Apelin Over-Expression Promotes Proliferation and Angiogenesis of Gastric Cancer Cells

Background: Apelin is a recently identied endogenous ligand associated with proliferation and angiogenesis of several cancers. However, only few studies have reported on the functions and the role of apelin in gastric cancer (GC). Therefore, in the present study, we investigated the association and the mechanisms underlying Apelin expression and proliferation of GC cells both in vitro and in vivo. Methods: We enrolled 178 postoperative care GC patients to investigate clinicopathological and immunohistochemical factors associated with Apelin expression. The relationship between Survival of patients and apelin expression was evaluated using Kaplan-Meier method and Cox regression analyses. The expression of apelin mRNA and its proteins in GC tissues and cell lines were analyzed using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), western blot and ELISA. The role and mechanisms underlying regulation of Apelin expression in human GC cells were evaluated through several in vitro and in vivo experiments. Results: Apelin was over expressed in human GC cells, relative to adjacent normal tissues. The over expression of apelin was associated with vessel invasion (P <0.01), lymph node metastasis (P <0.01), late-staged tumor (T) (P <0.05), worse pathological type (P <0.05), nerve invasion (P <0.05). In addition, expression of apelin strongly and positively correlated with that of vascular endothelial growth factor (VEGF). Over-expression of apelin promoted proliferation and invasion of MGC-803 cell via the ERK/Cyclin D1/MMP-9 signaling pathway. Apelin over-expression also promoted angiogenesis of GC cells, accelerating growth of subcutaneous xenograft of the cancer cells in vivo. Conclusions: Over-expression of apelin promotes proliferation and metastasis of GC cells via the ERK/Cyclin D1/MMP-9 signaling pathway and is associated with adverse events of the cancer. , saline, horseradish peroxidase, dimethyl Suberoylanilide hydroxamic acid, HDACI: histone deacetylase inhibitor, MAPK: mitogen-activated protein kinase kinase, P13K: phosphoinositide 3-kinase.


Background
Gastric cancer (GC) is the fth most prevalent and aggressive malignancy and the third most fatal cancer globally [1,2]. Despite the recent advances such as radical surgical resection, adjuvant chemoradiation and targeted therapy, the prognosis for GC patients remains poor, with the overall 5-year survival rate only between 20-40% [3][4][5]. Although several studies have identi ed numerous proteins associated with the clinical outcomes of GC patients, none of them has proposed early and accurate diagnostic as well as prognostic molecular marker for GC [6,7].
Apelin is a bioactive peptide recently identi ed as an endogenous ligand that binds APJ, a human G protein-coupled receptor [8]. Apelin, a member of the adipokine family secreted by adipose tissue, is expressed in numerous cell types [9]. The Apelin gene encodes a prepropeptide consisting of 77 amino acids that promotes proteolytic maturation and subsequently generate different bioactive peptide fragments: Apelin-17, Apelin-36 and Apelin-13, all predominant in human plasma [8,9]. Apelin/APJ signaling pathway, mostly associated with the cardiovascular system, participates in numerous physiological and pathological processes such as angiogenesis, heart failure, energy metabolism and cancer progression [9,10]. Dysfunctional and abnormal vasculature plays a critical role in the development and growth of tumors [10][11][12]. Accordingly, abnormal expression of VEGF and its receptor, associated in angiogenesis, have been therapeutic cancer targets for over 30 years [11,12]. Apelin overexpression stimulates proliferation of endothelial cells, enhances tumor vascularization and accelerating formulating capillary tubes demonstrated both in vivo and in vitro [13,14]. In addition, over expression of Apelin is associated with poor clinical outcomes in patients with certain cancers [15,16].
However, the role and molecular mechanisms of Apelin in GC is not well understood. Therefore, we investigated the correlation between Apelin expression and clinical outcomes of human GC and as well the mechanisms underlying Apelin expression and the occurrence of adverse events in GC cells.

Patients
We analyzed histopathologically con rmed GC cancer tissues of 178 patients attending the Binzhou Medical College A liated Hospital between January 2009 and December 2011. The patients underwent radical D2 lymphadenectomy followed by chemotherapy alone or in combination with radiotherapy. All

Immunohistochemical analysis
Immunohistochemical analyses were performed on tumor tissues previously xed in formalin and embedded in para n. The tissue sections (4 µm) were depara nized with xylene and rehydrated in several dilutions of alcohol. Heat-induced antigen retrieval was performed in 0.0l M citrate buffer (PH = 6) for 5 minutes, before heating the slides for 15 minutes at xyz°. The tissue sections were incubated in 3% H 2 O 2 -methanol solution at 37.0°C for 20 minutes to block endogenous peroxidase. Thereafter, the tissues were blocked for 10 minutes in albumen, before overnight incubation at 4°C with anti-Apelin, anti-VEGF or anti-CD34 antibodies at dilution ratios of 1:200, 1:100 or 1:200, respectively (Abcam, the USA). The tissues were stained and counter stained with diaminobenzidine and Mayer's modi ed hematoxylin respectively, and viewed using the EnVision-HRP detection system (Dako, Carpinteria, CA, the USA).
Tissues were then incubated at 37°C for 30 minutes with 80 µL horseradish peroxidase conjugated goat anti-rabbit or rabbit anti-mouse immunoglobulins G (IgG) (1:250) (Dako, Carpinteria, CA, the USA). The tissues were evaluated independently by two investigators, oblivious to the clinical data of the patients. Disagreements were resolved through discussion. Apelin or VEGF immunostaining score was calculated by multiplying the positive cell area score and the staining intensity score. An immunoexpression score > 3 was considered positive, with those ≤ 3 was considered negative. The staining intensity was classi ed into four levels: no staining (0), light staining (1), moderate staining (2) and deep staining (3). The positive cell area score was based on the percentage of positive cells, classi ed as follows: Negative (0), less than 10% positivity (1), 11-50% positivity (2), 51-75% positivity (3) and greater than 76% positivity (4) [15,16]. Microvessel densities (MVD) were established after labeling the capillaries (0.02-0.10 mm) with CD34 (Dako, Carpinteria, CA, the USA). Morphometric analysis of three sections per slide was performed using computer-aided CUE-2 software (Olympus Vanox, Tokyo, Japan) as previously described RNA isolation and quantitative real-time reverse transcription-polymerase chain reaction Total RNA from each human GC cell line was extracted using TRIzol (Invitrogen, Carlsbad, CA, the USA), following the manufacturer's protocol. Corresponding cDNA was synthesized through reverse transcription of the RNA, using the murine leukemia virus. The resultant DNA was ampli ed under the following cyclic conditions through 42 cycles: initial denaturation 95°C for 20 seconds, subsequent denaturations at 95°C for 5 seconds, annealing elongation at 60°C for 30 seconds. PCR products were stained with ethidium bromide, separated through gel (2%) electrophoresis and visualized under UV. The primer sequences for PCR were as follows: Apelin: 5'-GATGCCGCTTCCCGATG-3'(forward) and 5'-ATTCCTTGACCCTCTGGGCT-3'(reverse), β-actin; 5'-TGCTGTCCCTGTATGCCTCT-3' (forward) and 5'-AGGTCTTTACGGATGTCAACG-3' (reverse). The expression levels of mRNA for the above proteins were calculated based on the 2 −ΔΔCt method [18]. Gene expression level of Apelin mRNA in MGC-803 cell line was determined using the same method, 48 hours after transfection with control or Apelin-encoding pc DNA 3.1 vector.
Protein Extraction and Western Blot analysis Cultured cells at logarithmic growth phase were resuspended in lysis buffer containing 5µL Protease Inhibitor Cocktail (cOmplete, Sigma, Germany) before lysis as previously [19]. The concentration of xyz proteins in the supernatants was measured using the Bradford protein assay kit (Bio-Rad, Hercules, CA, the USA). Equal amounts of protein samples (80µg/lane) were fractionated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred onto polyvinylidene uoride (PVDF) membranes at 0.8mA/cm 2 for 20-30 minutes. The membranes were blocked for 2 hours at 37°C with Tris-buffered saline (TBS) containing 10% nonfat milk before overnight incubation at 4°C with primary antibodies against Apelin, β-actin, pERK, pAkt, Cyclin D1 and MMP-9. Membranes were washed four times with TBST and thereafter incubated for 2 hours at 37.0°C with horseradish peroxidase (HRP)-conjugated secondary antibodies. The proteins were visualized after staining with the ECL reagent and thereafter quanti ed using the Quantity One software. The dilutions for the primary antibodies were as follows To assess cell proliferation, cells (1 × 10 4 / well) well were seeded in at-bottomed 96-well microplates and cultured for 24 hours. Then the cells were washed twice using PBS and cultured in DMEM supplemented 0.5% FBS for 12 hours. The cells were treated with MTT (5mg/mL) for 4 hours at 37.0°C after 24 h incubation with different concentrations of Apelin-13 (0.02µmol/L, 0.1µmol/L, 0.5µmol/L, 2.5µmol/L 12.5µmol/L, 62.5 µmol/L), the cells were treated with. Thereafter, 150µL of dimethyl sulfoxide (DMSO) was added to each well to remove cells that were not reduced by MTT reagent. The absorbance of cell supernatants was measured at 570 nm using a microplate reader (MA, USA). The absorbance was also measure at 570 nm using the optimal concentration obtained from the previous experiment at different time points (6h, 12h, 24h, 36h and 48h). The absorbance was also measured at 570 nm at the aforementioned time points to validate change in cell proliferation after transfection with pcDNA 3.1 or pcDNA 3.1-Apelin vector.

Cell Migration and Invasion Analysis
The migration and invasion of cells were evaluated using the Transwell chambers (BD, the USA). Brie y, MGC-803 cells were divided into 4 groups: Non-treatment group, Apelin-13 treatment group, pcDNA 3.1transfection group and pcDNA 3.1-Apelin -trasnfection group. The cells (5 × 10 4 cells/ well) were seeded in the upper chamber pre-coated with serum free Matrigel (BD, the USA). The lower chamber contained 600µL of DMEM. After 24 h of incubation, cells that had not penetrated the membrane were removed.

Construction of MGC-803 cell stably expressing Apelin
To determine the optimal concentration of puromycin (Beijing, China), cells (2×10 4 cells/well) at the logarithmic phase were cultured overnight in 24-well microplates under different puromycin concentrations (0, 1, 2.5, 5, 7.5, 10, 15µg/mL ). We found 7.5µg/mL of puromycin (the lowest concentration) killed all MGC-803 cells after 72 hours of treatment, thus was selected for subsequent experiments. MGC-803 cells were cultured in 24-well plates containing FBS or antibiotic free DMEM. The cells were cultured to 80% con uent. The cells were further incubated at 37°C for 4 hours after addition of a mixture of 2mL polybrene (Beijing. China) ( nal concentration 6µg/mL) and viral supernatant (0.5mL). Thereafter, fresh DMEM was added in each well followed by another 24 h incubation at 37°C. Puromycin (7.5µg/mL) was added to each well after 3 days, followed by x h incubation at 37°C. The medium was changed every 2-3 days until colonies were formed. Negative colonies were removed, whereas the puromycin-resistant colonies were transferred to a 24-well microplate divide to more than half of the dish. mRNA and protein expression of target genes were determined using RT-PCR and Western blot assays, respectively.

Subcutaneous xenografts in vivo
Fourteen nude BALB/c nu/nu, female, 5-6 weeks old mice were purchased from the Laboratory Animal Resources of Chinese Academy of Sciences (Shanghai, China). The mice were raised in pathogen-free conditions. The xenograft tumors were established by subcutaneous injection of 0.2mL MGC-803 cells (1×10 7 /mL) stably expressing Apelin or control into the mice. The long diameter (a), short diameter (b) and weight of the tumors were measured twice a week by the same investigator. The tumor volumes (mm 3 ) was calculated based on V = ab 2 /2. The resultant volumes were used to plot a tumor growth curve.
Mice were euthanized after 35 days of xenograft for further analyses. The tumor proliferation rate was calculated as; (the averaged weight of xenograft tumors in the experiment -the averaged weight of control group)/ the averaged weight of xenograft tumors ×100%.

Immunohistochemistry of MVD in Vivo
Immunohistochemical staining was performed for MVD in murine tumors as described in the previous section.

Statistical Analysis
Data was analyzed using SPSS software, version 16.0 (NY, USA). The association between clinicopathologic factors and Apelin as well as VEGF expression was analyzed using chi-square test. Continuous variables such as MVD were expressed as mean ± S.E.M. Differences between groups were analyzed using Student t-test or nonparametric tests. The association between expression of Apelin and VEGF was analyzed using Spearman's rank correlation test. The association between Apelin and prognosis of GC was assessed using Kaplan-Meier, Cox proportional hazards survival analyses and logrank test. P < 0.05 was considered statistically signi cant.
The tumors were divided into well or moderately differentiated (82 cases), poorly differentiated (75 cases), mucinous adenocarcinoma (17 cases) and signet ring cell carcinomas (2 cases) based on the degree of differentiation, based on the AJCC/UICC TNM classi cation method, revised in 2010. In addition, 5, 98, 75 patients presented with I, II and staged III, respectively, of GC. Immunohistochemical analyses revealed Apelin was expressed in 53 of 178 normal gastric tissues adjacent to tumor tissues and 88 of 178 tumor tissues (29.78% vs 49.44%, P<0.001, Table 1.1). Additionally, immunostaining revealed that in tumor tissues, apelin was mostly distributed in the cytoplasm (Figure 1.1). On the other, VEGF was expressed in 55.62% of tumor cells, compared to 39.89% in adjacent normal tissues (P=0.003, Table 1.1). Similarly, just like apelin, VEGF proteins displayed cytoplasmic distribution (Figure 1.2). Chisquare test revealed that apelin expression was strongly and positively correlated with vessel invasion and lymph node metastasis (P <0.01), (P <0.01) ( Table 1.2). In addition, Apelin expression was associated with late-staged tumor (T), pathological type and nerve invasion (P <0.05). Contrarily, we found no signi cant associations between Apelin expression and gender, age and the site of primary lesion (P>0.05). Meanwhile, VEGF expression was signi cantly high during late T stage (P <0.01), vessel invasion and N stage (P <0.05). However, there was no signi cance difference in VEGF expression between gender, across age groups, site of primary lesion, pathological type and nerve invasion (P >0.05). Meanwhile, the MVD was greater in Apelin positive (33.086±7.862; P <0.05) than Apelin-negative (21.071±6.320) tumor tissues. Similarly, a statistically signi cant difference was observed between the VEGF-positive subgroup with high MVD expression (29.075±8.193) and VEGF-negative subgroup with low MVD expression (19.638±5.614; P <0.05) ( Table 1 Overall, Apelin expression was associated with poor OS and shorter PFS. Univariate Cox proportional hazards analysis revealed that except for gender, age and the site of primary tumor or nerve invasion, Apelin and VEGF expression, T stage, lymph node (N) stage, vessel invasion and histological type (all P <0.05) were all associated with poor prognosis of GC (Table 1.5). Further multivariate regression analysis revealed that lymph node metastasis (P<0.001), late-staged status (P=0.008), poor differentiation (P=0.027) and histological type (including mucinous adenocarcinoma, signet ring cell carcinoma and small cell carcinoma) were independent factors associated with poor prognostic and OS of GC (P=0.012) ( Table 1 Table 3.1). A maximum body weight of the mice and the mean tumor size of 2.96±0.61 g was observed in the mice over-expressing -Apelin, whereas the minimum body weight of the mice and the mean tumor size (1.70±0.43 g) was observed in the control group mice (Figure 3.4). Overall, these ndings suggest that Apelin promotes proliferation of gastric cancer cells both in vitro and in vivo. MVD assay by CD34 immunolabeling assays revealed that was performed to explore the mechanism underlying the differences in neoplastic growth velocity between over-expression of Apelin promotes angiogenesis of cancer cells. Xenograft tumors overexpressing apelin displayed greater MVD (168.833±35.078) relative to controls (112.333±29.859; t=-2.734, P=0.021) (Figure 3.5).

Discussion
Local recurrence and metastasis, the principal factors associated with poor prognosis, are regulated by numerous genes and cytokines [21,22]. The novel relationship between angiogenesis and proliferation, metastasis as well as recurrence of tumors has prompted greater research in various factors associated with angiogenesis. In particular, VEGF signaling pathway is thought to play key roles in tumor angiogenesis. Consequently, VEGF-targeted therapy has resulted in signi cant bene ts into the treatment of several tumors cancers such as lung and colon cancers [23,24]. Previous works have demonstrated that VEGF signaling pathway participates in tumorigenesis and development of GC and over expression of VEGF is associated with poor prognosis of GC [23-25]. However, the clinical bene ts of anti-VEGF drug in human GC have been sub-optimal [25]. Therefore, it is necessary to identify novel biomarker and new vascular therapeutic targets for GC.
Apelin is an endogenous ligand recently thought to regulate angiogenesis [9,13,14,26]. Previous studies have demonstrated dysregulated expression of Apelin in several human solid tumors [13,26]. However, only few studies have evaluated the role of Apelin in tumorigenesis and progression of GC. In our study, we found that VEGF was not only over-expressed in tumor tissues, but also its expression exhibited a strong positive correlation with Apelin. We also found that over expression of Apelin or VEGF predicated poor prognosis of GC. Nevertheless, further studies are needed to evaluate any synergistic relationship between Apelin and VEFG expression in promoting angiogenesis. Feng and colleagues demonstrated that expression of Apelin in tumor tissues, rather than serum, was associated with worse clinicopathological features of tumors. In addition, expression of apelin in tissues is an independent poor prognostic factor for GC [27]. Although we found Apelin was not an independent prognostic factor, multivariate Cox regression analysis revealed that over expression of Apelin was associated with poor prognosis. In addition, the strong correlation between Apelin over-expression and late -T and N stages, as well as vessel invasion suggests that Apelin promotes local recurrence and lymph node metastasis of tumors.
RT-PCR, western blot and ELISA further revealed that both Apelin proteins and its mRNA were under expressed in MGC-803 cells. However, previous works have demonstrated that Apelin proteins and its mRNA are expressed differently in colon adenocarcinomas [28]. A similar observation was made for human non-small lung cancer [15]. MGC-803 cell line is poorly differentiated, HGC-27 cell line is undifferentiated whereas SGC-7901 cell line exhibits metastatic properties. These distinct features have been linked to the differential expression of Apelin in the cells.
In the present study, we used the MGC-803 cell line. Overall, RT-PCR, western blot and ELISA analyses revealed that Apelin was over-expressed in MGC-803 cells transfected with pcDNA3.1-Apelin. The ndings suggested that Apelin expressed in cancer cells function in paracrine and autocrine manner. Furthermore, we found that both over-expressed exogenous and endogenous Apelin substantially modulated the SAHA, a member of histone deacetylase inhibitor, induces apoptosis in MGC-803 cells [20]. To our knowledge, the relationship between overexpression of Apelin and malignant biological behavior of human GC cells both in vitro and in vivo has not been reported. In this study, we found SAHA had no effect onMGC-803 cells transfected with pcDNA3.1-Apelin or treated with exogenous Apelin.
Studies shows that both mitogen-activated protein kinase kinase/(MAPK)/ERK and phosphoinositide 3kinase (PI3K)/Akt signaling pathways regulate the proliferation, migration, angiogenesis and inhibition of apoptosis of tumor cells, including the human GC cells [32,33]. Tumor cells promote ERK phosphorylation, inducing transcription of cyclin D1 that mainly promote growth of tumor cells via the ERK signaling pathway. Cyclin D1 promotes transition of cells from G1 to S phase and proliferation of cells [34]. Both MAPK/ERK and PI3K/Akt signaling pathways regulate invasion and metastasis of cancer cells primarily by up-regulating MMP-9 transcription [35,36]. The apoptosis is inhibited by the expression of Bcl-2 family regulated via the MAPK/ERK and PI3K/Akt signaling pathway or inhibition of the caspase signaling pathway.
Our ndings demonstrated that the expression of phosphorylated ERK (pERK), cyclin D1 associated with cell proliferation and MMP-9 linked to invasion of cancer cells were all over-expressed following Apelin treatment. Strikingly, there was no signi cant difference in the expression of pAkt between treatment and control groups. Apelin/APJ signaling pathway regulates apoptosis of cells through phosphorylation of Apelin [28]. MG132 is a proteasome inhibitor, which participates in inducing apoptosis of cells by downregulating the expression of pAkt in multiple tumor cells [37]. Apelin/APJ modulates apoptosis of colorectal cancer cells by inducing phosphorylation of Akt [28]. Remarkably, exogenous Apelin neither promoted nor modulated the expression of pERK in LoVo cells [28]. Yang et al. reported that Apelin-13 induces cell proliferation and promotes autophagy through phosphorylation of ERK. Phosphorylated ERK intern activates downstream transduction cascades in human lung cancer cell line A549 [29]. Moreover, exogenous Apelin promotes migration and inhibits apoptosis of cancer cells but not proliferation of cancer cells by inducing phosphorylation of ERK and Akt in human lymphatic endothelial cells [31].
On the other hand, Apelin had no effect on apoptosis in vitro induced by SAHA and phosphorylated Akt. There relationship between SAHA and the MAPK/ERK and PI3K/Akt signaling pathways is not straightforward. Apelin regulated apoptosis of GC cells via the MAPK/ERK signaling pathway. Therefore, further studies should be conducted to unravel the relationship between Apelin and SAHA expression. Notably, we observed a positive feedback between Apelin expression and ERK activation. Nevertheless, we did not down-regulate Apelin expression in the HGC-27 GC cell lines for lack of an effective SiRNA.
To the best of our knowledge, this is the rst study to establish human GC cell xenograft models overexpressing Apelin. Overall our ndings suggest that Apelin overexpression promotes growth and angiogenesis of human GC cells in vivo. Apelin is an angiogenic factor that stimulates proliferation and migration of vascular endothelial cells through extrinsic and intrinsic pathways. In addition, Apelin or APJ knockdown e ciently blocks angiogenesis [38]. Sorli et al. revealed that Apelin overexpression increased the formation of tumorous vessels and accelerated tumor growth and tumor neoangiogenesis in paracrine manner [39]. Kidoya et al. demonstrated that the Apelin/APJ signaling pathway regulates maturation and function of tumor vascular system [40]. MVD is the gold standard for estimating tumor angiogenesis, closely associated with tumor invasion and metastasis and is an independent prognosis factor for GC [39]. Berta et al. observed that Apelin overexpression stimulates tumor growth and increase MVD and capillary diameter [15]. Apelin-13 antagonist blocks tumor proliferation or angiogenic activity of hepatocellular carcinoma xenograft model, validating the role of Apelin overexpression in tumourigenesis through angiogenesis [41]. Overall, overexpression of Apelin promotes proliferation of tumor cells, while simultaneously enhancing the MVD.

Conclusion
In general, compared to normal adjacent cells, human gastric cancer tissues over-express apelin.

Consent for publication
All participants consented to this study.

Availability of data and materials
The data used or analyzed in this study are available subject on reasonable request.

Competing interests
The authors declare no con icts of interest.        Expression of Apelin protein in different GC cell lines by Western blot analysis. There was a statistical difference between two cell lines *P 0.05 ** P 0.01

Figure 8
Levels of secreted Apelin protein in different GC cell lines veri ed by ELISA. There was a statistical difference between two cell lines ** P 0.01 ***P 0.001 Figure 9 RT-PCR analysis of the relative expression of Apelin mRNA before and after transfection. When MGC-803 cells were transfected with pcDNA3.1-Apelin, the relative expression of Apelin was signi cantly elevated.
(** P 0.01  The relative expression levels of Apelin mRNA in vivo by RT-PCR analysis. There was a signi cant difference between stable transfected MGC-803 cells of over-expressed Apelin group and control *P 0.05

Figure 19
Page 39/40 The relative expression levels of Apelin-encoding protein by Western blot analysis in vivo. There was a signi cant difference between stable transfected MGC-803 cells of over-expressed Apelin group and control *P 0.05

Figure 20
The tumor growth curves within 24 days in vivo. The subcutaneous over-expressed Apelin and control were separately injected into the nude mice. (*P 0.05 ** P 0.01 Figure 21