N6-isopentenyladenosine a new potential anti-angiogenic compound that targets human microvascular endothelial cells in vitro

Article abstract of DOI:10.1080/15257770.2018.1503673

N6-isopentenyladenosine is an anti-proliferative and pro-apoptotic atypical nucleoside for normal and tumor cells. Considering the role of angiogenesis in various diseases, we investigated the cytotoxic effect of N6-isopentenyladenosine on human microvascular endothelial cells, protagonists in angiogenesis. Our results show that N6-isopentenyladenosine induced a significant reduction of cell viability, upregulated p21 and promoted caspase-3 cleavage in a dose dependent manner leading to apoptotic cell death as detected by FACS analysis. To understand structure-function relationship of N6-isopentenyladenosine, we investigated the effect of some N6-isopentenyladenosine analogs. Our results suggest that N6-isopentenyladenosine and some of its derivatives are potentially novel angiostatic agents and might be associated with other anti-angiogenic compounds for a better outcome.

Full text of DOI:10.1080/15257770.2018.1503673

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
N6-isopentenyladenosine a new potential anti-
angiogenic compound that targets human
microvascular endothelial cells in vitro
Sara Castiglioni, Valentina Romeo, Silvana Casati, Roberta Ottria, Cristiana
Perrotta, Pierangela Ciuffreda & Jeanette A. M. Maier
To cite this article: Sara Castiglioni, Valentina Romeo, Silvana Casati, Roberta Ottria, Cristiana
Perrotta, Pierangela Ciuffreda & Jeanette A. M. Maier (2018): N6-isopentenyladenosine a new
potential anti-angiogenic compound that targets human microvascular endothelial cells inꢀvitro,
Nucleosides, Nucleotides and Nucleic Acids
To link to this article: https://doi.org/10.1080/15257770.2018.1503673
Published online: 22 Nov 2018.
Submit your article to this journal
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=lncn20

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
https://doi.org/10.1080/15257770.2018.1503673
N6-isopentenyladenosine a new potential
anti-angiogenic compound that targets human
microvascular endothelial cells in vitro
Sara Castiglioni, Valentina Romeo, Silvana Casati, Roberta Ottria,
Cristiana Perrotta, Pierangela Ciuffreda, and Jeanette A. M. Maier
Dipartimento di Scienze Biomediche e Cliniche “L. Sacco”, Universit ꢀa degli Studi di Milano,
Milano, Italy
ABSTRACT
ARTICLE HISTORY
N6-isopentenyladenosine is an anti-proliferative and pro-apop-
totic atypical nucleoside for normal and tumor cells.
Considering the role of angiogenesis in various diseases, we
investigated the cytotoxic effect of N6-isopentenyladenosine
on human microvascular endothelial cells, protagonists in
angiogenesis. Our results show that N6-isopentenyladenosine
induced a significant reduction of cell viability, upregulated
p21 and promoted caspase-3 cleavage in a dose dependent
manner leading to apoptotic cell death as detected by FACS
analysis. To understand structure-function relationship of
N6-isopentenyladenosine, we investigated the effect of some
N6-isopentenyladenosine analogs. Our results suggest that N6-
isopentenyladenosine and some of its derivatives are poten-
tially novel angiostatic agents and might be associated with
other anti-angiogenic compounds for a better outcome.
Received 20 December 2017
Accepted 19 July 2018
KEYWORDS
N6-isopentenyladenosine;
HMEC; angiogenesis
Introduction
N6-isopentenyladenosine (iPA, 1a), an atypical nucleoside that belongs to
[1]
the cytokinin family, is found in transfer RNA (tRNA) of eukaryotic and
[2]
prokaryotic cells. In particular, in mammals iPA is part of tRNA[Ser]sec
and the cognate tRNA-isopentenyltransferase (TRIT1) has been hypothe-
[
3]
sized to be a putative tumor suppressor.
Several studies have demonstrated the anti-proliferative and pro-apop-
totic effects of iPA and some analogs in a broad variety of normal diploid
cells and in different tumor cell lines derived from carcinomas, sarcomas,
[
4–11]
and myeloma.
In HeLa cells, iPA-induced apoptosis was associated with the inhibition
of both AKT and transforming growth factor b–activated kinase 1 (TAK1)
activities. Since iPA binds the RING domain of tumor necrosis factor
CONTACT Roberta Ottria
roberta.ottria@unimi.it
Dipartimento di Scienze Biomediche e Cliniche “L.
Sacco”, Universit ꢀa degli Studi di Milano, Milano 20157, Italy
ß 2018 Taylor & Francis Group, LLC

2
S. CASTIGLIONI ET AL.
receptor–associated factor 6 (TRAF6), which, in turn, is involved in the
ubiquitination and activation of AKT and TAK1 signaling pathways, the
anti-proliferative effect of iPA in HeLa cells is probably due to its inter-
[12]
action with TRAF6.
In human endothelial cells derived from the umbil-
ical vein (HUVEC) iPA significantly impaired the viability through the
[
13]
activation of AMP-kinase (AMPK).
Endothelial cells are highly heterogeneous
[
14]
as the results of the differ-
ent mechanical and structural characteristics due to the different caliber, of
the signals originating in diverse microenvironments and also of the differ-
ent functions exerted by the endothelium in different tissues. HUVEC are
widely considered as a model of macrovascular endothelium but do not
reflect the highly specialized nature of microvascular endothelial cells,
which are crucial in regulating tissue homeostasis and are protagonists in
angiogenesis and inflammation. To test the potential anti-angiogenic activ-
ity of iPA and analogs, we utilized Human Microvascular Endothelial Cells
(
HMEC). We found that iPA and some of its analogs are cytotoxic and
promote apoptosis, thus suggesting that they might be useful as anti-angio-
genic molecules.
Materials and methods
Reagents
N6-isopentenyladenine (3c), trans-zeatin riboside (4), trans-zeatin (5), 6-
chloropurine riboside, 6-chloropurine, all available starting amines, the
other reagents and all solvents were purchased from Sigma-Aldrich (St.
Louis, MO, USA). iPA (1a) and the other molecules were prepared and
[
8,15,16]
purified as described previously.
Briefly, compounds (1a–e, 3b) were
synthesized as follow: to a solution of 6-chloropurine riboside or 6-chloro-
purine in absolute EtOH, Et N, and the appropriate amine were added.
3
ꢀ
The mixture was stirred at 80 C for 3 h, cooled to room temperature and
the solvent was removed under vacuum to leave syrupy residue. The add-
ition of dry Et O precipitated Et NHCl, which was filtered off. The crude
2
3
[15]
residue after evaporation was crystallized from MeOH (Scheme 1a).
For
inosine derivatives (2a,b) BaCO and 3,3-dimethylallylbromide or benzyl
3
bromide were added to a solution of inosine in DMF and the mixture was
stirred at room temperature for 24 h. The moisture was filtered using a
Celite pad and washed with DMF. The combined filtrate was evaporated to
a small volume and purified by column chromatography on silica gel
[16]
(Scheme 1b).
At last hypoxanthine derivative (3a) was synthesized in the
following manner: to a suspension of sodium hydride in dry tetrahydro-
furan, cooled with an ice bath, benzylalcohol was added slowly. After
20 min, 6-chloropurine was added and the solution refluxed for 24 h. After

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
Scheme 1. (a) (i) RNH , EtOH, Et N, r.t, 3 h; (b) (ii) RBr, BaCO , DMF, rt, 24 h; (c) (iii) benzylalco-
2
3
3
ꢀ
hol, THF dry, NaH, 0 C 20 min, then 24 h reflux.
removal of the solvent under vacuum, the residue was dissolved in a 10%
solution of NH Cl and extracted with dichloromethane and ethyl acetate,
4
[
8]
the crude was purified by colon chromatography (Scheme 1c). Purity
(
ꢁ99%) and structure of all compounds were verified by HPLC, NMR, as
[17,18]
described before,
and mass spectrometry measurements. Structures of
all compounds are presented in Figure 1. Stock solutions of these com-
pounds were prepared in DMSO (final concentration 10 mM) and kept at
ꢀ
-
20 C. Appropriate dilutions were freshly prepared in culture medium just

4
S. CASTIGLIONI ET AL.
Figure 1. Structures of all the compounds used.
prior the assays. The controls were added with the final concentrations of
DMSO (0.01%).
Cell culture
HMEC were obtained from Centers for Disease Control and Prevention
(
Atlanta, United States) and grown in MCDB131 (Invitrogen, Milan, Italy)
containing Epidermal Growth Factor (EGF) (10 ng/mL), hydrocortisone
[19,20]
(
1 lg/mL) and 10% FBS on 2% gelatin-coated dishes.
To evaluate the
effects of iPA and derivatives, MTT assay was used in HMEC seeded in 96
well/plates. Briefly, the medium was replaced with medium containing 3-
(
4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrasodium bromide (MTT, 0.5 mg/
ml) (Sigma Aldrich, Oakville, Ontario, Canada). At the end of the incuba-
tion, media were removed and formazan crystals generated by the cellular
[21]
reduction activity were dissolved in DMSO.
Absorbance was measured
at 550 nm. In dose dependent experiments, HMEC were exposed to differ-
ent concentrations of iPA, 1b and 1c and IC50 values were calculated by

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
non-linear regression analysis of Log[iPA or derivatives concentration]/cell
viability using GraphPad Prism 5. In some experiments, HMEC were
exposed also to human recombinant interferon (IFN)c (PromoKine, Bio-
Connect B.V., The Netherlands). Data represent the mean ± standard devi-
ation of three separate experiments in triplicates.
Cell cycle analysis by fluorescence-activated cell sorting (FACS)
HMEC cells were fixed in 70% ethanol for 30 min, centrifuged at 2000 rpm
for 10 min and washed with ice-cold PBS. The pellets were resuspended in
PBS containing 25 mg/ml propidium iodide (Sigma Aldrich), incubated in
ꢀ
the dark at 37 C for 30 min, and then analyzed using Coulter EPICS 753
[21]
flow cytometer (Beckman Coulter, Miami, FL).
The percentage of cells
in each phase of the cell cycle was determined by using a Multicycle soft-
ware version 2.53 (Phoenix Software, San Diego, CA).
Flow cytometric analysis of cell death
Phosphatidylserine exposure on the outer leaflet of the plasma membrane
was detected by analysis of cells stained for 15 min with Alexa Fluor 488-
labeled Annexin V (1 mg/ml) (Molecular Probes-Thermo Fisher Scientific,
Waltham, MA, USA) and Propidium Iodide (Sigma-Aldrich, Saint Louis,
MO, USA), using a Fluorescence-Activated Cell Sorter (Gallios, Beckman-
[
21]
Coulter, Brea, CA, USA) as described previously.
Western blot analysis
HMEC cells were lysed, separated on SDS-PAGE, and transferred to nitro-
cellulose sheets. Western analysis was performed using antibodies against
p21, actin (Santa Cruz Biotechnology, Dallas, Texas, USA) and caspase 3
(
Cell Signaling Technology, Danvers, MA, USA). Secondary antibodies
were labeled with horseradish peroxidase (GE Healthcare, Little Chalfont,
Buckinghamshire, UK). The SuperSignal chemiluminescence kit (Pierce,
Rockford, IL, USA) was used to detect immunoreactive proteins.
Statistical analysis
Statistical significance was determined using the Student’s T test and set at
p values less than 0.05. In the figures
ꢂꢂꢂ:
ꢂ: p < 0.05; ꢂꢂ: p < 0.01;
p < 0.001.

6
S. CASTIGLIONI ET AL.
Figure 2. iPA induces cytotoxicity on HMEC cells. (a) HMEC were exposed to various concentra-
tions of iPA for 72 h or (b) different cell densities were cultured in the presence of 10 mM of
iPA for 72 h. In a and b cell viability was measured by MTT assay and expressed as the percent-
age of control. Data are represented as the means ± standard deviation of at least three separ-
ꢂ
ꢂꢂ
ate experiments. p value was calculated vs untreated cells: p < 0.05, p < 0.01.
Results
iPA is cytotoxic for HMEC
We first studied the toxicity of iPA by culturing HMEC in a medium con-
taining increasing concentrations (from 0.1 to 10 mM) of this cytokinin for
72 hours. Using the MTT assay we analyzed the effect of this molecule on
cell vitality. iPA induces a significant dose dependent reduction of cell via-
bility (Figure 2a). Cytotoxicity was statistically significant at a concentration
of 0.1 lM with an Inhibitory Concentration 50 (IC50) ¼ 0.5 ± 0.2 lM.
[7–9]
Since 10 mM is cytotoxic for most neoplastic cells,
we studied the
effect of this concentration of iPA in HMEC seeded at different densities
2
(
from 7500 to 150000 cells/cm ). Indeed, under physiologic conditions the
endothelial cells form a confluent monolayer, but in tumor angiogenesis the
morphology of neo-formed vessels is chaotic and it is possible that the endo-
[22]
thelium is discontinuous or also absent.
On these bases, we performed an
MTT assay in sparse and confluent cells and found that HMEC were more sen-
sitive to the toxic effect of iPA when seeded at low density (Figure 2b).
iPA induces cell cycle arrest and apoptosis
To understand the mechanisms of iPA-induced cytotoxicity, HMEC were
cultured in the presence of iPA (10 mM) for 24 h and cell cycle distribution
was analyzed by FACS. As shown in Figure 3a, iPA induced an accumula-
tion of HMEC in G1 phase. This finding paralleled with the increase of the
G1/S-CDK blocker and DNA synthesis inhibitor p21 as detected by western
blot (Figure 3b). Figure 3a also revealed the decrease of the cells in the S
and G2/M phases. The most significant result was the increase of the per-
centage of the sub-G1 cell population in iPA-treated vs untreated cells

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
Figure 3. iPA induces cell cycle arrest. (a) HMEC exposed to iPA (10 mM) for 24 h were analyzed
by FACS to study the cell cycle distribution compared to untreated cells. The data are shown as
the percentage of cells in sub-G1, G1, S and G2/M phases. Each bar is the mean of three separ-
ate experiments in triplicate ± standard deviation. (b) HMEC were exposed to iPA (10 mM) for 8
and 24 h. Cells extracts (100 mg/lane) were processed for western blot using antibodies against
p21. Actin is used as control of loading. Densitometric analysis was performed by the ImageJ
ꢂ
software and p21/actin ratio was calculated on three separate experiments. p < 0.05,
p < 0.01,
ꢂꢂ
ꢂꢂꢂ
p < 0.001.
(
from 13% to 43%). The sub-G1 peak in the histogram identifies the rela-
tive amount of apoptotic cells. Using Annexin V/PI staining we confirmed
a marked increase of the apoptotic cell population in iPA treated cells vs
controls (Figure 4a). In particular, about 29 and 50% of 1 and 10 mM iPA
treated cells resulted apoptotic (Annexin V positive and PI negative
cells þ Annexin V and PI positive cells). This finding prompted us to inves-
tigate the processing of caspase-3, which is a critical executioner of apop-
tosis. Indeed, caspase-3 is activated by proteolytic cleavage from an inactive
zymogen into activated p17 and p19 fragments. Western blot shows that
24 h exposure to iPA (1 and 10 mM) induced caspase-3 cleavage in a dose-
dependent fashion (Figure 4b).
Cytotoxic effect of iPA derivatives
To better understand the relationship between iPA structure and its bio-
logical activity, we treated HMEC with some iPA derivatives to compare

8
S. CASTIGLIONI ET AL.
Figure 4. iPA induces apoptosis. HMEC were exposed to iPA (1 and 10 mM) for 24 h. (a)
Annexin V/PI staining was performed and the cells were analyzed by flow cytometry. A repre-
sentative experiment is shown. (b) Cells extracts (100 mg/lane) were processed for western blot
using antibodies against cleaved caspase 3. Actin and total caspase 3 are used as control of
loading. Densitometric analysis was performed by the ImageJ software and cleaved caspase 3/
ꢂ
ꢂꢂ
ꢂꢂꢂ
p < 0.001.
actin ratio was calculated on three separate experiments. p < 0.05, p < 0.01,
their cytotoxic effect. Compounds reported in Figure 1 have been selected
as the more active molecules from a library of iPA derivatives synthesized
and assessed for their biological activity on cancer cells in our laborato-
[8,9,15,16]
ries.
On the basis of the results obtained with iPA (Figure 2), the
different compounds were assessed on sparse and confluent HMEC. By
MTT assay, we show that some compounds (1b, 1c, 1d, 1e, 4) exert a sig-
nificant toxic effect that is dependent on HMEC density (Figure 5a).
Indeed, sparse HMEC cells resulted more sensitive to these compounds.
We selected compounds 1b and 1c, which induced 80 and 50% reduction
of cell viability on sparse HMEC, respectively, for further studies. HMEC
were exposed to different doses of 1b and 1c, from 0.1 to 10 mM. Our
results demonstrate that the reduction of cell viability was dose dependent

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
2
Figure 5. Cytotoxic effect of some iPA derivatives. (a) Sparse (7500 cells/cm ) and confluent
2
(
37500 cells/cm ) HMEC were cultured for 72 h in the presence of iPA. Viable cells were eval-
uated by MTT assay. (b) MTT assay was performed on HMEC cultured in the presence of differ-
ent doses of iPA and its derivatives 1b and 1c.
Figure 6. The additive effect of iPA and its derivatives with IFNc. (a) HMEC were treated with
IFNc (1, 10, 100 IU/ml) and (b) with iPA and its derivatives 1b and 1c (1 mM) with or without
IFNc for 72 h. In a and b cell viability was measured by MTT assay and expressed as the per-
centage of control. Data are represented as the means ± standard deviation of at least three
ꢂ
ꢂꢂ
separate experiments. p < 0.05, p < 0.01.
(
Figure 5b) with an IC50 of 0.4 ± 0.1 lM for 1b and 3.5 ± 0.2 lM for 1c.
Moreover, using Annexin V/PI staining we show that 17 and 48% of 1 and
0 mM 1b treated cells or 16 and 34% of 1 and 10 mM 1c treated cells were
1
apoptotic (Figure 4a)
The effect of iPA and its derivatives in association with IFNc
We also investigated the cytotoxicity of iPA and its derivatives added in
combination to the culture medium of HMEC. Indeed, among its multiple

1
0
S. CASTIGLIONI ET AL.
[23]
functions, IFNc inhibits endothelial growth and migration.
Initially, we
studied HMEC sensitivity to IFNc. IFNc significantly impairs HMEC via-
bility at a concentration of 100 IU/ml (Figure 6a). Using this concentration
we demonstrate a significant additive effect with iPA 1 mM and its deriva-
tives 1b and 1c (Figure 6b).
Discussion
In vivo microvascular endothelial cells are quiescent with a turnover rate of
[24]
approximately once every three years.
In certain conditions, these quies-
cent cells are activated and initiate angiogenesis, i.e. the formation of new
vessels from preexisting ones. Angiogenesis is fundamental in physiological
[25]
processes, from embryonic development to wound repair.
However,
excessive angiogenesis contributes to the pathogenesis of various diseases,
among which cancer, blinding retinopathies, psoriasis, arthritis, endometri-
[
25]
osis and Crohn’s disease.
Also virus and bacteria carry angiogenic genes
[26]
or upregulate angiogenic genes in the host.
Several antiangiogenic drugs
with various indications are available or in various phases of clinical trials,
but there is constant need of new molecules that target microvascular
endothelial cells. Considering the importance of angiogenesis for tumor
growth and progression and the significant anti-proliferative activity of iPA
on a large number of tumor models, we analyzed iPA effects on human
microvascular endothelial cells and found that it induces a dose dependent
reduction of their viability. This is particularly evident in sparse cells. It is
noteworthy that HMEC are very sensitive to the cytotoxic effect of iPA,
with an IC50 ¼ 0.5 lM, while tumor cells usually require higher concentra-
tions. For instance, iPA toxic effect in bladder cancer T24 cells is signifi-
[
8]
cant at 1 mM, with an IC50 around 3 mM. As previously shown in T24
[
8]
cells, also in HMEC, iPA promotes apoptosis as detected by FACS. In
agreement with these results, we observed dose dependent caspase-3 cleav-
age. Interestingly, p21, which is considered a marker of endothelial senes-
[
27]
cence,
is upregulated by iPA. It is noteworthy that senescent endothelial
cells are more susceptible to apoptosis than their young counterparts
because of the reduced activity of the anti-apoptotic endothelial nitric oxide
[28]
synthase (eNOS).
Therefore, we hypothesize that the induction of
apoptosis might take place in two steps. Some cells immediately undergo
apoptosis, others acquire a senescent phenotype that sensitizes them to
pro-apoptotic stimuli.
We also studied the toxic effects of a series of iPA derivatives with modi-
fication of the alkylation in N6-position and of the base moiety. HMEC
were exposed for 72 h to 11 different iPA derivatives and cell viability was
analyzed. Only five of the N6-substituted adenosine analogs tested affected

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
cell viability, while the other modifications of iPA structure abolished iPA
biological activity. In particular, compounds 1b and 1c induce apoptosis in
a dose dependent fashion as detected by FACS analysis after Annexin V/PI
staining. The active compounds are characterized by side chains containing
at least three carbon atoms and the presence of an unsaturation in the
chain. However, it is important to underscore that the five active com-
pounds are less cytotoxic than iPA.
It is noteworthy that iPA and its derivatives 1b and 1c show an additive
effect with IFNc, which is known to inhibit endothelial migration and
[
23]
growth, two crucial events in angiogenesis.
Our results suggest that iPA
and derivatives might be used in association with iPA to obtain a more
effective inhibition of endothelial cell proliferation.
On these bases, we conclude that iPA and some of its derivatives might
be interesting in an oncologic setting, since they hit both neoplastic and
microvascular endothelial cells. They might also be utilized in any other
condition characterized by excessive angiogenesis.
Acknowledgements and Funding
This work was partially supported by: “Universit ꢀa di Milano, Linea 2 – Azione A
2016-2017”.
Disclosure statement
No potential conflict of interest was reported by the author(s).
References
[
[
[
1] Faust, J. R.; Dice, J. F. Evidence for Isopentenyladenine Modification on a Cell
Cycle-Regulated Protein. J. Biol. Chem 1991, 266, 9961–9970.
2] Kersten, H. On the Biological Significance of Modified Nucleosides in tRNA. Prog.
Nucleic Acid Res. Mol. Biol. 1984, 31, 59–114.
3] Spinola, M.; Galvan, A.; Pignatiello, C.; Conti, B.; Pastorino, U.; Nicander, B.;
Paroni, R.; Dragani, T. A. Identification and Functional Characterization of the
Candidate Tumor Suppressor Gene TRIT1 in Human Lung Cancer. Oncogene 2005,
2
4, 5502–5509.
[
[
[
4] Bifulco, M.; Malfitano, A. M.; Proto, M. C.; Santoro, A.; Caruso, M. G.; Laezza, C.
Biological and Pharmacological Roles of N6-Isopentenyladenosine: An Emerging
Anticancer Drug. Anticancer Agents Med Chem. 2008, 8, 200–2004.
5] Spinola, M.; Colombo, F.; Falvella, F. S.; Dragani, T. A. N6-Isopentenyladenosine: A
Potential Therapeutic Agent for a Variety of Epithelial Cancers. Int. J. Cancer. 2007,
1
20, 2744–2748.
6] Ishii, Y.; Hori, Y.; Sakai, S.; Honma, Y. Control of Differentiation and Apoptosis of
Human Myeloid Leukemia Cells by Cytokinins and Cytokinin Nucleosides, Plant
Redifferentiation-Inducing Hormones. Cell. Growth Differ 2002, 13, 19–26.

1
2
S. CASTIGLIONI ET AL.
[
7] Castiglioni, S.; Casati, S.; Ottria, R.; Ciuffreda, P.; Maier, J. A. N6-
Isopentenyladenosine and Its Analogue N6-Benzyladenosine Induce Cell Cycle
Arrest and Apoptosis in Bladder Carcinoma T24 Cells. Anticancer Agents Med
Chem. 2013, 13, 672–678.
[
8] Casati, S.; Ottria, R.; Baldoli, E.; Lopez, E.; J. A. M.; Ciuffreda, P. Effects of
Cytokinins, Cytokinin Ribosides and Their Analogs on the Viability of Normal and
Neoplastic Human Cells. Anticancer Res 2011, 31, 3401–3406.
[
9] Ottria, R.; Casati, S.; Maier, J. A. M.; Mariotti, M. Ciuffreda P. Novel
Isopentenyladenosine Analogues: Synthesis, Characterization, and Evaluation of
Antiproliferative Activity on Bladder Carcinoma Cells. Nucleos. Nucleot. Nucl 2009,
2
8, 736–751.
[
10] Ranieri, R.; Ciaglia, E.; Amodio, G.; Picardi, P.; Proto, M. C.; Gazzerro, P.; Laezza,
C.; Remondelli, P.; Bifulco, M.; Pisanti, S. N6-Isopentenyladenosine Dual Targeting
of AMPK and Rab7 Prenylation Inhibits Melanoma Growth through the
Impairment of Autophagic Flux. Cell Death Differ. 2018, 25, 353–367.
[
11] Ciaglia, E.; Grimaldi, M.; Abate, M.; Scrima, M.; Rodriquez, M.; Laezza, C.; Ranieri,
R.; Pisanti, S.; Ciuffreda, P.; Manera, C.; et al. The Isoprenoid Derivative N6-
Benzyladenosine CM223 Exerts Antitumor Effects in Glioma Patient-Derived
Primary Cells through the Mevalonate Pathway. Br. J. Pharm 2017, 174, 2287–2301.
12] Li, M.; Qi, Y.; Wei, J.; Lu, L.; Zhao, X.; Zhou, L. N6-Isopentenyladenosine Promoted
HeLa Cell Apoptosis through Inhibitions of AKT and Transforming Growth Factor
b-Activated Kinase 1 Activation. Tumour. Biol. 2017, 39, 101042831769596.
13] Pisanti, S.; Picardi, P.; Ciaglia, E.; Margarucci, L.; Ronca, R.; Giacomini, A.;
Malfitano, A. M.; Casapullo, A.; Laezza, C.; Gazzerro, P.; Bifulco, M. Antiangiogenic
Effects of N6-Isopentenyladenosine, an Endogenous Isoprenoid End Product,
Mediated by AMPK Activation. Faseb J 2014, 28, 1132–1144.
[
[
[
14] Aird, W. C. Endothelial Cell Heterogeneity. Cold Spring Harb Perspect Med 2012, 2,
a006429
6
[15] Ottria, R.; Casati, S.; Baldoli, E.; Maier, J. A. M.; Ciuffreda, P. N -Alkyladenosines:
synthesis and Evaluation of in Vitro Anticancer Activity. Bioorg. Med. Chem 2010,
1
8, 8396–8402.
[
[
[
[
[
[
16] Ottria, R.; Casati, S.; Manzocchi, A.; Baldoli, E.; Mariotti, M.; Maier, J. A. M.;
Ciuffreda, P. Synthesis and Evaluation of in Vitro Anticancer Activity of Some
Novel Isopentenyladenosine Derivatives. Bioorg. Med. Chem 2010, 18, 4249–4254.
17] Casati, S.; Manzocchi, A.; Ottria, R.; Ciuffreda, P. 1H, 13C and 15N NMR Spectral
Assignments of Adenosine Derivatives with Different Amino Substituents at
6
C position. Magn. Reson. Chem. 2011, 49, 279–283.
18] Casati, S.; Manzocchi, A.; Ottria, R.; Ciuffreda, P. 1H, 13C and 15N NMR
Assignments for N6-isopentenyladenosine/inosine analogues . Magn. Reson. Chem.
2
010, 48, 745–748.
19] Castiglioni, S.; Caspani, C.; Cazzaniga, A.; Maier, J. A. Short- and Long-Term
Effects of Silver Nanoparticles on Human Microvascular Endothelial Cells. World J.
Biol. Chem. 2014, 5, 457–464.
20] Castiglioni, S.; Cazzaniga, A.; Maier, J. A. Potential Interplay between NFjB and
PPARc in Human Dermal Microvascular Endothelial Cells Cultured in Low
Magnesium. Magnes. Res 2014, 27, 86–93.
21] Castiglioni, S.; Cazzaniga, A.; Perrotta, C.; Maier, J. A. Silver Nanoparticles-Induced
Cytotoxicity Requires ERK Activation in Human Bladder Carcinoma Cells. Toxicol.
Lett. 2015, 237, 237–243.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
[
[
22] Reitan, N. K.; Thuen, M.; Goa, P. E.; de Lange Davies, C. Characterization of
Tumor Microvascular Structure and Permeability: Comparison between Magnetic
Resonance Imaging and Intravital Confocal Imaging. J. Biomed. Opt. 2010, 15,
036004.
23] Maier, J. A.; Morelli, D.; Lazzerini, D.; M ꢀe nard, S.; Colnaghi, M. I.; Balsari, A.
Inhibition of Fibronectin-Activated Migration of Microvascular Endothelial Cells by
Interleukin-1alpha, Tumour Necrosis Factor Alpha and Interferon Gamma. Cytokine
1
999, 11, 134–139.
[
[
[
24] Foreman, K. E.; Tang, J. Molecular Mechanisms of Replicative Senescence in
Endothelial Cells. Exp. Gerontol. 2003, 38, 1251–1257.
25] Tahergorabi, Z.; Khazaei, M. A Review on Angiogenesis and Its Assays. Iran J Basic
Med. Sci. 2012, 15, 1110–1126.
26] Harada, K.; Lu, S.; Chisholm, D. M.; Syrj €a nen, S.; Schor, A. M. Angiogenesis and
Vasodilation in Skin Warts. Association with HPV Infection. Anticancer Res. 2000,
2
0, 4519–4523.
[
27] Bernardini, D.; Ballabio, E.; Mariotti, M.; Maier, J. A. Differential Expression of
EDF-1 and Endothelial Nitric Oxide Synthase by Proliferating, Quiescent and
Senescent Microvascular Endothelial Cells. Biochim. Biophys. Acta 2005, 1745,
265–272.
[
28] Rippe, C.; Blimline, M.; Magerko, K. A.; Lawson, B. R.; LaRocca, T. J.; Donato,
A. J.; Seals, D. R. MicroRNA Changes in Human Arterial Endothelial Cells with
Senescence: relation to Apoptosis, eNOS and Inflammation. Exp. Gerontol 2012, 47,
45–51.