We evaluated the involvement of angiotensin II (AngII)-dependent pathways in melanoma growth, through the pharmacological blockage of AT1 receptor by the anti-hypertensive drug losartan (LOS).
We showed immunolabeling for both AngII and the AT1 receptor within the human melanoma microenvironment. Like human melanomas, we showed that murine melanomas also express the AT1 receptor. Growth of murine melanoma, both locally and at distant sites, was limited in mice treated with LOS. The reduction in tumor growth was accompanied by a twofold decrease in tumor-associated microvessel density and by a decrease in CD31 mRNA levels. While no differences were found in the VEGF expression levels in tumors from treated animals, reduction in the expression of the VEGFR1 (Flt-1) at the mRNA and protein levels was observed. We also showed downregulation of mRNA levels of both Flt-4 and its ligand, VEGF-C.
Together, these results show that blockage of AT1 receptor signaling may be a promising anti-tumor strategy, interfering with angiogenesis by decreasing the expression of angiogenic factor receptors.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Price excludes VAT (USA)
Tax calculation will be finalised during checkout.
Kim S, Iwao H (2000) Molecular and cellular mechanisms of angiotensin II-mediated cardiovascular and renal diseases. Pharmacol Rev 52:11–34
Brunner HR, Chang P, Wallach R et al (1972) Angiotensin II vascular receptors: their avidity in relationship to sodium balance, the autonomic nervous system, and hypertension. J Clin Invest 51:58–67
Tamarat R, Silvestre JS, Durie M et al (2002) Angiotensin II angiogenic effect in vivo involves vascular endothelial growth factor- and inflammation-related pathways. Lab Invest 82:747–756
Bell L, Madri JA (1990) Influence of the angiotensin system on endothelial and smooth muscle cell migration. Am J Pathol 137:7–12
Nadal JA, Scicli GM, Carbini LA et al (1999) Angiotensin II and retinal pericytes migration. Biochem Biophys Res Commun 266:382–385
Marshall RP, McAnulty RJ, Laurent GJ (2000) Angiotensin II is mitogenic for human lung fibroblasts via activation of the type 1 receptor. Am J Respir Crit Care Med 161:1999–2004
Otani A, Takagi H, Oh H et al (2000) Angiotensin II-stimulated vascular endothelial growth factor expression in bovine retinal pericytes. Invest Ophthalmol Vis Sci 41:1192–1199
Morrell NW, Upton PD, Kotecha S et al (1999) Angiotensin II activates MAPK and stimulates growth of human pulmonary artery smooth muscle via AT1 receptors. Am J Physiol 277:L440–L448
Sasaki K, Murohara T, Ikeda H et al (2002) Evidence for the importance of angiotensin II type 1 receptor in ischemia-induced angiogenesis. J Clin Invest 109:603–611
Timmermans PB, Wong PC, Chiu AT et al (1993) Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol Rev 45:205–251
Sauter M, Cohen CD, Wornle M et al (2007) ACE inhibitor and AT1-receptor blocker attenuate the production of VEGF in mesothelial cells. Perit Dial Int 27:167–172
Imai N, Hashimoto T, Kihara M et al (2007) Roles for host and tumor angiotensin II type 1 receptor in tumor growth and tumor-associated angiogenesis. Lab Invest 87:189–198
Krishnamoorthy S, Honn KV (2006) Inflammation and disease progression. Cancer Metastasis Rev 25:481–491
Triggle DJ (1995) Angiotensin II receptor antagonism: losartan—sites and mechanisms of action. Clin Ther 17:1005–1030
Gonçalves AR, Fujihara CK, Mattar AL et al (2004) Renal expression of COX-2, ANG II, and AT1 receptor in remnant kidney: strong renoprotection by therapy with losartan and a nonsteroidal anti-inflammatory. Am J Physiol Renal Physiol 286:F945–F954
Fujihara CK, Velho M, Malheiros DM et al (2005) An extremely high dose of losartan affords superior renoprotection in the remnant model. Kidney Int 67:1913–1924
Rivera E, Arrieta O, Guevara P et al (2001) AT1 receptor is present in glioma cells; its blockage reduces the growth of rat glioma. Br J Cancer 85:1396–1399
Fujita M, Hayashi I, Yamashina S et al (2005) Angiotensin type 1a receptor signaling-dependent induction of vascular endothelial growth factor in stroma is relevant to tumor-associated angiogenesis and tumor growth. Carcinogenesis 26:271–279
Lever AF, Hole DJ, Gillis CR et al (1998) Do inhibitors of angiotensin-I-converting enzyme protect against risk of cancer? Lancet 352:179–184
Deshayes F, Nahmias C (2005) Angiotensin receptors: a new role in cancer? Trends Endocrinol Metab 16:293–299
Christian JB, Lapane KL, Hume AL et al (2008) Association of ACE inhibitors and angiotensin receptor blockers with keratinocyte cancer prevention in the randomized VATTC trial. J Natl Cancer Inst 100:1223–1232
Wilop S, von Hobe S, Crysandt M et al (2009) Impact of angiotensin I converting enzyme inhibitors and angiotensin II type 1 receptor blockers on survival in patients with advanced non-small-cell lung cancer undergoing first-line platinum-based chemotherapy. J Cancer Res Clin Oncol 135:1429–1435
de Melo FH, Butera D, Medeiros RS et al (2007) Biological applications of a chimeric probe for the assessment of galectin-3 ligands. J Histochem Cytochem 55:1015–1026
Coutinho EL, Andrade LN, Chammas R et al (2007) Anti-tumor effect of endostatin mediated by retroviral gene transfer in mice bearing renal cell carcinoma. Faseb J 21:3153–3161
Rafii S, Lyden D, Benezra R et al (2002) Vascular and haematopoietic stem cells: novel targets for anti-angiogenesis therapy? Nat Rev Cancer 2:826–835
Dvorak HF (1986) Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. N Engl J Med 315:1650–1659
Ino K, Shibata K, Kajiyama H et al (2006) Angiotensin II type 1 receptor expression in ovarian cancer and its correlation with tumour angiogenesis and patient survival. Br J Cancer 94:552–560
Arrieta O, Pineda-Olvera B, Guevara-Salazar P et al (2008) Expression of AT1 and AT2 angiotensin receptors in astrocytomas is associated with poor prognosis. Br J Cancer 99:160–166
Egami K, Murohara T, Shimada T et al (2003) Role of host angiotensin II type 1 receptor in tumor angiogenesis and growth. J Clin Invest 112:67–75
Shen XZ, Li P, Weiss D et al (2007) Mice with enhanced macrophage angiotensin-converting enzyme are resistant to melanoma. Am J Pathol 170:2122–2134
Seghezzi G, Patel S, Ren CJ et al (1998) Fibroblast growth factor-2 (FGF-2) induces vascular endothelial growth factor (VEGF) expression in the endothelial cells of forming capillaries: an autocrine mechanism contributing to angiogenesis. J Cell Biol 141:1659–1673
Maragoudakis ME, Tsopanoglou NE, Andriopoulou P (2002) Mechanism of thrombin-induced angiogenesis. Biochem Soc Trans 30:173–177
Tsopanoglou NE, Maragoudakis ME (1999) On the mechanism of thrombin-induced angiogenesis. Potentiation of vascular endothelial growth factor activity on endothelial cells by up-regulation of its receptors. J Biol Chem 274:23969–23976
Fong GH, Rossant J, Gertsenstein M et al (1995) Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature 376:66–70
Dumont DJ, Fong GH, Puri MC et al (1995) Vascularization of the mouse embryo: a study of flk-1, tek, tie, and vascular endothelial growth factor expression during development. Dev Dyn 203:80–92
Olsen MW, Ley CD, Junker N et al (2006) Angiopoietin-4 inhibits angiogenesis and reduces interstitial fluid pressure. Neoplasia 8:364–372
Otani A, Takagi H, Oh H et al (2001) Angiotensin II induces expression of the Tie2 receptor ligand, angiopoietin-2, in bovine retinal endothelial cells. Diabetes 50:867–875
Oh SJ, Jeltsch MM, Birkenhager R et al (1997) VEGF and VEGF-C: specific induction of angiogenesis and lymphangiogenesis in the differentiated avian chorioallantoic membrane. Dev Biol 188:96–109
Pepper MS, Mandriota SJ, Jeltsch M et al (1998) Vascular endothelial growth factor (VEGF)-C synergizes with basic fibroblast growth factor and VEGF in the induction of angiogenesis in vitro and alters endothelial cell extracellular proteolytic activity. J Cell Physiol 177:439–452
Witmer AN, van Blijswijk BC, Dai J, Hofman P et al (2001) VEGFR-3 in adult angiogenesis. J Pathol 195:490–497
Thiele W, Sleeman JP (2006) Tumor-induced lymphangiogenesis: a target for cancer therapy? J Biotechnol 124:224–241
Wang L, Cai SR, Zhang CH et al (2008) Effects of angiotensin-converting enzyme inhibitors and angiotensin II type 1 receptor blockers on lymphangiogenesis of gastric cancer in a nude mouse model. Chin Med J (Engl) 121:2167–2171
Boehm T, Folkman J, Browder T et al (1997) Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 390:404–407
Xu L, Zuch CL, Lin YS et al (2008) Pharmacokinetics and safety of bevacizumab administered in combination with cisplatin and paclitaxel in cynomolgus monkeys. Cancer Chemother Pharmacol 61:607–614
Pande A, Lombardo J, Spangenthal E et al (2007) Hypertension secondary to anti-angiogenic therapy: experience with bevacizumab. Anticancer Res 27:3465–3470
Kaplan RN, Riba RD, Zacharoulis S et al (2005) VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature 438:820–827
Garcia AA, Hirte H, Fleming G et al (2008) Phase II clinical trial of bevacizumab and low-dose metronomic oral cyclophosphamide in recurrent ovarian cancer: a trial of the California, Chicago, and Princess Margaret Hospital phase II consortia. J Clin Oncol 26:76–82
Arafat HA, Gong Q, Chipitsyna G et al (2007) Antihypertensives as novel antineoplastics: angiotensin-I-converting enzyme inhibitors and angiotensin II type 1 receptor blockers in pancreatic ductal adenocarcinoma. J Am Coll Surg 204:996–1005
Khakoo AY, Sidman RL, Pasqualini R et al (2008) Does the renin–angiotensin system participate in regulation of human vasculogenesis and angiogenesis? Cancer Res 68:9112–9115
This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP, Grants 1998/14247-6, 2006/60200-0) and Conselho Nacional de Desenvolvimento Científico and Ministério da Saúde/DECIT(CNPq, Grants CNPq/DECIT 401030/05-9, 152083/06-5). We thank Prof. A. Colquhoun, Instituto de Ciências Biomédicas da Universidade de São Paulo for providing us both anti-VEGFR1 and anti-VEGFR2 antibodies.
Conflict of interest statement
The authors declare no conflict of interest.
Electronic supplementary material
Below is the link to the electronic supplementary material.
(A and B) Human tissues labeled with non-immune Ig, as negative control for the reaction with the anti-AT1 R. Arrows in A and B indicate non-stained vessels within the dermis (A) or within a melanoma tissue (B). In C, the anti-AT1 R was used. Note that smooth muscle cells from a large vascular structure stained positively for AT1 R (arrow in C). In D, the interface of a melanoma and the surrounding stroma is depicted. Dashed lines indicate the interface. Small vessels within the stroma were stained with the anti-AT1 R antibodies (arrows in D). Bars indicate 25 μm. (JPG 219 kb)
Tumor tissues from non-treated mice were carefully excised and RNA extraction was performed. RT-PCR reactions for AT1 receptor (control group, n = 8) were done and representative samples were showed. NO represents a PCR reaction run without adding cDNA. Reverse Transcriptase Polymerase Chain Reactions (RT-PCR) for the murine AT1 receptor were performed as follows. cDNA was synthesized using Superscript II RNase H Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) from 1μg of total RNA derived from murine melanomas. PCR reactions were then performed using specific primers which were designed using Primer3 Input (http://frodo.wi.mit.edu/primer3) and synthesized by IDT Inc. (Coralville, IA, USA). Primers for the murine AT1 receptor were; (1) forward primer, 5′-CAA AGC TTG CTG GCA ATG TA-3′; (2) reverse primer, 5′- AAA CAA GGT TCC TTG CCC TT-3′ (amplification product, 401 pb). Primers for the housekeeping gene, β-actin, were; (1) forward primer, 5′-TGT TAC CAA CTG GGA CGA CA-3′; (2) reverse primer, 5′-CTG GGT CAT CTT TTC ACG GT-3′ (amplification product, 139 pb). The amplification protocol consisted of an initial template denaturation step at 95°C for 5 min, followed by 35 cycles of 15 s at 94°C, 30 s at 60°C, and 60 s at 72°C, and a last primer extension at 72°C for 10 min. The reaction mixtures were subsequently analyzed by 2% agarose gel electrophoresis. (JPG 9 kb)
Supplemental Fig. 3. Immunohistochemical pattern of VEGFR1- and VEGFR2-positive vascular structures and VEGFR1- and VEGFR2-positive infiltrating mononuclear cells.
Sections were incubated with anti-VEGFR1 (A-D) and anti-VEGFR2 (E–H) antibodies followed by secondary-antibody/AP incubation, developed with a suitable chromogen (Fast Red, DAKO) and counterstained with Harris hematoxylin. The images were acquired with a Nikon Eclipse E600 microscope coupled to a Nikon DXM1200F capture system. Note the intense immunostaining for VEGFR1 and VEGFR2 displayed in the vessel walls (A and E, respectively) and infiltrating mononuclear cells (C and G, respectively) in the controls, compared to the weak reactivity and rarely positives structures in tumors from LOS-treated mice (anti-VEGFR1-labeled B and D; anti-VEGFR2-labeled, F and H). Scale bar 10 µm, LOS: tumors from losartan-treated mice. (JPG 107 kb)
About this article
Cite this article
Otake, A.H., Mattar, A.L., Freitas, H.C. et al. Inhibition of angiotensin II receptor 1 limits tumor-associated angiogenesis and attenuates growth of murine melanoma. Cancer Chemother Pharmacol 66, 79–87 (2010). https://doi.org/10.1007/s00280-009-1136-0