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

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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
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.
Florescent intensity of Mitoxantrone and
Doxorubicin in MDA-MB-435 Xenograft Tumors
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
None MDR 38.0 5.4 42 51 6.0 68

PSC833 WT 81.2 12.6 100 80 4.6 100
PSC833 MDR 75.8 5.3 94 76 6.3 94
Cytoxicity of mitoxantrone and doxorubicin and
modulation of drug resistance by PSC833 in human
MDA-MB-435 cancer cells
Pre-treatment MDA-MB -435 cell IC50 of MTX (µM) RFa IC50 of DOX (µM) RFa

None WT 0.2 0.01 1.0 0.60 0.04 1.0
None 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
PSC833 (3mg/ml) MDR 0.2 0.00 1.0 3.71 0.47 6.5
Statistically significant difference (p lt 0.05)
in fluorescent intensity of doxorubicin in
MDA-MB-435wt tumors and MDA-MB-435mdr tumors.

• Summary
• Human MDA-MB-435mdr cancer cells were 8- and
  9-fold more resistant to mitoxantrone and
  doxorubicin than the wild type cells.
• Accumulation of mitoxantrone and doxorubicin in
  MDA-MB-435mdr cells were 33 and 19 of that in
  the wild type cells.
• 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.
• Accumulation of mitoxantrone and doxorubicin in
  MDA-MB-435mdr xenograft tumors were 42 and 68
  of that in the wild type xenograft tumors.
• Pgp inhibitor, PSC833 increased the accumulation
  and uptake of mitoxantrone and doxorubicin in
  MDA-MB-435mdr cells.

Statistically significant difference (P lt 0.05)
in drug resistance a Resistance factor IC50 of
mitoxantrone and doxorubicin in MDR cells divided
by IC50 of same drug in the WT cells.