The Multi-Drug Resistant Mechanism And Reversal Of Mifepristone In Homoharringtonine-Multidrug-Resistant Human Leukemia Cell Line K562/HHT

Leukemia is one of the most common malignant tumors. Chemical therapy is the important way to treat leukemia. But multidrug resistance(MDR) has handicapped the effect of chemical therapy. Homoharringtonine(HHT) is a cephalotoxin alkaloid with anti-leukemic activity and had been used successfully in the treatment of acute myeloid leukemia. However, the drug resistance affected its effect. At present, the mechanism of resistance to HHT was unknown and it is essential to establish a drug resistant cell line for researching the mechanism of MDR. In this paper, K562 cell line was induced into MDR cell line K562/HHT by drug HHT. We also researched the characteristic, mechanism of drug resistance and reversal of drug resistance of K562/HHT. The major research procedures and results are as follows:1. Establishment of Homoharringtonine-multidrug-resistant cell line K562/HHT and its biological characteristics Human leukemia cell line K562 was induced into MDR cell line by intermittent administration of high dose of HHT. MTT assay was used to detect the sensitivity of these MDR cells to all sorts of chemotherapeutic agents. The result showed this MDR cell line possessed the ability of 462.6 fold resistance to HHT and cross-resistance to adriamycin, vincristine and etoposide. RT-PCR was used to detect the expression of mdr1 gene and glucosylceramide synthase (GCS) gene in these MDR cells. The result showed that the expression of mdr1 gene and GCS gene was increased in K562/HHT cells than in K562 cells. Flow cytometry was used to detect the expression of P-glucoprotein and the accumulative value of intracellular daunorubicin(DNR) in these MDR cells. The result indicated that the expression of P-glucoprotein was increased in K562/HHT cells than in K562 cells, while the accumulative value of intracellular DNR was decreased in K562/HHT cells. Immunohistochemistry was used to detect the expression of bcl-2, bax and caspase-3 in these MDR cells. The result indicated that the bcl-2/bax ratio was increased in K562/HHT cells than in K562 cells and the caspase-3 expression was decreased. The drug resistance of K562/HHT had no significant change after 6 months, which suggesting that this subline be classically multidrug resistance and could be good experimental modal for study of multidrug resistance mechanism of leukemia.2. Effects of progestogen antagonist mifepristone reversing multidrug resistance of K562/HHT cellsTo identify the effects of mifepristone (RU486) reversing multidrug resistance of K562/HHT and its mechanism, we observed effects of mifepristone on proliferation of K562/HHT and mifepristone reversing multidrug resistance of K562/HHT. The results indicated that RU486 could reverse multidrug resistance of K562/HHT cells. Flow cytometry was used to detect the accumulative value of intracellular daunorubicin(DNR) in these MDR cells with or without RU486. The result indicated that accumulative value of intracellular DNR was increased after the effect of RU486. Immunohistochemistry was used to detect the expression of bcl-2, bax and caspase-3 in these MDR cells after the effect of RU486. The result indicated that the bcl-2/bax ratio was decreased and the caspase-3 expression was increased after the effect of RU486. So RU486 may be a potential reversal agent of multidrug resistance for leukemia.