Caffeine Inhibits Cell Proliferation by G0/G1 Phase Arrest in JB6 Cells

[CANCER RESEARCH 64, 3344–3349, May 1, 2004]

Takashi Hashimoto,1 Zhiwei He,1 Wei-Ya Ma,1 Patricia C. Schmid,1 Ann M. Bode,1 Chung S. Yang,2 and Zigang Dong1 1Hormel Institute, University of Minnesota, Austin, Minnesota, and 2Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey

ABSTRACT radiation or other DNA-damaging agents (14–16). Thus, caffeine shows various effects on the cell cycle and may cause cell growth Caffeine is a major biologically active constituent in coffee and tea. arrest, resulting in anticarcinogenic effects. Because caffeine has been reported to inhibit carcinogenesis in UVB- Cells are believed to be quiescent or in G phase in most mammals exposed mice, the cancer-preventing effect of caffeine has attracted con- 0 siderable attention. In the present study, the effect of caffeine in quiescent under normal conditions. After stimulation with growth factors, cells progress through the cell cycle phase from G0 to G1 and then G1 to S. (G0 phase) cells was investigated. Pretreatment with caffeine suppressed cell proliferation in a dose-dependent manner 36 h after addition of fetal The D-type cyclin and cyclin-dependent kinase (cdk)4/6 complex bovine serum as a cell growth stimulator. Analysis by flow cytometry plays an important role in G0 to G1 and G1 to S transitions (17, 18).

showed that caffeine suppressed cell cycle progression at the G0/G1 phase, These complexes phosphorylate protein retinoblastoma (pRb), which i.e., 18 h after addition of fetal bovine serum, the percentages of cells in binds with transcriptional factors, including E2Fs, and the phosphory- G0/G1 phase in 1 mM caffeine-treated cells and in caffeine-untreated cells lation promotes the expression of target proteins such as cyclin A (17, were 61.7 and 29.0, respectively. The percentage of cells in G0/G1 phase at 18). pRb is a key regulator in the G0 to G1 transition, and its activity 0 h was 75.5. Caffeine inhibited phosphorylation of retinoblastoma protein is modulated by cyclin-cdk complexes. In addition, the consensus at Ser780 and Ser807/Ser811, the sites where retinoblastoma protein has motif for phosphorylation by the cyclin D1-cdk4 complex is different been reported to be phosphorylated by cyclin-dependent kinase 4 (cdk4). Furthermore, caffeine inhibited the activation of the cyclin D1-cdk4 com- from that for phosphorylation by the cyclin E-cdk2 complex, and the plex in a dose-dependent manner. However this compound did not directly Ser780 site in pRb is specifically phosphorylated by the cyclin D1- inhibit the activity of this complex. In addition, caffeine did not affect cdk4 complex in vivo and in vitro (19). p16INK4 or p27Kip1 protein levels, but inhibited the phosphorylation of In this study, to elucidate the mechanism for the anticarcinogenic protein kinase B (Akt) and glycogen synthase kinase 3␤. Our results activity of caffeine, the effect of caffeine in mouse epidermal JB6

showed that caffeine suppressed the progression of quiescent cells into the cells synchronized in G0 phase was investigated after stimulation with cell cycle. The inhibitory mechanism may be due to the inhibition of cell fetal bovine serum (FBS) as a growth factor. Caffeine inhibited growth signal-induced activation of cdk4, which may be involved in the FBS-stimulated cell proliferation without the induction of apoptosis, inhibition of carcinogenesis in vivo. apparently resulting from the suppression of the activation of the cyclin D1-cdk4 complex and the subsequent inhibition of pRb phos- INTRODUCTION phorylation. This inhibitory effect of caffeine on cell proliferation may be one of the mechanisms explaining the anticarcinogenic activ- Because it is present in numerous dietary sources, including coffee, ity of this compound in vivo. tea, cocoa beverages, and soft drinks, caffeine is the most widely consumed behaviorally active substance in the world (1). Caffeine has been the subject of intensive study, and various effects have been MATERIALS AND METHODS reported, including antagonistic effects on adenosine receptors (2), Reagents. Caffeine was purchased from Sigma-Aldrich (St. Louis, MO), inhibition of phosphodiesterases (3, 4), stimulation of muscle contrac- MEM was from Mediatech (Herndon, VA), FBS was from Gemini Bio- tion levels (5), and alterations in glucose metabolism (6–8). Topical Products (Woodland, CA), and the antibiotics (penicillin and streptomycin) ␮ ␮ application of caffeine (6.2 mol in 100 l of acetone, once a day, 5 were from Invitrogen (Carlsbad, CA). For cell cycle analysis, propidium iodide days a week, for 18 weeks) was reported recently to inhibit formation and RNase A were purchased from Sigma-Aldrich. of UVB-induced skin tumors in mice in vivo, and the induction of Cell Culture. The mouse epidermal cell line, JB6 Cl 41, was maintained apoptosis was suggested as one of the responsible mechanisms (9). with MEM, containing 5% FBS, 50 IU/ml penicillin G, and 50 ␮g/ml strep-

However, most of the mechanisms of action are as yet still unknown. tomycin, in an atmosphere of 95% air-5% CO2 at 37°C. Fifty thousand cells Recent studies showed that various concentrations of caffeine in- were seeded in a 60 mm dish and cultured for 24 h. The cells were serum- duce apoptosis in several cell lines, including 10 mM caffeine in starved in 0.1% FBS in MEM for 36 h to synchronize the cell cycle into G0 phase (20). The cells were treated with various concentrations of caffeine or human neuroblastoma cells (10), 4 mM in human pancreatic adeno- distilled water as a control for 1 h followed by addition of FBS (final 5% carcinoma cells (11), 5 mM in human A549 lung adenocarcinoma cells ␮ concentration) as a cell growth stimulator. (12), and 450 M in mouse epidermal JB6 Cl 41 cells (13). Besides Cell Proliferation Assay [3-(4,3-dimethylthiazol-2-yl)-5-(3-carboxy- apoptosis, suppression of cell proliferation is one of the most impor- methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium Assay]. Cell prolifera- tant strategies for anticarcinogenesis. Caffeine (5 mM) has been re- tion was analyzed using the CellTiter 96 AQueous One Solution Cell Prolifer- ported to induce TP53-independent G1 phase arrest in a human lung ation Assay (Promega, Madison, WI) following the instructions provided. In

adenocarcinoma cell line (12). On the other hand, this compound has brief, cells synchronized into G0 phase in a 96-well multiplate were treated

also been reported to prevent G2 phase arrest induced by exposure to with caffeine and then FBS was added. After an additional 36 h in culture, cells were incubated for2hat37°C with 3-(4,3-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium reagent in a hu- Received 11/4/03; revised 1/23/04; accepted 2/16/04. Grant support: The Hormel Foundation and NIH Grants CA81064 and CA88961. midified, 5% CO2 atmosphere. Then the absorbance of a colored formazan The costs of publication of this article were defrayed in part by the payment of page product catalyzed by dehydrogenase enzymes in metabolically active cells was charges. This article must therefore be hereby marked advertisement in accordance with recorded at 492 nm and with 690 nm as a background. Data were expressed as 18 U.S.C. Section 1734 solely to indicate this fact. the percentage of absorbance versus untreated control cells. Requests for reprints: Zigang Dong, Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912. Phone: (507) 437-9600; Fax: (507) 437-9606; Measurement of Apoptosis by Flow Cytometry. The apoptotic action of E-mail: [email protected]. caffeine was determined using the Annexin V-FITC Apoptosis Detection kit 3344

following the instruction protocol provided (MBL International, Woburn, MA). Briefly, 36 h after addition of FBS, cells were trypsinized, washed twice with ice-cold Dulbecco’s phosphate buffered saline (DPBS), and incubated at room temperature for 15 min in the dark with Annexin V-conjugated FITC and propidium iodide in the binding buffer provided. Stained cells were analyzed by flow cytometry using the FACSCalibur (BD Biosciences, San Jose, CA). Cell Cycle Analysis. Cell cycle was analyzed by a published method (20) with slight modifications. At different time points, various hours after stimulation with FBS, cells were trypsinized, washed with ice-cold DPBS, and fixed with ice-cold 70% ethanol at Ϫ20°C overnight. Cells were then washed twice with DPBS, incubated with 20 ␮g/ml RNase A and 200 ␮g/ml propidium iodide in DPBS at room temperature for 30 min in the dark, and subjected to flow cytometry using the FACSCalibur flow cytometer. Data were analyzed using ModFit LT (Verity Software House, Inc., Topsham, ME).

Western Blot and Antibodies. Cells synchronized into G0 phase were pretreated with caffeine for 1 h, and then FBS was added. Cells were washed twice with ice-cold DPBS and harvested with 20 mM Tris-HCl (pH 7.5), 150

mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium PPI,1mM ␤ ␮ -glycerophosphate, 1 mM Na3VO4,1 g/ml leupeptin, and 1 mM phenyl- methanesulfonyl fluoride (PMSF). Immunoblotting was performed by following the protocol provided by Cell Signaling Technology (Beverly, MA) and Santa Cruz Biotechnology (Santa Cruz, CA), with slight modifications. The sources of antibodies were as follows: phospho-retinoblastoma (Rb; Ser780), phospho-Rb (Ser807/Ser811), phospho-glycogen synthase kinase (GSK)-3␣/␤ (Ser21/9), GSK-3␤, phospho-Akt (Thr308), Akt, and p27, Cell Signaling Technology; and p16 and ␤-actin, Santa Cruz Biotechnology. Immunoprecipitation Kinase Assay. The cyclin D1-cdk4 kinase assay Fig. 2. Apoptotic effects of caffeine in quiescent JB6 cells. JB6 cells were prepared as described for Fig. 1, and apoptosis induced by caffeine was analyzed by flow cytometry was performed as described (21, 22) with modifications. Briefly, cells syn- using dual staining with propidium iodide and FITC-conjugated annexin V as described chronized into G0 phase were pretreated for 1 h with the indicated concentra- in “Materials and Methods.” Data shown are representative of triplicate experiments and tion of caffeine, and then FBS was added. Six h after the addition of FBS, cells values indicate the mean (n ϭ 3); bars, ϮSD. FBS, fetal bovine serum. were washed twice with ice-cold DPBS and harvested with a kinase buffer [25 mM Tris-HCl (pH 7.5), 5 mM ␤-glycerophosphate, 2 mM DTT, 0.1 mM ␮ Na3VO4,10mM MgCl2,and1mM PMSF]. Protein content was determined Signaling Technology) and 200 M ATP in a kinase buffer at 30°C for 30 min. using the modified Lowry method (23). Cyclin D1 was immunoprecipitated Phosphorylation of Rb-C fusion protein was detected by Western blotting with from 400 ␮g of the cell extract using an anticyclin D1 monoclonal antibody antiphospho-Rb (Ser780) and (Ser801/Ser811) polyclonal antibodies. (Cell Signaling Technology) diluted at 1:100 overnight. Then 20 ␮l of protein In Vitro Kinase Assay for the Cyclin D1-cdk4 Complex. Cells synchro-

A/G PLUS-Agarose (Santa Cruz Biotechnology) was added, incubated at 4°C, nized into G0 phase were treated with FBS for6htoallow the activation of the and then washed at 4°C four times with ice-cold kinase buffer. Immunopre- cyclin D1-cdk4 complex. Immunoprecipitated cyclin D1, which was prepared cipitated cyclin D1 was incubated with 1 ␮g Rb-C fusion protein (Cell as described in the section “Immunoprecipitation Kinase Assay,” was incubated with caffeine, 1 ␮g Rb-C fusion protein, and 200 ␮M ATP in a kinase buffer at 30°C for 30 min. Phosphorylation of the Rb-C fusion protein was detected by Western blotting with an antiphospho-Rb (Ser780) polyclonal antibody.

RESULTS Caffeine Inhibits Cell Proliferation in Quiescent JB6 Cells Stimulated with Serum. Treatment with caffeine for 24 h was demonstrated recently to induce apoptosis in JB6 cells after 72 h of serum starvation (13). In the present study, the effect of caffeine on cell proliferation in response to cell growth stimulation by FBS in quies-

cent JB6 cells synchronized into G0 phase by 36-h serum starvation (0.1% FBS) was investigated. As shown in Fig. 1, stimulation with 5% FBS for 36 h increased cell proliferation to 80% above control (100%), which was cultured without addition of 5% FBS for 36 h. Pretreatment with caffeine for 1 h before stimulation with FBS significantly inhibited cell proliferation in a dose-dependent manner (Fig. 1, solid circles). Caffeine at 1 mM inhibited cell proliferation ϳ75% ϳ Fig. 1. Caffeine inhibits cell proliferation in quiescent JB6 cells. JB6 Cl 41 mouse in response to FBS. The IC50 was 0.7 mM. On the other hand, epidermal cells were serum-starved with 0.1% fetal bovine serum (FBS) in MEM for 36 h caffeine only slightly decreased proliferation in control cells (Fig. 1, to synchronize the cell cycle into G0 phase. The cells then were treated with the indicated concentration of caffeine for 1 h. To stimulate cell growth, a final concentration of 5% open circles). FBS was then added to one set of caffeine-treated cells and 36 h later, cell proliferation Flow cytometry was used to determine, under the same conditions was estimated by the 3-(4,3-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- as for Fig. 1, whether the inhibition of proliferation is due to apoptosis sulfophenyl)-2H-tetrazolium assay as described in “Materials and Methods.” F, cells in 5% FBS. E, cells in 0.1% FBS. Values were normalized against untreated cells as a (Fig. 2). Pretreatment with 1 mM caffeine under 0.1% FBS medium -P Ͻ 0.001, versus JB6 conditions slightly increased the number of apoptotic cells. The per ,ء .control and data represent the mean (n ϭ 5 wells); bars, ϮSD ,ءء .cells untreated with caffeine and cultured in 5% FBS as determined by Student’s t test P Ͻ 0.001, versus JB6 cells untreated with caffeine and cultured in 0.1% FBS as centage of total apoptotic cells stained with Annexin V-FITC was determined by Student’s t test. 6.0 Ϯ 2.5 (sum of 4.21 Ϯ 2.25 and 1.87 Ϯ 0.28) and 10.1 Ϯ 0.5 (sum 3345

Fig. 3. Caffeine causes G0/G1 phase arrest in quiescent JB6 cells. A–C, JB6 cells with cell cycle synchronized into F the G0 phase were treated for1hwith1mM caffeine ( )or distilled water as a control (E), and a final concentration of 5% fetal bovine serum (FBS) was added to stimulate cell growth. Cell cycle analysis was performed by flow cytometry as described in “Materials and Methods.” Data represent P Ͻ 0.001, control JB6 cells ,ء .the mean (n ϭ 3); bars, ϮSD untreated with caffeine versus caffeine-treated JB6 cells as determined by Student’s t test. D, JB6 cells in G0 phase were treated with the indicated concentration of caffeine for 1 h. FBS was then added as a growth stimulant, and 16 h after addition of FBS, cell cycle analysis was performed. Data P Ͻ 0.001, versus ,ء .represent the mean (n ϭ 3); bars, ϮSD JB6 cells added 5% FBS and untreated with caffeine as determined by Student’s t test.

Ϯ Ϯ of 6.09 0.35 and 4.28 0.29) in the absence (Fig. 2, top left panel) cell line (12). Thus, we hypothesized that G0/G1 phase arrest caused or in the presence (Fig. 2, top right panel) of caffeine, respectively. by caffeine at low concentrations (Յ1mM) was specific to quiescent

However, 1 mM caffeine did not induce apoptosis in quiescent JB6 cells, and that these concentrations of caffeine might affect the G0 to cells stimulated with 5% FBS. The percentage of total apoptotic cells G1 transition but not the G1 to S transition. was 4.3 Ϯ 0.5 (sum of 3.54 Ϯ 0.42 and 0.79 Ϯ 0.16) and 4.4 Ϯ 0.2 Caffeine Inhibits the Activation of the Cyclin D1-cdk4 Com- Ϯ Ϯ (sum of 3.22 0.24 and 1.15 0.08) in the absence (Fig. 2, bottom plex. Phosphorylation of pRb plays an important role in the G0 to G1 left panel) or in the presence (Fig. 2, bottom right panel) of caffeine, transition (24). To investigate the effects of caffeine on the G0 to G1 respectively. transition, phosphorylation of pRb was examined at Ser780 and

Caffeine Causes G0/G1 Phase Arrest in Quiescent JB6 Cells. To Ser807/Ser811, the sites at which pRb has been reported to be phos- reveal the inhibitory mechanism of caffeine on cell proliferation, the phorylated by cdk4, a key kinase in the G0 to G1 transition (Fig. 5). effect of this compound on cell cycle was investigated. In control In control cells, these sites in pRb were phosphorylated 3–6 h after quiescent cells shown as open circles in Fig. 3, A and B, 14 h after stimulation by 5% FBS, and the phosphorylation was continued for at addition of 5% FBS, the percentage of cells in G0/G1 phase started least 9 h (Fig. 5A). Pretreatment of cells with 1 mM caffeine before the decreasing (Fig. 3A), and the percentage of cells in S phase began addition of FBS significantly inhibited the phosphorylation at these increasing (Fig. 3B), meaning that the G0/G1 to S progression was executed in response to FBS stimulation. In quiescent cells treated with 1 mM caffeine shown as closed circles in Fig. 3, A and B, the progression was inhibited significantly. Sequentially in control quiescent cells, 18 h after addition of 5% FBS, the percentage of cells in S phase started decreasing (Fig. 3B), and the percentage of cells in G2/M phase began increasing (Fig. 3C), meaning that the S to G2/M progression was subsequently executed. Caffeine also significantly inhibited this progression as shown as closed circles in Fig. 3, B and C. As shown in Fig. 3D, the inhibitory effects were dose-dependent, and ϳ the IC50 was 0.7 mM. To investigate whether the inhibitory effect of caffeine is specific to quiescent cells, JB6 cells without serum starvation were cultured with caffeine in 5% FBS-containing MEM for 24 h. Caffeine (Յ1mM) did not affect the phase distribution of the cell cycle, although a high concentration (5 mM) of caffeine caused G0/G1 phase arrest (Fig. 4). Caffeine at high concentrations seems to cause G0/G1 phase arrest Fig. 4. Caffeine at a 1-mM dose does not cause cell cycle arrest in active JB6 cells. Active JB6 Cl 41 cells without serum-starvation were cultured for 24 h with 0.5, 1.0, or through the inhibition of activation of cdk2, a key kinase in the G1 to 5.0 mM caffeine. Cell cycle analysis was performed by flow cytometry as described in ,P Ͻ 0.001 ,ء .S transition. A concentration of 5 mM caffeine has already been “Materials and Methods.” Data represent the mean (n ϭ 3); bars, ϮSD reported to inhibit the activation of cdk2 in a lung adenocarcinoma versus control group (caffeine ϭ 0mM) as determined by Student’s t test. 3346

chronized into G0 (quiescent) phase after stimulation with FBS as a cell growth signal (Figs. 1 and 3). Phosphorylation of pRb plays a

critical role in the G0 to G1 and G1 to S transitions (24). The phosphorylation leads to the disruption of the pRb-E2F transcription factor complex, and the release of active E2F additionally triggers the activation of a number of genes (18). Cdk4/6 and cdk2 are key

kinases, which phosphorylate pRb in the G0 to G1 and G1 to S transitions (29). Ser780 in pRb is a specific site for phosphorylation by cdk4 (19). Ser807 and Ser811 in pRb are also phosphorylated by cdk4 in vivo and in vitro (30). As shown in Fig. 5, caffeine inhibited the phosphorylation of Ser780 and Ser807/Ser811 after stimulation with FBS. Cdk4/6 forms a complex with D-type cyclins in response to growth factor stimulation, and the active complex phosphorylates pRb (25). Thus, the cyclin D1-cdk4 complex is a key factor in regulation

of the G0 to G1 transition (31). Pretreatment with caffeine resulted in an inhibition of cyclin D1-cdk4 complex activation in a dose-dependent manner (Fig. 6A). Taken together, these data suggest that caffeine

causes G0/G1 phase arrest accompanied by inhibition of cyclin D1- cdk4 complex activation and cell proliferation in response to cell growth stimulation. Some effects of caffeine on the cell cycle have Fig. 5. Caffeine suppresses phosphorylation of protein retinoblastoma (pRb). A, JB6 cells with cell cycle phase synchronized into G0 were treated for1hwith1mM caffeine already been reported, including abrogation of the G2 phase arrest that or distilled water as a control. Both sets of cells were then treated with 5% fetal bovine was observed in cells exposed to radiation or other DNA-damaging serum (FBS) for the indicated time. B, synchronized cells were treated with 0.25, 0.5, or agents (14–16) and suppression of the activation of cdk2 in a TP53- 1mM caffeine for 1 h and then cultured with 0.1% or 5% FBS in MEM for 6 h. The phosphorylation level of pRb was analyzed by Western blotting as described in “Materials independent mechanism (12). The present study shows a new effect of and Methods.” Equal loading of protein was confirmed by ␤-actin, and data shown are caffeine on cell cycle, the suppression of cyclin D1-cdk4 activation. representative of at least three independent experiments. Direct inhibition by caffeine of several kinases, ataxia-telangiecta- sites (Fig. 5A). The inhibitory effect on the phosphorylation was also sia mutated kinase (IC50, 0.2 mM), ATM- and Rad3-related kinase observed 6 h after addition of 5% FBS in JB6 cells pretreated with 0.25, 0.5, or 1 mM caffeine (Fig. 5B). The effect of caffeine on activation of cyclin D1-cdk4 was next investigated because cdk4 is required to form a complex with cyclin D1 for activation (25). Pretreatment with caffeine significantly inhibited the activation of cyclin D1-cdk4 in a dose-dependent manner

(Fig. 6A). These results indicate that caffeine inhibited the G0 to G1 transition through the suppression of activation of cyclin D1-cdk4. Caffeine Inhibits the Akt-GSK-3␤ Cell Growth Signal. Because cyclin D1-cdk4 is regulated by GSK-3␤ (26) and also the cdk inhibitors, p16INK4 (27) and p27Kip1 (28), the effect of caffeine on these proteins was investigated. Caffeine had no effect on the protein levels of p16 INK4 and p27Kip1, and over time the protein levels tended to decrease in a time-dependent manner after stimulation with FBS (Fig. 6B). In addition, caffeine did not appear to directly affect cyclin D1-cdk4 activity (Fig. 6C). On the other hand, caffeine markedly inhibited the time-dependent phosphorylation of protein kinase B (Akt Thr308) and its substrate, GSK-3␤ (Ser9; Fig. 7A). In addition, the inhibitory effect on the phosphorylation was also observed in JB6 cells pretreated with 0.25 and 0.5 mM caffeine 30 min after FBS stimulation (Fig. 7B). Finally, epidermal growth factor or platelet-derived growth factor was used as a cell growth stimulator instead of FBS. Pretreatment with caffeine (1 mM) for 1 h before addition of epidermal growth factor (10 Fig. 6. Caffeine inhibits fetal bovine serum (FBS)-stimulated activation of the cyclin ng/ml) or platelet-derived growth factor (5 ng/ml) inhibited the phos- D1-cdk4 complex. A, JB6 cells with cell cycle phase synchronized into G0 were treated ␤ with 0.25, 0.5, or 1 mM caffeine for 1 h and then cultured with 0.1% or 5% FBS in MEM phorylation of Akt and GSK-3 30 min after stimulation by these for 6 h. Cells were harvested with a kinase buffer and the immunoprecipitation kinase compounds (Fig. 8A). In addition, caffeine suppressed the change in assay for measuring the cyclin D1-cdk4 activity was performed as described in “Materials phase distribution in the cell cycle 18 h after stimulation with epider- and Methods.” Phosphorylation of Rb-C fusion protein was detected by Western blot. Data shown are representative of three independent experiments. B, JB6 cells with cell mal growth factor (10 ng/ml; Fig. 8B). Thus, caffeine may inhibit cell cycle phase synchronized into G0 were treated with 1 mM caffeine or distilled water proliferation in response to several growth factors through an inhibi- (control) for 1 h and then treated with 5% FBS for the indicated time. The protein level ␤ of p16INK4A and p27Kip1 was analyzed by Western blotting using the corresponding tion of the Akt-GSK-3 cell growth signal. antibody as described in “Materials and Methods.” Data shown are representative of three independent experiments. C, the active cyclin D1-cdk4 complex was immunoprecipitated DISCUSSION with an anticyclin D1 monoclonal antibody, and the in vitro kinase assay was performed as described in “Materials and Methods.” NC, the negative control without immunoprecipitated cyclin D1; PC, the positive control, phospho-Rb-C fusion protein provided by This study demonstrated that caffeine caused G0/G1 phase arrest Cell Signaling Technology. Data shown are representative of three independent experi- and inhibited proliferation in mouse epidermal JB6 Cl 41 cells syn- ments. 3347

Thr286 in cyclin D1, thereby triggering cyclin D1 turnover and decreasing the activity of cdk4 (26). Pretreatment with caffeine suppressed the phosphorylation of both Akt and GSK-3␤ as shown in Fig. 7. In addition, caffeine has already been reported to inhibit PI3K directly (33). Therefore, the inhibitory effects of caffeine on the cell growth signaling of Akt-GSK-3␤ may result from the direct inhibition of PI3K, which is upstream of Akt. In fact, the inhibition of PI3K by caffeine is believed to have several effects on the physiological functions of cells, including the inhibition of translocation of glucose transporter to the cell membrane in response to insulin stimulation (33). In this study, caffeine suppressed the Akt-GSK-3␤ signal stimulated by not only FBS but also by epidermal growth factor and

platelet-derived growth factor (Fig. 8A), and caused G0/G1 phase arrest (Fig. 8B). On the other hand, a cell growth signal might bifurcate to a signal leading to the ubiquitination of p27Kip1 and a signal leading to Akt-GSK-3␤ upstream from PI3K, because caffeine did not affect p27Kip1 in response to FBS stimulation (Fig. 6B). However, inhibition of the PI3K pathway has been reported to induce an increase in p27Kip1 in a senescence-like growth arrest, which is induced in mouse primary embryo fibroblasts by inhibitors of PI3K (38). This contradiction may be due to the existence of a signal pathway for p27Kip1 synthesis downstream from PI3K. Therefore, the Kip1 ␤ rate of disruption of p27 may be faster than the synthesis. The Fig. 7. Caffeine suppresses the Akt-glycogen synthase kinase (GSK)-3 cell growth Kip1 signal pathway. A, JB6 cells with cell cycle phase synchronized into G0 were treated with disruption of p27 occurs by an ubiquitin-proteasome pathway 1mM caffeine or distilled water (control) for 1 h and then treated with 5% fetal bovine (37), and the phosphorylation of Ser10 in p27Kip1 is required for the serum (FBS) for the indicated time. B, synchronized cells were treated with 0.25, 0.5, or disruption (39). However, the kinase responsible is still unknown. 1mM caffeine for 1 h and then cultured in 0.1% or 5% FBS in MEM for 30 min. The ϳ phosphorylation of Akt at Thr308 (p-Akt) and GSK-3␤ at Ser9 (p-GSK-3␤) was analyzed The ED50 at which caffeine causes G0/G1 phase arrest, was 0.7 by Western blotting using the corresponding antibody as described in “Materials and mM as estimated from Figs. 1 and 2. He et al. (13) showed that 0.45 Methods.” Equal loading of protein was monitored by total Akt and GSK-3␤, and data shown are representative of at least three independent experiments.

(IC50, 1.1 mM), mammalian target of rapamycin (IC50, 0.4 mM), Ͻ Ͻ DNA-dependent protein kinase (IC50, 10 mM), hChk1 (IC50, 5 Ј ␣ mM), phosphatidylinositol 3 -kinase (PI3K) p100 (IC50, 0.4 mM), ␤ ␥ PI3K p100 (IC50, 0.4 mM), PI3K p100 (IC50, 1.0 mM), and PI3K ␦ ␮ p100 (IC50,75 M; Refs. 32, 33) has been reported. The inhibitory effect may be due to an antagonistic effect on ATP because caffeine is a purine derivative. However, caffeine did not directly inhibit the activity of cdk4 (Fig. 6C). In addition, caffeine (8 mM) had no effect on the activity of active cdk2/cyclin E (Upstate, Lake Placid, NY; data not shown), although 5 mM caffeine has been reported to inhibit the activity of cdk2 by a TP53-independent mechanism in a lung adenocarcinoma cell line (12). Also in the present study, 5 mM, but not 1 mM, caffeine caused G0/G1 phase arrest in JB6 Cl 41 cells without serum starvation (Fig. 4), and the effect might be due to the inhibition of cdk4 as was reported previously (12). Furthermore, caffeine has been reported to enhance the activity of cdk1 in cells exposed to DNA-damaging agents (34, 35). Thus, caffeine is unlikely to directly inhibit cdks specifically. Cdks are regulated by cdk inhibitors, including the INK4 family and Cip/Kip family (36). In particular, the cyclin D1-cdk4 complex is regulated by p16INK4, a member of the INK4 family (25). In the current study, caffeine did not affect the protein level of p16INK4 (Fig. Kip1 6B). A member of the Kip family, p27 is also known to regulate Fig. 8. Caffeine inhibits epidermal growth factor (EGF)- and platelet-derived growth the cyclin D1-cdk4 complex (28), and to be exported from the nucleus factor (PDGF)-induced phosphorylation of Akt and glycogen synthase kinase (GSK)-3␤ and degraded by the ubiquitin-proteasome pathway (37). In this study, and cause G0/G1 phase arrest. JB6 cells with cell cycle synchronized into the G0 phase Kip1 were treated with 1 mM caffeine or distilled water for 1 h, and then a final concentration caffeine did not affect protein levels of p27 , although the levels of 10 ng/ml EGF or 5 ng/ml PDGF in MEM (0.1% fetal bovine serum; FBS) was added were time-dependently decreased after stimulation with FBS as a to stimulate cell growth. A, cells were harvested 30 min after the addition of EGF or ␤ ␤ growth signal (Fig. 6B). Therefore, the inhibitory effect of caffeine on PDGF, and the phosphorylation of Akt at Thr308 (p-Akt) and GSK-3 at Ser9 (p-GSK-3 ) was analyzed by Western blotting using the corresponding antibody as described in the activation of the cyclin D1-cdk4 complex is not due to the “Materials and Methods.” Data shown are representative of at least three independent regulation of p16INK4 and p27Kip1 levels. experiments. B, cells were trypsinized 18 h after addition of EGF, and cell cycle analysis was performed by flow cytometry as described in “Materials and Methods.” Data P Ͻ 0.01, versus JB6 cells added EGF and ,ء .Cell growth signals sequentially mediate Ras, PI3K, and Akt, represent the mean (n ϭ 5); bars, ϮSE resulting in the phosphorylation of GSK-3␤. GSK-3␤ phosphorylates untreated with caffeine as determined by Student’s t test. 3348

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