4(8):2009–2015. doi: 10.1002/j.1460-2075.1985.tb03885.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993 Apr 29;362(6423):801–809. doi: 10.1038/362801a0. [DOI] [PubMed] [Google Scholar]
  40. Rothman A., Wolner B., Button D., Taylor P. Immediate-early gene expression in response to hypertrophic and proliferative stimuli in pulmonary arterial smooth muscle cells. J Biol Chem. 1994 Mar 4;269(9):6399–6404. [PubMed] [Google Scholar]
  41. Rönnstrand L., Mori S., Arridsson A. K., Eriksson A., Wernstedt C., Hellman U., Claesson-Welsh L., Heldin C. H. Identification of two C-terminal autophosphorylation sites in the PDGF beta-receptor: involvement in the interaction with phospholipase C-gamma. EMBO J. 1992 Nov;11(11):3911–3919. doi: 10.1002/j.1460-2075.1992.tb05484.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Shichiri M., Hanson K. D., Sedivy J. M. Effects of c-myc expression on proliferation, quiescence, and the G0 to G1 transition in nontransformed cells. Cell Growth Differ. 1993 Feb;4(2):93–104. [PubMed] [Google Scholar]
  43. Simons M., Leclerc G., Safian R. D., Isner J. M., Weir L., Baim D. S. Relation between activated smooth-muscle cells in coronary-artery lesions and restenosis after atherectomy. N Engl J Med. 1993 Mar 4;328(9):608–613. doi: 10.1056/NEJM199303043280903. [DOI] [PubMed] [Google Scholar]
  44. Studzinski G. P., Shankavaram U. T., Moore D. C., Reddy P. V. Association of c-myc protein with enzymes of DNA replication in high molecular weight fractions from mammalian cells. J Cell Physiol. 1991 Jun;147(3):412–419. doi: 10.1002/jcp.1041470305. [DOI] [PubMed] [Google Scholar]
  45. Suzawa H., Kikuchi S., Arai N., Koda A. The mechanism involved in the inhibitory action of tranilast on collagen biosynthesis of keloid fibroblasts. Jpn J Pharmacol. 1992 Oct;60(2):91–96. doi: 10.1254/jjp.60.91. [DOI] [PubMed] [Google Scholar]
  46. Tamaoki T., Nomoto H., Takahashi I., Kato Y., Morimoto M., Tomita F. Staurosporine, a potent inhibitor of phospholipid/Ca++dependent protein kinase. Biochem Biophys Res Commun. 1986 Mar 13;135(2):397–402. doi: 10.1016/0006-291x(86)90008-2. [DOI] [PubMed] [Google Scholar]
  47. Thompson C. B., Challoner P. B., Neiman P. E., Groudine M. Levels of c-myc oncogene mRNA are invariant throughout the cell cycle. 1985 Mar 28-Apr 3Nature. 314(6009):363–366. doi: 10.1038/314363a0. [DOI] [PubMed] [Google Scholar]
  48. Tso J. Y., Sun X. H., Kao T. H., Reece K. S., Wu R. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res. 1985 Apr 11;13(7):2485–2502. doi: 10.1093/nar/13.7.2485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wilcox J. N., Smith K. M., Williams L. T., Schwartz S. M., Gordon D. Platelet-derived growth factor mRNA detection in human atherosclerotic plaques by in situ hybridization. J Clin Invest. 1988 Sep;82(3):1134–1143. doi: 10.1172/JCI113671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wisdom R., Lee W. The protein-coding region of c-myc mRNA contains a sequence that specifies rapid mRNA turnover and induction by protein synthesis inhibitors. Genes Dev. 1991 Feb;5(2):232–243. doi: 10.1101/gad.5.2.232. [DOI] [PubMed] [Google Scholar]

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES