Elsevier

European Journal of Pharmacology

Volume 781, 15 June 2016, Pages 60-68
European Journal of Pharmacology

Molecular and cellular pharmacology
Quercetin inhibits angiogenesis by targeting calcineurin in the xenograft model of human breast cancer

https://doi.org/10.1016/j.ejphar.2016.03.063 Get rights and content

Abstract

Vascular endothelial growth factor receptor 2 (VEGFR2) mediated calcineurin/nuclear factor of activated T-cells (NFAT) pathway is crucial in the angiogenesis of human breast cancer. Quercetin (Qu), a flavonoid known to possess anti-angiogenesis and antitumor properties, inhibited calcineurin activity in vitro. Herein, we performed a study in vivo to evaluate the effects of Qu on the angiogenesis in breast cancer. Female BALB/c nude mice were injected with MCF-7 cells into the mammary fat and were randomly divided into four groups. The animals were treated with vehicle solution, tamoxifen (TAM, 5.6 mg/kg), tacrolimus (FK506, 3 mg/kg), or Qu (34 mg/kg) for 21 days, respectively. The results showed that, similar to TAM and FK506, Qu decreased tumor growth, limited oncocyte proliferation and promoted tumor necrosis. Anti-angiogenic actions of Qu were demonstrated as decreased serum VEGF (P<0.01), and sparse microvessel density (P<0.05). Qu significantly inhibited tumor calcineurin activities, and the inhibitory rate was 62.73% in Qu treated animals, compared to that was 72.90% in FK506 group (P>0.05). Effects of Qu on calcineurin/NFAT pathway were confirmed as decreased subcellular located levels of VEGF (P<0.05), VEGFR2 (P<0.05) and NFATc3 (P<0.01), downregulated gene expression of VEGF (P<0.05), VEGFR2 (P<0.05) and NFATc3 (P<0.01), reduced protein levels of VEGF (P<0.05), VEGFR2 (P<0.05), and NFATc3 (P<0.01) in tumor tissues. These findings indicate that Qu inhibit angiogenesis of human breast cancer xenograft in nude mice, which was associated with suppressing calcineurin activity and its regulated pathway activation.

Introduction

Breast cancer is the most common malignant tumor in women (DeSantis et al., 2014). Angiogenesis plays an essential role in the development, invasion, and metastasis of breast cancer (Schneider and Miller, 2005). Developing novel anti-angiogenic agents is an important strategy for treating patients in clinic.

Vascular endothelial growth factor receptor 2 (VEGFR2) mediated calcineurin/nuclear factor of activated T-cells (NFAT) pathway plays leading roles in the angiogenesis of breast cancer (Hoeben et al., 2004). Calcineurin is a target enzyme in this pathway (Mancini and Toker, 2009). In endothelia, VEGFR2 activates calcineurin, triggers NFAT translocated into nucleus, and up-regulates angiogenic factors (Muller and Rao, 2010). Tacrolimus (FK506) is a calcineurin inhibitor, which blocks calcineurin/NFAT pathway, and consequently suppresses angiogenesis (Mancini and Toker, 2009, Muller and Rao, 2010). FK506 also inhibits breast tumor growth in vivo, and reduces vessel formation of human umbilical vein endothelial cells (HUVEC) in vitro (Siamakpour-Reihani et al., 2011). However, FK506 has not been used in clinic for cancer owing to its immunosuppressive effect, so the novel inhibitors targeting calcineurin with minor adverse effect might provide a major advantage in treating breast cancer.

Quercetin (Qu), a flavonoid abundant in fruits, plants, and vegetables, is characterized by a phenyl benzo(c)pyrone-derived structure (Nabavi et al., 2015). It is present in plants in many different glycosidic forms, with the most widespread form of quercetin-3-rutinoside (Erlund, 2004). It has exhibited antioxidant, cardioprotective (Nabavi et al., 2012b, Nabavi et al., 2012a), vasodilatory (Larson et al., 2012), live-protective (Nabavi et al., 2012a, Nabavi et al., 2012b), anti-inflammatory, and anti-cancer activities (Nabavi et al., 2015), with almost no human toxicity. Its anti-cancer action is associated with anti-proliferation, pro-apoptosis and anti-angiogenesis (Cheng et al., 2010, Chirumbolo, 2013). Qu induces breast cancer cell apoptosis via trigging extrinsic or mitochondria-related apoptotic pathways (de Oliveira et al., 2015). It interacts with the intracellular signaling cascades for cancer cell survival or proliferation, such as PI3K/AKT and Ras Raf/MEK/ERK1/2/RSK signaling pathways (Dajas, 2012). Qu can also eliminate damaged DNA, inhibit cancer associated inflammatory and obesity pathways (Burd, 2011).

In addition, previous studies have shown that Qu reduces serum VEGF levels of tumor bearing animals (Camargo et al., 2011) and inhibits VEGF secretion of human breast cancer MCF-7 cells (Oh et al., 2010). And in vitro observations find that Qu exerts a strong inhibitory effect on calcineurin (Lei et al., 2011). Therefore, in the present study, we hypothesize that calcineurin is the key target of Qu in the tumor angiogenesis. Anti-tumor and anti-angiogenesis effects of Qu were assessed in a human breast cancer xenograft model in nude mice. Furthermore, the underlying mechanisms of anti-angiogenic activities of Qu were demonstrated as calcineurin activity, endothelial NFAT activation, subcellular location and gene expression of molecules associated in VEGFR2 mediated calcineurin/NFAT pathway for angiogenesis.

Section snippets

Animals

Thirty two female BALB/c nude mice were obtained from Animal Centre of the Chinese Academy of Medical Sciences. All animals were kept under a 12-h-light/-dark cycle, temperature (25.0±0.2 °C) and humidity (45±2%) controlled specific pathogen free environment. The animals were acclimatized for one week, fed standard rodent pellets and allowed free access to filtered water. All experimental procedures were approved by the Ethics Committee of the Institute of Medicinal Plant Development, CAMS &

Quercetin inhibits MCF-7 breast cancer xenograft growth in mice

The effect of Qu on the growth of xenograft tumor in nude mice was evaluated. Volume ratios of tumors were the largest in model animals, and these were significantly decreased in TAM (P<0.01), FK506 (P<0.05) and Qu (P<0.01) treated animals. Similarly, the weight ratio of tumors in each treatment group is significantly decreased than that in model group. The inhibitory rates of tumor growth in TAM, FK506 and Qu group were 22.86%, 14.05% and 18.26%, respectively (Table 1).

Quercetin induces tumor necrosis

H&E staining showed

Discussion

Angiogenesis plays a critical role in the development of breast cancer (Schneider and Miller, 2005). Calcineurin/NFAT pathway regulated by VEGFR2 is highly activated in human breast tumor endothelia (Courtwright et al., 2009, Siamakpour-Reihani et al., 2011). NFAT is a transcription factor that mediates vascular development and angiogenesis, and calcineurin is the key enzyme inducing NFAT activation (Mancini and Toker, 2009). FK506 binds to immunophilin FKBP12 and inhibits calcineurin

Conclusions

In conclusion, this study demonstrated the antitumor and antiangiogenic effect of Qu on breast cancer in vivo. It indicated that Qu inhibited breast cancer angiogenesis, at least in part via targeting calcineurin/NFAT pathway. Therefore, this study provides for the further study of Qu as a novel antiangiogenic agent for the treatment of cancers. And other potential mechanisms of Qu on molecular links between inflammation and tumor angiogenesis might be identified. Furthermore, the most

Declaration of interest

The authors have declared no conflict of interest.

Acknowledgements

This work was supported by the Beijing Natural Science Foundation (Grant no. 7132150), Education Ministry Science Foundation of the People's Republic of China (Grant no. 108019), and National Natural Science Foundation of China (Grant no. 81541082).

References (38)

  • M. Yoeli-Lerner et al.

    Akt blocks breast cancer cell motility and invasion through the transcription factor NFAT

    Mol. Cell.

    (2005)
  • A.L. Armesilla et al.

    Vascular endothelial growth factor activates nuclear factor of activated T cells in human endothelial cells: a role for tissue factor gene expression

    Mol. Cell. Biol.

    (1999)
  • L. Carr et al.

    Calcineurin activity assay measurement by liquid chromatography-tandem mass spectrometry in the multiple reaction monitoring mode

    Clin. Chem.

    (2013)
  • S. Cheng et al.

    Quercetin induces tumor-selective apoptosis through downregulation of Mcl-1 and activation of Bax

    Clin. Cancer Res.

    (2010)
  • S. Chirumbolo

    Quercetin in cancer prevention and therapy

    Integr. Cancer Ther.

    (2013)
  • A. Courtwright et al.

    Secreted frizzle-related protein 2 stimulates angiogenesis via a calcineurin/NFAT signaling pathway

    Cancer Res.

    (2009)
  • C. DeSantis et al.

    Breast cancer statistics, 2013

    CA: Cancer J. Clin.

    (2014)
  • J. Duo et al.

    Quercetin inhibits human breast cancer cell proliferation and induces apoptosis via Bcl-2 and Bax regulation

    Mol. Med. Rep.

    (2012)
  • A. Greenhough et al.

    The COX-2/PGE2 pathway: key roles in the hallmarks of cancer and adaptation to the tumour microenvironment

    Carcinogenesis

    (2009)
  • Cited by (80)

    • Antioxidant, antibacterial, and antitumor activities of goat's stirred yoghurt fortified with carob molasses

      2022, Annals of Agricultural Sciences
      Citation Excerpt :

      Finally, the plain stirred yoghurt (Control) is moderate active agents (IC50 = 45.48 μg, 31.21 μg and 38.51 μg) against HCT116, Hela and MCF7 cell lines, respectively. These results agree with those reported by Angst et al. (2013) and Zhao et al. (2016), who reported that the carob is rich in phytochemical compounds that inhibited angiogenesis in pancreatic and breast cancer. The increase of carob molasses concentration decreased the relative viability of the three tested human tumor cell lines (Fig. 1).

    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text