טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentDaniella Yeheskely-Hayon
SubjectThe Role of Intracellular Binding of Chemotherapeutic
Drugs in Drug Uptake and MDR
DepartmentDepartment of Biology
Supervisor Professor Eytan Gera
Full Thesis textFull thesis text - English Version


Abstract

Multidrug resistance (MDR) is a resistance exhibited by cancerous cells to a wide variety of structurally and functionally unrelated anticancer chemotherapeutic drugs.

The main goal of the present study is to elucidate the potential effect of the intracellular binding of drugs to the active efflux by MDR pumps.

For this purpose, we studied the uptake of TMRM, a rhodamine dyes, which serve as an MDR probe. Rhodamines have previously shown to accumulate in the mitochondria. Therefore, the role played in MDR by intracellular binding can be studied by comparison of TMRM uptake and extrusion in the presence and the absence of mitochondrial inhibitors.

Surprisingly, TMRM was prevented from entering cells presumed to be sensitive to MDR-type drugs, in the presence of mitochondria inhibitors. An efflux mechanism active in these cells was inhibited by NBD-Cl and MK571 and to a lesser extent by ATP depletion, indomethacin, probenecid, and vanadate. All the inhibitors apart from NBD-Cl, are known to modulate MRP1. Since MRP1 was expressed in all the cell lines tested and the MRP1-mediated efflux was inhibited by NBD-Cl, both in sensitive and MRP1 over expressing cells, the innate resistance is mediated by MRP1. Surprisingly, the basal levels of MRP1 expressed in sensitive cells whose mitochondria were inactivated prevent uptake of even high concentrations of TMRM. Thus, basal expression levels of MRP1 cannot compete with TMRM tightly bound to internal sink.

We have extended our research to the anticancer drug family of anthracyclines which are used often in anticancer chemotherapy and revealed that MRP1 mediates anthracyclines resistance in a variety of sensitive cells. Moreover, a negative correlation between MRP1 success and the affinity of intracellular binding sites was demonstrated by a comparison of the uptake of the anthracycline, daunorubicin to its enantiomer WP900 which does not bind to the DNA, and by preventing anthracyclines sequestration into lysosomes. While basal level of MRP1 expressed in sensitive cells could not compete with intracellular binding of anthracyclines, overexpressed MRP1 successfully competed with these high affinity binding sites. Thus, the success of MRP1 in drug extrusion from cells depends not only on competition with drug influx, but also on competition with intracellular drug binding.

We have demonstrated that the innate resistance, mediated by MRP1, is not unique to cancer cells, but exists in non malignant tissues as well. We suggest that MRP1 may play an important role in protecting non malignant tissues from drug side effects.