Identification and characterization of [6]-shogaol from ginger as inhibitor of vascular smooth muscle cell proliferation

Scope Vascular smooth muscle cell (VSMC) proliferation is involved in the pathogenesis of cardiovascular disease, making the identification of new counteracting agents and their mechanisms of action relevant. Ginger and its constituents have been reported to improve cardiovascular health, but no studies exist addressing a potential interference with VSMC proliferation. Methods and results The dichloromethane extract of ginger inhibited VSMC proliferation when monitored by resazurin metabolic conversion (IC50 = 2.5 μg/mL). The examination of major constituents from ginger yielded [6]-shogaol as the most active compound (IC50 = 2.7 μM). In the tested concentration range [6]-shogaol did not exhibit cytotoxicity toward VSMC and did not interfere with endothelial cell proliferation. [6]-shogaol inhibited DNA synthesis and induced accumulation of the VSMC in the G0/G1 cell-cycle phase accompanied with activation of the nuclear factor-erythroid 2-related factor 2 (Nrf2)/HO-1 pathway. Since [6]-shogaol lost its antiproliferative activity in the presence of the heme oxygenase-1 (HO-1) inhibitor tin protoporphyrin IX, HO-1 induction appears to contribute to the antiproliferative effect. Conclusion This study demonstrates for the first time inhibitory potential of ginger constituents on VSMC proliferation. The presented data suggest that [6]-shogaol exerts its antiproliferative effect through accumulation of cells in the G0/G1 cell-cycle phase associated with activation of the Nrf2/HO-1 pathway.


Introduction
Ginger, Zingiber officinale Roscoe (Zingiberaceae), is widely used as a spice in foods and beverages, and has also a long history of use in traditional medicine for the treatment of inflammation, rheumatic disorder, indigestion, vomiting, and fever, among others [1-4]. Many pharmacological activities have been reported for this plant and its major pungent principles, including anti-inflammatory, anti-tumorigenic, anti-apoptotic, anti-hyperglycemic, cancer-chemopreventive, anti-lipidemic, and anti-emetic effects [4][5][6][7][8][9]. Cardiovascular disease is the number one cause of death in the world, mainly elicited by atherosclerosis together with hypertension [10]. Aberrant and accelerated vascular smooth muscle cell (VSMC) proliferation not only contributes to initial atherosclerotic plaque formation but also to restenosis (pathological renarrowing of the vessel lumen) after surgical interventions like percutaneous transluminal coronary angioplasty or bypass surgery. To overcome restenosis, drugeluting stents have been developed, aiming at inhibiting VSMC growth by the release of antiproliferative substances. The most prominent drugs used in drug-eluting stents so far have been paclitaxel (a microtubules stabilizing agent) and sirolimus (a mTOR inhibitor). These compounds, however, exhibit a number of unresolved drug-related issues such as impaired reendothelialization and delayed thrombosis induction [11,12], which makes the discovery of novel effective compounds and molecular mechanisms suppressing VSMC proliferation highly relevant.
Plant derived natural products proved to be an excellent resource for the identification of new lead compounds [13]. While ginger is reported to possess vasoprotective effects [14,15], its impact on VSMC proliferation in particular has not been studied so far. In this study, we therefore examined the antiproliferative potential of ginger extract and some of its major active components ([6]-gingerol, [6]-shogaol, zingerone, [6]-paradol, and rac-[6]-dihydroparadol) in VSMC, and characterized in more detail the cellular mode of action of the most active identified compound, [6]-shogaol.

Ginger extraction
Root material of Zingiber officinale (Zingiberaceae) was purchased from PLANTASIA (Oberndorf, Austria). A voucher sample is kept at the University of Graz, Department of Pharmacogonosy under reference number 730149. Ground root material (3.17 g) was mixed with 0.97 g diatomaceous earth (Dionex Corporation, Sunnyvale, CA, USA) before being extracted with HPLC grade dichloromethane using an Accelerated Solvent Extractor (ASE 200, Dionex Corporation, Sunnyvale, CA, USA). Extraction conditions were as follows: Temperature: 44ЊC, cell preheating time: 1 min, pressure: 68.9 bar, static time: 5 min, flush volume: 150%. Three extraction cycles were performed yielding 168 mg of dry extract (5.3%) after solvent evaporation.

Resazurin conversion assay
VSMC were seeded in 96-well plates at 5 × 10 3 cells/well. After 24 h, cells were serum-starved for 24 h to render them quiescent. Quiescent cells were pretreated for 30 min with ginger extract, compounds, or vehicle (0.1% DMSO) as indicated, and subsequently stimulated for 48 h with PDGF-BB (20 ng/mL). To measure the number of metabolically active VSMC by resazurin conversion [18,19], cells were washed with PBS and incubated in serum-free medium containing 10 g/mL resazurin for 2 h. Total metabolic activity was measured by monitoring the increase in fluorescence at a wavelength of 590 nm using an excitation wavelength of 535 nm in a 96-well plate reader (Tecan GENios Pro). HUVECtert cells [16,20] were seeded in 96-well plates at 5 × 10 3 cells/well. After 24 h, HUVECtert cells were treated with compounds or vehicle (0.1% DMSO) as indicated and incubated for 48 h. Then, cells were washed with PBS and incubated in culture medium containing 10 g/mL resazurin for 2 h. The detection step is performed as described above.

Crystal violet biomass staining
VSMC were seeded in 96-well plates at 5 × 10 3 cells/well. Twenty-four hours later, cells were serum starved for 24  to render them quiescent. Quiescent cells were pretreated for 30 min with ginger extract, compounds, or vehicle (0.1% DMSO) as indicated and subsequently stimulated for 48 h with PDGF-BB (20 ng/mL). To determine the total biomass by crystal violet staining, cells were then incubated in 100 L of crystal violet staining solution (0.5% crystal violet, 20% methanol) for 15 min and then washed with ddH 2 O. After drying, 100 L EtOH/Na-citrate solution were added (EtOH: 0.1M Na-citrate = 1:1) and the absorbance of the samples was measured at 595 nm in a 96-well plate reader (Tecan sunrise).

5-Bromo-2 -deoxyuridine (BrdU) incorporation assay
VSMC were seeded in 96-well plates at 5 × 10 3 cells/well. Twenty-four hours later, cells were serum starved for 24 h to render them quiescent. Quiescent cells were pretreated for 30 min with compounds, or vehicle (0.1% DMSO) as indicated and subsequently stimulated with PDGF-BB (20 ng/mL). To estimate de novo DNA synthesis in VSMC [21,22], BrdU was added 2 h after PDGF stimulation, and the incorporation amount was determined 22 h afterwards according to the manufacturer's instructions (Roche Diagnostics).

Assessment of cytotoxicity
VSMC were seeded in 96-well plates at 5 × 10 3 cells/well. Twenty-four hours later, cells were serum starved for 24 h to render them quiescent. Quiescent cells were pretreated for 30 min with compounds, or vehicle (0.1% DMSO) as indicated, and subsequently stimulated for 24 h with PDGF-BB (20 ng/mL). Loss of cell membrane integrity as a sign for cell death can be quantified by the release of the soluble cytosolic protein lactate dehydrogenase (LDH) [20,23]. For this, the supernatant of the treated cells was assessed for LDH activity. For estimation of the total LDH, identically treated samples were incubated for 45 min in the presence of 1% Triton X-100. The released and total LDH enzyme activity was measured for 30 min at the dark in the presence of 4.5 mg/mL lactate, 0.56 mg/mL NAD+, 1.69 U/mL diaphorase, 0.004% w/v BSA, 0.15% w/v sucrose, and 0.5 mM 2-p-iodophenyl-3nitrophenyl tetrazolium chloride. The enzyme reaction was stopped with 1.78 mg/mL oxymate and the absorbance was measured at 490 nm. Potential effects on cell viability were estimated as percentage of extracellular LDH enzyme activity. The cytotoxic natural product digitonin (100 g/mL) was used as a positive control.

Immunoblot analysis
Cells (MEF or VSMC) were seeded onto six-well plates ((3-4) × 10 5 cells/well). Then, cells were lysed and protein extracts were subjected to SDS-PAGE electrophoresis and immunoblot analysis as described [20,22]. Proteins were visualized using enhanced chemiluminescence reagent and an LAS-3000 luminescent image analyzer (Fujifilm) with AIDA software (Raytest) for densitometric evaluation.

Cell counting
VSMC and HUVECtert were seeded and treated as described in chapter 2.4 ("Resazurin conversion assay"). Cell numbers were determined at different time points (with time point zero corresponding to the treatment with the solvent vehicle, 0.1% DMSO) upon trypan blue staining with a ViCell counter (Beckman Coulter, Brea, CA).

Statistical analysis
Statistical analysis was performed by analysis of variance /Bonferroni test or by t test (when comparing just two experimental groups). The number of experiments is given in the figure legends, and a probability value < 0.05 was considered significant. All tests were performed using GraphPad PRISM software, version 4.03.

Ginger extract and its major bioactive components inhibit VSMC proliferation
For evaluating whether ginger contains compounds able to inhibit PDGF-induced proliferation of VSMC, a dichloromethane extract of Z. officinale roots was applied at different concentrations (0.3-30 g/mL; Fig. 1), and the total amount of metabolically active cells was measured after 48 h by the resazurin conversion method [18,19]. The extract suppressed VSMC proliferation concentration dependently with an IC 50 of 2.5 g/mL. The highest tested concentration (30 g/mL) decreased the signal to the basal level of the untreated growth-arrested cells (Fig. 1)   metabolic conversion of resazurin, which generally correlates well to the cell number but could be potentially sensitive to redox-active chemicals or treatments modulating the cellular metabolic capacity. To confirm the antiproliferative effects of the investigated compounds with a method independent of cell metabolism and redox reactions, we quantified total cellular biomass by crystal violet staining. The obtained results were in line with the data from the resazurin conversion assay (Table 1 and Fig. 3B). To further assure that the decreased VSMC number upon treatment with test compounds is not due to cytotoxicity, we also quantified cell death by measuring LDH inside cells and in cell supernatants. No significant changes in cell viability were detected in the investigated concentration range (Table 1 and Fig. 3C).
As vascular health also depends on a functional and intact endothelium, an optimal vasoprotective compound inhibits VSMC activation, but does not interfere with endothelial cell viability. We therefore investigated the impact of the four most active compounds on endothelial viability using metabolic activity as readout. In contrast to paclitaxel (1 M), none of the four ginger compounds that clearly influence VSMC proliferation showed a negative effect on endothelial cells when used in the same concentration range, (Table 1, Fig. 4). Interestingly, paclitaxel had an even stronger antiproliferative effect in endothelial cells (IC 50 = 4 nM) than in VSMC (IC 50 = 108 nM; not shown). To assure that the observed differences in the potency of ginger compounds and paclitaxel in VSMC and endothelial cells are not due to different growth rates between the two cell types, we have determined the doubling times of the two cell types under the used experimental conditions. Cell counting revealed comparable proliferation rates for the PDGF-BB stimulated Based on the observed promising antiproliferative profile of action on the VSMC, we have chosen the most potent identified compound, [6]-shogaol, for further mechanistic studies.

[6]-Shogaol leads to accumulation of VSMC in G 0 /G 1
In order to examine the mechanism of action of [6]-shogaol in VSMC, we first investigated whether it blocks DNA synthesis by quantification of BrdU incorporation in treated cells.
[6]-shogaol potently blunted PDGF-stimulated DNA synthesis in a concentration-dependent manner exhibiting an IC 50 of 3.0 M, which is in the same range as the IC 50 values obtained in the resazurin conversion and the crystal violet assay (Fig. 5A). In line with the observed inhibition of DNA synthesis, [6]-shogaol (10 M) counteracted the PDGF-induced transition of VSMC from the G 0 /G 1 cell-cycle phase to the G 2 /M phase, as revealed by flow cytometry analysis of PI stained nuclei (Fig. 5B).

[6]-Shogaol blocks VSMC proliferation by Nrf2-dependent HO-1 induction
In a consequent experiment the molecular events underlying the observed cell-cycle arrest in VSMC upon shogaol exposure were investigated. Among other bioactivities, [6]shogaol is reported to be an activator of nuclear factor E2 related factor 2 (Nrf2) [24,25]. Nrf2 is a transcription factor that is activated by a vast variety of stressors including oxidative insults, inadequate nutrient supply or electrophilic agents. Activated Nrf2 then launches a transcriptional program that mainly aims at detoxification and cellular stress resistance including cell-cycle control and metabolic adaptation (e.g. reviewed in [26,27]). Activated Nrf2 and elevated expression and activity of the Nrf2-target gene HO-1 have been linked with reduced VSMC proliferation and prevention of cardiovascular disease [28][29][30][31]. We therefore were prompted to analyse whether Nrf2 activation and subsequent HO-1 induction could possibly account for the growth arrest observed in shogaol-treated VSMC. For this, HO-1 expression in proliferating VSMC upon [6]-shogaol exposure was evaluated. Concentrations of 3 and 10 M [6]-shogaol significantly elevated HO-1 levels compared to vehicle treated cells (Fig. 6A). Moreover, using WT and isogenic Nrf2-/-fibroblasts, a strictly Nrf2-dependent increase of HO-1 upon [6]shogaol treatment (Fig. 6B) was observed, confirming Nrf2 activation by [6]-shogaol and excluding involvement of other transcription factors in the HO-1 induction in fibroblasts and most likely also in VSMC. In order to prove causality between the [6]-shogaol-induced HO-1 induction and proliferation stop in VSMC we made use of the HO-1 inhibitor tin protoporphyrin IX. As shown in Fig. 6C and D, [6]-shogaol loses its antiproliferative influence on VSMC in the presence of the HO-1 inhibitor. These findings indicate that [6]-shogaol induces HO-1 that then contributes to a reduced proliferation rate in VSMC.

Discussion
The present study reveals for the first time the inhibition of VSMC proliferation by a ginger extract and four major ginger constituents exhibiting IC 50  induces growth stop associated with accumulation of cells in G 0 /G 1 and induction of HO-1 expression. Ginger has been widely used as a culinary spice as well as in traditional oriental medicine for centuries. In recent years ginger attracted increasing attention due to a variety of newly described bioactivities promoting its use as a safe and effective medicinal plant [1,2,4,32]. The nonvolatile pungent components, such as gingerols, shogaols, paradols, and zingerone, have been reported to be responsible for many of the reported biological effects of the plant [33][34][35]. In fresh ginger, gingerols are the major pungent components, with [6]gingerol being the most abundant. Gingerols are not stable, and their dehydration may yield large amounts of shogaols during prolonged ginger storage. Zingerone also cooccurs with shogaols in stored ginger. Shogaols may undergo reduction to form paradols that are also present in ginger. Shogaols are minor components in fresh ginger, while predominant pungent constituents in the dried ginger, with the ratio of [6]-shogaol to [6]-gingerol being around 1:1 in dried ginger [32][33][34]36,37]. In commercially available ground ginger powders the amount of [6]-shogaol was reported to be in the range of 353-1459 g/g [36,38]. After a single oral administration of ginger oleoresin (300 mg/kg) to rats, the peak plasma concentration of [6]-shogaol was reported to be 0.11 g/L (equalling 0.40 M) [39]. It is important to note that the anti-proliferative effect of [6]-shogaol on VSMC was  (Fig. 3A), total biomass (Fig. 3B), and DNA synthesis (Fig. 5A), respectively. Therefore, although the here described anti-proliferative potential of [6]-shogaol might be of relevance upon local application (e.g. as a coating agent in drug-eluting stents), the question of whether acute or chronic oral administration of ginger could result in beneficial antirestenotic effects requires further research.
Since all investigated ginger constituents are structurally related (Fig. 2), a comparison of their IC 50 values (Table 1) allows deducting a structure-activity relationship (SAR). All compounds possess a vanillyl moiety (4-hydroxy-3-methoxyphenyl) and an alkyl side chain, as shown in Fig. 2. They differ in the substitution pattern of the side chain at positions C3 or C5.
[6]-gingerol, differing from [6]-paradol by having an additional hydroxy substituent at C5, showed even less potency. The activity of rac-[6]-dihydroparadol with a hydroxyl group at C3 was in a similar range as that of [6]-gingerol. Zingerone, also having a carbonyl group at C3, but a shortened and therefore less lipophilic alkyl side chain, showed little activity. Having all this in mind, the presence of an ␣,␤-unsaturated ketone and the length of the alkyl side-chain seems to have significant impact on the extent of the observed antiproliferative activity. Interestingly, the investigated compounds showed a similar SAR when being investigated for anti-oxidative and anti-inflammatory effects [40], cytotoxicity and apoptosis induction in human promyelocytic leukemia (HL-60) cells [41], and protection of neuronal cells from ␤amyloid insult [42].
Noteworthy, the Michael system of the ␣,␤-unsaturated carbonyl type is involved in Nrf2 activation by [6]-shogaol and other small molecules [25,43]. Moreover, HO-1 induction in VSMC by [6]-shogaol appears to be dependent on Nrf2 suggesting that the capacity of activating Nrf2/HO-1 is the basis for the observed SAR between the compounds.
Activation of Nrf2/HO-1 signaling also interferes with VSMC migration (e.g. [44]) and cholesterol accumulation in macrophages (e.g. [45]), as well as prevents endothelial dysfunction (e.g. [46]) and inflammation (e.g. [47]). These features make the hub an attractive target for the prevention of atherosclerosis by influencing different steps in the etiology of the disease. Analyzing whether [6]-shogaol could exert such pleiotropic vasoprotection by activation of Nrf2/HO-1 may therefore represent an interesting subject of future research. At this point it should be noted that although the causality between HO-1 induction and inhibited proliferation was demonstrated, it cannot be excluded that also other factors might be involved in the observed antiproliferative activity of [6]-shogaol. Likewise, inhibition of STAT3 or activation of PPAR␥ were reported for [6]-shogaol [48,49] and could also contribute to the cell-cycle arrest in VSMC [22,50].
Despite its potent effect on VSMC, [6]-shogaol did obviously not impair endothelial viability and proliferation (Fig. 4). Reendothelialization is a key step toward successful vascular healing after therapeutic interventions such as angioplasty or bypass surgery [51,52]. Currently, the only clinical available treatment for restenosis is drug-eluting stent coated with rapamycin or paclitaxel [53]. Both drugs not only inhibit VSMC, but also endothelial cells, causing impaired reendotheliazation and lengthening the healing of the wounded vessels [54,55]. Our results on the apparent preferential activity of ginger compounds towards VSMC (Table 1) may therefore inspire the future search for novel stent coating agents. Interestingly, [6]-shogaol exhibited its potent antiproliferation effect on VSMC without affecting the proliferation of endothelial HUVECtert cells.
In conclusion, the present study examines for the first time the inhibitory potential of ginger and its constituent [6]-shogaol towards VSMC proliferation. Furthermore, the structure-function relationship of several closely related ginger constituents is characterized and it is demonstrated that [6]-shogaol exerts its antiproliferative effect through accumulation of cells in G 0 /G 1 associated with the activation of Nrf2/HO-1 pathway.
The authors have declared no conflict of interest.