Inhibition of Vascular Smooth Muscle Cell Proliferation by Gentiana lutea Root Extracts

Gentiana lutea belonging to the Gentianaceae family of flowering plants are routinely used in traditional Serbian medicine for their beneficial gastro-intestinal and anti-inflammatory properties. The aim of the study was to determine whether aqueous root extracts of Gentiana lutea consisting of gentiopicroside, gentisin, bellidifolin-8-O-glucoside, demethylbellidifolin-8-O-glucoside, isovitexin, swertiamarin and amarogentin prevents proliferation of aortic smooth muscle cells in response to PDGF-BB. Cell proliferation and cell cycle analysis were performed based on alamar blue assay and propidium iodide labeling respectively. In primary cultures of rat aortic smooth muscle cells (RASMCs), PDGF-BB (20 ng/ml) induced a two-fold increase in cell proliferation which was significantly blocked by the root extract (1 mg/ml). The root extract also prevented the S-phase entry of synchronized cells in response to PDGF. Furthermore, PDGF-BB induced ERK1/2 activation and consequent increase in cellular nitric oxide (NO) levels were also blocked by the extract. These effects of extract were due to blockade of PDGF-BB induced expression of iNOS, cyclin D1 and proliferating cell nuclear antigen (PCNA). Docking analysis of the extract components on MEK1, the upstream ERK1/2 activating kinase using AutoDock4, indicated a likely binding of isovitexin to the inhibitor binding site of MEK1. Experiments performed with purified isovitexin demonstrated that it successfully blocks PDGF-induced ERK1/2 activation and proliferation of RASMCs in cell culture. Thus, Gentiana lutea can provide novel candidates for prevention and treatment of atherosclerosis

The consequences of soluble epoxide hydrolase deletion on tumorigenesis and metastasis in a mouse model of breast cancer

Epoxides and diols of polyunsaturated fatty acids (PUFAs) are bioactive and can influence processes such as tumor cell proliferation and angiogenesis. Studies with inhibitors of the soluble epoxide hydrolase (sEH) in animals overexpressing cytochrome P450 enzymes or following the systemic administration of specific epoxides revealed a markedly increased incidence of tumor metastases. To determine whether PUFA epoxides increased metastases in a model of spontaneous breast cancer, sEH-/- mice were crossed onto the polyoma middle T oncogene (PyMT) background. We found that the deletion of the sEH accelerated the growth of primary tumors and increased both the tumor macrophage count and angiogenesis. There were small differences in the epoxide/diol content of tumors, particularly in epoxyoctadecamonoenic acid versus dihydroxyoctadecenoic acid, and marked changes in the expression of proteins linked with cell proliferation and metabolism. However, there was no consequence of sEH inhibition on the formation of metastases in the lymph node or lung. Taken together, our results confirm previous reports of increased tumor growth in animals lacking sEH but fail to substantiate reports of enhanced lymph node or pulmonary metastases

Effect of PDGF-BB (20 ng/ml) on ERK1/2 and NO signaling.

<p>A) Time course of ERK1/2 activation in response to PDGF, B&C) Time course of generation of intracellular nitric oxide in response to PDGF, D) Activation of eNOS through phosphorylation of Ser¹¹⁷⁷ residue in response to PDGF and E) PDGF-induced expression of iNOS. Bar graphs summarize data for a minimum of four independent experiments. *P<0.05, **P<0.01 and ***P<0.001 versus control.</p

Role of ERK1/2-NO axis on PDGF-induced proliferation of RASMCs and effect of <i>G lutea</i> on PDGF induced NO index.

<p>A) Effect of ERK inhibitor 328000 (1 µmole/L) on PDGF induced cell proliferation, B) Effect of NOS inhibitor L-NAME (10 µmole/L) on PDGF induced cell proliferation, C) Effect of <i>G lutea</i> extract on generation of cellular NO in response to PDGF. *P<0.05 versus control and †<0.05, ††<0.01 and †††<0.001 versus corresponding PDGF treatment.</p

Effect of <i>G lutea</i> extract on PDGF-induced cell signaling in RASMCs.

<p>A) PDGFR-β phosphorylation, B) ERK1/2 activation, C) Phosphorylation of IKKα and D&E) Representative blot and bar graph indicating expression of cyclin D1, PCNA and iNOS for a minimum of three independent experiments. *<0.05, **P<0.01 and ***P<0.001 versus control and ††<0.01 and †††<0.001 versus PDGF treatment.</p

Effect of isovitexin on PDGF- induced RASMC proliferation.

<p>A) Docking analysis revealing binding pocket of isovitexin on MEK1, B) Effect of isovitexin (0.5–10 µmol/L) on PDGF induced ERK1/2 activation and cell cycle progress and C) Effect of isovitexin on PDGF induced cell proliferation measured through alamar blue assay. Bar graphs summarize data for a minimum of three independent experiments. **P<0.01 versus control and ††<0.01 and †††<0.001 versus PDGF treatment.</p

Predicted binding energies for <i>G lutea</i> constituents and known MEK1. inhibitors.

<p>Predicted binding energies for <i>G lutea</i> constituents and known MEK1. inhibitors.</p

IC₅₀ values for the <i>G. lutea</i> extract (1 mg/ml) on smooth muscle cells.

<p>A) Primary cultures of rat aortic smooth muscle cells (RASMCs), B) Rat aortic smooth muscle cell line A7r5 and C) Human aortic smooth muscle cell line ATCC-CRL-1999.</p

Summary figure depicting the pathway blocked by <i>G lutea</i> extract to prevent PDGF-induced RASMC proliferation.

<p>Summary figure depicting the pathway blocked by <i>G lutea</i> extract to prevent PDGF-induced RASMC proliferation.</p

Effect of <i>G. lutea</i> extract (1 mg/ml) on proliferation and apoptosis.

<p>A) Effect of extract on PDGF-BB (20 ng/ml, 24 hours) induced proliferation of primary cultures of rat aortic smooth muscle cells (RASMCs), B) A7r5 and C) percentage of apoptotic RASMCs in response to experimental treatments. *P<0.05 and ***P<0.001 versus control and †P<0.05 and †††<0.001 versus PDGF treatment.</p