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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 - Korean J. Physiol. Pharmacol. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Chungnam National University College of Pharmacy, Daejeon 305-764, Korea.

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 [(3)H]-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.

No MeSH data available.


Related in: MedlinePlus

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.
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Figure 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.

Mentions: It has been known that the cells reach a restriction (check) point in late G1 phase [22]. Beyond this point, the cells are committed to DNA replication, and further cell cycle progression proceeds independently of growth factor stimulation. pRb is a key component of the molecular network controlling this restriction point. Although several CDKs are known to phosphorylate pRb, suppression of CDK2 alone may be sufficient to prevent pRb hyper-phosphorylation [2324]. Hypo-phosphorylated pRb binds to E2F family transcription factors, and thus inhibits the transcription of E2F-responsive genes necessary for cell cycle progression. To examine the underlying mechanism of the murrayafoline A-induced cell cycle arrest, we measured the expression of cyclin D1, cyclin E, CDK2, and CDK4 using immunoblotting. Fig. 5 shows that murrayafoline A significantly inhibited the PDGF-BB-induced expression of cyclin D1/E and CDK2/4 significantly and in a concentration-dependent manner. Moreover, murrayafoline A induced the concentration-dependent inhibition of PDGF-BB-induced pRb hyper-phosphorylation. The expression of proliferating cell nuclear antigen (PCNA), synthesized as a phospho-pRb-mediated gene product in early G0/G1 and S phase of the cell cycle [25], was also inhibited by murrayafoline A in the same pattern as shown for the inhibition of pRb phosphorylation. Taken together, this observation indicates that murrayafoline A inhibits cell cycle progression from G0/G1 to S phase by inhibiting the expression of cyclin D1/E, CDK2/4, and PCNA, and the phosphorylation of pRb in PDGF-BB-stimulated VSMCs.


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 - Korean J. Physiol. Pharmacol. (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4553401&req=5

Figure 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.
Mentions: It has been known that the cells reach a restriction (check) point in late G1 phase [22]. Beyond this point, the cells are committed to DNA replication, and further cell cycle progression proceeds independently of growth factor stimulation. pRb is a key component of the molecular network controlling this restriction point. Although several CDKs are known to phosphorylate pRb, suppression of CDK2 alone may be sufficient to prevent pRb hyper-phosphorylation [2324]. Hypo-phosphorylated pRb binds to E2F family transcription factors, and thus inhibits the transcription of E2F-responsive genes necessary for cell cycle progression. To examine the underlying mechanism of the murrayafoline A-induced cell cycle arrest, we measured the expression of cyclin D1, cyclin E, CDK2, and CDK4 using immunoblotting. Fig. 5 shows that murrayafoline A significantly inhibited the PDGF-BB-induced expression of cyclin D1/E and CDK2/4 significantly and in a concentration-dependent manner. Moreover, murrayafoline A induced the concentration-dependent inhibition of PDGF-BB-induced pRb hyper-phosphorylation. The expression of proliferating cell nuclear antigen (PCNA), synthesized as a phospho-pRb-mediated gene product in early G0/G1 and S phase of the cell cycle [25], was also inhibited by murrayafoline A in the same pattern as shown for the inhibition of pRb phosphorylation. Taken together, this observation indicates that murrayafoline A inhibits cell cycle progression from G0/G1 to S phase by inhibiting the expression of cyclin D1/E, CDK2/4, and PCNA, and the phosphorylation of pRb in PDGF-BB-stimulated VSMCs.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Chungnam National University College of Pharmacy, Daejeon 305-764, Korea.

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 [(3)H]-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.

No MeSH data available.


Related in: MedlinePlus