Synergistic Inhibition of Endothelial Cell Proliferation,
Tube Formation, and Sprouting by Cyclosporin A and
Benjamin A. Nacev1,2, Jun O. Liu1,3*
1Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 2Medical Scientist
Training Program, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 3Department of Oncology, Johns Hopkins University
School of Medicine, Baltimore, Maryland, United States of America
Pathological angiogenesis contributes to a number of diseases including cancer and macular degeneration. Although
angiogenesis inhibitors are available in the clinic, their efficacy against most cancers is modest due in part to the existence
of alternative and compensatory signaling pathways. Given that angiogenesis is dependent on multiple growth factors and
a broad signaling network in vivo, we sought to explore the potential of multidrug cocktails for angiogenesis inhibition. We
have screened 741 clinical drug combinations for the synergistic inhibition of endothelial cell proliferation. We focused
specifically on existing clinical drugs since the re-purposing of clinical drugs allows for a more rapid and cost effective
transition to clinical studies when compared to new drug entities. Our screen identified cyclosporin A (CsA), an
immunosuppressant, and itraconazole, an antifungal drug, as a synergistic pair of inhibitors of endothelial cell proliferation.
In combination, the IC50dose of each drug is reduced by 3 to 9 fold. We also tested the ability of the combination to inhibit
endothelial cell tube formation and sprouting, which are dependent on two essential processes in angiogenesis, endothelial
cell migration and differentiation. We found that CsA and itraconazole synergistically inhibit tube network size and sprout
formation. Lastly, we tested the combination on human foreskin fibroblast viability as well as Jurkat T cell and HeLa cell
proliferation, and found that endothelial cells are selectively targeted. Thus, it is possible to combine existing clinical drugs
to synergistically inhibit in vitro models of angiogenesis. This strategy may be useful in pursuing the next generation of
Citation: Nacev BA, Liu JO (2011) Synergistic Inhibition of Endothelial Cell Proliferation, Tube Formation, and Sprouting by Cyclosporin A and Itraconazole. PLoS
ONE 6(9): e24793. doi:10.1371/journal.pone.0024793
Editor: Marc Tjwa, University of Frankfurt - University Hospital Frankfurt, Germany
Received September 10, 2010; Accepted August 19, 2011; Published September 28, 2011
Copyright: ? 2011 Nacev, Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by NCI [Grant RO1 CA122814], the National Center for Research Resources (NCRR), a component of the National
Institutes of Health (NIH) and NIH Roadmap for Medical Research [Grant UL1 RR 025005], the Flight Attendant Medical Research Institute Fund, the Keck
Foundation, the Walsh Prostate Cancer Fund, and the Commonwealth Foundation (J.O.L.), and by the NIH Medical Scientist Training Program [Grant T32GM07309]
(B.A.N). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Angiogenesis, the process of new blood vessel growth and
development, underlies a number of human diseases including
cancer, macular degeneration, psoriasis, rheumatoid arthritis,
diabetic retinopathy, and pulmonary hypertension . Inhibitors
of angiogenesis such as the anti-VEGF antibody bevacizumab are
used clinically to treat cancer. However, the experience with
existing therapies has been mixed. While they have shown efficacy,
their effects in terms of halting disease progression and improving
survival have been modest and often involve side effects including
hypertension and increased risk of stroke . Hence, although the
promise of angiogenesis inhibitors has been demonstrated, there is
a clear need for more effective anti-angiogenic therapies.
On average, the development of a clinically viable drug requires
an investment of roughly $800 million and takes about 12 years .
One strategy to accelerate drug development is to re-purpose
existing drugs . Because re-purposed drugs have already been
approved for clinical use, their pharmacodynamic and pharmaco-
kinetic properties are well established. In addition, existing drugs
have acceptable levels of toxicity and in many cases they have
known mechanisms, which makes their pharmacology amenable to
detailed molecular study. Thus, by focusing on existing drugs, many
hurdles in drug development are already cleared. The end result is a
discovered to have new applications. We have previously adopted
this approach when we assembled and screened the Johns Hopkins
Drug Library (JHDL) for inhibitors of angiogenesis and other
activities [5–9]. Presently, the JHDL contains ,3,300 drugs
approved by the US Food and Drug Administration or foreign
equivalent. The initial screen for angiogenesis inhibitors identified
221 compounds with .50% inhibition of human umbilical vein
endothelial cell (HUVEC) proliferation at a 10 mM dose.
A number of these hits had IC90doses above the peak plasma
level obtained under clinical dosing regimens or had dose-limiting
toxicities. One way to expand the clinical applicability of these
hits, we reasoned, was to find synergy between them, thereby
reducing the doses needed for those synergistic pairs to inhibit
angiogenesis in vivo. We have thus conducted a screen for synergy
among 741 binary combinations of 39 clinical drugs that were hits
PLoS ONE | www.plosone.org1September 2011 | Volume 6 | Issue 9 | e24793
from the initial screen. In addition to lowering the necessary dose
of otherwise toxic agents, combination therapy is often used to
limit the potential for drug resistance and to achieve synergistic
inhibition of multiple independent pathways that converge on a
single essential molecular process. For these reasons, the
simultaneous use of multiple drugs has been an effective strategy
to overcome diseases intractable to single agent therapies. For
instance, combination therapy is now the standard of care in
treating HIV infection and many neoplasms .
Synergy, or superadditivity, is often observed upon the inhibition
of multiple pathways that converge to promote a single biological
process such as proliferation. Thus, it is hypothetically possible that
synergistic inhibition of angiogenesis may be possible given that
signaling in angiogenesis is complex and involves multiple pathways
including those downstream of growth factors such as vascular
endothelial growth factor (VEGF) and basic fibroblast growth factor
(bFGF) [11,12]. In fact, the limitations of anti-VEGF therapy have
been attributed to the existence of redundant alternative pathways
in the in vivo pro-angiogenic signaling network . Thus, a more
effective strategy to inhibit angiogenesis may be to simultaneously
target multiple pathways. Just as anticancer regimens have evolved
to simultaneously utilize drugs with multiple mechanisms to achieve
synergy, so might anti-angiogenic regimens have to evolve to
provide additional efficacy. Thus, we sought to determine whether
there exist clinical drugs that synergistically inhibit endothelial cell
proliferation and tube formation.
Materials and Methods
Reagents and materials
Pooled HUVEC and EGM-2 bullet kit media were purchased
from Lonza. Jurkat T cells (a human acute T cell leukemia line)
and HeLa cells (a human cervical adenocarcinoma line) were from
the American Type Tissue Collection. Low and high glucose
DMEM, RPMI 1640, fetal bovine serum, and penicillin/
streptomycin were from Gibco. Recombinant human VEGF165
and bFGF146were purchased from R&D systems and reconsti-
tuted in 0.1% BSA in PBS as 100 mg/mL and 10 mg/mL stocks,
respectively. Methyl cellulose (4 cP) was purchased from Sigma
and used to prepare methocel as previously described .
Itraconazole (Ita) (Sigma), cyclosporin A (CsA) (LC labs), and
sunitinib (LC labs) were stored frozen in DMSO and added to cells
from 2006stocks. Calcein AM and Alamar Blue were purchased
from Invitrogen and [3H]-thymidine was from PerkinElmer. Glass
filtermats were obtained from Wallac. Phenol red free Matrigel
and rat tail collagen type I were from BD biosciences.
All cells were grown at 37uC with 5% CO2in a humidified
environment. HUVEC were grown in EGM-2 bullet kit media
and used between passages 2 and 8. Jurkat T cells were grown in
RMPI 1640 (+10% FBS, 1% penicillin/streptomycin), HeLa were
grown in low glucose DMEM (+10% FBS, 1% penicillin/
streptomycin), and HFF in high glucose DMEM (+10% FBS,
2000 HUVEC or HeLa/well or 16104Jurkat T cells/well were
seeded in a 96-well plate (Costar) in 199 mL media. After an
overnight recovery, drugs were added. For CsA+Ita combinations
the molar ratio was always 10:1. Following a 24-h incubation, cells
were pulsed with 0.9 mCi of [3H]-thymidine for 6 h, washed once
with PBS, trypsinized, and transferred to filtermats (Wallac) using
a Mach III M Harvester 96 (Tomtec). For Jurkat T cells, the
PBS wash and trypsinization steps were omitted. After drying,
[3H]-thymidine retention on the filtermats was determined by
scintillation counting using a 1450 Microbeta apparatus (Wallac).
Counts were normalized to that of control cells treated with
vehicle only. GraphPad Prism (v4.03) software was used to
determine IC50values using a four parameter logistic regression.
In the case of growth factor-dependent proliferation assays, the
cells were first seeded as above but in basal EBM-2 basal media
(Lonza) with 2% FBS added (hereafter referred to as ‘basal’
media). After an overnight recovery, the media was replaced with
either basal media, standard EGM-2 media, basal media with
100 ng/mL VEGF165or basal media with VEGF165vehicle alone.
Drugs were then added and the assay was continued as described
above. In processing the data for VEGF-dependent proliferation,
the [3H] counts for basal proliferation at each dose was subtracted
from that of basal+VEGF proliferation prior to normalizing the
data to vehicle control.
Cell Viability Assay
HUVEC and HeLa were plated at 2000 cells/well and HFF at
2500 cells/well in a 96-well plate in 199 mL of media. Cells were
incubated with drug for 30 h and then washed with 200 mL of PBS
and incubated for 30 min at 37uC with 100 mL of 1 mM Calcein
AM diluted from a 1 M stock in DMSO. The excess dye solution
was then aspirated and 100 mL PBS was added prior to reading
with a fluorescence plate reader (Fluostar Optima). The
fluorescence of drug treated samples was normalized to DMSO
controls. Three independent experiments were performed.
An Alamar Blue assay for Jurkat viability was used since it does
not require the media to be removed prior to analysis, which is
advantageous for cells grown in suspension. 16104Jurkat cells were
seeded per well of a 96-well plate in 200 mL of media. Following a
24-h incubation with drug, 20 mL of Alamar blue was added and
cells were incubated for an additional 6 h. The florescence of each
well was measuredat590 nmaftera 544 nmexcitation.Mediaonly
wells were included as blanks. Three independent experiments with
multiple technical replicates were performed.
Tube formation assay
For the synergy analysis, 230 mL of ice cold, phenol red-free
matrigel was added to a 24-well plate using a chilled pipet tip.
Following a 30–40 min incubation at 37uC, 76104HUVEC were
added in 500 mL of media. Cells were treated with drug for 18 h.
The cells were then gently washed with 500 mL PBS and followed
by incubation with 300 mL of 2 mM calcein AM (diluted in PBS
from a 1 M DMSO stock) for 30 min. The calcein AM was
replaced with 500 mL PBS and the tube network was photographed
using fluorescent microscopy. For the comparison of sunitinib and
CsA+Ita, the above experiment was scaled down to a 96-well plate
format. 50 mL of ice cold matrigel was used and 1.56104HUVEC
in 200 mL of media were seeded. Following an 18-h drug treatment,
the cells were washed with 150 mL of PBS and incubated for
30 minutesat 37uCwith100 mLofCalceinAMwhichwas replaced
with100 mL of PBS prior to imaging. In both cases, the imageswere
inverted and equal areas of the central or best focused region of the
image were cropped out using Adobe Photoshop (v 9.0.2). The tube
networks were quantified using Angioquant .
This assay was based on work by Korff and Augustin . We
modified the procedure by first coating the bottom of the tissue
culture surface with a collagen, growth factor, and drug solution to
VEGF165(30 ng/mL) and bFGF (30 ng/mL) or vehicle (0.1% BSA
in PBS) was added to a methocel:media (42:58) solution, followed by
Synergistic Inhibition of Endothelial Cells
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28. Sharpe RJ, Arndt KA, Bauer SI, Maione TE (1989) Cyclosporine inhibits basic
fibroblast growth factor-driven proliferation of human endothelial cells and
keratinocytes. Arch Dermatol 125(10): 1359–1362.
29. Benelli U, Ross JR, Nardi M, Klintworth GK (1997) Corneal neovascularization
induced by xenografts or chemical cautery. inhibition by cyclosporin A. Invest
Ophthalmol Vis Sci 38(2): 274–282.
30. Liu J, Farmer JD, Jr., Lane WS, Friedman J, Weissman I, et al. (1991)
Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506
complexes. Cell 66(4): 807–815.
31. Nacev B, Low WK, Huang Z, Su T, Su Z, et al. (2011) A calcineurin-
independent mechanism of angiogenesis inhibition by a non-immunosuppressive
cyclosporin A analog. J Pharmacol Exp Ther 338(2): 466.
32. Lamb DC, Kelly DE, Waterman MR, Stromstedt M, Rozman D, et al. (1999)
Characteristics of the heterologously expressed human lanosterol 14alpha-
demethylase (other names: P45014DM, CYP51, P45051) and inhibition of the
purified human and candida albicans CYP51 with azole antifungal agents. Yeast
33. Trosken ER, Adamska M, Arand M, Zarn JA, Patten C, et al. (2006)
Comparison of lanosterol-14 alpha-demethylase (CYP51) of human and candida
albicans for inhibition by different antifungal azoles. Toxicology 228(1): 24–32.
34. Xu J, Dang Y, Ren YR, Liu JO (2010) Cholesterol trafficking is required for
mTOR activation in endothelial cells. Proc Natl Acad Sci U S A 107(10):
35. Wei S, Nacev BA, Bhat S, Liu JO (2010) Imapct of absolute stereochemistry on
the antiangiogenic and antifungal activities of itraconazole. ACS Med Chem
Lett 1(4): 155.
36. Cruz MC, Goldstein AL, Blankenship JR, Del Poeta M, Davis D, et al. (2002)
Calcineurin is essential for survival during membrane stress in candida albicans.
EMBO J 21(4): 546–559.
37. Hartman JL, 4th, Garvik B, Hartwell L (2001) Principles for the buffering of
genetic variation. Science 291(5506): 1001–1004.
Synergistic Inhibition of Endothelial Cells
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