Development of rational in vitro models for drug resistance in breast cancer and modulation of MDR by selected compounds.
BACKGROUND
[corrected] The effectiveness of chemotherapy is limited by the emergence of multidrug resistance (MDR). MDR is caused by the activity of various ATP binding cassette (ABC) transporters that pump anticancer drugs out of the cells in an ATP-dependent manner. Additionally some other cellular mechanisms of MDR have been reported. The purpose of this study was to investigate mechanisms of MDR in drug resistant MCF-7 cell lines and to modulate P-glycoprotein (P-gp) and MRP1-based MDR.


METHODS
Paclitaxel (MCF-7/Pac), docetaxel (MCF-7/Doc), doxorubicin (MCF-7/Dox) and vincristine (MCF-7/Vinc) resistant sublines were developed from the parent MCF-7 cell line (MCF-7/S) by stepwise selection in dose increments over two years. Flow cytometry, MTT cytotoxicity assay, RT-PCR, caspase-3 activity assay and checkerboard combination assay were performed to investigate the degree of resistance developed in sublines and to reverse drug resistance phenotype.


RESULTS
The flow cytometry histograms of drug accumulation assays demonstrated that the drug-resistant cell lines are P-gp and MRPI positive. RT-PCR results showed that the resistant sublines express both MDR1 and MRP1 genes. Resistance indices of each subline to each anticancer drug were determined using the MTT cytotoxicity assay and it was found that all the sublines were resistant to their respective agents. Caspase-3 activities of the cell lines were also determined. Caspase-3 activity is an important indicator of apoptosis in the cell. The reversal of MDR was attempted by two cinnamylidene ketone and two organosilicon compounds. The results indicated that these compounds modulated P-gp effectively, but they were not very effective at reversing MRP1 activity in the MCF-7 sublines. Four selective anticancer drugs (paclitaxel, docetaxel, doxorubicin and vincristine) and four synthetic MDR modulators [2-(2-methoxycinnamylidene) indan-1-one (cinnamylidene-1), 2-(2- methoxycinnamylidene)-3, 4-dihydro-2H-naphthelen-1-one) (cinnamylidene-2), ALIS 409 and ALIS 421] were applied to the sublines in combination. The fractional inhibitory indices illustrated that combined applications of cinnamylidene ketones and organosilicon compounds with paclitaxel, docetaxel or vincristine exerted significant antiproliferative effects on the resistant sublines.


CONCLUSIONS
This report demonstrates the development of rational models for drug resistance MCF-7 cell lines and reversal of acquired drug resistance.
Kars
Meltem Demirel
MD
Middle East Technical University, Department of Biological Sciences, 06531, Ankara, Turkey.
Iseri
Ozlem Darcansoy
OD
Gündüz
Ufuk
U
Ural
Ali Ugur
AU
Arpaci
Fikret
F
Molnár
József
J
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000502:physiology; D000970:Antineoplastic Agents / Q000627:therapeutic use; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000473:pathology; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000627:therapeutic use; D019008:Drug Resistance, Neoplasm; D005434:Flow Cytometry; D006801:Humans; D008954:Models, Biological; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000502:physiology; D017239:Paclitaxel / Q000627:therapeutic use; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014750:Vincristine / Q000627:therapeutic use
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D004317:Doxorubicin; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D017239:Paclitaxel; D014750:Vincristine; C421109:multidrug resistance-associated protein 1