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Adv Biomed Res 2013,  2:83

Effect of DAPT, a gamma secretase inhibitor, on tumor angiogenesis in control mice

1 Department of Physiology, Students Research Center, Faculty of Medicine, Isfahan, Iran
2 Department of Anatomy, Isfahan University of Medical Sciences, Isfahan, Iran
3 Physiology Research Center, Isfahan University of Medical Sciences, Isfahan, Iran

Date of Submission13-Jan-2013
Date of Acceptance03-Mar-2013
Date of Web Publication30-Nov-2013

Correspondence Address:
Majid Khazaei
Department of Physiology, Faculty of Medicine, Isfahan University of Medical Sciences, HezarJarib Ave, Isfahan
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Source of Support: The Vice Chancellor of Isfahan University of Medical Sciences supported this study, Conflict of Interest: None

DOI: 10.4103/2277-9175.122498

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Background: Notch signaling is a key factor for angiogenesis in physiological and pathological condition and γ-secretase is the regulator of Notch signaling. The main goal of this study was to assess the effect of (N-[N-(3,5-Diflurophenaacetyl-L-alanyl)]-S-phenylglycine t-Butyl Ester) DAPT, a γ-secretase inhibitor, on serum angiogenic biomarkers, and tumor angiogenesis in control mice.
Materials and Methods: Tumor was induced by inoculation of colon adenocarcinoma cells (CT26) in 12 male Balb/C mice. When tumors size is reached to a 350 ± 50 mm 3 , the animals were randomly divided into two groups: control and DAPT (n = 6/group). DAPT was injected subcutaneously 10 mg/kg/day. After 14 days, blood samples were taken and the tumors were harvested for immunohistochemical staining.
Results: Administration of DAPT significantly increased serum nitric oxide concentration and reduced vascular endothelial growth factor receptors-1 (VEGFR1) concentration without changes on serum VEGF concentration. DAPT reduced tumor vascular density in control mice (280.6 ± 81 vs. 386 ± 59.9 CD31 positive cells/mm 2 ), although, it was not statistically significant.
Conclusion: It seems that γ-secretase inhibitors can be considered for treatment of disorders with abnormal angiogenesis such as tumor angiogenesis.

Keywords: Angiogenesis, obesity, tumor, γ-secretase

How to cite this article:
Kalantari E, Saeidi H, Kia NS, Tahergorabi Z, Rashidi B, Dana N, Khazaei M. Effect of DAPT, a gamma secretase inhibitor, on tumor angiogenesis in control mice. Adv Biomed Res 2013;2:83

How to cite this URL:
Kalantari E, Saeidi H, Kia NS, Tahergorabi Z, Rashidi B, Dana N, Khazaei M. Effect of DAPT, a gamma secretase inhibitor, on tumor angiogenesis in control mice. Adv Biomed Res [serial online] 2013 [cited 2020 Oct 21];2:83. Available from:

  Introduction Top

Angiogenesis is a regulated process, which requires several steps including degradation of basement membrane by protease, migration, and proliferation of endothelial cells and maturation of blood vessels. [1] Several angiogenic and anti-angiogenic factors are involved during angiogenesis. Vascular endothelial growth factor (VEGF) is the known angiogenic factor. The role of VEGF on angiogenesis has been documented in several in vivo and in vitro studies. [2] VEGF has two receptors: Vascular endothelial growth factor receptors-1 (VEGFR1) and VEGFR2. [3] VEGFR1 has higher affinity to VEGF than VEGFR2. VEGFR1 is a potent negative regulator of VEGFR2 action. [4] VEGFR1 is a negative regulator of angiogenesis. [5] VEGFR1 is the dominant receptor in tumor vasculature. [6] Nitric oxide (NO) is the main endothelium-relaxing factor, which has several effects on cardiovascular system. It also considered as an angiogenic factor, which is documented in different studies. [7]

Notch signaling is a key factor for angiogenesis in physiological and pathological condition including carcinoma. [8],[9] First time, the role of Notch signaling was shown in lymphoblastic leukemia. [10] γ-secretase is the regulator of Notch signaling, and is a key molecule in postnatal angiogenesis. γ-secretase is required for processing of several proteins involve in angiogenesis including Notch and CD44 and the drugs oppose angiogenesis by altering the processing of those proteins. [11] In this study, we used a γ-secretase inhibitor, (N-[N-(3,5-Diflurophenaacetyl-L-alanyl)]- S-phenylglycine t-Butyl Ester) DAPT (N-[N-(3,5-Diflurophenaacetyl-L-alanyl)]-S-phenylglycine t-Butyl Ester) to evaluate its effect on serum NO, VEGF, soluble form of VEGFR1 (sVEGFR1), and tumor angiogenesis in control mice.

  Materials and Methods Top

Animals and diets

Male Balb/C mice (n = 12) were purchased from Pasteur Institute (Tehran, Iran) and housed two per cage in standard animal house conditions, room temperature between 20 and 25°C, constant humidity and 12 h light/dark cycle. The experimental procedures were reviewed and approved by the ethical committee of the Isfahan University of Medical Sciences. The mice (at age of 20 weeks) were randomly divided into two groups: Control and DAPT.

Induction of tumor and drug administration

CT26 colon adenocarcinoma cells (5 × 10 5 cells) in 500 μl of Phosphate buffer solution (PBS) were inoculated subcutaneously into the dorsum of all mice using a syringe fitted with a 21 gauge needle. Tumor growth and development was then followed-up and monitored after inoculation. Once tumors became palpable and their sizes reached to approximately to 350 ± 50 mm 3 , the animals were treated by DAPT. [12] DAPT was prepared by dissolving in Dimethyl sulfoxide (DMSO) and subcutaneously injected (10 mg/kg/day). [11] Control group received the same amount of DMSO. After 14 days, the mice were sacrificed. Blood samples were collected by cardiac puncture and the serums were separated and kept at -30°C for further analysis.

Histological analysis

By the end of experiment, the induced tumors were collected and processed for histological analysis. The tumors were put in formalin solution. Paraffin-embedded tissues were sectioned at a thickness of 5 μm and stained with standard immunohistochemical protocol with a monoclonal rat anti-mouse CD31 antibody (Abcam Co., USA, Cat# Ab28364). CD31 positive cells were counted in 20 fields of 10 sections at ×40 magnification and reported per mm 2 .

Serum biochemical and angiogenic measurements

Blood glucose was measured by a glucometer. ELISA kits were used for determination of serum nitrite (Promega Corp, USA, Cat#G2930), the main metabolite of NO, VEGF, and sVEGFR1 (R and D systems, Mineapolis, USA) concentrations.


Data are presented as mean ± SEM. The significant differences between groups were tested by Students t-test (SPSS version 16). P < 0.05 was considered statistically significant.

  Results Top

Serum NO concentrations

[Figure 1] illustrates the changes of serum concentration in experimental groups. As shown, administration of DAPT significantly increased serum NO concentration in control mice (P < 0.05).
Figure 1: Serum nitric oxide concentration in experimental groups. *: P < 0.05 compare to other group

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Serum VEGF and sVEGFR1 concentrations

The changes of serum VEGF and sVEGFR1 concentrations in experimental groups are shown in [Figure 2]. Administration of DAPT significantly reduced serum sVEGFR1 while, could not change serum VEGF concentration in control mice.
Figure 2: Changes of serum vascular endothelial growth factor (a) and s vascular endothelial growth factor receptors1 (b) in experimental groups *: P <0.05 compare to other group

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Angiogenesis assay

Immunohistochemical study of the tumors showed that CD31 positive cells were reduced after DAPT administration (280.6 ± 81 vs. 386 ± 59.9 CD31 positive cells/mm 2 ), although it was not statistically significant [Figure 3]a. Samples of immunohistochemical staining are shown in [Figure 3]b.
Figure 3: capillary density in adenocarcinoma tumors expressed as CD31 positive cells per mm2 (a) Samples of immunohistochemical staining (×400) from tumor tissue in control (b) and control + DAPT (c) Arrows indicate CD31 positive cells

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  Discussion Top

This study investigated the impact of DAPT, a γ-secretase inhibitor on tumor angiogenesis and serum angiogenic factors in control mice. Results showed that DAPT increased serum NO, reduced serum sVEGFR1 and non-significantly reduced tumor vascular density.

Notch signaling has been implicated in for vascular development and angiogenic process. [9] γ-secretase is required for processing of several proteins involve in angiogenesis including Notch and CD44. Thus, the drugs oppose the γ-secretase, inhibits angiogenesis by altering the processing of those proteins. [11] In the present study, we used DAPT, a γ-secretase inhibitor and we expected that it reduced vascular density and angiogenesis in tumor cells. Our results showed that administration of DAPT reduced tumor vascular density, although it was not statistically significant.

Previous studies suggested that DAPT may be affect tumor growth by disturbance of angiogenesis. [13] Inhibition Notch signaling enzyme complex (γ-secretase) disrupt vascular structure and function, however it increases vascular density. [14] Paris et al. revealed that γ-secretase inhibitors inhibit angiogenesis and tumor growth in rat aortic ring model of angiogenesis and glioblastoma and human lung adenocarcinoma and they showed that DAPT dose dependently inhibited the sprouting of new capillaries in rat aortic ring model. [11] The mice lacking γ-secretase activity exhibits cerebral hemorrhage due to abnormal vessel formation. [15]

In this study, we found that administration of DAPT increased serum NO and reduced sVEGFR1 concentration and it seems that it may affect tumor angiogenesis through altering the angiogenic factors. An in vitro study in the mouse micro vascular endothelial cell line showed that DAPT increased endothelial nitric oxide synthase (eNOS) and VEGFR2 protein and expression and decreased VEGFR1 at both the expression and protein and they showed that DAPT up regulates NOS in a concentration-dependent manner. [16] They also indicated that DAPT regulates VEGF signaling via alteration of VEGF receptors expression. VEGF has two receptors: VEGFR-1 and VEGFR2. VEGFR1 is a negative regulator of angiogenesis and VEGFR1 is the dominant receptor in tumor vasculature. [6] DAPT reverse VEGFR1:VEGFR2 ratio and DAPT treatment down-regulate VEGFR1 but up-regulates VEGFR2. [17]

We conclude that γ-secretase inhibitors are associated with disruption of eNOS and VEGF signaling and may be considered for treatment of excessive angiogenesis disorders such as colon adenocarcinoma.

  Acknowledgment Top

The authors thank the Vice chancellor of Isfahan University of Medical Sciences for financial support.

  References Top

1.Carmeliet P. VEGF gene therapy: Stimulating angiogenesis or angioma-genesis? Nat Med 2000;6:1102-3.  Back to cited text no. 1
2.Ferrara N. The role of VEGF in the regulation of physiological and pathological angiogenesis. EXS 2005;94:209-31.  Back to cited text no. 2
3.Rahimi N. Vascular endothelial growth factor receptors: Molecular mechanisms of activation and therapeutic potentials. Exp Eye Res 2006;83:1005-16.  Back to cited text no. 3
4.Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L. VEGF receptor signalling-in control of vascular function. Nat Rev Mol Cell Biol 2006;7:359-71.  Back to cited text no. 4
5.Fong GH, Rossant J, Gertsenstein M, Breitman ML. Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature 1995;376:66-70.  Back to cited text no. 5
6.Zhang Z, Neiva KG, Lingen MW, Ellis LM, Nör JE. VEGF-dependent tumor angiogenesis requires inverse and reciprocal regulation of VEGFR1 and VEGFR2. Cell Death Differ 2010;17:499-512.  Back to cited text no. 6
7.Cooke JP, Losordo DW. Nitric oxide and angiogenesis. Circulation 2002;105:2133-5.  Back to cited text no. 7
8.Mailhos C, Modlich U, Lewis J, Harris A, Bicknell R, Ish-Horowicz D. Delta4, an endothelial specific notch ligand expressed at sites of physiological and tumor angiogenesis. Differentiation 2001;69:135-44.  Back to cited text no. 8
9.Rehman AO, Wang CY. Notch signaling in the regulation of tumor angiogenesis. Trends Cell Biol 2006;16:293-300.  Back to cited text no. 9
10.Ellisen LW, Bird J, West DC, Soreng AL, Reynolds TC, Smith SD, et al. TAN-1, the human homolog of the Drosophila notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms. Cell 1991;66:649-61.  Back to cited text no. 10
11.Paris D, Quadros A, Patel N, Delle Donne A, Humphrey J, Mullan M. Inhibition of angiogenesis and tumor growth by beta and gamma-secretase inhibitors. Eur J Pharmacol 2005;514:1-15.  Back to cited text no. 11
12.Dovey HF, John V, Anderson JP, Chen LZ, de Saint Andrieu P, Fang LY, et al. Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain. J Neurochem 2001;76:173-81.  Back to cited text no. 12
13.Masuda S, Kumano K, Suzuki T, Tomita T, Iwatsubo T, Natsugari H, et al. Dual antitumor mechanisms of signaling inhibitor in a T-cell acute lymphoblastic leukemia xenograft model. Cancer Sci 2009;100:2444-50.  Back to cited text no. 13
14.Noguera-Troise I, Daly C, Papadopoulos NJ, Coetzee S, Boland P, Gale NW, et al. Blockade of Dll4 inhibits tumour growth by promoting non-productive angiogenesis. Nature 2006;444:1032-7.  Back to cited text no. 14
15.Nakajima M, Yuasa S, Ueno M, Takakura N, Koseki H, Shirasawa T. Abnormal blood vessel development in mice lacking presenilin-1. Mech Dev 2003;120:657-67.  Back to cited text no. 15
16.Zou YH, Cao YQ, Wang LX, Zhang YH, Yue ZJ, Liu JM. γ-secretase inhibitor up-regulates vascular endothelial growth factor receptor-2 and endothelial nitric oxide synthase. Exp Ther Med 2011;2:725-729.  Back to cited text no. 16
17.Zou Y, Cao Y, Yue Z, Liu J. Gamma-secretase inhibitor DAPT suppresses glioblastoma growth via uncoupling of tumor vessel density from vessel function. Clin Exp Med 2012; [Epub ahead of print].  Back to cited text no. 17


  [Figure 1], [Figure 2], [Figure 3]

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