Biochimica et Biophysica Acta 1800 (2010) 912–918

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Beta-arrestin 2 modulates resveratrol-induced apoptosis and regulation of Akt/GSK3β pathways

Xiuli Sun a,b,⁎, Yi Zhang b, Jianliu Wang a, Lihui Wei a, Hui Li b, Gregory Hanley c, Miaoqing Zhao b,Yi Li b, Deling Yin b,⁎a Department of Obstetrics and Gynecology, Peking University People's Hospital, Beijing, Chinab Department of Internal Medicine, College of Medicine, East Tennessee State University, Johnson City, TN 37614, USAc Department of Laboratory Animal Resources, James Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA

Abbreviations: EC, endometrial cancer; RNAi, RNA iylated Akt; GSK-3β, glycogen synthase kinase-3β;transfected with β-arrestin 2 RNAi plasmid; Ishiktransfected with Arr2 full-length plasmid⁎ Corresponding authors. X. Sun is to be contacted at

Gynecology, Peking University People's Hospital, Beiji3756. D. Yin, Tel.: +1 423 439 8826.

E-mail addresses: sunxiuli918@126.com (X. Sun), yi

0304-4165/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.bbagen.2010.04.015

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 22 November 2009Received in revised form 22 March 2010Accepted 27 April 2010Available online 7 May 2010

Keywords:Endometrial cancerβ-Arrestin 2ResveratrolApoptosisAktGSK3β

Background: Resveratrol is emerging as a novel anticancer agent. However, the mechanism(s) by whichresveratrol exerts its effects on endometrial cancer (EC) are unknown. We previously reported that β-arrestin 2 plays a critical role in cell apoptosis. The role of β-arrestin 2 in resveratrol modulation ofendometrial cancer cell apoptosis remains to be established.Scope of Review: EC cells HEC1B and Ishikawa were transfected with either β-arrestin 2 RNA interfering(RNAi) plasmid or β-arrestin 2 full-length plasmid and control vector. The cells were then exposed todiffering concentrations of resveratrol. Apoptotic cells were detected by TUNEL assay. Expression of total andphosphorylated Akt (p-Akt), total and phosphorylated glycogen synthase kinase 3 beta (p-GSK3β), andcaspase-3 were determined by Western blot analysis. Our data demonstrate that inhibition of β-arrestin 2increases the number of apoptotic cells and caspase-3 activation. Additionally β-arrestin 2 exerted anadditive effect on resveratrol-reduced levels of p-Akt and p-GSK3β. Overexpression of β-arrestin 2 decreasedthe percentage of apoptosis and caspase-3 activation and attenuated resveratrol-reduced levels of p-Akt and

p-GSK3β. Taken together, our studies demonstrate for the first time that β-arrestin 2 mediated signalingplays a critical role in resveratrol-induced apoptosis in EC cells.Major Conclusions: Resveratrol primes EC cells to undergo apoptosis by modulating β-arrestin 2 mediatedAkt/GSK3β signaling pathways.General significance: These inspiring findings would provide a new molecular basis for further understandingof cell apoptotic mechanisms mediated by β-arrestin 2 and may provide insights into a potential clinicalrelevance in EC.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

β-arrestin 2, a member of arrestins family, is a multifunctionalscaffold/adaptor protein that plays a critical role in G protein-coupledreceptor (GPCR) regulation [1,2]. Both β-arrestin 1 and β-arrestin 2,two universally expressedmembers of arrestin family in many tissues[3–6], are key negative regulators and scaffolds of GPCR signaling[2,7]. Recent evidence reveals that β-arrestins function as adaptors toconnect the receptors to the cellular trafficking machinery, such asscaffolding GPCRswith Src andmediating GPCR activation of JNK3 and

nterference; p-Akt, phosphor-HEC1B/βarr2−, HEC1B cellsawa/βarr2+, Ishikawa cells

Department of Obstetrics andng, China. Tel.: +86 10 8242

n@etsu.edu (D. Yin).

ll rights reserved.

ERK1/2 pathways [8,9]. β-arrestin 2 interacts with these signalingmolecules by modulating the phosphorylation, ubiquitination and/orsubcellular distribution of their binding partners. Accumulatingevidences suggest that β-arrestin 2 function in anti-apoptoticpathway through impacting the activity of interacted kinases[2,10,11]. Furthermore, β-arrestin 2 has been shown to affect growthfactor receptor-mediated cell proliferation through the activation ofAkt [11]. In addition, β-arrestin 2 interacts with critical mediators ofToll-like receptors (TLRs) signaling, and acts as a negative regulator ofTLRs signaling [12]. We have previously reported that apoptosismediated by TLRs is negatively regulated by β-arrestin 2 [13].Overexpression of β-arrestin 2 inhibits cell apoptosis through TLRs[13]. However, the role of β-arrestin 2 in apoptosis of endometrialcancer cells is unknown.

Endometrial cancer is the fourth most prominent cancer among allfeminine cancers in the world [14,15]. Despite the frequent detectionof early-stage cancers and the evolving use of adjuvant chemotherapyfor advanced disease, the death rate from this malignancy has

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increased [14,15]. Resveratrol (3,4′,5-trihydroxy-trans-stilbene), anantioxidant found in food products such as grape and wine is anemerging novel anticancer agent [16,17]. Resveratrol activates pro-apoptotic signaling, such as p38 and p53 pathways in several types ofcancers [16,17]. For example, resveratrol inhibits cell growth in breastcancer and prostate cancer cells [17–19]. However, the mechanismsby which endometrial cancer is affected by resveratrol and the role ofβ-arrestin 2 in resveratrol-induced apoptosis are unknown. Weaddressed this question in our present studies.

Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinasethat refers to two isoforms—GSK3α and GSK3β, and has a broadlyregulatory influence on cellular functions, including apoptosis [20–23]. Of the mechanisms tightly regulating the activities of twoisoforms of GSK3, the most well-defined mechanism is the phospha-tidylinositol 3-kinase (PI3K)/Akt (also known as protein kinase B,PKB) signaling pathway. GSK3 activity is inhibited through PI3K/Aktsignaling by phosphorylation of serine-9 in GSK3β or serine-21 inGSK3α. Interestingly, recent studies showed that regulations of Aktand GSK3β by drugs in the brain were mediated by β-arrestin 2through certain cell membrane receptors [24]. Mice lacking β-arrestin2 showed no response to Akt-GSK3 phosphorylation of drugs,demonstrating apparent mechanisms on regulation of Akt/GSK3through β-arrestin 2 [25].

In the present study, we investigated the molecular mechanismsby which β-arrestin 2 modulates resveratrol-mediated apoptosis inhuman endometrial cancer cells. Specifically, we determined theinvolvement of the Akt/GSK3β signaling pathway.We found here thatβ-arrestin 2 acts as a negative regulator in resveratrol-inducedapoptosis in endometrial cancer cells.

2. Materials and methods

2.1. Reagents

Resveratrol was purchased from Sigma (St. Louis, MO). Theantibodies of β-arrestin 2, total GSK3β, phospho-GSK3β, total Akt,phosphor-Akt, total caspase-3, and cleaved caspase-3 were purchasedfrom Cell Signaling Technology (Beverly, MA). The antibodies of β-arrestin1, actin, and GAPDH were purchased from Santa CruzBiotechnology (Santa Cruz, CA).

2.2. Cell culture

Human endometrial cancer HEC1B cell line (estrogen receptor(ER) positive and progesterone receptor (PR) negative) [26] waspurchased from American Type Culture Collection. Human endome-trial cancer Ishikawa cell line (ER positive and PR positive) [27] waskindly provided by Dr. Jonathan Braun (David Geffen School ofMedicine, University of California, CA). Both of these cell lines weregrown in DMEM medium (Invitrogen Corporation, Carlsland, CA)supplemented with 10% heat-inactivated fetal bovine serum (AtlantaBiologicals, Lawrenceville, GA). Cultures were incubated at 37 °C and5% CO2 in a fully humidified incubator.

2.3. Cell transfection

Transient transfection of experimental cells was performed usingthe lipofectamine plus reagent (Invitrogen Corporation, Carlsland, CA)as described previously [13]. The β-arrestin 2 full-length plasmid andβ-arrestin 2 RNAi (RNA interference) plasmid and control vector [28]were kindly provided by Dr. Gang Pei (Chinese Academy of Sciences,Shanghai, China). After 24 h transfection, a transfection efficiency of90–95% was observed using a fluorescent microscopy (Motic Com-pany, Richmond, Canada). Forty-eight hours after transfection, themedium was changed with fresh DMEM containing 10% FBS for

further treatment with different concentrations of resveratrol asdescribed below.

2.4. Detection of apoptosis by TUNEL assay

The experimental cells were transfected with indicated plasmids.Forty-eight hours after transfection, the cells were treated withresveratrol at different concentrations. Nucleosomal DNA fragmenta-tion was determined by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay using an in situ apoptosisdetection kit (Roche Diagnostic, Indianapolis, IN) as described in ourprevious publication [29]. The number of apoptotic cells was countedin randomly selected fields to calculate the ratio of apoptotic cells andtotal cells.

2.5. Western blot analysis

Western blot analysis was performed as described in our previouspublications [30,31]. Briefly, the cellular proteinswere separated by SDS-polyacrylamide gel electrophoresis and transferred onto Hybond ECLmembranes (Amersham Pharmacia, NJ). The ECL membranes wereincubatedwith theappropriateprimaryantibodies. Theblotwasexposedto the SuperSignal West Dura Extented Duration substrate (PierceBiotechnology, Rockford, IL). The signals were quantified by scanningdensitometry using a Bio-Image Analysis System (Bio-Rad). The resultsfrom each experiment were expressed as relative integrated intensitycompared with that of control lymphocytes measured with the samebatch.

2.6. Statistical analysis

All data were represented as means±SEM. The data wereanalyzed using one-way analysis of variance (ANOVA) followed byBonferroni tests to determine where differences among groupsexisted. Differences were considered statistically significant for valuesof pb0.05.

3. Results

3.1. Decreased expression of β-arrestin 2, but not β-arrestin 1 inendometrial cancer cells following resveratrol treatment

Although it is established that resveratrol plays a pro-apoptoticeffect in various cancer cell types [16,17,19], the mechanisms bywhich resveratrol affects endometrial cancer cells remain to beelucidated. To determine the mechanisms, HEC1B and Ishikawaendometrial cancer cells were treated with resveratrol and theexpression of β-arrestin 2 and β-arrestin 1 was determined byWestern blot analysis. As shown in Fig. 1, resveratrol dramaticallydecreased the expression of β-arrestin 2 in both HEC1B (Fig. 1A) andIshikawa cells (Fig. 1B). However, resveratrol could not alter the levelof β-arrestin 1 in these cells (Fig. 1). Our subsequent studies focusedon the role of β-arrestin 2 in resveratrol-mediated endometrial cancercell apoptosis.

3.2. Effect of β-arrestin 2 on resveratrol-mediated cell proliferation andapoptosis

To determine the role of β-arrestin 2 in resveratrol-mediated cellproliferation, we investigated resveratrol-induced cell viability inHEC1B and Ishikawa cells.We transfectedβ-arrestin 2 RNAi plasmid inHEC1B cells (HEC1B/βarr2−) and Ishikawa cells (Ishikawa/βarr2−).In addition, we transfected β-arrestin 2 full-length plasmid in HEC1Bcells (HEC1B/βarr2) and Ishikawa cells (Ishikawa/βarr2). The cellswere then treatedwith resveratrol at different concentrations for 48 h.As shown in Fig. 2, inhibition of β-arrestin 2 caused significant

Fig. 1. Resveratrol inhibits the expression of β-arrestin 2. HEC1B cells (A) and Ishikawacells (B) were treated with resveratrol at 10 µM, or 100 µM for 48 h. Cell lysates wereprobed for β-arrestin 2 and β-arrestin 1 expression by Western blot. * pb0.01. Meanvalues were derived from three independent experiments.

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decreases in cell viability in HEC1B cells and Ishikawa cells followingresveratrol treatment. However, overexpression of β-arrestin 2dramatically enhanced cell viability in HEC1B cells and Ishikawa cellsfollowing resveratrol treatment compared to their control cells.

We next determined whether β-arrestin 2 modulates cellproliferation with resveratrol treatment was due to apoptosis usingTUNEL assay. We found that inhibition of β-arrestin 2 in HEC1B cells(Fig. 3A) and Ishikawa cells (Fig. 3B) significantly increased thepercentage of apoptotic cells following resveratrol treatment, com-pared with control cells. Furthermore, after resveratrol treatment for48 h, HEC1B/βarr2+ cells (Fig. 3A) and Ishikawa/βarr2+ cells(Fig. 3B) showed a significantly lower percentage of apoptotic cells.These results demonstrated that β-arrestin 2 plays an anti-apoptoticrole in resveratrol-induced apoptosis in endometrial cancer cells.

Fig. 2. Effect of β-arrestin 2 on resveratrol-mediated cell proliferation in HEC1B and Ishikawaβarr2+) or β-arrestin 2 RNAi plasmid (HEC1B/βarr2−) and control vector. Forty-eight hourswith resveratrol at different concentrations for 48 h and cell viability was measured by MTTcells were transfected with either β-arrestin 2 full-length plasmid (Ishikawa/βarr2+) or βarrestin 2 was determined by Western blot (C). Cells were then treated with resveratrol at

3.3. β-arrestin 2 inhibits resveratrol-induced caspase-3 activation

The cleaved caspase-3 is an established specific marker forapoptosis [32]. Thus, we examined caspase-3 activation in HEC1B/βarr2− cells and Ishikawa/βarr2+ cells. As shown in Fig. 4A, afterexposure to resveratrol for 24 h, HEC1B/βarr2− cells have asignificantly higher level of cleaved caspase-3 than control cells.Ishikawa/βarr2+ cells treated with resveratrol showed a significantlylower level of cleaved caspase-3 than control cells (Fig. 4B). Theseresults further demonstrated that β-arrestin 2 inhibits resveratrol-primed endometrial cancer cell apoptosis.

3.4. Effect of β-arrestin 2 on the levels of phosphorylated Akt followingresveratrol treatment

Recent evidence suggests that β-arrestin 2 modulates Akt [24,25].Akt is generally considered to promote cell survival and inhibits cellapoptosis [25]. To examine whether β-arrestin 2 can activate Aktsignaling following resveratrol treatment, we examined the levels ofphosphorylated Akt at Ser473 (p-Akt) in HEC1B/βarr2− cells andIshikawa/βarr2+ cells following resveratrol treatment. We observedthat the levels of p-Akt in HEC1B/βarr2− cells are significantly lowercompared to control cells (Fig. 5A). Furthermore, resveratrolreduction in the level of p-Akt was inhibited by transfection with β-arrestin 2 full-length plasmid in Ishikawa cells (Fig. 5B). These datasuggest that decreasing β-arrestin 2-mediated signaling will attenu-ate Akt activity.

3.5. β-arrestin 2 attenuates resveratrol-reduced levels of phospho-GSK3β

GSK3β is an important downstream target of the Akt signalingpathway [20,21]. Phosphorylation of GSK-3β on the inactivatingresidue serine-9 by Akt results in GSK-3β inactivation [21]. Wedetermined the effect of β-arrestin 2 on phosphor-GSK3β (p-GSK3β)with or without resveratrol treatment. As shown in Fig. 6A, the level ofp-GSK3β at serine-9 was significantly lower in HEC1B/βarr2- cellscompared to transfected control vector in the absence or presence of

cells. HEC1B cells were transfected with either β-arrestin 2 full-length plasmid (HEC1B/later, β-arrestin 2 expression was detected byWestern blot (A). Cells were then treatedmethod (B). Mean values were derived from three independent experiments. Ishikawa-arrestin 2 RNAi plasmid (Ishikawa/βarr2−) and control vector. The expression of β-different concentrations for 48 h. Cell viability was examined by MTT method (D).

Fig. 3. Role ofβ-arrestin 2 in resveratrol-induced apoptosis inHEC1B and Ishikawa cells. (A) HEC1B cells were transfectedwith eitherβ-arrestin 2 full-length plasmid (HEC1B/βarr2+) orβ-arrestin 2 RNAi plasmid (HEC1B/βarr2−) and control vector. After transfection for 48 h, the cells were treated with the indicated concentrations of resveratrol for 48 h. Apoptotic cellswere determined by TUNEL assay. Photographs of representative TUNEL-stained cells are shown at the top. Magnification 200×. The bar graph shows the percentage of apoptotic cells.Results represent mean±s.e.m. of three independent experiments. * pb0.01. (B) Ishikawa/βarr2+ cells and Ishikawa/βarr2− cells were treated with indicated concentrations ofresveratrol for 48 h. Apoptotic cells were determined by TUNEL assay. * pb0.01.

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resveratrol. In addition, overexpression of β-arrestin 2 in Ishikawa/βarr2+ cells diminished the resveratrol-reduced level of phosphor-GSK3β (Fig. 6B). Our results showed that resveratrol treatment couldnot alter the level of phosphor-GSK3β in HEC1B cells, but decreasedthe level of phosphor-GSK3β in Ishikawa cells. This may be due to the

Fig. 4. β-arrestin 2 attenuates resveratrol-induced caspase-3 activation. (A) HEC1B/βarr2− ccleaved caspase-3 and total caspase-3 were determined by Western blot. Data are represetreated with resveratrol at 0, 10 µM, or 100 µM for 24 h. Total and cleaved caspase-3 were

difference in expression levels of β-arrestin 2 since we have shownthat β-arrestin 2 modulates the level of phosphor-Akt/GSK3β. Sincephosphorylation of serine-9 is inhibitory for GSK3β activity, theseresults suggested that resveratrol decreases GSK3β activity through aβ-arrestin 2-dependent mechanism.

ells were treated with the indicated concentrations of resveratrol for 24 h. The levels ofntative of three independent experiments. * pb0.01. (B) Ishikawa/βarr2+ cells weredetermined by Western blot. * pb0.01.

Fig. 5. Role of β-arrestin 2 in resveratrol-mediated Akt phosphorylation. (A) inhibition of β-arrestin 2 enhances resveratrol—reduction of the level of Akt phosphorylation (p-Akt).HEC1B/βarr2− cells were treated with resveratrol at 0, 10 µM, or 100 µM for 24 h. Total and p-Akt were determined by Western blot. * pb0.01. (B) Overexpression of β-arrestin 2attenuates resveratrol—decreased the level of p-Akt. Ishikawa/βarr2+ cells were treated with indicated concentrations of resveratrol for 24 h. * pb0.01.

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4. Discussion

β-arrestin 2, functions as multifunctional scaffold/adaptor protein formost GPCRs and is widely involved in desensitization and endocytosis ofdifferent kinds of cell surface receptors [2,33,34]. Recent evidence sug-gests that β-arrestin 2 contributes to an anti-apoptotic effect [2,10,11].Our previous studies have shown that overexpression of β-arrestin 2significantly inhibits opioid-induced apoptosis [13], revealing thatβ-arrestin 2 acts as a negative regulator in opioid-induced apoptosis.

Resveratrol, a natural antioxidant found in red wine, exerts variousphysiological effects. At low concentrations normally occurring in food,resveratrol has been evaluated to produce neuroprotective effects [35].

Fig. 6. Effect of β-arrestin 2 on resveratrol-mediated GSK3β phosphorylation. (A) Inhibition of ββarr2− cells were treatedwith indicated concentrations of resveratrol for 24 h. The levels of tβ-arrestin 2 attenuated Res-reduced the level of p-GSK3β. Ishikawa/βarr2+ cells were trealevel of total and p-GSK3β are shown at the top. * pb0.05.

This effect is mostly attributed to its antioxidant properties. Morerecently, resveratrol has been shown tohaveotherhealthbenefits suchasanticancer activity. Indeed, resveratrol possesses anticancer activitywhenadministered at higher, non-physiological doses. In these conditions,resveratrol inhibits cell proliferation and induces cell apoptosis in severalcancers cell types [16]. In addition, resveratrol has been shown to inhibitangiogenesis and delay tumor growth in animalmodels of cancer [36,37].

Endometrial cancer constitutes a potential target for resveratrol.Resveratrol exerts the capacity to bind to the estrogen receptors (ERs),and ER is notably present and activated in the normal and neoplasticendometria [38,39]. Nonetheless, themechanisms bywhich resveratrolinduces endometrial cancer cells apoptosis remain largely unknown. It

-arrestin 2 increased resveratrol reduction of the level of p-GSK3β in HEC1B cells. HEC1B/otal and p-GSK3β at Ser9were examined byWestern blot. * pb0.01. (B) Overexpressionted with resveratrol at different concentrations for 24 h. Representative results of the

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has been shown that resveratrol contributes a pro-apoptotic role tovarious cancer cell types [16,17,19], however, the role of β-arrestin 2 inresveratrol-induced cell apoptosis remains to be elucidated. Wedetermined the effect of β-arrestin 2 on endometrial cancer cellsurvival/apoptosis. Our data have revealed that β-arrestin 2 plays ananti-apoptotic role on resveratrol-induced apoptosis. Our results havealso shown that inhibition of β-arrestin 2 significantly increases thenumber of apoptotic cells induced by resveratrol, indicating thatinhibition of β-arrestin 2 will enhance the anticancer effect ofresveratrol. We found that resveratrol induces caspase-3 activation.Interestingly, inhibition of β-arrestin 2 exerts an additive effect onresveratrol-induced caspase-3 activation. The caspase-3 activation actsas an active and lethal protease at themost distal stage of the apoptoticpathways and is the major caspase involved in cell apoptosis [40,41].

Recent studies have demonstrated that Akt contributes to an anti-apoptotic effect [42]. Akt regulates cellular activation, inflammatoryresponses, and apoptosis [42]. Activated Akt phosphorylates severaldownstream targets of the PI3K signaling pathway such as GSK3β[20,22,42]. GSK3β is a constitutively active enzyme that is inactivatedby Akt [20]. Recent evidence revealed that the Akt pathwaydifferentially modulates cytokine production in response to endotoxininhibition of GSK3β [21,22]. GSK3β plays a pivotal role in regulatingmany cellular functions, including cell survival and apoptosis [20,22].GSK3β has paradoxical pro- and anti-apoptotic actions [23]. GSK3βpromotes apoptosis mainly through the mitochondria-mediatedintrinsic apoptotic pathway. It is also established that GSK3 inhibitsthe death receptor-mediated extrinsic apoptosis pathways [22,23].

β-arrestin 2 not only terminates G-protein couple receptor signalingbut also regulates other signalingpathways [24,25].β-arrestin2 signalingcomplex with Akt/GSK-3β has been well established [24,25], whichillustrates the activation of GSK-3β by β-arrestin 2 through scaffoldingPP2A to Akt [25]. We determined the role of β-arrestin 2 in resveratrol-mediated Akt and GSK3β activities. We have shown that inhibition of β-arrestin 2 exerts an additive effect on resveratrol-reduced levels of Aktphosphorylation and inactivation of GSK3β. We also observed thatoverexpression of β-arrestin 2 attenuated resveratrol-reduced levels ofAkt phosphorylation and inactivation of GSK3β. Our data revealed thatresveratrol decreased GSK3β activity through a β-arrestin 2 dependentmechanism. These results suggest that β-arrestin 2 modulates resvera-trol-mediated apoptosis through the Akt/GSK3β signaling pathway. Thisevidence led us to propose that GSK3β activity plays an important role inresveratrol-induced endometrial cancer cell apoptosis.

In summary, the data presented herein demonstrated for the firsttime, to our knowledge, a key role for β-arrestin 2 in resveratrol-mediated apoptosis in endometrial cancer cells. It is widely acceptedthat resveratrol-mediated signaling mainly activates pro-apoptoticpathways [16,17]. This provides a possible target for treatment ofendometrial cancers. Our studies reveal that β-arrestin 2 inhibitsresveratrol-induced endometrial cancer cell apoptosis through theAkt/GSK3β signaling pathway. Further understanding of the mechan-isms mediated by β-arrestin 2 may provide insights into potentialtherapeutic interventions endometrial cancer.

Acknowledgements

This workwas supported by the National Institutes of Health (NIH)grant DA020120-03A1 to D. Yin. This work was also supported in partby the National Natural Science Foundation of China 30571937 to J.Wang and 30571938 to L. Wei.

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