The dietary phytochemical 3,3 0 -diindolylmethane induces G2/M arrest and apoptosis in oral squamous cell carcinoma by modulating Akt-NF-jB, MAPK, and p53 signaling Jing-Ru Weng a,⇑ , Li-Yuan Bai b,c , Chang-Fang Chiu c,d , Ying-Chu Wang a , Ming-Hsui Tsai b a Department of Biological Science and Technology, China Medical University, 91 Hsueh-Shih Road, Taichung 40402, Taiwan b College of Medicine, China Medical University, Taichung 40402, Taiwan c Division of Hematology and Oncology, Department of Internal Medicine, China Medical University Hospital, Taichung 40402, Taiwan d Cancer Center, China Medical University Hospital, Taichung 40402, Taiwan article info Article history: Received 31 October 2011 Received in revised form 14 January 2012 Accepted 14 January 2012 Available online 24 January 2012 Keywords: 3,3 0 -Diindolylmethane Oral cancer Apoptosis NF-jB p53 Indole-3-carbinol abstract In light of the growing incidence of oral cancer in Taiwan, this study is aimed at investigating the anti- tumor activity of 3,3 0 -diindolylmethane (DIM), an active metabolite of the phytochemical indole-3-carbi- nol (I3C), in oral squamous cell carcinoma (OSCC). DIM exhibited substantially higher antiproliferative potency than I3C in three OSCC cell lines with IC 50 values in SCC2095, SCC9, and SCC15 cells, respectively, of 22 versus 168 lM, 25 versus 176 lM, and 29 versus 300 lM. Flow cytometric analysis and Comet assay indicated that DIM suppressed the viability of SCC2095 cells by inducing apoptosis and G2/M arrest. Wes- tern blot analysis of various signaling markers revealed the ability of DIM to target pathways mediated by Akt, mitogen-activated protein (MAP) kinases, nuclear factor (NF)-jB, and p53, of which the concerted action underlined its antitumor efficacy. The concomitant inactivation of Akt and MAP kinases in response to DIM facilitated the dephosphorylation of the proapoptotic protein Bad at Ser-136 and Ser- 112, respectively. Through endoplasmic reticulum (ER) stress, DIM stimulated the activation of p53 via Ser-15 phosphorylation, leading to increased expression of the BH3-only proapoptotic Bcl-2 members Puma and Noxa. Together, these changes decreased the mitochondrial threshold for apoptosis. G2/M arrest might be attributable to the suppressive effect of DIM on the expression of cyclin B1 and cdc25c. As many downstream effectors of the Akt-NF-jB pathway, including glycogen synthase kinase 3b,IjB kinase a, and cyclooxygenase-2, have been shown to promote oral tumorigenesis, the ability of DIM to inhibit this signaling axis underscores its chemopreventive potential in oral cancer. Ó 2012 Elsevier Ireland Ltd. All rights reserved. 1. Introduction The growing incidence of oral cancer has emerged as an impor- tant public health issue in Taiwan as well as South and Southeast Asia due to the prevalent habit of betel quid chewing coupled with smoking and drinking [1]. More than two million people have the betel quid chewing habit which is associated with approximately 80% of oral cancer cases. Even with the use of chemotherapeutic agents including platinum, 5-fluorouracil, taxane, ifosfamide, and methotrexate, their therapeutic efficacy is often compromised by the development of drug resistance during the course of tumor progression, leading to poor clinical outcomes [2]. To reduce the incidence of oral cancer, development of effective, nontoxic che- mopreventive agents becomes an urgent issue for patients with oral epithelial dysplasia to reduce the incidence of oral cancer by blocking carcinogen-induced oral tumorigenesis. The use of dietary phytochemicals in cancer prevention has received much attention because of the pleiotropic effects of these agents on multiple carcinogen-activated oncogenic pathways, and equally important, excellent safety profiles [3,4]. Among various phytochemicals with chemopreventive potential, our research focused on indole-3-carbinol (I3C), one of the active phytonutri- ents of cruciferous vegetables (e.g., broccoli, cabbage, and cauli- flower) [5,6]. Substantial evidence indicates that increased consumption of cruciferous vegetables helps to reduce the risk for some types of cancer [7–10], and that this chemopreventive effect is, in part, attributable to the antiproliferative activity of I3C [5,6]. However, a major issue with I3C is its intrinsic instability in acidic milieu to undergo acid-catalyzed dehydration and poly- merization to generate a series of oligomeric products, of which the most noteworthy is 3,3 0 -diindolylmethane (DIM) [11]. This acid lability severely restricts the plasma concentrations of I3C that can be achieved, rendering its pharmacokinetic behavior unpredict- able. For example, a phase I trial in women showed that I3C was not detectable in plasma following oral doses even up to 0009-2797/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2012.01.003 ⇑ Corresponding author. Tel.: +886 4 22053366x2511; fax: +886 4 22071507. E-mail address: columnster@gmail.com (J.-R. Weng). Chemico-Biological Interactions 195 (2012) 224–230 Contents lists available at SciVerse ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint