Multidrug Resistance (MDR) is one of the major reasons for anti-cancer chemotherapy failure. The...
1
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 MDA-MB- 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. 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 Pre- t r e a t m e n t MDA-MB -435 cell IC 50 of MTX (µM) RF a IC 50 of DOX (µM) RF a None 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* PSC833 (3mg/ml) WT 0.2 ± 0.04 1.0 0.57 ± 0.04 1.0 MDR 0.2 ± 0.00 1.0 3.71 ± 0.47 6.5* Cytoxicity of mitoxantrone and doxorubicin and modulation of drug resistance by PSC833 in human MDA- MB-435 cancer cells *Statistically significant difference (P < 0.05) in drug resistance a Resistance factor: IC 50 of mitoxantrone and doxorubicin in MDR cells divided by IC 50 of same drug in the WT cells. Accum ulation ofm itoxantrone in the nuclei ofM D A-M B -435 cells 0 30 60 90 120 150 180 Tim e (m in) m dr nuc wtnuc m dr+PSC 833 Accum ulation of m itoxantrone in the cytoplasm of M DA-M B- 435 cells 50 60 70 80 90 100 110 120 0 30 60 90 120 150 180 Tim e(m in) Fluorescent intensity m drcyto wt cyto m dr+PSC833 5 uM MTX 3ug/ml PSC833 WT MDR 5 µM MTX 3 µg/ml PSC833 + 5 µM MTX * 0 20 40 60 80 100 120 140 WT cells MDR cells N= 15 cells/bar Fluorescent intensity of MTX Mitoxantrone accumulation in MDA-MB-435 cells Pretreatment MDA-MB- 435 tumors Fluorescent Intensity of MTX (Mean ± SE) % of WT tumors Fluorescent Intensity of DOX (Mean ± SE) % of WT tumors None WT 91.1 ± 27.3 100 75 ± 4.7 100 MDR 38.0 ± 5.4 42* 51 ± 6.0 68* PSC833 WT 81.2 ± 12.6 100 80 ± 4.6 100 MDR 75.8 ± 5.3 94 76 ± 6.3 94 Florescent intensity of Mitoxantrone and Doxorubicin in MDA-MB-435 Xenograft Tumors *Statistically significant difference (p < 0.05) in fluorescent intensity of doxorubicin in MDA-MB-435wt tumors and MDA-MB-435mdr tumors. Summary: 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. MDR WT Fluorescent intensity of Dox 5 uM DOX * 3ug/ml PSC833 0 10 20 30 40 50 60 70 80 90 100 Wt cells mdr cells 3 µg/ml PSC833 + 5 µM MTX 5 µM MTX Doxorubicin accumulation in MDA-MB-435 cells MDR
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Transcript of Multidrug Resistance (MDR) is one of the major reasons for anti-cancer chemotherapy failure. The...
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 MDA-MB-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.
Visualization and quantification of the reversal of MDR by PSC833 in vitro and in vivo
Fei Shen and Leonard C. EricksonDepartment of Pharmacology & Toxicology and IU Simon cancer center
Visualization and quantification of the reversal of MDR by PSC833 in vitro and in vivo
Fei Shen and Leonard C. EricksonDepartment of Pharmacology & Toxicology and IU Simon cancer center
Abstract
Pre-treatment MDA-MB -435 cell IC50 of MTX (µM) RFa IC50 of DOX (µM) RFa
NoneWT 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*
PSC833(3mg/ml)
WT 0.2 ± 0.04 1.0 0.57 ± 0.04 1.0
MDR 0.2 ± 0.00 1.0 3.71 ± 0.47 6.5*
Cytoxicity of mitoxantrone and doxorubicin and modulation of drug resistance by PSC833 in human MDA-MB-435 cancer cells
*Statistically significant difference (P < 0.05) in drug resistancea Resistance factor: IC50 of mitoxantrone and doxorubicin in MDR cells divided by IC50 of same drug in the WT cells.
Accumulation of mitoxantrone in the nuclei of MDA-MB-435 cells
50
60
70
80
90
100
110
0 30 60 90 120 150 180
Time (min)
Flu
oresc
en
t in
ten
sity
mdr nuc
wt nuc
mdr+PSC833
Accumulation of mitoxantrone in the cytoplasm of MDA-MB-435 cells
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80
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120
0 30 60 90 120 150 180
Time (min)
Flu
ores
cent
inte
nsit
y
mdr cyto
wt cyto
mdr+PSC833
5 uM MTX
3ug/ml PSC833
WT
MDR
5 µM MTX 3 µg/ml PSC833 + 5 µM MTX
*
0
20
40
60
80
100
120
140
WT cells
MDR cells
N= 15 cells/bar
Flu
ores
cen
t in
ten
sity
of
MT
X
Mitoxantrone accumulation in MDA-MB-435 cells
Pretreatment MDA-MB-435 tumors
Fluorescent Intensity of MTX
(Mean ± SE)
% of WT tumors
Fluorescent Intensity of DOX
(Mean ± SE)
% of WT tumors
NoneWT 91.1 ± 27.3 100 75 ± 4.7 100
MDR 38.0 ± 5.4 42* 51 ± 6.0 68*
PSC833WT 81.2 ± 12.6 100 80 ± 4.6 100
MDR 75.8 ± 5.3 94 76 ± 6.3 94
Florescent intensity of Mitoxantrone and Doxorubicin in MDA-MB-435 Xenograft Tumors
*Statistically significant difference (p < 0.05) in fluorescent intensity of doxorubicin in MDA-MB-435wt tumors and MDA-MB-435mdr tumors.
Summary:
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.
MDR
WT
Flu
ores
cen
t in
ten
sity
of
Dox
5 uM DOX
*
3ug/ml PSC833
0
10
20
30
40
50
60
70
80
90
100Wt cells
mdr cells
3 µg/ml PSC833 + 5 µM MTX5 µM MTX
Doxorubicin accumulation in MDA-MB-435 cells
MDR
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