**3.2 Resveratrol disrupts mitochondrial membrane potentials in a sirtuin 1-dependent manner**

Resveratrol is shown to regulate mitochondrial functions or energy metabolism by interacting with NAD-dependent deacetylase sirtuin 1 (Kaeberlein et al., 2005; Lagouge et al., 2006). Interestingly, resveratrol-induced MH7A cell death was inhibited by sirtinol (10 M), an inhibitor of sirtuin 1 (Fig. 2), suggesting that resveratrol induces MH7A cell death in a sirtuin 1-dependent manner. Moreover, the resveratrol effect was inhibited by tricostatin A (30 nM), an inhibitor of histone deacetylase (HDAC) (Fig. 2). This may account for the implication of sirtuin 1-regulated apoptosis-related gene transcription in the resveratrol effect.

To see whether resveratrol-induced MH7A cell apoptosis is mediated via the mitochondria, mitochondrial membrane potentials were monitored. For untreated cells, orange-red fluorescent signals alone were found (Fig. 3A,B). In contrast, resveratrol (100 M) accumulated green fluorescent signals without orange-red fluorescent signal (Fig. 3C,D), indicating that resveratrol disrupts mitochondrial membrane potentials in MH7A cells. The resveratrol effect on mitochondrial membrane potentials was abolished by sirtinol (10 M) (Fig. 3E,F). This implies that resveratrol disrupts mitochondrial membrane potentials in MH7A cells under the control of sirtuin 1. Taken together, these results indicate that resveratrol induces MH7A cell apoptosis by damaging the mitochondria in a sirtuin 1 dependent manner.

with TTBS (150 mM NaCl, 0.05% Tween20, and 20 mM Tris, pH 7.5) containing 5% BSA and subsequently reacted with an anti-cytochrome c antibody (1:400) (Chemicon, Billerica, MA, USA), followed by an HRP-conjugated goat anti-mouse IgG antibody. Immunoreactivity was detected with an ECL kit (GE Healthcare, NJ, USA) and visualized using a chemiluminescence detection system (FUJIFILM, Tokyo, Japan). Signal density was

Caspase activation was measured using a caspase fluorometric assay kit (Ac-Asp-Glu-Val-Asp-MCA for a caspase-3 substrate peptide; Ac-Ile-Glu-Thr-Asp-MCA for a caspase-8

In the MTT assay, resveratrol reduced MH7A cell viability in a concentration (1-200 M) and treatment time (24-72 h)-dependent manner (Fig. 1A), suggesting that resveratrol

Resveratrol increased TUNEL-positive cells in a concentration (100-200 M)-dependent manner (Fig. 1B), indicating that resveratrol induces MH7A cell apoptosis. DNA damage or apoptosis is recognized to stimulate phosphorylation of histone H2A.X. Resveratrol (100 M) significantly enhanced H2A.X phosphorylation, the extent reaching approximately 13 fold of control levels (Fig. 1C). This provides further evidence for resveratrol-induced

**3.2 Resveratrol disrupts mitochondrial membrane potentials in a sirtuin 1-dependent** 

Resveratrol is shown to regulate mitochondrial functions or energy metabolism by interacting with NAD-dependent deacetylase sirtuin 1 (Kaeberlein et al., 2005; Lagouge et al., 2006). Interestingly, resveratrol-induced MH7A cell death was inhibited by sirtinol (10 M), an inhibitor of sirtuin 1 (Fig. 2), suggesting that resveratrol induces MH7A cell death in a sirtuin 1-dependent manner. Moreover, the resveratrol effect was inhibited by tricostatin A (30 nM), an inhibitor of histone deacetylase (HDAC) (Fig. 2). This may account for the implication of sirtuin 1-regulated apoptosis-related gene transcription in

To see whether resveratrol-induced MH7A cell apoptosis is mediated via the mitochondria, mitochondrial membrane potentials were monitored. For untreated cells, orange-red fluorescent signals alone were found (Fig. 3A,B). In contrast, resveratrol (100 M) accumulated green fluorescent signals without orange-red fluorescent signal (Fig. 3C,D), indicating that resveratrol disrupts mitochondrial membrane potentials in MH7A cells. The resveratrol effect on mitochondrial membrane potentials was abolished by sirtinol (10 M) (Fig. 3E,F). This implies that resveratrol disrupts mitochondrial membrane potentials in MH7A cells under the control of sirtuin 1. Taken together, these results indicate that resveratrol induces MH7A cell apoptosis by damaging the mitochondria in a sirtuin 1-

substrate peptide; and Ac-Leu-Glu-His-Asp-MCA for a caspase-9 substrate peptide).

measured with Image Gauge software (FUJIFILM, Tokyo, Japan).

**2.9 Enzymatic assay of caspase activity** 

**3.1 Resveratrol induces MH7A cell apoptosis** 

**3. Results** 

induces MH7A cell death.

MH7A cell apoptosis.

the resveratrol effect.

dependent manner.

**manner** 

Fig. 1. Resveratrol induces apoptosis in MH7A cells. (**A**) MH7A cells were treated with resveratrol at concentrations as indicated for 24-72 h in serum-free culture medium, and cell viability was quantified with an MTT assay. In the graph, each point represents the mean (± SEM) percentage of basal levels (MTT intensities for cells untreated with resveratrol) (n=8). (**B**) Cells were treated with resveratrol at concentrations as indicated for 24 h in serum-free culture medium, and TUNEL-positive cells were counted. In the graph, each column represents the mean (± SEM) percentage of basal levels (TUNEL-positive cell numbers without resveratrol treatment) (n=3-6). *P* values, unpaired *t*-test. (**C**) Cells were treated with resveratrol at concentrations as indicated for 24 h in FBS-free culture medium, and H2A.X phosphorylation was quantified. In the graph, each column represents the mean (± SEM) ratio against basal levels (H2A.X phosphorylation without resveratrol treatment) (n=4). *P*  values, unpaired *t*-test.

Resveratrol: A Candidate Drug for Treating Rheumatoid Arthritis 275

downregulated expression of the Bcl-XL mRNA in MH7A cells from 1-h through 3-h treatment (Fig. 4C), while it had no effect on expression of mRNAs for Bcl-2 (Fig. 4B), Bad (Fig. 4D), Bax (Fig. 4E), and Bak (Fig. 4F). Collectively, resveratrol may disrupt mitochondrial membrane potentials by reducing Bcl-XL expression through a pathway

Fig. 4. Resveratrol upregulates expression of the sirtuin 1 mRNA and downregulates expression of the Bcl-XL mRNA. MH7A cells were untreated (Control) and treated with resveratrol (100 M) for 20-60 min or 0.5-3 h in serum-free culture medium, and then RT-PCR was carried out. In the graphs, each point represents the ratio against the intensity at 0

min/h regarded as 1. Note that similar results were obtained with 3 independent

experiments.

relevant to sirtuin 1-mediated transcription.

Fig. 2. Resveratrol-induced MH7A cell death is inhibited by a sirtuin 1 inhibitor or an HDAC inhibitor. MH7A cells were treated with resveratrol (100 M) in the absence and presence of sirtinol (10 M) or tricostatin A (TSA) (30 nM) for 48 h in serum-free culture medium, and cell viability was quantified with an MTT assay. In the graph, each column represents the mean (± SEM) percentage of basal levels (MTT intensities for cells untreated with any drug) (n=4). *P* values, unpaired *t*-test.

Fig. 3. Resveratrol disrupts mitochondrial membrane potentials in a sirtuin 1-dependent manner. MH7A cells were treated with resveratrol (100 M) in the absence and presence of sirtinol (10 M) for 24 h in serum-free culture medium, and mitochondrial membrane potentials were monitored. (**A,C,E**) Orange-red fluorescent images at an absorbance of 590 nm. (**B,D,F**) Green fluorescent images at an absorbance of 530 nm. Note that similar results were obtained with 3 independent experiments.

In the RT-PCR analysis, resveratrol (100 M) increased expression of the sirtuin 1 mRNA in MH7A cells in a treatment time (20-60 min)-dependent manner (Fig. 4A). This points to sirtuin 1 being a significant target in resveratrol-induced MH7A cell death. Accumulating evidence has shown that resveratrol upregulates or downregulates expression of the Bcl-2 family that includes Bcl-2 and Bcl-XL, to prevent from mitochondrial damage, and Bad, Bax, and Bak, to induce mitochondrial damage (Shakibaei et al., 2009). Resveratrol (100 M)

Fig. 2. Resveratrol-induced MH7A cell death is inhibited by a sirtuin 1 inhibitor or an HDAC inhibitor. MH7A cells were treated with resveratrol (100 M) in the absence and presence of sirtinol (10 M) or tricostatin A (TSA) (30 nM) for 48 h in serum-free culture medium, and cell viability was quantified with an MTT assay. In the graph, each column represents the mean (± SEM) percentage of basal levels (MTT intensities for cells untreated with any drug)

Fig. 3. Resveratrol disrupts mitochondrial membrane potentials in a sirtuin 1-dependent manner. MH7A cells were treated with resveratrol (100 M) in the absence and presence of sirtinol (10 M) for 24 h in serum-free culture medium, and mitochondrial membrane potentials were monitored. (**A,C,E**) Orange-red fluorescent images at an absorbance of 590 nm. (**B,D,F**) Green fluorescent images at an absorbance of 530 nm. Note that similar results

In the RT-PCR analysis, resveratrol (100 M) increased expression of the sirtuin 1 mRNA in MH7A cells in a treatment time (20-60 min)-dependent manner (Fig. 4A). This points to sirtuin 1 being a significant target in resveratrol-induced MH7A cell death. Accumulating evidence has shown that resveratrol upregulates or downregulates expression of the Bcl-2 family that includes Bcl-2 and Bcl-XL, to prevent from mitochondrial damage, and Bad, Bax, and Bak, to induce mitochondrial damage (Shakibaei et al., 2009). Resveratrol (100 M)

(n=4). *P* values, unpaired *t*-test.

were obtained with 3 independent experiments.

downregulated expression of the Bcl-XL mRNA in MH7A cells from 1-h through 3-h treatment (Fig. 4C), while it had no effect on expression of mRNAs for Bcl-2 (Fig. 4B), Bad (Fig. 4D), Bax (Fig. 4E), and Bak (Fig. 4F). Collectively, resveratrol may disrupt mitochondrial membrane potentials by reducing Bcl-XL expression through a pathway relevant to sirtuin 1-mediated transcription.

Fig. 4. Resveratrol upregulates expression of the sirtuin 1 mRNA and downregulates expression of the Bcl-XL mRNA. MH7A cells were untreated (Control) and treated with resveratrol (100 M) for 20-60 min or 0.5-3 h in serum-free culture medium, and then RT-PCR was carried out. In the graphs, each point represents the ratio against the intensity at 0 min/h regarded as 1. Note that similar results were obtained with 3 independent experiments.

Resveratrol: A Candidate Drug for Treating Rheumatoid Arthritis 277

Fig. 6. Resveratrol activates caspase-3 and -9 in a sirtuin 1-dependent manner. MH7A cells were treated with resveratrol (100 M) in the absence and presence of sirtinol (10 M) for 24 h in serum-free culture medium, and caspase activities were assayed. In the graph, each column represents the mean (± SEM) ratio against basal levels (caspase activities for cells

Fig. 7. Pathway underlying resveratrol-induced apoptosis in MH7A human rheumatoid

arthritis synovial cells.

untreated with any drug) (n=4-6). *P* values, unpaired t-test. *NS*, not significant.

#### **3.3 Resveratrol activates caspase-3 and -9 through mitochondrial damage in a sirtuin 1-dependent manner**

Mitochondrial damage allows release of apoptosis-related factors including cytochrome c. In the Western blot analysis using the mitochondrial and cytosolic component from MH7A cells, resveratrol (100 M) increased presence of cytosolic cytochrome c in parallel with a treatment time (3-24 h)-dependent decrease in the presence of mitochondrial cytochrome c (Fig. 5). This suggests that resveratrol stimulates release of cytochrome c from the mitochondria into the cytosol.

Fig. 5. Resveratrol stimulates cytochrome c release from the mitochondria. MH7A cells were treated with resveratrol (100 M) for 3-24 h in serum-free culture medium, followed by fractionation into the mitochondrial (M) and cytosolic component (C), and Western blotting was carried out. In the graph, each point represents the ratio against the immunoreactive intensity at 0 h for the mitochondrial component regarded as 1. cyto c, cytochrome c. Note that similar results were obtained with 3 independent experiments.

In the enzymatic assay of caspase activity, resveratrol (100 M) significantly activated caspase-3 and -9, but no activation of caspase-8 was obtained (Fig. 6). Resveratrol-induced activation of caspase-3 and -9 was inhibited by sirtinol (10 M) (Fig. 6). Consequently, the results indicate that resveratrol activates caspase-9 and the effector caspase-3 in association with mitochondrial damage allowing cytochrome c release in a sirtuin-dependent manner, to induce MH7A cell apoptosis.

Overall, resveratrol appears to downregulate Bcl-XL expression in a sirtuin 1-dependent manner, which promotes Bax-Bax complex, causing disruption of mitochondrial membrane potentials allowing cytochrome c release from the mitochondria. This is followed by activation of caspase-9 and the effector caspase-3, which, in turn, are responsible for apoptosis in MH7A human rheumatoid arthritis synovial cells (Nakayama et al., 2010) (Fig. 7).

**3.3 Resveratrol activates caspase-3 and -9 through mitochondrial damage in a sirtuin** 

Mitochondrial damage allows release of apoptosis-related factors including cytochrome c. In the Western blot analysis using the mitochondrial and cytosolic component from MH7A cells, resveratrol (100 M) increased presence of cytosolic cytochrome c in parallel with a treatment time (3-24 h)-dependent decrease in the presence of mitochondrial cytochrome c (Fig. 5). This suggests that resveratrol stimulates release of cytochrome c from the

Fig. 5. Resveratrol stimulates cytochrome c release from the mitochondria. MH7A cells were treated with resveratrol (100 M) for 3-24 h in serum-free culture medium, followed by fractionation into the mitochondrial (M) and cytosolic component (C), and Western blotting was carried out. In the graph, each point represents the ratio against the immunoreactive intensity at 0 h for the mitochondrial component regarded as 1. cyto c, cytochrome c. Note

In the enzymatic assay of caspase activity, resveratrol (100 M) significantly activated caspase-3 and -9, but no activation of caspase-8 was obtained (Fig. 6). Resveratrol-induced activation of caspase-3 and -9 was inhibited by sirtinol (10 M) (Fig. 6). Consequently, the results indicate that resveratrol activates caspase-9 and the effector caspase-3 in association with mitochondrial damage allowing cytochrome c release in a sirtuin-dependent manner,

Overall, resveratrol appears to downregulate Bcl-XL expression in a sirtuin 1-dependent manner, which promotes Bax-Bax complex, causing disruption of mitochondrial membrane potentials allowing cytochrome c release from the mitochondria. This is followed by activation of caspase-9 and the effector caspase-3, which, in turn, are responsible for apoptosis in MH7A

that similar results were obtained with 3 independent experiments.

human rheumatoid arthritis synovial cells (Nakayama et al., 2010) (Fig. 7).

**1-dependent manner** 

mitochondria into the cytosol.

to induce MH7A cell apoptosis.

Fig. 6. Resveratrol activates caspase-3 and -9 in a sirtuin 1-dependent manner. MH7A cells were treated with resveratrol (100 M) in the absence and presence of sirtinol (10 M) for 24 h in serum-free culture medium, and caspase activities were assayed. In the graph, each column represents the mean (± SEM) ratio against basal levels (caspase activities for cells untreated with any drug) (n=4-6). *P* values, unpaired t-test. *NS*, not significant.

Fig. 7. Pathway underlying resveratrol-induced apoptosis in MH7A human rheumatoid arthritis synovial cells.

Resveratrol: A Candidate Drug for Treating Rheumatoid Arthritis 279

Fig. 9. Effects of resveratrol and a variety of polyphenols on MH7A cell death. MH7A cells were treated with resveratrol and polyphenols as indicated at concentrations ranging from 10-100 M for 24-72 h in serum-free culture medium, and cell viability was quantified with an MTT assay. In the graphs, each point represents the mean (± SEM) percentage of basal

**3.5 Resveratrol increases expression of mRNAs for FOXO-1, FOXO-3, p21, p27, and** 

Sirtuin 1 regulates apoptosis-related gene transcription mediated by FOXO or NF-κB (Giannakou & Partridge, 2004; Salminen & Kaarniranta, 2009). In support of this, resveratrol induces growth arrest and apoptosis by activating FOXO in prostate cancer cells (Chen et al., 2010). In the real-time RT-PCR analysis, resveratrol (100 M) increased expression of mRNAs both for FOXO-1 and FOXO-3 in MH7A cells in a bell-shaped treatment time (20-60 min)-dependent manner, with the peak at 20-min treatment (Fig. 10A), indicating

Lines of studies have shown that resveratrol induces apoptosis in a variety of cells by activating p53 (Huang et al., 1999; Kuo et al., 2002; She et al., 2001). Resveratrol (100 M) here, however, had no effect on expression of the p53 mRNA in MH7A cells (Fig. 10B). This

Resveratrol is shown to upregulate expression of p21 and p27 (Ganapathy et al., 2010; Ragione et al., 2003). Resveratrol (100 M) increased expression of mRNAs both for p21 and p27 still in MH7A cells in a bell-shaped treatment time (20-60 min)-dependent manner, with

suggests that resveratrol induces MH7A cell apoptosis in a p53-independent manner.

levels (MTT intensities for cells untreated with resveratrol or polyphenols) (n=8).

involvement of FOXO in resveratrol-induced MH7A cell apoptosis.

**AIF in MH7A cells** 

#### **3.4 Resveratrol induces MH7A cell apoptosis with higher potency**

We examined the effect of other polyphenols such as piceatannol, rhapontin, (-)-catechin, (+)-catechin, (-)-epicatechin, (-)-gallocatechin, (-)-epigallocatechin, (-)-catechin gallate, (-) epicatechin gallate, (-)-gallocatechin gallate, and (-)-epigallocatechin gallate on MH7A cell death (Fig. 8). Of these, piceatannol, rhapontin, (-)-gallocatechin, (-)-epigallocatechin, (-) catechin gallate, (-)-epicatechin gallate, (-)-gallocatechin gallate, and (-)-epigallocatechin gallate induced MH7A cell death in a concentration (10-100 M)- and treatment time (24-72 h)-dependent manner, but with lesser potency than resveratrol, whilst no effect was observed for (-)-catechin, (+)-catechin, or (-)-epicatechin (Fig. 9). This indicates that resveratrol is capable of inducing apoptosis in MH7A human rheumatoid arthritis synovial cells, with much higher potency than other polyphenols.

Fig. 8. Chemical structures of resveratrol and a variety of polyphenols.

We examined the effect of other polyphenols such as piceatannol, rhapontin, (-)-catechin, (+)-catechin, (-)-epicatechin, (-)-gallocatechin, (-)-epigallocatechin, (-)-catechin gallate, (-) epicatechin gallate, (-)-gallocatechin gallate, and (-)-epigallocatechin gallate on MH7A cell death (Fig. 8). Of these, piceatannol, rhapontin, (-)-gallocatechin, (-)-epigallocatechin, (-) catechin gallate, (-)-epicatechin gallate, (-)-gallocatechin gallate, and (-)-epigallocatechin gallate induced MH7A cell death in a concentration (10-100 M)- and treatment time (24-72 h)-dependent manner, but with lesser potency than resveratrol, whilst no effect was observed for (-)-catechin, (+)-catechin, or (-)-epicatechin (Fig. 9). This indicates that resveratrol is capable of inducing apoptosis in MH7A human rheumatoid arthritis synovial

**3.4 Resveratrol induces MH7A cell apoptosis with higher potency** 

Fig. 8. Chemical structures of resveratrol and a variety of polyphenols.

cells, with much higher potency than other polyphenols.

Fig. 9. Effects of resveratrol and a variety of polyphenols on MH7A cell death. MH7A cells were treated with resveratrol and polyphenols as indicated at concentrations ranging from 10-100 M for 24-72 h in serum-free culture medium, and cell viability was quantified with an MTT assay. In the graphs, each point represents the mean (± SEM) percentage of basal levels (MTT intensities for cells untreated with resveratrol or polyphenols) (n=8).

#### **3.5 Resveratrol increases expression of mRNAs for FOXO-1, FOXO-3, p21, p27, and AIF in MH7A cells**

Sirtuin 1 regulates apoptosis-related gene transcription mediated by FOXO or NF-κB (Giannakou & Partridge, 2004; Salminen & Kaarniranta, 2009). In support of this, resveratrol induces growth arrest and apoptosis by activating FOXO in prostate cancer cells (Chen et al., 2010). In the real-time RT-PCR analysis, resveratrol (100 M) increased expression of mRNAs both for FOXO-1 and FOXO-3 in MH7A cells in a bell-shaped treatment time (20-60 min)-dependent manner, with the peak at 20-min treatment (Fig. 10A), indicating involvement of FOXO in resveratrol-induced MH7A cell apoptosis.

Lines of studies have shown that resveratrol induces apoptosis in a variety of cells by activating p53 (Huang et al., 1999; Kuo et al., 2002; She et al., 2001). Resveratrol (100 M) here, however, had no effect on expression of the p53 mRNA in MH7A cells (Fig. 10B). This suggests that resveratrol induces MH7A cell apoptosis in a p53-independent manner.

Resveratrol is shown to upregulate expression of p21 and p27 (Ganapathy et al., 2010; Ragione et al., 2003). Resveratrol (100 M) increased expression of mRNAs both for p21 and p27 still in MH7A cells in a bell-shaped treatment time (20-60 min)-dependent manner, with

Resveratrol: A Candidate Drug for Treating Rheumatoid Arthritis 281

therefore, may be a primary target of resveratrol-regulated apoptosis-related gene

Resveratrol induced MH7A cell apoptosis in a concentration (1-200 M)- and treatment time (24-72 h)-dependent manner. Two major pathways for apoptosis are well-recognized, i.e., oxidative stress-induced mitochondria-mediated apoptotic pathway and endoplasmic reticulum (ER) stress-induced apoptotic pathway. For the former pathway, the Bcl-2 family such as Bcl-2, Bcl-XL, Bad, and Bax plays a central role; Bcl-2 and Bcl-XL protect the mitochondria by capturing Bax, but Bad otherwise disrupts mitochondrial membrane potentials by releasing Bax from a Bcl-2/Bax complex or a Bcl-XL/Bax complex. Oxidative stress disrupts mitochondrial membrane potentials by making a Bax/Bax pore, thereby damaging the mitochondria to allow release of apoptosis-related proteins such as cytochrome c, AIF, Smac/DIABLO, Omi/HtrA2, and endonuclease G into the cytosol (Wang, 2001). Subsequently, released cytochrome c activates caspase-3 by forming an apoptosome complex together with apoptosis proteases activating factor-1 (Apaf-1) and caspase-9, leading to apoptosis (Wang, 2001). Resveratrol disrupted mitochondrial membrane potentials, stimulated cytochrome c release from the mitochondria into the cytosol, and activated caspase-3 and –9 in MH7A cells. This, taken together with the finding that resveratrol downregulated the Bcl-XL mRNA, accounts for mitochondriamediated caspase-dependent pathway in resveratrol-induced MH7A cell apoptosis. Of particular interest is that resveratrol-induced MH7A cell apoptosis, mitochondrial damage, and caspase-3/-9 activation were prevented by a sirtuin 1 inhibiter or an HDAC inhibitor. This confirms that sirtuin 1 is required for resveratrol-induced MH7A cell

Findings by Byun et al. (2008) suggest that resveratrol induces apoptosis in fibroblast-like synoviocytes derived from patients with rheumatoid arthritis by activating caspase-8 as a primary target, which cleaves Bid, causing mitochondrial damage that triggers activation of caspase-9 and the effector caspase-3, without affecting the levels of Bax, Bcl-XL, and Bcl-2. This observation contrasts with our finding that resveratrol does not activate caspase-8 in MH7A cells (Nakayama et al., 2010). Thus, resveratrol-induced MH7A cell apoptosis may be

In contrast, resveratrol upregulated expression of the AIF mRNA in MH7A cells. AIF induces apoptosis by causing chromatin condensation and DNA fragmentation. This suggests that resveratrol could induce MH7A cell apoptosis via an additional pathway, i.e.,

Resveratrol also increased expression of the p21 and p27 mRNAs in MH7A cells. p21 and p27 are recognized to inhibit cyclin E/Cdk2 that proceeds cell growth at the G1 phase of cell cycling. Resveratrol, consequently, could suppress MH7A cell growth by inhibiting cyclin

Like resveratrol, some other polyphenols induced apoptosis in MH7A human rheumatoid arthritis synovial cells. Of polyphenols examined here resveratrol induced MH7A cell apoptosis with the highest potency. This implies that, of the polyphenols, resveratrol could

In summary, the results of the present study show that resveratrol upregulates expression of FOXO and suirtuin 1 relevant to apoptosis-related gene transcription and its regulation in MH7A human rheumatoid arthritis synovial cells. Resveratrol downregulates expression of

be the best target for the development of new drugs for treating rheumatoid arthritis.

transcription.

apoptosis.

mediated via a novel apoptotic pathway.

E/Cdk2 under the control of p21 and/or p27.

mitochondria-mediated caspase-independent pathway.

the peak at 40-min treatment (Fig. 10B). This raises the possibility that resveratrol could suppress MH7A cell proliferation and growth by inhibiting cyclin-dependent protein kinases (Cdks) via control of p21 and p27.

Apoptosis-inducing factor (AIF) is released from damaged mitochondria and causes chromatin condensation and large-scale (~50 kbp) DNA fragmentation, leading to caspaseindependent apoptosis. Interestingly, resveratrol induces apoptosis in human lung adenocarcinoma ASTC-a-1 cells through mitochondria-mediated AIF release (Zhang et al., 2011). In the present study, a huge increase in the expression of the AIF mRNAs in MH7A cells was found after 40-min treatment with resveratrol (100 M) (Fig. 10C), suggesting that AIF also participates in resveratrol-induced MH7A cell apoptosis.

Fig. 10. Effects of resveratrol on mRNAs for FOXO-1, FOXO-3, p21, p27, p53, and AIF. MH7A cells were treated with resveratrol (100 M) for periods as indicated in serum-free culture medium, and then real-time RT-PCR was carried out. The mRNA quantity for each gene was calculated from the standard curve made by amplifying different amounts of the GAPDH mRNA, and normalized by regarding the average of independent basal mRNA quantity at 0 h as 1. In the graphs, each point represents the mean (± SEM) ratio (n=3 independent experiments).
