MCF-7 human breast adenocarcinoma cells (ATCC, Manassas, VA, USA) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 units penicillin and 100 µg streptomycin (all from Gibco-BRL, Gaithersburg, MD, USA). The cells were maintained in the growing medium in a humidified 5% CO₂ atmosphere at 37°C.

MCF-7 cells were cultured in 96-well plates (5×10⁴ cells) and were synchronized for 24 h in DMEM containing 0.1% FBS. After treatment with (0.01, 0.1 and 10 µM) apelin-13 or pre-treatment with PD98059 (Sigma, St. Louis, MO, USA) for 1 h followed by the addition of 10 µM apelin-13 for 24 h, the cells were incubated with MTT solution (Invitrogen Life Technologies, Carlsbad, CA, USA) for 4 h at 37°C. Non-reduced MTT was removed by aspiration and the formazan crystals were dissolved in dimethyl sulfoxide (150 µl/well) for 30 min at 37°C. The formazan was spectroscopically quantified at 490 nm using a Bio-Rad Microplate Reader (Bio-Rad, Hercules, CA, USA).

MCF-7 cells in 3 ml of DMEM with 10% FBS were plated in a 6-well plate at a density of 1×10⁶ cells/well and incubated at 37°C with 5% CO₂. After 24 h, the cells were treated with apelin-13 (0.01, 0.1 and 10 µM) in serum-free medium and incubated for 12 h under similar conditions. BrdU (Amresco LLC, Solon, OH, USA) was added to the culture medium 1 h before the end of the 24-h incubation period, cells were collected and each cell sample was subsequently analyzed by flow cytometry.

The invasion of MCF-7 cells was measured using Transwell chambers (BD Biosciences, Franklin Lakes, NJ, USA). After 12 h of serum depletion, MCF-7 cells were detached and resuspended with DMEM containing 0.1% bovine serum albumin (BSA) and added to the upper compartment with 2×10⁵ cells per each Transwell insert. The lower compartment of each well contained 0.5 ml of DMEM with 0.1% BSA with apelin-13 (0.01, 0.1 and 10 µM) or pre-treatment with PD98059 for 1 h followed by the addition of 10 µM apelin-13. After 6 h of incubation, the upper compartment was fixed and stained with 0.1% crystal violet (Zhuo Kang Biological Technology Co., Ltd., Shanghai, China) for 10 min. The number of invasion cells in 10 fields per filter was counted using a microscope (magnification, ×100). Crystal violet was eluted with 33% acetic acid, and the eluent optical density value was measured at 570 nm using the Bio-Rad Microplate Reader.

Total RNA from MCF-7 cells was extracted with TRIzol reagent (Invitrogen Japan K.K., Tokyo, Japan) following the manufacturer's instructions, and subsequently frozen at −20°C in RNase-free water [Takara Biotechnology (Dalian) Co., Ltd., Liaoning, China]. Single-stranded cDNA was reverse transcribed from total RNA (500 ng) using a First Strand cDNA Synthesis kit for RT-PCR [Takara Biotechnology (Dalian) Co., Ltd.] and a random primer. Reverse transcription conditions were 1 cycle of 30°C for 10 min, 45°C for 30 min, 95°C for 5 min, and finally 5°C for 5 min.

cDNA (1 µg) was reverse-transcribed according to the manufacturer's instructions [Takara Biotechnology (Dalian) Co., Ltd.]. The sequences for the sense and antisense primers respectively were: Cyclin D1, 5′-GATGCCAACCTCCTCAACGAC-3′ and 5′-CTCCTCGCACTTCTGTTCCTC-3′ (171 bp); MMP-1, 5′-CCTTCTACCCGGAAGTTGAG-3′ and 5′-TCCGTGTAGCACATTCTGTC-3′ (158 bp); AIB1, 5′-CCAGCTGCACACTCTTCTCA-3′ and 5′-TAAGAAAACCCTGCTGGGGC-3′ (488 bp); and GAPDH, 5′-TCGGAGTCAACGGATTTGGTCGTA-3′ and 5′-TGGCATGGACTGTGGTCATGAGTC-3′ (525 bp). The cyclin D1 and MMP-1 amplifi-cation program consisted of 5 min for initial denaturation at 95°C followed by 35 cycles at 94°C for 30 sec, 60°C for 30 sec, 72°C for 30 sec and 7 min for the final extension at 72°C. The AIB1 and GAPDH amplification program consisted of 5 min for initial denaturation at 95°C followed by 35 cycles at 94°C for 30 sec, 60°C for 30 sec, 72°C for 40 sec and 7 min for the final extension at 72°C. The PCR products underwent electrophoresis in a 1.5% (w/v) agarose gel (Biowest, Barcelona, Spain), with a thickness ~0.5 cm. The gel imaging was scanned and the optical density of the electrophoretic bands was determined using BandScan 5.0 software (Glyko, Novato, CA, USA).

MCF-7 cells were plated at a density of 2×10⁵ cells/well in 96-well plates. The concentrations of MMP-1 and cyclin D1 were quantified by ELISA kits (Invitrogen Life Technologies). The colorimetric reaction was measured using a Microplate Reader (Bio-Rad) at 450 nm wavelength.

To assay the levels of protein expression and phosphorylation of p44/42 ERK_1/2 and β-actin, MCF-7 cells were cultured in 6-well plates and treated with 0.01, 0.1 and 10 µM of recombinant apelin-13 or pre-treatment with PD98059 for 1 h followed by the addition of 10 µM apelin-13 for 12 h. For immunoblotting, cells were washed twice with ice-cold phosphate-buffered saline and subsequently lysed in radioimmunoprecipitation assay buffer with a cocktail of protease inhibitors on ice for 15 min. Following centrifugation at 13,000 × g for 20 min at 4°C, the quantity of protein in the supernatants was detected using the bicinchoninic acid Protein Assay kit (Beijing Solarbio Science & Technology Co., Ltd., Shanghai, China). The protein samples were separated by SDS-PAGE and were transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Following blocking with 5% non-fat milk for 1 h, the membranes were incubated with primary antibodies specific for phospho-p44/42 ERK_1/2 (1:2,000; #4370) and β-actin (1:2,000; #4970) (all from Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C. Blots underwent three 8 min washes with Tris-buffered saline with 0.1% Tween-20 and were incubated with secondary antibodies IgG (1:8,000; #14708; Cell Signaling Technology, Inc.) for 1 h. The membranes were washed as above and the bands were scanned by the Tanon 5500 fully automatic digital gel image analysis system (Tanon Science & Technology Co., Ltd., Shanghai, China).

The results are expressed as the mean ± standard deviation (SD). The mean and SD of each dataset were calculated using the SigmaPlot 10.0 software (Systat Software, San Jose, CA, USA). The differences between groups were assessed using the t-test to determine P-values (P<0.05 was considered to indicate a clear difference, and P<0.01 was considered to indicate a statistically significant difference).

Previous studies have suggested that apelin stimulates cell proliferation and invasion in several cell lines, including endothelial, lung and oral squamous cells (7,9,10). The present study examined the effects of apelin-13 on MCF-7 proliferation and invasion. Using the MTT and flow cytometry assay, the treatment of MCF-7 with apelin-13 was demonstrated to significantly increase cell proliferation (Fig. 1A and B). In order to explore the effect of apelin-13 on cyclins, the expression level of cyclin D1 was detected by ELISA and RT-PCR analysis. The results showed that the protein and mRNA expression levels of cyclin D1 were significantly increased after MCF-7 cells were incubated with apelin-13 for 12 h, suggesting that apelin-13 affects the cell cycle through increasing cyclin D1 expression, and subsequently induces MCF-7 cell proliferation (Fig. 1C and D).

Degradation of the stromal connective tissue and basement membrane components are critical elements in tumor invasion. This is particularly true regarding the interstitial collagens, which were degraded by MMPs. MMP-1 is a member of a family of enzymes that can degrade the majority of ECM macromolecules, particularly types I and III. Therefore, whether apelin-13 could promote MCF-7 cell invasion and MMP-1 expression was investigated. As hypothesized, the results revealed that the quantity of apelin-13-treated cells passing through the basement membrane into the lower compartment was significantly greater compared with that of the control group (Fig. 2A and B). This finding suggests that apelin-13 can promote the invasion of MCF-7 cells. Subsequently, the expression of MMP-1 was examined and the expression of the proteins was closely correlated with apelin-13 (Fig. 2C and D). The results showed that MMP-1 may be critically involved in the metastasis of tumors.

To evaluate the potential signaling pathways involved in apelin-13-induced MCF-7 cell proliferation and invasion, the role of apelin-13 on ERK_1/2 was examined by western blotting. As is shown in Fig. 3A, dose-dependent effects of apelin-13 on ERK_1/2 activation were determined. ERK_1/2 reached maximal phosphorylation upon treatment with apelin-13 at 10 µM. PD98059 (an inhibitor of ERK_1/2) significantly inhibited the overexpression of apelin-13-induced ERK_1/2 phosphorylation (Fig. 3B).

To further evaluate whether ERK_1/2 has any effect on apelin-13-induced MCF-7 cell proliferation, the protein and mRNA expression levels of cyclin D1 were detected. MTT analysis indicated that pre-treatment with PD98059 prevented apelin-13-induced MCF-7 proliferation (Fig. 4A). The data were confirmed by ELISA and RT-PCR. The levels of cyclin D1 expression were decreased following pre-incubation with PD98059 for 1 h (Fig. 4B and C). These results suggested that apelin-13 influenced MCF-7 cell proliferation, possibly via ERK_1/2 signaling cascades.

To address the functional significance of apelin-mediated MCF-7 cell invasion in the context of ERK_1/2 activation, the effect of PD98059 upon MCF-7 cell invasion and MMP-1 was examined. MCF-7 cell invasion and the expression of MMP-1 were reduced by PD98059 (Fig. 5). These results suggested that apelin-13-induced invasion of MCF-7 cells and ERK_1/2 may be involved in the signaling pathway.

AIB1 amplification and overexpression are associated with proliferation, invasion and poor prognosis in breast cancer. Therefore, AIB1 mRNA expression was detected by RT-PCR. Apelin-13 could promote the endogenous levels of AIB1 mRNA in a concentration-dependent manner. Additionally, the molecular mechanism of AIB1 overexpression was observed following treatment with the apelin-13. The result showed that PD98059 inhibited AIB1 expression. Together, these data suggested that apelin-13 promoted AIB1 expression via the ERK_1/2 signal pathway (Fig. 6).