(S)-[6]-Gingerol inhibits TGF-β stimulated biglycan synthesis but not

glycosaminoglycan hyperelongation

in vascular smooth muscle cells

aDanielle Kamato#, a,bHossein Babaahmadi Rezaei#, aRobel Getachew, aLyna Thach, aDaniel Guidone,

a,cNarin Osman, dBasil Roufogalis, dColin C Duke, dVan Hoan Tran eWenhua Zheng and a,cPeter J.

Little

# These two authors contributed equally to this work.

aDiscipline of Pharmacy, School of Medical Sciences and Diabetes Complications Group, Health

Innovations Research Institute, RMIT University, Bundoora, VIC 3083 Australia and bDepartment of

Clinical, Biochemistry, Ahvaz Jundishapur University of Medical Sciences, Ahvaz Iran,

cDepartments of Medicine, Nursing and Health Sciences and Immunology, Monash University

School of Medicine (Central and Eastern Clinical School, Alfred Health), Prahran VIC, 3004,

Australia, dFaculty of Pharmacy, A15, The University of Sydney, NSW 2006 Australia and eState

Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Centre and School of Pharmaceutical

Sciences, Sun Yat-sen University Guangzhou, China,

Running title: Gingerol and proteoglycan synthesis

Keywords: (S)-[6]-Gingerol, proteoglycans, biglycan, atherosclerosis, Smad signalling, Akt

Address correspondence to: Dr Narin Osman,Deputy Head, Diabetes Complications Laboratory, Discipline of Pharmacy, School of Medical Sciences,RMIT University, Melbourne, Victoria 3083 AustraliaTel: +61 3 9925 6686Fax : +61 3 0025 7063Email: narin.osman@rmit.edu.au

Page 1 of 30

Abstract

Objectives (S)-[6]-Gingerol is under investigation for a variety of therapeutic uses. Transforming

growth factor (TGF)-β stimulates proteoglycan synthesis leading to increased binding of Low

Density Lipoproteins which is the initiating step in atherosclerosis. We evaluated the effects of

(S)-[6]-gingerol on these TGF-β mediated proteoglycan changes to explore its potential as an anti-

atherosclerotic agent. Methods Purified (S)-[6]-Gingerol was assessed for its effects on proteoglycan

synthesis by 35S- sulfate incorporation into glycosaminoglycan chains and 35S-met/cys incorporation

into proteoglycans and total proteins. Biglycan expression was assessed by real-time quantitative

polymerase chain reactions and the effects of (S)-6-gingerol on TGF- β signalling by assessment of

the phosphorylation Smads and Akt by Western blotting. Key Findings (S)-[6]-Gingerol

concentration-dependently inhibited TGF-β stimulated proteoglycan core protein synthesis and this

was not secondary to inhibition of total protein synthesis. (S)-[6]-gingerol inhibited biglycan mRNA

expression. (S)-[6]-Gingerol did not inhibit TGF-β stimulated GAG hyperelongation,

phosphorylation of Smad 2 in either the carboxy terminal or linker region or Akt phosphorylation.

Conclusions The activity of (S)-[6]-gingerol to inhibit TGF-β stimulated biglycan synthesis suggests

a potential role for ginger in the prevention of atherosclerosis or other lipid binding diseases. The

signalling studies indicate a novel site of action of (S)-[6]-gingerol in inhibiting TGF-β responses.

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Introduction

(S)-[6]-Gingerol ((S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3-decanone) (Fig. 1) is the

principal bioactive phenolic constituent of the oleoresin from the rhizomes of the common fresh

natural food product, ginger (Zingiber officinale). (S)-[6]-Gingerol is rapidly absorbed and is of

generally low toxicity. The chemical structure is altered with manipulations such as drying and

cooking, generating derivatives such as shogoal and zingerone [1]. Ginger is chemically related to

capsaicin and it shares some biological activities with this compound. Ginger is pain relieving and

has been used for many years to relieve the symptoms of rheumatism and other inflammatory

conditions. The actions of (S)-[6]-gingerol on pain receptors also have lead to its use in various

forms of environmental and drug induced nausea. (S)-[6]-Gingerol has also been shown to have

beneficial actions in several models of cancer. (S)-[6]-Gingerol inhibits cell adhesion, invasion,

motility and activities of MMP-2 and MMP-9 in MDA-MB-231 human breast cancer cell lines [2].

Extracts have activity in the prevention of the parameters of the metabolic syndrome in animal

models and also demonstrate anti-hyperlipdemic activity in high fat fed rats [3, 4]. In vascular

biology (S)-[6]-gingerol inhibits angiogenesis and may be useful in the treatment of tumours and

other angiogenesis-dependent diseases. [5]. (S)-[6]-Gingerol and its derivatives show potent anti-

platelet activity [6]. Ginger and its pungent components including (S)-[6]-gingerol are thus

recognised as phytonutrients with therapeutic potential.

Cardiovascular disease is the largest single cause of premature mortality and its major

underlying pathology is the development, progression and final clinical sequlae of atherosclerosis

and plaque rupture [7, 8]. The acute rupture of unstable atherosclerotic plaques causes life

threatening heart attacks and strokes [9, 10]. Atherosclerosis commences with the trapping of lipids

in the blood vessel wall and considerable evidence suggests that this occurs due to binding and

retention of apolipoproteins by modified proteoglycans, particularly the small leucine rich

chondroitin sulfate-dermatan sulfate (CS/DS) proteoglycan, biglycan [11-14]. Modified biglycan,

Page 3 of 30

specifically the form with hyperelongated, meaning longer than naturally occurring,

glycosaminoglycan (GAG) chains, shows enhanced lipid binding in vitro and increases lipid

deposition in the vessel wall in animal models of atherosclerosis [15, 16] [17]. These early, pre-

inflammatory events can be prevented in animal models of atherosclerosis by drugs which inhibit

proteoglycan synthesis and GAG hyperelongation [15, 18].

Transforming growth factor (TGF)-β is a pleiotropic growth factor expressing both pro- and

anti-inflammatory effects at different stages of the disease process[19]. In the early pre-inflammatory

stage encompassed by the “response to retention” hypothesis TGF-β is pro-atherogenic through its

effects on proteoglycan synthesis and structure [20, 21]. TGF-β stimulates biglycan synthesis in

human vascular smooth muscle cells (VSMC) including increased expression of biglycan core

protein and hyperelongation of GAG chains the latter which results in increased binding to LDL [21,

22]. There is considerable knowledge about the signalling pathways through which TGF-β mediates

its effects on biglycan synthesis in vascular smooth muscle cells [23, 24]. Signalling involves the

TGF-β type I receptor (TβRI) also known as Activin-like Kinase V (AlkV) and phosphorylation of

both the C terminal and the linker region of the transcription factor, Smad2 [25, 26]. TGF-β

stimulated expression of biglycan mRNA and protein is also dependent upon phosphorylation of Akt

[22]. To the extent of our current knowledge, the signalling pathway for biglycan protein expression

is similar to that for cell cycle progression [22]. GAG hyperelongation has different signalling

pathways that have not been fully elucidated but involve phospho Erk 1/2 [18, 27]. Thus, we have

used proteoglycan synthesis in human VSMCs, being one critical component of an in vitro model of

atherogenesis, to study the pharmacology and potential anti-atherogenic actions of (S)-[6]-gingerol.

We demonstrate that (S)-[6]-gingerol inhibits the TGF-β stimulated proteoglycan core protein

expression and specifically the expression of biglycan but it does not block the action of TGF-β to

cause hyperelongation of GAG chains on biglycan. (S)-[6]-Gingerol does not block either of the

TGF-β signalling pathways being the TβRI pathway leading to carboxy terminal phosphorylation of

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Smad2 (pSmad2C) or the TβRI and the Mitogen Activated Protein (MAP) kinase pathway leading to

phosphorylation of Smad2 in its linker region (pSmad2L) nor does it block phosphorylation of the

serine/threoenine kinase, Akt. (S)-[6]-gingerol does not block total de novo protein synthesis.

Although (S)-[6]-gingerol lacks the major activity of blocking GAG chain hyperelongation it does

provide some mechanistic basis for the potential role of ginger in the prevention of atherosclerosis

and possibly other lipid binding diseases through the inhibition of growth factor stimulated biglycan

expression.

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Materials and Methods

Materials

The following chemicals were purchased from Sigma Aldrich (St Louis, MO, USA): benzamidine

hydrochloride, DEAE-Sephacel, proteinase K, chondroitin sulfate, SB431542 and penicillin and

streptomycin. Recombinant TGF-1 was from Cell Signaling Technology (Danvers, MA, USA).

Dulbecco’s modified eagle medium (DMEM), glutamine and fetal bovine serum (FBS) were

purchased form Invitrogen (Gibco) Grand Island, NY, USA). [35S]-Met/Cys was from MP

Biomedicals, (Irvine, CA, USA). Rainbow [14C] methylated protein molecular weight standard was

from Amersham Pharmacia (Buckinghamshire, England). Cetyl pyridinium chloride (CPC) was

from Unilab Chemicals and Pharmaceuticals (India). Whatman 3MM chromatography paper from

Biolab (Mulgrave, Australia). Instagel plus scintillation fluid and [35S]-sulfate were from Perkin-

Elmer (Waltham, Massachusetts, USA). Poly-Prep columns was purchased from (Bio-Rad, Hercules,

CA, USA). Freeze dried ginger powder (Grade A, Batch No. 9240089) was provided by Buderim

Ginger Pty Ltd (Queensland, Australia).

Culture of human VSMC

Human VSMCs were obtained by the explant method from otherwise discarded sections of the

saphenous veins from patients undergoing coronary artery bypass grafting at the Alfred Hospital

(Melbourne, Vic, Australia) with approval of the Alfred Health Ethics Committee [28]. VSMC were

grown in Dulbecco’s Modified Eagle Medium (DMEM) with 5 mM glucose, 10% fetal bovine serum

(FBS) and 1% penicillin-streptomycin solution. For experimentation, human VSMCs were seeded

into 24-well plates (proteoglycan experiments), 6-well plates (quantitative real-time PCR

experiments) and 60 mm diameter plates (Western blotting experiments), grown until confluency

then rendered quiescent by serum starvation (0.1% FBS) for 48h prior to experimentation.

Page 6 of 30

Purification of (S)-[6]-gingerol

Freeze dried ginger powder (1 kg) was extracted once with ethyl acetate (3 L) with stirring at room

temperature. The filtrate was collected and solvent evaporated under reduced pressure at 40°C to

afford a liquid residue (55 g) as total ginger extract. The extract (30 g) was fractionated by short

column vacuum chromatography (SCVC) on silica gel (Merck silica gel 60 H, 8 x 12 cm ID column)

using a stepwise gradient of hexane-EtOAc, with EtOAc concentrations of 5, 10, 15, 20, 25, 30 and

40%. Seven fractions (200 mL each) were collected, the solvent removed under reduced pressure,

and analysed by 1H-NMR. Fractions (F6 and F7) that contained mainly gingerols were combined and

further fractionated on the SCVC system as described above. The column was eluted with

dichloromethane (DCM) – hexane (8:1, 2 x 100 mL), then a stepwise gradient of DCM – EtOAC

with EtOAc concentrations of 0, 1, 2, 3, 4, 5, 6, 8 and 10% (2 x 100 mL each). Twenty fractions

were collected and monitored for (S)-[6]-gingerol (CAS 235130-14-6) content by TLC using a

mobile phase of DCM – EtOAc (6:1) then analysed by 1H-NMR. Fractions with similar profile with

respect to (S)-[6]-gingerol content were combined and the solvents evaporated under reduced

pressure to give a yellowish liquid (2.8 g). Purity was determined by analytical high-performance

liquid chromatography (HPLC) (Shimadzu UFLC system) on a C18 column (Hewlett-Packard,

Nucleosil 100 C18, 5 µm, 4 mm × 125 mm) eluted with methanol-water-acetic acid (62:37.8:0.2) at

1mL/min and detected at 230 nm with a UV-Vis detector (Shimadzu SPD-20A).

Quantitation and sizing of proteoglycans produced by human VSMCs

These methods were performed exactly as described in earlier papers from our laboratory [21, 29].

Quantitative real-time PCR

Human VSMCs were plated onto 6-well culture plates in DMEM supplemented with 5mM

glucose, 10% FBS and 1% antibiotics. Once confluent cells were serum deprived (0.1% FBS) for

Page 7 of 30

24h prior to treatment and then pre-incubated for 30 min with (S)-[6]-gingerol followed by TGF

(2ng/ml) stimulation for 24h. Total mRNA was extracted from 5×105 cells using RNeasy mini kit

according to the manufacturer’s instructions (Qiagen, Germany). Purity of RNA was qualified with

spectrophotometry (260/280nm) by using a Nanodrop spectrophotometer (Thermo Scientific 2000).

First-strand cDNA was synthesised from a total of 1µg RNA using QuantiTect reverse transcriptase

kit (Qiagen, Germany). Quantitative real-time PCR was performed with a Rotor Gene real-time

thermo cycler and SYBR Green PCR master mix (Qiagen, Germany). Specific primer sequences

used for amplification of biglycan were, sense: 5´- CTC AAC TAC CTG CGC ATC TCA G and

antisense: 5´- GAT GGC CTG GAT TTT GTT GTG. The 18S gene expression was used as the

endogenous loading control. To monitor the presence of nonspecific products and primer dimers,

analysis of melting curves and resolution of PCR products on a 2% agarose gel were performed.

Relative expression of mRNA level was quantified using comparative delta-delta Ct (ΔΔCt) method.

Western blotting

Total cell lysates were resolved on 10% SDS-PAGE and transferred onto PVDF membranes.

Membranes were blocked with 5% skim milk, incubated with primary rabbit monoclonal antibodies

(anti-phospho-Smad2C (Ser465/467) (1:1000), anti-phospho-Smad2L (Ser245/250/255) (1:1000),

anti-phospho-Akt (Ser473) and anti-GAPDH (14C10) (1:4000) as endogenous control followed by a

secondary HRP-anti-rabbit IgG (1:1000) (Cell Signalling Technology, MA, USA) and ECL detection

(Amersham). Blots from at least two experiments were quantified by densitometry using Image Lab

software (Bio-Rad).

Statistical analysis

Data was analysed for statistical significance using a 1-way analysis of variance (ANOVA).

Results were considered statistically significant at P<0.05 as indicated in the text and figure legends.

Page 8 of 30

Results

(S)-[6]-Gingerol was prepared by extraction of freeze dried ginger powder with ethyl acetate, which

yielded approximately 5.5% of total ginger extract. NMR and HPLC analysis (data not shown) of

this sample showed that (S)-[6]-gingerol was the major constituent with an approximate content of

15%. Isolation and purification of this (S)-[6]-gingerol were efficiently achieved by two short

column vacuum chromatographic steps as described in Methods which yielded approximately 60%

of (S)-[6]-gingerol as the main single component the structure of which is shown (Fig. 1). On

analytical HPLC analysis this sample was found to be 93.1±0.9% pure, which was considered

adequate for initial biological evaluation without further purification. Other components present in

this sample included (S)-[8]-gingerol (6.63±0.74%) and [6]-shogaol (0.24±0.18%).

We determined the effect of (S)-[6]-gingerol on TGF-β stimulated proteoglycan synthesis in

VSMCs using [35S]-sulfate incorporation and SDS-PAGE to assess the size of the proteoglycans as a

surrogate for the apparent size of the glycosaminoglycan chains [29, 30]. TGF-β (2ng/ml) stimulated

a two-fold increase in [35S]-sulfate incorporation which is consistent with our earlier observations

[21, 23, 24]. We evaluated proteoglycans synthesized in the presence of (S)-[6]-gingerol (0.1 – 10

µM). (S)-[6]-Gingerol had an initial inhibitory effect at approximately 1µM and a concentration

dependent inhibitory effect at higher concentrations (Fig. 2, upper panel). The half maximal

inhibitory concentration of (S)-[6]-gingerol was less than 3µM (Fig. 2, upper panel). We used the

well characterized Alk-V/TβRI inhibitor SB431542 (3µM) as a control inhibitor and it completely

blocked TGF-β stimulated [35S]-sulfate incorporation and reduced the rate to below control values

(Fig. 2, upper panel).

Our major interest is in growth factor mediated elongation of GAG chains on biglycan because

of its direct association with lipid binding and retention as an initiating step in atherosclerosis [13,

14]. We isolated the proteoglycans secreted in the same experiments above and subjected them to

SDS-PAGE. SDS-PAGE provides an estimate of apparent molecular weight and it correlates closely

Page 9 of 30

with measurements by size-exclusion chromatography [29, 30]. TGF-β treated cells secrete biglycan

molecules which are on average markedly larger than biglycan molecules secreted under basal

conditions (Fig. 2 lane 1 vs 2). In cells stimulated with TGF-β and treated with (S)-[6]-gingerol

(0.1-10µM) there was no effect of (S)-[6]-gingerol on this action of TGF-β (Fig. 2, lower panel lanes

3 – 7). As above, in cells treated with SB431542 (3µM) as an inhibitor of Alk-V/TβRI, the

stimulatory effect of TGF-β (2ng/ml) on the average apparent size of biglycan molecules was

completely prevented (Fig. 2, lower panel, lanes 1, 2 and 8). These data indicate that the inhibitory

effect of (S)-[6]-gingerol on [35S]-sulfate incorporation is due entirely to an effect on the synthesis of

proteoglycan core proteins and not on the GAG elongation effect of TGF-β.

TGF-β stimulated [35S]-sulfate incorporation into secreted proteoglycans is the result of a

combination of increased proteoglycan core protein synthesis (mostly biglycan in these cells) which

provides more initiation points for the addition of GAG chains and the elongation of GAG chains on

the core proteins where there is increased [35S]-sulfate due to additional monosaccharide units which

are sulfated [31]. That (S)-[6]-gingerol inhibited [35S]-sulfate incorporation but did not inhibit TGF-β

stimulated GAG elongation (see Fig. 2 upper and lower panel) suggests that the inhibition must arise

from blocking core protein synthesis. Such inhibition might be specific for proteoglycan core

proteins or secondary to inhibition of total de novo protein synthesis [32]. This can be evaluated by

measurement of proteoglycan core protein and total protein synthesis in the same experiment as we

have reported previously for studies of the actions of anti-diabetic biguanides on proteoglycan

synthesis in VSMCs [32]. In cells labelled with [35S]-met/cys, radioactivity can be assessed in the

culture media containing secreted proteoglycans and total de novo protein synthesis assessed by

evaluation of total cell protein. Proteoglycan core protein synthesis represents a modest (10-20%)

component of total protein synthesis in these cells. We determined [35S]-met/cys incorporation into

core proteins secreted into the media and isolated and quantitated by the CPC precipitation method

for proteoglycans. In three independent experiments conducted in triplicate, TGF-β treatment

Page 10 of 30

resulted in a small but statistically significant increase in [35S]-met/cys incorporation into

proteoglycan core proteins and this was blocked in a concentration dependent manner and to 100 per

cent inhibition at 10µM (S)-[6]-gingerol (Fig. 3 upper panel). (S)-[6]-Gingerol had no effect on the

basal rate of incorporation of [35S]-sulfate into proteoglycan core proteins (Fig. 3, upper panel). We

also assessed if the effect was due to (S)-[6]-gingerol acting as an inhibitor of total de novo protein

synthesis. Cells were treated with TGF-β (2ng/ml, 18h) in the presence of (S)-[6]-gingerol (1-10µM).

TGF-β stimulated an almost two-fold increase in total de novo protein synthesis in these VSMCs

(Fig 3, lower panel). (S)-[6]-gingerol had no statistically significant effect on total de novo protein

synthesis in these cells both under basal and stimulated conditions (Fig. 3 lower panel). The small

but not statistically significant effect on total protein synthesis most likely represents the inhibition of

the component of proteoglycan core protein synthesis as shown above (Fig. 3, upper panel). These

data indicate that (S)-[6]-gingerol inhibits proteoglycan core protein synthesis but this is not

secondary to inhibition of total protein synthesis in human VSMCs.

The major proteoglycan synthesised and secreted by these cells and also the major

proteoglycan which is increased in cells treated with TGF-β is the small leucine-rich lipid binding

proteoglycan, biglycan [33]. To further explore the action of (S)-[6]-gingerol on proteoglycan core

protein synthesis identified above, we assessed the effect of (S)-[6]-gingerol on TGF-β stimulated

expression of biglycan mRNA. VSMCs were treated with TGF-β (2ng/ml, 24h) in the presence of

various concentrations of (S)-[6]-gingerol (1-10µM) and SB431542 (10µM). We harvested and

quantitated biglycan mRNA as described in detail in the Methods section. TGF-β treatment resulted

in an approximately 1.5 fold increase in the level of expression of biglycan mRNA (Fig. 4); this is a

low level of expression but it is consistent with earlier reports from our laboratory [22]. (S)-[6]-

Gingerol across all concentrations caused an appreciable and statistically significant inhibition of

TGF-β stimulated biglycan expression but we were not able to show concentration dependence for

this response (Fig. 4). SB431542 inhibited TGF-β stimulated biglycan mRNA expression to below

Page 11 of 30

basal levels of expression which may indicate that there is some endogenous TGF-β release and

endocrine action occurring in these cells [23]. These data demonstrate that the inhibitory action of

(S)-[6]-gingerol on TGF-β stimulated proteoglycan core protein synthesis is targeted at the

expression of biglycan.

The basic signalling pathway through which TGF-β mediates its effects on proteoglycan

synthesis in human VSMCs has been well described in studies from our laboratory [34]; [21, 23, 24].

TGF-β stimulates proteoglycan synthesis via the canonical carboxy terminal Smad phosphorylation

pathway for the expression of biglycan and also the non-Smad pathways utilising the MAP kinases

(Erk1/2 and p38) downstream of Smad linker region phosphorylation for this effect on GAG

elongation [23]. We assessed the effect of (S)-[6]-gingerol on TGF-β signalling pathways in these

cells. Confluent serum-deprived human VSMCs were treated with TGF-β (2 ng/ml, 1h) and the

major Smad products, carboxy terminal phosphorylated Smad2 (pSmad2C) and the linker region

phosphorylated Smad 2 (pSmad2L) were assessed by Western blotting as described in Material and

Methods. As we have previously reported, TGF-β treatment resulted in a two fold increase in

pSmad2C levels which was blocked by SB431542 (Fig. 5). (S)-[6]-Gingerol (1-10µM) had no effect

on this response. TGF-β also stimulated approximately a two fold increase in the linker region

phosphorated product, pSmad2L, which was also inhibited by the control inhibitor SB431542 but

there was no effect of (S)-[6]-gingerol on this response (Fig. 5). These data indicate that the

inhibitory effect and target of (S)-[6]-gingerol does not reside in the upstream areas of the Smad or

early non-Smad signalling pathways in these cells.

We further explored the possible mechanism of the inhibitory action of (S)-[6]-gingerol by

examining its effect on TGF-β-stimulated phosphorylation of the ubiquitous signalling intermediate

kinase, Akt [35]. TGF-β activates phosphatidylinositol- 3- kinase (PI3K) leading in turn to

phosphorylation of Akt as part of the signalling pathway leading to the increased expression of

biglycan mRNA by TGF-β [22] as well as PDGF-mediated versican mRNA expression in vascular

Page 12 of 30

smooth muscle (Osman and Little, unpublished observations). VSMCs were stimulated with TGF-β

(2 ng/ml, 1h) in the presence of (S)-[6]-gingerol (1, 3 and 10 µM) and the inhibitor of Akt

phosphorylation, SN30978 (3 µM). Phosphorylated Akt (pAkt (Ser473)) was assessed by Western

blotting with an antibody directed at the Ser473 residue. As previously reported [22], TGF-β

treatment lead to a rapid increase (two fold) in pAkt (Fig. 6 lanes 1 v 2); (S)-[6]-gingerol at

concentrations up to 10 µM) had no effect on this response whereas the prototypical inhibitor

SN30978 (3 µM) completely blocked the increase in pAkt due to TGF-β (Fig. 6, lane 1, 2 v 5). .

These data indicate that inhibition of the phosphorylation of Akt is not the pathway through which

(S)-[6]-gingerol inhibits TGF-β-stimulated biglycan expression in these cells.

Discussion

We have been investigating the actions of drugs on the synthesis and structure of

proteoglycans, especially the CS/DS proteoglycan biglycan produced and secreted by human

vascular smooth muscle cells on the basis that modified proteoglycans are involved in the pre-

inflammatory stages of atherogenesis and preventing these modifications might yield a therapeutic

agent [36, 37]. We have investigated the action of (S)-[6]-gingerol the major pungent component of

fresh ginger in this model. TGF-β stimulates atherogenic changes in proteoglycan synthesis being

enhanced expression of biglycan and hyperelongation of the GAG chains which are responsible for

lipid binding [14, 16, 21]. (S)-[6]-Gingerol inhibited the expression of proteoglycan core proteins

specifically, biglycan, but did not block TGF-β mediated GAG elongation. The inhibition of TGF-β

stimulated proteoglycan core protein expression was interesting because it was not secondary to an

overall inhibitory effect on total protein synthesis, which is a phenomenon that we have previously

described for the action of anti-diabetic biguanides on proteoglycan synthesis in VSMCs [32].

Surprisingly, (S)-[6]-gingerol did not block either of the TGF-β signalling pathways being the

canonical TβRI pathway leading to carboxy terminal phosphorylation of Smad2 (pSmad2C) or the

Page 13 of 30

so-called non-Smad MAP kinase pathway leading to phosphorylation of Smad2 in its linker region

(pSmad2L) [34]. The latter observation is consistent with the absence of an inhibitory effect on GAG

elongation as we have previously demonstrated that this is due to signalling through Erk1/2 and p38

signalling pathways [23].

Our study used TGF-β stimulated bigylcan synthesis including GAG hyperelongation as an in

vitro model of atherogenesis [21]. Biglycan is a member of the small-leucine rich proteoglycans in

which the core protein is composed of tandem-linked repeats of around 25 amino acid long leucine-

rich repeats to which (usually) two GAG chains are attached. Biglycan itself contains 10 leucine-rich

repeats and its 40kDa core protein binds two chondroitin or dermatan sulfate GAG chains.

Hyperelongated GAG chains due to the action of multiple growth factors and hormones show

enhanced binding to positively charged regions of apoliopoproteins on LDL [13, 14] [14, 17, 38].

GAG synthesising enzymes are increased in experimental models of atherosclerosis. Biglycan levels

are elevated in experimental [39] and clinical [40] atherosclerosis. We have demonstrated that the

inhibition of the action of growth factors on biglycan synthesis and GAG hyperelongation by agents

such as kinase inhibitors prevents the increase in lipid binding in vitro [15, 18] and reduces lipid

deposition in the vessel wall ex vivo and in vivo [15, 18]. (S)-[6]-Gingerol inhibited biglycan

expression but it did not inhibit the distinct signalling pathways leading to GAG hyperelongation

[18]. Inhibition of GAG elongation is a clear pathway to an anti-atherosclerotic agent [15, 18] but

inhibitors of biglycan core protein expression, without inhibition of GAG elongation, might tend to

be anti-atherogenic due to a reduction in the levels of this lipid binding proteoglycan in the vessel

wall.

(S)-[6]-Gingerol inhibited TGF-β stimulated biglycan expression but it did not inhibit the two

major biglycan TGF-β signalling pathways being the Smad and non-Smad pathways [25, 26]. This

raises an interesting question of pharmacology and cell biology as to the mechanism of the inhibitory

action of (S)-[6]-gingerol. At this time we can only speculate on the mechanism of inhibition based

Page 14 of 30

by analogy on the effect of genistein on PDGF stimulated proliferation of VSMCs [41] and the

inhibition of proteoglycan synthesis [42, 43]. In this latter example, PDGF stimulation of VSMCs

leads to PDGF receptor phosphorylation and downstream a fall in the levels of p27kip1 as an

obligatory step in cell cycle progression [41]. Genistein inhibits proliferation by blocking the fall in

p27kip1 levels but it does not block PDGF receptor phosphorylation. Genistein blocks proteoglycan

synthesis in human VSMCs without blocking PDGF mediated PDGF receptor phosphorylation.

Thus, it is possible that (S)-[6]-gingerol blocks a biochemical process mediating biglycan expression

which is downstream of the Smad phosphorylation. TGF-β effects on biglycan expression might be a

good model for further studies on the cellular actions of (S)-[6]-gingerol.

After ruling out Smad phosphorylation as a target for the inhibitory action of (S)-[6]-gingerol

on TGF-β-stimulated biglycan expression in these VSMCs we considered further elements of the

signalling pathways controlling proteoglycan synthesis and structure in these cells. PI3K-mediated

phosphorylation of Akt is part of the TGF-β signalling pathway leading to the increased expression

of biglycan mRNA by VSMCs [22]. Activation of Akt is not part of the signalling pathway

associated with GAG hyperelongation, so it is a point of differentiation of the signalling pathways

controlling two of the structural elements, core protein expression and GAG elongation, of biglycan

in these cells. pAkt is thus a logical target to consider for the action of (S)-[6]-gingerol which inhibits

biglycan expression but not GAG elongation [14, 22]. In the current work we confirmed that TGF-β

stimulates Akt phosphorylation in these cells and we found that (S)-[6]-gingerol did not inhibit this

response. The biochemical signalling target of (S)-[6]-gingerol is other than the recognised Smad and

Akt phosphorylation pathways and thus there appears to be a novel site of action for (S)-[6]-gingerol.

Natural products in the category of traditional and complementary medicines are undergoing

intense investigation for their pharmacological activity and potential therapeutic uses. In addition,

much work is focused on determining the structure of individual components of the natural

medicines and the biological activity of the components. This is in apparent contrast to the

Page 15 of 30

understanding that the actions of complimentary or herbal medicines are based on the principle that

natural medicinal substances have synergistic constituents that allow their use at lower doses with a

resultant broader and safer therapeutic index [44]. Nevertheless, from the perspective of the use of

Western/modern mechanistic medicines, it is necessary to know the chemical structure and

understand the activity of individual component structures. It may well be that ultimately it will be

necessary to reconstitute multiple individual components into products that will mimic the activity of

natural products. In this context we have investigated the actions of (S)-[6]-gingerol being the most

prominent pungent component of fresh ginger.

In this study we have demonstrated that the study of natural products such a (S)-[6]-gingerol

can give considerable insights into biological processes such as the pathways controlling synthesis of

extracellular matrix molecules, specifically the proteoglycan biglycan, by human VSMCs. Further,

such broadening of our understanding of the pharmacology and therapeutic activities of natural

products can shed light on the mechanisms that underlie the therapeutic benefits ascribed to such

products. Where studies focus on the individual chemical components of natural products then there

is the potential to reveal structures which might form the basis of further studies to derive new

molecules with potent pharmacological activities compatible with the development and use of

Western mechanistic medicines. Greater knowledge of the pharmacology of molecules intended for

human therapeutic use can also point to potential toxic effects, an area which has seen the demise of

several important molecules in recent years [45, 46].

Conclusion

This study has revealed some new pharmacological actions of (S)-[6]-gingerol. We have

described the inhibition of the actions of the pro-fibrotic and pro-atheorgenic growth factor, TGF-β

on proteoglycan and synthesis in VSMCs. These actions occur without blocking the major known

Smad and Smad-independent signalling pathways nor the phosphorylation of Akt. As biglycan is a

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central mediator of lipid binding and retention in the early pre-inflammatory stage of atherosclerosis

then these findings underpin the potential use of ginger as a natural product medicine and the use of

(S)-[6]-gingerol as a reference molecule for the development of further molecules with potential

beneficial therapeutic actions.

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Acknowledgements

This work has been supported by research grants from the NHMRC Project Grant 2012 – 2014

(#1022800) (PJL and NO) and National Heart Foundation of Australia, 2011-2012 Grant-in-Aid for

Research (PJL and NO). We acknowledge support of an ARC-Linkage grant to Basil Roufogalis, van

Hoan Tran and Colin Duke. This work was also supported by funding from National Natural Science

Fund of China (No. 30670652; No. 30711120565; No. 30970935) and Science and Technology

Project of Guangdong Province (No. 2011B050200005; No. 2009B060700008) (WZ). We thank

Susan Holmes for assistance with the preparation of the manuscript. This paper is dedicated to the

memory of Elaine Margaret Little (mother of PJL) who passed away during the origins of the

collaboration that lead to the generation of this project.

Conflict of Interest

No conflict to disclose

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Figures

Figure 1. Chemical structure of (S)-[6]-gingerol ((S)-5-hydroxy-1-(4-hydroxy-3-

methoxyphenyl)-3-decanone) (CAS 235130-14-6).

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Figure 2. Effect of (S)-[6]-gingerol on proteoglycan synthesis in human vascular smooth muscle

cells.

VSMCs were preincubated for 30 minutes with (S)-[6]-gingerol (0.1-10M) or control inhibitor

SB431542 (3M) and then stimulated with TGF (2ng/ml) for 24h in the presence of [35S]-sulfate

(25 µCi/ml). Harvested media containing secreted proteoglycans was spotted onto Whatman paper

followed by CPC precipitation to assess radiolabel incorporation into proteoglycans. Results are the

mean ± S.E.M. of each treatment in triplicate (upper panel). Secreted proteoglycans were isolated

using DEAE ion-exchange chromatography followed by concentration using ethanol/potassium

acetate precipitation. Overall size of complete proteoglycans was assessed by SDS-PAGE over a 4-

13% acrylamide gradient gel. The gel reveals biglycan as the proteoglycan of interest (lower panel).

Figure 2.

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Figure 3. Effect of (S)-[6]-gingerol on proteoglycan core protein and total de novo protein

synthesis in VSMCs.

Confluent serum-deprived human VSMCs were treated with (S)-[6]-gingerol (1-10 μM) in the

presence and absence of TGF- (2ng/ml) for 18h. The treatment medium was then removed and

replaced with fresh medium containing 0.1% FBS and 10 Ci/ml [35S]-met/cys for 6 hours. The

culture medium was harvested and proteoglycans precipitated and quantitated on Whatman 3MM

paper using CPC precipitation to determine proteoglycan core protein synthesis (upper panel). The

cell layer from this was assessed for total cellular de novo protein synthesis using standard

techniques (lower panel). Results are the mean ± S.E.M. of data normalised to basal from three

separate experiments in triplicate. **, p<0.01 and *, p<0.05 vs. agonist and ##, p<0.01 vs. basal, using

a one-way ANOVA.

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Figure 3.

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Figure 4. Effect of (S)-[6]-gingerol on TGF-β stimulated biglycan expression in human VSMCs.

VSMCs were pre-incubated 30 min with (S)-[6]-gingerol (1-10M) and control inhibitor SB431542

(3M) followed by stimulation with TGF-β (2ng/ml) for 24h. Real-time quantitative PCR was

performed to determine expression of biglycan messenger and normalized against 18S. Comparative

delta-delta Ct method was used to quantify relative expression levels. Results are shown as mean ±

S.E.M of data combined and normalised to basal from three separate experiments in duplicate.

(#P<0.05 and ##P<0.01 vs. basal) (*P<0.05 and **p<0.01 vs. agonist).

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Figure 5. Effect of (S)-[6]-gingerol on TGF-β receptor (TβRI/AlkV) mediated Smad transcription factor phosphorylation in human VSMCs.A schema shows the relative positions of carboxy terminal (C) and linker region (L)

phosphorylation. After 24h of serum-deprivation (0.1% FBS), confluent human VSMCs were

pre-incubated for 30 min in the presence or absence of (S)-[6]-gingerol (0.3-10M) and control

inhibitor SB431542 (3M) followed by stimulation with TGF-β (2ng/ml) for 1 hour. Whole cell

protein lysates were resolved by 10% SDS-PAGE and transferred to PVDF membranes. Membranes

were blocked for 1h with 5% skim milk prior to incubation with primary monoclonal anti-phospho

Smad2C (Ser465/467) and anti-phospho Smad2L (Ser245/250/255) antibodies and ECL detection.

Blots were stripped and reprobed with GAPDH for loading control. Density analysis was performed

using Image Lab software from Bio-Rad (##P<0.01 vs. basal) (*P<0.05 and **p<0.01 vs. agonist).

Figure 5.

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Figure 6. Effect of (S)-[6]-gingerol of TGF-β stimulated Akt phosphorylation.

Confluent human VSMCs were serum-starved (0.1% FBS) for 24 h prior to 30 min pre-incubation

with or without of (S)-[6]-gingerol (1-10M) and control inhibitor, SN30978 (2M). Cells were

stimulated with TGF-β (2ng/ml) for 1 h and whole cell protein lysates were collected and resolved

by 10% SDS-PAGE and then transferred to PVDF membranes. Membranes were blocked for 1 h

with 5% skim milk prior to incubation with primary monoclonal anti-phospho Akt (Ser473) and ECL

detection. Blots were stripped and reprobed with GAPDH for loading control.

Page 27 of 30

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