Apigenin Induces Apoptosis in Human Leukemia Cells and Exhibits Anti-Leukemic Activity in Vivo

Published OnlineFirst November 14, 2011; DOI: 10.1158/1535-7163.MCT-11-0343

Molecular Cancer Preclinical Development Therapeutics

Apigenin Induces Apoptosis in Human Leukemia Cells and Exhibits Anti-Leukemic Activity In Vivo

Amit Budhraja1, Ning Gao1,5, Zhuo Zhang3, Young-Ok Son1, Senping Cheng1, Xin Wang1, Songze Ding1, Andrew Hitron1, Gang Chen2, Jia Luo2, and Xianglin Shi1,4

Abstract jun In this study, we investigated the functional role of Akt and c- -NH2-kinase (JNK) signaling cascades in apigenin-induced apoptosis in U937 human leukemia cells and anti-leukemic activity of apigenin in vivo. Apigenin induced apoptosis by inactivation of Akt with a concomitant activation of JNK, Mcl-1 and Bcl-2 downregulation, cytochrome c release from mitochondria, and activation of caspases. Constitutively active myristolated Akt prevented apigenin-induced JNK, caspase activation, and apoptosis. Conversely, LY294002 and a dominant-negative construct of Akt potentiated apigenin-induced apoptosis in leukemia cells. Interruption of the JNK pathway showed marked reductioninapigenin-inducedcaspaseactivation and apoptosis in leukemia cells. Furthermore, in vivo administration of apigenin resulted in attenuation of tumorgrowthinU937xenograftsaccompaniedbyinactivation of Akt and activation of JNK. Attenuation of tumor growth in U937 xenografts by apigenin raises the possibility that apigenin may have clinical implications and can be further tested for incorporating in leukemia treatment regimens. Mol Cancer Ther; 11(1); 132–42. 2011 AACR.

Introduction kinases leads to phosphorylation of PIP2 at 30-position at its inositol ring and converts PIP2 to PIP3 at plasma Apigenin is a flavonoid belonging to the flavones struc- membrane. Successively, PIP3 recruits Akt and PDK1 tural class and is chemically known as 40,5,7-trihydroxy- through their Pleckstrin homology domain. Full acti- flavone (1). It is present in abundance in fruits and vege- vation of Akt occurs when it is phosphorylated by tables such as oranges, grapefruit, celery, parsley, onions, PDK1 at Thr308 and by mTORC2 at Ser473 (14). Acti- chamomile, and wheat sprouts (1, 2). It has been reported vated Akt inactivates several proapoptotic factors that apigenin is a potent inhibitor of cell growth and including BAD, procaspase-9, and forkhead transcrip- inducer of apoptosis in various cancer cells including tion factors (14). Constitutively active Akt has been breast (3), prostate (4, 5), lung (6), and hematologic reported in various types of leukemia (15, 16) and is (7, 8). Studies have revealed that apigenin induces apop- responsible for uncontrolled proliferation and resis- tosis through different cellular signaling transduction tance to apoptosis in leukemia cells, providing a poten- pathways such as NFkB (9), p53 (10), MAPK (11), and tial therapeutic target in leukemia. jun PI3K/Akt (12, 13). The c- -NH2-kinase (JNK) belongs to the superfam- The PI3K/Akt signaling pathway plays an important ily of mitogen-activated protein kinases that are role in cell survival and apoptosis. Activation of phos- involved in various cellular processes such as prolifer- phoinositide 3-kinase (PI3K) through growth receptor ation, differentiation, and apoptosis. JNKs can promote apoptosis by different mechanisms. First, activated JNK translocates to the nucleus where it phosphorylates and transactivates c-Jun, which leads to the increased Authors' Affiliations: 1Graduate Center for Toxicology, 2Department of Internal Medicine, College of Medicine, 3Department of Preventive Med- expression of proapoptotic genes such as TNF-a, Fas-L, icine and Environmental Health, College of Public Health, 4Markey Cancer and Bak. Second, activated JNK can be translocated to Center, University of Kentucky, Lexington, Kentucky; and 5Department of Pharmacognosy, College of Pharmacy, Third Military Medical University, mitochondria where it can phosphorylate Mcl-1 and Bcl-2 Chongqing, China to antagonize their antiapoptotic activity. JNK can also c Note: Supplementary data for this article are available at Molecular Cancer stimulate the release of cytochrome from mitochondria Therapeutics Online (http://mct.aacrjournals.org/). through Bid-Bax–dependent mechanism, which leads to Corresponding Author: Xianglin Shi, Graduate Center for Toxicology, apoptosis (17). College of Medicine, University of Kentucky, Lexington, KY 40536. Phone: Apigenin exposure to different leukemia cells resulted 859-257-4054; Fax: 859-323-1059; E-mail: [email protected] in selective apoptosis in monocytic and lymphocytic doi: 10.1158/1535-7163.MCT-11-0343 leukemias (18). Exposure of human promyelocytic leu- 2011 American Association for Cancer Research. kemia HL60 cells to apigenin resulted in induction of

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cell-cycle arrest, caspase-3 and PARP cleavage (7, 19). Analysis of apoptosis Another study showed that apigenin induces apoptosis For Annexin V/propidium iodide (PI) assay, cells were in primary effusion lymphoma cells via suppression of stained with Annexin V-fluorescein isothiocyanate and PI Akt pathway (20). The relationships between apigenin- and apoptosis was evaluated by flow cytometry according induced apoptosis and cell signaling cascades have not to manufacturer’s protocol (BD Pharmingen) and yet been examined in-depth in human leukemia cells. In described previously (21). this study, we have elucidated the functional role of Akt and JNK pathways in apigenin-induced lethality in Measurement of Akt kinase activity leukemia cells. Our results suggest a hierarchical model U937 cells were seeded in 6-well plate and then treated of apigenin-induced apoptosis in human leukemia cells. with apigenin (40 mmol/L) for 24 hours. In vitro Akt kinase In this model, apigenin-induced Akt inactivation repre- assay was then used to measure Akt kinase activity as per sents a primary event resulting in JNK activation, manufacturer’s instruction. downregulation of Mcl-1 and Bcl-2, and culminating in caspase activation, and apoptosis. In addition, we Western blotting have shown that apigenin attenuated tumor formation Western blotting was carried out using NuPAGE Bis- in U937 xenograft in athymic nude mice, suggesting Tris electrophoresis system (Invitrogen). For tissue sec- that apigenin is not only effective in vitro but also tions, radioimmunoprecipitation assay (RIPA) buffer was in vivo. added to the sections and homogenized with electric homogenizer. After incubation for 20 minutes on ice, Materials and Methods samples were centrifuged for 30 minutes at 12,000 rpm at 4C and supernatant was collected as total cell Chemicals lysate. SDS-PAGE was carried out as described previously Apigenin was purchased from Sigma. LY294002 and (21). Blots shown were representative for 3 separate SP600125 were from EMD Biosciences. Antibodies against experiments. Akt1, phospho-JNK, JNK1, and b-actin were from Santa Cruz Biotechnology. Antibodies against Bad, Bax, cyto- U937 xenograft assay chrome c, and Mcl-1 were from BD Pharmingen. Cleaved To evaluate the therapeutic effect of apigenin in vivo, caspase-3, cleaved caspase-7, cleaved caspase-9, Bcl-XL, xenograft of human U937 cells were used. Athymic nude phospho-Akt (Ser473), phospho-Bad (Ser136) and Akt mice (nu/nu, 4 weeks old; The Jackson Laboratory) were kinase assay kit were from Cell Signaling Technology. housed in a specific pathogen-free room within the animal Antibodies against PARP and Bcl-2 were from Biomol and facilities at the University of Kentucky, Lexington, KY. DAKO, respectively. Animals were allowed to acclimatize to their new envi- ronment for 2 weeks prior to use. All animals were Cell culture and plasmid transfection handled according to the Institutional Animal Care and U937, Jurkat, and HL60 human leukemia cells were Use, University of Kentucky. U937 cells (2 106) were obtained from American Type Culture Collection and resuspended in serum-free RPMI-1640 medium with grown in RPMI-1640 medium with 10% FBS. Normal Matrigel basement membrane matrix (BD Biosciences) at peripheral blood mononuclear cells (NPBMNC) were a 1:1 ratio (total volume: 100 mL) and then were subcuta- obtained from AllCells and maintained in RPMI-1640 neously injected into the flanks of nude mice. Four days with 10% FBS. Dr. Ruth Craig (Dartmouth Medical School, after tumor inoculation, mice were randomly divided into Hanover, NH) kindly provided U937 cells stably over- 3 groups (n ¼ 6 in each group), and apigenin (0, 20, and 40 expressing Mcl-1 and their empty vector counterpart mg/kg body weight) was administered intraperitoneally (pCEP). Authors carried out no further cell line authen- in 150 mL of dimethyl sulfoxide/0.9% physiologic saline tication in the last 6 months. Dr. Bing-Hua Jiang (West (1:0.5) daily for 5 days a week for 4 weeks. Body weight Virginia University, Morgantown, WV) kindly provided and tumor mass were measured every 5 days throughout the constitutive active form of Akt (myristolated Akt) the study. Tumor volumes was determined by a caliper and dominant-negative Akt mutant (Akt-DN). U937 cells and calculated according to the formula (width2 were transfected with m-Akt and Akt-DN using Amaxa length)/2. The dose of the apigenin for in vivo study was Nucleofactor II (Lonza) as recommended by the selected as described previously (22). manufacturer. Statistical analysis RNA interference and transfection Statistical analysis was conducted using GraphPad U937 cells (1.5 106) were transfected with 100 nmol/L Prism version 3. For analysis of apoptosis, values were JNK1 double-stranded RNA interference oligonucleotide presented as mean SD. Statistical differences between with CAAAGAUCCCUGACAAGCAtt (sense) and control and treated groups were determined by the Stu- UGCUUGUCAGGGAUCUUUGgt (antisense) sequence dent t test for unpaired observations. Differences were (Ambion) by using Amaxa Nucleofactor II as recom- considered statistically significant for values of P < 0.05 or mended by the manufacturer. P < 0.01.

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A B 100 100 ** ** 12h Control ** Api (40 µmol/L) 24h ** Figure 1. Effects of apigenin (Api) on 75 ** 75 ** ** apoptosis, caspase activation, and PARP cleavage in U937 cells. A, U937 cells were treated with/ 50 ** 50 ** * without various concentrations of apigenin for 12 and 24 hours. B,

(Annexin V/PI) (Annexin 25 V/PI) (Annexin 25 U937 cells were treated with/

% of apoptotic cells % of apoptotic cells without 40 mmol/L apigenin for 0 0 indicated times. Cells were stained Api (µmol/L) 01020 30 40 010203040 Time (h) 0 5 10 15 20 25 with Annexin V/PI, and apoptosis was determined using flow 12h 24h C D cytometry as described in Api (µmol/L) 0 10 20 3040 010203040 Api (h) 0 1 2 4 6 9 12 24 Materials and Methods. The values PARP obtained from Annexin V assays Cleaved form PARP Cleaved form represent the means SD for 3 separate experiments. or , Cleaved-casp-3 Cleaved-casp-3 values for cells exposed to apigenin were significantly increased compared with values in Cleaved-casp-7 Cleaved-casp-7 control cells by the Student t test; P < 0.05 or P < 0.01. C and D, U937 Cleaved-casp-9 Cleaved-casp-9 cells were exposed to apigenin in dose- and time-dependent manners. Total cellular extracts β-Actin β-Actin were subjected to Western blotting E OH using indicated antibodies. E, structure of apigenin 0 HO O (4 ,5,7,-trihydroxyflavone).

4',5,7,-trihydroxyflavone OH O

Results Exposure of U937 cells to apigenin resulted in downregulation of Bcl-2 and Mcl-1 Apigenin induced apoptosis, activated caspases and Furthermore, we evaluated the expression of various cleaved PARP in dose- and time-dependent manners members of Bcl-2 family of proteins. A dose- and time- in U937 cells dependent exposure of U937 cells to apigenin showed Apigenin induced a dose-dependent apoptosis in cleavage of Bcl-2 and downregulation of Mcl-1 (Fig. 2A U937 cells. Moderate increase in apoptosis was observed and B). No changes in Bcl-XL, Bax, and XIAP expres- after 12 and 24 hours of exposure to apigenin at concen- sion were observed in U937 cells in dose- as well as tration of 20 mmol/L and marked increase in apoptosis time-dependent manners (Fig. 2A and B). These results was observed at concentrations of 30 mmol/L or higher suggested that exposure of leukemia cells to apigenin (Fig. 1A). Apigenin exposure of U937 cells at concentra- resulted in cleavage or downregulation of antiapoptotic tion of 40 mmol/L also caused apoptosis in a time-depen- members of Bcl-2 family members such as Bcl-2 and dent manner and a signiﬁcant increase in apoptosis Mcl-1. was observed as early as 6 hours after apigenin exposure (Fig. 1B). Exposure of U937 cells to apigenin resulted in Western blotting revealed that apigenin induced apo- inactivation of Akt and pronounced increase in JNK ptosis in a caspase-dependent manner. Exposure of activation U937 cells at indicated concentrations of apigenin for Next, we examined the effects of apigenin on cell 12 and 24 hours activates caspases-3, 7 and 9 and survival and stress-induced signaling pathways. A cleaved PARP (Fig. 1C). In addition, a time course study dose-dependent study showed that apigenin dephos- of U937 cells exposed to 40 mmol/L apigenin showed phorylates Akt at Ser473 and its downstream targets marked increase in activation of caspases-3, 7 and 9 and mTOR (Ser2448) and Bad (Ser136) at concentrations of PARP cleavage (Fig. 1D). Figure 1E shows structure of 30 mmol/L or higher. Total Akt1 and mTOR levels were apigenin. Together, these results indicate that apigenin also decreased (Fig. 2C). In addition, we observed that JNK induces apoptosis in dose- and time-dependent man- phosphorylation levels increased concomitantly with a ners in U937 cells. decrease in Akt phosphorylation in dose-dependent

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A 12h 24h B Api (h) 0 1 2 4 6 9 12 24 Api (µmol/L) 0102030 40 010203040 XIAP XIAP Mcl-1 Mcl-1 Bcl-2 Bcl-2 c-Bcl-2 c-Bcl-2 Bcl-XL Bcl-XL Bax Bax Figure 2. Effects of apigenin (Api) β β-Actin on Bcl-2 family proteins and stress- -Actin induced signaling. A and C, U937 C D cells were treated with the indicated 12h 24h Api (h) 0 1 2 4 6 9 12 24 concentrations of apigenin for Api (µmol/L) 0 10 20 30 40 0 10 20 30 40 12 and 24 hours. B and D, U937 cell p-Akt were treated with 40 mmol/L apigenin p-Akt for indicated times. Total cellular Akt 1 extracts were subjected to Western Akt 1 blotting using indicated antibodies. p-Bad E, U937 cells were treated with m p-Bad 40 mol/L apigenin for 24 hours. Akt Bad kinase activity was determined. p-Akt was immunoprecipitated (IP) Bad using immobilized p-Akt antibody p-mTOR and subjected to Western blotting p-mTOR (WB) using antibody against mTOR p-GSK-3a/b (Ser21/9). XIAP, mTOR X-linked inhibitor of apoptosis p-JNK protein. p-JNK JNK1

JNK1 β-Actin β-Actin

E Api (40 µmol/L) – + p-GSK-3α/β (Ser21/9)

IP-p-Akt (Ser473) WB-p-GSK-3α/β

manner whereas JNK1 levels remained unchanged Apigenin induced apoptosis in leukemia cells via (Fig. 2C). caspase-independent inactivation of Akt and A time course study showed that exposure of U937 cells activation of JNK to 40 mmol/L apigenin resulted in dephosphorylation of To determine whether inactivation of Akt and activa- Akt as early as 6 hours after drug exposure and a con- tion of JNK were secondary to caspase activation, we comitant increase in JNK phosphorylation, which reached treated U937 cells with 40 mmol/L apigenin in the pres- maximum level at 24 hours (Fig. 2D). ence or absence of the broad-spectrum caspase inhibitor Exposure of U937 cells to apigenin for 24 hours reduced Z-VAD-FMK at 20 mmol/L. Exposure of Z-VAD-FMK kinase activity of Akt as shown by decreased phosphor- to U937 cells attenuated apigenin-induced apoptosis ylation of GSK-3a/b (Ser21/9; Fig. 2E). Collectively, these (Supplementary Fig. S1A), PARP cleavage, and caspase results suggest that inactivation of Akt with a concomitant activation (Supplementary Fig. S1B). In addition, Z-VAD- activation of JNK may play an important role in apigenin- FMK failed to inactivate Akt, activate JNK, and down- induced apoptosis. regulate the expression of Mcl-1 (Supplementary Fig. S1C

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and S1D). Interestingly, Z-VAD-FMK inhibited apigenin- then to apigenin resulted in a sharp increase in apoptosis induced cleavage of Bcl-2 (Supplementary Fig. S1D), as compared with apigenin alone. Figure 3B revealed that suggesting that the cleavage of Bcl-2 was caspase depen- pretreatment with LY294002 and then apigenin exposure dent. Together these findings suggest that apigenin- resulted in a pronounced increase in activation of cas- induced inactivation of Akt, activation of JNK, and pases-3, 7, 9, and PARP cleavage. In addition, LY294002 Mcl-1 downregulation were caspase independent. potentiates apigenin-induced Akt inactivation, phosphorylation of JNK, Bcl-2 cleavage, and Mcl-1 downregulation. Apigenin induced apoptosis in leukemia cells via However, LY294002 showed no effect on total Akt1 and mitochondrial dependent mechanism JNK1 levels (Fig. 3C and D). Figure 3E shows structure of It has been reported that exposure of leukemia cells to LY294002. apigenin resulted in mitochondrial injury, release of cyto- To assess the functional significance of Akt inactivation chrome c into cytosol, and caspase activation (7). We in apigenin-induced apoptosis, we used Akt-DN con- investigated apigenin-induced mitochondrial alterations struct. As shown in Fig. 4A, Akt-DN–expressing cells in U937 cells by DiOC6 staining. As shown in Supplemen- were more sensitive to apigenin-induced apoptosis than tary Fig. S2A and S2B, exposure of cells to apigenin in pcDNA3.1 vector control cells. Western blotting revealed dose- and time-dependent manners increased the number that Akt-DN construct potentiates apigenin-induced acti- of cells with low mitochondrial transmembrane potential vation of caspases-3, 7, 9, and PARP cleavage as compared as compared with control. In addition, apigenin induced with vector control (Fig. 4B). Consistent with these find- release of cytochrome c from mitochondria to cytosol in ings, Akt-DN construct potentiates apigenin-induced Akt dose- and time-dependent manners (Supplementary Fig. inactivation and JNK activation with no change in the S2C and S2D). Such findings indicate that apigenin levels of total JNK1 (Fig. 4C). In addition, apigenin- induced mitochondrial injury in leukemia cells that leads induced Bcl-2 cleavage and Mcl-1 downregulation were to cytochrome c release, caspase activation, and apoptosis. enhanced in Akt-DN–expressing cells (Fig. 4D). Over- expression of Akt by a constitutively active m-Akt pre- Apigenin induced similar effects in other leukemia vented apigenin-induced apoptosis in U937 cells as com- cells but not in NPBMNCs pared with vector control (Fig. 4E). In addition, there was a To assess whether apigenin-induced effects are restrict- marked increase in levels of total Akt1 and ability of ed to monocytic (U937) leukemia cells, we conducted apigenin to dephosphorylate Akt was inhibited in cells similar studies in T-cell lymphoblastic leukemia cells expressing m-Akt (Fig. 4E, Western). Apigenin-induced (Jurkat) and acute promyelocytic leukemia (HL60). The activation of caspases-3, 7, 9, and PARP cleavage were cells also showed apoptotic effects on apigenin exposure drastically reduced in U937 cells expressing m-Akt (Fig. but were less sensitive than U937 cells (Supplementary 4F). Interestingly, the ability of apigenin to induce JNK Fig. S3A). Moreover, apigenin showed no significant activation was abrogated in cells expressing m-Akt (Fig. apoptotic effect on NPBMNCs at concentration of 40 4G). Furthermore, overexpression of Akt essentially abol- mmol/L (Supplementary Fig. S3B). Supplementary Figure ished apigenin-induced Bcl-2 cleavage and Mcl-1 down- S3C revealed that in HL60 cells there was very little or no regulation (Fig. 4G). Together, these findings indicate that PARP cleavage or caspase-3 activation or expression of inactivation of Akt plays a critical role in apigenin- phospho-Akt (Ser473). These observations suggest that induced apoptosis and that this event lies upstream of HL60 cells were less sensitive to apigenin-induced apo- Mcl-1 and Bcl-2 downregulation and JNK activation. ptosis at the same experimental condition. In addition, Mcl-1 expression was downregulated in all 3 cell lines. To Activation of JNK played an important role in elucidate the mechanism by which the NPBMNCs did not apigenin-induced caspase activation and apoptosis undergo apoptosis, we carried out Western blotting and To dissect the possible functional significance of JNK the results show that treatment of NPBMNCs with api- activation in apigenin-induced apoptosis, we used both genin at various concentrations neither cause cleavage of pharmacologic and genetic approaches. Pretreatment PARP nor dephosphorylation of Akt, downregulation of of U937 cells with JNK inhibitor SP600125 (15 mmol/L) Mcl-1, and activation of JNK (Supplementary Fig. S3D). for 1 hour diminished apigenin-induced apoptosis Collectively, these results show that apigenin induced (Fig. 5A). Figure 5B revealed that pretreatment with apoptosis in several leukemia cells but not in NPBMNCs. SP600125 blocked apigenin-induced caspases-3, 7, and 9 activation and PARP cleavage. In addition, SP600125 Inactivation of Akt is responsible for apigenin- reduced apigenin-induced phosphorylation of JNK and induced JNK and caspases activation and apoptosis showed no effect on phosphorylation of Akt, total Akt1 or Results in Fig. 2 show that inactivation of Akt may play JNK1 levels (Fig. 5C). Furthermore, interruption of JNK by an important role in apigenin-induced apoptosis. To test SP600125 inhibited apigenin-induced Bcl-2 cleavage and this hypothesis, we pretreated U937 cells with PI3K inhib- Mcl-1 downregulation (Fig. 5D). itor LY294002 (10 mmol/L) for 1 hour, followed by expo- To confirm the role of JNK in apigenin-induced apo- sure to apigenin (20 mmol/L) for 12 and 24 hours. As ptosis, short interfering RNA (siRNA) of JNK1 was used. shown in Fig. 3A, pretreatment of cells with LY294002 and Apigenin-induced apoptosis was sharply reduced in

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A B 12h 24h 100 Api (20 µmol/L) –+ –+++ – – 12h LY294002 (10 µmol/L) – –++++ –– 75 24h PARP ** Cleaved form Figure 3. Effects of LY294002 on 50 ** Cleaved-casp-3 apigenin (Api)-induced apoptosis in

U937 cells. A, U937 cells were V/PI) (Annexin 25 Cleaved-casp-7 pretreated with 10 mmol/L of % of apoptotic cells LY294002 for 1 hour, followed by the 0 Cleaved-casp-9 addition of 20 mmol/L of apigenin for Api (20 µmol/L) – –++++ – – 12 and 24 hours. Cells were stained LY294002 (10 µmol/L) – –++++–– β-Actin with Annexin V/PI, and apoptosis was determined using ﬂow C 12h 24h D 12h 24h cytometry. The values obtained from Annexin V assays represent the Api (20 µmol/L) – ++++–– – Api (20 µmol/L) – ++++––– LY294002 (10 µmol/L) LY294002 (10 µmol/L) means SD for 3 separate – – ++–– + + –– ++–– + + experiments. , values for cells p-Akt Mcl-1 treated with apigenin and LY294002 β-Actin in combination were signiﬁcantly Akt1 increased as compared with Bcl-2 apigenin alone by the Student t test; p-JNK c-Bcl-2 P < 0.01. B–D, total cellular extracts β-Actin were subjected to Western blotting JNK1 using indicated antibodies. β E, structure of LY294002 -Actin (2-morpholin-4-yl-8- phenylchromen-4-one; ref. 38). E O

O N O 2-Morpholin-4-yl-8-phenylchromen-4-4- one

JNK1 siRNA–transfected cells and that JNK1 levels were absence of apigenin failed to reduce apigenin-mediated reduced to one third (approximately) as compared with Mcl-1 downregulation (data not shown). control siRNA (Fig. 5E). As shown in Fig. 5F, JNK1 To further delineate the mechanism by which apigenin siRNA–transfected cells diminished apigenin-induced diminishes Mcl-1 expression in U937 cells, we exposed the caspases-3, 7, and 9 activation and PARP cleavage. Fur- cells to apigenin (40 mmol/L) for various intervals in the thermore, JNK1 siRNA cells showed no change in phos- presence or absence of the proteasome inhibitor MG132 phorylation of Akt and total Akt1 levels (Fig. 5G). Api- (10 mmol/L). As shown in Supplementary Fig. S4C, genin-induced Bcl-2 cleavage and Mcl-1 downregulation MG132 essentially blocked the downregulation of Mcl- were inhibited in JNK1 siRNA–transfected cells (Fig. 5G). 1. In addition, co-administration of protein synthesis Collectively, these results indicate that apigenin-induced inhibitor cycloheximide (20 mmol/L) hastened the rate of JNK activation played an important functional role in Mcl-1 downregulation (Supplementary Fig. S4D). Togeth- apoptosis and that the activation of JNK occurred down- er, these findings suggest that apigenin not only blocks stream of Akt inactivation. Mcl-1 transcription but also degrades Mcl-1 via proteasome-dependent mechanism. Apigenin-induced Mcl-1 downregulation proceeds via transcriptional and proteasome-dependent Overexpression of Mcl-1 substantially diminished mechanisms apigenin-induced apoptosis, caspases activation, and Results from Fig. 2 indicate that downregulation of Mcl- PARP cleavage in U937 cells 1 upon apigenin treatment is tightly connected to Akt To asses the functional significance of Mcl-1 in apigenin- dephosphorylation and JNK activation. Therefore, to elu- induced apoptosis, weused U937 cells overexpressing Mcl- cidate the mechanism underlying Mcl-1 downregulation 1. As shown in Supplementary Fig. S5A, overexpression of by apigenin, we used quantitative reverse transcriptase Mcl-1 substantially diminished the apigenin-induced apo- PCR analysis. As shown in Supplementary Fig. S4A and ptosis, whereas empty vector control (pCEP) cells were as S4B, exposure of U937 cells to apigenin resulted in sig- sensitive as parental cells. Treatment with apigenin dimin- nificant decrease in Mcl-1 mRNA levels in dose- and time- ished Mcl-1 expression in pCEP cells but failed to down- dependent manners. Inhibition of transcription by expos- regulate Mcl-1 in overexpressing cells (Supplementary Fig. ing cells to actinomycin D (5 mg/mL) in the presence or S5B). Supplementary Fig. S5C revealed that apigenin failed

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A BCpcDNA3.1 Akt-DN pcDNA3.1 Akt-DN 100 Control * Api 40 (µmol/L) Api (40 µmol/L) –+ – + Api (40 µmol/L) –+ –+ 75 PARP Cleaved form p-Akt 50 Cleaved-casp-3 Akt1

(Annexin V/PI) (Annexin 25 Cleaved-casp-7 p-JNK % of apoptotic cells 0 pcDNA3.1 Akt-DN Cleaved-casp-9 JNK-1

β -Actin β D pcDNA3.1 Akt-DN -Actin Api (40 µmol/L) –+ – + F PCB6 m-Akt Mcl-1 Api (40 µmol/L) –+–+ G PCB6 m-Akt Bcl-2 PARP Api (40 µmol/L) –+–+ c-Bcl-2 Cleaved form β-Actin Cleaved-casp-3 p-JNK

Cleaved-casp-7 JNK1

E Cleaved-casp-9 β-Actin

β-Actin Mcl-1 100 Control Api 40 (µmol/L) PCB6 75 m-Akt Bcl-2 c-Bcl-2 Api (40 µmol/L) –+–+ 50 β-Actin p-Akt ** (Annexin V/PI) (Annexin 25

% of apoptotic cells Akt1

0 β-Actin PCB6 m-Akt

Figure 4. Determination of functional significance of Akt in apigenin (Api)-induced apoptosis. A, U937 cells were transfected with an empty vector (pcDNA3.1) and Akt-DN as described in Materials and Methods. Akt-DN–expressing cells were treated with/without 40 mmol/L of apigenin for 24 hours, after which apoptosis was analyzed using Annexin V/PI assay. , values for Akt-DN cells treated with apigenin were significantly increased compared with those for pcDNA3.1 cells by the Student t test; P < 0.05. B–D, total cellular extracts were subjected to Western blotting using indicated antibodies. E, U937 cells were transfected with an empty vector (PCB6) and constitutively active m-Akt. m-Akt- and PCB6-expressing cells were treated with/without 40 mmol/L of apigenin for 24 hours, after which, apoptosis and p-Akt and Akt1 expressions were analyzed. , values for m-Akt cells treated with apigenin were significantly decreased compared with those for PCB6 cells by the Student t test; P < 0.01. F and G, total cellular extracts were subjected to Western blotting using indicated antibodies.

to activate caspases-3, -9 and cleave PARP in Mcl-1–over- injected with vehicle or apigenin (20 and 40 mg/kg expressingcells. Inaddition,apigenin-inducedcytochrome intraperitoneally) daily for 5 days a week for 4 weeks c release from mitochondria was diminished in Mcl-1– as described in Materials and Methods. As shown in Fig. overexpressing cells (Supplementary Fig. S5D). These ﬁnd- 6A and B, treatment of mice with 20 and 40 mg/kg ings show that apigenin-induced downregulation of Mcl-1 apigenin resulted in 58.4% and 71% inhibition of tumor is an essential event in apigenin-induced apoptosis in U937 growth as compared with control group on day 20. In cells. addition, no statistically signiﬁcant change in body weight was observed in control and apigenin-treated Apigenin inhibited tumor formation in xenografts of animals (Fig. 6C), indicating that apigenin was not U937 human leukemia cells toxic. Figure 6D and E revealed that apigenin (40 On the basis of the in vitro studies described in above mg/kg) dephosphorylated Akt and activated JNK as sections, we extend our studies to test the anti-leukemic compared with vehicle in tissue sections. PARP cleav- activity of apigenin in vivo in U937 human leukemia age, an indicator of apoptosis, was also increased in xenografts. Athymic nude mice were inoculated with treatment group (Fig. 6D). Downregulation in Mcl-1 U937 cells subcutaneously, after which mice were levels in apigenin-treated mice was consistent with

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A BC 12h 24h 12h 24h 100 12h 24h Api (40 µmol/L) ––––++ ++ Api (40 µmol/L) ––––++++ 75 SP (15 µmol/L) ––++ –– + + SP (15 µmol/L) ––++ –– + + PARP p-JNK 50 Cleaved form ** **

(Annexin V/PI) (Annexin 25 Cleaved-casp-3 JNK1 % of apoptotic cells p-Akt 0 Cleaved-casp-7 Api (40 µmol/L) ––+ +++ – – SP (15 µmol/L) – –––++ + + Cleaved-casp-9 Akt1

β-Actin β-Actin D 12h 24h –+–+++ – – Api (40 µmol/L) FGCon siRNA JNK1 siRNA Con siRNA JNK1 siRNA SP (15 µmol/L) – –––+ +++ Api (40 µmol/L) ––+ +– Api (40 µmol/L) ++– Mcl-1 PARP p-Akt Cleaved form β-Actin Cleaved-casp-3 Akt1 Bcl-2 c-Bcl-2 β-Actin Cleaved-casp-7 β-Actin Mcl-1 Cleaved-casp-9

E β Bcl-2 -Actin c-Bcl-2 100 Control β-Actin Api (40 µmol/L) Con siRNA JNK1 siRNA 75 Api (40 µmol/L) –+– + 50 JNK1 ** (Annexin V/PI) (Annexin 25 % of apoptotic cells β-Actin

0 Con siRNA JNK1 siRNA

Figure 5. Effects of pharmacologic and genetic inhibition of JNK on apigenin (Api)-induced apoptosis. U937 cells were pretreated with 15 mmol/L SP600125 for 1 hour followed by the addition of 40 mmol/L of apigenin for 12 and 24 hours. A, cells were stained with Annexin V/PI and apoptosis was determined using flow cytometry. The values obtained from Annexin V assays represent the means SD for 3 separate experiments. , values for cells treated with apigenin and SP600125 were significantly less than those obtained for cells treated with apigenin alone by the Student t test; P < 0.01. B–D, total cellular extracts were subjected to Western blotting using indicated antibodies. E, U937 cells were transfected with JNK1 siRNA oligonucleotides or control (con) siRNA and allowed to grow for 24 hours, after which, cells were treated with 40 mmol/L of apigenin for another 24 hours. Apoptosis was determined using the Annexin V/PI assay and JNK1 expression by Western blotting. , values for cells treated with apigenin after transfection with JNK1 siRNA oligonucleotides were significantly decreased compared with those for control cells treated with apigenin alone by the Student t test; P < 0.01. F and G, total cellular extracts were subjected to Western blotting using indicated antibodies. in vitro finding (Fig. 6F). Collectively, these findings exposure of apigenin to leukemia cells leads to mitochon- suggest that inactivation of Akt and activation of JNK drial injury, caspase activation, and apoptosis. In addi- signaling contributes to apigenin-induced apoptosis not tion, our results provide the mechanistic information of only in vitro but also in vivo. how apigenin exerts its proapoptotic effects in leukemia cells, that is, by inactivation of Akt and activation of JNK. Discussion Because phosphorylation by upstream kinases is required for complete activation of Akt (14), it is not Previous studies have shown that apigenin-induced surprising that various protein phosphatases dephos- apoptosis in several types of cancer cells such as breast, phorylate Akt. Activation of Akt generally involves PTEN prostate, lung, and hematologic (3–8). Presently, no infor- inactivation and results in attenuation of apoptosis (23). In mation is available about the functional importance of Akt our study, apigenin exposure resulted in dephosphory- and JNK pathways in apigenin-induced lethality in leu- lation of Akt. Thus, it would be enticing to relate this kemia cells. The results in the present study indicate that observation to PTEN activation. However, the absence of

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AB ) 3 4,000 Figure 6. In vivo anti-leukemic Day 10 activity of apigenin (Api) in U937 3,000 Day 20 xenografts. Eighteen athymic nude mice were inoculated with U937 6 2,000 cells (2 10 cells per mouse, * subcutaneously) and randomly * divided into 3 groups (n ¼ 6per 1,000 group) for treatment with vehicle/ apigenin as described in Materials * **

Api (mm Mean tumor volume 0 and Methods. A, representative 0 20 40 Api (mg/kg) (mg/kg) 0 20 40 animals from each group as indicated with tumor. B, average C 35 Control D tumor volume in vehicle control Api (40 mg/kg) mice and mice treated with 30 Api (mg/kg) 040 20 and 40 mg/kg apigenin on days PARP 10 and 20. Data are means SD 25 Cleaved form (n ¼ 18; 6 mice per group with tumors implanted on right ﬂank of 20 p-JNK each mouse). or , P < 0.05 or Body weight (g) Body weight P < 0.01 values were signiﬁcantly 15 different compared with vehicle 0 51015 20 25 β-Actin Day control by the Student t test. E C, body weight changes of mice F during 20 days of treatment. D–F, Api (mg/kg) 0 40 Api (mg/kg) 0 40 tissue sections subjected to Western blotting using indicated p-Akt Mcl-1 antibodies. β-Actin β-Actin

wild-type PTEN in U937 cells does not support this enhanced apigenin-induced lethality as compared with possibility (24). In addition, Akt can be dephosphorylated vector control. Last, overexpression of Akt by a constitu- by phosphatases such as PP2A and PHLPP (25, 26). It is tively active m-Akt prevented apigenin-induced caspase likely that apigenin increases the activity or expression of activation and apoptosis. In addition, we have shown that these phosphatases that in turn dephosphorylate Akt. In apigenin downregulated Akt kinase activity, suggesting fact, our unpublished results show that apigenin its reduced ability to phosphorylate downstream targets. enhanced the expression of PP2A in U937 cells. Addition- Furthermore, we elucidated the functional role of JNK al mechanistic studies are required to show the role of pathway in apigenin-induced apoptosis in leukemia cells. these phosphatases in apigenin-induced Akt dephos- We have shown a dose- and time-dependent correlation phorylation in leukemia cells. It is possible that apigenin, between Akt inactivation and JNK activation. Inhibition of through a mechanism not known yet, blocks the actions of JNK by SP600125 and JNK1 siRNA had no effect on Akt, PI3K. The ﬁndings that LY29004 augmented apigenin- suggesting that Akt lies upstream of JNK. Constitutively induced inactivation of Akt, caspase activation, and PARP active m-Akt prevented apigenin-induced JNK activation, cleavage were in agreement with this hypothesis. indicating that one of the mechanisms by which Akt Akt is a well-known substrate of caspases. For instance, pathway protects leukemia cells from apigenin-induced cleavage of Akt1 was caspase dependent in U937 and lethality is due to the inhibition of JNK. Conversely, Jurkat cells exposed to UV light, etoposide, and Fas inhibition of Akt pathway by LY29004 and Akt-DN poten- ligation (27). The results shown in the present study tiated apigenin-induced JNK activation and apoptosis. showed that pretreatment of leukemia cells with caspase The following evidences can explain the correlation inhibitor Z-VAD-FMK failed to prevent inactivation of between Akt inactivation and JNK activation. It has been Akt, suggesting that inactivation of Akt by apigenin was shown that Akt suppresses the JNK pathway by phos- caspase independent. phorylating and negatively regulating ASK-1 (apoptosis Results from the present study suggest that Akt inac- signal–regulating kinase-1), MLK3 (mixed-lineage pro- tivation by apigenin plays a functional role in apigenin- tein kinase), and MKK4/SEK1 (mitogen-activated pro- induced lethality in human leukemia cells. First, pretreat- tein/ERK kinase; refs. 28–30). In addition, Akt suppress ment with the LY29004 followed by treatment with api- JNK activation by directly interacting with JNK-interact- genin enhanced apigenin-induced caspases activation ing protein (JIP) and thus preventing the recruitment of and apoptosis. Second, U937 cells expressing Akt-DN upstream kinases to JNK (31).

140 Mol Cancer Ther; 11(1) January 2012 Molecular Cancer Therapeutics

Apigenin Induces Apoptosis In Vitro and In Vivo

JNK can induce apoptosis by phosphorylating and inhi- in phospho-JNK expression, PARP cleavage, dephos- biting antiapoptotic function of Bcl-2 and Mcl-1 (32, 33). phorylation of Akt (Ser473), and downregulation of In this study, we found that apigenin downregulated Mcl-1 in the apigenin-treated group compared with the Mcl-1 and induced Bcl-2 cleavage. We have also shown vehicle group, providing the apoptotic evidence in api- that inhibition of JNK decreased apigenin-induced Mcl-1 genin-treated U937 xenograft mice. The expression levels downregulation and Bcl-2 cleavage, suggesting that these of phospho-Akt, phospho-JNK, PARP cleavage, and Mcl- antiapoptotic proteins lie downstream of JNK. Collective- 1 in tissue sections of U937 xenograft tumor were closely ly, these findings showed an important functional role of correlated with the reduction of U937 tumor xenografts. JNK pathway in apigenin-induced apoptosis. In summary, the results obtained from the present Notably, apigenin exposure resulted in downregula- study provide an evidence that Akt and JNK signaling tion of Mcl-1, an antiapoptotic protein that may play an pathways are potential targets for apigenin-induced apo- important role in apoptosis in malignant hematopoietic ptosis in leukemia cells and in vivo. Further efforts are cells (34). Overexpression of Akt inhibited apigenin- required to understand the mechanism by which apigenin induced Mcl-1 downregulation, suggesting that Mcl-1 inactivates Akt and induces apoptosis in human leukemia downregulation is significant for apigenin-induced apo- cells and U937 tumor xenografts, which could provide a ptosis. This finding was supported by the evidence that reasonable evidence to incorporate apigenin in leukemia Mcl-1 is upregulated by PI3K/Akt pathway through a treatment regimens. transcription factor complex CREB (35). In addition, we have shown that apigenin inhibited Mcl-1 transcription, Disclosure of Potential Conflicts of Interest and overexpression of Mcl-1 substantially diminished apigenin-induced apoptosis, caspases activation, PARP No potential conflicts of interest were disclosed. cleavage, and mitochondrial injury (cytochrome c Grant Support release). Together, these findings suggest that apigenin- induced downregulation of Mcl-1 plays an essential role The study was supported by NIH grant RO1 ES015375 (X. Shi) in apigenin-induced apoptosis in leukemia cells. The costs of publication of this article were defrayed in part by the Apigenin has been shown to inhibit tumor growth of payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate prostate, ovarian, and lung xenografts by inducing apo- this fact. ptosis (36, 37). The present study shows that apigenin exhibits significant inhibitory effects on the growth of Received May 19, 2011; revised September 14, 2011; accepted October 19, U937 leukemia tumor xenograft. We found an increase 2011; published OnlineFirst November 14, 2011.

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142 Mol Cancer Ther; 11(1) January 2012 Molecular Cancer Therapeutics

Apigenin Induces Apoptosis in Human Leukemia Cells and Exhibits Anti-Leukemic Activity In Vivo

Amit Budhraja, Ning Gao, Zhuo Zhang, et al.

Mol Cancer Ther 2012;11:132-142. Published OnlineFirst November 14, 2011.

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