Korean J Physiol Pharmacol.  2015 Sep;19(5):421-426. 10.4196/kjpp.2015.19.5.421.

Murrayafoline A Induces a G0/G1-Phase Arrest in Platelet-Derived Growth Factor-Stimulated Vascular Smooth Muscle Cells

• Han JH
• Kim Y
• Jung SH
• Lee JJ
• Park HS
• Song GY
• Cuong NM
• Kim YH
• Myung CS

• KMID: 2285586
• DOI: http://doi.org/10.4196/kjpp.2015.19.5.421

Abstract

The increased potential for vascular smooth muscle cell (VSMC) growth is a key abnormality in the development of atherosclerosis and post-angioplasty restenosis. Abnormally high activity of platelet-derived growth factor (PDGF) is believed to play a central role in the etiology of these pathophysiological situations. Here, we investigated the anti-proliferative effects and possible mechanism(s) of murrayafoline A, a carbazole alkaloid isolated from Glycosmis stenocarpa Guillamin (Rutaceae), on PDGF-BB-stimulated VSMCs. Murrayafoline A inhibited the PDGF-BB-stimulated proliferation of VSMCs in a concentration-dependent manner, as measured using a non-radioactive colorimetric WST-1 assay and direct cell counting. Furthermore, murrayafoline A suppressed the PDGF-BB-stimulated progression through G0/G1 to S phase of the cell cycle, as measured by [3H]-thymidine incorporation assay and cell cycle progression analysis. This anti-proliferative action of murrayafoline A, arresting cell cycle progression at G0/G1 phase in PDGF-BB-stimulated VSMCs, was mediated via down-regulation of the expression of cyclin D1, cyclin E, cyclin-dependent kinase (CDK)2, CDK4, and proliferating cell nuclear antigen (PCNA), and the phosphorylation of retinoblastoma protein (pRb). These results indicate that murrayafoline A may be useful in preventing the progression of vascular complications such as restenosis after percutaneous transluminal coronary angioplasty and atherosclerosis.

Keyword

Murrayafoline A; Platelet-derived growth factor; Proliferation; Vascular smooth muscle cells; Cell cycle

MeSH Terms

Angioplasty, Balloon, Coronary
Atherosclerosis
Cell Count
Cell Cycle
Cyclin D1
Cyclin E
Cyclins
Down-Regulation
Muscle, Smooth, Vascular*
Phosphorylation
Phosphotransferases
Platelet-Derived Growth Factor
Proliferating Cell Nuclear Antigen
Retinoblastoma Protein
Rutaceae
S Phase
Cyclin D1
Cyclin E
Cyclins
Phosphotransferases
Platelet-Derived Growth Factor
Proliferating Cell Nuclear Antigen
Retinoblastoma Protein

Figure

• 

  Fig. 1 Effects of murrayafoline A on VSMC proliferation and viability. VSMCs cultured in serum-free medium were stimulated with 50 ng/ml PDGF-BB for 24 h and the effects of various concentrations of murrayafoline A (1-5 µM) on cell proliferation and viability were measured as described in the Experimental Section. (A) Optical densities at 450 nm, as determined in the WST-1 assay (n=3). (B) Cell numbers counted using a hemocytometer (n=4). (C) Viability as determined by the WST-1 assay (n=4) for various incubation times (0-48 h). All values are expressed as means±SEM. Statistical differences from the PDGF-BB control (PDGF-BB-stimulated, but no murrayafoline A) are indicated by *p<0.05 and **p<0.01.

• 

  Fig. 2 Effects of murrayafoline A on the PDGF-BB-stimulated activation of PDGF-Rβ, PLCγ1, Akt, ERK1/2, and STAT3. Quiescent VSMCs cultured in serum-free medium were stimulated with 50 ng/ml PDGF-BB, and the effects of various concentrations of murrayafoline A (1-5 µM) in changing the PDGF-BB-induced phosphorylation of PDGF-Rβ, PLCγ1, Akt, ERK1/2, and STAT3 were measured as described in the Experimental Section.

• 

  Fig. 3 Effects of murrayafoline A on DNA synthesis. VSMCs cultured in serum-starved medium were stimulated with 50 ng/ml PDGF-BB, and the effects of various concentrations of murrayafoline A (1-5 µM) on [3H]-thymidine incorporation were measured after the addition of 2 µCi/ml [3H]-thymidine, as described in the Experimental Section. The values are expressed as means±SEM (n=4), and statistical differences from the PDGF-BB control (PDGF-BB-stimulated, but no murrayafoline A) are indicated by **p<0.01.

• 

  Fig. 4 Effects of murrayafoline A on cell cycle progression. VSMCs cultured in serum-starved medium were stimulated with 50 ng/ml PDGF-BB, and the effects of various concentrations of murrayafoline A (1-5 µM) on cell cycle progression were measured as described in the Materials and methods.

• 

  Fig. 5 Effects of murrayafoline A on the inhibition of cell cycle regulatory proteins. Quiescent VSMCs cultured in serum-free medium were stimulated with PDGF-BB to express cell cycle regulatory proteins, and the effects of murrayafoline A on the expression of cyclin E, CDK2, cyclin D1, CDK4, and PCNA, and activation of pRb were assessed as described in the Experimental Section. β-Actin was used for normalization. Immunoblots were analyzed by densitometry and the values are given based on the control of 1.0. The results are an average of four similar experiments, expressed as means±SEM. The insets display representative blots of four similar independent experiments. Statistical differences from the PDGF-BB control (PDGF-BB-stimulated, but no murrayafoline A) are indicated by *p<0.05 or **p<0.01.

Reference

1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993; 362:801–809. PMID: 8479518.

2. Raines EW, Ross R. Smooth muscle cells and the pathogenesis of the lesions of atherosclerosis. Br Heart J. 1993; 69(1 Suppl):S30–S37. PMID: 8427762.

3. Millette E, Rauch BH, Kenagy RD, Daum G, Clowes AW. Platelet-derived growth factor-BB transactivates the fibroblast growth factor receptor to induce proliferation in human smooth muscle cells. Trends Cardiovasc Med. 2006; 16:25–28. PMID: 16387627.

4. Bornfeldt KE, Raines EW, Graves LM, Skinner MP, Krebs EG, Ross R. Platelet-derived growth factor. Distinct signal transduction pathways associated with migration versus proliferation. Ann N Y Acad Sci. 1995; 766:416–430. PMID: 7486687.

5. Sachinidis A, Locher R, Hoppe J, Vetter W. The platelet-derived growth factor isomers, PDGF-AA, PDGF-AB and PDGF-BB, induce contraction of vascular smooth muscle cells by different intracellular mechanisms. FEBS Lett. 1990; 275:95–98. PMID: 2262007.

6. Kondo T, Konishi F, Inui H, Inagami T. Differing signal transductions elicited by three isoforms of platelet-derived growth factor in vascular smooth muscle cells. J Biol Chem. 1993; 268:4458–4464. PMID: 8440728.

7. Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, Cobb MH. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev. 2001; 22:153–183. PMID: 11294822.

8. Bowman T, Broome MA, Sinibaldi D, Wharton W, Pledger WJ, Sedivy JM, Irby R, Yeatman T, Courtneidge SA, Jove R. Stat3-mediated Myc expression is required for Src transformation and PDGF-induced mitogenesis. Proc Natl Acad Sci U S A. 2001; 98:7319–7324. PMID: 11404481.

9. Simon AR, Takahashi S, Severgnini M, Fanburg BL, Cochran BH. Role of the JAK-STAT pathway in PDGF-stimulated proliferation of human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol. 2002; 282:L1296–L1304. PMID: 12003786.

10. Ahn HY, Hadizadeh KR, Seul C, Yun YP, Vetter H, Sachinidis A. Epigallocathechin-3 gallate selectively inhibits the PDGF-BB-induced intracellular signaling transduction pathway in vascular smooth muscle cells and inhibits transformation of sis-transfected NIH 3T3 fibroblasts and human glioblastoma cells (A172). Mol Biol Cell. 1999; 10:1093–1104. PMID: 10198059.

11. Heldin CH, Ostman A, Rönnstrand L. Signal transduction via platelet-derived growth factor receptors. Biochim Biophys Acta. 1998; 1378:F79–F113. PMID: 9739761.

12. Braun-Dullaeus RC, Mann MJ, Sedding DG, Sherwood SW, von der Leyen HE, Dzau VJ. Cell cycle-dependent regulation of smooth muscle cell activation. Arterioscler Thromb Vasc Biol. 2004; 24:845–850. PMID: 15016640.

13. Dzau VJ, Braun-Dullaeus RC, Sedding DG. Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies. Nat Med. 2002; 8:1249–1256. PMID: 12411952.

14. Kim TJ, Lim Y, Kim DW, Kwon JS, Son JH, Jin YR, Son DJ, Jung JC, Avery MA, Hong JT, Yun YP. Epothilone D, a microtubule-stabilizing compound, inhibits neointimal hyperplasia after rat carotid artery injury by cell cycle arrest via regulation of G1-checkpoint proteins. Vascul Pharmacol. 2007; 47:229–237. PMID: 17706465.

15. Cuong NM, Wilhelm H, Porzel A, Arnold N, Wessjohann L. 1-O-Substituted derivatives of murrayafoline A and their antifungal properties. Nat Prod Res. 2008; 22:1428–1432. PMID: 19023805.

16. Itoigawa M, Kashiwada Y, Ito C, Furukawa H, Tachibana Y, Bastow KF, Lee KH. Antitumor agents. 203. Carbazole alkaloid murrayaquinone A and related synthetic carbazolequinones as cytotoxic agents. J Nat Prod. 2000; 63:893–897. PMID: 10924160.

17. Cuong NM, Hung TQ, Sung TV, Taylor WC. A new dimeric carbazole alkaloid from Glycosmis stenocarpa roots. Chem Pharm Bull (Tokyo). 2004; 52:1175–1178. PMID: 15467229.

18. Chamley JH, Campbell GR, McConnell JD, Gröschel-Stewart U. Comparison of vascular smooth muscle cells from adult human, monkey and rabbit in primary culture and in subculture. Cell Tissue Res. 1977; 177:503–522. PMID: 402216.

19. Eid SY, El-Readi MZ, Wink M. Digitonin synergistically enhances the cytotoxicity of plant secondary metabolites in cancer cells. Phytomedicine. 2012; 19:1307–1314. PMID: 23062361.

20. Lee JJ, Yu JY, Zhang WY, Kim TJ, Lim Y, Kwon JS, Kim DW, Myung CS, Yun YP. Inhibitory effect of fenofibrate on neointima hyperplasia via G₀/G₁ arrest of cell proliferation. Eur J Pharmacol. 2011; 650:342–349. PMID: 21040719.

21. Schwaiberger AV, Heiss EH, Cabaravdic M, Oberan T, Zaujec J, Schachner D, Uhrin P, Atanasov AG, Breuss JM, Binder BR, Dirsch VM. Indirubin-3'-monoxime blocks vascular smooth muscle cell proliferation by inhibition of signal transducer and activator of transcription 3 signaling and reduces neointima formation in vivo. Arterioscler Thromb Vasc Biol. 2010; 30:2475–2481. PMID: 20847306.

22. Besson A, Dowdy SF, Roberts JM. CDK inhibitors: cell cycle regulators and beyond. Dev Cell. 2008; 14:159–169. PMID: 18267085.

23. Akiyama T, Ohuchi T, Sumida S, Matsumoto K, Toyoshima K. Phosphorylation of the retinoblastoma protein by cdk2. Proc Natl Acad Sci U S A. 1992; 89:7900–7904. PMID: 1518810.

24. Connell-Crowley L, Elledge SJ, Harper JW. G1 cyclin-dependent kinases are sufficient to initiate DNA synthesis in quiescent human fibroblasts. Curr Biol. 1998; 8:65–68. PMID: 9427630.

25. Tomita H, Osanai T, Toki T, Maeda N, Murakami R, Chen Z, Yamabe H, Osawa H, Yasujima M, Okumura K. Roxithromycin is an inhibitor of human coronary artery smooth muscle cells proliferation: a potential ability to prevent coronary heart disease. Atherosclerosis. 2005; 182:87–95. PMID: 16115478.

26. Yoshida Y, Mitsumata M, Ling G, Jiang J, Shu Q. Migration of medial smooth muscle cells to the intima after balloon injury. Ann N Y Acad Sci. 1997; 811:459–470. PMID: 9186623.

27. Jung F, Haendeler J, Goebel C, Zeiher AM, Dimmeler S. Growth factor-induced phosphoinositide 3-OH kinase/Akt phosphorylation in smooth muscle cells: induction of cell proliferation and inhibition of cell death. Cardiovasc Res. 2000; 48:148–157. PMID: 11033117.

28. Ross R. Cell biology of atherosclerosis. Annu Rev Physiol. 1995; 57:791–804. PMID: 7778883.

29. Liang KW, Yin SC, Ting CT, Lin SJ, Hsueh CM, Chen CY, Hsu SL. Berberine inhibits platelet-derived growth factor-induced growth and migration partly through an AMPK-dependent pathway in vascular smooth muscle cells. Eur J Pharmacol. 2008; 590:343–354. PMID: 18590725.

30. Dong LH, Wen JK, Miao SB, Jia Z, Hu HJ, Sun RH, Wu Y, Han M. Baicalin inhibits PDGF-BB-stimulated vascular smooth muscle cell proliferation through suppressing PDGFRβ-ERK signaling and increase in p27 accumulation and prevents injury-induced neointimal hyperplasia. Cell Res. 2010; 20:1252–1262. PMID: 20661261.