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Farrerol inhibited angiogenesis through Akt/mTOR, Erk and Jak2/Stat3 signal pathway.

ABSTRACT

Background: Farrerol is one of traditional Chinese medicines, isolated from Rhododendron dauricum L. It has been reported that Farrerol exerts multiple biological activities. Angiogenesis is an important drug target for cancer and inflammation therapy, the effect of Farrerol on angiogenesis is unknown. Hypothesis/purpose: We aimed to investigate whether Farrerol may have inhibitory effects against angiogenesis.

Study design/methods: Two kinds of endothelial cells, named human umbilical vein endothelia cell and human micro vessel endothelial cells, were used to examine the effect and mechanism of Farrerol on angiogenesis. MTT assay was used to detect cell proliferation, wound healing assay and boyden's chamber assay were used to examine cell migration, Matrigel was used as basement membrane substratum in tube formation assay, Annexin V-FITC/P1 dual staining assay and trypan blue staining were used to detect cell apoptosis, mouse aortic rings assay was performed as ex vivo assay, the expression of proteins involved in angiogenesis was tested using western blot, the binding of Farrerol to Stat3 was monitored by docking assay, molecular dynamics simulations and MM-GBSA method.

Results: Farrerol showed an inhibitory effect on proliferation, migration and tube formation of human umbilical vein endothelia cell and human micro vessel endothelial cells in a concentration-dependent manner. Farrerol induced cell cycle arrest and increased the apoptotic percentage of endothelial cells. Farrerol also suppressed the formation of new micro vessels from mouse aortic rings. Moreover, Farrerol reduced the phosphorylation levels of Erk, Akt, mTOR, Jak2 and Stat3 as well as protein expression of Bcl2 and Bcl-xl. Docking assay, molecular dynamics simulations and MM-GBSA method showed that Farrerol bound to domain of Stat3, Ser613,Gln635, Glu638 and Thr714 are the main residues in Farrerol binding sites with the binding free energy -7.3 ~ -9.0 kcal/mol.

Conclusions: In this study, we demonstrated that Farrerol inhibited angiogenesis through down regulation of Akt/mTOR, Erk and Jak2/Stat3 signal pathway. The inhibitory effect of Farrerol on angiogenesis suggested that this compound may be helpful to the angiogenesis-related diseases treatment, such as cancer and inflammations.

Keywords:

Farrerol

Angiogenesis

Endothelial cells

Signal pathway

Introduction

Angiogenesis, considered as the process of vascular growth by sprouting from existing vessels, is a hallmark of cancers and various inflammation diseases (Carmeliet and Jain, 2000; Nagasawa et al., 2014). So, angiogenesis inhibitors are helpful to conventional therapies in treatment of these diseases. Angiogenesis is regulated by growth factors such as VEGF and FGF, several signal proteins (including Akt, Erk, mTOR) and signal transducers and activators of transcriptions (Stat)(Gupta and Qin, 2003; Munoz-Chapuli et al., 2004; Yu et al., 2014). A series of inhibitors targeting these mechanisms were applied and researched in clinical trials (Carmeliet and Jain, 2011; Kerbel and Folkman, 2002),suggesting that inhibiting the formation of new vessels is a promising therapy for aberrant angiogenesis-related diseases.

Rhododendron dauricum L also known as 'Man shan hong', is a traditional Chinese herbal medicine with the activity of anti-chronic tracheitis and bronchitis (Xiong et al., 2013). Farrerol, a new kind of 2, 3-dihydro-flavonoid isolated from the leaves of R. dauricum L, has been to show multiple pharmacological activities such as anti-oxidant effects, anti-bacterial, anti-inflammatory effects, anti-cancer and anti-T lymphocyte activation (Ci et al., 2012; Liu et al., 2015; Qiu et al., 2011; Shi et al., 2010; Zhang et al., 2015; Zhu et al., 2007). Flavonoids are important natural compound and have been the focus of many researchers for a long time. Several flavonoids, such as nobiletin and hispidulin, had anti-angiogenic functions (Chen et al., 2015; Fotsis et al., 1997; He et al., 2011). As a new type of flavonoids, Farrerol inhibited the proliferation of rat thoracic aorta vascular smooth muscle cell (Li et al., 2011). Qingshan Li, et al demonstrated that Farrerol could protect human endothelium-derived EA.hy926 cells from apoptosis induced by hydrogen peroxide ([H.sub.2][O.sub.2]) (Li et al., 2013).In addition, Farrerol also could induce apoptosis of human gastric cancer SGC-7901 cells (Liu et al., 2015). However, the effect of Farrerol on angiogenesis has not been investigated as yet.

In current study, we chose two kinds of endothelial cells, human micro vascular endothelial cells (HMEC-1) and human umbilical vein endothelial cells (HUVEC), to investigate the effect of Farrerol on angiogenesis. The results displayed that Farrerol suppressed proliferation, migration and tube formation of endothelial cells. Farrerol also induced cell cycle arrest and increased the apoptotic percentage of endothelial cells, and suppressed the formation of new microvessels sprouting from mouse aortic rings. Further studies displayed that Farrerol exerted the inhibitory effect on angiogenesis through Akt/mTOR, Erk and Jak2/Stat3 signal pathway.

Materials and methods

Regents and cell culture

Human umbilical vein endothelia cell (HUVEC) and human micro vessel endothelial cell-1 (HMEC-1) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Both cell lines were cultured in completed endothelial cell medium provided by ScienCell Research Laboratories (Carlsbad, CA.USA). The antibodies used in this study including anti-Bcl-xl, Bcl-2, Stat3, Phos-Stat3, Akt, Phos-Akt, Erk, Phos-Erk, Phos-mTOR and [beta]-actin were purchased from Santa Cruz Biotechnology (Dallas, Texas USA). Medium was supplemented with 10% FBS, penicillin (100 units/ml) and streptomycin (100 units/ml). Farrerol ([greater than or equal to] 98%, purity by HPLC) was obtained from Dalian meilun biology technology co., LTD (Dalian, Liaoning, China).

Wound healing migration assay

For endothelial cell migration assay, cells were seeded in 6 well plates and grown to confluency. After the starvation 4-6 h, the cells were scratched with a pipette tip followed by the addition of Farrerol. Cells were fixed using 4% paraformaldehyde after treatment for 12 h and stained with 0.2% violet crystal. Images of wound area were taken by Leica microscope (Leica, Mannheim, Germany), the number of migrated endothelial cells was counted. During the migration assays, we did not find the obvious phenomenon of apoptosis.

Transwell migration assay

Boyden's chamber assay in 24-well cell culture plate with 8-[micro]m pore was used. Briefly, the fresh medium containing Farrerol was placed in the lower wells, and cells were suspended in the upper side of the filter at the final concentration of 4 x [10.sup.4]/well, and exposed to predetermined concentration of Farrerol. The plate was incubated in 37[degrees]C and 5% C[O.sub.2] for 8 h. Cells were fixed with 4% paraformaldehyde and stained with crystal violet. Non-invaded cells on the upper side of the filter were removed by cotton swabs, and the mounted cells that invaded to the lower side of the filter were captured with an inverted microscope.

Cell proliferation assay

The function of Farrerol on cell proliferation was determined by MTT assay. In brief, HUVEC or HMEC-1 was seeded in 96-well plates (5000 cells/well). For HUVEC, the plates were coated with 0.1% Gelatin dissolved in Millipore-Q water. After culture for 12 hours, cells were starved for 6 h and subsequently treated with various concentrations of Farrerol for 48 h. Then, 50 [micro]l of MTT solution was added to every well. After incubation for 4 h, the supernatants were discarded followed by the addition of 100 [micro]l DMSO. The plates were vortexed for 10 min at room temperature, and the absorbances were measured with a micro plate reader (Powerwave XS; Biotek, Winooski, VT, USA).

Cell cycle and apoptosis assay

For cell cycle analysis, cells were harvested and washed with PBS twice. Then, collected cells were re-suspended in 70% cold ethanol and fixed overnight at 4[degrees]C. After centrifugation, cells were resuspended with PBS containing RNase and stained by propidium iodide at 37[degrees]C in dark room for 30 min. The cell cycle distribution was analyzed by flow cytometry (BD Bioscience, Franklin Lakes, NJ. USA). Annexin V-FITC/PI dual staining assay and trypan blue staining were used to detect the effect of Farrerol on apoptosis. Cells were treated with various concentrations of Farrerol for 48 h, harvested and washed with PBS twice. After centrifugation, cells were re-suspended with binding buffer and incubated with annexin V and PI. Cells were incubated in dark room at room temperature for 15 min, followed by analysis through flow cytometry. For trypan blue staining, cells were plated onto 6-well plates and were exposed to Farrerol for 48 h. After treatment, all cells including the supernatant were collected and centrifuged to collect the viable, apoptotic, and dead cells. The cells were suspended in serum-free medium. Equal volume of trypan blue and cell suspension were mixed and analyzed by an inverted microscope (Leica).

Tube formation assay

Tube formation assay was performed as described previously (Dai et al., 2012). Briefly, 96-well plates were pre-cooled followed by the addition of Matrigel. After the incubation for more than 30 min, after cell starvation for 6 h, 1 x [10.sup.4] cells per well with various concentrations of Farrerol were seeded in 96-well plates. The cells were cultured at 37[degrees]C with 5% C[O.sub.2] for 7-10 hours, tube structures in eight randomly chosen fields were photographed and counted by scoring the enclosed networks.

Mouse aortic ring assay

All animal care and experimentation conformed to the Guide for the Care and Use of Laboratory Animals published by Henan University. Thoracic aortas from Balb/c mouse were sliced into rings of 1-1.5 mm in circumference, the rings were seed into 96-well plate coated with Matrigel. Once the rings were seed onto the Matrigel, the rings were coated with Matrigel again. MCDB131 medium with or without Farrerol was added to aortic rings coated with Matrigel. Medium with or without Farrerol was exchanged every 2 days. On day 7, micro vessel outgrowths were photographed and analyzed.

Western blot

After starvation and the subsequent treatment of Farrerol, cells were harvested and centrifuged, cell pellets were washed three times with ice-cold PBS and were lysed with RIPA buffer (Beyotime, Nantong, Jiangsu, China). The concentration of protein was examined using BCA assay kit. The total lysates were subjected to 5xSDS-loading buffer at 100[degrees]C for 10min followed by exposure to 8-12% SDS-PAGE for 1-2h. Seperated proteins were transferred onto PVDF membranes, and membranes were blocked by 5% dried skimmed milk at room temperature for 1 h. After blockage, membranes were incubated with specific primary antibodies at 4[degrees]C overnight. After incubation with determined HRP-conjugated secondary antibody, protein expression was detected by using the ECL plus reagents (Beyotime).

Cell morphology and DAPI staining

After Farrerol treatment for 48 h, HUVEC and HMEC-1 were stained with violet crystal; the cell morphology was observed and captured with an inverted microscope. To observe the shape of nucleus, cells were fixed using 4% paraformaldehyde followed by washes with PBS. Cells were stained with DAPI (4',6-diamidino-2-phenylindole), and images were taken by Leica DMi8 (Leica).

Binding assay

This assay is prepared based on the crystal structure of STAT3/DNA complex (PDB: 1BG1) by removing DNA. The Farrerol is docked into the active site by Sybyl 3.0 software to obtain the initial STAT3-substrate complex. In order to get the accurate binding mode and binding free energy, molecular dynamics simulations is employed. The protonation states of charged residues are determined by H++ package. The substrate is treated with Amber GAFF force field (GAFF), and the charge parameters are calculated by the restrained electrostatic potential (RESP) method at HF/6-31G(d) level by using Gaussian 03 package. The protein and water molecules are handled by Amber99SB force field and the TIP3P model, respectively. The system is solvated in a cubic water box with 10 [Angstrom], and neutralized by [Cl.sup.-] ions. The minimizations are carried out to obtain the favorable structure. Then the system is heated from 0 to 300 K in 50 ps, and the other 50 ps MD simulations are used to relax the model. After that, 10 ns NVT MD equilibrations are performed. The 12 [Angstrom] cutoff distance is used for the electrostatic interaction and van der Waals calculations. The temperature is controlled at 300 K by Langevin method. The last 2 ns snapshots are employed for binding free energy analysis by MM-GBSA method. All the simulations are carried out by using Amber12 software. The relevant pictures are depicted with Pymol and Sybyl software.

Statistical analysis

Data in this study were expressed as mean [+ or -] SD from at least three separate experiments. Unless otherwise noted, the differences between groups were analyzed using Student's t-test (two groups), p < 0.05 was set to be considered statistically significant, * p <0.05, ** p <0.01, and *** p <0.001.

Results and discussion

Farrerol inhibited endothelial cells growth

In angiogenesis study, endothelial cells were widely used in vitro model system (Bouis et al., 2001), and we chose two different vascular endothelial cell types, named human microvascular endothelial cell (HMEC-1) and human umbilical vein endothelial cell (HUVEC), respectively. Activation of the endothelial cell proliferation is one common feature of angiogenesis (Feng et al., 2012). So, inhibition of endothelial cells proliferation is one strategy for anti-angiogenesis. To explore the inhibitory effect of Farrerol (Fig. 1A) on endothelial cells proliferation in vitro, we performed MTT assay. As shown in Fig. 1(B), addition of Farrerol inhibited endothelial cell viability significantly in a concentration-dependent manner. The [IC.sub.50] in HMEC-land HUVEC was 20.04 [micro]M and 22.29 [micro]M, respectively. Meanwhile, we also observed that the density of endothelial cells decreased with the addition of Farrerol after 24 h and 48 h (Fig. 1C and D).

Farrerol induced G1 phase arrest and apoptosis of endothelial cells

Endothelial cell proliferation inhibition is partially due to arrest endothelial cell cycle progression. To examine the effect of Farrerol on endothelial cell cycle, we detect the cell cycle progression after addition of Farrerol. Flow cytometer analysis revealed that Farrerol induced G1-phase cell cycle arrest in HUVEC and HMEC-1 (data not shown). For tumor, induction of endothelial cell apoptosis could limit neovascularization of tumor, resulting in tumor necrosis (Dimmeler and Zeiher, 2000). Flow cytometer analysis showed that the percentage of HMEC-1 undergoing apoptosis in culture treated with 50 [micro]M Farrerol is 25.79% compared with 8.93% in the control population, and Farrerol elevated levels of apoptotic cells in a dose-dependent manner, as shown in Fig. 2(A). Since live cells could exclude trypan blue, and trypan blue staining is always used to detect apoptotic cells (Dong et al., 2002). In HUVEC, trypan blue staining results indicated that the percentage of the population of cells that were apoptotic in Farrerol-treated group was near 7 fold compared with the untreated group as shown in Fig. 2(B). When apoptosis occurs, some obvious features occurred, including shrinkage, nuclear fragments and apoptotic bodies (Ziegler and Groscurth, 2004). After Farrerol treatment, nuclear shrinkage, chromatin condensation, nuclear fragments and apoptotic bodies appeared in HMEC-1 and HUVEC as shown in Fig. 2(C).

Farrerol affected endothelial cell morphology and suppressed migration of endothelial cells

In Farrerol-treated HMEC-1 and HUVEC, a reduction in cell volume and shrunken cytoplasm were observed as shown in Fig. 3(A). Lamellipodia (LP) are thin, sheetlike protrusions in untreated cells characterized by periods of extension and retraction (indicated by dashed line), After Farrerol treatment, the size of cell showed shrink with minimal protrusive activity (indicated by dashed line). The changes of cell shape influences the cell motility, so we postulate that Farrerol might affect the endothelial cell migration, which is a key process during new capillaries formation (Carmeliet, 2000). Firstly, we performed wound healing assay, and found that the closure of wound area treated by Farrerol was wider compared with untreated group. Meanwhile, we found that Farrerol inhibited the migration of HMEC-1 and HUVEC significantly in a concentration-dependent manner as shown in Fig. 3(B). To confirm the inhibitory effect of Farrerol on endothelial cell migration, we also used transwell boyden's chamber. In transwell migration assay as shown in Fig. 3(C), Farrerol obviously suppressed endothelial cell migrating from the upper side to the lower side.

Farrerol inhibited tube formation of endothelial cells and suppressed micro vessels sprouting from mouse aortic rings

The in vitro formation of capillary-like tubes by endothelial cells on a basement membrane matrix is a powerful in vitro method to screen various factors that promote or inhibit angiogenesis (Arnaoutova and Kleinman, 2010). To detect the effect of Farrerol on tube formation, we chose Matrigel as a basement membrane substratum. After endothelial cells co-cultured with Farrerol, we observed that Farrerol inhibited the formation of capillary-like tubes on Matrigel in a concentration-dependent manner and the occurrence of intact tube was nearly not observed at 50 [micro]M Farrerol, as shown in Fig. 4(A and B). The vessels that grow out from aortic rings are anatomically similar to neovessels in vivo, so aortic ring assay is a beneficial tool to evaluate angiogenesis, and has become one of the most widely used models researching angiogenic and anti-angiogenic factors or agents (Baker et al., 2012; Bellacen and Lewis, 2009). Therefore, we used mouse aortic ring assays to examine the effect of Farrerol on angiogenesis, and found that few new micro vessels were formed from mouse aortic rings treated by Farrerol compared with untreated group, as shown in Fig. 4(C).

Farrerol inhibited the phosphorylation of Akt, mTOR and erk

Akt/mTOR pathway is critical for the growth, proliferation and migration of endothelial cells as well as cancer cells, and this pathway was considered as the target for antiangiogenic agents (Karar and Maity, 2011; Sabatini, 2006). To examine the effect of Farrerolon this pathway, we detected the phosphorylation levels of Akt and mTOR. After Farrerol treatment, the phosphorylation levels of Akt and mTOR were decreased in both of HMEC-1 and HUVEC, as shown in Fig. 5 A and B. Activation of Erk pathway in endothelial cells is required for angiogenesis (Mavria et al., 2006).So, blockage of Erk pathway is a choice of anti-angiogenesis therapy (Kim, 2014; Song et al., 2012). In this study, we found that Farrerol inhibited the phosphorylation of Erk in both of HMEC-1 and HUVEC, as shown in Fig. 5(A and B).

Farrerol inhibited Jak2/Stat3 signal pathway and bound to domain of Stat3 in docking assay

Angiogenesis response induced by growth factors, chemokines and receptor activation are mediated through Jak2/Stat3 pathway (Xiong et al., 2014). Therefore, inhibition of Jak2/Stat3 pathway could prevent the occurrence of angiogenesis in tumor growth (Siveen et al., 2014; Yu et al., 2014). In HMEC-1 and HUVEC cells, as shown in Fig. 6(A and B), Farrerol treatment reduced the phosphorylation level of Jak2 and Stat3.Previous studies have reported that activated Stat3 could participate in oncogenesis through dysregulation of genes encoding the antiapoptotic proteins Bcl-xl, Bcl-2 and so on (Jing and Tweardy, 2005). In contrast, decreased Stat3 activation could downregulated the expression of Bcl-xl as well as Bcl-2. Consistent with this notion, as shown in Fig. 6(C and D), the expression of Bcl-2 and Bcl-xl in HMEC-1 as well as HUVEC cells was decreased after Farrerol treatment.

Many natural compound-for example, resveratrol, curcumin or quercetin-can target many molecules (Ji et al., 2009), implying that natural products might be broad-spectrum inhibitors. In addition to the above mentioned signal pathway, as a natural compound, Farrerol might target other molecules. Based on the decrease of Bcl-2 and Bcl-xl expression, we postulated that Farrerol may bind to the activity domain of Stat3 resulting in inhibition of Stat3 transcriptional activity. To confirm this hypothesis, we performed docking assay. DNA-Stat3 interaction play a key role in regulating the Stat3 transcriptional activity (Becker et al., 1998), therefore, the docking assay is prepared based on the crystal structure of Stat3/DNA complex (PDB: 1BG1) by removing DNA.As shown in Fig. 6(E and F), Farrerol bound to the domain of Stat3. The structure of Stat3 bound to DNA has been encompassing the domains of Stat3 from residues 127 to 722 including the SH2 domain (Becker et al., 1998).In this model, the residues such as Lys591, Arg595, Arg609, Ser6l1, Glu612, Ser613, Thr620, lle634, and Gln635 constituted the binding sites to stabilize the Farrerol, suggesting that Farrerol disrupted the binding of Stat3 to DNA, which might result in the inhibition of transcriptional activity of Stat3.

The accurate binding mode and free energy of interaction between Farrerol and Stat3

In order to get the accurate binding mode and binding free energy, molecular dynamics simulations is employed. The STAT3-substrate complex after molecular dynamics simulations is depicted in Fig. 7(A) (left).Since the binding site is almost localized at the border of protein and solvent, Farrerol is floating on the surface of protein. In order to elucidate this status, several snapshots are selected randomly, and the binding sites are listed in Fig. 7(A) (middle), the corresponding electrostatic potential surfaces are drawn in Fig. 7(A) (right). A large number of water molecules around Farrerol, the electrostatic interaction, fluctuating hydrogen bond nets and fluctuating-related residues suggested that the binding free energies might be small.

To further confirm the conditions, MM-GBSA method has been used to estimate the relative binding free energy in different periods. As shown in Fig. 7(B), from 6 to 8 ns, the binding free energy is -9.0 kcal/mol, and the Ser613, Trp623, Gln635, Glu638, Val637, and Thr714 play an important role in binding substrate through the water molecules. From 8-10 ns, the binding free energy is--7.3 kcal/mol, and the Lys557, Ser613, Gln635, Glu638, and Thr714 are very crucial. These results showed that the contribution degrees of the residues are different in different molecular dynamics sampling stages. The residues in different periods also suggested that Ser613, Gln635, Glu638 and Thr714 are the main residues in Farrerol binding sites.

Farrerol significantly suppressed the phosphorylation of Jak2, resulting in the inhibition of Stat3 phosphorylation. Meanwhile, Farrerol bond to the activity domain of Stat3 with small force in constraining Farrerol, and Farrerol may be flee away from the binding sites. So, Farrerol inhibited the activity of Stat3 mainly by inhibition of Jak2 activation.

Taken together, Farrerol inhibited that phosphorylation of Akt, mTOR, Erk, Jak2 and Stat3, which are key molecules in regulating angiogenesis as mentioned in the above results. Many natural compound including resveratrol and curcumin could target many proteins or signal pathway, suggesting that natural products might be broad-spectrum inhibitors. Based on this, we postulated that all of these signal pathways such as Akt/mTOR, Erk and Jak2/Stat3 signal pathway in antiangiogenic activity of Farrerol played important roles.

Conclusions

In this study, we found that Farrerol inhibited angiogenesis through downregulation of Akt/mTOR, Erk and Jak2/Stat3 signal pathway. The inhibitory effect of Farrerol on angiogenesis suggested that this compound may be helpful to the angiogenesis-related diseases treatment, such as cancer and inflammations.

ARTICLE INFO

Article history:

Received 23 December 2015

Revised 1 March 2016

Accepted 19 March 2016

Conflict of interest

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Acknowledgments

This study was partially sponsored by Key Scientific Research Project of Henan Province (15A310004), Projects of Science and Technology of Henan (162300410231) and China Postdoctoral Science Foundation Funded Project (2015M582183).

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Fujun Dai (a), (1), *, Lei Gao (b), (1), Yuan Zhao (a), Chaojie Wang (a), Songqiang Xie (c), *

(a) The Key Laboratory of Natural Medicine and Immuno-Engineering, Henan University. Kaifeng 475004, China

(b) Joint Tomato Research Institute. School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China

(c) Institute of Chemical Biology, Pharmaceutical College of Henan University, Kaifeng 475004, China

Abbreviations: MD, Molecular dynamics; HMEC-1, Human microvascular endothelial cell; HUVEC, Human umbilical vein endothelial cell; Stat, Signal transducers and activators of transcriptions; mTOR, Mammalian target of rapamycin.

* Corresponding authors. Tel. +86 371 22864665; fax: +86 371 22864665.

E-mail addresses: fjdwl@hotmail.com (F. Dai), xiesq@henu.edu.cn (S. Xie).

(1) Common first authors: Fujun Dai (a) and Lei Gao (b)

http://dx.doi.Org/10.1016/j.phymed.2016.03.008
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Author:Dai, Fujun; Gao, Lei; Zhao, Yuan; Wang, Chaojie; Xie, Songqiang
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:1USA
Date:Jun 15, 2016
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