Un-Ho Jin, Seok-Jong Suh, Hyen Wook Chang, Jon-Keun Son, Seung Ho Lee, Kun-Ho Son, Young-Chae Change, Cheorl-Ho Kim
Abstract
Migration of smooth muscle cells (SMC) plays an important role in both normal angiogenesis and disease related remodeling that causes malformations, arteriosclerosis, and restenosis. Matrix Metalloproteinases (MMPs) play an important role in SMC proliferation and migration, and as such, the expression of MMPs have been thought to be a major indicator of angiogenesis. In the study, tanshinone IIA is examined as a potential inhibitor of SMC migration and inhibitor of the expression of MMPs, specifically MMP-9 . Tanshinone IIA is derived from a traditional herbal medicine used to treat cardiovascular diseases, and has been known to (as part of the hydrophobic pharmacological component of S. miltiorrhiza Bunge, the actual drug) effective treat atherosclerosis. Through the study, it was found that the drug inhibited IkBalpha phosphorylation, p65 nuclear translocation via inhibition of AKT phosphorylation, TNF-alpha-induced ERK and c-jun phosphorylation, but not other MAPKs (ex: JNK and p38). The study gave evidence that tanshinone IIA may one day be used therapeutically in the inhibition of SMC migration.
Materials and Methods
The tanshinone IIA, which was derived from the dried root of S. miltiorrhiza Bunge via an extraction with methanol, was dissolved in dimethyl sulfoxide (DMSO). Recombinant human TNF-alpha was obtained from R&D systems, monoclonal and polyclonal antibodies for p-ERK1/2, p-SAPK/JNK, and p-p38 were obtained from New England Biolabs, [γ32]P-ATP was obtained from Amersham Pharmacia Biotech, and NF-κB (p65), p-c-jun, p-IκB(, p-AKT, and AKT antibodies were purchased from Santa Cruz Biotechnology.
Human aortic smooth muscle cells (HASMCs) were purchased from Bio-Whittaker, and cultured in SMC growth medium-2 containing 10% FBS, 2ng/ml human basic fibroblast growth factor, 0.5 ng/ml human EGF, 50 µg/ml gentamicin, 50 µg/ml amphotericin-B, and 5 µg/ml bovine insulin. For each of the experiments, passage HASMCs were grown to about 80-90% confluence and made quiescent by serum starvation for at least 24 hours. The serum-free medium contained secreted proteins (ie: MMP-9). The amount of secreted proteins in media was estimated and quantified based on the number of cells, while the secreted albumin was used as a control to normalize the amount of total secreted proteins.
Cells were suspended in 0.4 ml of lysis buffer containing 10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 2.0 µg/ml leupeptin, and 2.0 µg/ml aprotinin, and allowed to incubate for 15 minutes, before adding 25 µl of 10% Nonidet P-40. The tube was then centrifuged, and the pellet resuspended in ice-cold nuclear extraction buffer containing 20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 2.0 µg/ml leupeptin, and 2.0 µg/ml aprotinin. Nuclear extract centrifuged, and supernatant used. Protein content was measured using the Bio-Rad protein assay kit, while Electrophoretic mobility shift assay (EMSA) was done using a gel shift assay system kit (Promega) according to manufacturer’s instructions. double-stranded oligonucleotides containing the consensus sequences for AP-1 (5′-CTGAC CCCTGAGTCAGCACTT-3′), and NF-κB (5′-CCAGTGGAATTCCCCAG-3′) were end-labeled with [γ-32P] ATP (3000 Ci/mmol; Amersham Pharmacia Biotech) using T4 polynucleotide kinase and used as probes for EMSA. The samples were also incubated with the gel shift binding buffer (4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 10 mM Tris–HCl (pH 7.5), and 0.05 mg/ml poly(deoxyinosine-deoxycytosine)). Each sample was then electrophoresed in a 4% nondenaturing polyacrylamide gel in 0.5x TBE buffer at 250V for 20 minutes.
To evaluate the cytotoxicity of tanshinone IIA on HASMCs, a proliferation kit (XTT II, Boehringer Mannheim) was used. The cells were placed in 96 well culture plates at a density of 1 × 104 cells/well in DMEM culture medium and allowed to attach for 24 h. After 24 h of culture, 50 µl of XTT reaction solution (sodium 3′-[1-(phenyl-aminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate and N-methyl dibenzopyrazine methyl sulfate; mixed in proportion 50:1) was added to the wells. The optical density was read at 490 nm wavelength in an ELISA plate reader after 4 h incubation of the plates with XTT in an incubator (37°C and 5% CO2 + 95% air). This was used as a way to determine an appropriate dosage of the drug to the HASMC.
A Matrigel invasion assay was performed as described by Chung et al. 2002. The Matrigel-coated filter inserts (8 µm pore size) that fit into 24-well invasion chambers were obtained from Becton–Dickinson. HASMCs (5 × 104 cells/well) to be tested for invasion were detached from the tissue culture plates, washed, resuspended in conditioned medium collected from without-TNF-α (experimental control) or TNF-α-treated or tanshinone IIA (0, 50, 100 µM) + TNF-α treated to HASMCs for 24 h, and then added to the upper compartment of the invasion chamber. Cells that invaded through the gel were counted, with 3-5 invasion chambers used per experimental condition.
Gelatin zymography was also done. Culture supernatants of HASMCs treated with or without TNF-α (100 ng/ml) were resuspended in a sample buffer containing 62.5 mM Tris–HCl (pH 6.8), 10% glycerol, 2% SDS, and 0.00625% (w/v) bromophenol blue and loaded without boiling in 7.5% acrylamide/bisacrylamide (29.2:0.8) separating gel containing 0.1% (w/v) gelatin. Electrophoresis was carried out at a constant voltage of 100 V. After electrophoresis, the gels were soaked in 0.25% Triton X-100 (twice for 30 min) at room temperature and rinsed in distilled pure water. After electrophoresis, the gels were washed, incubated, and the bands were stained for using a Coomassie stain.
All of the above data was evaluated statistically as well.
Results
From the above cytoxicity assay, it was determined that a concentration at hundred micromolars of tanshinone IIA has a slight cytotoxic effect on the HASMCs and therefore, were used in this experimental study.
In zymography, both MMP-2 and MMP-9 were detercted in the conditioned media of the HASMCs while TNF-alpha significantly increased MMP-9 activity. Tanshinone IIA treatment inhibited MMP-9 activity in a concentration dependent way.
Figure 2. Effect of tanshinone IIA on the MMP-2, and MMP-9 activity of TNF-α-induced HASMCs. A: Zymography was performed with conditioned media collected from HASMCs cultured in the presence or absence of TNF-α and tanshinone IIA (0, 15, 30, 45, 50, 100 µM). ST, MMP-2/9 marker (0.3 ng). The secreted albumin was served as a loading control to normalize the amount of total secreted proteins. B: The densitometric intensity of the zymography bands was estimated as described in Materials and Methods Section. The values are calculated by percent of control and expressed as means ± SE of three independent experiments.
TNF-alpha activates AKT, which then phosphorylates and activates IkB kinase, therefore promoting NF-kB function, which is an important transcription factor that regulates MMP-9 expression.
Figure 3. Inhibitory effect of tanshinone IIA on NF-κB signaling pathway. A: Inhibitory effect of tanshinone IIA on TNF-α-induced AKT phosphorylation. Cells were pretreated with or without 100 µM tanshinone IIA for 2 h and then stimulated with or without 100 ng/ml of TNF-α for 1 h. The cytosolic extract was prepared and analyzed by Western blot. AKT (total AKT) is served as a loading control. B: Inhibitory effect of tanshinone IIA on TNF-α-induced p65 nuclear translocation and IκBα degradation. Cells were pretreated with 0, 15, 30, 45, 50, 100 µM tanshinone IIA for 2 h and then stimulated with or without 100 ng/ml TNF-α for 1 h. Cytosolic and nuclear proteins were prepared and analyzed by Western blot. GAPDH is served as a loading control. C: Concentration-dependent inhibition by tanshinone IIA on TNF-α-induced NF-κB DNA binding activity. The nuclear extract was subjected to EMSA as described in Materials and Methods Section. nc, negative control; sc, specific competitor for NF-κB. The densitometric intensity of the autoradiography bands was represented in bar graphs. The values are the means ± SE of three independent experiments.
Also, since it is known that TNF-alpha activates NF-kB via IkB phosphorylation and degradation, followed by a p65 translocation, the effect of tanshinone IIA on this pathway was looked at. Figure 3B shows that TNF-alpha treatment caused a large increase in the phosphor-IkBalpha. However, when treated with tanshinone, the pathway is inhibited and both phosphorylation of IkBalpha and p65 translocation were affected based on concentration of the dosage.
Figure 4. Inhibitory effect of tanshinone IIA on AP-1 signaling pathway. A: Inhibitory effect of tanshinone IIA on TNF-α-induced c-jun phosphorylation in nucleus and (B) AP-1 DNA binding activity. Cells were pretreated with 0, 15, 30, 45, 50, 100 µM tanshinone IIA for 2 h and then stimulated with or without 100 ng/ml TNF-α for 1 h. Nuclear protein was prepared and analyzed by Western blot and EMSA as described in Materials and Methods Section. nc, negative control; sc, specific competitor for AP-1. The densitometric intensity of the Western blot and autoradiography bands was represented in bar graphs, respectively. The values are the means ± SE of three independent experiments.
Another major transcription factor that regulates the expression of MMP-9 is AP-1 with c-jun being a main component of this factor. It is known that a phosphor-c-jun in the nucleus is important in the binding and transcriptional activity of AP-1. TNF-alpha enhanced the phosphor-c-jun in a concentration dependent manner, while tanshinone inhibited this activity, also in a concentration dependent manner.
Figure 5. Effect of tanshinone IIA on TNF-α-induced ERK, JNK, and p38 phosphorylation. A: Cells were pretreated with or without 100 µM tanshinone IIA for 2 h and then stimulated with or without 100 ng/ml TNF-α for 0, 10, 20, 30, and 60 min, respectively. The treated cells were harvested and analyzed by Western blot. GAPDH is served as a loading control. B: The densitometric intensity of the p-ERK, p-JNK, and p-p38 bands was normalized to GAPDH bands and represented in bar graphs, respectively. The values are the means ± SE of three independent experiments.
Also, it can be noted the that MAPK pathways can affect AP-1 transactiviation. To determine which class of MAPK was involved in tanshinone IIA mediated inhibition of AP-1 was active, the effect of the drug on the phosphorylation and activation of ERK, JNK, and p38 kinase were examined. The results (see figure 5) show that the inhibition of ERK phosphorylation by the drug is an underlying mechanism iinvolved in the inhibition of AP-1 and downregulation of MMP-9.
Figure 6. Effect of tanshinone IIA on migration of TNF-α-induced HASMCs. HASMCs (5 × 104A: Microphotograph of migrated cells without TNF-α and tanshinone IIA (1), with only 100 ng/ml TNF-α (2), with 100 ng/ml TNF-α and 50 µM of tanshinone IIA (3), and with 100 ng/ml TNF-α and 100 µM of tanshinone IIA (4) were captured. B: The values were obtained and calculated by averaging the total number of migrated cells from three filters and expressed as means ± SE. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Based on the migration of the HASMCs, it can be seen (see figure 6) that tanshinone IIA inhibited the TNF-alpha induced migration of HASMCs in a concentration dependent manner.
Discussion
From the above results, it can be shown that tanshinone IIA purified from S. miltiorrhiza Bunge, when administered at a nontoxic dose, inhibits HASMC migration and MMP-9 activity. Furthermore, it was shown that the drug also inhibited the activity of PI-3K/AKT and ERK1/2 signal pathway, but not other MAPK pathway. It can also be noted that the concentration of tanshinone IIA administered to the cells was also important, as the level of inhibition was determined by the concentration of the doses. These finding support previous studies that demonstrated the tanshinone could effectivel prevent neointima formation, inhibit intima hyperplasia, which is characterized by the proliferation and migration of SMC due to abnormal hemodynamic changes.
Critique
This study proposes the use of Tanshinone IIA as an inhibitor of the dysfunctional mutations, proliferations, and migration of vascular smooth muscle cells (VSMC). The importance of the study is that VSMC migrations and the expression of matrix metalloproteinases (MMPs) is believed to be a major cause of many vascular diseases. This study provides an excellent start to analyze the possible effective use of the drug, by using controls and using different assays to gather as much relevant data as possible.
For this study, human aortic smooth muscles cells were cultured, and were put through Electrophoretic Mobility Shift Assay (EMSA), Cell Viability Assay (used to determine the appropriate dosages for cells), Invasion Assays, and Gelatin Zymography Assay. Statistical Analysis was then performed to determine and evaluate the significance of the data. The study looked at the expression different MMPs and different signaling processes. By comparing the resulting data of the western blots to the controls, percentages based off the controls gave a quantification of how effectiveness of the medication.
The results showed that at appropriate dosages, tanshinone IIA did in fact inhibit HASMC migration and matrix metalloproteinases (MMPs) growth, which are important for smooth muscle cell proliferation.
While the study does incorporate a lot of useful and practical information to answer its hypotheses, it seems overly crowded. The information is compartmentalized, but I still found the overload of information to be overwhelming. It took multiple readings to fully understand and to summarize the wealth of data that they reported. I would have definitely appreciated the simplification of the information, the data, and paper in general to be more approachable and easier to understand.
