THE ANATOMICAL RECORD 294:781–788 (2011)

Role of CD41CD251 Regulatory T Cellsin Melatonin-Mediated Inhibition

of Murine Gastric Cancer Cell GrowthIn Vivo and In Vitro

HUI LIU, LI XU, JIAN-EN WEI, MEI-RONG XIE,SHI-E WANG, AND RUI-XIANG ZHOU*

Department of Human Anatomy, Histology and Embryology, Neurobiology ResearchCenter, Fujian Medical University, Fuzhou, Fujian, People’s Republic of China

ABSTRACTMelatonin is an important immune modulator with antitumor func-

tions, and increased CD4þCD25þ regulatory T cells (Tregs) have beenobserved in tumor tissues of patients and animal models with gastric can-cer. However, the relationship between melatonin and Tregs remainsunclear. To explore this potential connection, we performed an in vivostudy by inoculating the murine foregastric carcinoma (MFC) cell line inmice and then treated them with different doses of melatonin (0, 25, 50,and 100 mg/kg, i.p.) for 1 week. The results showed that melatonin couldreduce the tumor tissue and decrease Tregs numbers and Forkhead boxp3 (Foxp3) expression in the tumor tissue. An in vitro study was also per-formed to test the effects of purified Tregs on melatonin-mediated inhibi-tion of MFC cells. The cell cultures were divided into three groups: 1)MFCþ Tregs; 2) MFC only; and 3) MFCþCD4þCD25� T cells. After treat-ment with different concentrations of melatonin (0, 2, 4, 6, 8, and 10 mM)for 24 h, a dose-dependent apoptosis and cell cycle arrest at the G2/Mphase was detected in melatonin-treated MFC at melatonin concentrationhigher than 4 mM. There were no significant differences in the rates ofapoptosis and cell cycle distributions of MFC among the three groups. Inconclusion, the antigastric cancer effect of melatonin is associated withdownregulation of CD4þCD25þ Tregs and its Foxp3 expression in the tu-mor tissue. Anat Rec, 294:781–788, 2011. VVC 2011 Wiley-Liss, Inc.

Keywords: Melatonin; gastric cancer; CD4+CD25+ Tregs; Foxp3

INTRODUCTION

Gastric cancer is one of the most common human ma-lignant tumors and causes a mortality rate, which rankssecond worldwide among malignant tumors (Crew andNeugut, 2006). Many studies have shown that the mech-anism by which cancer cells use to escape detection bythe body’s immune system plays a key role in the occur-rence and development of malignant tumors. Conse-quently, the production and development of CD4þCD25þ

regulatory T cells (Tregs) are gaining a great deal ofattention. CD4þCD25þ Tregs, first reported by Sakagu-chi in 1995 (Sakaguchi et al., 1995), are activated T cellsthat express the IL-2 receptor a chain (IL-2Ra, CD25).Moreover, they are known to induce anergy and possessimmunosuppressive properties and play an important

Grant sponsor: National Natural Sciences FoundationProjects of China; Grant numbers: 30840049, 30971541; Grantsponsor: Key Project of Science and Technology Commission ofFujian Province of China; Grant number: 2009Y0023.

*Correspondence to: Rui-Xiang Zhou, Department of HumanAnatomy, Histology and Embryology, Neurobiology ResearchCenter, Fujian Medical University, 88 Jiaotong Road, Fuzhou350004, Fujian, People’s Republic of China. Fax: þ86-591-22862273. E-mail: rxzhou@mail.fjmu.edu.cn

Received 3 October 2010; Accepted 7 January 2011

DOI 10.1002/ar.21361Published online 17 March 2011 in Wiley Online Library(wileyonlinelibrary.com).

VVC 2011 WILEY-LISS, INC.

role in maintaining immune tolerance, protection againstautoimmune diseases, as well as inhibiting the trans-plant rejection response. Foxp3 is a member of the fork-head/winged-helix transcription factor family andencodes a 48 kDa scurfin protein. As a nuclear transcrip-tion factor, it is a molecular determinant of differentia-tion and function of Tregs. Mutation or deletion of thegene encoding Foxp3 causes severe autoimmune diseasesin both human and mice, due to a malfunction ofCD4þCD25þ Tregs (Hori et al., 2003).

Many of the recent studies focused on changes inTregs in the tumor microenvironment (Curiel et al.,2004; Ghiringhelli et al., 2005; Enarsson et al., 2006).For example, Tregs are induced to differentiate and pro-liferate by immature or semimature antigen-presentingcells in the tumor-bearing host and suppress the accu-mulation of immune effector cells in the tumor localmicroenvironment. To date, some related studies haveshown a functional relationship between gastric cancerand Tregs, and a large number of Tregs are present inthe peripheral blood and tumor tissues of many patientswith malignancies, such as pancreatic, liver and gastro-intestinal cancer (Liyanage et al., 2002; Ichihara et al.,2003; Unitt et al., 2005). They further showed that Tregsharbor antigen-specific properties and inhibit tumor-spe-cific T cells, which accelerate tumor growth and decreasethe survival rate of patients. Therefore, inhibiting thefunction and migration of Tregs to tumor tissues canpotentially improve the treatment of human digestivetract tumors.

Melatonin (N-acetyl-5-methoxytryptamine) is mainlysecreted from the pineal gland and has many effects ona wide range of physiological functions, such as inhibi-tion of the inflammatory response, regulation of circa-dian rhythms, and activity of antioxidant enzymes. Inparticular, melatonin has antitumor effects (Martinet al.; Reiter, 2004; Joo and Yoo, 2009; Park et al., 2009;Reiter et al., 2009) and is an important immune modula-tor (Srinivasan et al., 2008b). Regarding the immunesystem, melatonin has been also localized in the thymusand in mast cells, natural killer (NK) cells, and eosino-philic leukocytes. However, there are few studies on therole of Tregs in melatonin-mediated inhibition of gastriccancer. In this study, we established an in vivo mousemodel bearing gastric cancer and an in vitro coculturesystem of murine foregastric cancer (MFC) cells withTregs or CD4þCD25�T cells to test the effects of differ-ent concentrations of melatonin on tumor/tumor cellgrowth. We demonstrate that melatonin inhibited thegrowth of experimental gastric cancer, and CD4þCD25þTregsplayed a role in the inhibition of gastric cancer growthby melatonin, our data support melatonin might regu-late antitumor immune responses.

MATERIAL AND METHODSMFC Cell Line Culture

The MFC cells are foregastric carcinoma cells derivedfrom the 615-mouse strain and purchased from the Chi-nese Academy of Sciences, Shanghai Institute for Biolog-ical Science. MFC cells were cultured in RPMI-1640medium supplemented with 10% fetal bovine serum(FBS). The cells were maintained at 37�C in a humidi-fied incubator with 5% CO2. The cells were passagedevery 3 days by using trypsin for dissociation. All cell

culture reagents were purchased from Gibco (Invitrogen,Carlsbad, CA).

Experimental Tumor Animal Model

Male and female 6–8-wk-old inbred mice of the 615-(H-2Kk) strain were used in these experiments. Thebody weights of the specific pathogen-free (SPF) grademice ranged from 20 to 25 g and purchased from TianjinInstitute of Hematology, the Chinese Academy of Medi-cal Science. All animal experiments were conducted inaccordance with the guidelines for the Care and Use ofLaboratory Animals in Fujian Medical University. Allanimals were housed in an environmentally controlledroom (temperature ¼ 21–22�C) with a 12-hr light/darkcycle (08:00–20:00 hr). Some mice were subcutaneouslyinoculated with 5 � 104 MFC cells under the rightaxilla. One week after inoculation, the tumor-bearingmice models were successfully established, and the ex-perimental animal groups were set up as follows: groupA: normal control mice (n ¼ 10); group B: tumor-bearingcontrol mice with daily intraperitoneal (i.p.) injection of100 mg/kg saline water (n ¼ 10); group C: tumor-bearingmice injected with 25 mg/kg (low dose) melatonin (SigmaChemical Company, St. Louis, Missouri) (n ¼ 10); groupD: tumor-bearing mice injected with 50 mg/kg (mediumdose) melatonin (n ¼ 17); and group E: tumor-bearingmice injected with 100 mg/kg (high dose) melatonin (n ¼14). Before use, the melatonin was dissolved in anhy-drous ethanol and combined with normal saline, and thefinal ethanol concentration was 0.1%. The melatoninsolution was kept at 4�C and away from light before use.Melatonin was given to the assigned groups at 17:00 hrevery day for 1 week via i.p. injection, after which thetumor tissues were excised and weighed. We, then,measured the long and short diameters of the tumors,and the tumor volume was calculated by using thefollowing formula (Tu et al., 2003): tumor volume V ¼ 4/3p � L/2 � (W/2)2, where L and W are the long and shortaxes, respectively.

Detection of CD41CD251 Tregsin Tumor Tissues

The tumor tissues were macerated with a syringeneedle before filtration through a 200 stainless steelmesh. The cells were then rinsed with phosphate-buf-fered saline (PBS) buffer before double staining with aFITC-conjugated rat anti-mouse CD4 (FITC-CD4) mono-clonal antibody and a PE-conjugated rat anti-mouseCD25 (PE-CD25) monoclonal antibody (BD Pharmingen).Stained cells were analyzed by a flow cytometer (XLBeckman Coulter). Control cells were double stained withrat (DA) IgG 2a, j-FITC and rat (LEW) IgG2b, j-PE.

Immunofluorescence Assay

The tumor tissues from mice in group B were doublestained and immunohistochemically analyzed for CD4þ

and CD25þ cells as follows. The tumor tissues were fixedin neutral formalin solution, embedded in conventionalparaffin and cut at 8 lm thickness. The sections wererehydrated through decreasing concentrations of ethanoland prepared for antigen retrieval in high pressure for 2min (pH 6.0 citrate buffer as repair solution). After

782 LIU ET AL.

rinsing with PBS buffer, the specimens were incubatedwith 3% H2O2 for 10 min, followed by incubation withblocking serum (5% FBS þ0.1% triton X-100 þ PBS) for30 min at room temperature (RT). Subsequently, thesamples were washed with PBS three times for 5 mineach. Then incubated with the primary antibodies (anti-CD4-FITC, anti-CD25-PE, working concentration at1:100 dilution) at 4�C overnight. After washing withPBS buffer, the slides were sealed and fixed in gly-cerol. The sections were then observed under a laserconfocal scanning microscope (Leica, Germany). For anegative control, PBS was used instead of the primaryantibody.

Quantitative Real-Time RT-PCR(Qrt-PCR) Analysis

Total RNA was isolated from tumor tissue using theTrizol reagent (Invitrogen), and the RNA was quantifiedby measuring the absorbance at 260/280 nm (OD) on aUV spectrophotometer. A total of 2-lg RNA was used inthe reverse transcription to generate cDNA, which wasthen used as a template for the PCR amplification.Quantitative PCR of murine samples was performedwith the Brilliant SYBR Green QPCR master mix (Stra-tagene). The real-time PCR reactions each contained thefollowing: 1 lL of the above reverse transcription prod-uct, 1 lL of each primer (5 pmol/lL), 6.7 lL ddH2O, 0.3lL DYE, and 10 lL 2 � Brillion II SYBR Green PCRbuffer. The total reaction volume was 20 lL, and eachsample was analyzed in triplicate. After mixing the reac-tion solution, the tubes were placed into a Quantitativereal-time PCR instrument (Applied Biosystems 7500model) for reaction and detection. The PCR reaction con-ditions were set as follows: predenaturation at 95�C for10 min, 40 cycles of denaturation at 95�C for 30 sec,annealing at 60�C for 30 sec, and extension at 72�C for30 sec. PCR products were normalized against thehousekeeping gene GAPDH, and measurements betweensamples were compared by the cycle threshold (Ct). Rel-ative gene expressions were calculated by the 2�DDCT

method. The Foxp3 and GAPDH primers were as fol-lows: for Foxp3, 50-CACTGGGCTTCTGGGTATGT-30 and50-AGACAGGCCAGGGGATAGTT-30; for GAPDH, 50-CCG-AGAATGGGAAGCTTGTC-30 and 50-TTCTCGTGGTTC-ACACCCATC-30.

Western Blot Analysis

Total protein was extracted from tumor tissues, andthe protein concentration was determined using theBCA assay (Sigma). A total of 30 lg of protein was ana-lyzed using 12% sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE). After electrophoresis,the proteins were transferred onto a polyvinylidene di-fluoride (PVDF) membrane. The membrane was blockedwith 5% nonfat milk dissolved in TBS-T buffer (Tris 50mM; NaCl 1.5%; Tween-20 0.05%; pH 7.5) and then incu-bated with a specific rat anti-mouse Foxp3 monoclonalantibody (1:200, Santa Cruz Biotechnology, Santa Cruz,CA) or anti-mouse b-actin (1:4,000, Sigma) overnight at4�C. After washing, alkaline phosphatase-conjugatedaffinity purified anti-mouse IgG (Invitrogen) was addedand incubated for 40 min. After washing, the chemo-fluorescence substrate (Invitrogen) was added for expo-

sure on X-ray film. For quantitative analysis, thedensity of each band was scanned and determined byusing a Image-Quant software (Molecular Dynamics,Sunnyvale, CA).

Purification of CD41CD251 Tregs andCD41CD252 T Cells

Spleens from 615 mice (6–8 week old) were each surgi-cally removed, crushed, and passed through a stainlesssteel mesh. The single cell suspensions were then proc-essed using the Mouse CD4þCD25þ regulatory T cell iso-lation kit (Miltenyi Biotec, Bergish Gladcach, Germany).Briefly, according to the manufacturer’s instructions, thesuspension containing spleen cells (ca. 108 total cells)were first incubated with a mixture of beads labeledwith anti-CD8 (Ly-2), CD11b (Mac-1), CD45R (B220),CD49b (DX5), Ter-119 antibodies, antibiotin beads, andCD25-PE, then were loaded onto a MACSVR column,which was placed in the magnetic field of a MACS Sepa-rator. The magnetically labeled non-CD4þ T cells wereretained in the column, whereas the unlabeled CD4þ

T cells ran through. For the isolation of CD4þCD25þ

T cells, the CD25þ PE-labeled cells in the enriched CD4þ

T cell fraction are magnetically labeled with Anti-PEmicrobeads. The cell suspension is again loaded onto acolumn. The magnetically labeled CD4þCD25þ T cellsare retained in the column, whereas the unlabeled cells(CD4þCD25� T cells) ran through. After removal of thecolumn from the magnetic field, the retainedCD4þCD25þ cells (about 106 total cells) were eluted with1-mL buffer by an attached quick plunger, and the cellpurity was analyzed by flow cytometry.

MFC Cell Apoptosis and CellCycle Phase Analyses

The cell subgroups of the in vitro study were designedas follows: group 1: MFC cells þ105 Tregs; group 2: MFCcells only; group 3: MFC cells þ 105 CD4þCD25� T cells.All cells were plated in culture medium containing se-rum into six-well plates (Falcon, Becton Dickinson Lab-ware) at the same time. After 12 hr, MFC cells (3 � 105)became adherent, the cultures were treated with differ-ent melatonin concentrations (0, 2, 4, 6, 8, and 10 mM)for 24 h. Each concentration was examined in duplicate,whereas the whole experiment was performed threetimes. After 24 h, the cells in the three groups were col-lected, washed twice with PBS, and fixed in 70% ethanolovernight at �20�C. Then, the ethanol was removed bycentrifugation. Cell density was adjusted to 5 � 105 cell/mL with PBS buffer, and 500 lL of mixed staining solu-tion was added to the cells for 30 min in the dark. Cellswere analyzed by flow cytometry for cell cycle distribu-tion and detection of apoptosis. The mixed staining solu-tion contained 0.125-g sodium citrate, 0.75-mL Triton X-100, 0.03-g PI, and 0.01-g RNAase in a final volume of250 mL (Sigma).

Statistical Analysis

Data were expressed as mean � SD. Data were ana-lyzed by analysis of variance (ANOVA) by using SPSS13.0 software. Differences in mRNA expression levels indifferent groups were analyzed by one-sample T test.

ANTITUMOR EFFECTS OF MELATONIN AND CD4þCD25þ TREGS 783

Student Newman–Keuls test was used for significanceanalysis and comparison between the groups. Differen-ces were considered statistically significant at the levelof P < 0.05.

RESULTSMelatonin Inhibits the Growth ofExperimented Gastric Cancer In Vivo

The antitumor effects of melatonin have been exten-sively described. In our study, we determined the effec-tive concentrations of melatonin that were required toinhibit the mouse gastric cancer and to induce MFC cellsdeath and apoptosis. Compared with the tumor-bearingcontrol mice (group B, the blank control), the tumorweights of the melatonin (medium and high doses)treated tumor-bearing mice (groups D and E) were sig-nificantly reduced (Fig. 1A). The tumor volumes frommelatonin (low, medium, and high doses)-treated tumor-bearing mice (groups C, D, and E) were also significantlyreduced (Fig. 1B).

Melatonin Reduces CD41CD251 TregsRatio in Gastric Cancer Tissue

To investigate the immunopotentiation activity of mel-atonin in vivo, we tested its effect on the Tregs ratio.The results showed that compared with the blank con-trol group, there were fewer Tregs in the tumor tissuesof high-dose melatonin-treated tumor-bearing mice (P <0.05). However, there were no significant differencesbetween the low- and medium-dose melatonin groups(Fig. 2).

Melatonin Decreases Foxp3 Expressionin Gastric Cancer Tissue

Foxp3 is specifically expressed in Tregs of mice andacts as a master molecule controlling the developmentand function of Tregs, so we also examined the Foxp3expression levels by real-time PCR and Western blot to

Fig. 2. Ratio of CD4þCD25þ Tregs in tumor-bearing control miceand tumor-bearing melatonin treated animals. The upper right quad-rant cells are Tregs in dual-parameter flow cytometric density dot plots(A). Melatonin (100 mg/kg, i.p.) induced a statistically significant

decrease of Tregs in tumor tissue. Results are represented as themean � SD of each independent experiment. Significant differencesfrom the control group are indicated by *P < 0.05.

Fig. 1. Comparison of tumor weight (A) and volume (B) between tu-mor-bearing control mice and tumor-bearing melatonin treated mice.The tumor volumes were calculated as described in Materials andMethods. Data were statistically analyzed with one-way ANOVA, andresults are represented as the mean � SD. Significant differencesfrom the control group are indicated by *P < 0.05.

784 LIU ET AL.

investigate whether they were affected by melatonintreatment, and whether the observed cancer inhibitoryeffects were related to a Foxp3-dependent immunoregu-lation pathway. Compared with the tumor-bearing con-trol mice, the levels of Foxp3 mRNA in high-dosemelatonin-treated tumor-bearing mice was significantlydecreased, whereas the levels of Foxp3 mRNA in low-and medium-dose melatonin-treated groups were not sig-nificantly different (Fig. 4). Compared with the blankcontrol mice, scurfin protein in low-, medium-, and high-dose melatonin-treated tumor-bearing mice was signifi-cantly reduced (P < 0.05), which indicated Foxp3 protein(scurfin) expression were implicated in function of Tregs(Fig. 5).

Role of Tregs in the Melatonin-MediatedInhibition of MFC Cell Growth In Vitro

The spleen CD4þCD25þ Tregs were isolated usingimmune magnetic-activated cell sorting (MACS). TheCD4þCD25þ Tregs (approximately 1% of the cells fromthe mice spleen before sorting) were enriched with a pu-rity reaching 88.4 � 1.2% after magnetic-activated cellsorting. The purity of the CD4þCD25� T cell fractionwas 96.0 � 2.4%. MCF cells were cultured alone (group2), with CD4þCD25þ Tregs (group 1), or with

CD4þCD25� T cells (group 3) and then treated with var-ious concentrations of melatonin (0–10 mM) as describedin Materials and Methods section. The apoptosis rateand cell cycle of the MFC cells were then detected byflow cytometry. As illustrated in Table 1, the apoptosisrates in groups 1 and 2 increased in a dose-dependentmanner beginning with a 6-mM melatonin dose. In

Fig. 3. CD4þCD25þTregs in the tumor-bearing control mice by dou-ble immunofluorescence staining assay. Detection of FITC-CD4 posi-tive cells (A). Detection of PE-CD25 positive cells (B). Merged imageof CD4 and CD25 double positive cells (in yellow) generated by using

the laser confocal scanning microscope software (C). The double posi-tive cells had a diffuse distribution in the tumor tissue, and the fluores-cent signals were distributed in the cytoplasm but not in the nucleus.(400�, bar ¼ 50 lm).

Fig. 4. Expression of Foxp3 mRNA in the tumor tissues of the tu-mor-bearing control mice and tumor-bearing melatonin-treated mice.Foxp3 mRNA expression in mouse tumor tissue. Data were statisti-cally analyzed with One-Sample T test, and results are represented asthe mean � SD. All data were compared with the blank control mice.Significant differences from control group are indicated by *P < 0.05.

ANTITUMOR EFFECTS OF MELATONIN AND CD4þCD25þ TREGS 785

group 3, this increase in apoptosis began at 4-mM mela-tonin. The differences were statistically significant com-pared with the blank control (P < 0.05). Regarding thecell cycle analysis, Table 2 shows that the percentage ofcells in the G1 phase of the cell cycle decreased in allthree groups starting at 4-mM melatonin, whereas thepercentage of cells in the S phase of the cell cycleincreased. The percentage of cells in the G2 phase of thecell cycle decreased at 2-mM melatonin and began toincrease at 6-mM melatonin. These differences werestatistically significant compared with the blank control(P < 0.05). However, the rate of MFC cell apoptosis andchanges in the percentage of cells in different phases ofthe cell cycle were not significantly different betweenthe three cell groups.

DISCUSSION

The pattern and levels of endogenous melatoninexpression in cancer patients and tumor-bearing animalmodels are reportedly abnormal (Bartsch et al., 1991;Kos-Kudla et al., 2002; Cos et al., 2006). Many articles(Mocchegiani et al., 1999; Saez et al., 2005; Sainz et al.,2005; Srinivasan et al., 2008a) reported that pinealec-

tomy accelerates the growth and metastasis of tumors inmost experimental animal models and that exogenousmelatonin inhibits the growth of tumors in human andanimal models. Our results indicate that melatonin hasa significant inhibitory effect on tumor volume thantumor weight. In the control tumor-bearing mice, thetumor capsule was incomplete, and the base of the tu-mor was wide, causing it to not be easily detached. Bycontrast, tumors of melatonin-treated tumor-bearingmice were reduced in size and had complete capsules,which were easy to remove. Therefore, our results con-firmed that melatonin has an antitumor effect, and thatthis effect was dose-dependent. Importantly, we wereinterested in knowing whether the antitumor effect ofmelatonin treatment was related to CD4þCD25þ Tregsactivities. A recent study (Perrone et al., 2008) showedthat the ratio of Tregs in the tumor tissues of gastriccancer was significantly higher than that in normal tis-sues. Using immunohistochemistry, Mizukami et al.(Mizukami et al., 2008) found that the distribution ofFoxp3, a characteristic Treg cell marker, in patientswith gastric cancer was closely related to prognosis. Thediffuse pattern of Foxp3 distribution in the tumor wasassociated with poorer prognosis than a pattern thatsurrounded the cancer. In our study, the results showedthat high-dose melatonin treatment achieved an antitu-mor effect through the downregulation of Tregs in tumortissues. Consistent with the clinical case study men-tioned above, the Tregs in group B (saline control) tumortissues showed a diffuse distribution (Fig. 3) while high-dose melatonin treatment significantly reduced the levelof Foxp3 mRNA in tumor tissues. These resultshighlight the relationship between the antitumor effectof melatonin and Foxp3. Melatonin at a differentdose could also downregulate Foxp3 protein (scurfin)expression. Recently, Lissoni et al.(Lissoni et al., 1989)achieved some beneficial results using a combination oflow-dose IL-2 and melatonin to treat advanced gastriccancer patients, who previously had poor responses tochemotherapy. Meanwhile, a combination of melatoninand a different regimen of chemotherapies resulted in abetter inhibitory effect on gastric cancer and a higher2-year survival rate than chemotherapy alone (Lissoni,2007). Hence, from these previous reports and ourpresent study, melatonin demonstrated antigastriccancer effects in vivo. Our work here suggests that onemechanism of its antitumor effects occurs through thedecrease of CD4þCD25þ Tregs numbers and Foxp3expression in the tumor tissue.

TABLE 1. Effect of different concentrations of melatonin on MFC apoptosis

Groups 1 (MFC þ Tregs) 2 (MFC only) 3 (MFCþCD4þCD25� T cells)

Blank control 2.03 � 3.52 0 0.77 � 1.332-mM melatonin 2.77 � 3.09 0 7.10 � 7.654-mM melatonin 4.23 � 4.20 2.73 � 4.73 10.10 � 8.80*6-mM melatonin 20.27 � 6.62* 14.33 � 5.70* 12.67 � 8.77*8-mM melatonin 26.93 � 12.91* 17.23 � 6.32* 19.50 � 9.82*10-mM melatonin 29.00 � 12.69* 19.03 � 6.19* 22.03 � 10.37*

Ratio of MFC cells apoptosis (%) in the three groups of cells treated with different concentra-tions of melatonin. The data were analyzed using a two-way ANOVA, and results are repre-sented as the mean � SD (n ¼ 3). The rates of MFC apoptosis were not significantly differentbetween the three cell groups.*P < 0.05 compared with the blank control group in each of the three cells groups.

Fig. 5. Expression of scurfin protein in the tumor tissues of the tu-mor-bearing control mice and tumor-bearing melatonin-treated mice.Columns and bars represent densitometric quantification of opticaldensity (OD) of specific protein signal normalized with the OD valuesof b-actin served as a loading control (mean � SD by one-wayANOVA). Significant differences from control group are indicated by *P< 0.05.

786 LIU ET AL.

Furthermore, in this study, we found that melatonintreatment at a concentration of 6 mM caused MFC cellcycle arrest at the G2/M phase and induced apoptosis ina dose-dependent manner. The checkpoint at G2/M isresponsible for determining whether a cell will undergomitosis. The arrest of MFC cells at the G2/M phase bymelatonin induced DNA damage, which led to apoptosis.These results are consistent with the findings reportedby Carbajo-Pescador et al. (Carbajo-Pescador et al.,2009), in which 1 and 2.5-mM melatonin caused theextension of the G2/M phase in HepG2 cells in a time-and dose-dependent manner. While confirming thatmelatonin inhibits the growth of MFC in vitro is impor-tant, the more compelling question to answer is whetherit is related to CD4þCD25þ Tregs. Recent studies (Yuanand Yankner, 2000) have found that melatonin at a con-centration of 10 mM had moderate cytotoxic effects inCMK, Jurkat and MOLT-4 cell lines. The apoptoticeffects of melatonin showed different apoptotic effects innormal cells (Jou et al., 2007) and tumor cells (Wenzelet al., 2005; Martinez-Campa et al., 2008; Bejaranoet al., 2009). In fact, for normal cells such as immunecells and nerve cells, melatonin inhibits apoptosis. Bycontrast, melatonin promotes apoptosis in cancer cells(Sainz et al., 2003). These opposing actions on the nor-mal versus malignant cells are valuable for the potentialuse of melatonin in treatment of tumors.

The purities of the isolated CD4þCD25þ Tregs andCD4þCD25� T cells in this study were sufficiently high.However, we did not find that Tregs could protect gastriccancer cells from melatonin-induced apoptosis in vitro,and the CD4þCD25� T cells did not increase apoptosis ingastric cancer cells. Tregs may be affected by two factorsin this coculture system. First, the cytokines or factorsreleased from the tumor could stimulate the prolifera-tion of Tregs. Liyanage et al. (Liyanage et al., 2002)detected varying effects of different immunogenic tumormicroenvironments on Tregs. By coculturing mousetumor cells with syngeneic spleen cells, they found thatlow immunogenic tumor cells secreted more inhibitorycytokines than those high immunogenic tumor cells,such as IL-10 and TGF-b, which induced proliferation ofTregs and downregulated the tumor immune response.Second, a high concentration of melatonin only hadslight toxic effects on Tregs, whereas it was more toxicto CD4þCD25� T cells. Some studies have reported(Curiel et al., 2004) that Tregs are enhanced and thatCD4þCD25� T cells are selectively reduced in cancerpatients. This discrepancy may be due to a difference inclone screening or a difference in sensitivity to apoptosis.In previous animal studies, Tregs have demonstrated aresistance to apoptosis-induced clonal depletion, whichwas virus superantigen- and Fas-dependent. Duringthe course of human malignant tumor development, thetumor-associated antigens induce apoptosis in theCD4þCD25� T cell subset but not in the Treg population.These scenarios may play out due to the inability ofCD4þCD25� T cell or Tregs to affect the melatonin-induced tumor cell process or the insufficient number ofTregs in vitro.

In summary, melatonin has a potent antigastric cancereffect in vivo, and the effect is associated with downreg-ulation of CD4þCD25þ Tregs and Foxp3 expression inthe tumor tissue. Although we did not observe that puri-fied CD4þCD25þ Tregs could protect gastric cancer cells

TABLE

2.Effectofdifferentconcentrationsofmelato

nin

on

MFC

cell

cycle

Group

1(M

FCþ

Tregs)

2(M

FC

only)

3(M

FCþC

D4þCD25�Tcells)

G1

SG2

G1

SG2

G1

SG2

Blankcontrol

68.4

�0.1

20.4

�1.8

11.3

�1.9

68.4

�5.0

25.7

�5.4

5.8

�4.6

62.3

�5.9

24.9

�7.2

12.1

�5.2

2-m

Mmelatonin

62.9

�10.7

28.6

�5.0*

5.1

�5.9*

72.2

�4.1

24.6

�6.6*

3.2

�2.8*

59.8

�6.4

40.2

�6.4*

0*

4-m

Mmelatonin

57.2

�4.4*

35.0

�4.4*

7.8

�0.1

55.1

�4.3*

36�

2.54*

4.0

�0.3

52.0

�1.1*

41.0

�0.9*

7�

0.28

6-m

Mmelatonin

50.4

�3.9*

34.5

�1.5*

13.0

�1.0*

49.9

�4.4*

31.3

�2.5*

18.9

�3.4*

46.2

�7.9*

41.2

�7.1*

12.5

�5.3*

8-m

Mmelatonin

46.8

�7.6*

38.5

�7*

12.3

�3.9*

49.6

�4.1*

34.1

�1.9*

16.3

�5.2*

48.8

�6.2*

35.5

�5.5*

15.8

�2.8*

10-m

Mmelatonin

47.8

�8.5*

36.8

�8.7*

15.4

�4.8*

46.7

�4.4*

35.1

�3.4*

18.1

�2.1*

48.7

�6.8*

33.9

�7.4*

6.7

�1.6*

Percentageof

MFC

cell

cycle(%

)in

thethreegroupsof

cellstrea

tedwith

differentconcentration

sof

melatonin;data

werestatisticallyanalyzedwith

atw

o-way

ANOVA,andresu

ltsare

representedasthemea

n�

SD

(n¼

3).Changes

inthepercentageof

cellsin

differentphasesof

thecellcyclewerenot

significantlydiffer-

entbetweenthethreecellgroups.

*P<

0.05comparedwiththeblankcontrol

groupin

each

ofthethreecellgroups.

ANTITUMOR EFFECTS OF MELATONIN AND CD4þCD25þ TREGS 787

in vitro, we clearly demonstrated a concentration-depend-ent effect of melatonin-induced apoptosis in the MFC gas-tric cancer cells in vitro. This apoptosis was also related tothe arrest of the cell cycle at the G2/M phase. Together, ourfindings support the immunomodulatory role of Tregs ingastric cancer and indicate that melatonin may inhibit thistumor via inhibition of Tregs. Thus, this study may furtherlead to the development of combination therapies includingmelatonin for gastric cancer.

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