Resveratrol (trans-3,5,4′-trihydroxystilbene; RSV), a natural polyphenol, exerts a beneficial effect on health and diseases. RSV targets and activates the NAD+-dependent protein deacetylase SIRT1; in turn, SIRT1 induces an intracellular antioxidative mechanism by inducing mitochondrial superoxide dismutase (SOD2). Most RSV found in plants is glycosylated, and the effect of these glycosylated forms on SIRT1 has not been studied. In this study, we compared the effects of RSV and two glycosyl RSVs, resveratrol-3-O-β-d-glucoside (3G-RSV; polydatin/piceid) and resveratrol-4′-O-β-d-glucoside (4′G-RSV), at the cellular level. In oxygen radical absorbance capacity and 2,2-diphenyl-1-picrylhydrazyl radical scavenging assays, the antioxidant activity of 3G-RSV was comparable to that of RSV, whereas the radical-scavenging efficiency of 4′G-RSV was less than 50% of that of RSV. However, 4′G-RSV, but not 3G-RSV, induced SIRT1-dependent histone H3 deacetylation and SOD2 expression in mouse C2C12 skeletal myoblasts; as with RSV, SIRT1 knockdown blunted these effects. RSV and 4′G-RSV, but not 3G-RSV, mitigated oxidative stress–induced cell death in C2C12 cells and primary neonatal rat cardiomyocytes. RSV and 4′G-RSV inhibited C2C12 cell proliferation, but 3G-RSV did not. RSV was found in both the intracellular and extracellular fractions of C2C12 cells that had been incubated with 4′G-RSV, indicating that 4′G-RSV was extracellularly deglycosylated to RSV, which was then taken up by the cells. C2C12 cells did not deglycosylate 3G-RSV. Our results point to 4′G-RSV as a useful RSV prodrug with high water solubility. These data also show that the in vitro antioxidative activity of these molecules did not correlate with their ability to protect cells from oxidative stress–induced apoptosis.
Resveratrol (RSV), a defense antioxidant molecule found in plants, has various target molecules in mammalian cells. Its health benefits include activity against cancer (Jang et al., 1997), metabolic disease (Baur et al., 2006; Lagouge et al., 2006), diabetes (Su et al., 2006), atherosclerosis (Wu and Hsieh, 2011), and muscular dystrophy (Hori et al., 2011), as well as cardioprotective (Tanno et al., 2010) and neuroprotective (Sun et al., 2010) effects. RSV targets and activates the NAD+-dependent protein deacetylase SIRT1 (Howitz et al., 2003). RSV not only acts as an antioxidant itself but also induces other intracellular antioxidative activity. We previously found that RSV decreases intracellular reactive oxygen species (ROS) levels by inducing mitochondrial superoxide dismutase (SOD2) via SIRT1 activation in C2C12 myoblast cells and cardiomyocytes (Tanno et al., 2010) and that long-term RSV treatment decreases ROS levels and significantly prolongs survival in cardiomyopathic TO2 hamsters (Tanno et al., 2010).
Plants produce various RSV derivatives, among which glycosyl RSVs, especially resveratrol-3-O-β-d-glucoside (3G-RSV; polydatin/piceid), are prominent (Romero-Perez et al., 1999). Because 3G-RSV resists oxidation by tyrosinases, its half-life may be longer than that of RSV in vivo (Regev-Shoshani et al., 2003). Glycosylation increases the water solubility of polyphenols (Vogt and Jones, 2000), allowing for more convenient, efficient administration either orally or parenterally. We recently synthesized 3G-RSV and 4′-O-β-d-glucoside RSV (4′G-RSV) (Weis et al., 2006) from RSV using a glucosyltransferase expressed in Escherichia coli (Ozaki et al., 2012). Although unmodified RSV and 3G-RSV have the same antioxidative capacity in vitro (Fabris et al., 2008), the characteristics of 4′G-RSV have not been determined. Likewise, it has not been determined whether glycosyl RSVs can activate SIRT1 or exert other biologic functions in cultured cells.
We found that resveratrol inhibits oxidative stress–induced cell death of C2C12 cells and cardiomyocytes (Tanno et al., 2010). In the present study, we used these cells to examine the properties of 3G-RSV and 4′G-RSV purified from cultured Phytolacca americana plant cells. Whereas RSV and 4′G-RSV exerted similar effects on myoblasts and cardiomyocytes, 3G-RSV had little or no effect on cellular function. We show here that although glycosyl RSVs are not transported into C2C12 cells, C2C12 cells can extracellularly deglycosylate 4′G-RSV to RSV, which then enters the cell.
Materials and Methods
RSV and Hoechst dye 33342 were purchased from Wako Pure Chemicals (Osaka, Japan). We synthesized 3G-RSV and 4′G-RSV (Fig. 1A) using RSV and P. americana plant cells; 3G-RSV and 4′G-RSV were purified as previously reported (Ozaki et al., 2012). The purity of RSV, 3G-RSV, and 4′G-RSV used in the experiments was confirmed by high-performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) (Supplemental Fig. 1). We used the following antibodies: rabbit polyclonal anti-acetyl-histone H3 (Calbiochem, San Diego, CA), rabbit polyclonal histone H3 (Abcam, Cambridge, MA), rabbit polyclonal anti-SOD2 (Millipore, Billerica, MA), rabbit polyclonal anti-SIRT1 (Sakamoto et al., 2004), mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH), mouse monoclonal anti-α-tubulin (Sigma-Aldrich, St. Louis, MO), and rabbit anti-active caspase-3 antibody (ab32042, 1:200 dilution) (Abcam). Other reagents were purchased from Wako Pure Chemicals or Sigma-Aldrich.
Oxygen Radical Absorbance Capacity Assay.
Oxygen radical absorbance capacity (ORAC) assay was based on a previous report by Prior et al. (2003). Test samples were dissolved in 99.5% ethanol. The assay was carried out on a Powerscan HT plate reader (DS Pharma Biomedical, Osaka, Japan). AAPH [2, 2′-azobis (2-amidinopropane) dihydrochloride] was used as a peroxyl generator and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) as a standard. Trolox standard solution (6.25, 12.5, 25, and 50 μM) or blank, fluorescein solution and AAPH solution were incubated in assay buffer at 37°C in a 96-well plate. The fluorescence (excitation 485 nm, emission 530 nm) was monitored every 2 minutes for 90 minutes. The net area under the curve was calculated by subtracting the area under the curve for the blank from that for the sample or standard. The ORAC values were calculated from the Trolox standard curve. The ORAC value was expressed as micromoles of Trolox equivalent per micromole of sample.
DPPH Radical-Scavenging Activity.
The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging activity was measured as reported by Morales and Jiménez-Pérez (2001), with a slight modification. Test samples in 500 μl of water were mixed with 500 μl of 99.5% ethanol containing 0.15 mM DPPH. This mixture was shaken and kept at room temperature for 30 minutes, and the absorbance of the mixture was measured at 517 nm. The results were calculated as percent inhibition according to the formula [(C –SB) – (S – SB)/(C – SB)] × 100, where S, SB, and C are the absorbance of the sample, blank sample, and control, respectively.
Cell Culture and Treatment.
C2C12 myoblast cells were cultured with Dulbecco’s modified Eagle’s medium (Wako Pure Chemical) supplemented with 1% antibiotic-antimycotic mixed stock solution (Nacalai Tesque, Kyoto, Japan) and 10% fetal bovine serum (MP Biomedicals, Solon, OH). Cells were seeded on glass slides for immunocytochemical studies. To examine histone deacetylation, C2C12 cells were pretreated for 18 hours with 10 nM trichostatin A, which inhibits class I and II histone deacetylases, and then incubated with vehicle (dimethyl sulfoxide) or 100 μM RSV, 3G-RSV, or 4′G-RSV for 24 hours. Cell samples were then analyzed by immunoblotting. SOD2 induction was examined by treating cells with vehicle or with 100 μM RSV, 3G-RSV, or 4′G-RSV for 24 hours; 100 μM antimycin A (AA) was added 6 hours before harvest to induce oxidative stress. Cell lysates were analyzed by immunoblotting, immunostaining, or quantitative reverse-transcription polymerase chain reaction.
Cells were lysed with CelLytic M Cell Lysis Reagent (Sigma-Aldrich) with 1% protease inhibitor cocktail (Nacalai Tesque) and centrifuged at 10,000g for 10 minutes at 4°C. The supernatant protein concentration was measured with the Protein Quantification Kit-Rapid (Dojindo, Kumamoto, Japan). Equal protein amounts per lane (20 μg) were analyzed by immunoblotting as described previously (Hori et al., 2011), using the following antibodies: anti-acetyl-histone H3 (1:4000), anti-histone H3 (1:50,000), anti-SOD2 (1:1000), anti-GAPDH (1:10,000), and anti-α-tubulin (1:10,000).
Samples were fixed with 4% paraformaldehyde and treated for 30 minutes with phosphate-buffered saline (PBS) containing 3% bovine serum albumin, 1% goat serum, and 0.1% Triton X-100. Next, samples were incubated with antibodies against SOD2 (1:1000 dilution) overnight at 4°C, washed with PBS, incubated with secondary antibodies (1:2000 dilution) for 4 hours at room temperature, and washed again with PBS. Samples were then incubated with Hoechst dye 33342 (1:1000 dilution) to stain the nucleus and were mounted with VECTASHIELD (Vector Laboratories, Burlingame, CA). SOD2 levels were compared by image fluorescence intensity and quantified with ImageJ Software (National Institutes of Health, Bethesda, MD); 8 independent fields were examined in each experiment, and the data from three independent experiments were compared. To stain activated caspase-3, C2C12 cells were treated with 30 μM RSV, 3G-RSV, or 4′G-RSV for 4 hours, and then cells were further incubated with 50 μM AA for 8 hours in the absence of serum. Fixed cells were stained with antiactive caspase-3 antibody and Hoechst dye 33342. Five fields for each treatment were examined, and the data from four independent experiments were compared.
Quantitative Reverse-Transcription Polymerase Chain Reaction.
Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA). First-strand cDNA was synthesized using SuperScript III (Invitrogen, Carlsbad, CA). DNA was amplified with TaqMan Universal Master Mix II with uracil-DNA glycosylase (UNG; also known as UDG) (Applied Biosystems, Foster City, CA) and TaqMan Gene Expression Assays for SOD2 (Mm00449726_m1) or GAPDH (Mm99999915_g1). Data from three independent experiments were compared.
Small interfering RNAs (siRNAs) for SIRT1 (SASI_Mm01_00105675) and control siRNAs from Sigma Genosys Japan (Ishikari, Japan) were used as described previously (Hori et al., 2011). We used a Nucleofector kit (Lonza, Basel, Switzerland) to electroporate siRNA (100 nM) into C2C12 cells twice, 24 hours apart, and the cells were used in experiments 24 hours after the second electroporation.
Apoptosis Assays in Neonatal Rat Cardiomyocytes.
Neonatal rat cardiomyocytes were isolated as previously reported (Tanno et al., 2010). Propidium iodide (PI) staining was used to identify dead cells before pharmacological modulation, and PI+ cells were eliminated for statistical analysis. Beginning 36 hours after isolation, cells were incubated with vehicle or 40 μM RSV, 3G-RSV, or 4′G-RSV for 36 hours; 100 μM AA was added 24 hours before harvest. Apoptotic cells were detected by terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling (TUNEL) staining and nuclear condensation as previously described (Tanno et al., 2010). Six independent experiments were carried out, and cells of 14 visual fields were counted in each experiment, except for one experiment in which the number of cells from seven visual fields was examined.
C2C12 cells were treated with vehicle or 30 μM RSV, 3G-RSV, or 4′G-RSV for 24 hours. Cells were fixed with 4% paraformaldehyde, stained by Hoechst dye 33342, and counted. Data from six independent experiments were compared.
Mass Spectrometry and High-Performance Liquid Chromatography.
ESI-MS was performed with a JMS-LCmate (JEOL, Tokyo, Japan) in positive ionization mode (ESI+) with a methanol carrier solution at a 0.5 ml/min flow rate and injection volume of 2 μl.
HPLC was performed with an MD-1510 and a reverse-phase CRESTPAK C18S column (150 × 4.6 mm; Jasco, Tokyo, Japan). Data were analyzed by DP-L1500W (Jasco). The mobile phase consisted of acetonitrile and H2O in a 15:85 ratio (v/v). The mobile phase was filtered through a 0.2-μm membrane and degassed by aspirator before analysis. A 20-μl sample was separated by the column at a 1.0 ml/min flow rate, and RSV and glycosyl RSVs were monitored at 323 nm. All experiments were performed at ambient temperature.
Intracellular and Extracellular RSV and Glycosyl RSV Measurement.
C2C12 cells cultured in a 10-cm dish were incubated with 100 μM RSV, 3G-RSV, or 4′G-RSV for 12 hours, after which 10 ml of culture medium was transferred to a tube containing 10 ml of methanol. The mixture was shaken vigorously for 30 minutes at room temperature, and the methanol layer, which contained the RSV or RSV derivatives, was isolated and evaporated; dried samples were stored at −80°C until use. After removing the culture medium, the cells were washed twice with PBS and lysed with CelLytic M Cell Lysis Reagent. The cell lysates were sonicated and centrifuged. The RSV and RSV derivatives were extracted from the supernatant fractions with equal volumes of methanol, and were dried. The samples were dissolved in 100 μl of methanol and examined by ESI-MS and HPLC in at least three independent experiments.
Results are presented as the mean ± S.E.M. All statistical analyses were carried out using SigmaStat (Systat Software, Chicago, IL). Differences were tested by a one-way analysis of variance. A P value <0.05 was considered significant.
3G-RSV and 4′G-RSV Antioxidative Activity In Vitro.
We compared the in vitro antioxidative activity of 3G-RSV and 4′G-RSV with that of RSV and found that, although RSV and 3G-RSV had similar ORAC values, those of 4′G-RSV were much lower (Table 1). The DPPH-scavenging capacity was almost the same for 3G-RSV and RSV, whereas the capacity for 4′G-RSV was approximately half that of RSV (Fig. 1B). The DPPH-scavenging half-maximal (50%) inhibitory concentrations of RSV and 3G-RSV were approximately 80 and 110 μM, respectively; however, 250 μM 4′G-RSV was needed to neutralize 50% DPPH radicals. Thus, the in vitro antioxidant activity of 3G-RSV was comparable to that of RSV, whereas 4′G-RSV was less able to eliminate ROS.
SIRT1-Dependent Induction of Histone H3 Deacetylation and SOD2 Expression by 4′G-RSV.
RSV promotes histone H3 deacetylation and increases SOD2 expression in a SIRT1-dependent manner (Tanno et al., 2010). The acetyl histone H3 level decreased significantly in C2C12 cells treated with RSV or 4′G-RSV but was unaffected by 3G-RSV (Fig. 2A). In addition, the SOD2 mRNA level increased in C2C12 cells treated with RSV or 4′G-RSV but not in those treated with 3G-RSV (Fig. 2B); immunostaining and immunoblotting experiments showed the same to be true for SOD2 protein levels (Fig. 2, C and D).
We next tested whether 4′G-RSV exerted these effects by activating SIRT1. The SIRT1 protein level of C2C12 cells treated with SIRT1 siRNA was 38% of that in the cells treated with control siRNA (Fig. 3A). RSV or 4′G-RSV promoted histone H3 deacetylation in cells transfected with control siRNA but not in SIRT1-knockdown cells (Fig. 3B). Knocking down SIRT1 also blunted the SOD2 protein increase induced by RSV or 4′G-RSV (Fig. 3C). Again, 3G-RSV treatment did not affect the SOD2 levels in cells transfected with either control siRNA or SIRT1 siRNA (Fig. 3C).
4′G-RSV Antiapoptotic and Antiproliferative Activity.
We next examined the antiapoptotic activity of the RSV derivatives. RSV inhibits oxidative stress–induced cell death in neonatal rat ventricular cardiomyocytes by activating SIRT1 (Tanno et al., 2010). Treatment with AA, which increases and releases ROS from mitochondria by inhibiting mitochondrial respiratory chain complex III, increased the number of C2C12 cells with condensed nuclei (Fig. 4A) and activated caspase-3–positive cells (Fig. 4B). Pretreating the cells with RSV or 4′G-RSV, but not 3G-RSV, significantly suppressed this increase (Fig. 4, A and B). We also examined apoptosis of neonatal rat ventricular cardiomyocytes by TUNEL staining (Fig. 4C). AA increased the number of TUNEL-positive apoptotic cells, and pretreatment with RSV or 4′G-RSV significantly suppressed this increase (Fig. 4C).
Since RSV arrests cell growth in some cells (Chen et al., 2010), we evaluated the effect of the glycosyl RSVs on cell growth by counting C2C12 cells after 24 hours of treatment with 30 μM RSV, 3G-RSV, or 4′G-RSV (Fig. 5). RSV significantly inhibited the C2C12 cell proliferation; the RSV-treated cell population was less than 50% of that of the control cells. The cell proliferation was moderately inhibited by 4′G-RSV; the 4′G-RSV–treated cell population was 73% of that of the control cells. Cell growth was not inhibited by 3G-RSV (Fig. 5).
Conversion of 4′G-RSV to RSV by C2C12 Cells.
Since these findings raised the possibility that 3G-RSV cannot permeate the C2C12 and cardiomyocyte cellular membranes, we next examined the cellular uptake of the glycosyl RSVs. C2C12 cells were treated with RSV, 3G-RSV, or 4′G-RSV for 12 hours; washed twice with PBS; and lysed, after which RSV or RSV derivatives were extracted from the cell lysates and analyzed by ESI-MS (Fig. 6, A–C). RSV was found in the intracellular fraction of RSV-treated cells (Fig. 6A). However, 3G-RSV could not be detected in the 3G-RSV–treated cells (Fig. 6B); thus, 3G-RSV poorly permeates the C2C12 cellular membrane. Interestingly, the intracellular fraction from 4′G-RSV–treated cells contained RSV but not 4′G-RSV (Fig. 6C). This raised the question of whether 4′G-RSV was transported into the cell and then deglycosylated into RSV or whether the 4′G-RSV was extracellularly deglycosylated and the resultant RSV taken up by the cell.
To address this question, C2C12 cells were incubated with culture medium and 100 μM RSV, 3G-RSV, or 4′G-RSV for 12 hours, and then cells and the culture medium were analyzed by an octadecylsilyl column. RSV, 3G-RSV, and 4′G-RSV were separated and identified with HPLC (Fig. 7) as shown by Ozaki et al. (2012) and the contents of RSV or RSV derivatives were shown (Table 2). Both the cell fraction and the culture medium of RSV-treated cells contained RSV [Fig. 7 (RSV) and Table 2]. Neither 3G-RSV nor RSV was detected in the cellular fraction of 3G-RSV–treated cells, although 3G-RSV was detected in the culture medium [Fig. 7 (3G-RSV) and Table 2]. Only RSV was detected in the intracellular fraction of 4′G-RSV–treated cells, although both 4′G-RSV and RSV were found in the culture medium [Fig. 7 (4′G-RSV) and Table 2]. When 4′G-RSV was incubated with culture medium in the absence of cells, however, RSV was not detected in the culture medium, indicating that 4′G-RSV was deglycosylated by the C2C12 cells (Supplemental Fig. 2). Our results suggest that C2C12 cells metabolized 4′G-RSV extracellularly and took in the resulting RSV.
Although glycosylating RSV should increase its bioavailability by increasing its water solubility and stability, it is not clear whether glycosyl RSV derivatives can retain the biologic activity of unmodified RSV. In this study, we found that the in vitro antioxidant activity of 3G-RSV was comparable to that of RSV, whereas the antioxidant activity of 4′G-RSV was less than 50% of that of RSV. However, treating cells with 4′G-RSV, but not 3G-RSV, induced histone deacetylation and SOD2 expression to the same degree as treatment with RSV. We also found that 4′G-RSV, but not 3G-RSV, suppressed apoptosis and inhibited cell proliferation. These data clearly indicate that RSV derivatives do not necessarily exert the same cellular effects as native RSV, and that a compound’s in vitro antioxidative activity does not necessarily reflect its ROS-scavenging capacity in vivo. In addition, the RSV glycosylation site is critical for the antioxidative function of a glycosyl RSV.
RSV cellular uptake occurs by passive diffusion without the participation of transporters or pumps in human intestinal Caco-2 cells (Henry et al., 2005) and in the rat intestinal perfusion model (Juan et al., 2010). We found that neither 3G-RSV nor 4′G-RSV could enter C2C12 cells (Figs. 6 and 7). The enhanced hydrophilic property of glycosyl RSVs compared with unmodified RSV may interfere with their diffusion across the lipid bilayer. As mentioned previously, 3G-RSV offered little or no protection against oxidative stress (Fig. 4, A–C), indicating that reducing the extracellular ROS is not sufficient to protect cells; RSV must enter the cells to improve cell survival, at least under the conditions we used. Although Caco-2 cells are able to take up 3G-RSV slowly, the accumulation rate is only 25% that of RSV (Henry et al., 2005). Pharmacological examination suggested that 3G-RSV is transported by the sodium-dependent glucose cotransporter 1 (Henry et al., 2005) that participates in glucose uptake in the brush-border membrane of the intestinal mucosa and the proximal tubule of the nephron (Wright et al., 2011). Wang et al. (2012) showed that 3G-RSV, administered intravenously, decreases the mitochondrial membrane potential and ATP level and inhibits mitochondrial swelling more effectively than RSV in arterial smooth muscle cells of a rat hemorrhage shock model. These results indicate that 3G-RSV and RSV differ in biologic function in vivo. Although sodium-dependent glucose cotransporter 11 is ubiquitously expressed in various tissues, including the heart (Wright et al., 2011), neonatal rat cardiomyocytes and C2C12 cells were insensitive to 3G-RSV in our experiments, and C2C12 cells could not take up 3G-RSV. The reason for this discrepancy is not known.
We found that C2C12 cells could metabolize 4′G-RSV, but not 3G-RSV, into RSV. Although β-glucosidase from almonds is reported to metabolize 3G-RSV to RSV (Krasnow and Murphy, 2004), our experiment indicated that 4′G-RSV, but not 3G-RSV, was a substrate for β-glucosidase in C2C12 cells. Since 4′G-RSV effectively protected rat neonatal cardiomyocytes against oxidative stress (Fig. 4C), cardiomyocytes may express a β-glucosidase similar to that in C2C12 cells. The mammalian β-glucosidase family consists of four members: cytosolic β-glucosidase (de Graaf et al., 2001); the lactase expressed in the intestinal brush-border membrane (Mantei et al., 1988); lysosomal β-glucosidase (β-glucocerebrosidase), which can cause Gaucher’s disease if defective (Kacher et al., 2008); and membrane-bound β-glucosidase. Among these, membrane-bound β-glucosidase, also known as β-glucosidase 2 (GBA2) or nonlysosomal glucosylceramidase, is expressed in the widest variety of tissues, including heart and skeletal muscles, and has the widest substrate specificity (Matern et al., 2001). Deglycosylated products are found in the culture medium of cells expressing GBA2 when substrate is added extracellularly (Boot et al., 2007). These results suggest that GBA2 is an exoplasmic glucosidase and that it catabolizes 4′G-RSV to RSV.
We found that the amount of intracellular RSV after incubation of C2C12 cells with 100 μM RSV was 5.34 ± 0.14 nmol (Table 2). Because the initial amount of RSV added to the culture medium was 1 μmol, only 0.5% of RSV was detected in the cells (Table 2). Accordingly, the resulting culture medium after incubation with RSV for 12 hours contained an unexpectedly low level of RSV, and only 1.7% of RSV was recovered from the culture medium (Table 2). Sodium bicarbonate in the culture medium is reported to degrade RSV, and 96% of RSV at a concentration of 200 μM is degraded in base-modified Eagle’s medium after 24 hours of incubation (Yang et al., 2010). Thus, extremely low levels of RSV and glycosyl RSVs in the culture medium and cells may be attributed to their degradation by the culture medium.
Whether SIRT1 is directly or indirectly activated by RSV is under debate (for a review, see Horio et al., 2011). As SIRT1 activation by RSV derivatives may provide clues to help resolve this debate, we also intended to examine whether glycosyl RSVs activated SIRT1 in the present study. However, the glycosyl RSVs failed to enter cells. We tried to determine the effects of the glycosyl RSVs on histone H3 deacetylation in cell homogenates of C2C12 cells, but neither glycosyl RSVs nor RSV promoted histone H3 deacetylation in vitro (data not shown).
Our experiments showed that 4′G-RSV may be a useful RSV prodrug. In natural plants, 3G-RSV is one of the predominant glycosyl RSVs found, unlike 4′G-RSV (Romero-Perez et al., 1999). However, E. coli cells expressing glucosyltransferase from P. americana produce 4′G-RSV and 3G-RSV from RSV at a ratio of 10:3, and the conversion rate of RSV to glycosyl RSVs is almost 100% after 24 hours of incubation (Ozaki et al., 2012). P. americana plant cell cultures also yielded high 4′G-RSV levels. Unlike RSV, which is highly hydrophobic, 4′G-RSV is hydrophilic and may be highly useful for medical use such as treatment of diabetes mellitus and muscular dystrophy.
Participated in research design: Horio, Kuno, Wakamiya, Hamada.
Conducted experiments: Hosoda, Kuno, Hori, Ohtani, Oohiro.
Performed data analysis: Kuno, Wakamiya, Hamada, Horio.
Wrote or contributed to the writing of the manuscript: Horio, Kuno, Hosoda.
This work was supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grant-in-Aid 22590245); by a National Project of the Knowledge Cluster Initiative (Second Stage); by Sapporo Biocluster Bio-S; by the Program for Developing the Supporting System for Upgrading Education and Research; and by the Regional R&D Proposal-Based Program from the Northern Advancement Center for Science & Technology of Hokkaido, Japan.
- antimycin A
- 2, 2′-diphenyl-1-picrylhydrazyl
- electrospray ionization mass spectrometry
- glyceraldehyde-3-phosphate dehydrogenase
- β-glucosidase 2
- high-performance liquid chromatography
- oxygen radical absorbance capacity
- phosphate-buffered saline
- propidium iodide
- reactive oxygen species
- small interfering RNA
- mitochondrial superoxide dismutase
- 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
- terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling
- Received July 31, 2012.
- Accepted October 4, 2012.
- Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics