Hesperetin Induced G1-Phase Cell Cycle Arrest in Human Breast ...

NUTRITION AND CANCER, 59(1), 115–119Copyright C○ 2007, Lawrence Erlbaum Associates, Inc.Hesperetin Induced G1-Phase Cell Cycle Arrest in Human BreastCancer MCF-7 Cells: Involvement of CDK4 and p21Eun Jeong ChoiAbstract: This study was to investigate the effects of hesperetinon cell proliferation and cell cycle arrest and exploredthe mechanism for these effects in breast carcinomaMCF-7 cells. Hesperetin significantly inhibited cell proliferationin a dose-dependent manner after treatment for 48 and72 h and resulted in significant cell cycle arrest in the G1phase. In the G1-phase related proteins, hesperetin downregulatesthe cyclin-dependent kinases (CDKs) and cyclinsand upregulates p21Cip1 and p27 Kip1 in cells treated withhesperetin for 48 h and 72 h. After 72 h treatment, these phenomenonswere more pronounced. Hesperetin treatment athigh concentration for 72 h resulted in a decrease in CDK2and CDK4 together with cyclin D. In addition, hesperetinincreases the binding of CDK4 with p21 Cip1 but not p27 Kip1or p57K ip2 . Taken together, our data suggest for the first timethat the regulation of CDK4 and p21 Cip1 may participatein the anticancer activity pathway of hesperetin in MCF-7cells.IntroductionFlavonoids are the most abundant natural antioxidantsin food. Several epidemiological studies have supported thehypothesis that the antioxidant action of flavonoids may reducethe risk of developing cancer and cardiovascular disease(1–4). Reports based on the in vitro action of flavonoids oncancer cells have found various anticancer effects such asthe inhibition of cell proliferation and kinase activity and theinduction of apoptosis (4,5–7).Flavonoids have been shown to inhibit the proliferationof cultured human cancer cell lines (8–9). The observed antiproliferativeproperty of flavonoids suggests that these compoundsmay inhibit the cell cycle or induce apoptosis (10).However, many of the mechanisms underlying the potentialanticancer activity of flavonoids are not fully understood.Hesperetin (3 ′ ,5,7-trihydroxy-4-methoxyflavanone), amember of the flavanone subclass of flavonoids, is foundin fruit sources including various citrus species (11).Hesperetin occurs as hesperidin (its glycoside form) innature. Dietary hesperidin is deglycosylated to hesperetinby intestinal bacteria prior to absorption (12), and hesperidinmay be considered a pro-drug, which is metabolized to hesperetin(13). In several animal studies of the absorption,bioavailability, and pharmacokinetics of hesperidin, hesperidinhas not been observed in plasma, bile, or urine (14–16). Several reports have described the beneficial biologicaleffects of hesperidin. However, these studies may have inadvertentlyinvestigated the biological effects of hesperidinbecause hesperidin is not found in the body. To understandthe biological activities of hesperidin, it is necessary to studythe molecule’s biologically active form. Moreover, relativelylittle has been reported about hesperetin in comparison withhesperidin, and only a few in vitro studies have assessed it.Hesperetin is reported to be a powerful radical scavengerand to promote cellular antioxidant defense-related enzymeactivity (17–18). Hesperetin has also shown potential anticarcinogenicactivity as an antiangiogenic agent in mES cells(19). Thus, hesperetin may be useful in nutritional therapyand chemotherapy. We investigated the possible anticarcinogeniceffects of hesperetin through the inhibition of cell proliferationand cell cycle arrest in human breast carcinomaMCF-7 cells, which are regarded as an in vitro breast cancermodel.Materials and MethodsThe Cells Culture and Hesperetin TreatmentMCF-7 cells were purchased from the Korean Cell LineBank (Seoul, Korea). Cells were routinely maintained inRPMI 1640 (Gibco BRL, MD), supplemented with 10%fetal bovine serum and antibiotics (50 U/ml of penicillinand 50 µg/ml streptomycin; Gibco) at 37 ◦ C in a humidifiedatmosphere containing 5% CO 2 . Cells were treatedwith hesperetin ranging from 1 to 100 µM for 24 h, 48 h,and 72 h. Hesperetin was purchased from Sigma and dissolvedin dimethyl sulfoxide (DMSO; final concentration0.1% in medium). All experiments were done in triplicate orquadruple.E. J. Choi is affiliated with the Plant Resources Research Institute, Duksung Women’s University, Seoul, South Korea.

Cell Proliferation and Cell Death AssayCell proliferation was determined using the methyl thiazolyltetrazolium (MTT) assay. At 24 h, 48 h, and 72 h point,the cells exposed to hesperetin were added to MTT. DMSOwas added 4 h later to each well to dissolve the resulting formazancrystals, and then absorbance was recorded at 490 nmin a microplate reader SpectraMax Plus; Molecular Devices,CA).Cell Cycle DistributionCells were then harvested, washed with cold phosphatebuffered solution (PBS), and processed for cell cycle analysis.Briefly, the cells were fixed in absolute ethanol and storedat –20 ◦ C for later analysis. The fixed cells were centrifugedat 1,000 rpm and washed with cold PBS twice. RibonucleaseA (20 µg/ml final concentration) and propidium iodidestaining solution (50 µg/ml final concentration) was addedto the cells and incubated for 30 min at 37 ◦ C in the dark.The cells were analyzed a FACS Calibur instrument (BDBiosciences, San Jose, CA) equipped with CellQuest version3.3 software. ModFit LT version 3.1 trial cell cycle analysissoftware was used to determine the percentage of cells in thedifferent phases of the cell cycle.Immunoblotting and Immunoprecipitation AssayCells were lysed in radio-immunoprecipitation assaybuffer (1% NP-40, 150 mM NaCl, 0.05% 4-chloro-2,5-dimethoxyamphetamine, 1% sodium dodecyl sulfate (SDS),50mM Tris, pH 7.5) containing protease inhibitor for 1 hat 4 ◦ C. The supernatant was separated by centrifugation,and protein concentration was determined by Bradfordprotein assay kit II (Bio-rad Laboratories, CA). Proteins(25 µg/well) denatured with sample buffer were separatedby 10% SDS-polyacrylamide gel. Proteins were transferredonto nitrocellulose membranes (0.45 µm). The membraneswere blocked with a 1% bovine serum albumin solutionfor 3 h, washed twice with PBS containing 0.2% Tween-20, and incubated with the primary antibody overnight at4 ◦ C. Antibodies against cyclin-dependent kinases (CDKs)CDK2, CDK4, CDK6, cyclin D, cyclin E, p16 INK4a , p18 INK4c ,p21 Cip1 , p27 Kip1 , p57 Kip2 , and β-actin were purchased fromSanta Cruz Biotechnology, Inc. (Santa Cruz, CA) and used toprobe the separate membranes. The next day, the immunoreactionwas continued with the secondary goat antirabbithorseradish-peroxidase–conjugated antibody after washingfor 2 h at room temperature. The specific protein bands weredetected by Opti-4CN Substrate kit (Bio-rad Laboratories).For CDK4-CDK inhibitors binding assay, cells weretreated with vehicle or 100 µM of hesperetin for 48 h and72 h. CDK4 from cell lysates (250 µg protein) was immunoprecipitatedusing anti-CDK4 antibody (4 µg) and preclearedprotein A/G-plus agarose beads (Santa Cruz) for overnightat 4 ◦ C. The precipitates were washed with lysis buffer, dena-Figure 1. Effect of hesperetin on cell proliferation of MCF-7 cells. Cellswere exposed to either vehicle (0.1% dimethyl sulfoxide in medium) orhesperetin (1–100 µM) and incubated for 24 h, 48 h, and 72 h. All dataare reported as the percentage change in comparison with the vehicle-onlygroup, which were arbitrarily assigned 100% viability. ∗ ,P

Figure 2. Effect of hesperetin on cell cycle distribution of MCF-7 cells. Values are expressed as percentage of the cell population in the G1, S, and G1/Mphase of cell cycle. Cells were exposed to either vehicle (0.1% dimethyl sulfoxide in medium) or hesperetin (1–100 µM) and incubated for 48 h and 72 h.∗ ,P

Figure 4. Effect of hesperetin on Bcl-2 and Bcl-2 associated x protein(Bax) expression of MCF-7 cells. Cells were exposed to either vehicle (0.1%dimethyl sulfoxide in medium) or hesperetin (1–100 µM) and incubated for48 h.in mouse embryonic stem cells, suggesting that its inhibitoryeffects may be associated with its prooxidant activity (19).Thus, hesperetin is a potential anticancer agent. Moreover,unlike other flavonoids that have prooxidant activity, nohesperetin-induced cytotoxicity was observed, even at highconcentrations (100 µM). Thus, we investigated the anticancereffects of hesperetin on breast carcinoma MCF-7 cellsin which hesperetin inhibits cell proliferation by causing cellcycle arrest.To assess whether the hesperetin-induced inhibition of cellgrowth was mediated via alterations in cell cycle progression,cells were exposed to either vehicle (0.1% DMSO) or variousconcentrations of hesperetin (1, 5, 10, 50, 100 µM). Hesperetinsignificantly inhibited cell proliferation in a dose- andtime-dependent manner. Hesperetin can inhibit the proliferationof both estrogen receptor-negative MDA-MB-435 andestrogen receptor-positive MCF-7 human breast cancer cellsin vitroin vitro (21–22). Our results supported these observations;the exposure of MCF-7 cells to hesperetin resultedin a significant inhibition of cell proliferation.We evaluated the mechanism by which hesperetin inhibitedcell proliferation using a cell cycle analysis. The eukaryoticcell cycle is controlled by catalytic complexes ofCDK/cyclin that coordinate internal and external signals atseveral key checkpoints (23). G1 arrest leads cells to undergorepair or to follow the apoptosis pathway (24). ActivatedCDK/cyclin complexes can be changed to an inactive stateby the binding of CDK inhibitory subunits (CKIs). The CKIscan be divided into 2 classes. One class contains p21 Cip1 ,p27 Kip1 , p57K ip2 , and related proteins, with a preference forCDK2 and CDK4/cyclin complexes; the other class containsthe INK4 family, including p16 INK4a , p15 INK4b , p18 INK4c , andp19 INK4d and closely related CKIs specific for CDK4 andCDK6/cyclin complexes (25–26).Hesperetin induced a decrease in cyclinD and CDK4, togetherwith an increase in p21 Cip1 and p27 Kip1 associated withCDK4. CDK4 appears to play a key role in the G1 cell cyclearrest observed in response to hesperetin.We assessed the effect of hesperetin on the interactionbetween CDK4 and its inhibitors using immunoprecipitation.An increase in the binding of CDK4p21 Cip1 , but not ofCDK4–p27 Kip1 or CDK4–p57 Kip2 , was observed in MCF-7cells exposed to hesperetin. Additionally, hesperetin inducedG1-phase cell cycle arrest through the regulation of CDK4and p21 Cip1 , indicating that the inhibition of cell proliferationand the contribution to the apoptotic process is a possiblemechanism by which hesperetin acts on cancer cells.Moreover, the G1-phase cell cycle arrest induced by hesperetinresulted in apoptosis in MCF-7 cells. Hesperetininduced a dose- and time-dependent decrease in Bcl-2 expressionand increase in Bax expression in MCF-7 cells.Apoptosis is a multistep process important in controlling cellnumber and proliferation as part of normal development;the Bax genes and members of the Bcl-2 family such asbcl-2 are involved in the control of apoptotic pathways. Decreasedexpression of Bcl-2 and increased expression of Baxare correlated with the response to anticancer compoundsfor chemotherapy in cancer cell lines. In addition, the lossof Bcl-2 expression can promote the induction of apoptosis(27–29). An anticancer drug that initiates apoptosis may beuseful as a new category of chemotherapeutic agent.In conclusion, our results indicate that hesperetin treatmentleads to the inhibition of cell proliferation, the inductionof cell cycle arrest at the G1 phase, and apoptosis. Additionally,our data suggest for the first time that the regulation ofCDK4 and p21 Cip1 may participate in the anticancer activityof hesperetin in MCF-7 cells. Therefore, we suggest thathesperetin is worthy of further study to assess its potentialas an anticancer drug; it remains to be determined whetherhesperetin has anticarcinogenic activity in vivo.Acknowledgments and NotesThis work was supported by the Korea Research Foundation Grant fundedby the Korean Government (MOEHRD, KRF–2006–311–F00127). Addresscorrespondence to EJ Choi, Ph.D., Plant Resource Research Institute,Duksung Women’s University, 419 Ssangmun-dong, Dobong-ku, 132–714,Seoul, South Korea. Phone: +82-2-901-8663. FAX: 82-2-901-8661. 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