|M.Sc Student||Renana Fischer-Weinberger|
|Subject||Active and Passive Transport of Anticancer Drugs in Cell|
|Department||Department of Biology||Supervisor||Professor Eytan Gera|
|Full Thesis text|
Multidrug resistance (MDR) is major impediment to a successful treatment of many human cancers. It is a resistance exhibited by malignant cells to a wide variety of anti-cancer chemotherapeutic drugs.
The main goal of our work is to investigate the role played by the membrane’s leaflets in the cellular uptake of dyes and drugs and in their extrusion from the cells by P-glycoprotein or MRP1.
For this purpose, the dyes or drugs accumulation was followed and measured via fluorescence resonance energy transfer (FRET) from1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene (TMA-DPH) located at the outer leaflet to the drugs or dyes located either at the outer or the inner leaflets of the membrane.
Initially, we have used the MDR probe- rhodamine 6G. Then we extended the research to drugs (anthracyclines).
Similar conclusions were obtained for rhodamine 6G and for the anthracyclines. Slow flip-flop rate resulted in agent accumulation mainly in the outer leaflet of the membrane. The kinetic barrier in their uptake was the transbilayer movement across the membrane.
High flip-flop rate resulted in agent accumulation in the outer and the inner leaflets simultaneously. The accumulation rate in the inner leaflet was higher than in the cytoplasm. Therefore, there is an additional kinetic barrier- between the inner leaflet of the membrane and the cytoplasm.
Intermediate flip-flop rates resulted in gradual accumulation in the inner leaflet of the membrane. There are two kinetic barriers in their uptake into the cells: the flip-flop across the membrane and their release from the inner leaflet into the cell’s interior.
The accumulation of rhodamine 6G in the inner leaflet of the membrane was delayed by its active uptake into respiring mitochondria. In addition, the accumulation of daunorubicin was prevented by its uptake into the nucleus. Since there are no reports of direct contact of mitochondria or nuclear DNA with the membrane, we have to assume that they accumulate the daunorubicin or rhodamine not directly from the membrane, but from the cytoplasm adjacent to the membrane. Therefore, the secondary barrier is not located at the inner surface of the membrane, but rather at a location more interior.
Pgp was found better in removing rhodamine 6G or anthracyclines from the inner leaflet of the membrane when their accumulation there was gradual, and it probably removed them also from the cytoplasm when their accumulation in the inner leaflet was instantaneous. MRP1 was found better in removing rhodamine 6G from the cytoplasm.