Gastric cancer is one of the most common human malignant tumors and causes a mortality rate, which ranks second worldwide among malignant tumors (Crew and Neugut,2006). Many studies have shown that the mechanism by which cancer cells use to escape detection by the body's immune system plays a key role in the occurrence and development of malignant tumors. Consequently, the production and development of CD4+CD25+ regulatory T cells (Tregs) are gaining a great deal of attention. CD4+CD25+ Tregs, first reported by Sakaguchi in1995 (Sakaguchi et al.,1995), are activated T cells that express the IL-2 receptor α chain (IL-2Rα, CD25). Moreover, they are known to induce anergy and possess immunosuppressive properties and play an important role in maintaining immune tolerance, protection against autoimmune diseases, as well as inhibiting the transplant rejection response. Foxp3 is a member of the forkhead/winged-helix transcription factor family and encodes a 48 kDa scurfin protein. As a nuclear transcription factor, it is a molecular determinant of differentiation and function of Tregs. Mutation or deletion of the gene encoding Foxp3 causes severe autoimmune diseases in both human and mice, due to a malfunction of CD4+CD25+ Tregs (Hori et al.,2003).
Many of the recent studies focused on changes in Tregs in the tumor microenvironment (Curiel et al.,2004; Ghiringhelli et al.,2005; Enarsson et al.,2006). For example, Tregs are induced to differentiate and proliferate by immature or semimature antigen-presenting cells in the tumor-bearing host and suppress the accumulation of immune effector cells in the tumor local microenvironment. To date, some related studies have shown a functional relationship between gastric cancer and Tregs, and a large number of Tregs are present in the peripheral blood and tumor tissues of many patients with malignancies, such as pancreatic, liver and gastrointestinal cancer (Liyanage et al.,2002; Ichihara et al.,2003; Unitt et al.,2005). They further showed that Tregs harbor antigen-specific properties and inhibit tumor-specific T cells, which accelerate tumor growth and decrease the survival rate of patients. Therefore, inhibiting the function and migration of Tregs to tumor tissues can potentially improve the treatment of human digestive tract tumors.
Melatonin (N-acetyl-5-methoxytryptamine) is mainly secreted from the pineal gland and has many effects on a wide range of physiological functions, such as inhibition of the inflammatory response, regulation of circadian rhythms, and activity of antioxidant enzymes. In particular, melatonin has antitumor effects (Martin et al.; Reiter,2004; Joo and Yoo,2009; Park et al.,2009; Reiter et al.,2009) and is an important immune modulator (Srinivasan et al.,2008b). Regarding the immune system, melatonin has been also localized in the thymus and in mast cells, natural killer (NK) cells, and eosinophilic leukocytes. However, there are few studies on the role of Tregs in melatonin-mediated inhibition of gastric cancer. In this study, we established an in vivo mouse model bearing gastric cancer and an in vitro coculture system of murine foregastric cancer (MFC) cells with Tregs or CD4+CD25−T cells to test the effects of different concentrations of melatonin on tumor/tumor cell growth. We demonstrate that melatonin inhibited the growth of experimental gastric cancer, and CD4+CD25+Tregs played a role in the inhibition of gastric cancer growth by melatonin, our data support melatonin might regulate antitumor immune responses.
MATERIAL AND METHODS
MFC Cell Line Culture
The MFC cells are foregastric carcinoma cells derived from the 615-mouse strain and purchased from the Chinese Academy of Sciences, Shanghai Institute for Biological Science. MFC cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). The cells were maintained at 37°C in a humidified incubator with 5% CO2. The cells were passaged every 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. The body weights of the specific pathogen-free (SPF) grade mice ranged from 20 to 25 g and purchased from Tianjin Institute of Hematology, the Chinese Academy of Medical Science. All animal experiments were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals in Fujian Medical University. All animals were housed in an environmentally controlled room (temperature = 21–22°C) with a 12-hr light/dark cycle (08:00–20:00 hr). Some mice were subcutaneously inoculated with 5 × 104 MFC cells under the right axilla. One week after inoculation, the tumor-bearing mice models were successfully established, and the experimental animal groups were set up as follows: group A: normal control mice (n = 10); group B: tumor-bearing control mice with daily intraperitoneal (i.p.) injection of 100 mg/kg saline water (n = 10); group C: tumor-bearing mice injected with 25 mg/kg (low dose) melatonin (Sigma Chemical Company, St. Louis, Missouri) (n = 10); group D: tumor-bearing mice injected with 50 mg/kg (medium dose) melatonin (n = 17); and group E: tumor-bearing mice injected with 100 mg/kg (high dose) melatonin (n = 14). Before use, the melatonin was dissolved in anhydrous ethanol and combined with normal saline, and the final ethanol concentration was 0.1%. The melatonin solution was kept at 4°C and away from light before use. Melatonin was given to the assigned groups at 17:00 hr every day for 1 week via i.p. injection, after which the tumor tissues were excised and weighed. We, then, measured the long and short diameters of the tumors, and the tumor volume was calculated by using the following formula (Tu et al.,2003): tumor volume V = 4/3π × L/2 × (W/2)2, where L and W are the long and short axes, respectively.
Detection of CD4+CD25+ Tregs in Tumor Tissues
The tumor tissues were macerated with a syringe needle before filtration through a 200 stainless steel mesh. The cells were then rinsed with phosphate-buffered saline (PBS) buffer before double staining with a FITC-conjugated rat anti-mouse CD4 (FITC-CD4) monoclonal antibody and a PE-conjugated rat anti-mouse CD25 (PE-CD25) monoclonal antibody (BD Pharmingen). Stained cells were analyzed by a flow cytometer (XL Beckman Coulter). Control cells were double stained with rat (DA) IgG 2a, κ-FITC and rat (LEW) IgG2b, κ-PE.
The tumor tissues from mice in group B were double stained and immunohistochemically analyzed for CD4+ and CD25+ cells as follows. The tumor tissues were fixed in neutral formalin solution, embedded in conventional paraffin and cut at 8 μm thickness. The sections were rehydrated through decreasing concentrations of ethanol and prepared for antigen retrieval in high pressure for 2 min (pH 6.0 citrate buffer as repair solution). After rinsing with PBS buffer, the specimens were incubated with 3% H2O2 for 10 min, followed by incubation with blocking serum (5% FBS +0.1% triton X-100 + PBS) for 30 min at room temperature (RT). Subsequently, the samples were washed with PBS three times for 5 min each. Then incubated with the primary antibodies (anti-CD4-FITC, anti-CD25-PE, working concentration at 1:100 dilution) at 4°C overnight. After washing with PBS buffer, the slides were sealed and fixed in glycerol. The sections were then observed under a laser confocal scanning microscope (Leica, Germany). For a negative control, PBS was used instead of the primary antibody.
Quantitative Real-Time RT-PCR (Qrt-PCR) Analysis
Total RNA was isolated from tumor tissue using the Trizol reagent (Invitrogen), and the RNA was quantified by measuring the absorbance at 260/280 nm (OD) on a UV spectrophotometer. A total of 2-μg RNA was used in the reverse transcription to generate cDNA, which was then used as a template for the PCR amplification. Quantitative PCR of murine samples was performed with the Brilliant SYBR Green QPCR master mix (Stratagene). The real-time PCR reactions each contained the following: 1 μL of the above reverse transcription product, 1 μL of each primer (5 pmol/μL), 6.7 μL ddH2O, 0.3 μL DYE, and 10 μL 2 × Brillion II SYBR Green PCR buffer. The total reaction volume was 20 μL, and each sample was analyzed in triplicate. After mixing the reaction solution, the tubes were placed into a Quantitative real-time PCR instrument (Applied Biosystems 7500 model) for reaction and detection. The PCR reaction conditions were set as follows: predenaturation at 95°C for 10 min, 40 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec. PCR products were normalized against the housekeeping gene GAPDH, and measurements between samples were compared by the cycle threshold (Ct). Relative gene expressions were calculated by the 2−ΔΔCT method. The Foxp3 and GAPDH primers were as follows: for Foxp3, 5′-CACTGGGCTTCTGGGTATGT-3′ and 5′-AGACAGGCCAGGGGATAGTT-3′; for GAPDH, 5′-CCGAGAATGGGAAGCTTGTC-3′ and 5′-TTCTCGTGGTTCACACCCATC-3′.
Western Blot Analysis
Total protein was extracted from tumor tissues, and the protein concentration was determined using the BCA assay (Sigma). A total of 30 μg of protein was analyzed using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% nonfat milk dissolved in TBS-T buffer (Tris 50 mM; NaCl 1.5%; Tween-20 0.05%; pH 7.5) and then incubated with a specific rat anti-mouse Foxp3 monoclonal antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA) or anti-mouse β-actin (1:4,000, Sigma) overnight at 4°C. After washing, alkaline phosphatase-conjugated affinity purified anti-mouse IgG (Invitrogen) was added and incubated for 40 min. After washing, the chemofluorescence substrate (Invitrogen) was added for exposure on X-ray film. For quantitative analysis, the density of each band was scanned and determined by using a Image-Quant software (Molecular Dynamics, Sunnyvale, CA).
Purification of CD4+CD25+ Tregs and CD4+CD25− T Cells
Spleens from 615 mice (6–8 week old) were each surgically removed, crushed, and passed through a stainless steel mesh. The single cell suspensions were then processed using the Mouse CD4+CD25+ regulatory T cell isolation kit (Miltenyi Biotec, Bergish Gladcach, Germany). Briefly, according to the manufacturer's instructions, the suspension containing spleen cells (ca. 108 total cells) were first incubated with a mixture of beads labeled with anti-CD8 (Ly-2), CD11b (Mac-1), CD45R (B220), CD49b (DX5), Ter-119 antibodies, antibiotin beads, and CD25-PE, then were loaded onto a MACS® column, which was placed in the magnetic field of a MACS Separator. The magnetically labeled non-CD4+ T cells were retained in the column, whereas the unlabeled CD4+ T cells ran through. For the isolation of CD4+CD25+ T cells, the CD25+ PE-labeled cells in the enriched CD4+ T cell fraction are magnetically labeled with Anti-PE microbeads. The cell suspension is again loaded onto a column. The magnetically labeled CD4+CD25+ T cells are retained in the column, whereas the unlabeled cells (CD4+CD25− T cells) ran through. After removal of the column from the magnetic field, the retained CD4+CD25+ cells (about 106 total cells) were eluted with 1-mL buffer by an attached quick plunger, and the cell purity was analyzed by flow cytometry.
MFC Cell Apoptosis and Cell Cycle Phase Analyses
The cell subgroups of the in vitro study were designed as follows: group 1: MFC cells +105 Tregs; group 2: MFC cells only; group 3: MFC cells + 105 CD4+CD25− T cells. All cells were plated in culture medium containing serum into six-well plates (Falcon, Becton Dickinson Labware) at the same time. After 12 hr, MFC cells (3 × 105) became adherent, the cultures were treated with different 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 three times. After 24 h, the cells in the three groups were collected, washed twice with PBS, and fixed in 70% ethanol overnight at −20°C. Then, the ethanol was removed by centrifugation. Cell density was adjusted to 5 × 105 cell/mL with PBS buffer, and 500 μL of mixed staining solution was added to the cells for 30 min in the dark. Cells were analyzed by flow cytometry for cell cycle distribution and detection of apoptosis. The mixed staining solution 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 of 250 mL (Sigma).
Data were expressed as mean ± SD. Data were analyzed by analysis of variance (ANOVA) by using SPSS 13.0 software. Differences in mRNA expression levels in different groups were analyzed by one-sample T test. Student Newman–Keuls test was used for significance analysis and comparison between the groups. Differences were considered statistically significant at the level of P < 0.05.
Melatonin Inhibits the Growth of Experimented Gastric Cancer In Vivo
The antitumor effects of melatonin have been extensively described. In our study, we determined the effective concentrations of melatonin that were required to inhibit the mouse gastric cancer and to induce MFC cells death and apoptosis. Compared with the tumor-bearing control mice (group B, the blank control), the tumor weights of the melatonin (medium and high doses) treated tumor-bearing mice (groups D and E) were significantly reduced (Fig. 1A). The tumor volumes from melatonin (low, medium, and high doses)-treated tumor-bearing mice (groups C, D, and E) were also significantly reduced (Fig. 1B).
Melatonin Reduces CD4+CD25+ Tregs Ratio in Gastric Cancer Tissue
To investigate the immunopotentiation activity of melatonin in vivo, we tested its effect on the Tregs ratio. The results showed that compared with the blank control group, there were fewer Tregs in the tumor tissues of high-dose melatonin-treated tumor-bearing mice (P < 0.05). However, there were no significant differences between the low- and medium-dose melatonin groups (Fig. 2).
Melatonin Decreases Foxp3 Expression in Gastric Cancer Tissue
Foxp3 is specifically expressed in Tregs of mice and acts as a master molecule controlling the development and function of Tregs, so we also examined the Foxp3 expression levels by real-time PCR and Western blot to investigate whether they were affected by melatonin treatment, and whether the observed cancer inhibitory effects were related to a Foxp3-dependent immunoregulation pathway. Compared with the tumor-bearing control mice, the levels of Foxp3 mRNA in high-dose melatonin-treated tumor-bearing mice was significantly decreased, whereas the levels of Foxp3 mRNA in low- and medium-dose melatonin-treated groups were not significantly different (Fig. 4). Compared with the blank control mice, scurfin protein in low-, medium-, and high-dose melatonin-treated tumor-bearing mice was significantly reduced (P < 0.05), which indicated Foxp3 protein (scurfin) expression were implicated in function of Tregs (Fig. 5).
Role of Tregs in the Melatonin-Mediated Inhibition of MFC Cell Growth In Vitro
The spleen CD4+CD25+ Tregs were isolated using immune magnetic-activated cell sorting (MACS). The CD4+CD25+ Tregs (approximately 1% of the cells from the mice spleen before sorting) were enriched with a purity reaching 88.4 ± 1.2% after magnetic-activated cell sorting. The purity of the CD4+CD25− T cell fraction was 96.0 ± 2.4%. MCF cells were cultured alone (group 2), with CD4+CD25+ Tregs (group 1), or with CD4+CD25− T cells (group 3) and then treated with various concentrations of melatonin (0–10 mM) as described in Materials and Methods section. The apoptosis rate and cell cycle of the MFC cells were then detected by flow cytometry. As illustrated in Table 1, the apoptosis rates in groups 1 and 2 increased in a dose-dependent manner beginning with a 6-mM melatonin dose. In group 3, this increase in apoptosis began at 4-mM melatonin. The differences were statistically significant compared with the blank control (P < 0.05). Regarding the cell cycle analysis, Table 2 shows that the percentage of cells in the G1 phase of the cell cycle decreased in all three groups starting at 4-mM melatonin, whereas the percentage of cells in the S phase of the cell cycle increased. The percentage of cells in the G2 phase of the cell cycle decreased at 2-mM melatonin and began to increase at 6-mM melatonin. These differences were statistically significant compared with the blank control (P < 0.05). However, the rate of MFC cell apoptosis and changes in the percentage of cells in different phases of the cell cycle were not significantly different between the three cell groups.
Table 1. Effect of different concentrations of melatonin on MFC apoptosis
1 (MFC + Tregs)
2 (MFC only)
3 (MFC+CD4+CD25− T cells)
Ratio of MFC cells apoptosis (%) in the three groups of cells treated with different concentrations of melatonin. The data were analyzed using a two-way ANOVA, and results are represented as the mean ± SD (n = 3). The rates of MFC apoptosis were not significantly different between the three cell groups.
P < 0.05 compared with the blank control group in each of the three cells groups.
Table 2. Effect of different concentrations of melatonin on MFC cell cycle
1 (MFC+ Tregs)
2 (MFC only)
3 (MFC+CD4+CD25− T cells)
Percentage of MFC cell cycle (%) in the three groups of cells treated with different concentrations of melatonin; data were statistically analyzed with a two-way ANOVA, and results are represented as the mean ± SD (n = 3). Changes in the percentage of cells in different phases of the cell cycle were not significantly different between the three cell groups.
P < 0.05 compared with the blank control group in each of the three cell groups.
The pattern and levels of endogenous melatonin expression in cancer patients and tumor-bearing animal models 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 pinealectomy accelerates the growth and metastasis of tumors in most experimental animal models and that exogenous melatonin inhibits the growth of tumors in human and animal models. Our results indicate that melatonin has a significant inhibitory effect on tumor volume than tumor weight. In the control tumor-bearing mice, the tumor capsule was incomplete, and the base of the tumor was wide, causing it to not be easily detached. By contrast, tumors of melatonin-treated tumor-bearing mice were reduced in size and had complete capsules, which were easy to remove. Therefore, our results confirmed that melatonin has an antitumor effect, and that this effect was dose-dependent. Importantly, we were interested in knowing whether the antitumor effect of melatonin treatment was related to CD4+CD25+ Tregs activities. A recent study (Perrone et al.,2008) showed that the ratio of Tregs in the tumor tissues of gastric cancer was significantly higher than that in normal tissues. Using immunohistochemistry, Mizukami et al. (Mizukami et al.,2008) found that the distribution of Foxp3, a characteristic Treg cell marker, in patients with gastric cancer was closely related to prognosis. The diffuse pattern of Foxp3 distribution in the tumor was associated with poorer prognosis than a pattern that surrounded the cancer. In our study, the results showed that high-dose melatonin treatment achieved an antitumor effect through the downregulation of Tregs in tumor tissues. Consistent with the clinical case study mentioned above, the Tregs in group B (saline control) tumor tissues showed a diffuse distribution (Fig. 3) while high-dose melatonin treatment significantly reduced the level of Foxp3 mRNA in tumor tissues. These results highlight the relationship between the antitumor effect of melatonin and Foxp3. Melatonin at a different dose could also downregulate Foxp3 protein (scurfin) expression. Recently, Lissoni et al.(Lissoni et al.,1989) achieved some beneficial results using a combination of low-dose IL-2 and melatonin to treat advanced gastric cancer patients, who previously had poor responses to chemotherapy. Meanwhile, a combination of melatonin and a different regimen of chemotherapies resulted in a better inhibitory effect on gastric cancer and a higher 2-year survival rate than chemotherapy alone (Lissoni,2007). Hence, from these previous reports and our present study, melatonin demonstrated antigastric cancer effects in vivo. Our work here suggests that one mechanism of its antitumor effects occurs through the decrease of CD4+CD25+ Tregs numbers and Foxp3 expression in the tumor tissue.
Furthermore, in this study, we found that melatonin treatment at a concentration of 6 mM caused MFC cell cycle arrest at the G2/M phase and induced apoptosis in a dose-dependent manner. The checkpoint at G2/M is responsible for determining whether a cell will undergo mitosis. The arrest of MFC cells at the G2/M phase by melatonin induced DNA damage, which led to apoptosis. These results are consistent with the findings reported by Carbajo-Pescador et al. (Carbajo-Pescador et al.,2009), in which 1 and 2.5-mM melatonin caused the extension of the G2/M phase in HepG2 cells in a time- and dose-dependent manner. While confirming that melatonin inhibits the growth of MFC in vitro is important, the more compelling question to answer is whether it is related to CD4+CD25+ Tregs. Recent studies (Yuan and Yankner,2000) have found that melatonin at a concentration of 10 mM had moderate cytotoxic effects in CMK, Jurkat and MOLT-4 cell lines. The apoptotic effects of melatonin showed different apoptotic effects in normal cells (Jou et al.,2007) and tumor cells (Wenzel et al.,2005; Martinez-Campa et al.,2008; Bejarano et al.,2009). In fact, for normal cells such as immune cells and nerve cells, melatonin inhibits apoptosis. By contrast, melatonin promotes apoptosis in cancer cells (Sainz et al.,2003). These opposing actions on the normal versus malignant cells are valuable for the potential use of melatonin in treatment of tumors.
The purities of the isolated CD4+CD25+ Tregs and CD4+CD25− T cells in this study were sufficiently high. However, we did not find that Tregs could protect gastric cancer cells from melatonin-induced apoptosis in vitro, and the CD4+CD25− T cells did not increase apoptosis in gastric cancer cells. Tregs may be affected by two factors in this coculture system. First, the cytokines or factors released from the tumor could stimulate the proliferation of Tregs. Liyanage et al. (Liyanage et al.,2002) detected varying effects of different immunogenic tumor microenvironments on Tregs. By coculturing mouse tumor cells with syngeneic spleen cells, they found that low immunogenic tumor cells secreted more inhibitory cytokines than those high immunogenic tumor cells, such as IL-10 and TGF-β, which induced proliferation of Tregs and downregulated the tumor immune response. Second, a high concentration of melatonin only had slight toxic effects on Tregs, whereas it was more toxic to CD4+CD25− T cells. Some studies have reported (Curiel et al.,2004) that Tregs are enhanced and that CD4+CD25− T cells are selectively reduced in cancer patients. This discrepancy may be due to a difference in clone screening or a difference in sensitivity to apoptosis. In previous animal studies, Tregs have demonstrated a resistance to apoptosis-induced clonal depletion, which was virus superantigen- and Fas-dependent. During the course of human malignant tumor development, the tumor-associated antigens induce apoptosis in the CD4+CD25− T cell subset but not in the Treg population. These scenarios may play out due to the inability of CD4+CD25− T cell or Tregs to affect the melatonin-induced tumor cell process or the insufficient number of Tregs in vitro.
In summary, melatonin has a potent antigastric cancer effect in vivo, and the effect is associated with downregulation of CD4+CD25+ Tregs and Foxp3 expression in the tumor tissue. Although we did not observe that purified CD4+CD25+ Tregs could protect gastric cancer cells in vitro, we clearly demonstrated a concentration-dependent effect of melatonin-induced apoptosis in the MFC gastric cancer cells in vitro. This apoptosis was also related to the arrest of the cell cycle at the G2/M phase. Together, our findings support the immunomodulatory role of Tregs in gastric cancer and indicate that melatonin may inhibit this tumor via inhibition of Tregs. Thus, this study may further lead to the development of combination therapies including melatonin for gastric cancer.