**4. Discussion**

Accumulating evidence has shown that resveratrol induces apoptosis or suppresses cell growth and proliferation by interacting with FOXO, NF-kB, and p53 involving wide-range of gene transcriptions such as those for apoptosis-related proteins, p21, p27, and AIF (Chen et al., 2010; Ganapathy et al., 2010; Huang et al., 1999; Kuo et al., 2002; Ragione et al., 2003; She et al., 2001). Notably, resveratrol is shown to increase expression of sirtuin 1, a mammalian NAD+-dependent deacetylase (class III HDAC) (Franco et al., 2010). In the present study, resveratrol upregulated expression of mRNAs for FOXO-1, FOXO-3, p21, p27, AIF, and sirtuin 1 in MH7A cells but otherwise downregulated expression of the Bcl-XL mRNA, without affecting expression of mRNAs for p53, Bcl-2, Bad, Bax, or Bak. FOXO belongs to the O subclass of the forkhead family of transcription factors, which includes FOXO-1, FOXO-3, FOXO-4, and FOXO-6. Evidence has pointed to the role of sirtuin 1 in the regulation of apoptosis-related gene transcription mediated by FOXO or NF-B (Giannakou & Partridge, 2004; Salminen & Kaarniranta, 2009). FOXO and/or sirtuin 1,

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

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

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

Accumulating evidence has shown that resveratrol induces apoptosis or suppresses cell growth and proliferation by interacting with FOXO, NF-kB, and p53 involving wide-range of gene transcriptions such as those for apoptosis-related proteins, p21, p27, and AIF (Chen et al., 2010; Ganapathy et al., 2010; Huang et al., 1999; Kuo et al., 2002; Ragione et al., 2003; She et al., 2001). Notably, resveratrol is shown to increase expression of sirtuin 1, a mammalian NAD+-dependent deacetylase (class III HDAC) (Franco et al., 2010). In the present study, resveratrol upregulated expression of mRNAs for FOXO-1, FOXO-3, p21, p27, AIF, and sirtuin 1 in MH7A cells but otherwise downregulated expression of the Bcl-XL mRNA, without affecting expression of mRNAs for p53, Bcl-2, Bad, Bax, or Bak. FOXO belongs to the O subclass of the forkhead family of transcription factors, which includes FOXO-1, FOXO-3, FOXO-4, and FOXO-6. Evidence has pointed to the role of sirtuin 1 in the regulation of apoptosis-related gene transcription mediated by FOXO or NF-B (Giannakou & Partridge, 2004; Salminen & Kaarniranta, 2009). FOXO and/or sirtuin 1,

kinases (Cdks) via control of p21 and p27.

independent experiments).

**4. Discussion** 

AIF also participates in resveratrol-induced MH7A cell apoptosis.

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

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 apoptosis.

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 mediated via a novel apoptotic pathway.

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., mitochondria-mediated caspase-independent pathway.

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

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

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

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