1006 Protein kinase CK2 modulates apoptosis induced by resveratrol and epigallocatechin-3-gallate in prostate cancer cells Kashif A. Ahmad,1,2 Nathan H. Harris,1 Introduction 1 1,2 Andrew D. Johnson, Hans C.N. Lindvall, In recent years, many bioactive polyphenolic compounds Guixia Wang,1,2 and Khalil Ahmed1,2,3 have gained recognition as candidate agents in chemo- 1 prevention and/or in cancer chemotherapy. Of this class, Cellular and Molecular Biochemistry Research Laboratory, resveratrol and epigallocatechin-3-gallate (EGCG)are two Veterans Affairs Medical Center and 2Department of Laboratory Medicine and Pathology and 3Cancer Center, University of of the most widely studied compounds (1–3). Resveratrol Minnesota, Minneapolis, Minnesota is a naturally occurring trans-3,5,4¶-trihydroxystilbene that is a constituent of various plants, such as grapes, berries, and peanuts. Of note is its consumption in red wine that Abstract has been suggested to prevent cardiovascular diseases Resveratrol and epigallocatechin-3-gallate (EGCG) are im- (commonly called the French Paradox; ref. 4). In addition portant candidates as chemopreventive agents by virtue of to its cardioprotective effects, resveratrol has been shown their ability to induce apoptosis in cancer cells. Casein kinase to suppress proliferation in a wide variety of tumor cells, 2 (CK2) is a ubiquitous protein ser/thr kinase that plays including those of the lymphoid, breast, colon, stomach, diverse roles in cell proliferation and apoptosis. We have and prostate. Resveratrol signaling is complex, and its previously shown that overexpression of CK2 suppresses action modulates a wide range of cellular activities and apoptosis induced by a variety of agents, whereas down- pathways (1, 5). In this context, it is noteworthy that regulation of CK2 sensitizes cells to induction of apoptosis. resveratrol has been used as a sensitizer in drug-induced We therefore investigated whether or not CK2 played a role apoptosis (6). Likewise, EGCG is a polyphenolic compound in resveratrol and EGCG signaling in androgen-sensitive present in green tea. It induces apoptosis in a variety of (ALVA-41) and androgen-insensitive (PC-3) prostate cancer tumor cells and has been proposed as a chemopreventive cells. Resveratrol- and EGCG-induced apoptosis is associat- agent for prostate cancer (2, 3, 7). The functional role of ed with a significant down-regulation of CK2 activity and resveratrol and EGCG has generated a considerable interest protein expression in both the ALVA-41 and PC-3cells. in identifying their cellular targets. A Overexpression of CK2 protected prostatic cancer cells Casein kinase 2 (CK2)is a ubiquitous protein serine/ against resveratrol- and EGCG-induced apoptosis. Relatively threonine kinase consisting of a heterotetrameric complex M low doses (10 mol/L) of resveratrol and EGCG induced a of a, a¶, and h subunits, such that the catalytic a subunits modest proliferative response in cancer cells that could be are linked via two regulatory h subunits to form config- switched to cell death by moderate inhibition of CK2. These a h aa¶h a¶ h urations, such as 2 2, 2,or 2 2, depending upon the findings characterize, for the first time, the effects of cell type. CK2 is a multifunctional protein kinase that is polyphenolic compounds on CK2 signaling in androgen- involved in cell growth, proliferation, and survival (8–12). sensitive and androgen-insensitive prostatic carcinoma cells CK2 has also been shown to act as a potent suppressor of and suggest that resveratrol and EGCG may mediate their apoptosis in cancer cells (13–15). The significance of these cellular activity, at least in part, via their targeting of CK2. functions of CK2 relates to the observations that CK2 is Further, the data hint at the potential of using these consistently dysregulated in various cancer, including polyphenols alongside CK2 inhibitors in combination che- prostate cancer (8, 11, 16, 17). Recently, CK2 has also been motherapy. [Mol Cancer Ther 2007;6(3):1006–12] proposed as a target for cancer therapy (13, 18–20). We decided to examine the role of CK2 as a possible target for the effects of resveratrol and EGCG in prostate cancer Received 8/14/06; revised 10/20/06; accepted 1/22/07. cells. Thus far, with the exception of one report on the effect Grant support: U.S. Department of Veterans Affairs MedicalResearch of resveratrol on COP9 signalosome–associated CK2 (21), Fund and NationalCancer Institute, Department of Healthand Human no detailed investigations have been undertaken to relate Services USPHS research grant CA-15062. resveratrol and EGCG signaling to CK2. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Our findings suggest that CK2 is an upstream target advertisement in accordance with 18 U.S.C. Section 1734 solely to wherein down-regulation leads to apoptosis, whereas its indicate this fact. overexpression impedes cell death signal (15, 22). In the Requests for reprints: Khalil Ahmed, Cellular and Molecular Biochemistry present study, we hypothesized that resveratrol and EGCG Research Laboratory (151), Veterans Affairs MedicalCenter, One Veterans Drive, Minneapolis, MN 55417. Phone: 612-467-2594; signaling, at least in part, may occur via an effect on CK2 in Fax: 612-725-2093. E-mail: [email protected] prostate cancer cells. Prostate cancer is the second most Copyright C 2007 American Association for Cancer Research. common cancer in males, and initially the disease starts as doi:10.1158/1535-7163.MCT-06-0491 an androgen-responsive tumor that eventually progresses MolCancerTher2007;6(3).March2007 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Molecular Cancer Therapeutics 1007 to the androgen-resistant phenotype (23). Thus, in this 100 AL of fresh media containing 100 AL/mL WST-1, and report we have used both androgen-sensitive (ALVA-41), incubation was carried out at 37jC for an additional androgen-insensitive (PC-3), and BPH-1 cell lines to 60 min. An automated plate reader (Molecular Devices, determine whether they show a differential response to Sunnyvale, CA)was used to measure absorbance at resveratrol and EGCG with regard to CK2 signaling. Our 450 nm. The results were confirmed in at least three results indicate that resveratrol- and EGCG-induced apo- independent experiments. ptosis is associated with down-regulation of CK2 in Determination of Caspase-3 Activity prostate cells, and that the level of cellular CK2 exerts a Caspase-3 activity was determined using the fluorescent significant effect on resveratrol and EGCG signaling. Both caspase substrate from BioMol (Plymouth, PA). PC-3 and ALVA-41 and PC-3 cells showed similar qualitative ALVA-41 cells (0.25 Â 106)were plated in six-well plates response to the action of resveratrol and EGCG with and treated with 100 Amol/L resveratrol or EGCG for 18 h. respect to CK2 signal. This study is the first to delineate the Cells were washed with 1 Â PBS and suspended in chilled role of CK2 in resveratrol and EGCG apoptotic signaling in cell lysis buffer (50–200 AL per sample)on ice for 15 min. A the androgen-sensitive and androgen-insensitive prostate 50-AL aliquot of 2Â reaction buffer (10 mmol/L HEPES, carcinoma cell lines. 2 mmol/L EDTA, 10 mmol/L KCl, 1.5 mmol/L MgCl2, 10 mmol/L DTT)with 5 AL of the conjugate substrate (DEVD-AFC)was added to cell lysates. Caspase activity Materials and Methods was determined by the relative fluorescence intensity at Cell Lines and Reagents 505 nm after excitation at 400 nm using a spectrofluorom- Prostate cancer cells (ALVA-41 and PC-3)were grown in eter (Molecular Devices). RPMI 1640 (Invitrogen/Life Technologies, Carlsbad, CA) Preparation of Whole-Cell Lysates supplemented with 2 mmol/L L-glutamine and 6% or 10% After the desired treatment, ALVA-41, PC-3, and BPH-1 fetal bovine serum for ALVA-41 and PC-3 cells, respec- cells (0.25–2.5 Â 106)were collected and washed twice with tively. BPH-1 cells were obtained form Dr. Simon Hayward 1 Â PBS by centrifuging at 100 Â g for 5 min. Cell pellets (Vanderbilt University, Nashville, TN)and were grown in were resuspended in ice-cold 1Â radioimmunoprecipita- RPMI 1640 supplemented with 10% bovine growth serum tion assay buffer lysis and lysates were used for immuno- and 2 mmol/L L-glutamine. Resveratrol and EGCG were blot analysis. purchased from Sigma (St. Louis, MO). 4,5,6,7-Tetrabro- CK2 ActivityAssay mobenzotriazole (TBB), a chemical inhibitor of CK2, was CK2 activity in cell lysates was determined by using the purchased from Calbiochem (San Diego, CA). CK2 assay kit (MBL International, Woburn, MA). Cell Treatment and Transfection Immunoblot Analysis For cell viability studies, ALVA-41, PC-3, and BPH-1 cells PC-3, ALVA-41, and BPH-1 cells were plated in six-well were treated for 24 h with 10 Amol/L and 100 Amol/L plates at a concentration of 0.25 Â 106 and subjected to concentrations of resveratrol or EGCG. Down-regulation of desired treatments. Whole-cell lysates were prepared as CK2 by resveratrol and EGCG was studied in parallel described previously (15). Membranes were probed with experiments. For sensitization of cells to low levels of mouse anti-CK2a primary antibody (1:1,000; BD Bioscien- Â 6 resveratrol and EGCG, PC-3 and ALVA-41 cells (0.25 10 ) ces, San Diego, CA). The membranes were then probed were first treated with 40 Amol/L TBB for 6 h or small with goat antimouse IgG (1:50,000; Pierce, Rockford, IL) interfering RNA–CK2a (0.25 Ag/mL)using N-[1-(2,3- and analyzed for chemiluminescence (Western Pico).