38th FEBS Congress Maxim L. Bychkov Poster for abstract: "DR5-B – DR5-selective mutant variant of cytokine TRAIL overcomes resistance of cancer cells to TRAIL" Abstract book, p. 595

1. HCT 116
Agent: EC 50, ng/ml Death*, %
Control
TRAIL 0,82±0,23 38,85±3,31
DR5-B 1,17±0,14 67,38±1,98
Taxol
(5 nM)
TRAIL 0,47±0,29 57,29±4,43
DR5-B 0,48±0,12 88,49±3,89
Bortezomib
(1 nM)
TRAIL 1,07±0,13 97,50±1,27
DR5-B 0,01±0,02 97,95±1,27
Doxorubicin
(100 nM)
TRAIL 0,56±0,25 43,00±2,21
DR5-B 1,05±0,10 93,03±3,06
Jurkat
Agent: EC 50, ng/ml Death*, %
Control
TRAIL 0,30 ± 0,06 45,14±0,40
DR5-B 0,08 ± 0,03 67,57±3,28
Taxol
(0,1 nM)
TRAIL 0,1 ± 0,05 67,41±3,07
DR5-B 0,07 ± 0,02 88,17±6,17
Bortezomib
(0,1 nM)
TRAIL 0,07 ± 0,05 55,87±3,26
DR5-B 0,05 ± 0,02 76,47±4,12
Doxorubicin
(0,1 nM)
TRAIL 0,1±0,05 63,49±2,04
DR5-B 0,09±0,03 75,46±3,47
U 937
Agent: EC 50, ng/ml Death*, %
Control
TRAIL 0,81±0,03 19,14±4,08
DR5-B 0,08±0,02 39,57±4,34
Taxol
(1 nM)
TRAIL 0,07±0,01 54,96±4,25
DR5-B 0,068±0,03 69,54±3,00
Bortezomib
(1 nM)
TRAIL 0,08±0,01 66,03±1,67
DR5-B 0,1±0,06 95,47±6,74
Doxorubicin
(100 nM)
TRAIL 0,1±0,02 60,45±5,05
DR5-B 0,072±0,05 96,65±0,89
A 549
Agent: EC 50, ng/ml Death*, %
Control
TRAIL 1,03 ± 0,08 16,41±1,05
DR5-B 0,12 ± 0,03 35,55±3,22
Taxol
(100 nM)
TRAIL 1,00 ± 0,07 44,37±3,34
DR5-B 0,09 ± 0,04 74,02±3,19
Bortezomib
(50 nM)
TRAIL 0,90 ± 0,07 62,12±3,42
DR5-B 0,09 ± 0,03 88,52±5,01
Doxorubicin
(100 nM)
TRAIL 0,073±0,03 42,22±3,19
DR5-B 0,070±0,04 65,91±3,35
HT 29
Agent: EC 50, ng/ml Death*, %
Control
TRAIL 0,72±0,02 5,43±0,06
DR5-B 0,54±0,22 40,64±0,09
Taxol
(1 nM)
TRAIL 0,09±0,06 37,62±2,54
DR5-B 0,08±0,05 82,12±1,34
Bortezomib
(1 nM)
TRAIL 0,10±0,09 45,31±3,35
DR5-B 0,10±0,05 82,01±4,28
Doxorubicin
(100 nM)
TRAIL 0,07±0,02 46,51±1,70
DR5-B 0,08±0,05 75,27±4,08
MCF-7
Agent: EC 50, ng/ml Death*, %
Control
TRAIL 1,16±0,07 11,02±0,40
DR5-B 0,45±0,12 53,52±2,02
Taxol
(100 nM)
TRAIL 0,078±0,01 52,33±4,03
DR5-B 0,08±0,05 83,28±4,52
Bortezomib
(10 nM)
TRAIL 0,08±0,02 38,23±3,09
DR5-B 0,095±0,03 96,62±4,23
Doxorubicin
(100 nM)
TRAIL 0,96±0,02 39,18±3,03
DR5-B 0,5±0,05 69,63±3,33
0
5
10
15
20
25
HCT116 U937 Jurkat A549 HT29 MCF-7
FluorescenceG-mean
DR5
DR4
DcR1
DcR2
-5
5
15
25
35
45
55
0 0,1 0,5 1 5 10 50 100 5001000
Celldeath,%
TRAIL / DR5-B, ng/ml
HT 29 (colorectal carcinoma)
TRAIL
DR5-B
0
10
20
30
40
50
60
70
80
90
0 0,1 0,5 1 5 10 50 100 5001000
Celldeath,%
TRAIL / DR5-B, ng/ml
HCT116 (colorectal carcinoma)
TRAIL
DR5-B
0
10
20
30
40
50
60
70
0 0,1 0,5 1 5 10 50 100 5001000
Celldeath,%
TRAIL / DR5-B, ng/ml
Jurkat (T cell leukemia)
TRAIL
DR5-B
0
10
20
30
40
50
0 0,1 0,5 1 5 10 50 100 5001000
Celldeath,%
TRAIL / DR5-B, ng/ml
A 549 (lung carcinoma)
TRAIL
DR5-B
0
10
20
30
40
50
60
70
80
0 0,1 0,5 1 5 10 50 100 5001000
Celldeath,%
TRAIL / DR5-B, ng/ml
MCF-7 (breast adenocarcinoma)
TRAIL
DR5-B
DR5-B – DR5-selective mutant variant of cytokine TRAIL
overcomes resistance of cancer cells to TRAIL.
Conclusions:
1.Recombinant preparation of DR5-selective TRAIL mutant variant DR5-B induces
apoptosis of TRAIL sensitive cells more effectively than wild type TRAIL.
2.Utilization of DR5-selective TRAIL mutant variant DR5-B can overcome the
resistance of TRAIL-insensitive colon, breast and lung cancer cells.
3.Chemotherapeutic agents (bortezomib, taxol, and doxorubicin) greatly increase
the efficacy of TRAIL and DR5-B on different TRAIL-sensitive and TRAIL-
insensitive cancer cell lines.
4.TRAIL and DR5-B preparations are non-toxic to normal cells even in
concentrations of 15 µg/ml.
Introduction
Cytokine TRAIL (Apo2L) induces apoptosis by binding to death receptors
DR4 and DR5 in broad spectrum of cancer cells while sparing normal cells (1), so
recombinant TRAIL preparations are considered as potential anti-cancer drugs (2).
However, many tumor cell lines are resistant to TRAIL, despite of DR4 and DR5
expression, partially due to the competition between death and decoy receptors
(membrane-bound DcR1, DcR2 and soluble OPG) for binding to TRAIL (3).
Recently, unique DR5-selective mutant variant of TRAIL (DR5-B) was generated in
our laboratory. DR5-B practically doesn't interact with decoy receptors and DR4
(4).
The aim of this work was to investigate the cytotoxic activity of recombinant
preparations of wild type TRAIL and its DR5-selective mutant variant DR5-B on
TRAIL-sensitive and resistant cancer cell lines, and also to study cytotoxicity of
these preparations on normal human cell lines.
Methods
Surface expression of TRAIL receptors was examined by flow cytometry (Becton
Dickinson), cytotoxicity of recombinant TRAIL and DR5-B preparations was studied
using MTT assay. Apoptotic cell death was confirmed by fluorescent microscopy
(Hoerst 33342 cell nuclei staining).
Thus, DR5 selective mutant variant of TRAIL DR5-B can be used as a powerful tool in therapy of wild type TRAIL resistant cancers.
Maxim L. Bychkov*, Marine E. Gasparian**, Dmitry A. Dolgikh*, Mikhail P. Kirpichnikov* (Moscow, Russia)
TRAIL
variants
KD,10-9 M
DR5 DR4 DcR1 DcR2 OPG
Wild type 0.51 ± 0.028 0.46 ± 0.014 1.09 ± 0.069 0.36 ± 0.009 0.99 ± 0.017
DR5-B 0.71 ± 0.013 NB NB 101 ± 7.0
143 ± 13*
221 ± 19*
Table. 1. Dissociation constants of TRAIL and DR5-B, measured by SPR (acc. to 4).
* Determined by equilibrium plots, NB – not binding
References:
1. Ashkenazi A., Rai R.C., Fong S. et al. (1999). Safety and antitumor
activity of recombinant soluble Apo2 ligand. J. Clin. Invest.; 104: 155
– 162.
2. Micheau O., Shirley S., Dufour F. (2013). Death receptors as targets
in cancer. Br J Pharmacol. 2013 May 3 . [Epub ahead of print].
3. Wajant H, Gerspach J, Pfizenmaier K. 2011. Engineering death
receptor ligands for cancer therapy. Cancer Lett. 2011 Jan 13. [Epub
ahead of print].
4. Gasparian M.E., Chernyak B.V., Dolgikh D.A. et al. (2009).
Generation of new TRAIL mutants DR5A and DR5B with improved
selectivity to death receptor 5. Apoptosis; 14: 778 – 787.
Table 2. Effective concentrations of TRAIL and
DR5-B on Jurkat, U 937 and HCT 116 cells alone
and in co-incubation with chemotherapeutic
agents (24-hour incubation).
Table 3. Effective concentrations of TRAIL and
DR5-B on MCF-7, HT 29 and A 549 cells alone
and in co-incubation with chemotherapeutic
agents (24-hour incubation).
Fig. 5. Viability of MCF-7, HT 29 and
A 549 cell lines after 24-hour
incubation with TRAIL and DR5-B.
Fig. 4. Viability of Jurkat, U 937 and
HCT 116 cell lines after 24-hour
incubation with TRAIL and DR5-B.
Fig. 1. Expression of TRAIL membrane receptors on surface of different cancer cell lines.
0
1
2
3
4
5
6
7
HUVEC HFF
FluorescenceG-mean
DR5
DR4
DcR1
DcR2
0
20
40
60
80
100
120
0
0,1
1
10
100
500
1000
2500
5000
7500
10000
15000
Cellviability,%
TRAIL/DR5-B, ng/ml
Human Foreskin Fibroblasts
TRAIL
DR5-B
0
20
40
60
80
100
120
0
0,1
1
10
100
500
1000
2500
5000
7500
10000
15000
Cellviability,%
TRAIL/DR5-B, ng/ml
HUVEC (Endothelial cells)
TRAIL
DR5-B
Fig. 3. Viability of normal fibroblasts and
endothelium cells after 24-hour incubation
with TRAIL and DR5-B.
Fig. 2. Expression of TRAIL membrane receptors
on surface of normal cell lines.
0
10
20
30
40
50
60
0 0,1 0,5 1 5 10 50 100 5001000
Celldeath,%
TRAIL / DR5-B, ng/ml
U 937 (monoblastic leukemia)
TRAIL
DR5-B
Results
DR5-B induced apoptosis 1.3-10-fold more effectively than wild type TRAIL either
in TRAIL-sensitive or in TRAIL-resistant cell lines which express all TRAIL membrane
receptors. Combinational treatment of cancer cell lines with chemotherapeutic agents
such as doxorubicin, taxol and bortezomib (1-100nM) and TRAIL or DR5-B preparations
resulted in more than 90 % apoptosis of Jurkat, HCT116 and U937 cells. Chemotherapeutic
agents enhanced DR5-induced apoptosis of TRAIL-resistant cell lines A549, HT29 and MCF-
7 much more than wild type TRAIL-induced cell death (maximal cell death 65-98 % and 35-
60 % for DR5-B and wild type TRAIL respectively). Both preparations of DR5-B and wild
type TRAIL were nontoxic to normal fibroblasts and endothelial cells (HUVEC) expressing
all TRAIL receptors.
These results demonstrate that DR5-selective TRAIL mutant variant DR5-B can
be used for therapy of TRAIL-resistant cancers.
1. TRAIL receptors are expressed either on TRAIL-sensitive or on TRAIL-resistant cell
lines.
3. DR5-B is 3-4 fold more active than wild type TRAIL on different cancer cell lines alone, as well as in combination with
chemotherapeutic agents (bortezomib, taxol and doxorubicin).
2. TRAIL and DR5-B preparations
are non-toxic to normal cells,
expressing all membrane TRAIL
receptors.
*Lomonosov Moscow State University. Biological faculty, department of bioengineering. 119234, Moscow, Russia. e-mail: maksim.bychkov@gmail.com
**Federal State Institution of science Institute of bioorganic chemistry RAS. Laboratory of protein engineering. 117997, Moscow, Russia. +7 (929) 910 86 26
*Death after 24-hour incubation with 100 ng/ml of TRAIL or DR5-B *Death after 24-hour incubation with 100 ng/ml of TRAIL or DR5-B