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Inhibition of angiotensin II receptor 1 limits tumor-associated angiogenesis and attenuates growth of murine melanoma

Abstract

Purpose

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).

Results

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.

Conclusions

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.

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Abbreviations

AngII:

Angiotensin II

LOS:

Losartan

MVD:

Microvascular density

RAS:

Renin–angiotensin system

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Acknowledgments

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.

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Correspondence to Roger Chammas.

Electronic supplementary material

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Supplemental Fig. 1. Immunolabeling of AT1 receptor in human melanoma.

(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)

Supplemental Fig. 2. Murine melanomas also express the AT1 receptor.

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)

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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

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Keywords

  • Melanoma
  • Angiogenesis
  • AT1 receptors
  • Angiotensin II
  • Losartan