Knockdown of RAGE expression inhibits colorectal cancer cell invasion and suppresses angiogenesis in vitro and in vivo

Cancer Letters 313 (2011) 91–98

Contents lists available at SciVerse ScienceDirect

Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Knockdown of RAGE expression inhibits colorectal cancer cell invasion and suppresses angiogenesis in vitro and in vivo Huasheng Liang a,⇑, Yuhua Zhong a, Shaobi Zhou a, Liang Peng b a

Beihai Institute of Endocrine and Metabolic Diseases, Beihai, Guangxi 536000, China Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China

b

a r t i c l e

i n f o

Article history: Received 5 May 2011 Received in revised form 24 August 2011 Accepted 24 August 2011

Keywords: RAGE VEGF Angiogenesis Colorectal cancer

a b s t r a c t The receptor for advanced glycation end-products (RAGE) is a transmembrane receptor in cells, and the interaction of RAGE with ligands results in pro-inflammatory gene activation. Aberrant RAGE activation was reported to promote the pathogenesis of colorectal cancer. This study aimed to investigate the effects of RAGE on the regulation of cell viability, invasion, and angiogenesis, as well as the underlying molecular mechanisms regulating these interactions in colorectal cancer cells. The RAGE mRNA and protein were evaluated in five colorectal cancer cell lines and in 45 cases of colorectal cancer tissue specimens (using immuohistochemistry). RAGE expression was then knockdown using RAGE shRNA for assessing cell viability and invasion assays as well as for tube formation and CAM assays in human umbilical vein endothelial cells and chick embryos, respectively. RAGE was highly expressed in colorectal cancer tissues, and was associated with increased microvessel density. Two of the four RAGE shRNA constructs were able to significantly knockdown RAGE expression in SW480 cells. RAGE knockdown inhibited invasion capacity of SW480 cells, but did not significantly affect cell viability. Furthermore, the conditioned growth medium from stable RAGE shRNA-transfected cells suppressed tube formation of human umbilical vein endothelial cells and angiogenesis of chicken embryos. Knockdown of RAGE inhibited expression of VEGF and SP1 protein in colorectal cancer cells. In summary, these data suggest that silence of RAGE expression could effectively inhibit colorectal cancer angiogenesis in vitro and in vivo. Ó 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The receptor for advanced glycation end-products (RAGE) was originally identified and characterized from bovine lung and found to be present on the surface of endothelial cells where it mediated the binding of AGEs (advanced glycation end-products) [1,2]. RAGE is a 35kD transmembrane receptor of the immunoglobulin super family, and ligand mediated RAGE activation has been

⇑ Corresponding author. E-mail address: fl[email protected] (H. Liang). 0304-3835/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2011.08.028

thought to lead to pro-inflammatory gene activation [1,2]. AGEs are the result of a chain of chemical reactions after an initial glycation reaction by nonenzymatic glycation, oxidation, and further irreversible rearrangements. AGE interaction with RAGE activates cellular signal transduction pathways, such as NF-jB, and Ras-MAPK cascade activation [3] RAGE can also serve as a receptor for amyloid-b peptide, a cleavage product of the b-amyloid precursor protein that accumulates and has been found to play a pathogenic role in diseases [4] such as diabetes, Alzheimer’s disease, and other inflammatory diseases. More recent data suggest that aberrant RAGE activation leads to the pathogenesis of colitis-associated cancer in the colon through carboxylated

92

H. Liang et al. / Cancer Letters 313 (2011) 91–98

glycans-mediated S100A8/A9 and RAGE binding [5]. Furthermore, overexpression of RAGE stimulates tumor cell proliferation and migration, in addition to induction of Erk1/2 phosphorylation and NF-jB activation in colorectal cancer cells [6,7]. In contrast, suppression of RAGE expression inhibits the growth and metastasis of implanted tumors and tumors developing spontaneously in susceptible mice [8]. In addition, RAGE is the only cellular receptor identified for extracellular HMGB1 [8–10], which is associated with growth, migration, and invasion of colorectal cancer cells [9], as well as with metastatic prostate cancer [9,10]. Overexpression of RAGE is associated with prostate cancer development. Blockade of the interaction between HMGB1 and RAGE reduces tumor growth and metastasis [11]. Furthermore, angiogenesis, a physiological process involving the growth of new blood vessels from pre-existing vessels, plays a key role in tumor growth and progression. Targeting of tumor angiogenesis has been shown to be an effective approach to suppress tumor growth and metastasis [12,13] Vascular endothelial growth factor (VEGF) has been demonstrated to be a major contributor to angiogenesis [14,15]. A randomized phase III clinical trial using bevacizumab, a humanized monoclonal antibody against VEGF together with conventional chemotherapy, increased the survival of patients with metastatic colorectal cancer [16]. Inhibition of VEGF expression or VEGF-mediated signaling pathways in endothelial cells can effectively suppress tumor angiogenesis [17,18]. Our research has been focused on novel molecular mechanisms responsible for colorectal carcinogenesis, and we were the first group to link RAGE expression to angiogenesis in the context of colorectal cancer. In this study, we first evaluated RAGE expression and the association with microvessel density in colorectal cancer tissue specimens, and then investigated the effects of RAGE on the regulation of cell viability, invasion, and angiogenesis. We then examined the underlying molecular mechanisms mediated by RAGE in colorectal cancer cells. Our data showed that RAGE expression was associated with increased microvessel density in colorectal cancer tissues and that knockdown of RAGE expression inhibited invasion capacity of SW480 cells, but did not significantly affect cell viability. Moreover, the conditioned growth medium from stable RAGE shRNA-transfected cells suppressed tube formation of human umbilical vein endothelial cells (HUVEC) and angiogenesis of chicken embryos. Knockdown of RAGE reduced VEGF and SP1 protein levels in colorectal cancer cells. These data suggest that targeting of RAGE expression could effectively control colorectal cancer in the future.

2. Materials and methods 2.1. Immuohistochemical staining A protocol for the use of patient samples in this study was approved by our institutional review board, which included paraffin block samples from 45 consecutive patients with colorectal adenocarcinoma who had undergone

surgery without preoperative chemotherapy or radiotherapy between January 2009 and December 2010 at Beihai Institute of Endocrine and Metabolic Diseases, Guangxi, China. For immunohistochemical analysis, the paraffin blocks were dewaxed in xylene and rehydrated with distilled water. The sections were then subsequently incubated with RAGE (1:300, Santa Cruz Biotechnologies) or CD31 (1:400, Miltenyi Biotec, Germany) primary antibodies overnight at 4 °C. The reaction was carried out using a standard ABC system and DAB substrate was used as the chromogen (Maixin Bio, China) following by hematoxylin counterstaining. Microvessel density (MVD) was evaluated by CD31 staining and scoring, i.e., after immunostaining, the sections were first reviewed under a microscope with the low power magnification (40) and selected for three areas with the most intense neovascularization (hot spots) for microvessel counts using high power magnification (200). The data were summarized for the mean microvessel counts from the three most vascularized areas and presented as MVD (microvessels per mm2). 2.2. Cell line and culture Human colorectal cancer SW480, HCT116, SW620, LOVO, and Colo205 cell lines were obtained from The American Type Culture Collection (ATCC; Manassas, VA). HUVECs were also obtained from ATCC. All of these cell lines were cultivated in RPMI 1640 growth medium supplemented with 10% fetal bovine serum and 100 U/ml penicillin and streptomycin (all from Invitrogen, Carlsbad, CA, USA) at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. 2.3. Stable gene transfection Stable RAGE shRNA gene transfection was used to knockdown RAGE expression. Briefly, SW480 cells were trypsinized and resuspended in RPMI 1640 with 10% FBS, but without antibiotics. 3  104 the cells were seeded into 24-well plates and grown overnight. The following day, the cells reached a density of 30–50%, at which time they were transfected with RAGE shRNA expression vectors or the negative shRNA control vector using the lipofectamine™ 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Thirty-six hours later, the cells were cultured in RPMI 1640 medium containing 1200 lg/ml G418 for selection of stable clones. After one and half months of selection culture, stable cells were analyzed for expression of RAGE mRNA and protein using real-time RT-PCR and Western blot analysis, respectively. The positive cell clones were maintained in RPMI 1640 medium containing 10% FBS and 600 lg/ml G418. RAGE shRNA sequences were designed, synthesized, and cloned into the pGPFU/GFP/Neo vectors by Genephmar (Shanghai, China). After the company amplified, sequence-confirmed, and tested these vectors, we purchased them for our experiments. The targeting sequences of RAGE shRNA were: shRNA-1,5-GCTGATCCTCCCTGAGATA-3 (ARGE-879nt); shRAN-2, 5-GCAATGAACAGGAATGGAA3 (ARGE-325nt); shRNA-3, 5-GGCTGGAATGGAAACTGAA-3 (RAGE-167nt); and shRNA-4 5-GAGTATCTGTGAAGGAACA-

H. Liang et al. / Cancer Letters 313 (2011) 91–98

3 (RAGE-533nt). Sequences for the negative control shRNA were 5-GTTCTCCGAACG- TGTCACGT-3.

2.4. Real-time reverse transcription-polymerase chain reaction (qRT-PCR) qRT-PCR was used to detect RAGE mRNA lYYYYYYYYUUUUUUUevels in SW480, HCT116, SW620, LoVo, and Colo205 cell lines. Briefly, total RNA was extracted from cells using Trizol (Invitrogen, UK) and cDNA was synthesized using reverse transcriptase. RAGE mRNA was amplified by real-time PCR with SYBR green (Invitrogen). PCR was initiated by a 5 min denaturation at 95 °C, followed by 40 cycles of 95 °C for 1 min, 60 °C for 1 min and a final elongation of 72 °C for 10 min using an Applied Biosystems 7900HT Sequence Detection System (Foster City, CA, USA). Gene expression was normalized to GAPDH mRNA expression and presented as fold-change compared to the control experiments. The PCR primers used to amplify the RAGE gene [19] were 5-GGAATGGAAAGGAGACCAAG-3 (forward) and 5-CTGGTGCCTAATGAGAAGGG-3 (reverse). GAPDH (a housekeeping gene) primers were 5-GGTATCGTGGAAGGACTCAT-3 (forward) and 5-ACCACCTGGTGGTCAGTGTA-3 (reverse).

2.5. Protein extraction and Western blotting Total cellular protein was isolated form the cells using a lysis buffer containing PMSF and RAPI. 25 lg of protein samples were incubated with denaturing buffer (0.3 M Tris pH 6.8, 10% 2-mercaptoethanol, 40% glycerol, 20% SDS, and 0.02% bromophenol blue) at 95 °C for 5 min, loaded onto a 10% SDS–polyacrylamide gel for electrophoresis, transferred onto PVDF membranes, and then blocked in 5% non-fat milk/TBS-Tween buffer for 1 h at room temperature. Membranes were then incubated overnight at 4 °C in a primary antibody against RAGE (1:500, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), SP1 (1:250, Abcam, USA), VEGF (1:200, Santa Cruz Biotechnologies), or a mouse alpha-tublin antibody as a loading control (1:1000, Cell Signaling Technologies, Danvers, MA, USA). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Immunoreactivity was detected by using chemiluminescence (Pierce, Rockford, IL, USA).

2.6. Cell viability assay We analyzed cell viability in order to assess the effects of RAGE knockdown on colorectal cell phenotype. Briefly, the cells were grown and plated at a density of 4  103 and then treated with different agents for up to 72 h. The cell viability was then analyzed using a CCK-8 assay (Beyotime Institute of Biotech, China) and measured by using a microplate reader at an absorbance of 450 nm. The change in cell viability was calculated as % of control using the following formula: number of the treated cells/number of control culture  100%.

93

2.7. Tubular formation assay To determine the effects of RAGE on angiogenesis, we collected the conditioned medium after 24 h culture of the stable RAGE shRNA-1- and the negative control shRNA-transfected SW480 cells in RPMI 1640 with 10% FBS and 600 lg/ml G418. Conditioned media was filtered using a 0.22 lm sterile Millipore filter, prior to use in the tube formation assay. 4  103 HUVEC cells were seeded onto the Matrigel of pre-coated 96-well culture dishes for 4 h at 37 °C. The conditioned medium was subsequently added and the cells were cultured for 6 h. At the end of experiment, HUVEC branching points and tubes were counted. The experiment was performed in triplicate and repeated four times.

2.8. Chicken embryo CAM assay Fertilized chicken eggs were purchased from Shanghai Poultry Breeding. For the chicken embryo CAM assay, embryonic eggs were incubated at 38.5–39 °C with a relative humidity of 65–70%. Five days later, a 1–2 cm2 window was made on egg shells and the shell membrane was removed to expose the CAM (chicken chorioallantoic membrane). A 6-mm-diameter Whatman filter paper disk was saturated with conditioned medium and was then placed onto the CAM. Filter paper with RPMI 1640 only was used as a negative control. The window was then sealed and eggs were incubated for additional 4 days. At the end of the experiment, the CAM was observed under a stereomicroscope and the neovascularization was quantified. The experiments were performed in triplicate and repeated three times.

2.9. Matrigel invasion assay Stable RAGE shRNA-1 and the negative control shRNAtransfected SW480 cells were grown and then serumstarved for 24 h. Cells were trypsinized, and 4  104 cells were seeded in the upper chamber precoated with 20 lg Matrigel (Transwell from Millipore, Billerica, MA, USA) with 200 ll serum-free medium. The lower chamber containing growth medium plus 10% FBS served as the chemoattractant. After 24–48 h of culture, non-invading cells in the upper chamber were removed with cotton swabs. Invading cells in the lower chamber were fixed with 4% formalin and stained with 0.1% crystal violet. The number of the cells from five microscope fields per filter was counted (magnification at 100). Each experiment was performed in triplicate and repeated at least once.

2.10. Statistical analysis Statistical analysis was performed using SPSS 13.0 software (SPSS, Chicago, IL, USA). The data were summarized as mean ± S.E.M. Differences/correlations between groups were calculated using a Student’s t-, Pearson-, or Wilcoxon-test. A p-value < 0.05 was considered statistically significant.

94

H. Liang et al. / Cancer Letters 313 (2011) 91–98

3. Results 3.1. RAGE expression was associated with induction of microvessel density in colorectal cancer tissues We first analyzed RAGE expression in 45 colorectal cancer tissue specimens using immunohistochemistry and found that 71% (32/45) of tumor tissues expressed RAGE protein. We then evaluated CD31 expression in these tissue samples, which specifically stains blood vessels. The data indicated that the average number of microvessels was increased in tissues positive for RAGE expression compared with RAGE negative tissues (Fig. 1).

3.2. RAGE expression in colorectal cancer cells We next analyzed RAGE expression in colorectal cancer SW480, HCT116, SW620, Lovo, and Colo205 cell lines and found that RAGE protein expression and mRNA levels were highest in SW480 cells (Fig. 2).

3.3. Characterization of stable RAGE-knockdown SW480 cells We stably transfected four different RAGE shRNA vectors and a negative control shRNA vector into SW480 cells. After obtaining stable cell populations, we performed Western blotting to analyze RAGE protein expression, and found that RAGE was reduced in shRNA-3 and shRNA-4 stable cell populations, but not in shRNA-1 and shRNA-2-transfected cells. Furthermore, RAGE expression was not reduced in the negative control vector-transfected cells (Fig. 3).

3.4. Reduced RAGE expression inhibited invasion capacity, but did not affect proliferation of colorectal cancer cells We performed cell viability and invasion assays to evaluate phenotypic changes exhibited by colorectal cancer cells after RAGE knockdown. We found that the RAGE shRNA-4-transfected cells had significantly reduced invasion capacity compared to the negative control shRNA-transfected cells (Fig. 4A; p < 0.01). However, cell viability was not significantly different in RAGE shRNA-4-transfected cells compared to negative control shRNA-transfected cells (Fig. 4B; p > 0.05).

Fig. 2. RAGE expression in different colorectal cancer cell lines. Colorectal cancer SW480, HCT116, SW620, LoVo, and Colo205 cell lines were grown, and total cellular RNA and protein were extracted. They were then subjected to qRT-PCR and Western blot analysis for detection of RAGE mRNA (A) and protein (B) expression, respectively.

Fig. 1. Expression of RAGE protein was associated with microvessel density in colorectal cancer tissue specimens, as detected by immunohistochemistry. 45 cases of colorectal cancer tissue specimens were immunostained with anti-RAGE or anti-CD31 antibody and then reviewed, scored, and summarized in the right side of the figure (p < 0.01). 100 Magnification.

95

H. Liang et al. / Cancer Letters 313 (2011) 91–98

from the RAGE knockdown SW480 cells significantly inhibited HUVEC tube formation, indicating that RAGE and its gene pathway can regulate angiogenesis. Moreover, we found that the conditioned medium from the RAGE knockdown cells inhibited blood vessel formation in chicken embryos. As shown in Fig. 5B, blood vessel formation in chick embryos was inhibited when RAGE is silenced. The avascular area within the 6mm-diameter circular area (about 150 mm2) defined by the Whatman filter paper disk, the number of newly developed blood vessels (black marker) was significantly reduced in the RAGE shRNA-4 group compared to the control.

3.6. Knockdown of RAGE expression reduced VEGF expression through SP1

Fig. 3. Expression of RAGE protein in four different RAGE shRNAtransfected SW480 cells and negative control shRNA-transfected cells. SW480 cells were stably transfected with four different RAGE shRNA constructs and a negative shRNA vector and total cellular protein was extracted and subjected to Western blot analysis. RAGE expression was then normalized to tubulin expression.

3.5. Knockdown of RAGE expression suppressed angiogenesis We collected conditioned media from RAGE shRNA-4-transfected cells and negative control shRNA-transfected cells in order to evaluate HUVEC tube formation. As shown in Fig. 5A, the conditioned medium

To understand the role of RAGE in regulation of angiogenesis, we explored whether the effects of RAGE shRNA were mediated through VEGF modulation. Western blotting data indicated that of RAGE knockdown reduced levels of VEGF protein in the stable RAGE shRNA-transfected cells compared to the negative control shRNA-transfected cells. Since the VEGF gene promoter contains a SP1 binding region, we also analyzed SP1 expression. Western blot analysis revealed that the SP1 protein was decreased in RAGE shRNA-transfected cells (Fig. 6).

4. Discussion Colorectal cancer is a highly invasive and metastatic tumor, and its worldwide mortality rate has increased in recent years [19]. Surgical resection and systemic chemotherapy are the main therapeutic strategies for the management of colorectal cancer. However, these approaches are lack effectiveness and may be associated with significant adverse events [20]. Novel approaches are

A

P<0.01

Cell viability (100%)

B

24h

48h

Fig. 4. Knockdown of RAGE expression inhibited invasion of, but not viability of colorectal cancer cells. (A) Transwell invasion assay. The stable RAGE shRNA-4-transfected SW480 cells or the control cells were seeded in the upper chamber of pre-coated Matrigel 0.8 lm transwell and grown for 24 and 48 h. Number of the invaded cells were counted and summarized ⁄p < 0.01. (B) Cell viability assay. The stable RAGE shRNA-4-transfected SW480 cells or control cells were seeded in 96-well plates and grown for 24 and 48 h and subjected to CCK-8 assay. ⁄p > 0.05; ⁄⁄p > 0.05.

96

H. Liang et al. / Cancer Letters 313 (2011) 91–98

Fig. 5. Knockdown of RAGE expression inhibited angiogenesis. (A) HUVEC tube formation assay. HUVEC (4  103) cells were seeded in 96-well plates that were coated with Matrigel and incubated for 4 h at 37 °C. Conditioned medium from the RAGE knockdown SW480 cells was then added. 48 h later, photomicrographs of tubule formation were taken from the control and the shRNA-4-conditioned media and tubular structures were then counted. ‘‘Branch point cells’’ were averaged and statistically analyzed (p < 0.01, compared to the control cultures). (B) Chicken embryo CAM assay. Chicken embryos were grown in incubator and the control and the shRNA-4-conditioned medium were added into the shell window (see Section 2). Four days later, the CAM was observed under a stereomicroscope and the neovascularization was quantified (p < 0.05, compared to the control embryos). Experiments were performed in triplicate and repeated three times.

Fig. 6. Knockdown of RAGE expression reduced VEGF and SP1 expression. The stable RAGE shRNA-4-transfected SW480 cells and the negative control cells were grown for 5 days with 600 lg/ml G418 and total cellular protein was then extracted and subjected to Western blot analysis to evaluate VEGF and SP1 expression. Compared to the control cells, expression of VEGF and SP1 was decreased in RAGE shRNA-4-transfected SW480 cells (⁄p < 0.01; ⁄⁄p < 0.01).

H. Liang et al. / Cancer Letters 313 (2011) 91–98

needed to effectively control colorectal cancer and the associated mortality, as well as the side effects of chemotherapy. RAGE has been reported to play a role in regulating cancer cell proliferation, invasion, and chemoresistant in many types of cancer [21,22]. Therefore, in the current study, we first determined RAGE expression and evaluated its association with microvessel density in colorectal cancer tissue specimens, and then investigated the effects of RAGE on regulation of cell viability, invasion, and angiogenesis in colorectal cancer cells. We found that colorectal cancer tissues expressed high levels of RAGE protein, which was associated with increased microvessel density. To further elucidate the relationship between RAGE expression and microvessel density, we knocked down RAGE expression in colorectal cancer cells using RAGE shRNA expression vectors. Knockdown of RAGE expression in SW480 cells inhibited tumor cell invasion capacity but did not significantly affect tumor cell viability. Furthermore, the conditioned growth medium from stable RAGE shRNA-transfected cells suppressed tube formation in HUVECs and angiogenesis in chicken embryos. At the molecular level, knockdown of RAGE inhibited expression of VEGF and SP1 protein in colorectal cancer cells. These data demonstrate the usefulness of RAGE knockdown for controlling of colorectal cancer progression. Future studies and potentially clinical trials will further investigate the effects of RAGE knockdown in vivo. Tumor cell invasion promotes tumor progression and metastasis, which is an important and aggressive characteristic of malignancy. Effective control of tumor cell invasion and metastasis will increase patient survival and lead to an improved quality of life. Tumor cell aggressiveness and local environment are required to induce tumor invasion and metastasis. The latter includes a number of factors, such as the extracellular matrix (ECM) and neoangiogenesis. These factors provide a favorable environment for tumor growth and spreading. In the current study, our data demonstrated for the first time that suppression of RAGE expression inhibits colorectal cancer cell invasion, although it did not affect tumor cell viability. The underlying molecular mechanism remains to be defined and further studies are warranted to further verify the effect of RAGE knockdown in other colorectal cancer cells in vitro and in vivo. Angiogenesis is a multistep process that includes cell proliferation, migration, and tube formation and is especially important in tumor progression and metastasis. It has been shown that blockade of any step of this process can inhibit the formation of new blood vessels [23] and in turn suppresses tumor progression and distant metastasis [24]. Our current study showed that the conditioned medium from colorectal cancer cells after RAGE silencing can significantly inhibit HUVEC tube formation and angiogenesis in chicken embryos. These data suggest that RAGE expression regulates angiogenesis-related gene expression and secretion in colorectal cancer cells. Indeed, our data demonstrate that RAGE knockdown inhibited VEGF expression in colorectal cancer cells. Indeed, VEGF plays a key role in physiological and pathological blood vessel formation [12]. During embryonic development, VEGF creates new blood vessel, and in adult

97

tissues VEGF promotes formation of new blood vessels after injury. During cancer development, cancers cells can express VEGF to adjust the need for growth and metastasis since solid tumors cannot grow beyond a limited size without maintenance of an adequate blood supply. Indeed, numerous studies have shown a decreased overall survival and disease-free survival in tumors that over-express VEGF. The overexpression of VEGF is also an early step in the metastatic process. Suppression of VEGF expression decreases tumor angiogenesis and delays tumor progression [14]. Our current data showed that RAGE expression was associated with increased microvessel density in colorectal cancer tissue specimens and that the conditioned medium from colorectal cancer cells with the silence of RAGE expression was able to reduce VEGF expression, suggesting that RAGE plays a role in VEGF expression and blood vessel formation in colorectal cancer. Furthermore, the proximal region of the VEGF gene promoter contains high GC-rich motifs, which can be regulated by the specificity protein 1 (SP1) transcriptional factor [17,25]. We also found that RAGE silencing resulted in suppression of SP1 expression, which corroborates results found in a previous study [2]. Collectively, our current study demonstrated that silencing RAGE alters multiple steps of colorectal cancer angiogenesis, partially through reducing SP1 and VEGF expression. References [1] A. Bierhaus, P.P. Nawroth, Multiple levels of regulation determine the role of the receptor for AGE (RAGE) as common soil in inflammation, Immune responses and diabetes mellitus and its complications, Diabetologia 52 (2009) 2251–2263. [2] S.G. Cho, Z. Yi, X. Pang, T. Yi, Y. Wang, J. Luo, Z. Wu, D. Li, M. Liu, Kisspeptin-10, a KISS1-derived decapeptide, inhibits tumor angiogenesis by suppressing Sp1-mediated VEGF expression and FAK/Rho GTPase activation, Cancer Res. 69 (2009) 7062–7070. [3] J. Li, A.M. Schmidt, Characterization and functional analysis of the promoter of RAGE, the receptor for advanced glycation end products, J. Biol. Chem. 272 (1997) 16498–16506. [4] B. Kuhla, C. Loske, S. Garcia De Arriba, R. Schinzel, J. Huber, G. Munch, Differential effects of ‘‘Advanced glycation endproducts’’ and betaamyloid peptide on glucose utilization and ATP levels in the neuronal cell line SH-SY5Y, J. Neural. Transm. 111 (2004) 427–439. [5] O. Turovskaya, D. Foell, P. Sinha, T. Vogl, R. Newlin, J. Nayak, M. Nguyen, A. Olsson, P.P. Nawroth, A. Bierhaus, N. Varki, M. Kronenberg, H.H. Freeze, G. Srikrishna, RAGE carboxylated glycans and S100A8/A9 play essential roles in colitis-associated carcinogenesis, Carcinogenesis 29 (2008) 2035–2043. [6] M.K. Fuentes, S.S. Nigavekar, T. Arumugam, C.D. Logsdon, A.M. Schmidt, J.C. Park, E.H. Huang, RAGE activation by S100P in colon cancer stimulates growth, migration, and cell signaling pathways, Dis. Colon. Rectum. 50 (2007) 1230–1240. [7] A. Bierhaus, S. Schiekofer, M. Schwaninger, M. Andrassy, P.M. Humpert, J. Chen, M. Hong, T. Luther, T. Henle, I. Kloting, M. Morcos, M. Hofmann, H. Tritschler, B. Weigle, M. Kasper, M. Smith, G. Perry, A.M. Schmidt, D.M. Stern, H.U. Haring, E. Schleicher, P.P. Nawroth, Diabetes-associated sustained activation of the transcription factor nuclear factor-kappa B, Diabetes 50 (2001) 2792–2808. [8] A. Taguchi, D.C. Blood, G. del Toro, A. Canet, D.C. Lee, W. Qu, N. Tanji, Y. Lu, E. Lalla, C. Fu, M.A. Hofmann, T. Kislinger, M. Ingram, A. Lu, H. Tanaka, O. Hori, S. Ogawa, D.M. Stern, A.M. Schmidt, Blockade of RAGE-amphoterin signalling suppresses tumour growth and metastases, Nature 405 (2000) 354–360. [9] H. Kuniyasu, Y. Chihara, H. Kondo, Differential effects between amphoterin and advanced glycation end products on colon cancer cells, Int. J. Cancer 104 (2003) 722–727. [10] H. Kuniyasu, Y. Chihara, H. Kondo, H. Ohmori, R. Ukai, Amphoterin induction in prostatic stromal cells by androgen deprivation is associated with metastatic prostate cancer, Oncol. Rep. 10 (2003) 1863–1868.

98

H. Liang et al. / Cancer Letters 313 (2011) 91–98

[11] H. Kuniyasu, Y. Chihara, T. Takahashi, Co-expression of receptor for advanced glycation end products and the ligand amphoterin associates closely with metastasis of colorectal cancer, Oncol. Rep. 10 (2003) 445–448. [12] P. Carmeliet, VEGF as a key mediator of angiogenesis in cancer, Oncology 69 (2005) 4–10. [13] N.G. de la Torre, H.E. Turner, J.A. Wass, Angiogenesis in prolactinomas: regulation and relationship with tumour behavior, Pituitary 8 (2005) 17–23. [14] H.L. Shen, W.L. Xu, Z.Y. Wu, Y.Y. Qi, X.M. Zhong, W.B. Huang, M. Xiao, Inhibition of vascular endothelial growth factor gene expression and proliferation of leukemia cells by RNA interference, Zhonghua Xue Ye Xue Za Zhi 26 (2005) 710–714. [15] M.P. Barr, D.J. Bouchier-Hayes, J.J. Harmey, Vascular endothelial growth factor is an autocrine survival factor for breast tumour cells under hypoxia, Int. J. Oncol. 32 (2008) 41–48. [16] M.S. Gordon, D. Cunningham, Managing patients treated with bevacizumab combination therapy, Oncology 69 (2005) 25–33. [17] G. Finkenzeller, A. Sparacio, A. Technau, D. Marme, G. Siemeister, Sp1 recognition sites in the proximal promoter of the human vascular endothelial growth factor gene are essential for platelet-derived growth factor-induced gene expression, Oncogene 15 (1997) 669– 676. [18] T. Yi, Z. Yi, S.G. Cho, J. Luo, M.K. Pandey, B.B. Aggarwal, M. Liu, Gambogic acid inhibits angiogenesis and prostate tumor growth by suppressing vascular endothelial growth factor receptor 2 signaling, Cancer Res. 68 (2008) 1843–1850.

[19] J. Weitz, M. Koch, J. Debus, T. Hohler, P.R. Galle, M.W. Buchler, Colorectal cancer, Lancet 365 (2005) 153–165. [20] J. Li, M.W. Saif, Current use and potential role of bevacizumab in the treatment of gastrointestinal cancers, Biologics 3 (2009) 429– 441. [21] R. Abe, Y. Fujita, S. Yamagishi, Angiogenesis and metastasis inhibitors for the treatment of malignant melanoma, Mini. Rev. Med. Chem. 7 (2007) 649–661. [22] K.A. Varker, J.E. Biber, C. Kefauver, R. Jensen, A. Lehman, D. Young, H. Wu, G.B. Lesinski, K. Kendra, H.X. Chen, M.J. Walker, W.E. Carson, 3rd A randomized phase 2 trial of bevacizumab with or without daily low-dose interferon alfa-2b in metastatic malignant melanoma, Ann. Surg. Oncol. 14 (2007) 2367–2376. [23] A. Csiszar, Z. Ungvari, Endothelial dysfunction and vascular inflammation in type 2 diabetes: interaction of AGE/RAGE and TNF-alpha signaling, Am. J. Physiol. Heart Circ. Physiol. 295 (2008) H475–476. [24] C.R. Pradeep, E.S. Sunila, G. Kuttan, Expression of vascular endothelial growth factor (VEGF) and VEGF receptors in tumor angiogenesis and malignancies, Integr. Cancer Ther. 4 (2005) 315– 321. [25] M. Abdelrahim, R. Smith, R. Burghardt 3rd, S. Safe, Role of Sp proteins in regulation of vascular endothelial growth factor expression and proliferation of pancreatic cancer cells, Cancer Res. 64 (2004) 6740– 6749.