Regulation of p53 and cell proliferation by resveratrol and its derivatives in breast cancer cells: an in silico and biochemical approach targeting integrin avb3 Tze-Chen Hsieh * , Christina Wong * , Dylan John Bennett and Joseph M. Wu Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY Resveratrol is a grape polyphenol with cancer preventative activities in tissue culture and animal model studies. Potential of resveratrol as a broad-based chemopreventive agent have been questioned by its limited bioavailability. The bioefficacy of resveratrol was compared with its derivatives, triacetyl-resveratrol (trans-3,5,4 0 -triacetylstilbene) and trimethoxy-resveratrol (trans-3,5,4 0 -trimethoxystilbene) in both estrogen receptor-a (ERa)-positive MCF-7 and ERa-negative MDA-MB-231 breast cancer cells. Binding to integrin avb3 and control of cell proliferation and p53 were chosen as targets for comparative analysis using an in silico and biochemical approach. Resveratrol and triacetyl-resveratrol interacted avidly and specifically with integrin avb3 through binding at the site targeted by the high affinity cyclic Arg-Gly-Asp (RGD) peptide. In contrast, binding of trimethoxy-resveratrol to this site was substantially less robust. Moreover, the different stilbenes also elicited diverse cellular and signaling responses in MCF-7 and MDA-MB-231 cells, as evidenced by analysis of colony formation, cell proliferation, cell cycle phase transition, the extent of phosphorylation of p53 at Ser15 and p53-inducible proteins, p21 and p53R2, respectively. Further, stilbene-elicited signaling cascade leading to p53 activation was examined in MCF-7 cells and results showed that resveratrol and triacetyl-resveratrol induced both ERK and p38 phosphorylation, whereas only marginal changes in state of phosphorylation in these two kinases were observed in trimethoxy-resveratrol-treated cells. Taken together, these results support that resveratrol and triacetyl-resveratrol regulate proliferation and gene expression in breast cancer cells by utilizing largely similar signaling molecules and pathways and cellular events, which appear quite distinct from those targeted by trimethoxy-resveratrol. Resveratrol (3,5,4 0 -trihydroxystilbene) is a phytoalexin with a stilbenoid core present abundantly in grapes, berries, and pea- nuts, which has been shown to inhibit cell proliferation and sup- press malignant tumor formation and progression. 1,2 Resveratrol and its effects on proliferation have been studied using estab- lished cell lines and animal models of carcinogenesis. Antiproli- ferative activities of resveratrol have been reported in numerous cancer cells including murine epidermal, and human colorectal and epidermoid carcinoma cells. 3–5 However, equivocal suppres- sion of proliferation has also been observed in established cells including those from breast cancer; high dose of resveratrol inhibited growth of estrogen receptor-a (ERa)-positive MCF-7, ERa-negative MDA-MB-231, 6,7 KPL-1, MKL-F, and T47-D breast cancer cells, 8–10 while low dose of resveratrol potentiated MCF-7 and T47-D proliferation. 11,12 These results suggest that cell type and dose of exposure may contribute to signaling path- ways and mechanisms used by resveratrol to control prolifera- tion and gene expression in breast cancer cells. Another aspect regarding the anticarcinogenic potential of resveratrol is its ability to induce apoptosis, mediated in part by activation of p53 3,13 and shown to occur secondary and downstream of activation of the Ser/Thr MAPK kinases, respectively, ERK, JNK and p38 kinase, as reported by Dong and coworkers 13 in mouse JB6 cells. Apparently p53 activation by resveratrol is coordinated with its increased phosphoryla- tion at Ser15; studies have shown that modification at this site of p53 is concomitant with stabilization and apoptosis-compe- tent activation of p53 while mutations preventing Ser15 phos- phorylation abrogated p53 induction of apoptosis. 14 Other insights on activation of p53 by resveratrol have been revealed by studies of Lin et al. 15 by using biochemical, pharmacological and siRNA-directed gene knockdown approaches, and show- ing that resveratrol significantly interacted with integrin avb3 by binding to the b3 subunit as its cognate receptor that led to ERK activation, followed by phosphorylation at Ser15 and acti- vation of p53, and finally apoptosis. 15 Because of its limited bioavailability, there is uncertainty regarding the use of resveratrol as a broad, diet-based Key words: resveratrol, resveratrol derivatives, MCF-7 breast cancer cells, cell cycle control, p53 Grant sponsor: Susan G. Komen Breast Cancer Foundation; Grant number: BCTR0600943 *T.-C.H. and C.W. contributed equally to this work DOI: 10.1002/ijc.25930 History: Received 12 Aug 2010; Accepted 30 Dec 2010; Online 10 Jan 2011 Correspondence to: Dr. Joseph M. Wu, Room 147, Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA, Tel.: +1 914 594 4891, Fax: þ1 914 594 4058, E-mail: joseph_wu@nymc.edu Cancer Therapy Int. J. Cancer: 129, 2732–2743 (2011) V C 2011 UICC International Journal of Cancer IJC