Transcript Visualization and quantification of the reversal of

Visualization and quantification of the reversal of MDR by PSC833 in vitro and in vivo
Fei Shen and Leonard C. Erickson
Department of Pharmacology & Toxicology and IU Simon cancer center
Abstract
Doxorubicin accumulation in MDA-MB-435 cells
Multidrug Resistance (MDR) is one of the major reasons for anti-cancer
chemotherapy failure. The molecular mechanisms of MDR in cancer cells are
involved in the over-expression of ATP-Binding Cassette (ABC) transporters
on cell membranes. These transporters mediate the efflux of the structurally
and functionally unrelated anti-neoplastic drugs from cells and thereby
decrease intracellular drug accumulation. Using confocal microscopy we
have conducted in vitro and in vivo studies on the roles of P-glycoprotein
(Pgp), one of the ABC transporters, in the uptake and efflux of doxorubicin
(DOX) and mitoxantrone (MX), and on the effects of Pgp modulators in
MDR transduced human cancer MDA-MB-435 cells (MDR). IC50s of DOX
and MX in MDA-MB-435 wild type (WT) cells are 0.60 ± 0.04 µM and 0.16
± 0.008 µM, respectively. The MDR cells were about 9- and 8-fold more
resistant to DOX and MX than the WT cells. Intracellular accumulation of
DOX and MX in the MDR cells was only 19% and 33% of that in the WT
cells. Lower DOX net uptake in the nuclei and stronger DOX efflux in the
cytoplasm were the main reasons for the decreased DOX intracellular
accumulation in the MDR cells. Compared to the WT cells, MDR cells had
lower net uptake of MX in both nuclei and cytoplasm, which was the major
cause of the reduced MX intracellular accumulation in the cells. In MDAMB-435 tumor xenografts in living mice, the accumulation of DOX and MX
in MDR tumors was 68% and 42% of that in WT tumors. Pgp inhibitor,
PSC833 increased the accumulation of DOX and MX in MDR cells to 65 %
and 85% of that in the WT cells in vitro and reversed the fluorescent intensity
of DOX and MX in MDR tumors to 94 % of that in the WT tumors in vivo.
Taken together, Pgp causes reduced intracellular accumulation by decreasing
drug net uptake and/or increasing drug efflux and the Pgp inhibitor PSC833
reverses the effects of Pgp in cancer cells. In addition, Pgp processes
individual anticancer compounds differently, which may be related to the
anti-neoplastic mechanisms of these drugs.
5 uM DOX
3ug/ml PSC833
Accumulation of mitoxantrone in the nuclei
of MDA-MB-435 cells
Accumulation of mitoxantrone in the cytoplasm of MDA-MB435 cells
WT
mdr cyto
120
wt cyto
100
100
Fluorescent intensity
Fluorescent intensity
MDR
110
mdr+PSC833
110
MDR
90
80
70
60
90
80
70
60
Wt cells
100
50
mdr cells
90
Fluorescent intensity of Dox
m dr n u c
wt n u c
m dr+PS C 833
50
0
80
30
60
70
90
120
150
180
0
30
90
120
150
Ti m e (m i n )
Time (min)
60
60
50
40
30
Florescent intensity of Mitoxantrone and Doxorubicin in MDA-MB-435
Xenograft Tumors
*
20
10
0
5 µM MTX
Fluorescent
Intensity of MTX
(Mean ± SE)
% of WT
tumors
Fluorescent
Intensity of DOX
(Mean ± SE)
% of WT
tumors
None
WT
MDR
91.1 ± 27.3
38.0 ± 5.4
100
42*
75 ± 4.7
51 ± 6.0
100
68*
PSC833
WT
MDR
81.2 ± 12.6
75.8 ± 5.3
100
80 ± 4.6
76 ± 6.3
100
94
3 µg/ml PSC833 + 5 µM MTX
Pretreatment
Mitoxantrone accumulation in MDA-MB-435 cells
5 uM MTX
MDA-MB435 tumors
3ug/ml PSC833
Cytoxicity of mitoxantrone and doxorubicin and modulation of drug
resistance by PSC833 in human MDA-MB-435 cancer cells
94
WT
IC50 of DOX (µM)
WT
0.2 ± 0.01
1.0
0.60 ± 0.04
1.0
MDR
1.6 ± 0.13
8.0*
5.29 ± 0.85
8.8*
WT
0.2 ± 0.04
1.0
0.57 ± 0.04
1.0
None
PSC833
(3mg/ml)
MDR
0.2 ± 0.00
1.0
3.71 ± 0.47
*Statistically significant difference (p < 0.05) in fluorescent intensity of doxorubicin in MDA-MB-435wt tumors
and MDA-MB-435mdr tumors.
RFa
Summary:
MDR
140
6.5*
*Statistically significant difference (P < 0.05) in drug resistance
a Resistance factor: IC of mitoxantrone and doxorubicin in MDR cells divided by IC of
50
50
same drug in the WT cells.
1. Human MDA-MB-435mdr cancer cells were 8- and 9-fold more resistant to mitoxantrone and
doxorubicin than the wild type cells.
2. Accumulation of mitoxantrone and doxorubicin in MDA-MB-435mdr cells were 33% and 19%
of that in the wild type cells.
3. Uptake of mitoxantrone in the cytoplasm and nuclei of MDA-MB-435mdr cells was less and
slower than that in the same intracellular compartments of MDA-MB-435wt cells.
4. Accumulation of mitoxantrone and doxorubicin in MDA-MB-435mdr xenograft tumors were
42% and 68% of that in the wild type xenograft tumors.
5. Pgp inhibitor, PSC833 increased the accumulation and uptake of mitoxantrone and doxorubicin
in MDA-MB-435mdr cells.
WT cells
Fluorescent intensity of MTX
Pre-treatment MDA-MB -435 cell IC50 of MTX (µM)
RFa
MDR cells
120
N= 15 cells/bar
100
80
60
*
40
20
0
5 µM MTX
3 µg/ml PSC833 + 5 µM MTX
180