New Hybrids Derived from Podophyllic Aldehyde and Diterpenylhydroquinones with Selectivity toward Osteosarcoma Cells

Article abstract of DOI:10.1021/acsmedchemlett.7b00493

A new family of molecular hybrids, between cyclolignans related to podophyllic aldehyde and several diterpenylnaphthohydroquinones (DNHQ), was prepared and its biological activity evaluated in several human solid tumor cell lines, which are representative of the most prevalent solid tumors in the Western world. Both cyclolignan and quinone fragments were linked through aliphatic or aromatic spacers. The new hybrid family was evaluated for its cytotoxicity, and it was found that the hybrids were several times more potent against the osteosarcoma cell line MG-63 than against MCF-7 and HT-29 cell lines. The presence of an aromatic ring in the linker gave the most potent and selective agent, improving the cytotoxicity of the parent compounds. Cell cycle studies demonstrated that this hybrid induces a strong and rapid apoptotic effect and arrests cells at the G2/M phase of the cell cycle, in the same way that the parent compound podophyllic aldehyde does.

Full text of DOI:10.1021/acsmedchemlett.7b00493

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NEW HYBRIDS DERIVED FROM PODOPHYLLIC
ALDEHYDE AND DITERPENYLHYDROQUINONES WITH
SELECTIVITY TOWARDS OSTEOSARCOMA CELLS
Angela P. Hernández, Paula Díez, Pablo A. García, José M. Miguel del Corral, Martín
Pérez-Andrés, David Diez, Arturo San Feliciano, Manuel Fuentes, and Mª Angeles Castro
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00493 • Publication Date (Web): 08 Mar 2018
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NEW HYBRIDS DERIVED FROM PODOPHYLLIC ALDEHYDE
AND DITERPENYLHYDROQUINONES WITH SELECTIVITY
TOWARDS OSTEOSARCOMA CELLS
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a,b
c,d
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a
Ángela P. Hernández , Paula Díez , Pablo A. García , José M. Miguel del Corral , Martín Pérez-
c
b
a
c,d
a
Andrés , David Díez , Arturo San Feliciano , Manuel Fuentes * and Mª Ángeles Castro *
a
Departamento de Ciencias Farmacéuticas, Área de Química Farmacéutica, Facultad de Farmacia, CIETUS/IBSAL, Campus
Miguel de Unamuno, University of Salamanca, 37007 Salamanca, Spain.
b
Departamento de Química Orgánica, Facultad de Ciencias Químicas, University of Salamanca, 37008 Salamanca, Spain.
c
Department of Medicine and General Cytometry Service-Nucleus, Cancer Research Centre (IBMCC/CSIC/USAL/IBSAL).
37007 Salamanca, Spain.
d
Proteomics Unit. Cancer Research Centre (IBMCC/CSIC/USAL/IBSAL), 37007 Salamanca, Spain
KEYWORDS Cyclolignans; hybrids; podophyllic aldehyde; diterpenylnaphthohydroquinones; cytotoxicity; apoptosis; cell cy-
cle; osteosarcoma.
ABSTRACT: A new family of molecular hybrids, between cyclolignans related to podophyllic aldehyde and several diterpenylnaph-
thohydroquinones (DNHQ), was prepared and its biological activity evaluated in several human solid tumor cell lines, which are
representatives of the most prevalence solid tumors in Western world. Both, cyclolignan and quinone fragments, were linked
through aliphatic or aromatic spacers. The new hybrid family was evaluated for its cytotoxicity and it was found that the hybrids
were several times more potent against the osteosarcoma cell line MG-63 than against MCF-7 and HT-29 cell lines. The presence
of an aromatic ring in the linker gave the most potent and selective agent, improving the cytotoxicity of the parent compounds.
Cell cycle studies demonstrated that this hybrid induces a strong and rapid apoptotic effect and arrests cells at the G2/M phase of
the cell cycle, in the same way that the parent compound podophyllic aldehyde does.
Hybridation is a useful strategy to generate new multifunctional
compounds by combining diverse chemical structures or frag-
ments. The junction of different bioactive molecules can provide
an unlimited number of combinations, which could greatly in-
previous cytotoxicity studies with two different types of natural
14,15,16
17,18,19
products, cyclolignans
showed a promising selectivity against colon carcinoma cell
and terpenylhydroquinones,
14,15
17,18
lines
and melanoma and ovarian carcinoma cell lines
1
crease the chemo-diversity of drug-like molecules. In this con-
respectively.
text, natural products are promising and efficient starting mate-
rials for the discovery and development of new drugs particularly
in the anticancer field; more than 70% of the current anticancer
Scheme 1: Structure of podophyllotoxin (1), podophyllic alde-
hyde (2), myrceocommunic acid (3), and some diterpenylhydro-
quinone derivatives (4-7) previously prepared by us.
2
drugs are natural or derived of natural products with a variety of
structures and mechanisms of action that would increase when
3
they are combined into new hybrid entities. The synergistic or
additive effect of those new chemical entities is a promising
4-10
approach in diverse diseases, particularly in cancer therapy,
for example to avoid resistances or discover new applications for
1
1
future treatments.
Among all the cancer types described, osteosarcoma is a very
rare bone cancer that appears in children and young adults. This
disease is particularly aggressive, presenting a high risk of metas-
1
2
tasis. Current treatments for this cancer combine surgery with
chemotherapy. Despite the improvements related to immuno-
modulation or early diagnosis, selective treatments continue
13
playing an important role in the therapy of this disease. Our
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To synthesize our objective compounds, the presence of a car-
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boxylic group in each precursor is necessary to join the linkers.
About the cyclolignans, podophyllotoxin, 1, can be easily trans-
formed into picropodophyllic acid, as previously described by
16
us. In order to avoid the re-lactonization to the corresponding
cis-lactone during esterification, picropodophyllic acid was pro-
24
tected as acetonide to get the acid 8. This protecting group can
be easily removed after condensation with the alkylated DNHQ
9
-11 (Scheme 2).
With regards to DNHQs, compound 5 was obtained by Diels-
Alder condensation of 3 and p-benzoquinone, followed by acety-
17,19
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lation.
Aromatization of 5 with DDQ gave 7 that was treated
with 1,6-dibromohexane, to obtain the DNHQ 10 in low overall
yield (21% from 5). We observed that aromatization to give 7
became a limiting step, which could be due to possible interfer-
ences between the free carboxylic group and the work up used
to remove the DDQ. In view of that, we decided to incorporate
the linker before aromatization. Thus, DNHQ 5 was treated with
the dibromoderivatives and subsequent aromatization of the
naphthalene core yielded the expected DNHQs 9-11 in accepta-
ble overall yields (43-61% from 5).
This fact prompted us to explore their effects on other cancer
lines, as osteosarcoma, and design a new family of hybrids,
named “lignohydroquinones”, by junction of both structures
looking for a dual mechanism of action. Previous cyclolignan
docking experiments revealed that bulky groups at C-9/C-9’
position of the cyclolignan skeleton can adopt different confor-
mations when interact with the colchicine-binding site, leaving a
free space that can allow to accommodate larger groups as those
The last steps of the synthetic route include the condensation of
the two fragments of the hybrid compounds and the final trans-
formation of the cyclolignan fragment into the α,β-unsaturated
aldehyde to get the lignohydroquinones, analogues of the podo-
phyllic aldehyde, 2. Thus, DNHQs 9-11 reacted with the ace-
tonide-acid 8 in DMF and potassium carbonate to get the conju-
gates 12-14 that were deprotected with p-TsOH and oxidized
20, 21
designed in this work.
Podophyllotoxin, 1, was the starting material for the cyclolignan
16
fragment, from which podophyllic aldehyde, 2, was obtained.
Additionally, the terpenoid myrceocommunic acid, 3, was used
19
previously to obtain the DNHQs 4-7, (Scheme 1). Both precur-
sors showed antineoplastic activity with different mechanisms of
action. Podophyllic aldehyde arrests the cell cycle in the G2/M
16
under Swern conditions to obtain the final hybrids 15-17 in
acceptable yields.
1
6
phase at sub-micromolar concentrations whereas DNHQs, as 6,
are supposed to work through a potent pro-oxidant effect that
Scheme 2: Synthesis of the lignohydroquinones 15-17 from the
corresponding natural products
22
induces cell death.
Preparation of the new lignohydroquinones and the synergistic
effect and improvement of the cytotoxicity against osteosarcoma
cell line MG-63 are presented here.
The general structure of the prepared lignohydroquinones is
depicted in Scheme 2. As it can be seen, both precursors are
connected through aliphatic or aromatic linkers by ester bonds,
maintaining the α,β-unsaturated aldehyde of the cyclolignan
fragment and the naphthohydroquinone diacetate moiety, due
to the high relevance of these features in their antineoplastic
cytotoxicity.
Thus, the natural compound podophyllotoxin, 1, isolated from
the resin of rhizome of Podophyllum emodi Wall. ex Hook.f. &
14
Thomson, (fam. Berberidaceae) and myrceocommunic acid, 3,
isolated from berries of Juniperus oxycedrus L. (fam. Cupres-
17
saceae), were the very starting materials of the new family of
lignohydroquinones.
Among the possible strategies to prepare the final lignohydro-
quinone hybrids, two were considered depending on whether
the linker is firstly connected to the cyclolignan and then con-
densed with the DNHQ or the linker is firstly joined to the DNHQ
and condensed later with the cyclolignan. Bearing in mind our
23
previous expertise with lignan-hybrids, it was decided to
choose the synthetic route that begins alkylating the DNHQ-
carboxylic acid with the linker, followed by reaction with the
cyclolignan 8, leaving for the last step the formation of the alde-
hyde group by oxidation under Swern conditions. As linker rea-
gents, α,ω-dibromoderivatives (1,3-dibromopropane, 1,6-
dibromohexane and α,α’-dibromoxylene) have been chosen in
order to analyse the effect of the spacer size and flexibility on the
bioactivity of the new hybrids.
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Reagents and conditions: (i) (1) KOH/MeOH (2) HCl/H O, pH=4
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(3) PTS, 2,2-DMP/Acetone; (ii) (1) p-BQ, BF /Et O (2) Ac O/pyr
P
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(3) Br-linker-Br, K CO /DMF (4) DDQ/Toluene; (iii) K CO /DMF;
2
3
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(iv) (1) p-TsOH, H O/Acetone (2) Swern
2
To determine the efficiency of the hybridation strategy in the
design of new drugs, the cytotoxicity of the new hybrids was
evaluated by MTT assay and then, flow cytometry experiments
were performed to deepen into the mechanism of action of the
new conjugates. In both cases, three tumor cell lines where
tested: MCF-7 (breast cancer), MG-63 (osteosarcoma) and HT-29
(
colon adenocarcinoma). These hybrids maintain the α,β-
1
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unsaturated aldehyde function present in the podophyllic alde-
hyde that has been the lead compound for this work. The precur-
sors podophyllic aldehyde, 2, DNHQ 6 and the starting cyclo-
lignan podophyllotoxin, 1, were also included in the assays for
comparison purposes.
Table 1: Cytotoxicity (IC , ꢀM) of the evaluated compounds by MTT assay and flow cytometry.
5
0
(
A) MTT assay (72 h)
MG-63
(B) Flow cytometry (24 h)
Comp
MCF-7
HT-29
MCF-7
MG-63
HT-29
1
2
6
0.028 ± 0.003
0.48 ± 0.03
0.007 ± 0.005
0.06 ± 0.02
0.41 ± 0.08
2.23 ± 0.40
0.066 ± 0.002
0.029 ± 0.006
0.018 ± 0.002
1.51 ± 0.43
>100
>100
18.3 ± 1.4
1.23 ± 0.06
1.16 ± 0.47
58.2 ± 1.0
>100
>100
0.015 ± 0.001
2.21 ± 0.05
29.9 ± 0.7
>100
5.65 ± 0.07
>100
1
5
1
1
6
7
1.00 ± 0.06
1.03 ± 0.14
2.06 ± 0.72
1.91 ± 0.95
1.44 ± 0.30
>100
>100
44.6 ± 0.2
57.6 ± 0.1
>100
0.15 ± 0.01
0.50 ± 0.29
25
By MTT assay, all the compounds were cytotoxic on any of the
three cell lines tested and several considerations of structure-
activity relationship can be deduced (Table 1). The precursors, 1,
and 6, were highly cytotoxic in MTT assay (72 h of incubation),
reaching even nM levels in most of the cases, which is in accord-
mediates 9-11 were cytotoxics are the ꢀM range (Table S1) Con-
sidering the lignohydroquinone hybrids synthesized in this work,
15-17, all of them were cytotoxic on the tumor cell lines evaluat-
ed, although the nature and size of the linker have influenced its
potency. Lignohydroquinones with aliphatic linkers presented
similar IC50 values at the ꢀM range in all of the three tumor cell
2
16,17
ance with our previous reports
(Table 1, A). Also, the inter-
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lines tested. Interestingly, lignohydroquinone 17, with the aro-
cytotoxic potency of the hybrids. The differences in potency for
this lignohydroquinone respect to the other hybrids is clearly
observed when the cellular viability (%) of each cell line is plotted
against drug concentrations (from 0.1 ꢀM to 100 ꢀM) (Figure1)
In order to go deeply into the mechanism of action of the new
selective lignohydroquinone 17 in comparison with the parent
compounds, the apoptotic ability of precursors and final hybrids
was evaluated in MG-63 using the annexin/PI assay, which al-
lowed the differentiation among cells in early apoptosis (annex-
in+/PI-), late apoptosis and death (annexin+/PI+), and living cells
(annexin-/PI-) (Figure 2). The ratios for living cells (annexin-/PI-)
and total apoptotic cells (annexin+) calculated for podophyllotox-
in, 1, and podophyllic aldehyde, 2, were in agreement with our
previous results in which a delayed apoptosis caused by inhibi-
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matic linker, showed a cytotoxicity in MG-63 cell line at the sub-
ꢀM level (IC50 = 0.15 ꢀM), being almost seven to ten times more
potent against this cell line than against MCF-7 and HT-29 lines
(IC = 1.03 ꢀM and 1.44 ꢀM, respectively). These results indicat-
50
ed a certain degree of selectivity that could be associated with an
aromatic linker and, so that it merits further studies on its mech-
anism of action. Since the cytotoxicity of DNHQs was also ob-
17
served earlier, we decided to analyse the cell growth inhibitory
26, 27
effect
over a shorter time of incubation (24 h) by flow cy-
tometry, with the aim to know if the new hybrids maintain or
even enhance the properties and potency of their precursors
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(
Table 1, B). In this assay, only the DNHQ 6 was cytotoxic on the
tree cell lines tested but none of the cyclolignans or hybrids were
cytotoxic on MCF-7 and HT-29 cells at the maximum concentra-
tion tested; only the hybrid 16, with the longer aliphatic spacer,
16
tion of tubulin polymerization was observed. In our assays (Fig
2 and Table S2), hybrids 15 and 16 lacked important apoptotic
effect, while 17 induced a strong and rapid apoptosis as deduced
from the percentage of total apoptotic cells (89.96 %, Table S2).
Furthermore, the ratio between cells in early apoptosis (annex-
in+/PI-) and late apoptosis (annexin+/PI+) for compound 17 was
similar as that found for 2, (Table S2) which could be associated
with similar mechanism of action but with a quicker effect, prob-
ably related with the molecule conformation because of the
aromatic linker. Finally, cell cycle assays allow the characteriza-
tion of drug action mechanisms when involving changes in the
normal cell cycle progression. It is well known that cyclolignans
showed a moderate cytotoxicity against HT-29 cell line (IC50
=
5
7.6 ꢀM). However, all of them showed from moderate to very
good cytotoxicity against MG-63 osteosarcoma, which was the
most sensitive cell line to these compounds. In fact, the lead
cyclolignan 2 was nearly 15x fold more potent than natural prod-
uct 1 against MG-63 (IC50 = 1.23 ꢀM and 18.3 ꢀM, respectively);
and regarding the synthesized hybrids, those with the most
flexible aliphatic linkers, 15 and 16, were moderate active (IC50
=
5
8.2 and 44.6 ꢀM, respectively), and conjugate 17, which had a
more rigid aromatic spacer, was the most potent in the assay
with an IC50 under ꢀM (IC50 = 0.50 ꢀM). This IC50 value has im-
proved those found for the precursors 1, 2 and 6, and highlighted
the importance of the size and flexibility of the linker on the
16
as podophyllotoxin, 1, or podophyllic aldehyde, 2, induce tubu-
lin polymerization inhibition what is translated in a G2/M arrest,
while quinones hardly show any influence in cell cycle.
Figure 1: Cell viability results. Comparison of the effect of the lignohydroquinone hybrids (15-17) on the three studied tumour cell lines
HT-29, MCF-7, MG-63) at different drug concentrations (0.1-100 ꢀM) during 24 h.
(
To determine the presence of significant alterations in cell cycle
phases, our hybrid 17 was incubated for 24 h in MG-63 cells and
compared to the precursors and the other hybrids at 1 ꢀM con-
centration. The cell cycle assay revealed differential cell cycle
patterns depending on the drug. These results are depicted in
Figure 3 and Table S3.
The hybrid 16 showed practically the same cell cycle profile than
the control while the compound 15 and the DNHQ precursor 6
present a G2/M arrest, but it is not as remarkable as the effect of
hybrid 17. Treatment with 17 demonstrated to increase G2/M
cell cycle arrest by 5 folds (P < 0.001) compared to the control.
Indeed, all the cells treated with this drug were arrested in this
phase of the cell cycle. This increase in G2/M population and the
complete reduction in G0/G1 and S populations in 17 and podo-
phyllic aldehyde, 2, confirmed that the hybrid preserved the
tubulin inhibition effect associated to cyclolignans.
highlighted again the potency of the linker used to synthetize the
hybrid. Additionally, on line analysis of the drug-
likeness/ADME/toxicity properties for the hybrid 17, performed
through PreADMET algorithms (http://preadmet.bmdrc.org/),
showed interesting results related to the practical absence of
carcinogenicity (mouse, rat), mutagenicity (Ames test), environ-
mental and developmental (algae, insects, minnow fish, medaka
embryos) risks, and only low cardiac risk due to hERG inhibition,
while ensuring high human intestinal absorption, and adequate
permeability values on Caco2 adenocarcinoma cells and through
skin (Fig. S1-3). Thus, from the standpoint of the design, the
hybridation appears as a good strategy to discover new drugs
from the junction of different natural products.
The comparison of the G2/M population identified after the
incubation with hybrid 17 and the other compounds, 15 and 16,
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Figure 2: Effect of the cyclolignan precursors (1 and 2) and the
final hybrids (15-17) on the apoptosis of MG-63 cells treated with
1 ꢀM of the indicated compound and control with vehicle
(DMSO, <0.001%) for 24 h. Apoptosis was determined by annexin
V-PI staining by flow cytometry. (A) Annexin/PI diagrams. (B)
Percentage of Anexxin+ total and the corresponding ratio PI-/PI+.
The combination and even improvement of the precursor effects
were observed for compound 17 that bear an aromatic linker,
particularly against osteosarcoma cells in comparison with the
other cell lines tested. These results demonstrated the im-
portance of considering different linkers to join the two frag-
ments that form the hybrid.
Interestingly, this new family of hybrids could open new perspec-
tives for the treatment of osteosarcoma due to the good results
in terms of antineoplastic cytotoxicity and selectivity found in
this work. Obviously, much more medicinal chemistry research
including molecular design, calculation/prediction of physico-
chemical/ADME-Toxicity properties, followed by further synthe-
sis and re-assaying would be necessary. In this sense, several
studies are in progress towards the synthesis of better lignohy-
droquinones.
Figure 3: Effect of the precursors (2 and 6) and the lignohydro-
quinone hybrids (15-17) on DNA-ploidy determined by flow
cytometric analysis in MG-63 cell cultures. The cells were incu-
bated with 1 ꢀM of the indicated compound and control with
vehicle (DMSO, <0.001%) for 24 h. *** significantly different
from control at p < 0.001. (A).DNA content analyzed by fluores-
cence flow cytometry. (B) Percentage of the cell population at
the phase of the cell cycle (G0/G, S and G2/M).
ASSOCIATED CONTENT
The supporting Information is available free of charge via the
Internet at http://pubs.acs.org.
Synthetic procedures, characterization data for final com-
pounds and intermediates, biological assay methods and
cell cycle analysis.
AUTHOR INFORMATION
Corresponding Author
*E-mail: macg@usal.es
Author Contribution
All the authors have given approval to the final version of the
manuscript.
ACKNOWLEDGMENT
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Financial support came from Spanish MINECO (CTQ2015-68175-
R, AGL2016-79813-C2-2-R), Junta de Castilla León
(BIO/SA07/15) and ISCIII-RICET Network (RD06/0022/1004) co-
financed by the Fondo Social Europeo of the European Union
biological evaluation of new selective cytotoxic cyclolignans derived
from podophyllotoxin. J. Med. Chem. 2004, 47, 1214-1222.
15) Gordaliza, M.; Castro, M. A.; San Feliciano, A.; Miguel del Corral,
J. M.; López-Vázquez, M.L.; Gárcia-Grávalos, M. D.; “Antineoplasic
cyclolignan derivatives”. 1996, EP711767B1; US5834507A.
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y
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(
Fondos FEDER –EU. Fundación Solórzano (FS/23-2015) has also
(
16) Castro, M. A.; Miguel del Corral, J. M.; García, P. A.; Rojo, M. V.;
sponsored this work. The Proteomics Unit belongs to ProteoRed,
PRB2-ISCIII, supported by grant PT13/0001, of the PE I+D+I 2013-
de la Iglesia-Vicente, J.; Mollinedo, F.; Cuevas, C.; San Feliciano, A.
Synthesis and biological evaluation of new podophyllic aldehyde
derivatives with cytotoxic and apoptosis-inducing activities. J. Med.
Chem. 2010, 53, 983-993.
2
016, funded by ISCIII and FEDER. A.P.H. and P.D. are supported
by JCYL-EDU/346/2013 Ph.D. scholarships.
ABBREVIATIONS
BQ, benzoquinone; DDQ, 2,3-Dichloro-5,6-dicyano-p-
benzoquinone; 2,2-DMP, 2,2-dimethoxypropane; DNHQ,
diterpenylnaphthohydroquinones; PI, propidium iodide; PPTS,
pyridinium p-toluensulfonate.
(
17) Miguel del Corral, J. M.; Gordaliza, M.; Castro, M. A.; Mahíques,
M. M.; Chamorro, P.; Molinari, A.; García-Grávalos, M. D.; Broughton,
H. B.; San Feliciano, A. New selective cytotoxic diterpenylquinones
and diterpenylhydroquinones. J. Med. Chem. 2001, 44, 1257-1267.
(18) García-Grávalos, M. D.; Gordaliza, M.; Miguel del Corral, J. M.;
Mahíques, M. M.; San Feliciano, A. 1996, EP0731078-B1;
WO9604230-A1.
(19) Miguel del Corral, J. M.; Castro, M. A.; Rodríguez, M. L.; Chamo-
rro, P.; Cuevas, C.; San Feliciano, A. New cytotoxic diter-
penylnaphthohydroquinone derivatives obtained from a natural
diterpenoid. Bioorg. Med. Chem. 2007, 15, 5760-5774.
(20) A. San Feliciano; Castro, M. A.; López-Pérez, J.L.; del Olmo, E.
The importance of structural manipulation of natural compounds in
drug discovery and development. In Plant Bioactives and Drug
Discovery. John Wiley & Sons, 2012; pp. 127-160.
(21) Escudero-Martínez, J.M.; Pérez-Pertejo, Y.; Reguera, R.M.;
Castro, M.A.; Rojo, M.V.; Santiago, C.; Abad, A.; García, P.A.; López-
Pérez, J.L.; San Feliciano A. Int. J. Parasitol.:Drug and Drugs Resist.
2017, 7, 272-285.
(22) Lu, J. J.; Bao, J. L.; Wu, G. S.; Xu, W. S.; Huang, M. Q.; Chen, X. P.;
Wang, Y. T. Quinones derived from plant secondary metabolites as
anti-cancer agents. Anticancer Agents Med. Chem. 2013, 13, 456-
463.
(23) Castro, M. A.; Miguel del Corral, J. M.; García, P. A.; Rojo, M. V.;
Bento, A. C.; Mollinedo, F.; Francesch, A. M.; San Feliciano, A. Ligno-
purines: a new family of hybrids between cyclolignans and purines.
Synthesis and biological evaluation. Eur. J. Med. Chem. 2012, 58, 377-
389.
(24) Prokhorevich, K. N.; Bekish, A. V. Preparation of Isopropylidene
Acetals from Butane-1,2,4-Triol and its Cyclopropane Congeners
Tetrahedron Lett. 2010, 51, 4073-4075.
(25) Ferrari, M.; Fornasiero, M. C.; Isetta, A. M. MTT colorimetric
assay for testing macrophage cytotoxic activity in vitro. J. Immunol.
Methods 1990, 131, 165-172.
(26) Zaritskaya, L.; Shurin, M. R.; Sayers, T. J.; Malyguine, A. M. New
flow cytometric assays for monitoring cell-mediated cytotoxici-
ty. Expert Rev. Vaccines 2010, 9, 601-616.
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