1. No category

Lysosomal sequestration of chemotherapeutics
triggers lysosomal biogenesis and lysosomedependent cancer multidrug resistance
Prof. Yehuda G. Assaraf, Dean
Faculty of Biology,
Head, The Fred Wyszkowski Cancer Research Lab,
Technion, Haifa, Israel.
Shapira A, …Assaraf YG (2011)
Drug Resist ance Updates
Decreased
influx
Increased
efflux
Increased DNA repair
The most common cause of MDR is reduced drug accumulation due to
the overexpression of MDR efflux transporters.
Three major MDR efflux transporters
P-glycoprotein (P-gp/ABCB1)
Multidrug resistance-protein 1
(MRP1/ABCC1)
Breast cancer resistance
protein (BCRP/ABCG2)
Schematic structure and function of ABC transporters
A549/K1.5 cells
A hydroxyl group at position R1-3 is crucial for ABCG2 substrate recognition
Bram EE., Adar Y, et al (2009) Mol Pharmacol
OH
A549/K1.5 cells
IA nuclear staining
Planar, polycyclic hydrophobic
weak bases bearing intrinsic
fluorescence
A
!! Capable of DNA
intercalation
!! Topoisomerase II inhibitors
!! C-1311, Symadex - lead
compound completed
phase II
TA structure
clinical trials for the treatment
of colon and breast cancer.
!!
!ABCG2-PE Ab
R6
O
NH(CH2)nNRa,b
OH
O
HN
N
OH
O
HN
H
N
OH
R1
R2
N
R3
R14
NH
OH
IA structure
N
Mitoxantrone
Bram EE, AdarNHY., etHNal (2009) Mol Pharmacol
O
N
Net Fluorescence
Exclusion of IAs from MDR cells in the presence or absence of the
ABCG2 transport inhibitor fumitremorgin C (FTC)
A.
1000
Group A
1000
800
800
600
600
+FTC
-FTC
400
200
0
0
0,1
+FTC
-FTC
400
200
1
10
100
1000
0,1
1
Group A
45
30
15
0
100
Resistance fold
Accumulation fold
60
C.
Group B
10
100
1000
[C-1176] (!M)
[C-1584] (!M)
75
Group B
B.
Group A
D.
10
Group B
1
0.1
IA derivative
0.1
1
10
Accumulation fold
100
Aller, SG …and Chang, G (2009) Science.
Structural formulas of Imidazoacridinones (IAs) and
various polycyclic chemotherapeutic agents
A
O
NH(CH2)nNRa,b
R6
O
NH(CH2)nNRa,b
OH
O
HN
N
OH
O
HN
H
N
OH
R1
R1
R2
N
N
N
R3
R14
N
NH
OH
TA structure
HO
IA structure
N
O
N
HN
NH
N
O
O
N
O
O
B
Pheophorbide A (PheA)
HO
NH
N
H2N
N
OH
OH O
SN-38
Mitoxantrone
N
N
(CH3)2N
O
N
H
NH2
Methotrexate (MTX)
O
OH
OH
N(CH3)2
OH
OMe O
NH
OH
O
O
CH3O
H3C
NH2
OH
NHSO2CH3
m-AMSA
O
HO
O
N
O
O
Tetramethylrosamine
Doxorubicin
Imidazoacridinone
Log P ~ 0.5-1
Hydrophobic
pKa ~ 8-10
Weak base
IAs that are not MDR efflux transporter
substrates, undergo sequestration in acidic
organelles including lysosomes and endosomes
Colocalization
6.3 fold
!!
"-hexosaminidase, a lysosomal enzyme was previously used
as a bona fide functional indicator for the number of
lysosomes per cell. (Jansen, G. and Assaraf YG. Mol Pharmacol,
1999)
!!
MCF7 cells were exposed to increasing nanomolar
concentrations of Mitoxantrone. "-Hexosaminidase activity
was determined colorimetrically using 4-Nitrophenyl Nacetyl-"-D-glucosaminide as a chromogenic substrate.
treatment
treatment
GNS expression
Fold increas
!!
MCF7 Cells were
treated with
mitoxantrone for 72
hr.
Quantitative real-time
PCR was used to
examine the
expression levels of
Glucosamine (NAcetyl)-6-Sulfatase
(GNS) and Cathepsin D
(CTSD), two lysosomal
genes that were shown
to be up- regulated
upon activation of
lysosomal biogenesis.
(Palmieri, M. et al.,
2011)
5
4,5
4
3,5
3
2,5
2
1,5
1
0,5
0
0
30
100
Mitoxantrone [nM]
CTSD expression
Fold increas
!!
2
1,8
1,6
1,4
1,2
1
0,8
0,6
0,4
0,2
0
0
30
Mitoxantrone [nM]
100
!!
Lysosomal biogenesis
was found to be
triggered by the
nuclear translocation
of transcription factor
EB (TFEB). (Sardiello, M.
et al., 2009)
!!
Stable TFEB
transfectants termed
MCF7 TFEB-3XFLAG
cells were exposed to
a 24 hr pulse of 0.5
µM doxorubicin
(second row), 0.5 µM
Mitoxantrone (third
row) or 100 µM
Chloroquine (forth
row). Cells were fixed,
and stained with DAPI
(blue) and an antiFLAG antibody (green).
What is the driving force for the sequestration
of IA in lysosomes?
Lysosome
Cytoplasm
pH ~ 7.3
B
B
B
B
B
H+
B
BH+
B
B
B
ATP
H+
H+
ADP+Pi
V-ATPase
BH+
BH+
H+
BH+
H+
BH+
BH+
H+
BH+
B = Imidazoacridinone
H+
BH+
pH ~ 5
H+
A549/K1.5
A549
C-1330
Ammonium Chloride
Bafilomycin A1
+
-
+
+
-
+
+
A’
B’
C’
10 s
60 s
A
B
C
D
E
F
A549/K1.5
A549
0s
D’
E’
F’
Photosensitizer
(excited state)
Tissue oxygen
Fluorescence
(singlet oxygen, hydroxyl radical)
Reactive Oxygen Species
(ROS)
C-1330
Light
470nm (blue light)
Photosensitizer
(ground state)
Lysosomal rupture
Cytoxicity
Non illuminated
Three hrs post-illumination
Adar Y, Stark M., …Assaraf YG (2012) Cell Death and Dis
A549/K1.5
Cell Growth (% of control)
Lysosomal photodesruction markedly increases
the cytotoxic effect elicited by IAs
100
Non illuminated
illuminated
80
60
14 fold
40
20
0
0,1
1
10
100
1000
10000
100000
A549
Cell Growth (% of control)
[C-1330] (nM)
100
80
60
6 fold
40
20
0
0,1
1
10
100
1000
[C-1330] (nM)
10000
100000
Three major MDR efflux
transporters
P-glycoprotein (P-gp)
Multidrug resistanceprotein 1 (MRP1)
Breast cancer resistance
protein (BCRP)
Expanding the cytotoxic impact of IA photoexcitation
to other MDR tumor cell lines
Illumination
2008/MRP1
Growth inhibitory effect of C-1375 on parental tumor cells and their MDR
sublines.
C1375 LD50 (nM)
P-gp
MRP1
Fold
Survival at 1000 nM* (%)
Cell line
Control
Illumination
sensitization
Control
Illumination
Delta
SW1573
5535 ± 596
233 ± 5
24
68 ± 6
11 ± 2
57
SW1573/2R160
4384 ± 125
330 ± 56
13
74 ± 3
24 ± 4
50
2008
3280 ± 345
90 ± 9
36
72 ± 0.8
11 ± 4
61
2008/MRP1
5115 ± 889
101 ± 0.1
51
80 ± 2
13 ± 3
67
In vivo photodynamic therapy (PDT) after intravenous injection
of IA in CAM vasculature and in ovarian cancer grafted CAMs
!!As
hydrophobic weak bases, IAs undergo a
dramatic differential sequestration within
acidic lysosomes and endosomes.
!!Likewise,
approved hydrophobic weak base
anticancer drugs including doxorubicin and
sunitinib also undergo a marked
compartmentalization in lysosomes.
!!Lysosomal
sequestration of hydrophobic
weak base cytotoxic agents is an endogenous
mechanism of cell defense and inherent drug
resistance.
!! Sequestration
of photosensitizers such as IAs within
lysosomes can be efficiently exploited as a Trojan
Horse approach to hypersensitize MDR cells which
frequently contain an increased number of
lysosomes, upon illumination.
!! Lysosomal
ROS.
photodestruction occurs via generation of
!!IA-dependent
lysosomal photodestruction
proved efficient in eradication of human
ovarian cancer xenografts and their
associated vasculature in vivo using the CAM
model.
Acknowledgements
Yamit Adar
Dr. Eran Bram
Prof. Hubert van den Bergh (Luasanne)
Dr. Patrycja Nowak-Sliwinska (Luasanne)
Dr. Michal Stark
Prof. Arjan Griffioen (Amsterdam)
Benny Zhitomirsky
Prof. Tadeusz Sarna (Krakow)
Dr. Vicky Goler-Baron
Prof. Andrzej Skladanowski (Gdansk)
Dr. Nitzan Gonen
Dr. Inbal Lasry