Dig Dis Sci (2008) 53:163–168 DOI 10.1007/s10620-007-9838-9
ORIGINAL PAPER
Angiotensin II Type 1 Receptor Expression in Human Gastric Cancer and Induces MMP2 and MMP9 Expression in MKN-28 Cells Wei Huang Æ Li-Fen Yu Æ Jie Zhong Æ Min-Min Qiao Æ Feng-Xiang Jiang Æ Fang Du Æ Xiang-Long Tian Æ Yun-Lin Wu
Received: 17 November 2006 / Accepted: 30 March 2007 / Published online: 8 May 2007 Springer Science+Business Media, LLC 2007
Abstract Angiotensin II (Ang II), a main effector peptide in the renin–angiotensin system, acts as a growth-promoting and angiogenic factor via angiotensin II receptor1 (AT1R). In this study, we investigated the expression of angiotensin II type1 receptor (AT1R) in gastric cancer and the effects of Ang II on the expression of MMP2 and MMP9 in the human gastric cancer cell line MKN-28 cells. The expression of the Ang II type I receptor was examined by western and immunocytochemistry in gastric cancer cell lines and detected by real-time PCR and immunohistochemistry in normal and gastric cancer tissues. The expression of MMP2 and MMP9 were detected by realtime PCR and western after treatment with Ang II and/or AT1R antagonist. AT1R were expressed in all human gastric cancer lines and the expression of AT1R was significantly higher in cancer tissues than that in normal gastric tissues (P < 0.01). Furthermore, Ang II promoted the expression of MMP2 and MMP9 in MKN-28 cells, and the stimulatory effects of Ang II could be blocked by AT1R antagonist. These results suggest that AT1R is involved in the progression of gastric cancer and may promote the angiogenesis of gastric cancer cell line (MKN28), and these effects may be associated with the upregulation of MMP2 and MMP9.
W. Huang L.-F. Yu J. Zhong M.-M. Qiao F.-X. Jiang X.-L. Tian Y.-L. Wu (&) Department of Gastroenterology, Ruijin Hospital, Jiaotong University School of Medicine, Shanghai 200025, China e-mail:
[email protected] F. Du Department of immunology, Renji Hospital, Jiaotong University School of Medicine, Shanghai 200025, China
Keywords Gastric cancer Angiotensin II type 1 receptor Matrix metalloproteinases
Introduction Angiotensin II (Ang II), a main effector peptide in the renin– angiotensin system (RAS), plays a fundamental role as a vasoconstrictor in controlling cardiovascular function and renal homeostasis. Ang II receptors, primarily of the AT1R subtype, are expressed on tumor and endothelial cells, and are up-regulated in many cancer tissues. AT1R are also expressed at inflammatory sites and up-regulate the levels of VEGF, suggesting a role for Ang II in vascular permeability and cellular infiltration in addition to its role in angiogenesis [1–3]. It is well known that matrix degradation by MMPs is critical for angiogenesis, tumor invasion and metastasis [4]. The importance of degradation of the ECM by matrix metalloproteinase during tumor invasion, metastasis and angiogenesis has long since been established. In gastric cancer, MMP2 and MMP9 play an important role in tumor invasion and metastasis and the extent of MMP2 and MMP9 expression has been shown to correlate with tumor grade and stage [5]. Ang II can activate MMP-2, MMP-9 in VSMCs [6, 7]; however, the role of Ang II on the expression of MMP-2 and MMP9 in gastric cancer has not been studied. Captopril (ACEI) was shown to inhibit the activity of zinc metalloproteinases MMP-2 and MMP-9, which play a major role in matrix degradation and invasion suggesting a direct Ang II-independent effect on MMP activity [8]. Based on these findings, we investigated the expression of AT1R in gastric cancer and the effects of Ang II on the expression of MMP2 and MMP9 in gastric cancer cell line (MKN-28) to explore the molecular mechanism of Ang II in the progression of gastric cancer.
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Materials and methods Reagents and chemicals Angiotensin II and non-peptide AT1 receptor agonist were purchased from Sigma (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide(DMSO). RPMI-1640, fetal bovine serum, penicillin and streptomycin were purchased from GIBCO. For primary antibodies, we obtained rabbit polyclonal AT1R antibody (Santa Cruz), rabbit polyclonal MMP2 antibody (Cell Signaling), rabbit polyclonal MMP9 antibody (Cell Signaling), and mouse monoclonal actin antibody (Sigma), goat anti-rabbit IgG HRP (Santa Cruz).
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used PBS instead of the primary antibody. Study specimens were evaluated independently by two investigators and reviewed by a pathologist. Angiotensin II and non-peptide AT1 receptor agonist treatment We investigated the effects of the angiotensin II and nonpeptide AT1 receptor agonist on the expression of MMP2 and MMP9 in MKN-28. For experiments, cells were plated onto 100-mm dishes at 5 · 105 cells/dish, allowed to growth to confluency (5–7 days), and then changed to serum-free RPMI-1640 for 24 h prior to experiments. After treatment the cells were harvested.
Cell lines and tissue specimens RNA purification and RT-PCR Four human gastric cancer cell lines (SGC-7901, AGS, MKN-28, MKN-45) were preserved in our laboratory and maintained in RPMI 1640 with 10% FBS. Pancreatic cancer cell line (Panc-1) and human leukemia cell line (HL-60) were used as a positive and negative control, respectively. About 23 gastric cancer specimens and 20 normal gastric mucosa specimens were collected from routine upper gastrointestinal endoscopy in our hospital from 2005 to 2006. None of the patients had received preoperative radiotherapy or chemotherapy. The gastric cancer patients’ sex, age, tumor size, histological type of the neoplasm, and TNM stage were obtained from surgical and pathological records. Immunocytochemistry and immunohistochemistry For immunocytochemistry, gastric cancer cells were grown on glass cover slips and the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min. The cells were washed and incubated for 1 h with the primary antibody at a 1:250 dilution. After washing with PBS, they were incubated for 1 h with FITC labeled anti-rabbit IgG. The cover slips were washed with PBS, mounted on glass slides and viewed with a confocal laser scanning microscope (Leica-laser-technik GmbH, Germany). Gastric tissue specimens were fixed in 10% formalin and paraffin embedded by conventional techniques. Freshly cut 4-lm sections were deparaffinized in xylene, and the slides were bathed in 0.01 mol/l sodium citrate and heated in a microwave oven for 12 min. After microwave treatment, endogenous peroxidase activity and non-specific binding was suppressed, and the sections were incubated with a rabbit polyclonal AT1R antibody (1:100) overnight at 4C. For the secondary developing reagents, a labeled streptavidin-biotin kit (DAKO, CA, USA) was used. Slides were developed with diaminobenzaminidine and counterstained with hematoxylin. For non-immune staining, we
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Total cellular RNA was extracted with Rneasy mini kit and was reverse-transcribed into cDNA using the Superscript RT kit (Invitrogen, Grand Island, NY). RNA from gastric tissues was extracted from gastric tissues using Trizol reagents (Invitrogen, Grand Island, NY) and detected by quantitative real-time PCR. The PCR reactions were performed in a 10 ll reaction mixture with two gene-specific primers. The primer sequences of MMP2, MMP9 and GAPDH were as follows: MMP2: 5¢-TACTGAGTGGCCGTGTTTGC-3¢(sense), 5¢-AGGGAGCAGAGATTCGGACTT-3¢(antisense); MMP9: 5¢-CAGTACCGAGAGA AAGCCTATTTCTG-3¢(sense), 5¢-TAGGTCACGTAGCCCACTTGGT-3¢(antisense); GAPDH: 5¢-GAAGGTGAA GGTCGGAGTC-3¢(sense), 5¢-GAAGATGGTGATGGGATTTC-3¢(antisense); sybrgreen mix 5 ll, cDNA 0.5 ll. The cycling conditions were: 50 C for 2 min, 95 C for 10 min, 40 cycles of 95 C for 15 s, 60 C for 1 min, and 1 cycle of 95 C for 15 s, 60 C for 15 s, 95 C for 15 s. All the reactions were performed with two negative controls for NES1 mRNA expression. Following the protocol of the manufacturer, the amount of NES1 expression, normalized to a human GAPDH endogenous reference is given by: 2–DDCT. QRT-PCR was repeated at least three times for each specimen, and mean was obtained. Western blot Cell lysates were made by standard methods. The protein concentration of each sample was measured using a BCA kit. For SDS-PAGE, 20 lg of protein from each sample was loaded on 10% polyacrylamide gels. Proteins were transferred to a polyvinylidene difluoride membrane with a tank transfer system (Bio-Rad Laboratory), then blocked with buffer containing 5% low fat skim milk and 0.1% Tween-20 in Tris-buffered saline (TBST) at room temperature for 1 h. All primary antibodies were diluted in
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TBST containing 5% skim milk. The membrane was incubated with primary antibody overnight at 4 C. After washing three times with TBST, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody (0.02 lg/ml in TBST) for 1 h at room temperature. Detection of chemiluminescence was performed with ECL Western blot detection kits (Amersham, Little Chalfont, UK) according to the supplier’s recommendation. Statistical analysis Statistical analyses were performed using SPSS11.0 software. The data are expressed as median (range). Wilcoxon signed rank test was used to analyze the AT1R expression in paired cancer specimens and corresponding normal specimens. P < 0.05 was considered statistically significant.
Results AT1 receptor expression in human gastric cancer cell lines We investigated the role of AT1R in gastric cancer cell lines. As shown in Fig. 1, AT1R proteins were observed in all gastric cell lines (AGS, MKN-45, MKN-28, SGC7901). And then we examined the localization of AT1R protein in these cells using immunofluorescence staining. Immunoreactive green signals were detected in the cell membrane and cytoplasm of these cells (Fig. 2). No nuclear staining was observed. It is reported that the AT1Rs are internalized within the cell and recycled to the cell surface. AT1 receptor expression in gastric cancer tissues The AT1R mRNA expression in GC tissue and the corresponding normal mucosa are shown in Fig. 3. The median AT1R/GAPDH of 23 cases of GC tissue was 2.301, which was much higher than that of the corresponding normal gastric tissue 0.180 (P < 0.01). The AT1R protein expression of 23 cases of CRC specimens and corresponding normal specimens were evaluated by immunoHL-60
AGS
MKN-45
Fig. 2 Immunocytochemical staining of AT1R in gastric cancer cell lines. Cells were grown on cover slips, fixed and stained with a FITClabeled anti-AT1 receptor antibody. Immunostaining was predominantly in the plasma membrane. The picture shows representative data in MKN28 and MKN-45 cells. Similar findings were observed in other gastric cancer cell lines
histochemistry. A negative or very weak immunostaining for AT1R was observed in gastric mucosal sections. In contrast, gastric cancer cells showed a variety of immunoreactivity in the cells (Fig. 4). These findings suggested that AT1R was up-regulated in gastric cancer. Angiotensin II induced MMP2 and MMP9 mRNA and protein expression in a dose dependent manner in MKN-28 cells. Total RNA and protein were isolated from cells that were treated with Ang II (10–10–10–7 mol/l) for 3 h. The levels of MMP2 and MMP9 expression were measured by QRT-PCR and Western blotting (Fig. 5A, B). Ang II induced MMP2 and MMP9 expression in dose-dependent manner which peaked 10–7 mol/l. The stimulatory effect of Ang II could be block by AT1R antagonist Fig. 6 Angiotensin II induced MMP2 and MMP9 mRNA and protein expression in a time-dependent manner in MKN-28 cells. To test the effects of Ang II on MMP mRNA and protein expression, MKN-28 were exposed to Ang II (10– 7 mol/l) for various periods of time. MMP-2 and MMP-9 levels were subsequently measured by Real-time PCR and Western Blotting. Ang II (10–7 mol/l) stimulated MMP2 and MMP9 expression in a time-dependent manner in MKN-28. MMP9 protein was also enhanced by Ang II (10–7 mol/l) but the time-dependent manner is not very apparent. All of the above findings suggested that an Ang
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Fig. 1 Western blot analysis of AT1 receptor expression in gastric cancer cell lines. All gastric cancer cell lines expression of AT1
receptor. A pancreatic cancer cell line (Panc-1) was used as positively control, and an HL-60 leukemia cell line was used as negative control
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Fig. 3 Relative AT1R expression levels in gastric cancer detected by quantitative RT-PCR in tumor tissue and corresponding normal tissues
Fig. 4 Expression of AT1R protein in paraffin-embedded specimens of (a) colorectal cancer tissue; (b) normal colorectal mucosa. Sectioned tissues were stained with an anti-AT1R antibody. Strong staining of AT1R protein was observed with brown in carcinoma cells, but negative or weak staining was observed in corresponding normal mucosal cells. Original magnification ·400
II-AT1 receptor be involved in the induce expression of MMP-2 and MMP-9.
Discussion Recent studies have shown the activation of the local RAS in various tumor tissues, including the abundant generation of Ang II by angiotensin-converting enzyme (ACE) and the upregulation of AT1R expression. Thus, considerable attention has been paid to the role of the RAS in cancer and its blockade as a new approach to the treatment of cancer. It has been reported that AT1R was expressed and upregulated in various human malignant tumor tissues, including breast cancer, skin squamous cell carcinoma, pancreatic cancer, laryngeal carcinoma , prostate cancer, cervical carcinoma and ovarian cancer et al. [9–15]. However, there have been no reports analysing the expression of AT1R in human gastric cancers. Evidence for the involvement of AT1R in tumor progression, such as growth, metastasis and angiogenesis, has accumulated in various animal models
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[16, 17]. Our results show that AT1R mRNA and protein was up-regulated in gastric cancer compared with normal gastric tissues, suggesting AT1R might play an important role in the progression of gastric cancer. Angiogenesis is essential for the growth and metastasis of solid tumors. Matrix metalloproteases have been implicated in gastric carcinogenesis by their ability to degrade extracellular matrix proteins, thereby facilitating tumor cell proliferation, invasion and metastasis. In gastric cancer MMP-2 and MMP-9 are linked to tumor invasion and metastasis as well as poor prognosis. Ang II is generated not only by ACE, but also by other enzymes, including mast cell chymase. Keisaku Kondo [18] recently reported that expression of chymase-positive cells in gastric canceris correlated with the angiogenesis and chymase-induced angiogenesis via activation of Ang II which could be significantly inhibited by an angiotensin converting enzyme inhibitor (ACEI) suggesting the role of Ang II play in the angiogenesis of gastric cancer. Ang II can activates MMP-2, MMP-9 in VSMC, however the effects of Ang II on the expression of MMP-2 and MMP9 in human cancer have not been studied. Our study is the first study to investigate the expression of AT1R in human gastric cancer and to explore the role of Ang II and AT1R antagonist on the expression of MMP2 and MMP9 in gastric cell line. Our study show that Ang II can up-regulate the expression of MMP2 and MMP9 in MKN-28 cells and these stimulatory effects of Ang II can be black by AT1R antagonist. Thus, as AT1R is overexpressed in gastric cancer, it is not unreasonable to speculate that it might be involved in the promotion of metastasis, directly of indirectly by activation the expression of MMP2 and MMP9. Although the underlying biological mechanism of Ang II involvement in the progression of gastric cancer is currently unknown, it is plausible that these effects may be associated with the upregulation of MMP2 and MMP9. Mast cell chymase is involved in local generation of Ang II from AngI in an ACE-independent manner. Therefore, the use of ACE inhibitor alone could not completely block the Ang II-mediated effects on tumor cells in vivo. As the tumor-
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Fig. 5 Angiotensin II induced MMP2 and MMP9 mRNA and protein expression in a dose dependent manner in MKN-28 cells. (A) Representative quantative real-time PCR results. (B) Representative western blotting results. Cells were harvested after being incubated withangiotensin II and/or its receptors antagonists for 3 h. Lanes 1–5
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show cells incubated angiotensin II at 0 mol/l, 10–10 mol/l, 10–9 mol/l, 10–8 mol/l, 10–7 mol/l, respectively; lane 6 shows cell pretreated with AT1 receptor antagonist 10–6 mol/l pior to stimulation with angiotensin II 10–7 mol/l. The data are shown as mean ± SD
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Fig. 6 Angiotensin II induced MMP2 and MMP9 mRNA and protein expression in a time-dependent manner in MKN-28 cells. (A) Representative quantative real-time PCR results. Cells were harvested after being incubated with angiotensin II (10–7 mol/l) for 0 min (lane1); 30 min (lane2); 1 h (lane3); 2 h (lane4); 3 h (lane5); 6 h
(lane6); The data are shown as mean ± SD. (B) Representative western blotting results. Cells were harvested after being incubated with angiotensin II (10–7 mol/l) for 0 h (lane1); 6 h (lane2); 12 (lane3); 24 h (lane 4); 48 h (lane5)
stimulatory effects of Ang II on most types of tumors are mediated through AT1R, selective AT1R blockade may be more advantageous than ACE inhibition. Further studies to determine the mechanisms associated with growth inhibition by the AT1R antagonist are needed to understand this process.
References
Acknowledgments The authors thank Dr. Fang Du for the technical assistance. This study was supported by a Grant-in-Aid for the Ministry of Education.
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